English:
[0012] Currently, there are therapeutic approaches under clinical evaluation that interfere with the immunological mechanisms underlying allergen-induced pathology, for example, an anti-IgE immunoglobulin, which directly targets IgE serum antibodies, thus inhibiting the central mechanism of immediate type hypersensitivity reactions. In addition, there has been interest in developing allergen-specific immunotherapy due to its potential to cure allergic diseases. However, both anti-IgE immunoglobulins and allergen-specific immunotherapy are limited by high costs and the necessity for permanent or every-season treatment.
[0047] Figure 1 is a schematic of IgE-mediated signal transduction events.
[0049] Figure 3 is an illustration of a 40 kDa branched PEG.
[0050] Figure 4 is an illustration of a 40 kDa branched PEG attached to the 5 'end of an aptamer.
[0051] Figure 5 is an illustration depicting various PEGylation strategies representing standard mono-PEGylation, multiple PEGylation, and dimerization via PEGylation.
[0053] Figure 7 shows the direct binding curves and binding affinities for ARC445 (SEQ ID NO 101) and derivatives thereof, depicting that modification yielded increased proportion binding to h-IgE.
[0054] Figure 8 depicts the ion exchange HPLC trace analysis of anti-IgE aptamer ARC445 (SEQ ED NO 101) and several derivatives thereof.
[0055] Figure 9 is a graph showing an increase of NMM fluorescence in ARC445 (SEQ ED NO 101), and a decrease in NMM fluorescence in ARC445 derivatives, ARC909-911 (SEQ ED NOs 191-193), which contain 7-deaza-G substitutions for dG.
[0056] Figure 10 is a graph showing an increase of NMM fluorescence in ARC 183, (positive experimental control) and a decrease in NMM fluorescence in ARC 1346 (negative experimental control).
[0057] Figure 11 shows the direct binding curves for ARC445 (SEQ ED NO 101) compared to derivatives thereof, depicting that substituting dG with 7-deaza-G (as in ARC909-911 (SEQ ED NOs 191-193)) significantly reduces proportion binding to h-IgE.
[0058] Figure 12 is a graph showing a decrease in NMM fluorescence in ARC445 derivatives ARC1641, 1642, and 1666 (SEQ ED NOs 212, 213, and 216 respectively), which contain inosine substitutions for dG, as compared to ARC 1384 (SEQ ED NO 181), an ARC445 derivative containing 2'-O-methyl and phosphorothioate substitutions but no inosine substitutions for dG. (SEQ TD NO 101).
[0059] Figure 13 is a schematic of predicted secondary structures for rRfY, dRmY and DNA minimized aptamers showing highest potency in IgE:FcεRl binding inhibition by FACS. Fig. 13(A)shows the rRfY clone according to SEQ ID NO 91, outlined residues are 2'-F; Fig. 13(B) shows ARC445 (SEQ ID NO 101), a dRmY clone, black residues are 2'-deoxy, grey residues are 2' -OMe; Fig. 13(C) shows ARC475 (SEQ ED NO 151), DNA clone, underlined residues are 2 '-deoxy.
[0060] Figure 14 is a plot showing ARC445 (SEQ ED NO 101) and ARC656 (SEQ ED NO 157) blocking of h-IgE-induced histamine release in SX38 cells.
[0061] Figure 15A is a graph depicting the % full length ARC1384 and ARC1666 present in human and rat plasma as a function of incubation time. Figure 15B is a graph depicting the % full length ARC 1384, ARC 1572 and ARC 1573 present in human and rat plasma a function of incubation time.
[0062] Figure 16 is a table outlining the design of a pharmacokinetics study of PEGylated anti-IgE aptamers ARC1785 (SEQ TD NO 295), ARC1787 (SEQ ED NO 293), ARC1788 (SEQ ED NO 294), and ARC 1790 (SEQ ED NO 296) administered intravenously to mice at 10 mg/kg.
[0063] Figure 17 is a graph showing the pharmacokinetic profile of PEGylated anti-IgE aptamers ARC1785 (SEQ TD NO 295), ARC1787 (SEQ ED NO 293), ARC1788 (SEQ ED NO 294), ARC 1790 (SEQ ED NO 296) after intravenous (TV) administration to mice at 10 mg kg.
[0064] Figure 18 is a table outlining the design of a pharmacokinetics study of PEGylated anti-IgE aptamers ARC1785 (SEQ TD NO 295), ARC1787 (SEQ ED NO 293), ARC1788 (SEQ ED NO 294), and ARC 1790 (SEQ ED NO 296) administered sub-cutaneously to mice at 10 mg/kg.
[0071] The random sequence portion of the oligonucleotide can be of any length and can comprise ribonucleotides and/or deoxyribonucleotides and can include modified or non-natural nucleotides or nucleotide analogs. See, e.g., U.S. Patent No. 5,958,691; U.S. Patent No. 5,660,985; U.S. Patent No. 5,958,691; U.S. Patent No. 5,698,687; U.S. Patent No. 5,817,635; U.S. Patent No. 5,672,695, and PCT Publication WO 92/07065. Random oligonucleotides can be synthesized from phosphodiester-linked nucleotides using solid phase oligonucleotide synthesis techniques well known in the art. See, e.g., Froehler et al, Nucl. Acid Res. 14:5399- 5467 (1986) and Froehler et al, Tet. Lett. 27:5575-5578 (1986). Random oligonucleotides can also be synthesized using solution phase methods such as triester synthesis methods. See, e.g., Sood et al, Nucl. Acid Res. 4:2557 (1977) and Hirose et al, Tet. Lett., 28:2449 (1978). Typical syntheses carried out on automated DNA synthesis equipment yield 1014-1016 individual molecules, a number sufficient for most SELEX experiments. Sufficiently large regions of random sequence in the sequence design increases the likelihood that each synthesized molecule is likely to represent a unique sequence.
[0079] The core SELEXTM method has been modified to achieve a number of specific objectives. For example, U.S. Patent No. 5,707,796 describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. Patent No. 5,763,177 describes SELEXTM based methods for selecting nucleic acid ligands containing photo reactive groups capable of binding and/or photo-crosslinking to and/or photo-inactivating a target molecule. U.S. Patent No. 5,567,588 and U.S. Patent No. 5,861,254 describe SELEXTM based methods which achieve highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. Patent No. 5,496,938 describes methods for obtaining improved nucleic acid ligands after the SELEXTM process has been performed. U.S. Patent No. 5,705,337 describes methods for covalentiy linking a ligand to its target.
[0081] Counter-SELEXTM is a method for improving the specificity of nucleic acid ligands to a target molecule by eliminating nucleic acid ligand sequences with cross-reactivity to one or more non-target molecules. Counter- SELEX is compnsed ofthe steps of: (a) preparing a candidate mixture of nucleic acids; (b) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder ofthe candidate mixture; (c) partitioning the increased affinity nucleic acids from the remainder ofthe candidate mixture; (d) dissociating the increased affinity nucleic acids from the target; (e) contacting the increased affinity nucleic acids with one or more non-target molecules such that nucleic acid ligands with specific affinity for the non-target molecule(s) are removed; and (f) amplifying the nucleic acids with specific affinity only to the target molecule to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity and specificity for binding to the target molecule. As described above for SELEX , cycles of selection and amplification are repeated as necessary until a desired goal is achieved.
[0085] En further embodiments, the oligonucleotides comprise modified sugar groups, for example, one or more ofthe hydroxyl groups is replaced with halogen, aliphatic groups, or functionalized as ethers or amines. In one embodiment, the 2'-position ofthe furanose residue is substituted by any of an O-methyl, O-alkyl, O-allyl, S-alkyl, S-allyl, or halo group. Methods of synthesis of 2'-modified sugars are described, e.g., in Sproat, et al, Nucl. Acid Res. 19:733- 738 (1991); Cotten, et al, Nucl. Acid Res. 19:2629-2635 (1991); and Hobbs, et al, Biochemistry 12:5138-5145 (1973). Other modifications are known to one of ordinary skill in the art. Such modifications may be pre-SELEXTM process modifications or post-SELEX process modifications (modification of previously identified unmodified ligands) or may be made by incoφoration into the SELEXTM process. [0086] Pre- SELEXTM process modifications or those made by incoφoration into the SELEX process yield nucleic acid ligands with both specificity for their SELEX target and improved stability, e.g., in vivo stability. Post-SELEX process modifications made to nucleic acid ligands may result in improved stability, e.g., in vivo stability without adversely affecting the binding capacity ofthe nucleic acid ligand.
[0092] Aptamers that contain 2 '-O-methyl ("2 '-OMe") nucleotides, as provided herein, overcome many of these drawbacks. Oligonucleotides containing 2' -OMe nucleotides are nuclease-resistant and inexpensive to synthesize. Although 2'-OMe nucleotides are ubiquitous in biological systems, natural polymerases do not accept 2 '-OMe NTPs as substrates under physiological conditions, thus there are no safety concerns over the recycling of 2'-OMe nucleotides into host DNA. The SELEXTM method used to generate 2'-modified aptamers is described, e.g., in U.S. Provisional Patent Application Serial No. 60/430,761, filed December 3, 2002, U.S. Provisional Patent Application Serial No. 60/487,474, filed July 15, 2003, U.S. Provisional Patent Application Serial No. 60/517,039, filed November 4, 2003, U.S. Patent Application No. 10/729,581, filed December 3, 2003, and U.S. Patent Application No. 10/873,856, filed June 21, 2004, entitled "Method for in vitro Selection of 2'-O-methyl Substituted Nucleic Acids", each of which is herein incoφorated by reference in its entirety.
[0096] Generally, it has been found that under the conditions disclosed herein, the Y693F mutant can be used for the incoφoration of all 2 '-OMe substituted NTPs except GTP and the Y639F/H784A mutant can be used for the incoφoration of all 2'-OMe substituted NTPs including GTP. It is expected that the H784A mutant possesses properties similar to the Y639F and the Y639F/H784A mutants when used under the conditions disclosed herein.
[0098] A prefeπed embodiment includes any combination of 2'-OH, 2'-deoxy and 2'-OMe nucleotides. A more prefeπed embodiment includes any combination of 2' -deoxy and 2' -OMe nucleotides. An even more prefeπed embodiment is with any combination of 2 '-deoxy and 2'- OMe nucleotides in which the pyrimidines are 2'-OMe (such as dRmY, mRmY or dGmH).
[00103] Another important factor in the incoφoration of 2'-OMe substituted nucleotides into transcripts is the use of both divalent magnesium and manganese in the transcription mixture. Different combinations of concentrations of magnesium chloride and manganese chloride have been found to affect yields of 2'-O-methylated transcripts, the optimum concentration ofthe magnesium and manganese chloride being dependent on the concentration in the transcription reaction mixture of NTPs which complex divalent metal ions. To obtain the greatest yields of maximally 2' substituted O-methylated transcripts (i.e., all A, C, and U and about 90% of G nucleotides), concentrations of approximately 5 mM magnesium chloride and 1.5 mM manganese chloride are prefeπed when each NTP is present at a concentration of 0.5 mM. When the concentration of each NTP is 1.0 mM, concentrations of approximately 6.5 mM magnesium chloride and 2.0 mM manganese chloride are prefeπed. When the concentration of each NTP is 2.0 mM, concentrations of approximately 9.6 mM magnesium chloride and 2.9 mM manganese chloride are prefeπed. In any case, departures from these concentrations of up to two-fold still give significant amounts of modified transcripts.
[00111] For each ofthe above (a) transcription is preferably performed at a temperature of from about 20 °C to about 50 °C, preferably from about 30 °C to 45 °C, and more preferably at about 37 °C for a period of at least two hours and (b) 50-300 nM of a double stranded DNA transcription template is used (200 nM template is used in round 1 to increase diversity (300 nM template is used in dRmY transcriptions)), and for subsequent rounds approximately 50 nM, a 1/10 dilution of an optimized PCR reaction, using conditions described herein, is used). The prefeπed DNA transcription templates are described below (where ARC254 and ARC256 transcribe under all 2'-OMe conditions and ARC255 transcribes under rRmY conditions).
[00121] The HEPES buffer concentration can range from 0 to 1 M. The present invention also contemplates the use of other buffering agents having a pKa between 5 and 10 including, for example, Tris-hydroxymethyl-aminomethane.
[00122] The DTT concentration can range from 0 to 400 mM. The methods ofthe present invention also provide for the use of other reducing agents including, for example, mercaptoethanol.
[00124] The PEG-8000 concentration can range from 0 to 50 % (w/v). The methods ofthe present invention also provide for the use of other hydrophilic polymer including, for example, other molecular weight PEG or other polyalkylene glycols.
[00125] The Triton X-100 concentration can range from 0 to 0.1% (w/v). The methods ofthe present invention also provide for the use of other non-ionic detergents including, for example, other detergents, including other Triton-X detergents.
[00126] The MgCl2 concentration can range from 0.5 mM to 50 mM. The MnCl2 concentration can range from 0.15 mM to 15 mM. Both MgCl2 and MnCl2 must be present within the ranges described and in a prefeπed embodiment are present in about a 10 to about 3 ratio of MgCl2:MnCl2, preferably, the ratio is about 3-5:1, more preferably, the ratio is about 3- 4:1.
[00127] The 2'-OMe NTP concentration (each NTP) can range from 5 μM to 5 mM.
[00128] The 2'-OH GTP concentration can range from 0 μM to 300 μM.
[00129] The 2'-OH GMP concentration can range from 0 to 5 mM.
[00147] The terms "sequence identity" or "% identity" in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum coπespondence, as measured using one ofthe following sequence comparison algorithms or by visual inspection. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., infra).
[00153] Recognition of bacterial DNA by the vertebrate immune system is based on the recognition of unmethylated CG dinucleotides in particular sequence contexts ("CpG motifs"). One receptor that recognizes such a motif is Toll-like receptor 9 ("TLR 9"), a member of a family of Toll-like receptors (~10 members) that participate in the innate immune response by recognizing distinct microbial components. TLR 9 binds unmethylated oligodeoxynucleotide ("ODN") CpG sequences in a sequence-specific manner. The recognition of CpG motifs triggers defense mechanisms leading to innate and ultimately acquired immune responses. For example, activation of TLR 9 in mice induces activation of antigen presenting cells, up regulation of MHC class I and II molecules and expression of important co-stimulatory molecules and cytokines including IL-12 and IL-23. This activation both directly and indirectly enhances B and T cell responses, including robust up regulation ofthe TH1 cytokine EFN- gamma. Collectively, the response to CpG sequences leads to: protection against infectious diseases, improved immune response to vaccines, an effective response against asthma, and improved antibody-dependent cell-mediated cytotoxicity. Thus, CpG ODNs can provide protection against infectious diseases, function as immuno-adjuvants or cancer therapeutics (monotherapy or in combination with a mAb or other therapies), and can decrease asthma and allergic response.
[00155] En group two, the strategies are directed to identifying or generating aptamers comprising a CpG motif and/or other sequences that are bound by the receptors for the CpG motifs (e.g., TLR9 or the other toll-like receptors) and upon binding stimulate an immune response. The first strategy of this group comprises performing SELEX to obtain an aptamer to a target known to bind to CpG motifs or other immunostimulatory sequences and upon binding stimulate an immune response using an oligonucleotide pool wherein a CpG motif has been incoφorated into each member ofthe pool as, or as part of, a fixed region, e.g., in some embodiments the randomized region ofthe pool members comprise a fixed region having a CpG motif incoφorated therein, and identifying an aptamer comprising a CpG motif. The second strategy of this group compnses performing SELEX to obtain an aptamer to a target known to bind to CpG motifs or other immunostimulatory sequences and upon binding stimulate an immune response and then appending a CpG motif to the 5' and/or 3' end or engineering a CpG motif into a region, preferably a non-essential region, ofthe aptamer. The third strategy of this group comprises performing SELEX to obtain an aptamer to a target known to bind to CpG motifs or other immunostimulatory sequences and upon binding stimulate an immune response wherein during synthesis ofthe pool, the molar ratio ofthe various nucleotides is biased in one or more nucleotide addition steps so that the randomized region of each member ofthe pool is enriched in CpG motifs, and identifying an aptamer comprising a CpG motif. The fourth strategy of this group comprises performing SELEXTM to obtain an aptamer to a target known to bind to CpG motifs or other immunostimulatory sequences and upon binding stimulate an immune response and identifying an aptamer - comprising a CpG motif. The fifth strategy of this group comprises performing SELEXTM to obtain an aptamer to a target known to bind to CpG motifs or other immunostimulatory sequences, and identifying an aptamer which upon binding, stimulate an immune response but which does not comprise a CpG motif.
[00172] In this context, the quantity of active ingredient and volume of composition to be administered depends on the host animal to be treated. Precise amounts of active compound required for administration depend on the judgment ofthe practitioner and are peculiar to each individual.
[00173] A minimal volume of a composition required to disperse the active compounds is typically utilized. Suitable regimes for administration are also variable, but would be typified by initially administering the compound and monitoring the results and then giving further controlled doses at further intervals.
[00174] For instance, for oral administration in the form of a tablet or capsule (e.g., a gelatin capsule), the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incoφorated into the mixture. Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyπolidone, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum starches, agar, alginic acid or its sodium salt, or effervescent mixtures, and the like. Diluents, include, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine.
[00180] Furthermore, prefeπed compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, inhalants, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. Other prefeπed topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of active ingredient would typically range from 0.01% to 15%, w/w or w/v.
[00187] Effective plasma levels ofthe compounds ofthe present invention range from 0.002 mg/mL to 50 mg/mL.
[00201] PAGs are polymers which typically have the properties of solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity. One use of PAGs is to covalentiy attach the polymer to insoluble molecules to make the resulting PAG-molecule "conjugate" soluble. For example, it has been shown that the water-insoluble drug paclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, et al, J. Org. Chem., 60:331-336 (1995). PAG conjugates are often used not only to enhance solubility and stability but also to prolong the blood circulation half-life of molecules.
[00202] Polyalkylated compounds ofthe invention are typically between 5 and 80 kDa in size however any size can be used, the choice dependent on the aptamer and application. Other PAG compounds ofthe invention are between 10 and 80 kDa in size. Still other PAG compounds ofthe invention are between 10 and 60 kDa in size. For example, a PAG polymer may be at least 10, 20, 30, 40, 50, 60, or 80 kDa in size. Such polymers can be linear or branched. In some embodiments the polymers are PEG. In some embodiment the polymers are branched PEG. In still other embodiments the polymers are 40kDa branched PEG as depicted in Figure 3. In some embodiments the 40 kDa branched PEG is attached to the 5' end ofthe aptamer as depicted in Figure 4.
[00205] Production of high molecular weight PEGs (>10 kDa) can be difficult, inefficient, and expensive. As a route towards the synthesis of high molecular weight PEG-nucleic acid conjugates, previous work has been focused towards the generation of higher molecular weight activated PEGs. One method for generating such molecules involves the formation of a branched activated PEG in which two or more PEGs are attached to a central core carrying the activated group. The terminal portions of these higher molecular weight PEG molecules, i.e., the relatively non-reactive hydroxyl (-OH) moieties, can be activated, or converted to functional moieties, for attachment of one or more ofthe PEGs to other compounds at reactive sites on the compound. Branched activated PEGs will have more than two termim, and in cases where two or more termini have been activated, such activated higher molecular weight PEG molecules are refened to herein as, multi-activated PEGs. In some cases, not all termini in a branch PEG molecule are activated. In cases where any two termini of a branch PEG molecule are activated, such PEG molecules are refeπed to as bi-activated PEGs. In some cases where only one terminus in a branch PEG molecule is activated, such PEG molecules are refeπed to as mono-activated. As an example of this approach, activated PEG prepared by the attachment of two monomethoxy PEGs to a lysine core which is subsequently activated for reaction has been described (Harris et al, Nature, vol.2: 214-221, 2003).
[00212] High molecular weight PAG-nucleic acid-PAG conjugates can be prepared by reaction of a mono-functional activated PEG with a nucleic acid containing more than one reactive site. In one embodiment, the nucleic acid is bi-reactive, or bi-activated, and contains two reactive sites: a 5'-amino group and a 3'-amino group introduced into the oligonucleotide through conventional phosphoramidite synthesis, for example: 3'-5'-di-PEGylation as illustrated in Figure 5. In alternative embodiments, reactive sites can be introduced at internal positions, using for example, the 5-position of pyrimidines, the 8-position of purines, or the 2'- position of ribose as sites for attachment of primary amines. In such embodiments, the nucleic acid can have several activated or reactive sites and is said to be multiply activated. Following synthesis and purification, the modified oligonucleotide is combined with the mono-activated PEG under conditions that promote selective reaction with the oligonucleotide reactive sites while minimizing spontaneous hydrolysis. In the prefened embodiment, monomethoxy-PEG is activated with succinimidyl propionate and the coupled reaction is carried out at pH 8.3. To drive synthesis ofthe bi-substituted PEG, stoichiometric excess PEG is provided relative to the oligonucleotide. Following reaction, the PEG-nucleic acid conjugate is purified by gel electrophoresis or liquid chromatography to separate fully, partially, and un-conjugated species.
[00215] All publications and patent documents cited herein are incoφorated herein by reference as if each such publication or document was specifically and individually indicated to be incoφorated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date ofthe same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples below are for pmposes of illustration and not limitation ofthe claims that follow. EXAMPLES
[00231] The dRmY h-IgE selection was enriched for h-IgE binding vs. the naϊve pool after 6 rounds of selection. At round 6 and round 7 the pool KD was approximately 4 nM. The round 6 pool was cloned using TOPO TA cloning kit (Invitrogen) and 31 individual sequences were generated. There were two dominant clones, represented by 8 and 3 ofthe 31 sequences, and 21 singletons. Figure 6 shows a plot of fraction bound versus h-IgE concentration for the round 6 and 7 pools.
[00244] Efforts were taken to minimize the IgE aptamers described above in Example 1 A while maintaining, preferably improving, binding affinity. In order to identify the core structural elements required for h-IgE binding, the 3 '-boundaries of several ofthe high affinity h-IgE binders were determined. RNA transcripts were labeled at the 5 '-end with γ-32P ATP and T4 polynucleotide kinase. Radiolabeled ligands were subjected to partial alkaline hydrolysis and then selectively bound in solution to h-IgE at 500 nM before being partitioned over nitrocellulose filters. Retained oligonucleotides were resolved on 8 % denaturing polyacrylamide gels. The smallest oligonucleotide bound to h-IgE defined the 3'- boundary. The 3'- boundaries of selected clones are described in Table 8. On the basis ofthe boundary experiments as well as visual inspection of predicted folds, truncated constructs were prepared and oligos were ordered from Integrated DNA Technologies (Coralville, IA). Minimized versions ofthe parent clones with nucleic acid sequences according to SEQ TD NO 11, SEQ ED NO 18, and SEQ ED NO 21 showed significant protein binding, measured by the sandwich filter binding assay previously described. Minimer binding data are shown in Table 8, while the coπesponding sequences are shown in Table 9.
[00263] The first step in the process of replacing deoxy containing residues with 2'-O methyl containing residues was the synthesis and assay for binding activity of ARC1250 - ARC1264 (SEQ TD NOs 158-172) each of which is equivalent to ARC445 with the addition of a 3'- inverted-dT (3T) and the replacement of a single 2'-deoxy residue with a 2'-O methyl residue. As can be seen from the binding data in Table 17, some positions readily tolerate substitution of a deoxy residue for a 2'-O methyl residue, while others do not. Interestingly, replacement of a 2 '-deoxy residue with a 2'-O methyl residue confeπed a significant improvement in affinity at position 10 (ARC1256) (SEQ TD NO 164).
[00266] During the synthesis of ARC445 and its derivatives, peaks were observed on ion exchange HPLC that appeared to conespond to multimeric aggregates of ARC445. Figure 8 is an example ofthe HPLC trace analysis of ARC445 and several derivatives which shows the multiple peaks due to aptamer aggregation. ARC445 contains 2 runs of guanosine residues at positions 6-9 and 16-21. While not intending to be bound by theory, either or both of these runs may be the cause of aptamer aggregation. It has been reported that in much the same way that ethidium bromide fluorescence is increased upon binding to duplex RNA and DNA, that N- methylmesoporphyrin EX (NMM) fluorescence is increased upon binding to G-quartet structures (Arthanari et al, Nucleic Acids Research, 26(16): 3724 (1996); Marathais et al, Nucleic Acids Research, 28(9): 1969 (2000); Joyce et al, Applied Specfroscopy, 58(7): 831 (2004)). Thus as shown in Figure 9, we used NMM fluorescence to establish that ARC445 does in fact adopt a G-quartet structure. According to the literature protocols, 100 microliter reactions containing ~1 micromolar NMM and ~ 2 micromolar aptamer in Dulbecco's PBS containing magnesium and calcium were analyzed using a SpectraMax Gemini XS fluorescence plate reader. Fluorescence emission spectra were collected from 550 to 750 nm with and excitation wavelength of 405 nm. The G-quartet structure ofthe anti-thrombin DNA aptamer ARC 183 has long been established in the literature (Macaya et al., Proc. Natl. Acad. Sci., 90: 3745 (1993)) and thus it was used as a positive control in this experiment (Figure 10). ARC 1346 shown in Figure 10 is an aptamer of a similar size and nucleotide composition as ARC445 that is not predicted to have a G-quartet structure and is therefore a negative control in the experiment (Figure 10). As can be seen Figures 9 and 10, ARC 183 and ARC445 show a significant increase in NMM fluorescence relative to NMM alone while the negative control, ARC 1346 does not.
[00293] Two hundred thousand (200,000) SX38 cells per well were plated in MEM plus 16% FBS in a 24 well plate one day prior to the experiment. The next day, appropriate concentrations of aptamer (8 point titration of 2-fold serial dilutions beginning with lμM) plus 3 ug/mL of h-IgE (Athens Research and Technology, Athens, GA) were incubated together for 30 minutes in MEM plus 16% FBS. The mixture was then added to the SX38 cells and incubated at 37 °C, 5% CO2 for 30 minutes. Each concentration was tested in quadruplicate. The cells were washed three times with MEM plus 16% FBS, then incubated for an additional 5.5 hours in MEM plus 16% FBS. An h-IgE cross-linking antibody (QED Biosciences, San Diego, CA) was added to the media on the cells at a concentration of 1 ug/mL and incubated for an additional 2 hours. The supematants were collected and frozen at -20 °C until use in the histamine ELISA. The histamine ELISA was used according to manufacturer's recommendations. h-IgE alone and h-IgE versus the naive pool were included as positive and negative controls, respectively. IgE aptamer activity was measured by percent inhibition of histamine release into the supematants as shown in Figure 14 (wherein ARC445 is SEQ TD NO 101, ARC656 is SEQ ED NO 157 and ARC714 is a non-binding negative control).
[00294] Several modified derivatives of ARC445 (SEQ ED NO 101) (described in Example 2E), along with the DNA aptamer, ARC656 (SEQ ED NO 157) (described in Example 2D) were also tested for their ability to inhibit histamine release in SX38 cells as described above. The calculated IC5oS for the ARC445 derivatives tested are summarized in Table 25.
[00300] Plasma concentration versus time data appeared was subjected to noncompartmental analysis (NCA) using the industry standard pharmacokinetic modeling software WinNonLin TM v.4.0 (Pharsight Corp., Mountain View, CA). Estimates were obtained for the following primary pharmacokinetic parameters: maximum plasma concentration, Cmax; area under the concentration-time curve, AUC; terminal half-life, tl/2; terminal clearance, CI; and volume of distribution at steady state, Vss. Noncompartmental analysis (NCA) ofthe data using WinNonLinTM v.4.0 (Pharsight Corp., Mountain View, CA)., yielded the estimates for the primary pharmacokinetic parameters listed in Figure 20.
[00304] The invention having now been described by way of written description and example, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the description and examples above are for purposes of illustration and not limitation ofthe following claims.

中文:
[0012]目前一些临床评估的治疗方法干预潜在的致过敏性病理免疫机制,比如,一种抗IgE的免疫球蛋白,它直接靶向于IgE血清抗体,从而抑制速发型超敏反应的主要机制。此外,目前人们的兴趣在于开发过敏原特异性免疫治疗方法,因为它具有治疗过敏症的潜力。但是,无论抗IgE的免疫球蛋白还是过敏原特异性免疫治疗都受到高成本、永久治疗或每个季节治疗的限制。
[0047]图1是一个IgE介导的信号传递活动示意图。
[0049]图3显示一个40kDa的分支状PEG。
[0050]图4显示40kDa的已经附着到适体5’末端的分支状PEG。
[0051]图5是一个描绘各种PEG化策略的插图,表示了标准单聚乙二醇化、多聚乙二醇化,和通过聚乙二醇化完成的二聚作用。
[0053]图7显示了对ARC445(序列号101)及其衍生物的直接结合力曲线和结合亲合力,描绘了修饰产物对h-IgE结合能力增加的比例。
[0054]图8描绘了抗IgE适体ARC445(序列号101)和其衍生物的离子交换HPLC痕量分析。
[0055]图9显示在ARC445(序列号101)中NMM荧光增加,在ARC445衍生物与ARC909-911(序列号191-193)中NMM荧光的降低,ARC909-911包括脱氧鸟嘌呤(dG)的7-脱氮-G取代物。
[0056]图10显示在ARC 183中(阳性对照)的增加,在ARC1346中(阴性对照)NMM荧光的降低。
[0057]图11显示了与其衍生物比较,ARC445(序列号101)的直接结合曲线,描述了用7-脱氮-G取代脱氧鸟嘌呤(ARC909-911(序列号191-193))明显的减少了与h-IgE结合的比例。
[0058]图12表示了与ARC1384(序列号181),包括2′-氧-甲基和硫代硫酸盐取代物,但没有dG的次黄嘌呤取代物(序列号101)的ARC445衍生物相比较,以下适体在NMM荧光中降低:ARC445衍生物ARC 1641,1642,和1666(序列号分别是212,213,和216),它们包括dG的次黄嘌呤取代物。
[0059]图13是一种结构示意图预测rRfY,dRmY和DNA最小化适体的二级结构,DNA最小化适体表明IgE:FcεR1之间的最高结合力,该力受到FACS的抑制。图13(A)显示根据序列号91的rRfY克隆,残基是2′-F,图13(B)显示ARC445(序列号101),一个rRfY克隆,黑色残基是2′-脱氧,灰色残基是2’-OMe,图13(C)显示ARC475(序列号151),DNA克隆,下划线是灰色残基是2′-脱氧。
[0060]图14显示SX38细胞中,ARC445(序列号101)和ARC656(序列号157)阻碍h-IgE诱导组氨酸的释放。
[0061]图15A描述存在于人类和小鼠血浆中作为孵育功能的ARC1384和ARC1666全长的百分率。图15B描述存在于人类和小鼠血浆中作为孵育功能的ARC1384,ARC1572和ARC1573全长的百分率。
[0062]图16是表格,给出对小鼠静脉给药10毫克/公斤后PEG化抗-IgE适体ARC1785(序列号295),ARC1787(序列号293),ARC1788(序列号294),和ARC1790(序列号296)的药代动力学设计。
[0063]图17是对小鼠静脉(IV)给药10毫克/公斤后,PEG化抗-IgE适体ARC1785(序列号295),ARC1787(序列号293),ARC1788(序列号294),ARC1790(序列号296)的药代动力学图形。
[0064]图18是对小鼠皮下给药10毫克/公斤后,PEG化抗-IgE适体ARC1785(序列号295),ARC1787(序列号293),ARC1788(序列号294),和ARC1790(序列号296)的药代动力学研究设计概括表格。
[0071]寡核苷酸的随机序列部分可以是任意长度,并且可包括核苷酸和/或脱氧核苷酸,还可包括修饰或非天然核苷或核苷类似物。参见美国专利第5,958,691号;美国专利第5,660,985号;美国专利第5,958,691号;美国专利第5,698,687号;美国专利第5,817,635号;美国专利第5,672,695号;和PCT刊物WO92/07065号。使用本领域已知的固相寡核苷酸合成技术从磷酸二脂键连接的核苷中合成随机寡核苷酸。参见Froehler等人在Nucl Acid Res.14:5399-5467(1986)上发表的文献和Froehler等人在Tet.Lett.27:5575-5578(1986)上发表的文献。也可使用溶液方法如三酯合成法合成随机寡核苷酸。参见例如Sood等人在Nucl.Acid Res.4:2557(1977)上发表的文献和Hirose等人在Tet.Lett.,28:2449(1978)上发表的文献。在自动DNA合成仪器中进行的经典合成产生了1014-1016个独立的分子,数量上足够进行大多数SELEXTM实验。序列设计中随机序列足够大的区域增加了每个被合成分子代表特异序列的可能。
[0079]核心SELEXTM方法被修改以获得许多特异目标。例如,美国专利第5,707,796号描述了联合使用SELEXTM和凝胶电泳筛选结构特异的核酸分子,如,弯曲DNA。美国专利第5,763,177号;描述了基于SELEXTM的方法,筛选含有反应基团的核酸配体,该反应基团能结合靶分子,和/或交叉连接到靶分子,和/或激活靶分子。美国专利第5,567,588号和第5,861,254描述了基于SELEXTM的方法,高效分离与靶点有高亲和力和低亲和力的寡核苷酸。美国专利第5,496,938描述了SELEXTM过程进行之后获取改进核酸配体的方法。美国专利第5,705,337号描述了将配体共价结合靶点的方法。
[0081]Counter-SELEXTM是一种通过减少能与一个或多个非靶点分子发生交叉反应的核酸配体序列从而改进核酸配体和靶点特异性的方法。Counter-SELEXTM的步骤包括步骤:(a)制备核酸侯选混合物(b)侯选混合物与靶点结合,其中相对于侯选混合物,对靶点有增加的亲和力的核酸,可能从侯选混合物的剩余物中分离出来;(c)从候选混合物的剩余物中分离出亲和力增加的核酸;(d)从靶点中分离出亲和力增加的核酸;(e)将亲和力增加的核酸与一个或一个以上非靶分子相互接触,对非靶点分子有特异亲和力的核酸配体可被清除掉;和(f)扩增只对靶分子有特异亲和力的核酸产生核酸混合物,富集于对靶分子有相对较高亲和力和特异性的核酸序列。如上述对SELEXTM的描述,筛选和扩增的循环数不断被重复直到达到目的。
[0085]在进一步的实施方案中,寡核苷酸包括修饰的糖基,如,一个或一个以上羟基被卤素基团,脂肪族基团或有酯或氨基功能的基团所取代。在一个实施方案中,2′位的呋喃糖残基被O-甲基,O-烷基,O-烯基,S-烷基,S-烯基,或卤素基团任意一个取代。2′修饰的糖的合成方法在例如,即,Sproat,等人的文献Nucl.Acid Res.19:733-738(1991);cotton等人的文献Nucl.Acid Res.19:2629-2635(1991)和Hobbs等人的文献Biochemistry 12:5138-5145(1973)中进行了描述。另一些修饰对本领域普通技术人员是已知的。这种修饰可能是pre-SELEXTM过程修饰或post-SELEXTM过程修饰(前面鉴定未修饰配体的修饰)或通过向SELEXTM中加入修饰。
[0092]这里提供的含有2′-氧-甲基(′′2′-OMe′′)核苷的适体克服了上述很多弊端。含有2’-氧-甲基核苷的寡核苷酸可抵制核酶降解,廉价合成。虽然2’-氧-甲基核苷在生物系统中普遍存在,天然聚合酶在生理条件下不接受2’-氧-甲基NTPs作为底物,因此对循环的2’-氧-甲基核苷酸进入宿主的DNA后,没有安全上的顾虑。产生2′-修饰适体的SELEX方法已作描述,例如,2002年12月3日提出的美国临时专利申请编号第60/430,761号,2003年7月15日提出的美国临时专利申请编号60/487,474;2003年11月4日提出的美国临时专利申请编号60/517,039;美国专利申请编号10/729,581;2004年7月21日提出的美国临时专利申请编10/873,856标题′′体外筛选2′-氧-甲基取代核酸的方法′′,这里提到的每个专利通过在此引证全部并入本文。
[0096]通常,在此公开的条件下,Y693F突变体可用于结合2’-OMe取代的除GTP外核苷三磷酸NTPs,Y639F/H784A突变体可用于所有2’-OMe取代的包括GTP在内的NTPs。可以预见,在此公开的条件下,突变体H784A与Y639F和突变体Y639F/H784A性质相似。
[0098]优选的实施方案包括任何组合的2′-OH,2′-脱氧和2′-OMe核苷。较好优选实施方案包括任何组合2′-脱氧和2′-OMe核苷。更优选实施方案包括任何组合的2′-脱氧,和2′-OMe核苷,其中嘧啶2′-OMe如(dRmY,mRmY或dGmH)。
[00103]影响2’-氧-甲基取代核苷渗入转录物的另一个重要因素是转录混合物中的二价镁和锰。已有发现氯化镁和氯化锰以不同的浓度组合影响2′-氧-甲基转录的产量,氯化镁和锰的最适浓度取决于其在含有二价金属离子以NTPs转录反应混合物的浓度。为了获取2′取代的氧甲基化转录物的最大产量(即所有的A,C和U和大约90%G核苷),当每种NTP的浓度是0.5mM时,优选的氯化镁的浓度大约是5mM,而优选的氯化锰的浓度大约是1.5mM。当每种NTP的浓度是1..0mM时,优选的氯化镁的浓度大约是6.5mM,而优选的氯化锰的浓度大约是2.0mM。当每种NTP的浓度是2.0mM时,优选的氯化镁的浓度大约是9.6mM,而优选的氯化锰的浓度大约是2.9mM。在任何情况下,非二倍浓度的情况下仍然能显著提高修饰转录产物的量。
[00111]对于上述每种情况(a)转录优选在20°C到50°C,优选于30°C到45°C,更优选37°C进行至少两个小时(b)50-300nM的双链DNA转录模板被使用(在第一轮反应中使用200nM模板用以增加多样性(300nM的模板用于dRmY转录)),在随后几轮反应中大约50nM,以1/10的比例稀释用来优化PCR反应,使用在此所述的条件。优选DNA转录模板如下(其中ARC254和ARC256在全2′-氧甲基的条件下转录而ARC255在rRmY的条件下转录)。
[00121]HEPES缓冲液的浓度范围为0到1M。本发明也考虑使用其他pKa值介于5-10的缓冲液试剂。例如,Tris-羟甲基-氨基甲烷。
[00122]DTT浓度范围介于0-400mM。本发明的方法也提供了其他还原试剂的使用方法,包括,巯基乙醇。
[00124]PEG-8000浓度范围为0到50%(w/v)。本发明的方法也提供了亲水聚合体使用方法,包括其他分子量的PEG或其他亲水的聚乙二醇。
[00125]Triton X-100浓度范围为0到0.1%(w/v)。本发明的方法也提供了非离子去垢剂的使用,包括其他去垢剂和其他Triton-X去垢剂。
[00126]MgCl2的浓度范围为0.5mM到50mM。MnCl2的浓度范围为0.15mM到15mM。MgCl2和MnCl2必须在此所述的范围内,在优选实施方案中MgCl2∶MnCl2的比例在大约10到3左右,优选的,MgCl2∶MnCl2的比例是大约3-5∶1,更优选MgCl2∶MnCl2的比例是大约3-4∶1。
[00127]2’-氧-甲基NTP浓度(每种NTP)范围为5μM-5mM。
[00128]2′-OH GTP浓度范围为0μM-300μM。
[00129]2′-OH GMP浓度范围为0-5mM。
[00147]文章里出现的关于核苷酸或蛋白质序列的术语′′序列一致性′′或′′一致性%′′指的是在使用下面一种序列对比运算方法或靠视觉观测来比较或比对最大吻合度时,两个或多个序列或子序列其氨基酸残基序列或核苷酸序列相同,或有一定比例的序列相同。序列对比时,通常,其中一个序列作为参照序列,其他序列与之进行对比。当用到一种数列对比运算方法时,参照序列和检测序列输入计算机,如有必要还可设定子序列对等物,还可设定序列运算法则程序中的参数。序列对比运算法则然后计算出检测序列相对于参照序列的的相同序列百分比。可对要比较序列做最佳比对,如通过Smith & Waterman,Adv.Appl.Math.2:482(1981)中的局部同源比对,通过NeedIeman & Wunsch,J MoI.Biol.48:443(1970)中同源比对运算法则,通过Pearson & Lipman,Proc.Nat′l.Acad.Sci.USA 85:2444(1988)的相似方法研究和这些运算法则的计算机化(Wisconsm GeneticsSoftware Package,Genetics Computer Group,575 Science Dr.,Madison,Wis中的GAP,BESTFIT,FASTA,和TFASTA)或通过视觉观测(参见Ausubel等人的文献,同上)。
[00153]脊椎动物免疫系统对细菌性DNA的识别是基于对特定序列背景下未甲基化CG二核苷酸的识别(′′CpG基序′′)。识别这种基序的一个受体是Toll样受体9(′′TLR9′′),是Toll样受体家族的成员(大约10个成员)内之一。Toll样受体通过识别特异细菌性成分参与先天免疫应答。TLR9与未甲基化寡核苷酸(′′ODN′′)CpG序列以一种序列特异方式结合。CpG基序的识别触发了引起先天免疫反应防御机制,最终获得了免疫应答。例如,激活小鼠体内TLR9将引发抗原呈现细胞的激活,I和II类MHC分子的上调以及重要共调节分子和细胞激酶包括IL-12和IL-23等的表达。这种激活都直接或介接加强了B和T细胞应答,包括TH1细胞激酶IFN-γ的有力上调。全体的,对CpG序列的应答引起:抵御感染疾病,改进对疫苗的免疫反应,对哮喘有效应答,改进抗体依赖细胞介导的细胞激酶。因此CpG ODNs可抵抗感染性疾病,起到免疫辅助作用或癌症治疗作用(单独疗法或与或其他药物联合疗法),并能减少哮喘和过敏反应。
[00155]第二组中,策略是针对鉴定或生成包括CpG基序和/或其他被CpG基序的受体(如,TLR9或其他toll形受体)结合的并触发免疫应答的序列的适体。本组的第一种策略包括进行SELEXTM获得对已知能与CpG基序或其他免疫触发序列相结合并刺激免疫应答发生的靶标的适体,使用CpG基序已经被加入到每一个成员中作为或者部分作为其固定区域的寡核苷酸库,例如在某些实施例中,寡核苷酸库成员的随机区域包括加入了CpG基序的固定区域,以及识别包括了CpG基序的适体。本组的第二种策略包括进行SELEXTM获得对已知可与CpG基序或其他免疫触发序列相结合的靶标的适体,这种结合一旦发生能够刺激免疫反应,然后将CpG基序添加到适体5′和/或3′末端或将其工程构造到适体的一种区域,优选非必需区域中。本组的第三种策略包括进行SELEXTM获得对已知与CpG基序或其他免疫触发序列相结合的靶标的适体,这种结合一旦发生能够刺激免疫反应,其中,在寡核苷酸库的合成过程中,不同核苷酸的摩尔比偏向一种或多种核苷酸添加步骤,使库中每一成员的随机化区域富集CpG基序,以及识别包括了CpG基序的适体。本组的第四种策略包括进行SELEXTM获得对已知与CpG基序或其他免疫触发序列相结合的靶标的适体,这种结合一旦发生能够刺激免疫反应,以及识别包括了CpG的适体。本该组的第五种策略包括进行SELEXTM以获得对已知与CpG基序结合或与其他免疫触发序列结合的靶标的适体,以及识别适体,该适体虽不包括CpG基序,但是,一旦结合就刺激免疫应答。
[00172]在这种背景下,活性成分的量和给予的组合物的体积取决于要处理的宿主动物。给药所需活性化合物需要的精确的量取决于执行者所做的判断并因个体不同而有差异。
[00173]通常使用分散活性化合物所需要的最小的体积的组合物。虽然适宜的给药方案也不同,但典型的给药方案是先执行化合物给药并监测结果然后再按一定的时间间隔上进一步控制药物剂量。
[00174]例如,对片剂或胶囊(例如明胶胶囊)形式的口服给药,活性药物成分可以与乙醇,甘油,水或这类能口服的非毒性药理学可容受的内载体一起给药。而且,根据需要还可在混合物中添加合适的连接物,润滑剂,崩解剂和着色剂。合适的连接物包括淀粉,硅酸锰铝,淀粉糊,明胶,甲基纤维素,羧甲基纤维素钠和/或聚乙烯吡咯烷酮,例如葡萄糖和beta-乳糖的天然糖,玉米甜料,天然或合成树脂,如阿拉伯胶,黄芪胶或藻酸钠,聚乙烯乙二醇,蜡或此类。在这些剂型中所使用的润滑剂包括油酸钠,硬脂酸钠,硬脂酸镁,安息香酸钠,醋酸钠,氯化钠,无水硅石,滑石,硬脂酸及其镁或钙盐和/或聚乙二醇,和此类。崩解剂包括,但不止限于,淀粉,甲基纤维素,琼脂,黏土,黄原胶树脂淀粉,藻酸及其钠盐,或生泡混合物,和此类。稀释剂包括乳糖,葡萄糖,蔗糖,甘露糖,山犁糖,纤维素和/或氨基乙酸。
[00180]此外,本发明的优选组合物可以使用本领域普通技术人员已知的方式通过适当的鼻用工具,吸入器鼻部局部给药,或通过透皮贴剂形式由真皮途径鼻内给药。皮肤贴剂通过透皮途径给药,在该领域里都是为人熟知的技艺。使用真皮运输系统的形式给药,整个剂量方案中剂量给药应该是连续的而不是间断。另一些优选的局部制剂包括乳剂,药膏,洗剂,气雾剂和凝胶体,其中活性成分典型的浓度在0.01%至15%,w/w或w/v范围内。
[00187]本发明化合物的有效血浆水平在0.002mg/mL至50mg/mL范围内。
[00201]PAGs是在水溶液和有机溶液中有良好溶解性,无毒性,无免疫原性的聚合物。PAGs可将聚合体共价连接到不溶性分子上使PAG-分子连接体变成可溶的。例如,水溶性药物 紫杉醇连接到PEG上就变成水溶性的。Greenwald等人,J.Org.Chem.60:331-336(1995)。PAG连接体不仅用于增加溶解性和稳定性也用于延长分子在血液循环中的半衰期。
[00202]本发明的聚烷基化合物代表性的大小在5-80kDa之间,尽管可以使用任意大小,但是还是要根据适体和应用来确定。发明的另一些PAG复合物大小在10-80kDa之间。还有其他发明的PAG复合物大小在10-60kDa之间。例如,一个PAG聚合体大小可能至少是10,20,30,40,50,60,或80kDa。这种聚合体可以是线形化的或分支的。在一些实施方案中,聚合体是PEG。在一些实施方案中,聚合体是分支PEG。在另一些实施方案中,如图3所述,聚合物是40kDa的支链PEG。在一些实施方案中,如图4所述,40kDa的支链PEG连接到适体的5′端。
[00205]高分子量PEGs(>10kDa)的生成是困难的,效率低下,且价格昂贵。作为合成高分子量PEG核酸连接体的一条途径,先前的工作目的集中于生成更高的分子量激活的PEGs。生成这种分子的一种方法包括形成激活的分支PEG,其中两个或多个PEGs连接到带有激活基团的中心核。这些高分子量PEG分子的末端部分,如相对的非反应羟基(-OH),可以激活或转化为功能基团,以便于一个或多个PEGs连接到其他组合物的反应位点上。分支的活化的PEGs可以多于两个作用位点。当两个或多个末端被激活,这种激活的高分子量PEG分子在此称为多激活PEGs。在一些情况中,分支的PEGs分子中并不是所有的作用位点都被激活。在任意两个位点都被激活的情况下的PEG分子被称为双激活PEGs。在只有一个位点激活的情况下,这种PEG分子被称为单激活PEGs。这种途径的一个例子是,通过连接单甲氧基PEGs到赖氨酸中心来制备激活PEG,该中心被激活以利于随后的反应(Harris等人,Nature,vol.2:214-221,2003)。
[00212]高分子量PAG-核酸-PAG连接体可通过单功能激活的PEG与含有多个反应位点核酸的反应来制备。在实施方案中,核酸是双反应的或双激活的含有多个反应位点:通过传统的脱氧核苷亚磷酰胺合成方法引入到寡核苷酸上的一个5′-氨基酸基团和一个3′-氨基酸基团,例如:在图5所示的3′,5′二PEG化产物。在另一些实施方案中,可在内部引入反应位点,如在嘧啶的第5位,嘌呤的第8位,或核糖的2′位点引入主要氨基酸的结合位点。在这些实施方案中,核酸可有几个激活的或反应位点,有人说可被多激活。合成和纯化后,在促进与寡核苷酸反应选择性反应同时也减少了自发水解的条件下,修饰寡核苷酸与单位点激活的PEG连接。在优选实施方案中,单甲氧化PEG由羟基琥珀酰亚胺丙酸盐激活,连接反应在pH8.3进行。为了促进双取代PEG的合成,提供了相对于寡核苷酸计量过量的PEG。反应之后,PEG-核酸连接体经凝胶电泳或液相色谱分离出完全,部分和非连接样本。
[00215]所有在此引用的专利和刊物都被在此引证而全部合并与本文,就如同每个专利文件和刊物都被分别明确逐个的引证而合并于本文。引用刊物和专利文件并不表明其是现有技术文献,也不表明对其内容或日期的认可。本发明已通过书面描述的方式进行描述,本领域技术人员将意识到本发明可在多种不同实施方案中实践。前面的描述和下面的实施例都是用以阐述的而不是对所附权利要求的限制。
[00231]筛选第6轮后,相对于自然序列库,dRmY h-IgE富集以与h-IgE连接。在第6和7轮后,序列库的KD接近于4nM。使用TOPO TA克隆仪(Mvitrogen)克隆第6轮混合物,产生了31个序列。31个序列中8和3是其中的两个主要代表序列,和21单件。图6显示是显示第6和7轮混合物相对于h-IgE浓度的结合比率图。
[00244]尽量使实施例1A中描述的IgE适体最小化,同时保持,优选改善适体的结合亲和力。为了鉴定h-IgE结合所必需的核心结构元件,确定了其中几个高亲和力h-IgE粘合物的3′边界。MA转录产物在5′末端以γ-32P ATP和T4多聚核苷酸激酶标记。放射性标记的配体部分碱水解并在硝化纤维过滤分开之前选择性的在溶液中连接到h-IgE 500nM。保留的寡核苷酸在8%的变性聚丙烯酰胺凝胶上被分解。最小的寡核苷酸连接到结合到h-IgE定义的3′封端。经过选择的克隆的3′封端在表8中描述。在边界实验和预测折叠视觉观测的基础上,制备部分结构并从整合DNA技术(Coralville,IA)上排序寡核苷酸片段。通过前述的夹层过滤结合试验可以检测到核酸序列与序列号11,序列号18和序列号21一致的母克隆的最小化产物显示了明显的蛋白质结合力。表8中显示小型结合数据在,而表9中显示了相应的序列。
[00263]用2′-O甲基残基替换脱氧残基过程的第一步是合成和检测ARC 1250-ARC 1264(序列号158-172)的结合活力,其中每个都与增加了3′-反向-dT(3T)和用2′-O-甲基残基替换了2′-脱氧残基的ARC445结合活性相当。从表17中的结合数据可以看出,一些位置容易容受2′-O-甲基残基替换脱氧残基,而另一些位置则不是。有趣的是,在位点10(ARC1256)(序列号164)以2′-O-甲基残基替换2′-脱氧残基显著改善亲和力。
[00266]ARC445和其衍生物的合成过程中,在离子交换高效能液体色谱中观察到的峰与ARC445多亚基聚合体一致。图8是ARC445和几个衍生物高效能液体色谱微量分析的实施例,由于适体的集合显示多重峰。ARC445在位置6-9和16-21包含了2串鸟苷残基。尽管不受任何理论的约束,这2串中的任何一个或两者都可能引起适体集合。根据报道,以基本同样的方式,结合了双链MA和DNA溴化乙啶荧光性就增加了,结合了G-四连体(G-quartet)结构后N-甲基中卟啉IX(NMM)荧光性也增加。(Arthanari等人,Nucleic Acids Research,26(16):3724(1996);Marathais等人,Nucleic Acids Research,28(9):1969(2000);Joyce等人,Applied Spectroscopy,58(7):831(2004))。如图9中显示,我们使用NMM荧光性建立了理论ARC445实际上采用了G-quarte结构。根据文献方案,使用SpectraMaX Gemmi XS荧光板阅读器分析了含有大约1微摩尔NMM100微升反应物和含有镁和钙的Dulbecco′s PBS中大约2微摩尔的适体。收集550到750nm和405nm波长刺激下的荧光发散色谱。抗凝血酶DNA适体ARC183的G-quartet结构已经在文献(Macaya等人,Proc.Natl.Acad.Sci.,90:3745(1993))中建立起来,因此在本实验中可作为阳性对照(图10)。图10显示了与ARC445大小相似核苷成分相似适体并没有被预测含有G-quartet结构,因此在本实验中用做阴性对照(图10)。从图9和10可以看出,相对于NMM单独作用相比,ARC183和ARC445的NMM荧光性明显增加,而ARCl 346则没有增加。
[00293]实验前一天在24孔板上每个含有MEM和16�S孔中都铺入(200,000)SX38细胞。第二天,适宜浓度的适体(1μM开始2倍稀释系溶液8点滴定)和3ug/mL h-IgE(AthensResearch and Technology,Athens,GA)在MEM和16�S中培育30分钟。然后将混合液加入到SX38细胞中在37°C,5%CO2中培育30分钟。每种浓度都检测四分之一。使用MEM和16�S洗涤细胞三次,然后再在MEM加16�S中再培育5.5小时。h-IgE交叉连接抗体(QED Biosciences,San Diego,CA)加入到1ug/mL浓度的细胞培养基上再培育2小时。在组氨酸ELISA使用前收集上清在-20°C冷冻。组氨酸ELISA按照使用推荐用法。单独h-IgE和h-IgE相对于天然混合物被分别作为阳性和阴性对照。通过测定组氨酸释放入上清的抑制百分比来测定IgE适体活性,如图14显示(其中ARC445是序列号101,ARC656是序列号157,ARC714是非结合阴性对照)。
[00294]ARC445(序列号101)(实施例2E描述)和DNA适体,ARC656(序列号157)(实施例2D描述)中几个修饰衍生物也被用来测验其抑制上述SX38细胞组氨酸释放的能力,测量得到ARC445衍生物的IC50概括在表25中。
[00300]血浆浓度和时间数据要使用工业标准药代动力学模型软件WmNonLmTM v.4.0(Pharsight Corp.,Mountam View,CA)进行非划分分析(NCA)。可以估计出下面主要药代动力学参数:最大血浆浓度,Cmax;浓度-时间曲线下面积AUC;终末半衰期,t1/2;终末清楚率,Cl;稳定状态分布体积,Vss。使用WmNonLmTM v.4.0(Pharsight Corp.,Mountam View,CA)。分析(NCA)非划分分析数据,获得了下图20列出的主要药代动力学参数的估计值。
[00304]本发明已经被用书面描述和实施例的形式描述了,本领域普通技术人员能认识到本发明可以在多种实施方案中应用,并且上面的描述和实施例都是为了说明而并不是所附权利要求的限制。