High temperature selection of nucleotide-supported carbohydrate vaccines and resulting glycosylated oligonucleotides 机翻标题: 暂无翻译,请尝试点击翻译按钮。

公开号/公开日
WO2015084846 A1 2015-06-11 [WO201584846] / 2015-06-11
申请号/申请日
2014WO-US68158 / 2014-12-02
发明人
KRAUSS ISAAC J;
申请人
BRANDEIS UNIVERSITY;
主分类号
IPC分类号
C07H-015/22C07H-021/04C12N-015/10
摘要
(WO201584846) The invention relates to an oligonucleotide including one or more modified nucleoside bases having the structure — B— L— A wherein for each of the modified nucleosides A is independently a monosaccharide or oligosaccharide, L is a linker molecule, and B is independently a pyrimidine or pyridine base linked to the sugar-phosphate backbone of the oligonucleotide; and wherein the oligonucleotide binds specifically to a carbohydrate-binding monoclonal antibody with an affinity of less than 100 nM.  Immunogenic conjugates that include the oligonucleotide, and pharmaceutical compositions that include the oligonucleotide or the immunogenic conjugate are also disclosed.  Various method of using the oligonucleotides, immunogenic conjugates, and pharmaceutical compositions are disclosed, including inducing an immune response, inhibiting viral or bacterial infection, treating a cancerous condition, and detecting a neutralizing antibody.  A method is also disclosed for selecting the oligonucleotides using an alternative Selection of Modified Aptamers (SELMA).
机翻摘要
暂无翻译结果,您可以尝试点击头部的翻译按钮。
地址
代理人
代理机构
;
优先权号
2013US-61910769 2013-12-02
主权利要求
(WO201584846)  WHAT IS CLAIMED:    1.  An oligonucleotide comprising one or more modified nucleoside bases having the structure:    - B- L- A   wherein for each of the modified nucleosides    A is independently a monosaccharide or oligosaccharide,    L is a linker molecule, and    B is independently a pyrimidine or pyridine base linked to the sugar-phosphate backbone of the oligonucleotide; and   wherein the oligonucleotide binds specifically to a carbohydrate-binding monoclonal antibody with an affinity of less than 100 nM.   2.  The oligonucleotide according to claim 1, wherein the oligonucleotide is an   oligodeoxynucleotide.   3.  The oligonucleotide according to claim 1, wherein the oligonucleotide is an   oligoribonucleotide.   4. The oligonucleotide according to claim 1, wherein the oligonucleotide comprises one or more phosphorothioate-linked nucleotides, or 2'-fluoro-, 2'-amino, 2'-0-methyl-, 5 '-iodo-, or 5 '- bromo-modified nucleotides.   5. The oligonucleotide according to any one of claims 1 to 4, wherein the oligonucleotide has a length of about 15 to about 100 nucleotides.   6. The oligonucleotide according to any one of claims 1 to 4, wherein the oligonucleotide comprises not more than one modified base.   7. The oligonucleotide according to any one of claims 1 to 4, wherein the oligonucleotide comprises from 2 to about 10 of said modified nucleoside bases.   8. The oligonucleotide according to claim 7, wherein the oligonucleotide comprises from 2 to 4 of said modified nucleoside bases. 9.  The oligonucleotide according to claim 7, wherein two or more of said modified nucleoside bases are at adjacent positions in the oligonucleotide.    10. The oligonucleotide according to claim 7, wherein two or more of said modified nucleoside bases are at nonadjacent positions in the oligonucleotide.   11.  The oligonucleotide according to any one of claims 1 to 10, wherein A is a   monosaccharide.   12.  The oligonucleotide according to any one of claims 1 to 10, wherein A is an   oligosaccharide.   13. The oligonucleotide according to claim 12, wherein the oligosaccharide comprises at least about 3 saccharide moieties.   14. The oligonucleotide according to claim 13, wherein the oligosaccharide is a branched or unbranched oligosaccharide of at least about 3 saccharide moieties up to about 20 saccharide moieties.   15. The oligonucleotide according to claim 13, wherein the oligosaccharide consists of 9 saccharide moieties.   16. The oligonucleotide according to claim 13, wherein the oligosaccharide is branched and consists of 9 mannose moieties.   17. The oligonucleotide according to any one of claims 1 to 13, wherein L is a linker molecule that comprises    , wherein each of Ri and R2 is optionally a direct link or independently selected from the group consisting of a linear or branched Ci to C18 hydrocarbon that is saturated or mono- or poly-unsaturated, optionally interrupted by one or more non-adjacent -0-, -C(=0)-, or -NR4-; a substituted or unsubstituted C3 to C10 cycloalkandiyl, a substituted or unsubstituted aryl diradical; a substituted or unsubstituted heteroaryl diradical; a monosaccharide diradical; or a disaccharide diradical; R3 is optional and can be -0-, -S-, or -NR4-; and R4 is independently H or a Ci to C10 alkyl.    18.  The oligonucleotide according to any one of claims 1 to 17, wherein B is selected from the group of   19. The oligonucleotide according to any one of claims 1 to 18, wherein the oligonucleotide binds specifically to a carbohydrate-binding monoclonal antibody with an affinity of less than 50 nM. 20.  The oligonucleotide according to any one of claims 1 to 19, wherein the oligonucleotide binds specifically to the carbohydrate-binding monoclonal antibody with an affinity that is substantially the same as or lower than the affinity of the carbohydrate-binding, neutralizing monoclonal antibody to its naturally occurring binding partner. 21.  The oligonucleotide according to any one of claims 1 to 19, wherein the carbohydrate- binding monoclonal antibody is neutralizing against a pathogen.   22.  The oligonucleotide according to claim 21, wherein the carbohydrate-binding   neutralizing monoclonal antibody binds specifically to N-glycosylated HIV gpl20, N- glycosylated HIV gp41, a combination of N-glycosylated HIV gpl20 and N-glycosylated HIV gp41 , or N-glycosylated HSV-2 gD.   23. The oligonucleotide according to claim 22, wherein the carbohydrate-binding, neutralizing monoclonal antibody is 2G12, PG9, PG16, PGT121, PGT122, PGT123, PGT125, PGT126, PGT127, PGT128, PGT129, PGT130, PGT131, PGT135, PGT136, PGT137, PGT141, PGT142, PGT143, PGT144, PGT145, PGT151, PGT152, PGT153, PGT154, PGT155, PGT156,    PGT157, PGT158, CHOI, CH02, CH03, CH04, 10-1074, 10-996, 10-1146, 10-847, 10-1341, 10- 1121, 10-1130, 10-410, 10-303, 10-259, 10-1369, or E317.   24. The oligonucleotide according to any one of claims 1 to 19, wherein the carbohydrate- binding monoclonal antibody is cytotoxic against a cancer cell. 25.  The oligonucleotide according to claim 24, wherein the carbohydrate-binding cytotoxic monoclonal antibody binds specifically to O-glycosylated cancer-specific human podoplanin; abberantly O-glycosylated cancer-specific MUC1, abberantly O-glycosylated cancer-specific Integrin .alpha.3.beta. 1, or N-glycosylated cancer-specific antigen RAAG12.   26. The oligonucleotide according to claim 25, wherein the carbohydrate-binding cytotoxic monoclonal antibody is LpMab-2, 237 MAb, RAVI 2, BCMabl, DF3, 115D8, and GOD3-2C4.   27. The oligonucleotide according to claim 23, wherein the carbohydrate-binding, neutralizing monoclonal antibody is 2G12 and the oligonucleotide comprises the sequence NGNAACCNACGGANA (SEQ ID NO: 103) where N is any nucleoside base and N is one of said modified nucleoside bases.   28. The oligonucleotide according to claim 23, wherein the carbohydrate-binding, neutralizing monoclonal antibody is 2G12 and the oligonucleotide comprises the sequence CNNNNNGNAACCNACGGANA (SEQ ID NO: 104) where N is any nucleoside base and N is one of said modified nucleoside bases.   29.  The oligonucleotide according to claim 27 or 28, wherein said oligonucleotide comprises the sequence:   Sequence SEQ ID NO:    AGACCCACG GNG C AAC CNAC G G ANA 84    AG AC C C AC AGNGC AAC CNAC GG ANA 85    AGACCCCCG GNG C AAC CNAC G G ANA 8 6    ACACCCACGGNGCAACCNACGGANA 87    AG AC C C AC AANGC AAC CNAC GG ANA 88    AGACGCACG GNG C AAC CNAC G G ANA 8 9    NGACCCACG GNG C AAC CNAC G G ANA 90    GAGNCCCAG GNG AAAC CNAC G G ANA 91    AG AC C CNC G GNG C AAC CNAC G G ANA 92    AG AC C C ANG GNG C AAC CNAC G G ANA 93    AG AC C C ANAGNG C AAC CNAC G G ANA 94    AGAC - CACG GNGNAAC CNAC G G ANA 95    AGANCCACG GNGNAAC CNAC G G ANA 96    30.  The oligonucleotide according to any one of claim 27 to 29, wherein said oligonucleotide is an oligodeoxynucleotide and said modified nucleoside bases comprises the structure according to formula (IV)   wherein A represents the monosaccharide or oligosaccharide.   31. The oligonucleotide according to claim 30, wherein A represents a branched or unbranched oligosaccharide consisting of 9 saccharide moieties.   32.  The oligonucleotide according to claim 30, wherein A represents a branched   oligosaccharide consisting of 9 mannose moieties.   33.  An oligonucleotide comprising three to five modified nucleoside bases   having the structure:    - B- L- A   wherein for each of the modified nucleosides    A is a branched-chain Man oligosaccharide,    L is a linker molecule, and    B is independently a pyrimidine or pyridine base linked to the sugar-phosphate backbone of the oligonucleotide; and   wherein the oligonucleotide binds specifically to HIV neutralizing monoclonal antibody 2G12 with a IQ value that is lower than 20 nM.    34. The oligonucleotide according to claim 33, wherein the oligonucleotide binds specifically to the HIV neutralizing monoclonal antibody 2G12 with a 3/4 value that is lower than lower than 5 nM.   35. The oligonucleotide according to claim 33 or 34, wherein the oligonucleotide comprises the sequence NGNAACCNACGGANA (SEQ ID NO: 103), where N is any nucleoside base and N is the modified nucleoside base.   36.  The oligonucleotide according to claim 35, wherein each said modified nucleoside base comprises the structure according to formula (IV)   The oligonucleotide according to claim 36, wherein said oligonucleotide comprises the   Sequence SEQ ID NO:    AGACCCACG GNG C AAC CNAC G G ANA 8 4    AG AC C C AC AGNGC AAC CNAC GG ANA 8 5    AGACCCCCG GNG C AAC CNAC G G ANA 8 6    ACACCCACGGNGCAACCNACGGANA 8 7    AG AC C C AC AANGC AAC CNAC GG ANA 8 8    AGACGCACG GNG C AAC CNAC G G ANA 8 9    GAGNCCCAG GNG AAAC CNAC G G ANA 91    AG AC C CNC G GNG C AAC CNAC G G ANA 92    AG AC C C ANG GNG C AAC CNAC G G ANA 93    AG AC C C ANAGNG C AAC CNAC G G ANA 94    38. An immunogenic conjugate comprising an oligonucleotide according to one of claims 1 to 37 covalently or non-covalently bound to an immunogenic carrier molecule.   39. The immunogenic conjugate according to claim 38, wherein the immunogenic carrier molecule is selected from the group consisting of bovine serum albumin, chicken egg ovalbumin, keyhole limpet hemocyanin, tetanus toxoid, diphtheria toxoid, thyro globulin, a pneumococcal capsular polysaccharide, CRM 197, and a meningococcal outer membrane protein.   40. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an oligonucleotide according to one of claims 1 to 36 or an immunogenic conjugate according to claim 38 or 39.   41. The pharmaceutical composition according to claim 40, wherein the oligonucleotide is present.   42. The pharmaceutical composition according to claim 40, wherein the immunogenic conjugate is present.   43. The pharmaceutical composition according to claim 40 further comprising an adjuvant.   44. The pharmaceutical composition according to claim 43, wherein the adjuvant comprises aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid, Quil A, non-infective Bordetella pertussis, QS-21, monophosphoryl lipid A, an alpha-galactosylceramide derivative, or PamCys-type lipids. 45.  The pharmaceutical composition according to one of claims 40 to 44, wherein the pharmaceutically acceptable carrier is a buffered saline solution.   46. The pharmaceutical composition according to one of claims 40 to 44, further comprising one or more additives or preservatives, or both.   47. The pharmaceutical composition according to one of claims 40 to 44, wherein the composition is present as a single dosage unit comprising about 1 .mu.g to about 5 mg of the oligonucleotide.    48. A method of inducing an immune response in an individual comprising: administering to an individual an oligonucleotide according to one of claims 1 to 37, an immunogenic conjugate according to claim 38 or 39, or a pharmaceutical composition according to one of claims 40 to 47, wherein said administering is effective to induce an immune response against the oligonucleotide.   49. The method according to claim 48, wherein said administering is effective to induce a carbohydrate-binding, neutralizing antibody response. 50.  The method according to claim 49, wherein the induced carbohydrate-binding, neutralizing antibody response is protective against a pathogen.   51. The method according to claim 50, wherein the pathogen is a virus or bacterium.   52. The method according to claim 48, wherein said administering is effective to induce a carbohydrate-binding, cytotoxic antibody response. 53.  The method according to claim 52, wherein the induced carbohydrate-binding, cytotoxic antibody response is cytotoxic against a cancer cell.   54. The method according to claim 53, wherein the cancer cell is a solid tumor cell.   55. The method according to claim 53 or 54, wherein the cancer cell expresses O- glycosylated cancer-specific human podoplanin; aberrantly O-glycosylated cancer-specific MUC 1 , aberrantly O-glycosylated cancer-specific integrin .alpha.3 .beta. 1 , or N-glycosylated cancer- specific antigen RAAG12.   56. The method according to claim 48, 49, or 52, wherein said administering is carried out orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraarterially, intralesionally, transdermally, intra- or peri-tumorally, by application to mucous membranes, or by inhalation.   57. The method according to claim 48, 49, or 52, wherein said administering is repeated.   58. The method according to claim 48, 49, 52, or 57, wherein the oligonucleotide or immunogenic conjugate is administered at a dose of about 1 .mu.g to about 5 mg.    59. The method according to any one of claims 48 to 58, wherein the individual is a mammal.   60. The method according to any one of claims 48 to 58, wherein the individual is a human.   61.  A method of inhibiting viral or bacterial infection comprising:    administering to an individual an oligonucleotide according to one of claims 1 to 37, an immunogenic conjugate according to claim 38 or 39, or a pharmaceutical composition according to one of claims 40 to 47, wherein said administering is effective to induce a neutralizing immune response against a virus or bacterial pathogen.   62. The method according to claim 61 , wherein the pathogen is a virus.   63. The method according to claim 62, wherein the individual has an active infection by the virus.   64. The method according to claim 62, wherein the individual is free of infection by the virus.   65.  The method according to claim 62, wherein the virus is selected from the group of Calicivirus, Chikungunya virus, Cytomegalovirus, Dengue virus, Eastern Equine Encephalitis virus, Ebola virus, Epstein-Barr virus, Hantaan virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Herpes simplex virus, Human   Immunodeficiency virus (HIV-1 or HIV -2), Human Papillomavirus, Influenza virus, Japanese encephalitis virus, Junin virus, Lassa virus, Marburg virus, Measles virus, Metapneumovirus, Nipah virus, Newcastle disease virus, Norwalk virus, Parainfluenza virus, Poliovirus, Rabies virus, Respiratory Syncytial virus, Rift Valley Fever virus, Rotavirus, Rubella virus, Sendai virus, Severe Acute Respiratory Syndrome (SARS Co-V), Tick-borne Encephalitis virus, Varicella zoster virus, Venezuelan Equine Encephalitis virus, Yellow Fever virus, Western Equine Encephalitis virus, and West Nile virus.   66. The method according to claim 62, wherein the virus is HIV-1.   67. The method according to claim 66, wherein the oligonucleotide comprises the sequence NGNAACCNACGGANA (SEQ ID NO: 103) where N is any nucleoside base and N is one of said modified nucleoside bases.    68. The method according to claim 66, wherein the oligonucleotide comprises the sequence CNNNNNGNAACCNACGGANA (SEQ ID NO: 104) where N is any nucleoside base and N is one of said modified nucleoside bases. 69.  The method according to claim 67 or 68, wherein the oligonucleotide comprises the sequence:    Sequence SEQ ID NO:    AGACCCACG GNG C AAC CNAC G G ANA 84    AG AC C C AC AGNGC AAC CNAC GG ANA 85    AGACCCCCG GNG C AAC CNAC G G ANA 8 6    ACACCCACGGNGCAACCNACGGANA 87    AG AC C C AC AANGC AAC CNAC GG ANA 88    AGACGCACG GNG C AAC CNAC G G ANA 8 9    NGACCCACG GNG C AAC CNAC G G ANA 90    GAGNCCCAG GNG AAAC CNAC G G ANA 91    AG AC C CNC G GNG C AAC CNAC G G ANA 92    AG AC C C ANG GNG C AAC CNAC G G ANA 93    AG AC C C ANAGNG C AAC CNAC G G ANA 94    AGAC - CACG GNGNAAC CNAC G G ANA 95    AGANCCACG GNGNAAC CNAC G G ANA 96   70.  The method according to one of claims 67 to 69, wherein the oligonucleotide is an oligodeoxynucleotide and said modified nucleoside bases comprises the structure according to formula (IV)     wherein A is as previously defined.    71. The method according to claim 70, wherein A represents a branched or unbranched oligosaccharide consisting of 9 saccharide moieties.   72. The method according to claim 70, wherein A represents a branched oligosaccharide consisting of 9 mannose moieties.   73. The method according to claim 61 , wherein the pathogen is a bacterium.   74.  The method according to claim 73, wherein the bacterium is Bacillus anthracis,   Bordetella pertussis B, Borrelia burgdorferi, Chlamydia trachomatis, Clostridium difficile,  Clostridium tetani, Candida albicans, Corynebacterium diphtheriae, Cryptococcus neoformans, Entamoeba histolytica, Escherichia coli, Francisella tularensis, Haemophilus influenzae   (nontypeable), Helicobacter pylori, Histoplasma capsulatum, Moraxella catarrhalis,   Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrheae, Neisseria   meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Methicillin-resistant    Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, or Yersinia pestis.   75. The method according to claim 73, wherein the individual has an active bacterial infection.   76. The method according to claim 73, wherein the individual is free of infection by the bacterium. 77.  The method according to any one of claims 61 to 76, wherein the individual is a mammal.   78. The method according to any one of claims 61 to 76, wherein the individual is a human.   79.  The method according to any one of claims 61 to 78, wherein said administering is carried out orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation,   intraarterially, intralesionally, transdermally, by application to mucous membranes, or by inhalation. 80.  The method according to any one of claims 61 to 79, wherein said administering is repeated.    81. The method according to any one of claims 61 to 79, wherein the oligonucleotide is administered at a dose of about 1 .mu.g to about 5 mg.   82. The pharmaceutical composition according to one of claims 40 to 47, wherein the carbohydrate-binding, cytotoxic monoclonal antibody binds specifically to a cancer cell that expresses O-glycosylated cancer-specific human podoplanin; aberrantly O-glycosylated cancer- specific MUC1, aberrantly O-glycosylated cancer-specific integrin .alpha.3.beta. 1, or N-glycosylated cancer- specific antigen RAAG12. 83.  A method of treating a cancerous condition comprising:    administering to an individual an oligonucleotide according to claim 24 to 26, an immunogenic conjugate comprising the oligonucleotide covalently or non-covalently bound to an immunogenic carrier molecule, or a pharmaceutical composition according to claim 82, wherein said administering is effective to induce an anti-tumor immune response against a cancer cell expressing a glycosylated cancer-specific protein.   84. The method according to claim 83, wherein the glycosylated cancer-specific protein is O- glycosylated human podoplanin; aberrantly O-glycosylated MUC1, aberrantly O-glycosylated integrin .alpha.3.beta. 1, or N-glycosylated antigen RAAG12.   85. The method according to claim 83 or 84, wherein the cancer cell is a solid tumor.   86. The method according to claim 85, wherein the solid tumor is a colorectal, gastric, ovarian, breast, or pancreatic cancer.   87. The method according to claim 85, wherein the solid tumor is a squamous cell carcinoma, lung and esophageal carcinoma, testicular seminoma, malignant brain tumor, fibrosarcoma, malignant mesothelioma, bladder cancers, and testicular cancers. 88.  The method according to claim 85, wherein the solid tumor is a bladder   89. The method according to claim 85, wherein the solid tumor is a breast cancer, ovarian cancer, lung cancer, pancreatic cancer, or prostate cancer. 90.  The method according to claim 83 or 84, wherein the cancer is a form of leukemia.   91. The method according to any one of claims 83 to 90, wherein the individual is a mammal.    92. The method according to any one of claims 83 to 90, wherein individual is a human.   93.  The method according to any one of claims 83 to 92, wherein said administering is carried out orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation,   intraarterially, intralesionally, intratumorally, transdermally, intra- or peri-tumorally, by application to mucous membranes, or by inhalation.   94. The method according to any one of claims 83 to 93, wherein said administering is repeated.   95. The method according to any one of claims 83 to 94, wherein the oligonucleotide is administered at a dose of about 1 .mu.g to about 5 mg. 96.  The method according to any one of claims 83 to 95, further comprising administering a chemotherapeutic agent, a radiation therapy, or an immunotherapeutic agent.   97.  A method for detecting a neutralizing antibody in serum comprising:    providing an oligonucleotide according to any one of claims 1 to 37;    contacting the oligonucleotide with serum from an individual; and    detecting whether the oligonucleotide binds specifically to an antibody present in the serum, wherein said detecting is carried out using a label.    98.  The method according to claim 97, wherein the label is a radiolabel, fluorescent label, enzymatic label, chemiluminescent marker, biotinyl group, an epitope recognized by a secondary reporter, a magnetic agent, or a toxin.   99.  The method according to claim 97 or 98, wherein said detecting is carried out in an assay selected from the group consisting of ELISA, radioimmunoassay, gel-diffusion precipitation reaction assay, immunodiffusion assay, agglutination assay, fluorescent immunoassay,   Immunoelectrophoresis assay, surface plasmon resonance assay, or biolayer interferometry assay.   100. The method according to claim 97 or 98, wherein said detecting is carried out using a secondary antibody that carries the label, the secondary antibody binding to the antibody bound specifically to the oligonucleotide.    101. The method according to claim 97 or 98, wherein the oligonucleotide is bound to a solid surface.   102.  A method for selecting a glycosylated oligonucleotide that binds to a target protein comprising:    providing a pool of modified, single-strand-double-strand hybrid oligonucleotides that are glycosylated within the single-strand region;    combining the pool with a target protein to form a mixture;    incubating the mixture at a temperature above 20 deg.C for a period of time sufficient to allow any target protein to bind to one or more of the modified, single-strand-double-strand hybrid oligonucleotides; and    isolating from the mixture the modified, single-strand-double-strand hybrid   oligonucleotides that bind to the target protein, thereby identifying a plurality of selected oligonucleotides.   103. The method according to claim 102, wherein the provided pool comprises greater than 1010 modified, single-strand-double-strand hybrid oligonucleotides.   104. The method according to claim 102, wherein the provided pool comprises greater than 1011 modified, single-strand-double-strand hybrid oligonucleotides.   12  105. The method according to claim 102, wherein the provided pool comprises greater than 10 modified, single-strand-double-strand hybrid oligonucleotides.   13 106.  The method according to claim 102, wherein the provided pool comprises about 10 modified, single-strand-double-strand hybrid oligonucleotides.   107. The method according to claim 102, wherein said incubating is carried out at a temperature of greater than 22 deg.C.   108. The method according to claim 102, wherein said incubating is carried out at a temperature of greater than 27 deg.C.   109. The method according to claim 108, wherein the temperature is from about 32 deg.C to about 42 deg.C.    110.  The method according to claim 102, wherein said isolating comprises:    exposing the mixture to magnetic beads labeled with an affinity reagent that binds to a portion of the target protein; and    recovering magnetic beads bound to modified, single-strand-double-strand hybrid oligonucleotide/target protein complexes.   111.  The method according to claim 102, further comprising:    amplifying the plurality of selected oligonucleotides, thereby forming a plurality of complementary oligonucleotides; and    regenerating a second pool of the modified, single-strand-double-strand hybrid    oligonucleotides that are glycosylated within the single-strand region using the plurality of complementary oligonucleotides, one or more modified nucleoside bases, and a modified oligosaccharide. 112.  The method according to claim 111, wherein said regenerating comprises:    forming stem-loop oligonucleotides comprising one or more modified nucleoside bases; reacting a modified oligosaccharide with the one or more modified nucleoside bases to form glycosylated stem-loop oligonucleotides; and    synthesizing a complementary strand, using the glycosylated stem-loop oligonucleotides as templates, a primer that hybridizes to a portion of the loop, dNTPs, and a polymerase having strand displacement activity, thereby forming the second pool of modified, single-strand-double- strand hybrid oligonucleotides that are glycosylated within the single-strand region.   113. The method according to claim 112, wherein one of the modified nucleoside bases and the modified oligosaccharide comprise an azide group or a thiol group, and the other of the modified nucleoside bases and the modified oligosaccharide comprises an alkynyl group or alkenyl group.   114. The method according to claim 112, wherein the modified nucleoside base comprises an alkynyl or alkenyl group and the modified oligosaccharide comprises an azide group or thiol group.   115. The method according to any one of claims 112 to 114, wherein the reaction conditions for said reacting includes copper catalysis or ruthenium catalysis or strain-promoted alkyne-azide cycloaddition.    116. The method according to any one of claims 112 to 114, wherein said reacting includes copper catalysis.   117. The method according to claim 111, wherein said amplifying comprises a polymerase chain reaction.   118. The method according to claim 111 or 112 further comprising repeating said combining using the second pool, said incubating, and said isolating to identify a second plurality of second oligonucleotides.   119. The method according to claim 118, wherein said incubating is carried out for the second pool using different conditions than said incubating for the first pool.   120. The method according to claim 119, wherein said conditions comprise one or more of target protein concentration, temperature, duration, introduction of competitor molecules, and increasing the number or condition of wash steps.   121.  The method according to claim 119, wherein said repeating comprises:    performing a negative selection step to remove oligonucleotides which bind to target protein without being glycosylated, remove oligonucleotides or glycosylated oligonucleotides that bind directly to a solid support.   122. The method according to any one of claims 102 to 121, wherein the target protein is an antibody.   123. The method according to claim 122, wherein the carbohydrate-binding monoclonal antibody is neutralizing against a pathogen.   124. The method according to claim 123, wherein the carbohydrate-binding neutralizing monoclonal antibody binds specifically to N-glycosylated HIV gpl20, N-glycosylated HIV gp41, a combination of N-glycosylated HIV gpl20 and N-glycosylated HIV gp41, or N-glycosylated HSV-2 gD.   125.  The method according to claim 124, wherein the carbohydrate-binding, neutralizing monoclonal antibody is 2G12, PG9, PG16, PGT121, PGT122, PGT123, PGT125, PGT126,  PGT127, PGT128, PGT129, PGT130, PGT131, PGT135, PGT136, PGT137, PGT141, PGT142, PGT143, PGT144, PGT145, PGT151, PGT152, PGT153, PGT154, PGT155, PGT156, PGT157,    PGT158, CHOI, CH02, CH03, CH04, 10-1074, 10-996, 10-1146, 10-847, 10-1341, 10-1121, 10- 1130, 10-410, 10-303, 10-259, 10-1369, or E317.   126. The method according to claim 122, wherein the carbohydrate-binding monoclonal antibody is cytotoxic against a cancer cell.   127. The method according to claim 126, wherein the carbohydrate-binding cytotoxic monoclonal antibody binds specifically to O-glycosylated cancer-specific human podoplanin; aberrantly O-glycosylated cancer-specific MUC1, aberrantly O-glycosylated cancer-specific Integrin .alpha.3 .beta. 1 , or N-glycosylated cancer-specific antigen RAAG 12.   128. The method according to claim 127, wherein the carbohydrate-binding cytotoxic monoclonal antibody is LpMab-2, 237 MAb, RAVI 2, BCMabl, DF3, 115D8, or GOD3-2C4.   A method, comprising the steps of:    (a) combining a plurality of oligonucleotides, a first DNA polymerase, and a plurality of deoxyribonucleotide triphosphates,    wherein    the oligonucleotides comprise a first primer binding site on the 5 ' end, a randomized region, and a stem-loop region;    the randomized region is located between the first primer binding site and the stem-loop region;    the stem-loop region comprises a second primer binding site; and at least one of the deoxyribonucleotide triphosphates comprises a reactive substituent;    thereby forming a plurality of extended oligonucleotides comprising an original strand and an extended strand, wherein the extended strand comprises at least one reactive substituent;    (b) combining a plurality of modifying compounds and the plurality of extended oligonucleotides under reaction conditions,    thereby forming a plurality of modified extended oligonucleotides comprising the original strand and a modified e(...)
法律状态
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