Patent Application
20080076670
The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
In the drawings:
a-d are schematic illustrations showing the construction of a programmable artificial receptor device according to a second preferred embodiment of the present invention.
a-d are a sequence of schematic illustrations showing successive stages in the manufacture of electrode layers to enable each layer to have a separate electrical contact. Note the receding shutter whose purpose is to leave exposed conducting segments of the conducting layers for later electrical contact;
a-c depict selective binding of the scFv fragment to the crystalline facets.
The present invention relates to an artificial receptor capable of binding specific biological moieties, and more particularly, to methods of using same for typing ligands, determining binding domains in proteins, targeted delivery of drug molecules, electronic capture and release of drugs, and electronic triggering and suppression of biological reactions such as gene expression.
The principles and operation of an artificial receptor device according to the present invention may be better understood with reference to the drawings and accompanying descriptions.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
Specific binding of biological moieties (i.e., biomolecules) to artificial substrates has been previously shown. For example, in recent years, following the work by S. Brown (S. Brown, 1997, Nature Biotechnolog 15: 269-272) several authors have shown that the remarkable molecular recognition capabilities of peptides can be harnessed to the specific recognition of non-biological entities and to the direction of metallic and semiconducting nano-particles synthesis (For a recent review see M. Sarikaya et al., 2003, Naturematerials 2: 577-585). Using combinatorial phage and cell-surface display libraries researchers were able to evolve peptides that bind selectively to different crystallographic planes of gold (Brown S., 2000, J. Mol. Biol. 299: 725-732), silver (Naik R. R. et al., 2002, Nature materials 1: 169-172), silica (Naik R. R. et al, 2002, J. Nanosci. Nanotechnol. 2: 1-6), zeolites (Scembri, M. A., et al., 1999, FEMS Microbiol. Lett. 170: 363-371), metal oxides (S. Brown, 1992, Proc. Natl. Acad. Sci. USA 89: 8651-8655; K. Kjacrgaard et al., 2000, Appl. Env. Microbiol. 66: 10-14), minerals (Gaskin, D. J. H., et al., 2000, Biotech. Lett. 22, 1211-1216), C60 (Braden, B. C. et al., 2000, PNAS 97 12193) and carbon nanotubes (Wang, S., et al., 2003, Nat. Mat. 2: 196). The selected peptides or phage display peptides displayed good discrimination between different crystal types and planes. Moreover some of them were found to catalyze the growth of specific crystal structures. It is anticipated that the evolved peptides will facilitate defect free, low temperature synthesis and assembly of inorganic materials for nanotechnological applications. Indeed, very recently such peptides, displayed on a virus, were successfully utilized to direct synthesis of semiconducting and magnetic nano-wires (Mao C., et al., 2004, Science 303: 213-217). Other studies by Whaley (Whaley, S. R. et al., 2000, Nature 405, 665) and. Lee (Lee, S -W., et al., 2002, Science 296: 892 (As and zinc sulfidedemonstrated differential binding of phage display peptides to specific facets of Ga (ZnS)semiconductors. However, when the amino acid sequence of one of the isolated phage display peptides was further synthesized and tested for its binding ability to GaAs facets, no selectivity was shown between the (100) and the (110) facets (Goede K., Nano Letters, 4, 2115-2120, 2004; specifically
It is noted that peptide selectivity to crystal structure and orientation implies that even atomically flat surfaces provide enough details for selectivity. It is important to appreciate that the distance between the binding sites on the peptide are longer than the crystal lattice constant.
Several groups have demonstrated in recent years electrical control over the electrostatic interaction between surfaces. Using AFM to measure forces they could control the repulsion between a charged colloid and a surface, and even turn the repulsion into attraction. More recently, two groups demonstrated electrical control over surface hydrophobicity and its turning into mild hydrophilicity. Electrical control over the electrostatic interaction between a charged colloid and a semiconducting surface is now available, tuning it continuously between repulsion and attraction.
Modeling of e.g. gold binding peptides teaches that the separation between binding sites typically measures 1-2 nanometers. Present fabrication technologies, most notably the Focused Ion Beam, are at the verge of this scale and may eventually provide such features. The more stringent constraint might potentially be a requirement for atomically accurate distance between any two binding sites.
However, to date the use of artificial surfaces has never been suggested for gaining electrical control over biological processes.
While reducing the present invention to practice, the present inventors have uncovered that individually contacted nano-scale electrodes, each biased to an individual set potential, can be used as artificial receptors capable of binding biological moieties and that such receptors can be used in any application which is based on molecular recognition involving electrostatic interactions such as to type ligands, identify binding domains of proteins and target delivery of drugs.
As is shown in Examples 1-4 of the Examples section which follows, the present inventors have shown that a specific facet of a semiconductor can differentially bind phage display antibodies as well as soluble antibodies (which are detached from the phages) and thus can be used to control the binding of biological moieties thereto.
Thus, according to one aspect of the present invention there is provided an artificial receptor. The artificial receptor comprising a non-biological surface having an extent, the surface having unique surface electrical properties that vary over the extent, the electrical properties being such as to configure an electrical field about the surface to provide specific binding for a target moiety.
The phrase “artificial receptor” which is interchangeably used herein also as “an electrode” refers to a specific device made of a material (e.g., crystal) such as a semiconducting or a conducting material, which is configured to exhibit a unique surface electrical properties to provide specific binding as described hereinbelow for a target moiety such as a target biological moiety.
The embodiments use electric fields which are configured to specifically bind biological moieties. A first embodiment has a permanent electric field pattern preset for a specific molecule. A second and a third embodiment are programmable to bind and release specific molecules at pre-determined times.
Referring now to the drawings,
Surface 12 of device 10 preferably includes a plurality of regions 16-26, each having a predetermined electrostatic field strength. Regions 16-26 within device 10 are constructed with built-in static electric fields that between them give the overall contour 14. The unique contour 14 is capable of binding a specific biological moiety as described hereinbelow (e.g., a protein). More particularly, the field binds a specific sub-region of the protein just as an antibody binds an antigen (e.g., epitope) or an enzyme's active site binds a corresponding site on a substrate.
It will be appreciated that since the specific interaction between biological moieties such as proteins often involves a unique three-dimensional binding site having a size of a few nanometers up to a few Angstroms, the regions defining the surface of the artificial receptor of the present invention are preferably in the Angstrom order of magnitude, more preferably, in the nanometer or tens of nanometer order of magnitude, so as to enable specific binding of biological moieties thereto.
Surface 12 of device 10 can be made of various materials having selected electrostatic properties. Non-limiting examples of such materials include ceramics and semiconductors (e.g., crystals or polycrystals such as PZT, GaAs and silicon). It will be appreciated that when a specific region is made of a crystal, the size of the region is preferably in the magnitude of five to ten lattice constants.
For example, device 10, is preferably constructed from ceramic bearing ferroelectric particles, allowing the ceramic to be pre-electrified with the desired field strength.
The result is an artificial receptor whose surface has defined and unique electrical properties that vary over its extent, the properties giving rise to an electrical field over the surface which provides specific binding for the target moiety such as a biological moiety or a chemical moiety (e.g., polymer and/or small molecule).
It should be appreciated that the electrodes need not be planar. They may comprise, for instance, carbon nanotubes and/or silicone nanowires sticking out of the plane. The same is true for the gaps between electrodes.
Binding of the biological moiety to the surface is of a proximity and orientation which mimics the equivalent biological binding pair and the resultant affinity is of a KD range of preferably 10−5-10−15 M, preferably at least 10−6 M, preferably at least 10−7 M, preferably at least 10−8 M, preferably at least 10−9 M, preferably at least 10−10 M preferably at least 10−11 M, preferably at least 10−12 M, preferably at least 10−13 M, preferably at least 10−14 M, preferably at least 10−15 M.
It will be appreciated that determination of such affinity can be performed using methods known in the art, such as, by scatchard analysis.
It will be appreciated that to enable the binding of a wide selection of molecules (e.g., biological moieties or chemical moieties) to the artificial receptor of the present invention and to control the binding of such molecules, the artificial receptor of the present invention is preferably programmable to provide different electrical fields as desired.
Such programming can be achieved by configuring at least one electrode capable of being biased to a unique electrical property.
An example for such configuration is presented in
d illustrates an insulating coating layer 32, which may be applied on the surface of the artificial receptor. Cavitations 34, which are holes in the insulating coating allow the binding of the molecules to the surface. An example of coating layer 32 is a glass. Preferably cavitations 34 are in the Angstrom or nanometer scale.
The glass is preferably passivated against protein binding. Using conventional methods in microelectronics the A layers are all contacted electrically away from the cleaved edge. The result is a large array of cavitations in the coating layer, each over an A/B interface. The conducting, A side of all spots can then be biased relative to the solution. The exposed A/B interface is the target for the antibodies. Since the peptide binding sites are typically either charged or polarized, the local electrostatic potential created by a different biasing of the A electrode should affect differently various antibody molecules.
The surface of the artificial receptor may comprise zero dimension, one dimension, two dimensions and three dimensions.
The surface of the artificial receptor may comprise electronically controllable hydrophobic coating to allow controllable hydrophobic properties for each independently electrifiable layer.
According to preferred embodiments of the present invention, variable electrical fields are applied to the regions. Such electrical fields can be controlled by adjusting the voltage and/or current supplied to the electrode or the layer as described hereinabove and those of skills in the art are capable of controlling the electrical fields.
Thus, the electrically biased artificial receptor is capable of binding various targets depending on the resultant electrical field provided at each time.
According to preferred embodiments of the present invention, the surface comprises switchable wiring, such wiring being switchable to provide the unique electrical properties. Preferably, such wiring is variably switchable, thereby providing specific binding to different target biological moieties as desired.
Reference is now made to
Preferably, the wafer layers are connected to the switching device or selector 38, which allows voltage levels to be electronically selected for each of the independently electrifiable layers. The electrical field may thus be varied to provide specific binding to different targeted biological moieties. Typically an overall power source 40, which may be a battery or a main fed power supply, supplies a series of voltage regulated power sources 42.1 . . . 42.n, each set at different voltage levels. A switching matrix 44 then connects any one of the layers 46.1 . . . 46.n to any one of the regulated power sources. Preferably the switching matrix is controlled by software. It will be appreciated that the switching device described with respect to
An alternative approach to control the microscopic electric field landscape of the artificial receptor of the present invention relies on the use of materials which upon application of an electric field, their unit cell or molecular structure is changed, resulting in a change in the electric field produced thereon. Such materials are for example, ferroelectric substances (e.g., PLZT) which exhibit high dielectric constants and are therefore changeable following application of changes in their electric field.
One example for a ferroelectric material is a Perovskite-like crystal, in which a high valence cation is encapsulated in an oxygen octahedron. The oxygen together with the A atoms form a face center cubic (fcc) crystal with the latter atoms at the corners. The high temperature phase is cubic and, hence, lacks electric moments. As the temperature is reduced the material may undergo a series of structural phase transitions to lower symmetry structures accompanied by large local electric moments. Since the central cation has a large charge and relatively broad energy minima, the electrical susceptibility is very large and the dielectric constant can approach values as high as 1000-5000. The corresponding polarization fields are enormous. One such crystal, PLZT, is particularly attractive for the scope of the present invention. At a ratio of 9/65/35 the virgin ceramics maintains an isotropic cubic phase. An application of a moderate electric field shifts the crystal to the rhombohedral or tetragonal phases characterized by enormous local electric dipoles. When the external field is removed the polar phase relaxes instantaneously back to its unpolarized cubic phase. The magnitude of the generated dipoles depends on the applied field. The large dielectric constant guarantees extremely large local electric moments.
For example, as described in
In the preferred embodiment, different target molecules (e.g., antibodies) bind selectively to the same crystal under different applied fields and released under other fields.
The physics of PLZT is fully understood and the phase diagram as a function of composition, temperature, and applied field is tabulated.
It will be appreciated that the surface of the artificial receptor of the present invention can be further modified by attaching materials or molecules capable of modifying the electrical property of the surface, as well as the hydrophilic or hydrophobic properties of the surface which may affect the capacity of the surface to form hydrophobic interactions, hydrogen bonding and van der Waals interactions with biological moieties.
Several types of substances, molecules and/or monolayers of molecules can be used to change the electric field of the surface according to this aspect of the present invention. These include molecules and materials which following the application of an electric field, mechanical stress and/or change in a temperature are capable of modifying the electric field generated thereupon. Substances which may affect the hydrophobic or hydrophilic properties of the surface may be, for example, charged peptides, phospholipids and the like, which following the application of an electric field can fold or change their relative orientation with respect to the surface.
For example, organic molecules such as hydroquinone, Rotaxane and charged organic (e.g., ferrocene) or biological polymers, undergo atomic and/or molecular changes following the application of an electric field.
Hydroquinone looses two hydrogen atoms following the application of an electric filed. The hydroquinone molecule may be attached to a substrate via an alkane tail. In either case the molecule may be switched between two stable states by electro-protonation. An antibody molecule selective to one of the configurations is attracted or released from the Hydroquinone depending on its state. The Hydroquinone transduces in this case the electronic signal to a change that is readily recognizable by antibodies.
Rotaxane is a linear dumbbell shaped molecule inserted into a mobile molecular ring having two redox states. The ring may rest in one of two positions along the molecule depending on the oxidation state. The latter is controlled by application of a bias between the substrate to which the dumbbell molecule is bound and the solution. It is very likely that antibodies can be selected to the two different configurations of the molecule, namely for the two positions of the ring along the molecule, hence providing an electrical control over which antibody binds the surface.
The ferroelectric materials (e.g., PLZT) which are described hereinabove represent an example of non-organic materials which upon the application of electric field exhibit structural changes that affect the electric field and, hence, the target for antibodies and other bio-molecules.
Pyroelectric materials can also be used to modify the electric field of the surface of the artificial receptor of the present invention. Following the application of a temperature change, the material undergoes structural changes of the unit cell or a molecular change which result in a modified electric field. Non-limiting examples for such materials which can be used along with the present invention include, Li—Ta—O3 and triglycine sulfate (TGS).
Piezoelectric materials can also be used to modify the electric field of the surface of the artificial receptor of the present invention. Following the application of electric voltage, the material undergoes conformational changes which result in a change in the unit cell. Reversal to the original conformation usually requires the application of an opposite voltage since the material is somewhat hysteretic. Non-limiting examples for such materials which can be used along with the present invention include, PZT and polyvinylidene fluoride (PVDF).
Ferroelastic materials can be also used to modify the electric field landscape of the surface of the artificial receptor of the present invention. Following the application of mechanical stress, the material undergoes conformational changes which result in a change in the electric field. A non-limiting example for such materials which can be used along with the present invention is LaAlO3.
It will be appreciated that several biological moieties can be also attached to the surface of the artificial receptor of the present invention in order to modify its electric field. For example, peptides can be attached to the surface and following the application of an electric field the peptide can change its configuration, e.g., can form a cyclic molecule or can attach along the surface. Such a change in configuration results in a change of the electrical properties of the surface or hydrophobic nature, or display of certain groups which can be used to selectively bind biological moieties.
It will be appreciated that such materials and substances which following structural and molecular changes (as a result of mechanical stress, temperature change and/or electric field change) are capable of modifying their electric field can be used to form the regions of the artificial receptor instead of the crystals or semiconductors.
While further reducing the present invention to practice, the present inventors have devised an artificial receptor having a surface with switchable electrical conductive tracks, the conductive tracks being switchable to configure an electrical field about the surface to provide specific binding for a target moiety (e.g., a target biological moiety).
According to preferred embodiments of the present invention, the artificial receptor further comprises a switching control for switching the conductive tracks.
Reference is now made to
Device 50 may include additional elements such as a covering layer over the semiconductor surface, as described above.
Elaborate Artificial Receptor (Electrode) Set
It will be appreciated that for activation or suppression of a single bio-process or even for the selection between two bio-processes a single electrode with two states is enough. For selection between multiple pathways or activation/suppression of multiple processes an elaborate electrode set with numerous biasing configurations may be required. The electrode size and separation may vary according to the application. Electrodes larger than ˜10 nanometer may be fabricated by conventional methods of micro and nanoelectronics such as electron beam lithography and focused ion beam. Smaller electrodes and/or smaller spacing between electrodes, still with an individual electrical contact to each of the electrodes can be realized by the utilization of metal/metal oxide alternating layers grown by molecular beam epitaxy. Separate contacts to the individual layers can be achieved by using a moving shutter during growth and later use of photolithography to contact the exposed metallic layers, this being illustrated in
An alternative approach relies on extension of the distance between the biological binding sites. The simplest construction would be an IgG antibody composed of two halves, each selective to a different bias. Such a construction should be selective to two electrodes spaced by a few nanometers. If two antibodies are fused at their tails (as occurs naturally with IgA) that distance can increase to 10-20 nm. The latter distance is easily accessible by present nanotechnology and it should be possible to contact independently two interfaces spaced by such a distance. One may obviously utilize other artificial or natural constructs like IgM pentamers.
Floating Electrodes
The electrodes as described above need not necessarily be fabricated on a supporting substrate nor need they be exclusively biased by an external power supply, although in several preferred embodiments they are so biased. In one embodiment the electrodes may comprise a microscopic p-n junction realized in a semiconducting nanocrystal. Due to the rectifying nature of such a junction, application of radiation to the nanocrystal results in generation of bias between the two poles of the p-n junction. This bias is equivalent to an external bias applied to electrodes by connecting them to an external source. The possibility to apply bias from a distance by radiation at a desired timing facilitates, for instance, drug release by external radiation. For that the drug is fused to the antibody and the latter is released by the induced voltage as described above.
Other bias sources include, e.g. a microscopic battery or even a chemical cell whose output depends on certain biochemical parameters, such as pH. A source like that may trigger antibody binding or release from the electrodes based on the presence of certain chemicals. Such an arrangement facilitates e.g. localized drug release depending on certain biochemical conditions.
The devices (or the electrodes) described hereinabove can be connected together in any desired way in order to provide three-dimensional fields. Furthermore, numerous devices (or electrodes) may be connected together to form arrays. Thus, the electrodes in the array may be identical, designed to fish out the maximum number of a target molecule, or the arrays may comprise electrodes with different fields or electrodes programmed differently, so that a range of target molecules can be searched for.
The following section describes possible embodiments of the electrodes set. The processes mentioned in the text are known to practitioners in the fields of micro and nanoelectronics, crystal growth, surface chemistry, etc.
Electrodes material—The electrode material used by the method of the present invention can be any type of material or materials combination. Following is a list of preferred materials:
(a) Metal electrodes—deposited on an insulating substrate (planar geometry) on a substrate such as glass, alumina, sapphire, etc. Examples for metals include gold, platinum, silver, aluminum, etc. The electrodes may be defined either by first depositing or epitaxially growing a metal layer (e.g. by molecular beam epitaxy, chemical vapor deposition, atomic layer deposition, electrochemistry) and then patterning it by e.g. electron beam lithography or focused ion beam (FIB). Alternatively, the electrodes may be deposited or grown on the substrate already in their patterned form, for instance by patterned epitaxial growth or by FIB deposition.
(b) Semiconductor electrodes—deposited on an insulating substrate (planar geometry) on a substrate such as glass, alumina, sapphire, etc. Examples for. semiconductors include silicon, GaAs, InAs, CuO, etc. The electrodes may be defined either by first depositing or epitaxially growing a semiconductor layer (e.g. by molecular beam epitaxy, chemical vapor deposition, atomic layer deposition, electrochemistry) and then patterning it by e.g. electron beam lithography or focused ion beam (FIB). Alternatively, the electrodes may be deposited or grown on the substrate already in their patterned form, for instance by patterned epitaxial growth or by FIB deposition.
(c) Conducting polymers electrodes—deposited on an insulating substrate (planar geometry) on a substrate such as glass, alumina, sapphire, etc. Examples for conducting polymers include PPV, polyanilin, etc. The electrodes may be defined either by first depositing or epitaxially growing a polymer layer (e.g. by molecular beam epitaxy, chemical vapor deposition, atomic layer deposition, electrochemistry) and then patterning it by e.g. electron beam lithography or focused ion beam (FIB). Alternatively, the electrodes may be deposited or grown on the substrate already in their patterned form, for instance by patterned epitaxial growth or by FIB deposition. The conducting polymers may be deposited or grown either parallel to the surface or angled to it.
(d) Semiconductor superlattice (vertical geometry)—Alternating layers of various semiconductor materials are grown on a substrate. In the simplest embodiment depicted in
(e) Metal/insulator superlattice (vertical geometry)—Alternating layers of various metals and insulating layers, e.g. metal oxides or ceramics, are grown on a substrate. In the simplest embodiment depicted in
(f) Conducting polymers (vertical geometry)—Alternating layers of various conducting polymers and insulating layers, e.g. metal oxides, ceramics, or insulating molecules and polymers are grown on a substrate. The structure may be grown by molecular beam epitaxy (MBE), chemical vapor deposition (CVD), metalo-organic molecular beam epitaxy (MOMBE), liquid phase epitaxy (LPE), chemical deposition, electrochemistry, atomic layer deposition, etc. The wafer is then cleaved as described in
(g) Molecular conductors and semiconductors—like carbon and other nanotubes provide in a natural way nanometer scale electrodes. Carbon nanotubes can be spin coated on an insulating layer or grown from metal catalysts. A particular attractive approach in the growth of multiple tubes from patterned catalysts islands. Other molecular conductors like sericonductor nanowires, nanorods, and dots can be used. The latter include also self assembled semiconductor dots defined by strain on a semiconductor substrate.
(h) Functionalized or unfunctionalized biomolecules—see E. Braun, Y. Eichen, U. Sivan and G. Ben Yoseph, DNA templated assembly and electrode attachment of conducting silver wire, Nature, 391, 775 (1998); K. Keren, M. Krueger, R. Gilad, G. Ben-Yoseph, U. Sivan and E. Braun, Sequence-Specific Molecular Lithography on Single DNA Molecules, Science, 297, 72 (2002); and K. Keren, R. S. Berman, E. Buchstab, U. Sivan, and E. Braun, DNA-Templated Carbon-Nanotube Field Effect Transistor, Science 302, 1382 (2003), which are fully incorporated herein by reference.
Ferroelectric and high dielectric constant electrodes—Ferroelectric and high dielectric constant ceramics and organics provide an efficient way for the creation of large local electric moments. The principle is presented hereinabove and in
Electrode functionalization and coating—The electrodes can be functionalized by biological, non-biological, or organic molecules. The latter may serve to modify the surface properties such as hydrophobicity/hydrophilicity, charge, stability, roughness, compatibility with the solutions and the molecules in solution, non-specific binding, etc. Functionalization may also provide an electronic control over surface properties as detailed e.g. in Frechette and Vanderlick, Langmuir 17, 7620 (2001); Barten et al. Langmuir 19, 1133 (2003). The electrodes can also be coated with polymers, gels, etc. for protecting them or the antibodies against chemical processes such as oxidation or reaction and for increasing the effective electrode area. Certain substances such as agarose provide a convenient environment to the biomolecules. The electrodes can also be modified with thin insulating layers such as silica and alumina. They can also be covered with colloids and beads.
Contacting individual electrodes—Individual electrodes are contacted by conventional microelectronics techniques. In the case of semiconductor superlattice access to the individual conducting layers may be provided either by post-growth selective etching or by masking parts of the layers during the crystal growth as depicted in
Thus, as described hereinabove, the artificial receptor of the present invention can be configured using any material/substance and method known in the art.
As mentioned, the artificial receptor surfaces of the present invention can interact with biological moieties (binding molecules). Such interaction can be monitored by an atomic force microscope (AFM) adapted for force measurements. For example, the antibody or peptide are attached to the AFM tip and substrate, respectively, and the tip deflection is monitored as a function of the separation between the tip and the artificial receptor for different fields applied between the substrate and/or tip and the solution. Since the tip spring constant is measured independently, the deflection can be translated directly to force.
It will be appreciated that the artificial receptor of the present invention can specifically bind a biological moiety. As is shown in Table 2 and is described in Example 3 of the Examples section which follows, while the GaAs (100) surface specifically bound the F10 and D3 clones, the GaAs (111A) surface specifically bound the E1, F1, C7 and EB clones. Thus, the predetermined electrostatic field of the GaAs crystal when cut at the 100 plane is different than that formed on the 111A plane and thus, various biological moieties specifically bind to each predetermined electrostatic field.
It will be appreciated that variation of the electrical field as described hereinabove is likely to alter the binding capacity of the surface, thus resulting in different biological moieties attaching thereto.
Thus, the teachings of the present invention can be used to gain electrical control over biological processes, namely, to trigger or suppress a selected biological pathway by an electronic signal presented to the system. A given antibody can bind a given set of electrodes biased to a certain voltage pattern and avoid binding to the same electrode set when biased in a different pattern. The latter pattern may, in turn, attract a different antibody. The same set of electrodes biased in different ways thus specifically bind different target molecules from the solution and, hence, act as a programmable artificial receptor.
Thus, the artificial receptor of the present invention can be used to provide specific binding for a target moiety (e.g., a biological moiety).
The phrase “specific binding” as used herein, refers to binding of a biological moiety via electrostatic, hydrophobic, hydrogen bonds and van der Waals interactions to an artificial receptor having a surface with unique electrical properties.
As used herein, the phrase “biological moiety” refers to any naturally occurring or synthetic macromolecule having a biological function. Examples include, but are not limited to DNA, RNA, protein, peptide (e.g., antigen, epitope), carbohydrate, antibodies and fragments thereof. It will be appreciated that the biological moiety used by the present invention can be isolated or included in a prokaryotic (e.g., bacteria, viruses) or eukaryotic organism (e.g., mammals). Non-limiting examples of biological moieties which can be used along with the present invention, include, growth factors, cytokines, transcription repressors, transcriptions enhancers, promoters (e.g., DNA, RNA and/or proteins), and any other molecule which can trigger, suppress, control or regulate any biological process.
The term “antibody” as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab′)2, Fv or single domain molecules such as VH and VL to an epitope of an antigen. These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab′, the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (3) (Fab′)2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (5) Single chain antibody (“SCA”), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule; and (6) Single domain antibodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen.
Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).
Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab′ fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10,: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).
The term “peptide” as used herein encompasses native peptides (either degradation products, synthetically synthesized peptides, or recombinant peptides), peptidomimetics (typically, synthetically synthesized peptides), and the peptide analogues peptoids and semipeptoids, and may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to: N-terminus modifications; C-terminus modifications; peptide bond modifications, including but not limited to CH2—NH, CH2—S, CH2—S═O, O═C—NH, CH2—O, CH2—CH2, S═C-NH, CH═CH, and CF═CH; backbone modifications; and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Ramsden, C. A., ed. (1992), Quantitative Drug Design, Chapter 17.2, F. Choplin Pergamon Press, which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinbelow.
The peptides of the present invention may be synthesized by any techniques that are known to those skilled in the art of peptide synthesis. For solid phase peptide synthesis, a summary of the many techniques may be found in: Stewart, J. M. and Young, J. D. (1963), “Solid Phase Peptide Synthesis,” W. H. Freeman Co. (San Francisco); and Meienhofer, J (1973). “Hormonal Proteins and Peptides,” vol. 2, p. 46, Academic Press (New York). For a review of classical solution synthesis, see Schroder, G. and Lupke, K. (1965). The Peptides, vol. 1, Academic Press (New York).
Further description of peptide synthesis is disclosed in U.S. Pat. No. 6,472,505. A preferred method of preparing the peptide compounds of the present invention involves solid-phase peptide synthesis, utilizing a solid support. Large-scale peptide synthesis is described by Andersson Biopolymers 2000, 55(3), 227-50.
The DNA or RNA molecules of the present invention can be used in the form of oligonucleotide or polynucleotide molecules.
The term “oligonucleotide” refers to a single-stranded or double-stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally occurring bases, sugars, and covalent internucleoside linkages (e.g., backbone), as well as oligonucleotides having non-naturally occurring portions, which function similarly to respective naturally occurring portions.
Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art, such as enzymatic synthesis or solid-phase synthesis. Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example: Sambrook, J. and Russell, D. W. (2001), “Molecular Cloning: A Laboratory Manual”; Ausubel, R. M. et al., eds. (1994, 1989), “Current Protocols in Molecular Biology,” Volumes I-III, John Wiley & Sons, Baltimore, Md.; Perbal, B. (1988), “A Practical Guide to Molecular Cloning,” John Wiley & Sons, New York; and Gait, M. J., ed. (1984), “Oligonucleotide Synthesis”; utilizing solid-phase chemistry, e.g. cyanoethyl phosphoramidite followed by deprotection, desalting, and purification by, for example, an automated trityl-on method or HPLC.
The oligonucleotide of the present invention is of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40, bases specifically hybridizable with sequence alterations known in the art.
The oligonucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3′-to-5′ phosphodiester linkage.
Oligonucleotides may be modified either in backbone, internucleoside linkages, or bases, using methods known in the art.
For example, the teachings of the present invention can be used to identify ligands of specific artificial receptors.
Thus, according to yet another aspect of the present invention there is provided a method and a kit for typing ligands.
As used herein, the phrase “typing ligands” refers to identifying ligands, i.e., biological moieties as described hereinabove which are capable of binding a specific surface or region of the artificial receptor of the present invention.
“Identifying” according to this aspect of the present invention refers to determining the amino acid sequence, nucleic acid sequence, and/or carbohydrate structure of the biological moiety which binds to the artificial receptor of the present invention. It will be appreciated when phage display libraries are utilized, determination of the DNA sequence of the clones generating the displayed peptide or protein molecules is preferably effected.
The method is effected by (a) exposing a plurality of biological moieties to the artificial receptor of the present invention; and (b) isolating at least one biological moiety of the plurality of biological moieties capable of the specific binding to the artificial receptor, the at least one biological moiety being the ligand capable of specifically binding the artificial receptor; thereby typing the ligands.
Preferably, the method is effected by subjecting the biological moieties (the ligands) to a plurality of surfaces of the artificial receptor of the present invention using conventional screening or panning methods which are optimized to fit the artificial receptor of the present invention. Thus, the kit for typing ligands further includes reagents (as described hereinbelow) for qualifying binding of the ligands to the plurality of artificial receptors.
Preferably, the method of this aspect of the present invention further comprising step (c): exposing the plurality of biological moieties to an additional surface of the artificial receptor, the additional surface having a unique surface electrical properties enabling a specific binding of an additional ligand thereto.
Preferably, step (c) may be effected prior to or following step (a).
It will be appreciated that step (c) is performed in order to increase the specificity of binding to the artificial receptor of the present invention. Thus, by exposing the plurality of the biological moieties to an additional surface of the artificial receptor, the non-specific binders of the artificial receptor of the present invention (e.g., molecules which bind to the material consisting the receptor and not to the specific electrical property of its surface) are depleted from the plurality of biological moieties exposed to the desired artificial receptor (i.e., the artificial receptor whose ligands are to be typed). A description of such a depletion step is provided under “General Materials and Experimental Methods” and in Examples 1-5 of the Examples section which follows.
Preferably, step (c) is performed at least twice, more preferably, at least three times, more preferably, at least four times, even more preferably, step (c) (i.e., the depletion step) is performed prior to each panning cycle. For depletion (binding to another surface of the artificial receptor), the electrodes of the artificial receptor are preferably switched or biased with a different voltage than provided for the panning. As for the semiconductor surfaces described in Example 1 of the Examples section which follows, no wash step is needed between the depletion step and the subsequent panning step.
Phage display is a powerful technology designed to evolve, from an initial library, peptides and antibody fragments having high affinity to a certain antigen. The most widely used library methodology is based on the filamentous phage, a bacteriophage that infects male Escherichia coli. Filamentous phage display is based on cloning DNA fragments encoding millions of variants of certain ligands (e.g. peptides, proteins or fragments thereof) into the phage genome, fused to the gene encoding one of the phage coat proteins (usually pIII, but also pIV, pVI or pVIII). Upon expression, the coat protein fusion is incorporated into new phage particles that are assembled in the periplasmic space of the bacterium. Expression of the gene fusion product and its subsequent incorporation into the mature phage coat results in the ligand being presented on the phage surface, while its genetic material resides within the phage particle. This connection between genotype and phenotype allows the enrichment of specific phage by selection on an immobilized affinity target. The phages are caused to interact with th e target antigen while the latter is immobilized. Phages that display a relevant ligand are retained by virtue of their binding to the target, while non-adherent phages are washed away. Bound phages are recovered from the surface, used to re-infect bacteria and reproduced for further enrichment followed by another affinity assay. With an appropriate starting library, several such cycles usually lead to satisfactory selectivity and binding affinity.
Antibodies selected from phage libraries may not be optimal for direct application. In many cases, manipulation of the antibody affinity, valency, specificity, or stability is required. In this case, phage display technology is applied in a manner similar to the production of synthetic libraries and selection of the best binders from them. Such secondary libraries contain variants of the antibodies isolated initially with mutations introduced either randomly or following a rational design. Mutations are introduced into the antibody genes using one of several methods: site-directed mutagenesis, error-prone PCR, chain shuffling, DNA shuffling, or mutator E. coli strains. Using one of these approaches it is possible to obtain antibodies having high affinities, (for biological antigens these methods yield affinities well below 100 pM) and good selectivity.
Any method that separates phages that bind from those that do not, can be used for phage selection, and indeed, many different selection methods can be used. The popular selection methods include affinity selection (also called biopanning) on immobilized antigen coated onto solid supports, columns or BIAcore sensor chips.
Antibodies in the form of recombinant antibody fragments were the first proteins to be successfully displayed on the surface of a phage. This was achieved by fusing the coding sequence of the antibody variable (V) regions encoding for a single-chain Fv (scFv) fragment to the amino terminus of the phage gene III, coding for the phage minor coat protein pIII. Initial attempts to display Fab′ fragments fused to pVIII, the phage major coat protein, were also successful. However, the pVIII site, although very popular for peptide phage display, is not suitable for the efficient display of large polypeptides such as antibodies. For this reason, most antibody phage-display systems utilize the pIII site.
Antibodies were first displayed using a phage vector, based on the genome of fd-tet and its gene III as fusion partner. In this vector, the genes coding for antibody scFv fragments were cloned in-frame with gene III and downstream of the gene III signal sequence, which normally directs the export of the phage-coat protein to the periplasm. Here, the antibody VH and VL domains may fold correctly, both stabilized by an intramolecular disulfide bridge, and pair to form a functional scFv.
The success of ligand phage display hinges on the synthesis of a large combinatorial repertoire on the phage and efficient selection and enrichment strategies.
Affinity Selection of Initial Library (Biopanning)
For affinity selection, the phage display antibody library is exposed to the artificial receptor or the array of electrodes described hereinabove. The following description refers to one embodiment of the present invention. In a first step, antibodies selective to the artificial receptor of the present invention such as the structure depicted in
It will be appreciated that blocking of non-specific binding to the artificial receptor of the present invention can be achieved by incubating the artificial receptor with conventional blocking reagents such as bovine serum albumin, goat serum albumin or milk (e.g., 1%).
In addition, in order to increase the specificity of binding to the artificial receptor of the present invention, at least one depletion cycle (as described hereinabove) is effected prior to panning.
Tuning washing times and stringency helps to determine the selection efficiency and to discriminate between phages with different affinities for the target. At times it pays to perform the initial rounds of selection under low stringency, so as not to lose rare binders, and to employ more stringent conditions in later rounds.
Elution of bound phages from the artificial surfaces of the present invention can be effected using basic (e.g., TEA at pH 12) or acidic (e.g., Glycine-HCl, at pH 2.2) conditions, depending on the surfaces used (for details see “General Materials and Experimental Methods” of the Examples section which follows). Alternatively, elution of bound phages from the electrifiable electrodes or the biased artificial receptor of the present invention can be effected by simply switching the wiring or varying the voltage supplied to the electrodes. Thus, a specific biased surface which specifically binds a ligand can be biased to release such ligand into the solution to thereby elute the desired phage display antibody.
Binding of monoclonal scFv-displaying phage in ELISA can be conventionally detected by primary rabbit anti-M13 antisera in combination with a horseradish peroxidase (HRP) conjugated anti-rabbit antibody. Alternatively, an HRP-anti-M13 conjugate may be used. As is shown in Examples 1 and 2 of the Examples section which follows, the same methods can be used on the artificial receptor surfaces of the present invention. Polyclonal phage ELISA on biased A, B, and A/B crystals can be performed to differentiate between specific and non-specific binders as well as for identification and quantification of the various selective binders.
Monoclonal Phage ELISA
To identify monoclonal phage antibodies the phagemids need to be rescued individually. Growth of phagemid containing cells and helper phage rescue is carried out in sterile 96 well, flat bottomed tissue-culture plates, essentially as described under “General Materials and Experimental Methods” of the Examples section which follows. In principle, colonies picked from the last panning cycle are expected to yield the most positive binders. However, it will be appreciated that since in the late cycles the phage population becomes dominated by a few (or even one) binders, clones from the outputs of earliest panning cycles that test positive in the polyclonal phage ELISA are also preferably selected for further analysis.
Production and ELISA Analysis of Soluble scFvs
This test is carried out in parallel with phage ELISAs to analyze individual clones for antigen binding. The phages is infected into HB2151, that does not carry an amber suppressor tRNA and then induced to give soluble expression of antibody fragments for ELISA.
Second Generation Libraries
Antibodies selected from phage libraries may not be optimal for direct application. In many cases, manipulation of the antibody affinity, valency, specificity, or stability is required. In this case, phage display technology is applied in a manner similar to the production of synthetic libraries and selection of the best binders from them. Such secondary libraries contain variants of the antibodies isolated initially with mutations introduced either randomly or following a rational design. Mutations can be introduced here into the antibody genes using one of several methods: site-directed mutagenesis, error-prone PCR, chain shuffling, DNA shuffling, or mutator E. coli strains. Using one of these approaches it should be possible to obtain antibodies having high affinities, (for biological antigens these methods yield affinities well below 100 pM) and good selectivity.
Antibodies for Different Bias Values
The same procedure is applied to the same multi-layer structure biased at different potentials relative to the solution. An optimal antibody for a certain bias might turn an excellent starting point for the generation of a phage display library to be screened by the same interface biased differently. At each bias, antibodies for the A and B crystals as well as for the A/B interface are evolved as described above. The binding regions are sequenced and compared. The sequences displayed by antibodies optimized for different bias values differ from each other. It is extremely interesting to test how large the bias difference should be in order to obtain two different binding. regions for two bias values. Strong affinity to a certain bias value is just one aspect of the desired antibodies. The complimentary aspect is selectivity which can be improved by interacting good binders for a certain bias with the same structure biased to a different value. Antibodies that bind also to a different bias can be depleted this way from the library (for further description of depletion steps, see “General Materials and Experimental Methods” of the Examples section which follows).
It will be appreciated that the end result of the screening described hereinabove is a collection of vials, each containing monoclonal antibodies with optimal selectivity to one of the biased surfaces of the artificial receptor of the present invention, e.g., the A, B, or A/B structures as described in
Screening of Carbohydrate Ligands Using the Artificial Receptors of the Present Invention
Due to their inherent bond type complexity, synthesizing complex carbohydrate combinatorial libraries necessitates a plurality of distinct synthesis reactions. Carbohydrate libraries can be synthesized employing the “one bead-one molecule” approach, in which the diversity is created by a split-and-pool synthesis or the dynamic combinatorial chemistry (DCC) approach (see for example, Schullek J R, et al., 1997, Anal. Biochem. 246: 20-9; U.S. Pat. Appl. No. 20040146941 to Zhang Biliang et al. Ramstrom 0, Lehn J M. Chembiochem. 2000 1: 41-8, which are fully incorporated herein by reference). Such libraries can be screened on the artificial surface of the present invention such that the carbohydrate structures interact with a specific surface are identified, isolated and analyzed for composition.
It will be appreciated that the ligands and corresponding artificial receptors can be used in various biological applications. For example, such a ligand and artificial receptor can be used for targeting delivery of a drug molecule.
Thus, according to an additional aspect of the present invention there is provided a method of controlling a delivery of a drug molecule to a tissue of a subject. The method is effected by (a) implanting a device body including at least one electrode configured capable of a unique surface electrical property enabling a specific binding of a ligand thereto, the ligand being attached to the drug; (b) modifying the unique surface electrical property to thereby control a binding or a release of the ligand and thereby controllably deliver the drug molecule to the tissue.
As used herein the term “implanting” refers to administering the artificial receptor of the present invention to a subject in need thereof, i.e., a subject having a pathology requiring the treatment of the drug molecule. Implanting is effected by surgically or minimally invasively inserting the device body within a human body, e.g., subcutaneously, subdermally, intramuscularly, intraperitoneally, intra brain, and the like.
As used herein, the term “drug” refers to any substance which can be used to trigger, enhance, suppress, control or regulate any biological process (e.g., cell proliferation, differentiation, expansion, apoptosis, secretion, absorption, transmission and the like). For example, such a drug can be a chemical, an organic molecule, a biological moiety (e.g., which is made of nucleic acids, ribonucleic acids, oligosaccharides, carbohydrates, fatty acids, amino acids) and the like.
For example, a drug molecule capable of treating a heart disease is covalently attached to a ligand capable of binding a specific surface (electrical property) of the artificial receptor of the present invention. Then, the artificial receptor of the present invention is implanted in the subject. For example, the artificial receptor (i.e., the electrode) can be subcutaneously implanted near the heart, similar to a heart pacemaker. Following implantation, the drug molecule is intravenously or orally administered to the subject. However, due to the specific electrical properties configured on the electrode of the artificial receptor of the present invention, the drug molecule preferably binds the implanted electrode. A release of the drug molecule is further effected by modifying the unique surface electrical property of the artificial receptor. As a result, the drug molecule is released in situ (i.e., at the site to be treated) and is thus far more efficient in treating the pathology.
The device preferably includes an internal power source and a micro receiver and an acoustic transducer. Modification of the unique surface electrical property can be effected essentially as described in U.S. Pat. No. 6,628,989 to Penner et al., by transmission of one or more external acoustic energy waves or signals from an external source into the subject's body, e.g., generally towards the location of the implanted device until the signal is received by the acoustic transducer. Upon excitation by the acoustic wave(s), the acoustic transducer produces an electrical output configured to generate a voltage capable of modifying the electrical properties of the electrodes in the artificial receptor. It will be appreciated that other types of activation of EM energy can also be used, such as RF, etc.
In one possible application, a variation of the present embodiments is used to provide controlled drug release. A quantity of the drug to be released is held in a reservoir, and in the meantime a molecule for which the artificial receptor has an affinity is released into the bloodstream. The molecule that is released has a magnetic particle attached thereto, thus enabling the attachment of the particle to be sensed at the device. The molecule with the magnetic particle reaches the artificial receptor and binds thereto. The magnetic particle is detected via its magnetic field. Detection of the magnetic particle triggers release of the drug. The reservoirs can be placed with the devices deep inside the body at the points where drug administration is required. The particles can then be systematically administered to control release of the drug at the device. The particles can be used to ensure that a given quantity of the drug is released using timing based say on the half-life of the drug within the body. An example is provided in
In a further possible application, an endogenous ligand binds to the artificial receptor. Binding of the ligand affects the electric field of the device temporarily and may cause a temporary signal spike which may be detected following suitable noise reduction. The ligand may be selected to be representative of biological activity that it is desired to monitor. For example the ligand may be an antibody, and the presence or level of too many of the antibodies may indicate a certain condition. The condition may be treatable with a given drug which can be part of a controllable release feature as before. An example is provided in
Thus detection of binding of the given ligand may be used to trigger controlled drug release as in the previous application. Assuming that the drug operates to calm down the condition and reduce the number of the given ligands, a system of negative feedback in fact becomes available for the condition.
It will be appreciated that the artificial receptor of the present invention can be also used to identify small molecules capable of mimicking large molecules (e.g., proteins) or cells.
Thus, according to yet an additional aspect of the present invention, there is provided a method of identifying a small molecule capable of mimicking a binding function of a ligand.
As used herein, the phrase “binding function” refers to the result of binding of a ligand (e.g., an antibody or any other biological moiety) to the artificial receptor of the present invention.
The method is effected by (a) exposing the ligand to at least one electrode configured capable of a unique surface electrical property enabling a specific binding of the ligand thereto, thereby identifying at least one electrode capable of specifically binding the ligand; and (b) identifying a small molecule of a plurality of small molecules capable of binding the at least one electrode being identified as capable of specifically binding the ligand, the small molecule being capable of mimicking the binding function of the ligand.
The method is based on the specific binding of a ligand which is any biological moiety as described hereinabove (e.g., an antibody) to the artificial receptor of the present invention (e.g., at least one electrode as described hereinabove). Following the identification of a specific surface or region in the artificial receptor which bind the ligand, the same region or surface is exposed to a plurality of small molecules, e.g., peptides, using for example, a phage display peptide library as described in Example 5 of the Examples section which follows. Thus, a small molecule which specifically binds to the same surface or region as the ligand is capable of mimicking the ligand binding function.
Thus the teachings of the method according to this aspect of the present invention can be used to identify the binding domains responsible for interactions between antibodies to foreign intruding antigen molecules, hormones and receptors, proteins capable of binding specific receptors on cancer cells and the like.
For example, a peptide, identified as described hereinabove, which is capable of mimicking the binding of a large protein (e.g., a hormone) to a receptor present on a cancer cell (e.g., estrogen receptor present in breast cancer cells) can be used as a targeting vehicle to deliver a drug molecule to the cancer cells, thus preventing and/or treating the subject having cancer. Briefly, a drug molecule (e.g., an agent capable of preventing cell division) is covalently attached to the peptide mimicking the large protein and is administering to the subject (e.g., using intravenous administration). The peptide is specifically recognized by the receptor on the cancer cells and the drug molecule enters the cancer cell and prevents cell proliferation.
Similarly, peptides mimicking the binding of proteins such as growth factors and cytokines can be used as agents for controlling the proliferation and/or differentiation of cells in vivo (for determination of factor effects), ex vivo (for preparation of cells prior to transplantation in a body) and even in vivo (for direct effect in the body).
It will be appreciated that peptides mimicking proteins which are capable of binding specific cell receptors, such as receptors on heart cells, can be used in facilitating the homing of stem cells to specific cells expressing such receptors. Briefly, the peptides are expressed on ex vivo expanded pluripotent or partially committed progenitor cells using an expression vector and known molecular biology techniques. The ex vivo expanded cells are then transplanted in a recipient subject and the peptide, which is displayed on the transplanted cell membrane as part of a cellular receptor is likely to home to heart cells which present the specific electric field generated by the artificial receptor used in the identification of such a peptide.
Once specific antibodies or peptides with appropriate specificity and affinity are generated, such antibodies or peptides can be engineered to contain a fused moiety that triggers or suppresses a biochemical or biological reaction.
To demonstrate triggering of a biochemical reaction, chimeric proteins are preferably prepared by fusing an enzyme such as peroxidase or alkaline phosphatase to the recombinant antibody and expressing such a construct in E. coli. Alternatively, the antibody is engineered with a specific peptide tag at the C-terminus for site specific biotinylation which enables further manipulation through biotin-streptavidin interaction. To demonstrate activation of a biological process the selected antibodies are fused with antigens capable of triggering different immune responses. Alternatively they are fused with a DNA binding protein and used to affect in-vitro gene expression.
It will be appreciated that the artificial receptor of the present invention can be can be used to characterize a binding site of a ligand. Briefly, such characterization can be achieved by first exposing the ligand to a plurality or an array of electrodes of the artificial receptor of the present invention and isolating and electrically characterizing at least one electrode exhibiting specific binding to the ligand.
As used herein the phrase “electrically characterizing” refers to determining the electric field generated by a surface of the at least one electrode. Such an electric field can be characterized in terms of field strength and/or field shape or spatial distribution on or above the surface (two dimensional or three dimensional).
Altogether, in contrast to a conventional (chemical or biological) receptor, the electronic or artificial receptor of the present invention can be reconfigured in real time to select a desired molecule out of a certain collection. Since each of the antibodies can be fused to an additional molecule having a certain biological function, elution of the bound molecules after rinsing all other molecules may be designed to trigger a desired process corresponding uniquely to the original electrode biasing pattern. Alternatively, binding of selected antibodies to the electrodes removes these molecules from the solution and blocks their corresponding biological processes. The present invention thus takes the interface between manmade electronics and molecular biology a giant leap forward. The teachings of the present invention can be used to facilitate activation or suppression of specific biological pathways based on electronically programmable signals.
Thus, according to an additional aspect of the present invention there is provided a method of activating or suppressing a biological pathway in cells of a subject. The method is effected by: (a) implanting a device body including at least one electrode configured capable of a unique surface electrical property enabling a specific binding of a ligand thereto, the ligand being capable of activating or suppressing the biological pathway; (b) modifying the unique surface electrical property to thereby control a binding or a release of the ligand and thereby controllably activating or suppressing the biological pathway in the cells of the subject.
Preferably, modifying is effected using a remote switching unit as described hereinabove.
It will be appreciated that the ligand can be an endogenous molecule (present in the cells of the subject) or can be an exogenous molecule which is further administered to the subject.
The implications of such an interface are far reaching. At one end of the spectrum it can be harnessed to electrically control biological processes. For instance, the fused segments may comprise antigens that activate a certain immune response or gene expression. One may envision sensors that probe several biological parameters and feed these signals into electronics that processes the data and activates the required biological pathway using the proposed artificial receptor. The ease of. computation by electronic logic provides unparalleled flexibility compared with pure biology alternatives. Moreover, since electronic signals can be generated in the electrodes in response to an electronically transmitted signal, the proposed approach also facilitates remote activation of biological processes. At the other end of the spectrum the scheme provides a promising strategy for closing feedback loops from electronic functionality of biologically constructed nanoelectronics to the construction process itself. To clarify the latter point consider the efforts in recent years to harness molecular biology for the self-assembly of molecular scale electronics. DNA molecules and related proteins were used to scaffold the assembly of a functional transistor made out of non-biological ingredients. Once the transistor was made, there was no way to test its electronic functionality in situ and feedback to the biological assembly process. Without such feedback it is hard to imagine self-assembly of significantly more complex circuits. Feedback from electronic functionality to the assembly process is hence the bottleneck for large scale integration.
The presently preferred embodiments add a new dimension to the interface between nano-electronics and biology and may dramatically affect both fields. The electrode device of
Finally, in the spirit of previous experiments on harnessing molecular biology to the self-assembly of molecular scale electronics, a direct recognition of biased nanoelectronic components by the biological moieties provides unparalleled flexibility and adds a revolutionary new concept to the tools developed thus far.
It is expected that during the life of this patent many relevant switching devices and field carrying materials will be developed and the scopes of the corresponding terms herein are intended to include all such new technologies a priori.
As used herein the term “about” refers to ±10%.
Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., Ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (Eds.) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., Ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., Ed. (1994); Stites et al. (Eds.), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (Eds.), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., Ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., Eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., Ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
Semiconductor crystals—Galium Arsenite (GaAs) (American Xtal technology Cat. No. 5129327) or silicone (Wacker chmitronic GMBH) crystals, were cut through two planes: The 100 plane which is parallel to one of the surface plane of the crystal and the 111 plane of the crystal. The semiconductor crystals used were: silicon (100), GaAs (100), GaAs (111A) and GaAs (111B). The crystals at the 100 plane were round with a diameter of about 5 cm; The crystals at the 111 plane were triangles. The width of all crystal wafers was 0.5 mm, regardless of its plane.
For panning (i.e., selecting for antibodies or peptides which bind the semiconductor surface), each crystal disc of the 100 crystals was cut into a square of 1×1 cm2 and each of the 111 crystals was cut to a 1 cm long triangle. The four rounds of panning were performed in Eppendorf tubes using a 360° C. tube rotator (Labquake Labotal).
For the ELISA assay, the crystal wafers were cut into small squares which fit the 96-well ELISA plate.
Selection of antibody molecules against specific crystal facets of semiconductor—A human phage display library, Ronit 1, was used for the selection. The library was constructed by Ronit Azriel and Itai Benhar from the faculty of life sciences at Tel Aviv University, Israel (kindly provided by Prof. Itai Benhar) and contains 1×109 independent scFv clones [Azriel-Rosenfeld, R., Valensi, M & Benhar, I., A human synthetic combinatorial library of arrayable single-chain antibodies based on shuffling in vivo formed CDRs into general framework regions, JMB 335, 177-192, (2003)]. The library is composed of different human synthetic single chain Fv fragments, with variable VH and VL genes in the CDR3 region and is known to generate specific binders to a host of biological targets. The RONIT 1 library consists of a principal in which in vivo formed complementarity determining regions were shuffled combinatorially onto germline-derived human variable-region frameworks. The arraying of library-derived scFvs is facilitated by a unique display/expression system, where scFvs are expressed as fusion proteins with a cellulose-binding domain. This library was screened against a number of peptides, proteins, and peptide-protein complexes and yielded antibody fragments exhibiting dissociation constants in the low nanomolar range.
In principle, the selection protocol was similar to that used for selecting biological targets (e.g., as described for the antibodies against the scMHC-peptide complexes) as detailed in Denkberg G., et al., 2002; The J. of Immunology 169: 4399-4407, except that some optimization was performed to adjust for the semiconductor surfaces.
Optimization of the scanning conditions for the semiconductor surfaces Crystal specific pH elution conditions—For the GaAs (100) and GaAs (111A), elution was superior in basic conditions (e.g., TEA at pH 12) rather than acidic conditions. On the other hand, for the silicone crystals (both 100 and 111), elution was performed under acidic conditions (e.g., Glycine-HCl, at pH 2.2).
Blocking of non-specific binding to semiconductor surfaces—GaAs substrates [GaAs (111A), (111B), (100)] showed extensive non-specific binding of the phages to the substrate via their coat proteins. Although the binding energy per protein was small, their excess number when compared to the number of expressed antibodies dominated the panning assay. To reduce the non-specific binding, the semiconductor surfaces were blocked for 1 hour at room temperature in Tris-buffered saline (TBS), 1% milk and then rinsed six times in TBS to wash excess milk.
Depletion cycles—In order to isolate specific binders for specific semiconductor surfaces, the phage display library was exposed to one type of surface [e.g., GaAs (111)] prior to being exposed to another surface [e.g., GaAs (100)], thus the clones capable of binding the first surface [e.g., GaAs (111)] were depleted from the library. It is worth mentioning that no wash step was performed between the depletion step [e.g., panning on the GaAs (111)] and the following panning step [e.g., panning on the GaAs (100)].
Selection of Phage-Antibodies—naïve library—Bound phages were eluted from surfaces using TEA at pH 12 [for GaAs (100) and GaAs (111A)] or Glycine-HCl, at pH 2.2 (for the silicone crystals). Eluted phages were used to infect TG1 E. Coli cells (at OD600 nm=0.5) for 60 minutes at 37° C., following which the infected bacteria were plated on 2YT plates containing 100 μg/ml ampicilin (2YT/A/G) and 1% glucose (Sigma G5767).
Panning—The semiconductor substrates were exposed to the Ronit1 library (which includes about 50% of the M13K07) or to M13KO7 helper phage (devoid of scFv, as a control experiment) in TBS, 1% milk. After rocking for 1 hour at room temperature, the surfaces were washed 10 times with TBS, 0.1% TWEEN-20.
Elution—The phages were eluted from the surfaces by adding tri-ethyl amine (pH 12, 0.1M) for 10 minutes, transferred to a fresh tube and then neutralized with Tris (HCl) (pH 7).
Quantification—The eluted phages were titrated in dilution series on solid agar and colonies were counted to estimate the number of adsorbed phages. Briefly, for each panning step colonies from bound phages were collected from the 2YT/A/G plates and diluted 1:100 in 25 ml of 2YT/A/G medium. Cells were grown to O.D600=0.5 and M13KO7 helper phage [5×1011 colony forming unit (cfu)] was added to 25 ml of the culture. After incubation for 60 minutes at 37° C., the cells were centrifuged, resuspended in 25 ml of 2YT/Ampicillin (100 μg/ml)/Kanamycin (50 μg/ml) and grown overnight at 30° C. Phages were collected from culture supernatants and purified for the next round of panning by PEG precipitation. This procedure repeated 4 times. The diversity of the selected antibodies was determined by DNA fingerprinting (using restriction enzymes such as BstNI). Non-specific binding was estimated by titrating the Kanamycin resistant M13KO7 helper phages on Kanamycin plates.
Rescue of phages from individual colonies—To rescue phage, single ampicillin-resistant colonies resulting from infection of TG1 bacteria with phages, were inoculated into 100 μl of 2×TY containing 100 μg/ml Ampicillin and 1% glucose (2×TY-Amp-glucose) in 96-well plates and grown overnight at 37° C. Five to twenty microlitters of the overnight cultures were inoculated into 150 μl fresh 2×TY-Amp, to an OD600 nm=0.5 (about 1.5-2 hours). The M13KO7 helper phage [25 μl of 2×TY-Amp-glucose containing 109 plaque forming unit (p.f.u.)] was then added to each well, and the plate was incubated without agitation for 60 minutes at 37° C. The plate was then shaken at 37° C. for one hour after which the cells were pelletted at 4000 rpm for 15 minutes. The cells were then resuspended in 200 μl 2×TY containing 100 μ/ml Amp and 25 μg/ml Kan (2×TY-Amp-Kan) and grown overnight at 30° C. The cells were pelletted as above, and the supernatants containing the phages (monoclonal scFv-displaying phages) were tested for their binding to the semiconductor surface using the ELISA assay.
ELISA conditions of monoclonal scFv-displaying phages on semiconductor surfaces—For ELISA assay, the semiconductor surfaces [GaAs (111A) and GaAs (100)] were cut into small squares of 4×4 mm to fit the 96-well ELISA plate, incubated for 60 minutes with 4% milk in TBS, and washed 6 times with TBS (200 μl each wash). In each well, about 60 μl of a monoclonal phage suspension (isolated following 4 panning as described above) was mixed with TBS to reach a final volume of 150 μl/well. The reaction (between the monoclonal scFv-displaying phages and the semiconductor surfaces) was incubated for one hour at room temperature while shaking. To remove unbound monoclonal scFv-displaying phages, the fluid was aspirated, and the wells including the semiconductor surfaces were washed 6 times in TBS containing 0.1% of Tween-20. To reveal antibody-bound surfaces, an anti M13 monoclonal antibody conjugate (Amersham 27-9421-01) (which is capable of specifically binding to the monoclonal scFv-displaying M13 phages) was added (0.125 μl per well), incubated for 1 hour in the presence of 1% milk, following which the wells were washed 6 times with TBS and the TMB One Step Substrate (S-159985 DAKO Cytomation, Denmark) was added. The ELISA reactions were read at 450 nm. The DNA of positive phage clones (i.e., phage clones displaying OD values of 0.750 or higher in the ELISA assay) was further characterized by BstNI restriction analysis and/or sequencing.
Expression and purification of soluble scFv antibodies—The scFv gene is rescued from the phage clone by PCR and is then subcloned into the phagemid vector pCANTAB6 by using the SfiI-NotI cloning sites. A Myc and hexahistidine tags are fused to the C-terminus of the scFv gene. The scFv antibody is expressed in BL21 λDE3 cells and purified from the periplasmic fraction by metal-ion affinity chromatography:
Soluble ScFv are purified from the periplasmic fraction of BL21 cells using the hexahistidine tag. An overnight starter culture of Fab specific clones is grown at 30° C. Bacterial cells are diluted 1:100 into 500 ml of 2YT/A/G, grown to OD600 nm=0.8-1.0 and induced to express the recombinant ScFv antibody by the addition of 1 mM IPTG for 3 hours at 30° C. The bacterial cells are centrifuged and the pellet is resuspended in 5 ml of a B-PER solution (Pierce) to release periplasmatic content. After 30 minutes of rotated incubation at room temperature, the solution is centrifuged (15000 rpm, 15 minutes) and the supernatant is incubated with 0.5 ml of pre-washed TALON beads suspension (Clontech) for 45 minutes at room temperature. The solution is applied onto a BioRad (Hercules, Calif.) disposable column, and after sedimentation the beads are washed three times with 10 ml of PBS/0.1% Tween 20 (pH 8.0). The bound ScFvs are eluted using 0.5 ml of 100 mM Imidazole in PBS. To remove residual imidazole, the eluted scFv are dialyzed twice against PBS (overnight, 4° C.). The homogeneity and purity of the purified ScFv/Fabs is determined by analysis on non-reduced and reduced SDS-PAGE.
ELISA assay of soluble scFv antibodies on semiconductor surfaces—4×4 mm squares of GaAs (111A) and GaAs (100) were placed in a 96 well ELISA plate with 4% milk for 60 minutes and then washed 6 times with PBS. The soluble scFv antibodies were then added in PBS containing 2% milk, and allowed to complex with the surface. Unbound soluble scFv antibodies were washed away and labeled secondary anti human HRP (Goat anti Human IgG F(ab)2-HRP; Jackson ImmunoResearch Laboratories. Inc. Cat. No. 109035097) were added to the wells. Binding of the scFv to the substrates was quantified by reaction with TMB colorimetric substrate.
GaAs etching—GaAs (100) was etched in H3PO4:H2O2:H2O 1:13.8:13.2 mixture in a —12° C. bath for 30 minutes.
Panning of semiconductor surfaces with a phage display peptide library—The Ph.D.-7 Phage Display Peptide Library Kit (New England Bio Labs Inc., Beverly, Mass., USA) was used to screen for positive phages displaying peptides capable of binding to the following semiconductor surfaces: GaAs (100), and GaAs (111A). The panning protocol was exactly as recommended by the kit's manufacturer except that panning was performed on crystal surfaces as described hereinabove for panning of scFv phages.
Studies by Whaley, S. R., (2000, Supra), have shown that phage display peptides bind GaAs (100) preferentially to GaAs (111A) and (111B). However, when one of these peptides was later synthesized and applied to GaAs, no selectivity was found between the (100) and (110) facets [Goede, K., 2004 (Supra)]. To test the reasons for such a discrepancy the present inventors have studied the non-specific binding of M13 phages, carrying no peptides or antibodies, to GaAs (100), GaAs (111A) and GaAs (111B), as follows.
Experimental Results
Binding of helper phage to semi conductors—Phages and the GaAs substrates were interacted and following panning and elution the density of phages was estimated from a count of colonies on agar plates. As is shown in
Thus, these results demonstrate that a panning protocol which includes a depletion step can be used to prevent non-specific binding of phages to a semiconductor facet, similar to the depletion step used in other screening methods. In addition, these results may suggest that experiments with free peptides are needed in order to confirm or disprove semiconductor facet recognition by short peptides.
To test the possibility that a human antibody library (e.g., scFv library) contains binders with preferred selectivity to a specific surface of a semiconductor such as GaAs, the present inventors have performed several cycles of panning and counted the number of phages eluted following each panning step, as follows.
Experimental Results
Selection of scFv binders to GaAs (111A)—To select scFv phage binders to GaAs (111A), about 1011 phages (˜100 copies of each library clone) were applied to the semiconductor crystal (the panning step). After washing the unbound phages, the bound ones were recovered by rinsing the sample in an alkaline solution. The recovered phages were then quantified by infecting bacteria and plating dilution series on Petri dishes. The amplified sub-library was applied again to the target crystal facet and so on. It typically took three to four panning rounds to isolate excellent binders to the target. As evident from
A depletion step enables selective binding of antibodies to semiconductor facets—Preferential binding to a given crystalline facet was achieved by a slight modification of the process which includes a depletion step. Thus, the phages recovered from the first panning on GaAs (111A) were amplified in E. coli bacteria and subsequently applied to GaAs (100). The unbound phages [which were not bound to GaAs (100)] were collected and applied in a second panning step to GaAs (111A).
Table 1, hereinbelow, summarizes the panning results.
Depletion cycles on a certain semiconductor facet increased binding affinity of phage display antibodies towards the other facet—As is shown in Table 1, hereinabove, at the second and third rounds of panning, the selectivity of binding to the GaAs (111A) surface increased by about an order of magnitude following depletion steps over the GaAs (100) surface. Similarly, at the second and third rounds of panning on the GaAs (100) surface, the selectivity of binding to the GaAs (100) surface increased by an order of magnitude following depletion steps on the GaAs (111A) surface.
In addition, when the pool of the phage display antibodies was depleted by panning on cycle 2 and 3 on the GaAs (100) surface, the phage display antibodies selected following the forth panning on the GaAs (111) exhibited a significant higher binding efficacy towards the GaAs (111) surface than towards the GaAs (100) surface. As evident from the results presented in
Thus, these results demonstrate that a significant increase in binders to a specific GaAs facet can be achieved by increasing the panning cycle and including at least one depletion step between the panning cycles.
Altogether, these unexpected results demonstrate that phage display antibodies which specifically bind to semiconductor surfaces (i.e., binders) can be isolated, preferably following one or two depletion cycles. Thus, specific binders to the GaAs (111A) were isolated following 2 depletions on the GaAs (100) surface.
The polyclonal population of selected phages contains different scFv fragments, each characterized by different affinity and selectivity to the two crystalline facets. To correlate specificity with a specific amino acid sequence, the binding selectivity of individual clones was analyzed. Monoclonal binders were isolated by infecting E. coli bacteria with the sub-library and plating them on solid agar. Since each bacterium can be infected by a single phage, all bacteria in a given colony carry DNA coding for the same scFv fragment. Infection of the colony with helper phages resulted in release of phages displaying the same scFv on their PIII coat proteins. The isolated monoclonal phages were then analyzed by ELISA against GaAs (111A) and (100), as follows.
Experimental Results
ELISA analysis confirmed selectivity of phage display antibodies to the semiconductor surfaces—After 4 rounds of panning and 2 rounds of depletion, several clones (monoclonal scFv-displaying phages) were subject to an ELISA assay. About 60% of the clones showed markedly enhanced binding to their target compared to the control group. The readings for the monoclonal scFv-displaying phages are summarized in Table 2, hereinbelow.
The ELISA results for a soluble scFv antibody of clone EB (which was found in another scFv phage library) are presented in
Altogether, clones E1, F1, C7 and EB were selective to GaAs (111A), clones F10 and D3 were selective to GaAs (100), and clones B7, E11, D11 and A3 bind GaAs (111A) and (100) equally well.
To further confirm the specificity of binding to the surface, the DNA of all ten clones (including EB) was sequenced for the VH CDR3 and VL CDR3 genes. Table 3, hereinbelow, presents the sequences of the VL and VH CDR3 genes of the isolated clones.
The sequences of CDR1 and 2 can be found in Table 4, hereinbelow.
Monoclonal binders of semiconductor facets share sequence homology—The sequences of CDR1, 2, and 3 of the monoclonal scFv-displaying phages was obtained and is presented in Tables 3 and 4, hereinabove. It should be noted, that although the EB clone is derived from another library, the sequence of the VH CDR3 of this clone (the EB clone) shares similarities with the same region in the Ronit1 library clones. The sequence of the EB VL CDR3 is identical, except a single amino acid, to the VL of b7. Inspection of the sequences reveals significant similarities between the different clones, some of which can be traced to conserved amino acids in the library. However, other similarities are attributed to the selection process itself, most notably; the abundance of positively charged amino acids in the VH CDR3 is twice as high compared with the frequency of such residues at those positions in a random sample of library clones. Out of 47 clones sampled randomly from the library, only one VH contained a positively charged amino acid in the first position and only eleven displayed such an amino acid in the second position. The 10 selected binders, on the other hand, contained three positively charged amino acids in the first position and five in the second position. The sequences presented in Table 3, hereinabove, were further compared to those of scFv selected on gold (data not shown). The abundance of positively charged amino acids in CDR3 VH, CDR1 VH, CDR1 VL of the latter was less than half compared with the former and no negatively charged amino acid was found in the first six positions of CDR3 VL. Those were replaced by polar uncharged amino acids, predominantly serine. The comparison with the library and anti-gold scFv sequences thus indicates the importance of positively charged amino acids in positions 1-3 of CDR3 VH and the negatively charged amino acids in CDR3 VL for binding GaAs.
Altogether, in contrast to Whaley et al., [Whaley, 2000 (Supra)] the antibody used in the present study was also tested and found selective to crystal orientation when detached from the phage (i.e., as a soluble scFv antibody).
Using phage display technology the present inventors demonstrate in vitro isolation of scFv that bind GaAs (111A) facets almost hundred times better than GaAs (100). Thus, the findings presented here demonstrate the remarkable selectivity of antibodies to the very simple structure of semiconductors compared with biomolecules. More generally, these findings imply that antibody molecules may fmd application in the assembly of nanoelectronics (Keren, K. et. al., 2002; Keren, K., et al., 2003; Braun, E., et al., 1998), in producing templates for localizing nanoparticles (Seeman, N. C., 2003), or for biosensors (Mirkin, C. A., et al., 1996).
The present inventors have tested the capability of the soluble EB scFv antibodies isolated from the monoclonal EB scFv-phage clone to discriminate between different crystalline facets of a GaAs semiconductor crystal, an almost flat target, unfamiliar to the immune system, as follows.
Experimental Results
Binding of soluble scFv antibodies to the semiconductors facets—Using conventional photolithography and H3PO4:H2O2:H2O etch, a long trench has been defined on a GaAs (100) substrate in the (110) direction (
Altogether, the results presented in
Thus, the results presented in
Prior studies applied specific crystal facets of metals, oxides, minerals, and semiconductors for the selective binding of peptides. The present inventors screen a phage display peptide library for selective binders of the GaAs (100) and GaAs (111A) surfaces, as follows.
It should be noted that in contrast to the panning process performed by Whaley, 2000 (Supra), which did not include a depletion step, the panning process employed to screen for phage display peptides included at least one depletion step as described under “General Materials and Experimental Methods”, hereinabove.
Experimental Results
Selection of peptides against specific crystal facets of semiconductors—The New England Biolabs random peptide library is an exhaustive collection of linear heptapeptide (1.28×109 different peptides). The randomized peptide sequences are expressed at the N-terminus of the minor coat protein pIII, resulting in a valency of 5 copies of the displayed peptide per virion.
The GaAs (100) and GaAs (111A) were screened using the Ph.D-7 phage display peptide library using 4 rounds of panning.
Clones selected following panning on the GaAs 100 surface—Clones were sequenced after each panning cycle on the GaAs (100) crystal. As seen in Tables 5, 6, and 7, hereinbelow, the third panning round (Table 7) produced 10 identical clones (which an amino acid sequence as set forth in SEQ ID NO:86) and another subset of two identical clones. Hence, the binding of other peptides to GaAs (100) must be significantly less efficient.
Clones selected following panning on the GaAs 111A surface—Clones were sequenced after each panning cycle on the GaAs (111) crystal. As seen in Tables 8 and 9, hereinbelow, the sequences selected to GaAs (111) show a clear consensus sequence.
These results clearly demonstrate that both the GaAs 111A and GaAs 100 surfaces are capable of selectively binding specific phage display peptides sharing a consensus sequence.
Analysis and Discussion
Phage display is a powerful technology designed to isolate from an initial library peptides or antibody fragments having high affinity to a certain antigen. The most widely used library methodology is based on the filamentous phage M13 (Smith, G. P., 1985), a bacteriophage infecting male Escherichia coli. Filamentous phage display is based on cloning DNA fragments encoding billions of variants of certain ligands into the phage genome, fused to the gene encoding one of the phage coat proteins. Expression of the gene fusion product and its subsequent incorporation into the mature phage coat, results in the ligand being presented on the phage surface, while its genetic material resides within the phage particle.
The Ronit1 scFv antibody phage library Azriel-Rosenfeld, R., et al., 2003), used in the present study, is a phagemid library comprising 2×109 different human semi-synthetic single chain Fv fragments, where in vivo formed complementarity determining regions (CDR loops) were shuffled combinatorially onto germline-derived human variable region framework regions of the heavy (VH) and light (VL) domaims.
Little is known on the interaction between bio-molecules and inorganic surfaces, let alone recognition of such surfaces by antibody molecules. The GaAs surface is modified by surface reconstruction, oxidation, and possibly other chemical reactions. Moreover, it displays atomic steps and possibly surface defects. It is therefore difficult to estimate how much of the underlying crystalline order manifests itself in the recognition process. Unfortunately, no experimental tools capable of determining these parameters with atomic resolution exist for the moment. The recognition mechanism is hence unclear except the indications for the importance of structural rigidity discussed in he introduction. The discrimination between the two crystalline facets may reflect the different underlying crystalline structure, may stem from the different surface chemistry of the two facets or may result from global properties such as atom density and different electro-negativity. The latter factor has been found to be important for the differential binding of specific peptides to different semiconductors (Goede, K., 2004). The unusually high abundance of positively charged amino acids in the heavy chain of CDR1 and CDR3 and the light chain of CDR1 may indicate affinity to the exposed Gallium atoms. The negatively charged amino acid in CDR3 VL(missing in anti-gold scFv isolated from the same library) combined with the positively charged CDR3 VH may match the polar nature of GaAs. may also be provided separately or in any suitable subcombination.
The 7 and 12 mer peptides used in most in vitro selection of binders to inorganic crystals are typically too short to assume a stable structure. Antibodies on the other hand, display a rigid 3D structure which is potentially essential for high affinity selective binding (Perl-Treves, D., et al., 1996; Bromberg, R., et al., 1998). Moreover, the recognition site in the latter case involves six amino acid sequences grouped in three complementarity determining regions (CDR). Altogether these CDRs form a large, structured binding site spanning 3×3 nm.
On a more fundamental level it should be noted that the remarkable selectivity reported here may hint to the importance of a rigid 3D structure for surface selectivity.
Another hint to the importance of rigidity for facet recognition is provided by the rigid structure characterizing antifreeze peptides that target specific ice facets (Knight C. A., et al., 1991). It has also been shown that the stable helical structure of a 31 mer peptide catalyzing calcite crystallization is essential for inducing directed crystal growth along a preferred axis (DeOliviera D. B. and Laursen R. A., 1997), possibly due to its differential binding to the various facets. Structure rigidity may thus turn central to facet recognition by biomolecules, underscoring the importance of antibody libraries as a promising source for selective binders.
Selective binding to specific crystalline facets can be directly utilized for numerous micro and nanotechnological applications including positioning of nanocrystals at a well defined orientation, governing crystal growth and forcing it to certain directions, and positioning nanometer scale objects at specific sites on a substrate marked by certain crystalline facets. An application of one of the soluble antibodies identified by the present study to the latter task is demonstrated in
In summary, the remarkable variety of binding sequences produced by the immune system, as represented in a semi-synthetic antibody library displayed on the surface of phages, were shown to include single chain Fv discriminating between different facets of the same semiconductor. This finding generates an intimate interface between manmade materials and biology. Beyond applications to the positioning of nanometer scale objects at desired sites or direction of crystal growth, the effect of semiconductor binding on the antibody may be harnessed to trigger biological processes, similar to those resulting from binding of biological antigens. In that respect, antibody binding is biologically more accessible than peptide binding. Imagine for instance the task of localizing a semiconductor nanocrystal at a given site. In a free running process confirmation of such assembly is practically impossible. However, the triggering of a certain biological process by the binding of the nanocrystal to the antibody may be engineered to signal successful assembly and even trigger the next assembly step. Recognition of manmade materials by antibodies thus opens new opportunities for a functional interface between these two alien worlds, far beyond what was exercised up till now.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications and GenBank Accession numbers mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application or GenBank Accession number was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.
This Application is a Continuation-In-Part (CIP) of PCT Patent Application No. PCT/IL2005/000333, filed on Mar. 24, 2005, which claims the benefit of U.S. Provisional Patent Application No. 60/555,670, filed on Mar. 24, 2004, the contents of which are incorporated herein by reference
| Number | Date | Country | |
|---|---|---|---|
| 60555670 | Mar 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IL2005/000333 | Mar 2005 | US |
| Child | 11524264 | US |
