WO2017190020A1 - Conjugués oligonucléotidiques et leurs utilisations - Google Patents

Conjugués oligonucléotidiques et leurs utilisations Download PDF

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Publication number
WO2017190020A1
WO2017190020A1 PCT/US2017/030146 US2017030146W WO2017190020A1 WO 2017190020 A1 WO2017190020 A1 WO 2017190020A1 US 2017030146 W US2017030146 W US 2017030146W WO 2017190020 A1 WO2017190020 A1 WO 2017190020A1
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Prior art keywords
oligonucleotide
component
conjugate
antibody
sequence
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PCT/US2017/030146
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English (en)
Inventor
Kristopher Lancaster NAZOR
Nicholas Joseph SCHORK
Devon Michael CAYER
Mohammad Reza GHADIRI
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The Scripps Research Institute
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Publication of WO2017190020A1 publication Critical patent/WO2017190020A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • Malignant tumors are the second leading cause of death in the United States, after heart disease (Boring et al., CA Cancel J. Clin. 43 :7 (1993)). Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue that proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites via a process called metastasis. In a cancerous state, a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
  • transmembrane or otherwise membrane-associated polypeptides that are specifically expressed on the surface of one or more particular types of cancer cells as compared to normal, non-cancerous cells. Often, such membrane-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to non-cancerous cells, and thus provide a mechanism for identifying and distinguishing cancer cells from non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction.
  • cancerous cells are highly variable, making treatment of cancer highly dependent on the ability of targeted therapeutic agents to bind tumorigenic tissue.
  • targeted therapies antibody- drug-conjugates, CAR T-cells, or bispecific antibodies
  • the ability to quickly and cost-effectively track and adjust therapy depending on how cancerous cells mutate and gain resistance to particular therapies would allow for maximal treatment efficacy with minimal side effects.
  • the ability to determine whether circulating cancerous cells are present in a patient prior to manifestation of cancer with metastatic potential would enable early-treatment of cancer that is isolated in select tissues. The present disclosure addresses these and other needs.
  • Oligonucleotide conjugates and uses thereof are provided.
  • An aspect of the subject oligonucleotide conjugates includes a targeting component, a linker component, a cleavage component, and an oligonucleotide component.
  • Methods of making and using the subject oligonucleotide conjugates in the diagnosis, prevention and/or treatment of cancer and other diseases are also provided.
  • An aspect of the disclosure includes an oligonucleotide conjugate, comprising: a) a targeting component, b) a linker component, c) a cleavage component, and d) an oligonucleotide component.
  • the targeting component comprises a polymer.
  • the polymer comprises an amino acid, a nucleic acid, or a polysaccharide.
  • the nucleic acid is selected from the group consisting of: a morpholino, a peptide nucleic acid (PNA), a thioester peptide nucleic acid (tPNA), a locked nucleic acid (LNA), a phosphorothioate, a phosphonoacetate (PACE) phosphoramidite, a ribonucleic acid (RNA), and a deoxyribonucleic acid (DNA).
  • the targeting component is selected from the group consisting of: an antibody, an antibody fragment, an enzyme, a small molecule, a lectin, and a carbohydrate.
  • the linker component comprises a polypeptide.
  • the linker component does not comprise a polypeptide.
  • the linker component is selected from the group consisting of: a tetrazine ligation linker, a strain-promoted-azide-alkylene (SPAAC) linker, a maleimide linker, a succinimide linker, a tyrosine linker, a chemoenzymatic linker, a hydrazone linker, and a hydrazine linker.
  • the cleavage component comprises a light- cleavable cleavage component.
  • the cleavage component comprises a chemically- cleavable cleavage component.
  • the cleavage component comprises an
  • the oligonucleotide of the cleavable component comprises a restriction site.
  • the restriction site comprises a number of nucleic acid residues ranging from about 6 to about 12.
  • the length of the oligonucleotide component comprises a number of nucleic acid residues ranging from about 16 to about 120.
  • the oligonucleotide component comprises a first universal sequence, a second universal sequence, and an identifier sequence, and wherein the identifier sequence identifies a binding target of the targeting component.
  • the targeting component binds to a protein, a lipid, a carbohydrate, or a small molecule.
  • the small molecule is a folate molecule.
  • the targeting component binds to a protein.
  • the protein is a lectin or a cytokine.
  • the protein is an intracellular protein, an extracellular protein, or a cell surface protein.
  • the protein is a cell surface protein.
  • the cell surface protein is HER2, FOLR1, NCAM1 (CD56), CD274 (PD-L2), CD278 (ICOS), CD9, CD 104 or CD119.
  • the first and the second universal sequences are different.
  • the first and the second universal sequences are the same.
  • the length of the first universal sequence comprises a number of nucleic acid residues ranging from about 12 to about 60.
  • the length of the second universal sequence comprises a number of nucleic acid residues ranging from about 12 to about 60.
  • the length of the identifier sequence comprises a number of nucleic acid residues ranging from about 5 to about 15.
  • the length of the identifier sequence comprises about 8 nucleic acid residues.
  • the first universal sequence, the identifier sequence, and the second universal sequence are in order from a 5' end to a 3' end of the oligonucleotide component.
  • the cleavage component is located at the 3' end of the oligonucleotide component.
  • the cleavage component is linked to the targeting component via the linker component.
  • the first universal sequence, the identifier sequence, the second universal sequence, and the restriction site are in order from a 5' end to a 3 ' end of the oligonucleotide component, and wherein the 3' end of the oligonucleotide component is linked by a strain- promoted-azide-alkylene (SPAAC) linker to an antibody.
  • SPAAC strain- promoted-azide-alkylene
  • the first universal sequence, the identifier sequence, the second universal sequence, and the restriction site are in order from a 5' end to a 3 ' end of the oligonucleotide component, and wherein the 3' end of the oligonucleotide component is linked by a strain- promoted-azide-alkylene (SPAAC) linker to a small molecule.
  • SPAAC strain- promoted-azide-alkylene
  • the first universal sequence, the identifier sequence, the second universal sequence, and the restriction site are in order from a 5' end to a 3 ' end of the oligonucleotide component, and wherein the 3' end of the oligonucleotide component is linked by a strain- promoted-azide-alkylene (SPAAC) linker to a lectin.
  • SPAAC strain- promoted-azide-alkylene
  • the first universal sequence, the identifier sequence, the second universal sequence, and the restriction site are in order from a 5' end to a 3 ' end of the oligonucleotide component, and wherein the 3' end of the oligonucleotide component is linked by a strain- promoted-azide-alkylene (SPAAC) linker to a folate molecule.
  • SPAAC strain- promoted-azide-alkylene
  • the first universal sequence, the identifier sequence, the second universal sequence, and the restriction site are in order from a 5' end to a 3 ' end of the oligonucleotide component, and wherein the 3' end of the oligonucleotide component is linked by a tetrazine ligation linker to an antibody.
  • the first universal sequence, the identifier sequence, the second universal sequence, and the restriction site are in order from a 5' end to a 3' end of the oligonucleotide component, and wherein the 3' end of the oligonucleotide component is linked by a tetrazine ligation linker to a small molecule.
  • the first universal sequence, the identifier sequence, the second universal sequence, and the restriction site are in order from a 5' end to a 3 ' end of the oligonucleotide component, and wherein the 3' end of the oligonucleotide component is linked by a tetrazine ligation linker to a lectin.
  • the first universal sequence, the identifier sequence, the second universal sequence, and the restriction site are in order from a 5' end to a 3 ' end of the oligonucleotide component, and wherein the 3' end of the oligonucleotide component is linked by a tetrazine ligation linker to a folate molecule.
  • An aspect of the disclosure includes a method for determining the presence of a binding target in a sample, the method comprising: a) contacting the sample with an
  • the oligonucleotide conjugate comprises: i) a targeting component that binds to the binding target; ii) a linker component; iii) a cleavage component; and iv) an oligonucleotide component, wherein the oligonucleotide component comprises a first identifier sequence that identifies the binding target; b) cleaving the cleavage component with a cleaving agent to release the first identifier sequence from the oligonucleotide conjugate; and c) detecting the first identifier sequence to determine the presence of the binding target in the sample.
  • the sample comprises a cell.
  • the binding target is a surface protein on the cell.
  • a second identifier sequence is linked to the first identifier sequence after the first identifier sequence has been released from the oligonucleotide conjugate.
  • the surface protein is HER2, FOLR1, NCAM1 (CD56), CD274 (PD-L2), CD278 (ICOS), CD9, CD104 or CD119.
  • the cleaving agent comprises a restriction enzyme.
  • the cleaving agent is a light beam.
  • detecting the first identifier sequence comprises sequencing at least a portion of the first identifier sequence.
  • a method comprises detecting the second identifier sequence.
  • detecting the second identifier sequence comprises sequencing at least a portion of the second identifier sequence.
  • the sequencing comprises high-throughput sequencing.
  • the sequencing comprises parallel sequencing by synthesis.
  • the detection is qualitative. In one aspect, the detection is quantitative.
  • An aspect of the disclosure includes a method for determining the presence of a plurality of binding targets in a sample, the method comprising: a) contacting the sample with a plurality of oligonucleotide conjugates, wherein each oligonucleotide conjugate comprises: i) a targeting component that binds to a different binding target; ii) a linker component; iii) a cleavage component; and iv) an oligonucleotide component, wherein the oligonucleotide component comprises a first identifier sequence that identifies the binding target; b) cleaving the cleavage component of each oligonucleotide conjugate with a cleaving agent to release the first identifier sequence from each oligonucleotide conjugate; and c) detecting the first identifier sequences to determine the presence of the plurality of binding targets in the sample.
  • the sample comprises a cell.
  • the binding target is a surface protein on the cell.
  • a second identifier sequence is linked to each of the first identifier sequences after step b).
  • detecting the first identifier sequence comprises sequencing at least a portion of the first identifier sequence.
  • a method comprises detecting the second identifier sequence.
  • detecting the second identifier sequence comprises sequencing at least a portion of the second identifier sequence.
  • the sequencing comprises high-throughput sequencing.
  • the sequencing comprises parallel sequencing by synthesis.
  • the detection is qualitative. In one aspect, the detection is quantitative.
  • aspects of the disclosure include a system for determining the presence of a plurality of binding targets in a sample, the system comprising: a) a plurality oligonucleotide conjugates, wherein each oligonucleotide conjugate comprises a targeting component that binds to a binding target, a linker component, a cleavage component, and an oligonucleotide component, wherein the oligonucleotide component comprises a first identifier sequence that identifies the binding target; and b) a sample analysis component, comprising: i) a controller, ii) a processor, and iii) a computer readable medium comprising instructions that, when executed by the processor, cause the controller to: contact the sample with the plurality of oligonucleotide conjugates; cleave the cleavage component with a cleaving agent to release the first identifier sequence from each oligonucleotide conjugate; and detect the pluralit
  • the sample comprises a cell.
  • the binding target is a surface protein on the cell.
  • An aspect of the disclosure includes a method of diagnosing a subject for cancer, the method comprising: a) administering to the subject i) a first oligonucleotide conjugate, wherein the first oligonucleotide conjugate comprises a targeting component that binds to a target on a cancer cell; a linker component; and an oligonucleotide component; and ii) a second
  • the targeting component comprises a polymer.
  • the polymer comprises an amino acid, a nucleic acid, or a polysaccharide.
  • the nucleic acid is selected from the group consisting of: a morpholino, a peptide nucleic acid (PNA), a thioester peptide nucleic acid (tPNA), a locked nucleic acid (LNA), a phosphorothioate, a phosphonoacetate (PACE) phosphoramidite, a ribonucleic acid (RNA), and a deoxyribonucleic acid (DNA).
  • the targeting component is selected from the group consisting of: an antibody, an antibody fragment, an enzyme, a small molecule, a lectin, and a carbohydrate.
  • the linker component comprises a polypeptide.
  • the linker component does not comprise a polypeptide.
  • the linker component is selected from the group consisting of: a tetrazine ligation linker, a strain-promoted-azide-alkylene (SPAAC) linker, a maleimide linker, a succinimide linker, a tyrosine linker, a chemoenzymatic linker, a hydrazone linker, and a hydrazine linker.
  • the length of the oligonucleotide component of the first oligonucleotide conjugate comprises a number of nucleic acid residues ranging from about 16 to about 120. In one aspect, the length of the oligonucleotide component of the second
  • the oligonucleotide conjugate comprises a number of nucleic acid residues ranging from about 16 to about 120.
  • the target on the cancer cell is a cell surface protein.
  • the cell surface protein is HER2, FOLRl, NCAMl (CD56), CD274 (PD-L2), CD278 (ICOS), CD9, CD 104 or CD119.
  • the targeting component of the first oligonucleotide conjugate comprises a protein linked to the oligonucleotide component by a strain-promoted-azide- alkylene (SPAAC) linker.
  • SPAAC strain-promoted-azide- alkylene
  • the targeting component of the first oligonucleotide conjugate comprises a protein linked to the oligonucleotide component by a tetrazine ligation linker.
  • the detectable moiety comprises a fluorophore.
  • An aspect of the disclosure includes a kit comprising a plurality of oligonucleotide conjugates, wherein each oligonucleotide conjugate comprises: (i) a targeting component; (ii) a linker component; (iii) a cleavage component; and (iv) an oligonucleotide component; wherein the oligonucleotide component comprises a first identifier sequence that identifies the binding target of each targeting component.
  • a kit comprises a plurality of second oligonucleotide conjugates, wherein each second oligonucleotide conjugate comprises a detectable moiety and an oligonucleotide component, and wherein at least a portion of the oligonucleotide component of each second oligonucleotide conjugate is complementary to at least a portion of the oligonucleotide component of a first oligonucleotide conjugate.
  • a kit comprises a cleaving agent that cleaves the cleavage component.
  • An aspect of the disclosure includes a method for synthesizing an oligonucleotide conjugate, the method comprising: a) immobilizing an oligonucleotide on a first solid support; b) contacting the oligonucleotide with a first reagent to create a functionalized oligonucleotide; c) immobilizing a targeting component on a second solid support; d) contacting the targeting component with a second reagent to create a functionalized targeting component; e) reacting the functionalized oligonucleotide with the functionalized targeting component to create an oligonucleotide conjugate; and f) isolating the oligonucleotide conjugate.
  • the functionalized oligonucleotide is separated from the first solid support and is reacted with the functionalized targeting component while the functionalized targeting component is
  • the functionalized targeting component is separated from the second solid support and is reacted with the functionalized oligonucleotide while the functionalized oligonucleotide is immobilized on the first solid support.
  • both the functionalized oligonucleotide and the functionalized targeting component are separated from the first and second solid supports before being reacted with one another.
  • the targeting component is selected from the group consisting of: an antibody, an antibody fragment, an enzyme, a small molecule, a lectin, and a carbohydrate.
  • the first reagent is selected from the group consisting of: a tetrazine ligation reagent, a strain- promoted-azide-alkylene (SPAAC) reagent, a maleimide reagent, an N-hydroxysuccinimide (NHS) reagent, a tyrosine ligation reagent, a chemoenzymatic attachment reagent, a hydrazone ligation reagent, and a hydrazine ligation reagent.
  • SPAAC strain- promoted-azide-alkylene
  • NHS N-hydroxysuccinimide
  • the second reagent is selected from the group consisting of: a tetrazine ligation reagent, a strain-promoted-azide-alkylene (SPAAC) reagent, a maleimide reagent, an N-hydroxysuccinimide (NHS) reagent, a tyrosine ligation reagent, a chemoenzymatic attachment reagent, a hydrazine ligation reagent, and a hydrazine ligation reagent.
  • the oligonucleotide is non-covalently immobilized on the first solid support.
  • the targeting component is non-covalently immobilized on the second solid support.
  • the first solid support comprises a cationic affinity resin.
  • the cationic affinity resin comprises diethylaminoethanol (DEAE) beads.
  • the second solid support comprises an affinity resin.
  • the affinity resin is selected from the group consisting of: a protein A resin, a protein G resin, a protein M resin, and a protein L resin.
  • a method comprises attaching a moiety to the targeting component.
  • the moiety is a detectable moiety.
  • the moiety is a therapeutic moiety.
  • isolating the oligonucleotide conjugate comprises contacting the oligonucleotide conjugate with a magnetic bead.
  • An aspect of the disclosure includes a method of treating a subject for cancer, the method comprising administering to the subject: a) a first oligonucleotide conjugate, wherein the first oligonucleotide conjugate comprises: (i) a targeting component that binds to a target on a cancer cell; (ii) a linker component; and (iii) an oligonucleotide component; and b) a therapeutic secondary oligonucleotide conjugate, wherein the therapeutic secondary oligonucleotide conjugate comprises: (i) a therapeutic moiety; and (ii) an oligonucleotide component, wherein at least a portion of the oligonucleotide component of the therapeutic secondary oligonucleotide conjugate is complementary to at least a portion of the oligonucleotide component of the first oligonucleotide conjugate.
  • the targeting component comprises a polymer.
  • the polymer comprises an amino acid, a nucleic acid, and a polysaccharide.
  • the nucleic acid is selected from the group consisting of: a morpholino, a peptide nucleic acid (PNA), a thioester peptide nucleic acid (tPNA), a locked nucleic acid (LNA), a
  • the targeting component is selected from the group consisting of: an antibody, an antibody fragment, an enzyme, a small molecule, a lectin, and a carbohydrate.
  • the linker component comprises a polypeptide. In one aspect, the linker component does not comprise a polypeptide.
  • the linker component is selected from the group consisting of: a tetrazine ligation linker, a strain-promoted-azide- alkylene (SPAAC) linker, a maleimide linker, a succinimide linker, a tyrosine linker, a chemoenzymatic linker, a hydrazone linker, and a hydrazine linker.
  • the length of the oligonucleotide component of the first oligonucleotide conjugate comprises a number of nucleic acid residues ranging from about 16 to about 120.
  • the length of the oligonucleotide component of the therapeutic secondary oligonucleotide conjugate comprises a number of nucleic acid residues ranging from about 16 to about 120.
  • the target on the cancer cell is a cell surface protein.
  • the cell surface protein is HER2, FOLR1, NCAM1 (CD56), CD274 (PD-L2), CD278 (ICOS), CD9, CD104 or CD119.
  • the targeting component of the first oligonucleotide conjugate comprises a protein linked to the oligonucleotide component by a strain-promoted-azide-alkylene (SPAAC) linker.
  • SPAAC strain-promoted-azide-alkylene
  • the targeting component of the first oligonucleotide conjugate comprises a protein linked to the oligonucleotide component by a tetrazine ligation linker.
  • the therapeutic moiety is selected from the group consisting of: a protein, a toxin, an antibody, an antibody-drug conjugate, an antibody fragment, an ADC fragment, an enzyme, a cell, and a small molecule.
  • the therapeutic moiety comprises an enzyme.
  • the method further comprises administering a prodrug to the subject, and wherein the prodrug is activated by the enzyme.
  • a method comprises: a) collecting a sample from the subject for determining the presence of one or more surface proteins on a cell in the sample; b) contacting the cell with a plurality of first oligonucleotide conjugates, wherein each first oligonucleotide conjugate comprises: i) a targeting component that binds to a different surface protein; ii) a linker component; iii) a cleavage component; and iv) an oligonucleotide component, wherein the oligonucleotide component comprises a first identifier sequence that identifies a surface protein; c) cleaving the cleavage component with a cleaving agent to release the first identifier sequence from each of the first oligonucleotide conjug
  • a second identifier sequence is linked to the first identifier sequence after the first identifier sequence has been released from the first oligonucleotide conjugate.
  • detecting the first identifier sequence comprises sequencing at least a portion of the first identifier sequence.
  • a method comprises detecting the second identifier sequence.
  • detecting the second identifier sequence comprises sequencing at least a portion of the second identifier sequence.
  • the sequencing comprises high-throughput sequencing.
  • the sequencing comprises parallel sequencing by synthesis.
  • the detection is qualitative. In one aspect, the detection is quantitative.
  • An aspect of the disclosure includes a kit, comprising: a) a first oligonucleotide conjugate, wherein the first oligonucleotide conjugate comprises: (i) a targeting component; (ii) a linker component; and (iii) an oligonucleotide component; and b) a therapeutic secondary oligonucleotide conjugate, wherein the therapeutic secondary oligonucleotide conjugate comprises: (i) a therapeutic moiety; and (ii) an oligonucleotide component, wherein at least a portion of the oligonucleotide component of the therapeutic secondary oligonucleotide conjugate is complementary to at least a portion of the oligonucleotide component of the first
  • oligonucleotide conjugates comprising: (a) a targeting component that targets a particular target antigen, and (b) an oligonucleotide component that is attached to the targeting component at the oligonucleotide component 3' end comprising, (i) a first universal sequence at the 3' end of the oligonucleotide component, (ii) a second universal sequence that is 5' to the first universal sequence, (iii) a first identifier sequence that is between the first universal sequence and the second universal sequence, and (iv) a second identifier sequence that is 5' to the second universal sequence.
  • the first identifier sequence identifies the targeting component.
  • the first identifier sequence ranges in length from about 5 nucleic acids to about 15 nucleic acids.
  • the first identifier sequence is 8 nucleic acids in length.
  • the first identifier sequence comprises a sequence according to SEQ ID NOS: 1- 96 or SEQ ID NOS: 205-300.
  • the second identifier sequence identifies the targeting component.
  • the second identifier sequence ranges in length from about 10 nucleic acids to about 30 nucleic acids.
  • the second identifier sequence is 20 nucleic acids in length.
  • the second identifier sequence is hybridized to a secondary oligonucleotide conjugate comprising a second oligonucleotide component that is attached to a diagnostic moiety.
  • the diagnostic moiety is selected from the group consisting of a reporter molecule, an enzyme, a radioisotope, a hapten, a fluorescent label, a phosphorescent molecule, a chemiluminescent molecule, a chromophore, and a photoaffinity molecule, or a biotin.
  • the oligonucleotide component is attached to the targeting component through a linker component.
  • the linker component is selected from the group consisting of a tetrazine ligation linker, a strain-promoted-azide-alkylene (SPAAC) linker, a maleimide linker, a succinimide linker, a tyrosine linker, a chemoenzymatic linker, a hydrazone linker, and a hydrazine linker.
  • the linker component is attached to the oligonucleotide component through a cleavage component.
  • the cleavage component comprises a light-cleavable cleavage component.
  • the cleavage component comprises a chemically-cleavable cleavage component. In some embodiments, the cleavage component comprises an oligonucleotide. In some embodiments, the oligonucleotide of the cleavage component comprises a restriction enzyme site. In some embodiments, the targeting component is selected from the group consisting of: an antibody fragment, an enzyme, a small molecule, a lectin, and a carbohydrate. In some embodiments, the targeting component is a small molecule. In some embodiments, the small molecule is a folate molecule. In some embodiments, the targeting component binds to a protein, lipid, a carbohydrate, or a small molecule.
  • the protein is a lectin or a cytokine. In some embodiments, the protein is an intracellular protein, an extracellular protein, or a cell surface protein. In some embodiments, the protein is a cell surface protein. In some embodiments, the cell surface protein is HER2, FOLR1, NCAM1 (CD56), CD274 (PD-L2), CD278 (ICOS), CD9, CD104 or CD119.
  • each oligonucleotide conjugate in the plurality comprises: (i) a targeting component that targets a particular target antigen, and (ii) an oligonucleotide component that is attached to the targeting component at the oligonucleotide component 3' end comprising, (1) a first universal sequence at the 3' end of the oligonucleotide component, (2) a second universal sequence that is 5' to the first universal sequence, (3) a first identifier sequence that is between the first universal sequence and the second universal sequence, and (4) a second identifier sequence that is 5' to the second universal sequence, (b) removing oligonucleotide conjugates that are not bound to target antigens in the biological sample, and (c) performing an assay
  • the assay comprises PCR amplification. In some embodiments, the assay comprises sequencing the first identifier sequence. In some embodiments, sequencing comprises performing next generation sequencing. In some embodiments, the assay comprises performing qPCR or fluorescence in situ hybridization. In some embodiments, the second identifier sequence hybridizes to a second identifier sequence primer for qPCR. In some embodiments, the method further comprises quantifying each target antigen that is detected in the biological sample to obtain an amount of each target antigen that is in the biological sample. In some embodiments, the amount of each target antigen that is in the biological sample is compared with a standard value. In some embodiments, if the amount of each target antigen is more than the standard value, then those target antigens are identified as a therapeutic target.
  • the first identifier sequence identifies the targeting component. In some embodiments, first identifier sequence ranges in length from about 5 nucleic acids to about 15 nucleic acids. In some embodiments, the first identifier sequence is 8 nucleic acids in length. In some embodiments, the first identifier sequence comprises a sequence according to SEQ ID NOS: 1-96 or SEQ ID NOS: 205-300. In some embodiments, the second identifier sequence identifies the targeting component. In some embodiments, the second identifier sequence ranges in length from about 10 nucleic acids to about 30 nucleic acids. In some embodiments, the second identifier sequence is 20 nucleic acids in length.
  • the second identifier sequence is hybridized to a secondary oligonucleotide conjugate comprising a second oligonucleotide component that is attached to a diagnostic moiety.
  • the diagnostic moiety is selected from the group consisting of a reporter molecule, an enzyme, a radioisotope, a hapten, a fluorescent label, a phosphorescent molecule, a chemiluminescent molecule, a chromophore, and a photoaffinity molecule, or a biotin.
  • the oligonucleotide component is attached to the targeting component through a linker component.
  • the linker component is selected from the group consisting of a tetrazine ligation linker, a strain-promoted-azide-alkylene (SPAAC) linker, a maleimide linker, a succinimide linker, a tyrosine linker, a chemoenzymatic linker, a hydrazone linker, and a hydrazine linker.
  • the linker component is attached to the oligonucleotide component through a cleavage component.
  • the cleavage component comprises a light-cleavable cleavage component.
  • the cleavage component comprises a chemically-cleavable cleavage component. In some embodiments, the cleavage component comprises an oligonucleotide. In some embodiments, the oligonucleotide of the cleavage component comprises a restriction enzyme site. In some embodiments, the targeting component is selected from the group consisting of: an antibody fragment, an enzyme, a small molecule, a lectin, and a carbohydrate. In some embodiments, the targeting component is a small molecule. In some embodiments, the small molecule is a folate molecule. In some embodiments, the targeting component binds to a protein, lipid, a carbohydrate, or a small molecule.
  • the protein is a lectin or a cytokine. In some embodiments, the protein is an intracellular protein, an extracellular protein, or a cell surface protein. In some embodiments, the protein is a cell surface protein. In some embodiments, the cell surface protein is HER2, FOLR1, NCAM1 (CD56), CD274 (PD-L2), CD278 (ICOS), CD9, CD104 or CD119.
  • a primary identifier oligonucleotide conjugate comprising (i) a targeting component that binds to a target on a cell, and (ii) a first oligonucleotide component that is attached to the targeting component at the 3 'end of the first oligonucleotide component, and (b) a secondary therapeutic
  • the disease is cancer or an infectious disease.
  • the therapeutic moiety is selected from the group consisting of a protein, a toxin, an antibody, an antibody-drug conjugate, an antibody fragment, an ADC fragment, an enzyme, a cell, and a small molecule.
  • the therapeutic moiety is a protein.
  • the therapeutic moiety is a toxin. In some embodiments, the therapeutic moiety is an antibody. In some embodiments, the therapeutic moiety is an antibody-drug conjugate. In some embodiments, the therapeutic moiety is an antibody fragment. In some embodiments, the therapeutic moiety is an ADC fragment. In some embodiments, the therapeutic moiety is an enzyme. In some embodiments, the therapeutic moiety is a cell. In some embodiments, the therapeutic moiety is a small molecule. In some embodiments, the cell is a cancer cell. In some embodiments, the target is a cell surface protein. In some embodiments, the cell surface protein is HER2, FOLR1, NCAM1 (CD56), CD274 (PD-L2), CD278 (ICOS), CD9, CD104 or CD119.
  • the oligonucleotide component of the primary identifier oligonucleotide, the secondary therapeutic oligonucleotide, or a combination thereof is selected from the group consisting of: a morpholino, a peptide nucleic acid (PNA), a thioester peptide nucleic acid (tPNA), a locked nucleic acid (LNA), a phosphorothioate, a phosphonoacetate (PACE) phosphoramidite, a ribonucleic acid (RNA), and a deoxyribonucleic acid (DNA).
  • the oligonucleotide component of the primary identifier oligonucleotide, the secondary therapeutic oligonucleotide, or a combination thereof is a PNA.
  • bispecific antibody conjugates comprising (a) a primary identifier oligonucleotide conjugate comprising (i) a first antibody that binds to a target on a cell, and (ii) a first oligonucleotide component that is attached to the the first antibody at the end 3 'end of the first oligonucleotide component, and (b) a secondary therapeutic oligonucleotide conjugate comprising (i) a second oligonucleotide component that hybridizes to the first oligonucleotide component of the primary identifier oligonucleotide conjugate, and (ii) a second antibody that is attached to the 3' end of the second oligonucleotide conjugate.
  • the cell is a cancer cell or an infectious disease cell.
  • antibody-drug conjugates comprising (a) a primary identifier oligonucleotide conjugate comprising (i) an antibody that binds to a target on a cell, and (ii) a first oligonucleotide component that is attached to the antibody at the end 3 'end of the first oligonucleotide component, and (b) a secondary therapeutic oligonucleotide conjugate comprising (i) a second oligonucleotide component that hybridizes to the first oligonucleotide component of the primary identifier oligonucleotide conjugate, and (ii)a small molecule that is attached to the 3' end of the second oligonucleotide conjugate.
  • the cell is a cancer cell or an infectious disease cell.
  • antibody-protein conjugates comprising (a) a primary identifier oligonucleotide conjugate comprising (i) an antibody that binds to a target on a cell, and (ii) a first oligonucleotide component that is attached to the antibody at the end 3 'end of the first oligonucleotide component, and (b) a secondary therapeutic
  • the cell is a cancer cell or an infectious disease cell.
  • antibody-fluorophore conjugates comprising (a) a primary identifier oligonucleotide conjugate comprising (i) an antibody that binds to a target on a cell, and (ii) a first oligonucleotide component that is attached to the antibody at the end 3 'end of the first oligonucleotide component, and (b) a secondary therapeutic oligonucleotide conjugate comprising (i) a second oligonucleotide component that hybridizes to the first oligonucleotide component of the primary identifier oligonucleotide conjugate, and (ii) a fluorophore moiety that is attached to the 3' end of the second oligonucleotide conjugate.
  • the cell is a cancer cell or an infectious disease cell.
  • FIG. 1A illustrates an oligonucleotide conjugate molecule that comprises an antibody as the targeting component, a tetrazine ligation linker as the linker component, a restriction site as the cleavable component, and an oligonucleotide component that comprises a universal sequence A, a universal sequence B, and an identifier sequence.
  • FIG. IB depicts an example synthesis process for the oligonucleotide conjugate shown in FIG. 1A.
  • FIG. 2 depicts various steps in an example diagnostic method that is carried out using the subject oligonucleotide conjugates.
  • FIGS. 3A-3B depict various examples of oligonucleotide conjugate molecules.
  • FIG. 3A illustrates a library of therapeutic oligonucleotide conjugates.
  • FIG. 3B illustrates a first oligonucleotide conjugate having bound to a cell surface marker and a secondary
  • oligonucleotide conjugate wherein the secondary oligonucleotide conjugate is used to recruit (as depicted from left to right) either a small molecule, an enzyme, a second antibody conjugate that binds to a cell surface marker on another cell, or immune cells with oligonucleotide conjugates directly incorporated into their cell membranes capable of inducing cytotoxicity or having some therapeutic effect.
  • FIG. 4 depicts various steps in an example therapeutic method that is carried out using the subject oligonucleotide conjugates.
  • FIG. 5 depicts the results of a molecular weight analysis carried out on various different conjugated and unconjugated molecules.
  • FIG. 6A depicts three different oligonucleotide conjugates (Herceptin-DA, Herceptin- DA*, and Herceptin-nsDA), as well as two different labeling compositions (IDE and goat anti- human HRP).
  • FIG. 6B depicts the interaction of the Herceptin-DA oligonucleotide conjugate with cells that express HER2 (SK-BR3 cells) and to cells that do not express HER2 (MDA-MB- 231 cells).
  • FIG. 6C depicts the quantification of surface-bound oligonucleotide conjugate using an IDE label on different cells.
  • FIG. 6D depicts the quantification of surface-bound
  • FIG. 6E depicts the results of quantification of surface-bound oligonucleotide conjugate using goat-anti-human HRP to visualize the bound oligonucleotide conjugate on the surface of SK-BR3 cells.
  • FIGS. 7A-7C depict confocal microscopy images of Herceptin-DA oligonucleotide conjugate recruitment to the surface of SK-BR3 (HER2+) cells.
  • FIG. 7A depicts location of the Herceptin-DA conjugate in SK-BR3(HER2+) cells in the cell periphery via hybridization of a fully complementary secondary oligonucleotide conjugate with a fluorescent diagnostic moiety, DI-(F).
  • DI-(F) fluorescent diagnostic moiety
  • FIG. 7B depicts Herceptin-nsDA conjugate is unable to recruit DI-(F) to the surface of SK-BR3(HER2+) cells due to a lack of sequence complementarity between the oligonucleotide component of Herceptin-nsDA and the oligonucleotide component of DI-(F).
  • FIG. 7C depicts Herceptin-DA conjugate is unable to recruit DI-(F) to the surface of the MDA-MB-231 (HER2-) cell line.
  • FIG. 8A illustrates viability of SK-BR3 cells as a function of concentration for different Herceptin oligonucleotide conjugates and peptidyl prodrug molecules.
  • FIG. 8B illustrates viability of SK-BR3 cells as a function of concentration for different Herceptin oligonucleotide conjugates and peptidyl prodrug molecules.
  • MDA-MB-231 cells illustrates viability of MDA-MB-231 cells as a function of concentration for different Herceptin oligonucleotide conjugates and peptidyl prodrug molecules.
  • FIG. 9A depicts three different oligonucleotide conjugates (Folate-DA, Folate-nsDA, and DA), as well as two different labeling compositions (IDE and goat anti-human HRP).
  • FIG. 9B depicts the interaction of the Folate-DA oligonucleotide conjugate with cells that express folate receptor (FR+) and to cells that do not express FR (FR-).
  • FIG. 9C depicts the
  • FIG. 9D depicts the quantification of surface-bound oligonucleotide conjugate using goat-anti- human HRP to visualize the bound oligonucleotide conjugate on FR+ and FR- cells. For each pair of bars, the first bar corresponds to KB (FR+) and the second bar corresponds to A549 (FR- ) ⁇
  • FIGS. 10A-10F depict confocal microscopy images of Folate-DA oligonucleotide conjugate recruitment to the surface of folate receptor positive (FR+) cells.
  • FIG. 10A depicts Folate-DA recruited DI-(F) to the cell periphery in the FR + cell line.
  • FIG. 10B depicts Folate- nsDA was unable to recruit DI-(F) to the cell surface of KB cells.
  • FIG. IOC depicts Cy5-Folate was able to stain the cellular periphery of FR + KB Cells.
  • FIG. 10D depicts Folate-DA was unable to recruit DI-(F) to the cell periphery of the FR " cell line, A549.
  • FIG. 10A depicts Folate-DA recruited DI-(F) to the cell periphery in the FR + cell line.
  • FIG. 10E depicts Folate- nsDA was unable to recruit DI-(F) to the cell periphery of the FR " cell line, A549.
  • FIG. 10F depicts Cy5-Folate was unable to stain the cellular periphery of the FR " cell line, A549.
  • FIG. 11A depicts three different oligonucleotide conjugates (Folate-DA, Folate-nsDA, and DA), as well as a labeling composition (IDE).
  • FIG. 11B depicts the interaction of the Folate-DA oligonucleotide conjugate with cells that express folate receptor (FR+).
  • FIG. 11C depicts HPLC data from doxorubicin products from treatment on FR+ KB cells.
  • FIG. 11D depicts the various doxorubicin products from each treatment.
  • FIG. HE depicts viability results from labeling with IDE and application of peptidyl prodrug (PD) with KB cells.
  • FIG. 12A illustrates an oligonucleotide conjugate molecule that comprises an antibody as the targeting component, a linker component, a cleavable component, and an oligonucleotide component that comprises a universal site 1, a first identifier sequence, a universal site 2, and a second identifier sequence.
  • FIG. 12B depicts an example synthesis process for the
  • FIG. 13 illustrates examples of the use of oligonucleotide conjugate molecules for both soluble antigen profiling and cellular antigen profiling.
  • FIG. 14 illustrates examples of the PCR amplification of the cleaved oligonucleotide component using either universal site 1 primer and universal site 2 primer to produce a next generation sequencing readout, or universal site 1 primer and the 2 nd identifier sequence primer to produce a qPCR readout.
  • FIG. 15 illustrates a soluble protein detection method
  • FIG. 16A illustrates a solid phase conjugation method to synthesis an AOC.
  • FIG. 16B illustrates gel electrophoresis of unconjugated antibodies and corresponding AOCs under non- reducing conditions.
  • FIG. 16C illustrates gel electrophoresis of unconjugated antibodies and corresponding AOCs under reducing conditions.
  • FIG. 17 illustrates an oligonucleotide conjugate molecule that comprises an antibody as the targeting component, a linker component, a cleavable component, and an oligonucleotide component that comprises a universal site 1, a first identifier sequence, a universal site 2, and a second identifier sequence.
  • An additional 5' biotin allows for analysis of AOCs via ELISA- HRP.
  • FIG. 18A illustrates the horseradish peroxidase (HRP) signal indicating cross reactivity between the AOC-biotin and unconjugated detection antibodies. Each well is reacted with the protein denoted on the x-axis, at a concentration of 250 pM. In the detection step, the y- axis identifies the target of AOC that is added across the entire row and are responsible for the signal detected upon adding HRP.
  • FIG. 18B illustrates a graphical representation of the on- target interaction indicated by the arrow in FIG. 18A.
  • FIG. 18C is the same as FIG. 18A, but with on-target interactions removed to accentuate off target binding.
  • FIG. 18D illustrates a graphical representation of the off-target interaction indicated by the arrow in FIG. 18C.
  • FIG. 19 illustrates whole proteome antibody characterization.
  • FIG. 20A represents the ELISA experiment setup and results in plate format.
  • ELISA plates were coated with capture antibody and recombinant proteins were incubated with their corresponding capture antibody across a 1 :4 dilution series, from InM to lpM, and analyzed in duplicate. Signal is generated by reacting streptavidin-HRP with the 5' biotin modifications on AOC oligos.
  • FIG. 20B illustrates aggregation of all ELISA standard curve data for all 16 sandwich ELSIA pairs.
  • FIG. 20C illustrates limit of detection data for individual soluble proteins via AOC-qPCR. ELISA plates were coated with capture antibodies in the same manner and detection of recombinant proteins was tested along a 1 : 10 serial dilution from InM to lOfM.
  • FIG. 21A illustrates two identifier sequences comprising trinucleotide repeats and hybridization of said identifier sequences.
  • FIG. 21B illustrates the hybridization of identifier sequences of two oligonucleotide conjugates to produce a variety of therapeutic and diagnostic compounds, including a bispecific antibody, an antibody-drug conjugate, an antibody-protein conjugate, and an antibody-fluorophore conjugate.
  • FIG. 22 illustrates use of the oligonucleotide conjugates to detect a unique surface protein of an abnormal cell of a patient relative to a normal cell of the patient followed by subsequent use of the unique surface protein as a target for a therapeutic compound comprising an oligonucleotide conjugate targeting the unique surface protein hybridized to a secondary oligonucleotide conjugate comprising a drug with cytotoxic activity.
  • Oligonucleotide conjugates and uses thereof are provided.
  • An aspect of the subject oligonucleotide conjugates includes a targeting component, a linker component, a cleavage component, and an oligonucleotide component.
  • Methods of making and using the subject oligonucleotide conjugates in the diagnosis, prevention and/or treatment of cancer and other diseases are also provided.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • targeting component is used herein to refer to a portion of an
  • oligonucleotide conjugate having a structure or binding determinant that binds to or has specificity for a binding target.
  • linker component is used herein to refer to a portion of an oligonucleotide conjugate that conjugates or links two or more different components of an oligonucleotide conjugate together.
  • a targeting component and an oligonucleotide component can be linked to one another by a linker component.
  • cleavage component is used herein to refer to a portion of an
  • oligonucleotide conjugate that is configured to be cleaved, or severed, under cleavage-promoting conditions.
  • artificial is used herein to refer to a molecule, component, or composition that does not occur in nature.
  • biopolymer or “biological polymer” as used herein refers to repeating units of biological or chemical moieties.
  • Representative biopolymers include, but are not limited to, nucleic acids, oligonucleotides, amino acids, proteins, peptides, hormones, oligosaccharides, lipids, glycolipids, lipopolysaccharides, phospholipids, synthetic analogues of the foregoing, including, but not limited to, inverted nucleotides, peptide nucleic acids, Meta-DNA, and combinations of the above.
  • immunoglobulin or "antibody” as used interchangeably herein refers to a basic 4-chain heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain has an N-terminus and a C-terminus, and also has regularly spaced intrachain disulfide bridges. In the case of IgG antibodies, each H chain has at the N- terminus a variable domain (V H ) followed by three constant domains (C H 1, C H 2 and C H 3).
  • V H variable domain
  • C H 1 constant domains
  • Each L chain has at the N-terminus a variable domain (V L ) followed by one constant domain (C L ).
  • V L is aligned with the V H and the C L is aligned with the first constant domain of the heavy chain (C H I).
  • C H I constant domain of the heavy chain
  • immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the ⁇ and a classes are further divided into subclasses on the basis of relatively minor differences in C H sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.
  • variable region or “variable domain” of an immunoglobulin refers to the N- terminal domains of the H or L chain of the immunoglobulin.
  • the variable domain of the H chain can be referred to as "V H "
  • the variable domain of the light chain can be referred to as "V L .” These domains are generally the most variable parts of an immunoglobulin and contain the antigen-binding sites.
  • variable refers to the fact that certain segments of the variable domains differ extensively in sequence among immunoglobulins.
  • the V domain mediates antigen binding and defines specificity of a particular immunoglobulin for its particular antigen.
  • variable domains consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long.
  • FRs framework regions
  • hypervariable regions regions of extreme variability
  • the variable domains of native H and L chains each comprise four FRs, largely adopting a ⁇ -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of immunoglobulins (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).
  • the constant domains are not involved directly in binding an immunoglobulin to an antigen, but exhibit various effector functions, such as participation of the immunoglobulin in antibody dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).
  • An "intact" immunoglobulin is one that comprises an antigen-binding site as well as a C L and at least H chain constant domains of the particular antibody class (e.g., IgG, IgA, IgM, IgD or IgE heavy chain constant domains).
  • the constant domains can be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.
  • An intact immunoglobulin can have one or more effector functions.
  • immunoglobulin fragments comprise a portion of an intact immunoglobulin, preferably the antigen binding or variable region of the intact immunoglobulin.
  • immunoglobulin fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear immunoglobulins (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain immunoglobulin molecules; and multispecific
  • an immunoglobulin fragment comprises an antigen binding site of the intact immunoglobulin and thus retains the ability to bind antigen.
  • Fab antigen-binding fragments
  • Fc residual fragment
  • the Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V H ), and the first constant domain of one heavy chain (C H I).
  • V H variable region domain of the H chain
  • C H I first constant domain of one heavy chain
  • Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.
  • F(ab')2 immunoglobulin yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen.
  • Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the C H 1 domain including one or more cysteines from the immunoglobulin hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab')2 immunoglobulin fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of
  • immunoglobulin fragments are also known.
  • the Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides.
  • the effector functions of immunoglobulins are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
  • Fv is the minimum immunoglobulin fragment which contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light- chain variable region domain in tight, non-covalent association.
  • scFv single-chain Fv
  • one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the immunoglobulin.
  • variable domain or half of an Fv comprising only three CDRs specific for an antigen
  • Single-chain Fv also abbreviated as “sFv” or “scFv” are immunoglobulin fragments that comprise the V H and V L immunoglobulin domains connected into a single polypeptide chain.
  • the sFv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding.
  • sFv see Pliickthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.
  • immunoglobulin or "antibody” specifically includes native human and non-human IgG (e.g., IgGl, IgG2, IgG3, IgG4), IgE, IgA (e.g., IgAl, IgA2), IgD and IgM antibodies, including naturally occurring variants thereof.
  • polypeptide e.g., an antibody or
  • immunoglobulin is used herein to refer to a polypeptide having a sequence that occurs in nature, regardless of its mode of preparation.
  • non-native with reference to a polypeptide (e.g., an antibody or immunoglobulin) is used herein to refer to a polypeptide having a sequence that does not occur in nature.
  • polypeptide is used herein in the broadest sense and includes peptide sequences.
  • peptide generally describes linear molecular chains of amino acids containing up to about 30, preferably up to about 60 amino acids covalently linked by peptide bonds.
  • the term "monoclonal” as used herein refers to an antibody or immunoglobulin molecule obtained from a population of substantially homogeneous immunoglobulins, i.e., the individual immunoglobulins comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts.
  • immunoglobulins are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal immunoglobulin is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the character of the immunoglobulin as being obtained from a substantially homogeneous population of immunoglobulins, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal immunoglobulins in accordance with the present disclosure can be made by the hybridoma method first described by Kohler and Milstein (1975) Nature 256:495, or can be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).
  • the monoclonal immunoglobulins herein specifically include “chimeric"
  • immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6851-6855).
  • Humanized forms of non-human e.g., rodent, e.g., murine or rabbit
  • immunoglobulins are immunoglobulins which contain minimal sequences derived from non- human immunoglobulin. For the most part, humanized immunoglobulins are human
  • immunoglobulins in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, hamster, rabbit, chicken, bovine or non-human primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, hamster, rabbit, chicken, bovine or non-human primate having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are also replaced by corresponding non-human residues.
  • humanized antibodies can comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized immunoglobulin will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the
  • hypervariable loops correspond to those of a non-human immunoglobulin and all or
  • substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized immunoglobulin optionally also will comprise at least a portion of an
  • Fc immunoglobulin constant region
  • human immunoglobulin as used herein, is intended to include
  • human immunoglobulins having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human immunoglobulins of the disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • human immunoglobulin as used herein, is not intended to include immunoglobulins in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • An "isolated" immunoglobulin is one which has been separated and/or recovered from a component of its natural environment, e.g., within a recombinant host cell. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses, and can include enzymes, hormones, and other proteinaceous or non- proteinaceous solutes, as well as undesired byproducts of the production.
  • an isolated immunoglobulin or oligonucleotide conjugate herein will be purified (1) to greater than 95% by weight, or greater than 98% by weight, or greater than 99% by weight, as determined by SDS-PAGE or SEC-HPLC methods, (2) to a degree sufficient to obtain at least 15 residues of N- terminal or internal amino acid sequence by use of an amino acid sequencer, or (3) to
  • an isolated immunoglobulin or oligonucleotide conjugate will be prepared by at least one purification step.
  • binding refers to the binding of a binding moiety to a binding target, such as the binding of a targeting component (e.g., an immunoglobulin) to a target antigen, e.g., an epitope on a particular polypeptide, peptide, or other target (e.g. a glycoprotein target), and means binding that is measurably different from a non-specific interaction (e.g., a non-specific interaction can be binding to bovine serum albumin or casein).
  • a targeting component e.g., an immunoglobulin
  • target antigen e.g., an epitope on a particular polypeptide, peptide, or other target (e.g. a glycoprotein target)
  • target antigen e.g., an epitope on a particular polypeptide, peptide, or other target (e.g. a glycoprotein target)
  • target antigen e.g., an epitope on a particular polypeptide, peptide, or other target (
  • specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non- labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.
  • the term "specific binding” or “specifically binds to” or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target of at least about 200 nM, alternatively at least about 150 nM, alternatively at least about 100 nM, alternatively at least about 60 nM, alternatively at least about 50 nM,
  • the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
  • Binding affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an immunoglobulin) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., immunoglobulin and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd).
  • the Kd can be about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or stronger.
  • Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art.
  • Kd or “Kd value” refers to a dissociation constant measured by a technique appropriate for the immunoglobulin and target pair, for example using surface plasmon resonance assays, for example, using a Biacore XI 00 or a Biacore T200 (GE).
  • conjugation refers to any and all forms of covalent or non-covalent linkage, and include, without limitation, direct genetic or chemical fusion, coupling through a linker or a cross-linking agent, and non-covalent association.
  • fusion is used herein to refer to the combination of amino acid sequences of different origin in one polypeptide chain by in-frame combination of their coding nucleotide sequences.
  • fusion explicitly encompasses internal fusions, i.e., insertion of sequences of different origin within a polypeptide chain, in addition to fusion to one of its termini.
  • fusion is used herein to refer to the combination of amino acid sequences of different origin.
  • epitope includes any molecular determinant capable of specific binding to a targeting component (e.g., an immunoglobulin).
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in one aspect, can have specific three dimensional structural characteristics, and/or specific charge characteristics.
  • An epitope is a region of an antigen that is bound by a targeting component (e.g., an immunoglobulin).
  • a "binding region” is a region on a binding target bound by a binding molecule.
  • target or “binding target” is used in the broadest sense and specifically includes polypeptides, without limitation, nucleic acids, carbohydrates, lipids, cells, and other molecules with or without biological function as they exist in nature.
  • antigen refers to an entity or fragment thereof, which can bind to an immunoglobulin or trigger a cellular immune response.
  • An immunogen refers to an antigen, which can elicit an immune response in an organism, particularly an animal, more particularly a mammal including a human.
  • antigen includes regions known as antigenic determinants or epitopes, as defined above.
  • An "antigen-binding site” or “antigen-binding region” of an immunoglobulin of the present disclosure typically contains six complementarity determining regions (CDRs) within each variable domain, and which contribute in varying degrees to the affinity of the binding site for antigen. In each variable domain there are three heavy chain variable domain CDRs
  • CDRH1, CDRH2 and CDRH3 three light chain variable domain CDRs
  • CDRL1, CDRL2 and CDRL3 three light chain variable domain CDRs
  • antibody/antigen complexes are also included within the scope of the disclosure.
  • functional antigen binding sites comprised of fewer CDRs (i.e., where binding specificity is determined by three, four or five CDRs). Less than a complete set of 6 CDRs can be sufficient for binding to some binding targets. Thus, in some instances, the CDRs of a V H or a V L domain alone will be sufficient.
  • certain antibodies might have non-CDR-associated binding sites for an antigen. Such binding sites are specifically included within the present definition.
  • a nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence.
  • DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • the terms "individual,” “patient,” or “subject” are used interchangeably. None of the terms require or are limited to situation characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly, or a hospice worker). Further, these terms refer to human or animal subjects.
  • a health care worker e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly, or a hospice worker. Further, these terms refer to human or animal subjects.
  • Treating refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a targeted pathologic condition or disorder.
  • Those in need of treatment include those already with the disorder, as well as those prone to have the disorder, or those in whom the disorder is to be prevented.
  • a subject or mammal is successfully "treated” for cancer, if, after receiving a therapeutic amount of a subject oligonucleotide conjugate according to the methods of the present disclosure, the subject shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (i.e., slowing to some extent and preferably stopping) of cancer cell infiltration into peripheral organs, including the spread of cancer into soft tissue and bone; inhibition (i.e., slowing to some extent and preferably stopping) of tumor metastasis; inhibition, to some extent, of tumor growth; and/or relief to some extent of one or more of the symptoms associated with the specific cancer; reduced morbidity and/or mortality, and improvement in quality of life issues.
  • An aspect of the disclosure includes oligonucleotide conjugates that include a targeting component, a linker component, a cleavage component, and an oligonucleotide component. Each of these features is described in greater detail herein.
  • An aspect of the disclosure includes targeting components that are configured to bind to a binding target.
  • Targeting components in accordance with an aspect of the disclosure can include any of a variety of suitable compositions that are configured to specifically interact with a binding target, e.g., to specifically bind to, or be bound by, a binding target.
  • a targeting component is an antibody, or an antigen-binding fragment thereof.
  • Oligonucleotide conjugates that include an antibody, or an antigen-binding fragment of an antibody, as a targeting component can be referred to herein as "antibody oligonucleotide conjugates" or "AOCs".
  • An antibody targeting component generally includes two identical light chains, as well as two identical heavy chains. Each light chain and each heavy chain includes an N-terminus and a C-terminus. Each light chain includes a variable domain, designated as V L , as well as a constant domain, designated as C L .
  • a light chain comprises a kappa light chain. In one aspect, a light chain comprises a lambda light chain.
  • each heavy chain includes a variable domain, designated as V 3 ⁇ 4 as well as a constant domain designated as C H 1, followed by one or more heavy chain Fc region domains.
  • Fc region domains on a heavy chain can include Fc region domains that are specific to a particular immunoglobulin type or subtype, including but not limited to Fc region domains from an IgG (such as an IgGl, IgG2, IgG3 or IgG4), IgA (such as an IgAl or IgA2), IgM, IgE or IgD antibody.
  • an immunoglobulin molecule can contain a native polypeptide sequence that occurs in nature.
  • an immunoglobulin molecule can contain a non-naturally occurring polypeptide sequence (i.e., an artificial sequence that does not occur in nature).
  • variable and constant domains along the light chain generally proceeds from the N-terminus to the C-terminus as V L -C L .
  • organization of the variable and constant domains along the heavy chain generally proceeds from the N-terminus to the C-terminus as V H -C H 1-FC.
  • An aspect of the subject antibody targeting components includes a variable domain with antigen binding functionality.
  • V L and V H sequences of the subject antibody targeting components are selected to specifically bind to a binding target, such as, e.g., an antigen on a tumor cell.
  • a targeting component can include an antigen-binding fragment of an antibody.
  • Antigen-binding fragments of antibodies include, but are not limited to: Fab, Fab', F(ab) 2 , F(ab') 2 , Fv, scFv, and single domain antibodies.
  • a targeting component is an enzyme that specifically binds to a binding target.
  • a targeting component is a small molecule that specifically binds to, or is specifically bound by, a binding target.
  • small molecules that specifically bind to or are bound by a binding target include, but are not limited to, folate.
  • a targeting component is a lectin that specifically binds to, or is specifically bound by, a binding target.
  • a targeting component is a carbohydrate that specifically binds to, or is specifically bound by, a binding target.
  • a targeting component comprises a polymer that specifically binds to, or is specifically bound by, a binding target.
  • Polymeric targeting components in accordance with an aspect of the disclosure include, but are not limited to, polypeptides (polymers of amino acid residues), polynucleotides (polymers of nucleic acids) and polysaccharides (polymers of sugar molecules).
  • a polymeric targeting component comprises a morpholino.
  • a morpholino also known as a phosphorodiamidate morpholino oligomer (PMO) is a nucleic acid analog that can bind to a target nucleic acid sequence (e.g., an RNA molecule, e.g., an mRNA molecule).
  • a target nucleic acid sequence e.g., an RNA molecule, e.g., an mRNA molecule.
  • a polymeric targeting component comprises a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • An individual PNA includes an N-(2-aminoethyl)-glycine backbone component, and in a polymer made of a plurality of PNAs, the individual PNAs (e.g., the repeating N-(2- aminoethyl)-glycine units) are linked together by peptide bonds.
  • a PNA is a thioester peptide nucleic acid (tPNA).
  • a polymeric targeting component comprises a polynucleotide that includes a locked nucleic acid (LNA).
  • LNA locked nucleic acid
  • An LNA comprises a modified RNA nucleotide wherein the ribose moiety is modified with an extra bond connecting the 2' oxygen and the 4' carbon.
  • the locked ribose conformation enhances base stacking and backbone pre-organization thereby increasing hybridization properties.
  • a polymeric targeting component comprises a polynucleotide that includes a phosphorothioate.
  • a phosphorothioate is a variant of normal DNA, wherein one of the non-bridging oxygen atoms is replaced by a sulfur atom. The sulfurization of the
  • internucleotide bond increases nuclease resistance, and can increase solubility in lipid bilayers.
  • a polymeric targeting component comprises a polynucleotide that includes a phosphonoacetate (PACE) phosphoramidite.
  • PACE phosphonoacetate
  • a PACE phosphoramidite contains a phosphoacetate linkage in place of the standard phosphodiester linkage. Oligonucleotides containing this modification have increased nuclease resistance and increased solubility in lipid bilayers.
  • a polymeric targeting component comprises a polynucleotide that includes a ribonucleic acid (RNA). In one aspect, a polymeric targeting component comprises a polynucleotide that includes a deoxyribonucleic acid (DNA).
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • a targeting component of the disclosure can be modified to contain one or more non-proteinaceous moieties that are known in the art and readily available.
  • a moiety suitable for derivatization of a targeting component e.g., an antibody
  • a water soluble polymer is a water soluble polymer.
  • Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either
  • polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the targeting component may vary, and if more than one polymer is attached, they can be the same or different molecules.
  • the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the targeting component to be used, whether the targeting component will be used in a diagnostic or therapeutic application, etc. Accordingly, a moiety suitable for derivatization of a targeting component can be used to attach diagnostic or therapeutic agents to a targeting component, for use as capture agents or competitors in competitive assays.
  • an aspect of the disclosure includes targeting components that specifically bind to, or are bound by, a binding target.
  • Binding targets in accordance with an aspect of the disclosure generally include any molecules that are associated with a particular disease pathology, i.e., are indicative of the presence of a disease or disorder, or that mediate signaling through one or more biological signaling pathways that are involved with a disease or disorder.
  • a binding target can be a protein.
  • a binding target can be a soluble protein (e.g., an extra-cellular protein).
  • a binding target can be a cell surface protein.
  • cell surface proteins that find use as binding targets in accordance with an aspect of the disclosure include, but are not limited to, HER2 (ERBB2), folate receptor 1 (FOLR1), NCAM1 (CD56), CD274 (PD-L2), CD278 (ICOS), CD9, CD104 and CD119.
  • a binding target is a cell surface protein that is a tumor-associated antigen.
  • tumor-associated antigens are known for virtually any type of cancer.
  • Specific tumor-associated antigen binding targets that can be targeted by a binding target in accordance with an aspect of the disclosure include, but are not limited to, FOLR2, CD138, CD19, CD79A, CD79B, ROR1, ROR2, FCRM, CS1, GPA33, MSLN, CD52, CD20, CD3, CD4, CD8, CD21, CD22, CD23, CD30, CD33, CD37, CD38, CD44, CD56, CD70, cyCD79a, CD80, BCMA, BMP6, IL12A, ILIA, IL IB, IL2, IL24, INHA, TNF, TNFSFIO, BMP6, EGF, FGF1, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, F
  • MACMARCKS MT3 (metallothionectin-III), MUC1 (mucin), PTGS2 (COX-2), RAC2
  • a binding target can be an intracellular protein.
  • intracellular proteins that find use as binding targets in accordance with an aspect of the disclosure include, but are not limited to, CREB1, CTNNBl, JUN, AKTl, AKT2, ATF2, CCND1, CHUK, FOS, JAK2, MAP2K2, MAPK1, MAPK14, RELA, RPS6KA2, SMAD3, STAT1, STAT2, STAT3, STAT5A, STMN1, and TP53.
  • Additional examples of intracellular proteins include, but are not limited to: HER2, FOLR1, NCAM1 (CD56), PD-L1, CD274 (PD-L2), CD278 (ICOS), CD9, CD104 and CD119.
  • a targeting component of a subject oligonucleotide conjugate can include an antibody or an antigen-binding fragment thereof.
  • the amino acid sequences of a variable domain region which provides antigen binding functionality to an antibody molecule, can include chimeric, humanized, or human amino acid sequences. Any suitable combination of such sequences can be incorporated into a variable domain of a subject antibody targeting component.
  • Antigen-binding variable region sequences can be selected from various monoclonal antibodies that are capable of binding specific targets and are well known in the art. These include, but are not limited to: anti-TNF antibody (U.S. Pat. No. 6,258,562), anti-IL-12 and or anti-IL-12p40 antibody (U.S. Pat. No.
  • anti-IL-18 antibody US 2005/0147610 Al
  • Antigen-binding variable region sequences can also be selected from various therapeutic antibodies approved for use, in clinical trials, or in development for clinical use.
  • therapeutic antibodies include, but are not limited to, RITUXAN®,
  • IDEC/Genentech/Roche see for example U.S. Pat. No. 5,736, 137
  • a chimeric anti-CD20 antibody approved to treat Non-Hodgkin's lymphoma HUMAX-CD20®
  • an anti-CD20 currently being developed by Genmab an anti-CD20 antibody described in U.S. Pat. No.
  • trastuzumab HERCEPTIN®, Genentech
  • trastuzumab HERCEPTIN®, Genentech
  • pertuzumab rhuMab-2C4, OMNITARG®
  • cetuximab (ERBITUX®, Imclone) (U.S. Pat. No. 4,943,533; PCT WO 96/40210), a chimeric anti-EGFR antibody in clinical trials for a variety of cancers; ABX-EGF (U.S. Pat. No. 6,235,883), currently being developed by Abgenix-Immunex-Amgen; HUMAX- EGFRTM (U.S. Ser. No. 10/172,317), currently being developed by Genmab; 425, EMD55900, EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864; Murthy et al. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al., 1987, J Cell Biochem. 35(4):315-20;
  • CAMPATH® Millennium
  • muromonab-CD3 Orthoclone OKT3®
  • an anti-CD3 antibody developed by Ortho Biotech/Johnson & Johnson
  • ibritumomab tiuxetan ZEVALUSr®
  • an anti-CD20 antibody developed by IDEC/Schering AG
  • gemtuzumab ozogamicin MYLOTARG®
  • an anti-CD33 (p67 protein) antibody developed by
  • REMICADE® an anti-TNF alpha antibody developed by Centocor, adalimumab
  • HUMIRA® an anti-TNF alpha antibody developed by Abbott, HUMICADE®, an anti- TNF alpha antibody developed by Celltech, etanercept (ENBREL®), an anti-TNF alpha Fc fusion developed by Immunex/Amgen, ABX-CBL, an anti-CD147 antibody being developed by Abgenix, ABX-IL8, an anti-IL8 antibody being developed by Abgenix, ABX-MAl, an anti- MUC18 antibody being developed by Abgenix, Pemtumomab (R1549, 90Y-muHMFGl), an anti-MUCl in development by Antisoma, Therex (R1550), an anti-MUCl antibody being developed by Antisoma, AngioMab (AS1405), being developed by Antisoma, HuBC-1, being developed by Antisoma, Thioplatin (AS 1407) being developed by Antisoma, ANTEGREN® (natalizumab), an anti-alpha-4-beta-l (VLA4)
  • LYMPHOCIDE® (Epratuzumab), an anti-CD22 antibody being developed by Immunomedics, AFP-Cide, being developed by Immunomedics, MyelomaCide, being developed by
  • Immunomedics LkoCide, being developed by Immunomedics, ProstaCide, being developed by Immunomedics, MDX-010, an anti-CTLA4 antibody being developed by Medarex, MDX-060, an anti-CD30 antibody being developed by Medarex, MDX-070 being developed by Medarex, MDX-018 being developed by Medarex, OSIDEM® (IDM-1), and anti-Her2 antibody being developed by Medarex and Immuno-Designed Molecules, HUMAX®-CD4, an anti-CD4 antibody being developed by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody being developed by Medarex and Genmab, CNTO 148, an anti-TNFa antibody being developed by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokine antibody being developed by
  • ICM-1 CD54 antibodies
  • FGFR-3 fibroblast growth factor receptor 3
  • NUVION® visilizumab
  • an anti-CD3 antibody being developed by Protein Design Labs, HUZAF®
  • an anti -gamma interferon antibody being developed by Protein Design Labs
  • Anti-a 5 ⁇ 1 Integrin being developed by Protein Design Labs
  • anti-IL-12 being developed by Protein Design Labs, ING-1
  • an anti-Ep-CAM antibody being developed by Xoma
  • XOLAIR® Opizumab
  • MLN01 an anti-Beta2 integrin antibody being developed by Xoma.
  • Non-protein binding targets include, for example, lectins, cytokines, lipids, carbohydrates, and small molecules (e.g., folate).
  • An aspect of the disclosure includes linker components (or “linkers”) that are configured to link a targeting component to an oligonucleotide component.
  • Linkers in accordance with an aspect of the disclosure can include one or more linker components that are adapted or configured to link a targeting component to an oligonucleotide component by forming a covalent linkage between the targeting component and the oligonucleotide
  • linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala- phe”), p-aminobenzyloxycarbonyl (a "PAB”), and those resulting from conjugation with linker reagents: N-Succinimidyl 4-(2-pyridylthio) pentanoate forming linker moiety 4- mercaptopentanoic acid (“SPP”), N-succinimidyl 4-(N-maleimidom ethyl) cyclohexane-1 carboxylate forming linker moiety 4-((2,5-dioxopyrrolidin-l-yl)methyl)cyclohexanecarboxylic acid (“SMCC”, also referred to herein as "MCC”), 2,5-dioxopyrroli
  • a linker can be a cleavable linker that is configured decouple a targeting component form an oligonucleotide component under cleavage- promoting conditions.
  • an acid-labile linker e.g., a hydrazine linker
  • protease- sensitive e.g., a peptidase-sensitive
  • photolabile linker dimethyl linker or disulfide- containing linker
  • a linker component can comprise a polypeptide.
  • polypeptide linker components include, but are not limited to: Tobacco Etch Virus proteolytic cleavage site, ENLYFQ ⁇ G (SEQ ID NO: 200), (where ⁇ denotes the cleavage site);
  • Enteropeptidase DDDDKA (SEQ ID NO: 201); Thrombin, LVPR ⁇ GS (SEQ ID NO: 202); Factor Xa, LVPRAGS (SEQ ID NO: 203); and Rhinovirus 3C protease, LEVLFQ ⁇ GP (SEQ ID NO: 204).
  • a linker comprises a chemical composition that does not include amino acid residues (e.g., does not comprise a polypeptide).
  • a linker comprises a tetrazine ligation linker.
  • a non-limiting example of a tetrazine ligation linker is provided in formula (I), below:
  • a linker comprises a strain-promoted-azide-alkylene (SPAAC) linker (also referred to herein as a dibenzylcycootyne (DBCO) linker).
  • SPAAC strain-promoted-azide-alkylene
  • DBCO dibenzylcycootyne
  • a linker comprises a maleimide linker, which comprises the chemical formula H 2 C 2 (CO) 2 H.
  • a linker comprises a succinimide linker, which comprises the chemical formula C 4 H 5 N0 2 .
  • a linker comprises a hydrazone linker, which comprises the chemical formula
  • a linker comprises a hydrazine linker, which comprises the chemical formula H 2 N H 2 .
  • a linker comprises a tyrosine linker.
  • a non-limiting example of a tyrosine linker is provided in formula (III), below:
  • a linker comprises a chemoenzymatic linker wherein a primary amine reacts with an LLQG acceptor site on a modified polypeptide or protein.
  • a chemoenzymatic linker is provided in formula (IV), below:
  • An aspect of the disclosure includes cleavage components that are located between a targeting component and an oligonucleotide component, and that are capable of being cleaved under cleavage-promoting conditions to separate an oligonucleotide component from one or more other components of an oligonucleotide conjugate.
  • cleavage-promoting conditions include, but are not limited to, the application of energy (e.g., in the form of electromagnetic radiation (i.e., photons) or heat), or the application of suitable chemical conditions (e.g., pH conditions (e.g., acidic or basic conditions), enzymatic cleavage conditions (e.g., incubation with one or more proteases or restriction endonucleases)), and the like.
  • energy e.g., in the form of electromagnetic radiation (i.e., photons) or heat
  • suitable chemical conditions e.g., pH conditions (e.g., acidic or basic conditions
  • enzymatic cleavage conditions e.g., incubation with one or more proteases or restriction endonucleases
  • a cleavage component comprises a polypeptide sequence.
  • a cleavage component that comprises a polypeptide sequence is configured to be cleaved by an enzyme that recognizes and specifically binds to at least a portion of the polypeptide sequence and cleaves the polypeptide sequence.
  • an enzyme that binds to a polypeptide sequence is a protease.
  • Non-limiting examples of polypeptide cleavage components include ENLYFQS (SEQ ID NO: 198) (cleavable by TEV protease);
  • polypeptide linker components include, but are not limited to: Tobacco Etch Virus proteolytic cleavage site, ENLYFQ ⁇ G (SEQ ID NO: 200), (where ⁇ denotes the cleavage site); Enteropeptidase, DDDDKA (SEQ ID NO: 201); Thrombin, LVPRAGS (SEQ ID NO: 202); Factor Xa, LVPRAGS (SEQ ID NO: 203); and Rhinovirus 3C protease, LEVLFQ ⁇ GP (SEQ ID NO: 204).
  • a cleavage component comprises a polynucleotide sequence.
  • a polynucleotide cleavage component is configured to be cleaved by a restriction endonuclease that specifically binds to polynucleotide sequence (referred to herein as a
  • restriction site and cleaves the polynucleotide sequence.
  • a number of nucleotides in a restriction site ranges from about 6 to about 12 nucleotides, such as about 7, 8, 9, 10, or 11 nucleotides.
  • a restriction endonuclease is functional at room temperature and in conditions that are non-cytotoxic to cells.
  • a restriction endonuclease can act on a double-stranded DNA substrate, requiring only a single nucleotide spacer from the terminal 5' or 3' ends.
  • Restriction endonucleases are generally known in the art and include, without limitation, Apal, which is operable at room temperature and in conditions that are non-cytotoxic to cells, and cuts a DNA segment that contains the sequence, from 5' to 3', GGGCCC (SEQ ID NO: 199).
  • An aspect of the disclosure includes oligonucleotide components that comprise a plurality of nucleic acid residues.
  • an oligonucleotide component has a number of nucleic acids that ranges from about 16 to about 120, such as about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, or about 115 nucleic acids.
  • an oligonucleotide component comprises an identifier sequence that contains a predetermined sequence of nucleic acids and that provides identifying information regarding a particular binding target.
  • an oligonucleotide conjugate includes a predetermined identifier sequence that identifies a binding target to which the targeting component of an oligonucleotide conjugate binds. The identifier sequence is therefore "matched" with the targeting component in a particular oligonucleotide conjugate, and is capable of providing information regarding the binding target of the targeting component.
  • the identifier sequence is a single stranded oligonucleotide.
  • an oligonucleotide component comprises a first identifier sequence and a second identifier sequence that contain a predetermined sequence of nucleic acids and that provides identifying information regarding a particular binding target.
  • an oligonucleotide conjugate includes a predetermined first identifier sequence and a second identifier sequence that identifies a binding target to which the targeting component of an oligonucleotide conjugate binds. The first identifier sequence and the second identifier sequence are therefore "matched" with the targeting component in a particular oligonucleotide conjugate, and are capable of providing information regarding the binding target of the targeting component.
  • the first identifier sequence is a single stranded
  • the first identifier sequence is a single stranded DNA or a single stranded PNA In some embodiments, the first identifier sequence identifies the targeting component. In one aspect, a first identifier sequence ranges in length from about 5 to about 15 nucleic acids, such as about 6, 7, 8, 9, 10, 11, 12, 13 or 14 nucleic acids. In one aspect, a first identifier sequence is 8 nucleic acids in length. In one aspect, the first identifier sequence comprises at least one trinucleotide repeat (FIG. 21A). In one aspect, the first identifier sequence does not comprise a trinucleotide repeat.
  • the second identifier sequence is a single stranded oligonucleotide.
  • the second identifier sequence is a single stranded DNA or a single stranded PNA
  • the second identifier sequence identifies the targeting component.
  • a second identifier sequence ranges in length from about 10 to about 30 nucleic acids, such as about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleic acids.
  • a second identifier sequence is 20 nucleic acids in length.
  • first identifier sequences include, but are not limited to, SEQ ID NOS: 1-96 and SEQ ID NOS: 205-300, as shown in Table 1.
  • second identifier sequences include, but are not limited to, SEQ ID NOS: 301-396, as shown in Table 2.
  • the first identifier and the second identifier comprise identical sequences. In some embodiments, the first identifier and the second identifier comprise non- identical sequences. In some embodiments, the first identifier is not contained within any portion of the second identifier sequence.
  • Table 1 Example first identifier sequences.
  • Table 2 Example second identifier sequences.
  • TCTCTTATCACCTCGGTCGG 319 TA AG C A ATCTCTG AG G CG CC 320
  • an oligonucleotide component comprises first universal sequence and second universal sequence that are configured for sequencing analysis.
  • a first universal sequence ranges in length from about 12 to about 60 nucleic acid residues, such as about 15, 20, 25, 30, 35, 40, 45, 50, or 55 nucleic acid residues.
  • a second universal sequence ranges in length from about 12 to about 60 nucleic acid residues, such as about 15, 20, 25, 30, 35, 40, 45, 50, or 55 nucleic acid residues.
  • a first and a second universal sequence are the same, whereas in one aspect, a first and a second universal sequence are different. Examples of first identifier sequences include, but are not limited to, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 397, and SEQ ID NO: 398, as shown in Table 3. Table 3: Example universal sequences.
  • an oligonucleotide component of an oligonucleotide conjugate is arranged such that a first universal sequence, an identifier sequence, and a second universal sequence are arranged in order from a 5' end to a 3 ' end of an oligonucleotide component.
  • an oligonucleotide component of an oligonucleotide conjugate is arranged such that a first universal sequence, a first identifier sequence, a second universal sequence, and a second identifier sequence are arranged in order from a 5' end to a 3 ' end of an oligonucleotide component.
  • an oligonucleotide component of an oligonucleotide conjugate is arranged such that a first universal sequence, an identifier sequence, and a second universal sequence are arranged in order from a 3 ' end to a 5' end of an oligonucleotide component.
  • an oligonucleotide component of an oligonucleotide conjugate is arranged such that a first universal sequence, a first identifier sequence, a second universal sequence, and a second identifier sequence are arranged in order from a 3 ' end to a 5' end of an oligonucleotide component.
  • a cleavage component is located at a 3 ' end of an oligonucleotide component.
  • An oligonucleotide component is generally configured such that one or more primers can be used in a sequencing analysis to determine the sequence of the identifier sequence or the first identifier sequence, thereby providing information about the binding target of the targeting component on the oligonucleotide conjugate.
  • the term "5BioSG” as used in connection with an oligonucleotide sequence denotes a 5' biotin molecule.
  • the term “3AmMO" as used in connection with an oligonucleotide sequence denotes a 3 ' amino modifier. Examples of full oligonucleotide component sequences include, but are not limited to, the full
  • oligonucleotide component sequences in Table 4 examples include, but are not limited to, SEQ ID NOS: 100-195 and SEQ ID NOS: 399-446, as shown in Table 4.
  • AAGAGACTTAGTGACCGGCG AACGAATCCACCCGCAG GATACCAG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 428
  • an oligonucleotide conjugate can include a spacer sequence that is placed in between two different features of an oligonucleotide component to provide a suitable separation between the components.
  • a spacer sequence can be placed between a universal sequence and an identifier sequence.
  • a spacer sequence can be placed at the 5' or 3' end of an oligonucleotide conjugate.
  • One example spacer sequence includes: AC AC AC A (SEQ ID NO: 99).
  • the oligonucleotide component of the oligonucleotide is selected from the group consisting of: a morpholino, a peptide nucleic acid (PNA), a thioester peptide nucleic acid (tPNA), a locked nucleic acid (LNA), a phosphorothioate, a
  • phosphonoacetate (PACE) phosphoramidite phosphoramidite
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • diagnostic moiety refers to any moiety that can be detected using techniques that are known in the art, e.g., an assay, an imaging technique, etc.
  • diagnostic moieties include reporter molecules, such as enzymes, radioisotopes, haptens, fluorescent labels, phosphorescent molecules, chemilluminescent molecules, chromophores, photoaffinity molecules, and colored particles or ligands, such as biotin.
  • Diagnostic moieties generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as diagnostic imaging protocols. Many appropriate imaging agents are known in the art, as are methods for their attachment to a targeting component (e.g., an antibody).
  • imaging moieties include paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.
  • Non-limiting examples of paramagnetic ions include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III).
  • Ions useful in other contexts, such as X-ray imaging include but are not limited to lanthanum (III), gold (III), lead (II), and bismuth (III).
  • Non-limiting examples of radioactive isotopes include 211 astatine, 14 carbon,
  • Radioactively labeled targeting components can be produced according to methods that are well known in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
  • Monoclonal antibodies can be labeled with 99m technetium by a ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column.
  • direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNC1 2 , a buffer solution such as sodium-potassium phthalate solution, and the antibody.
  • Intermediary functional groups that are often used to bind radioisotopes that exist as metallic ions to an antibody are diethylenetriaminepentaacetic acid (DTP A) or ethylene diaminetetracetic acid (EDTA).
  • DTP A diethylenetriaminepentaacetic acid
  • EDTA ethylene diaminetetracetic acid
  • Non-limiting examples of fluorescent labels include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY- TRX, Cascade Blue, Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate (FITC), HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.
  • FITC Fluorescein Isothiocyanate
  • HEX HEX
  • 6-JOE Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue
  • REG Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or
  • a diagnostic moiety is intended primarily for use in vitro, where the targeting component is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate.
  • suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase.
  • binding ligands include biotin, avidin and streptavidin compounds.
  • attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a
  • DTP A diethylenetriaminepentaacetic acid anhydride
  • ethylenetriaminetetraacetic acid N- chloro-p-toluenesulfonamide
  • tetrachloro-3a-6a-diphenylglycouril-3 attached to the targeting component
  • Targeting components can also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
  • imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p- hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.
  • linkers such as methyl-p- hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.
  • derivatization of targeting components is accomplished by selectively introducing sulfhydryl groups into the targeting component.
  • sulfhydryl groups can be selectively introduced into the Fc region using reaction conditions that do not alter the ability of the antibody to bind to its binding target.
  • Antibody conjugates produced according to this methodology exhibit improved longevity, specificity and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).
  • the diagnostic moiety is biotin (FIG. 17).
  • the diagnostic moiety is displaced by hybridization of an oligonucleotide that is fully complementary to the entire oligonucleotide component of the first oligonucleotide conjugate.
  • diagnostic moiety is cleaved from the first oligonucleotide conjugate using a nuclease.
  • the nuclease is a DNase.
  • the DNase is a double strand specific DNase.
  • An aspect of the disclosure includes therapeutic and diagnostic secondary
  • oligonucleotide conjugates that comprise an oligonucleotide component and a therapeutic or a diagnostic moiety.
  • a secondary oligonucleotide conjugate comprises a linker component that links the oligonucleotide component to the therapeutic or diagnostic moiety.
  • the second identifier sequence is hybridized to a secondary oligonucleotide conjugate.
  • the secondary oligonucleotide conjugate comprises a second oligonucleotide component that is attached to a diagnostic moiety.
  • the oligonucleotide component of the secondary oligonucleotide is selected from the group consisting of: a morpholino, a peptide nucleic acid (PNA), a thioester peptide nucleic acid (tPNA), a locked nucleic acid (LNA), a phosphorothioate, a
  • phosphonoacetate (PACE) phosphoramidite phosphoramidite
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • an oligonucleotide component of a secondary oligonucleotide conjugate is configured to hybridize with an oligonucleotide component of an oligonucleotide conjugate, as described above. Hybridization between the oligonucleotide components results in localization of a secondary oligonucleotide conjugate to a site within a subject where a targeting component of an oligonucleotide conjugate is bound. As such, a therapeutic activity of a therapeutic moiety or a diagnostic activity of a diagnostic moiety on a secondary oligonucleotide conjugate can be exerted at the site of localization, thereby facilitating targeted delivery of the therapeutic or diagnostic effects.
  • an aspect of the disclosure includes diagnostic secondary oligonucleotide conjugates that comprise a diagnostic moiety.
  • a secondary diagnostic oligonucleotide comprising a diagnostic moiety wherein the secondary diagnostic oligonucleotide further hybridizes with a primary identifier oligonucleotide include, but are not limited to, an antibody-fluorophore conjugate (FIG. 21B) or an antibody-biotin conjugate (FIG. 17).
  • an aspect of the disclosure includes therapeutic secondary oligonucleotide conjugates that comprise a therapeutic moiety.
  • Therapeutic moieties generally comprise molecules having a desired activity, e.g., cytotoxic or cytostatic activity,
  • Non-limiting examples of therapeutic moieties include thrombogenic agents, toxins, anti-tumor agents, therapeutic enzymes, lymphocyte binding domains, radionuclides, cytokines, growth factors, oligo- or
  • polypeptides polypeptides, antibodies, antigen-binding fragments of antibodies, antibody- drug conjugates (ADCs), and cells.
  • ADCs antibody- drug conjugates
  • Examples of a secondary therapeutic oligonucleotide comprising a therapeutic moiety, wherein the secondary therapeutic oligonucleotide further hybridizes with a primary identifier oligonucleotide include, but are not limited to, a bispecific antibody, an antibody-drug conjugate, or an antibody-protein conjugate (FIG. 21B).
  • the therapeutic moiety is selected from the group consisting of a protein, a toxin, an antibody, an antibody-drug conjugate, an antibody fragment, an ADC fragment, an enzyme, a cell, and a small molecule.
  • bispecific antibody conjugates comprising (a) a primary identifier oligonucleotide conjugate comprising (i) a first antibody that binds to a target on a cell, and (ii) a first oligonucleotide component that is attached to the first antibody at the end 3 'end of the first oligonucleotide component, and (b) a secondary therapeutic oligonucleotide conjugate comprising (i) a second oligonucleotide component that hybridizes to the first oligonucleotide component of the primary identifier oligonucleotide conjugate, and (ii) a second antibody that is attached to the 3' end of the second oligonucleotide conjugate.
  • the cell is a cancer cell.
  • antibody-drug conjugates comprising (a) a primary identifier oligonucleotide conjugate comprising (i) an antibody that binds to a target on a cell, and (ii) a first oligonucleotide component that is attached to the antibody at the end 3 'end of the first oligonucleotide component, and (b) a secondary therapeutic oligonucleotide conjugate comprising (i) a second oligonucleotide component that hybridizes to the first oligonucleotide component of the primary identifier oligonucleotide conjugate, and (ii) a small molecule that is attached to the 3' end of the second oligonucleotide conjugate.
  • the cell is a cancer cell.
  • the small molecule comprises an auristatin, a maytansine, a maytansinoid, a taxane, a calicheamicin, cemadotin, a duocarmycin, a pyrrolobenzodiazepine (PBD), a tubulysin, an anthracycline, a methotrexate, a vindesine, a trichothecene, or a derivative thereof.
  • the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).
  • the maytansinoid comprises DM1 (mertansine) or DM4.
  • the DM1 monomethyl auristatin E
  • DM4 monomethyl auristatin F
  • pyrrolobenzodiazepine is a pyrrolobenzodiazepine dimer.
  • the taxane is taxol, docetaxel, paclitaxel, larotaxel, tesetaxel, or orataxel.
  • the anthracycline is daunorubicin, doxorubicin, epirubicin, idarubicin, nemorubicin, pixantrone, sabarubicin, valrubicin, richothecane, a CC1065or derivatives or combinations thereof.
  • Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization.
  • Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division and have anticancer activity.
  • the calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations.
  • antibody-protein conjugates comprising (a) a primary identifier oligonucleotide conjugate comprising (i) an antibody that binds to a target on a cell, and (ii) a first oligonucleotide component that is attached to the antibody at the end 3 'end of the first oligonucleotide component, and (b) a secondary therapeutic
  • oligonucleotide conjugate comprising (i) a second oligonucleotide component that hybridizes to the first oligonucleotide component of the primary identifier oligonucleotide conjugate, and (ii) a protein that is attached to the 3' end of the second oligonucleotide conjugate.
  • the cell is a cancer cell.
  • the protein is an enzyme or a toxin.
  • the protein is derived from a bacteria, fungus, plant, or animal.
  • Non-limiting examples of enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • diphtheria A chain nonbinding active fragments of diphtheria toxin
  • exotoxin A chain from Pseudomonas aeruginosa
  • ricin A chain abrin A chain
  • the toxin is a bacterial toxin, fungal toxin, plant toxin, animal toxin, or a fragment thereof. In some embodiments, the toxin is an isolated component of a bacterial toxin, fungal toxin, plant toxin, animal toxin, or a fragment thereof. In some embodiments, the bacterial toxin is a diphtheria toxin or a Pseudomonas exotoxin. In some embodiments, the fungal toxin is a aflatoxin, an ochratoxin, a citrinin, an ergot alkaloid, a patulin, a fusarium, or a derivative thereof.
  • the fusarium is is a fumonisin, a trichothecene, or a zearalenone.
  • the plant toxin is a ribosome-inactivating protein (RIP).
  • the RIP is a type 1 RIP or a type 2 RIP.
  • the type 1 RIP is PAP (pokeweed antiviral protein), gelonin, saporin, curcin, curcin-L, or derivatives thereof.
  • the saporin is saporin-S6.
  • the type 2 RIP is ricin, abrin, modeccin, pulchellin, mistletoe lectin I, or volkensin
  • the animal toxin is a snake venom or an arthropod venom.
  • the snake venom is a venom from a snake in the family Crotalid, Elapid, or Viperid.
  • the arthropod is a scorpion, a bee, a wasp, a spider, an ant, a centipede, or a caterpillar
  • the toxin is an isolated component of an animal toxin.
  • the isolated component of an animal toxin is chlorotoxin, bengalin, melittin, phospholipase A 2 (PLA 2 ), mastoparan, Polybia MP-I, Polybia-MP-II, Polybia-MP-III, quinone, 7,8-seco-para- ferruginone (SPF), NVP(l), phospholipase-D, oxyopinin, psalmotoxin 1, pancrati statin (PST), solenopsin A, gomesin, or cecropin.
  • the toxin is a small molecule toxins such as geldanamycin A.
  • a toxin can impart a cytotoxic and/or cytostatic effect by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.
  • Additional non-limiting examples of toxins include ricin A-chain (Burbage, 1997), diphtheria toxin A (Massuda et al., 1997; Lidor, 1997), pertussis toxin A subunit, E.
  • the enzyme is a therapeutic enzyme.
  • the enzyme is an aminopeptidase, an alkaline phosphatase (AP), a glycosidase, a beta galactosidase, horseradish peroxidase (HRP), a penicillin amidase, a ⁇ -lactamase, a cytosine deaminase, a nitroreductase,
  • AP alkaline phosphatase
  • HRP horseradish peroxidase
  • penicillin amidase a ⁇ -lactamase
  • cytosine deaminase a nitroreductase
  • an enzyme works in conjunction with a prodrug by converting the pro-drug to an active composition in the vicinity of the enzyme.
  • antibody-fluorophore conjugates comprising (a) a primary identifier oligonucleotide conjugate comprising (i) an antibody that binds to a target on a cell, and (ii) a first oligonucleotide component that is attached to the antibody at the end 3 'end of the first oligonucleotide component, and (b) a secondary therapeutic oligonucleotide conjugate comprising (i) a second oligonucleotide component that hybridizes to the first oligonucleotide component of the primary identifier oligonucleotide conjugate, and (ii) a fluorophore moiety that is attached to the 3' end of the second oligonucleotide conjugate.
  • the cell is a cancer cell.
  • the fluorophore moiety is a xanthene, a cyanine, a squaraine, a naphthalene, a coumarin, an oxaidazole, an anthracene, a pyrene, and oxazine, an acridine, an arylmethine, a tetrapyrrole, or a derivative thereof.
  • the xanthene derivative is fluorescein, rhodamine, Oregon green, eosin, or Texas red.
  • the fluorophore moiety is a fluorescent label as described herein.
  • a therapeutic moiety is a thrombogenic agent.
  • thrombogenic agents include tissue factor (or a tissue factor peptide), cancer thrombogenic factor (CTF), doxorubicin, factor VIII, thalidomide, and homocysteine.
  • a thrombogenic therapeutic moiety can be a human tissue factor peptide having a length that ranges from about 5 to about 100 amino acids, such as about 5 to about 50 amino acids in length, or about 5 to about 25 amino acids in length.
  • a therapeutic moiety is an anti-angiogenic agent.
  • anti-angiogenic agents include drugs that block the pro-angiogenic function of vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • Other examples include SU5416, AG3340, endostatin, angiostatin, squalamine, thalidomide, CAI, Neovastat, and 2-methoxyestradiol.
  • a variety of radionuclides can be used as therapeutic moieties.
  • Non-limiting examples include 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.
  • Attachment of a therapeutic moiety to a therapeutic secondary oligonucleotide conjugate can be accomplished using a variety of bifunctional coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tol
  • a ricin immunotoxin can be prepared as described in Vitetta et al. (1987).
  • Carbon- 14-labeled lisothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of a radionucleotide.
  • an oligonucleotide conjugate comprises one or more artificial components.
  • an oligonucleotide conjugate comprises a biopolymer.
  • an oligonucleotide component comprises an antibody (immunoglobulin).
  • an oligonucleotide conjugate comprises an antibody that comprises a variable region.
  • an antibody comprises an antigen-binding site.
  • an oligonucleotide conjugate comprises an intact immunoglobulin.
  • an oligonucleotide conjugate comprises an immunoglobulin fragment.
  • an oligonucleotide conjugate comprises an Fv immunoglobulin fragment.
  • an oligonucleotide conjugate comprises an scFv immunoglobulin fragment.
  • an oligonucleotide conjugate comprises a native polypeptide.
  • an oligonucleotide conjugate comprises a monoclonal antibody.
  • an oligonucleotide conjugate comprises a chimeric antibody.
  • an oligonucleotide conjugate comprises a humanized antibody.
  • an oligonucleotide conjugate comprises a human antibody.
  • an oligonucleotide conjugate comprises an isolated antibody.
  • an antibody specifically binds to a binding target with a binding affinity.
  • an oligonucleotide conjugate comprises two or more polypeptide components that have been linked by fusion.
  • a targeting component binds to an epitope on a binding target.
  • an antibody binds to an antigen.
  • two or more portions or components of an oligonucleotide conjugate are operably linked to one another.
  • an oligonucleotide conjugate comprises an antibody that is linked to an oligonucleotide component by a SPAAC linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, an identifier sequence, and a second universal sequence having the sequence of SEQ ID NO: 398.
  • an oligonucleotide conjugate comprises an antibody that is linked to an oligonucleotide component by a SPAAC linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, a first identifier sequence, a second universal sequence having the sequence of SEQ ID NO: 398, and a second identifier sequence.
  • an oligonucleotide conjugate comprises an anti-HER2 antibody that is linked to an oligonucleotide component by a SPAAC linker, and the
  • oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, an identifier sequence, and a second universal sequence having the sequence of SEQ ID NO: 398.
  • an oligonucleotide conjugate comprises an anti- HER2 antibody that is linked to an oligonucleotide component by a SPAAC linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, a first identifier sequence, a second universal sequence having the sequence of SEQ ID NO: 398, and a second identifier sequence.
  • an oligonucleotide conjugate comprises an anti-FOLRl antibody that is linked to an oligonucleotide component by a SPAAC linker, and the
  • oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, an identifier sequence, and a second universal sequence having the sequence of SEQ ID NO: 398.
  • an oligonucleotide conjugate comprises an anti- FOLRl antibody that is linked to an oligonucleotide component by a SPAAC linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, a first identifier sequence, a second universal sequence having the sequence of SEQ ID NO: 398, and a second identifier sequence.
  • an oligonucleotide conjugate comprises small molecule that is linked to an oligonucleotide component by a SPAAC linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, an identifier sequence, and a second universal sequence having the sequence of SEQ ID NO: 398.
  • an oligonucleotide conjugate comprises small molecule that is linked to an oligonucleotide component by a SPAAC linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, a first identifier sequence, a second universal sequence having the sequence of SEQ ID NO: 398, and a second identifier sequence.
  • an oligonucleotide conjugate comprises a folate molecule that is linked to an oligonucleotide component by a SPAAC linker, and the
  • oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, an identifier sequence, and a second universal sequence having the sequence of SEQ ID NO: 398.
  • an oligonucleotide conjugate comprises a folate molecule that is linked to an oligonucleotide component by a SPAAC linker, and the
  • oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, a first identifier sequence, a second universal sequence having the sequence of SEQ ID NO: 398, and a second identifier sequence.
  • an oligonucleotide conjugate comprises an antibody that is linked to an oligonucleotide component by a tetrazine ligation linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, an identifier sequence, and a second universal sequence having the sequence of SEQ ID NO: 398.
  • an oligonucleotide conjugate comprises an antibody that is linked to an oligonucleotide component by a tetrazine ligation linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, a first identifier sequence, a second universal sequence having the sequence of SEQ ID NO: 398, and a second identifier sequence.
  • an oligonucleotide conjugate comprises an anti-HER2 antibody that is linked to an oligonucleotide component by a tetrazine ligation linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, an identifier sequence, and a second universal sequence having the sequence of SEQ ID NO: 398.
  • an oligonucleotide conjugate comprises an anti- HER2 antibody that is linked to an oligonucleotide component by a tetrazine ligation linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, a first identifier sequence, a second universal sequence having the sequence of SEQ ID NO: 398, and a second identifier sequence.
  • an oligonucleotide conjugate comprises an anti-FOLRl antibody that is linked to an oligonucleotide component by a tetrazine ligation linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, an identifier sequence, and a second universal sequence having the sequence of SEQ ID NO: 398.
  • an oligonucleotide conjugate comprises an anti- FOLRl antibody that is linked to an oligonucleotide component by a tetrazine ligation linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, a first identifier sequence, a second universal sequence having the sequence of SEQ ID NO: 398, and a second identifier sequence.
  • an oligonucleotide conjugate comprises small molecule that is linked to an oligonucleotide component by a tetrazine ligation linker, and the
  • oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, an identifier sequence, and a second universal sequence having the sequence of SEQ ID NO: 398.
  • an oligonucleotide conjugate comprises small molecule that is linked to an oligonucleotide component by a tetrazine ligation linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, a first identifier sequence, a second universal sequence having the sequence of SEQ ID NO: 398, and a second identifier sequence.
  • an oligonucleotide conjugate comprises a folate molecule that is linked to an oligonucleotide component by a tetrazine ligation linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, an identifier sequence, and a second universal sequence having the sequence of SEQ ID NO: 398.
  • an oligonucleotide conjugate comprises a folate molecule that is linked to an oligonucleotide component by a tetrazine ligation linker, and the oligonucleotide component comprises a first universal sequence having the sequence of SEQ ID NO: 397, a first identifier sequence, a second universal sequence having the sequence of SEQ ID NO: 398, and a second identifier sequence.
  • An aspect of the disclosure includes methods of synthesizing the oligonucleotide conjugates described herein.
  • the subject synthesis methods involve immobilizing one or more components of an oligonucleotide conjugate on a solid support.
  • affinity resins include cationic or anionic affinity resins, or protein-based affinity resins, e.g., protein A, protein G, protein M, and protein L affinity resins.
  • an affinity resin is a cationic affinity resin that comprises diethylaminoethanol (DEAE) beads.
  • a component of an oligonucleotide conjugate is covalently immobilized on a solid support (i.e., covalent bonds are formed between the component of the
  • a component of an oligonucleotide conjugate is non-covalently immobilized on a solid support (i.e., non- covalent bonds are formed between the component of the oligonucleotide conjugate and the molecules of the solid support).
  • a first component of an oligonucleotide conjugate is covalently immobilized on a first solid support, and a second component of the
  • oligonucleotide conjugate is non-covalently immobilized on a second solid support.
  • both a first and a second component of an oligonucleotide conjugate are non-covalently immobilized on a first and a second solid support.
  • a component of an oligonucleotide conjugate can be contacted with one or more reagents that chemically modify the component.
  • an oligonucleotide component is immobilized on a solid support and is contacted with a reagent to create a functionalized oligonucleotide component.
  • an immobilized oligonucleotide component is contacted with a tetrazine ligation reagent, a strain-promoted-azide-alkylene (SPAAC) reagent, a maleimide reagent, an N-hydroxysuccinimide (NHS) reagent, a tyrosine ligation reagent, a chemoenzymatic attachment reagent, a hydrazine ligation reagent, or a hydrazine ligation reagent.
  • SPAAC strain-promoted-azide-alkylene
  • NHS N-hydroxysuccinimide
  • an immobilized targeting component is contacted with a tetrazine ligation reagent, a strain-promoted-azide-alkylene (SPAAC) reagent, a maleimide reagent, an N- hydroxysuccinimide (NHS) reagent, a tyrosine ligation reagent, a chemoenzymatic attachment reagent, a hydrazine ligation reagent, or a hydrazine ligation reagent.
  • SPAAC strain-promoted-azide-alkylene
  • NHS N- hydroxysuccinimide
  • An aspect of a subject synthesis method involves contacting a targeting component with an oligonucleotide component under reaction conditions that are suitable for the formation of an oligonucleotide conjugate.
  • a functionalized targeting component is contacted with an oligonucleotide component to generate an oligonucleotide conjugate.
  • a functionalized oligonucleotide component is contacted with a targeting component to generate an oligonucleotide conjugate.
  • a functionalized targeting component is contacted with a functionalized oligonucleotide component to generate an oligonucleotide conjugate.
  • a first component of an oligonucleotide conjugate is separated from a solid support before it is reacted with a second component of the oligonucleotide conjugate.
  • a targeting component is immobilized on a solid support and is contacted with a reagent to create a functionalized targeting component, and is then separated from the solid support and reacted with an oligonucleotide component to create and
  • a first component of an oligonucleotide conjugate is separated from a solid support, and is reacted with a second component of an oligonucleotide conjugate while the second component remains immobilized on a solid support.
  • a targeting component is separated from a solid support and is contacted with an oligonucleotide component while the oligonucleotide component remains immobilized on the solid support.
  • an oligonucleotide component is separated from a solid support and is contacted with a targeting component while the targeting component remains immobilized on the solid support.
  • both a first and a second component of an oligonucleotide conjugate are separated from their solid supports before they are contacted with one another to form an oligonucleotide conjugate.
  • a targeting component is separated from a first solid support and an oligonucleotide component is separated from a second solid support, and the targeting component and the oligonucleotide component are then contacted with one another (e.g., in solution) to form an oligonucleotide conjugate.
  • a subject method of synthesis involves attaching (e.g., conjugating) a moiety to a targeting component of an oligonucleotide conjugate.
  • a method involves attaching a detectable moiety to a targeting component.
  • a method involves attaching a therapeutic moiety to a targeting component.
  • a moiety is attached to a targeting component while the targeting component is immobilized on a solid support.
  • a moiety is attached to a targeting component when the targeting component is separated from a solid support (e.g., in solution).
  • a subject method of synthesis involves isolating an oligonucleotide conjugate to create a substantially pure composition that comprises the oligonucleotide conjugate. Isolating a subject oligonucleotide conjugate results in a substantially pure preparation that comprises greater than about 80%, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or more of the isolated oligonucleotide conjugate.
  • an oligonucleotide conjugate can be combined with one or more additional components, e.g., one or more buffers or stabilizers, to create a composition that is suitable for diagnostic (e.g., laboratory) use.
  • isolating an oligonucleotide conjugate involves contacting the oligonucleotide conjugate with a magnetic bead.
  • the oligonucleotide conjugate interacts with or is bound to a magnetic bead, and one or more unwanted contaminants are separated or purified away.
  • the magnetic bead can be isolated using a magnet, and then the oligonucleotide conjugate can be removed or separated from the magnetic bead.
  • oligonucleotide conjugates in accordance with an aspect of the disclosure can be formulated into pharmaceutical compositions.
  • a therapeutic composition is formulated into a pharmaceutical composition.
  • a pharmaceutical composition can be administered by any of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the target disease or condition and the desired results. To administer a compound of the disclosure by certain routes of administration, it can be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
  • a compound can be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent.
  • Pharmaceutically-acceptable diluents include, but are not limited to, saline and aqueous buffer solutions.
  • Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.
  • compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and/or dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and/or dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It can also be
  • compositions of the present disclosure can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • a selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a composition must be sterile and fluid to the extent that the composition is deliverable by syringe.
  • a pharmaceutical carrier preferably comprises an isotonic buffered saline solution.
  • An aspect of the disclosure includes the use of an oligonucleotide conjugate in one or more diagnostic methods to determine the presence of one or more binding targets in a sample.
  • the subject methods can be used, e.g., to determine the presence of one or more cancer or infectious disease biomarkers in a sample.
  • An aspect of the disclosure includes the use of an oligonucleotide conjugate in one or more diagnostic methods to quantify the amount of one or more binding targets in a sample.
  • An aspect of the disclosure includes the use of an
  • oligonucleotide conjugate in one or more diagnostic methods to generate an antigen profile of a subject, e.g. a soluble antigen profile or a cellular antigen profile.
  • An aspect of the disclosure includes the use of one or more oligonucleotide conjugates in therapeutic methods for the treatment of a disease or disorder, e.g., a cancer, in a mammalian subject.
  • An aspect of the disclosure includes the use of one or more oligonucleotide conjugates in therapeutic methods for the targeted delivery of a therapeutic compound. In some embodiments, targeted delivery comprises tissue-specific delivery.
  • An aspect of the disclosure also includes the use of an oligonucleotide conjugate in quality assurance assays, e.g. for drug or food manufacturing.
  • use of an oligonucleotide conjugate in a quality assurance assay comprises use of the oligonucleotide conjugate for detecting the presence of a contaminant.
  • the contaminant is a microbe.
  • the microbe is a bacterium, fungi, virus, protozoan, or a combination thereof.
  • An aspect of the disclosure includes methods for determining the presence of one or more binding targets in a sample using the subject oligonucleotide conjugates.
  • the subject oligonucleotide conjugates comprise a targeting component, a linker component, a cleavage component, and an oligonucleotide component.
  • an oligonucleotide component comprises an identifier sequence that identifies a binding target of a targeting component.
  • a sample that is suspected of containing a binding target is contacted with an oligonucleotide conjugate that comprises an identifier sequence that identifies the binding target of the targeting component.
  • the targeting component of the oligonucleotide conjugate binds to the binding target (if present), and any unbound oligonucleotide conjugates are removed.
  • the cleavage component is cleaved with a cleaving agent to separate the oligonucleotide component of the oligonucleotide conjugate from the targeting component of the oligonucleotide conjugate.
  • the separated oligonucleotide component is then analyzed and the identifier sequence of the oligonucleotide component is used to determine the presence of the binding target in the sample.
  • the cleavage component is not cleaved.
  • the sample is determined to contain the binding target. If an identifier sequence for a given binding target is found to be absent, then the sample is determined not to contain the binding target.
  • the identifier sequence is a first identifier sequence and the oligonucleotide component further comprises a second identifier sequence.
  • the subject diagnostic methods are particularly useful in the identification of cell surface proteins in a sample.
  • oligonucleotide conjugate comprises an antibody that binds to a cell surface protein.
  • the cell surface protein is a biomarker for cancer.
  • a sample comprising a tumor cell is contacted with the oligonucleotide conjugate, and the antibody binds to the cell surface protein on the tumor cell.
  • Unbound oligonucleotide conjugates are removed from the sample.
  • the cleavage component of the bound oligonucleotide conjugate is cleaved to separate the oligonucleotide component from the antibody.
  • the oligonucleotide component is then analyzed to determine the presence of an identifier sequence that identifies the binding target of the antibody (i.e., the cancer biomarker). If the identifier sequence is present, then it is determined that the cell surface protein (i.e., the cancer biomarker) is present on the tumor cell.
  • a sample is then contacted with a second oligonucleotide conjugate that comprises an oligonucleotide component having a sequence that is complementary to at least a portion of the oligonucleotide component of the first oligonucleotide conjugate, as well as a detectable moiety.
  • the oligonucleotide component of the second oligonucleotide conjugate hybridizes with the oligonucleotide component of the first oligonucleotide conjugate, and any unbound second oligonucleotide conjugate is removed. The presence of the detectable moiety on the second oligonucleotide conjugate is then detected to determine the presence of the binding target in the sample.
  • a subject method involves contacting a sample with a plurality of different oligonucleotide conjugates to determine the presence of a plurality of binding targets in the sample.
  • a plurality of binding targets e.g., a plurality of cell surface proteins on a cancer cell.
  • This information can be utilized, e.g., to diagnose specific types of cancer that involve the expression of specific combinations or patterns of biomarkers.
  • a sample is simultaneously contacted with a plurality of different oligonucleotide conjugates, each containing a targeting component that binds to a different binding target. Unbound oligonucleotide conjugates are removed from the sample. In some embodiments, the cleavage components of the bound oligonucleotide conjugates are cleaved to separate the oligonucleotide components from the targeting components. In some embodiments, the cleavage components of the bound oligonucleotide conjugates are not cleaved. The oligonucleotide components are then analyzed to determine the presence of any identifier sequences that identify the various binding targets of the targeting components.
  • a sample is simultaneously contacted with a number of different oligonucleotide conjugates that ranges from about 2 up to about 300, such as about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, or about 300 different oligonucleotide conjugates.
  • An aspect of a subject method includes PCR-based sequence analysis assays, including but not limited to, qPCR assays and sequencing assays that incorporate next-generation sequencing (NGS) technologies
  • FIG. 14 Another aspect of a subject method involves the use of oligonucleotide primers to conduct PCR-based analyses. Examples of oligonucleotide primer sequences are provided below.
  • a primer includes an additional 5' sequence.
  • the 5' sequence comprises an NGS adapter, a sample identifier sequence, or a combination thereof.
  • Table 5 Example primer sequences.
  • An aspect of the disclosure includes methods for quantifying the presence of one or more binding targets in a sample using a plurality of oligonucleotide conjugates, each oligonucleotide conjugate comprising a targeting component, a linker component, a cleavage component, and an oligonucleotide component.
  • an oligonucleotide component comprises an identifier sequence that identifies a binding target of a targeting component.
  • a sample that is suspected of containing a binding target is contacted with an oligonucleotide conjugate that comprises an identifier sequence that identifies the binding target of the targeting component.
  • an oligonucleotide component comprises a first identifier sequence and a second identifier sequence that identifies a binding target of a targeting component.
  • a sample that is suspected of containing a binding target is contacted with an oligonucleotide conjugate that comprises a first identifier sequence and a second identifier sequence that identifies the binding target of the targeting component.
  • the targeting component of the oligonucleotide conjugate binds to the binding target or targets (if present), and any unbound oligonucleotide conjugates are removed.
  • the cleavage component is cleaved with a cleaving agent to separate the oligonucleotide component of the oligonucleotide conjugate from the targeting component of the oligonucleotide conjugate. In some embodiments, the cleavage component is not cleaved.
  • the methods for quantifying the presence of one or more binding targets in a sample using a plurality of oligonucleotide conjugates comprise the use of quantitative PCR (qPCR) (FIG. 14).
  • Quantitative PCR (qPCR) or real-time PCR, monitors the amplification of a nucleic acid molecule during PCR rather than at its end, as in conventional PCR.
  • the methods described herein comprise qPCR to quantify one binding target.
  • the method further comprises administering to the oligonucleotide component to be quantified by qPCR an intercalating fluorescent dye prior to beginning qPCR.
  • the intercalating fluorescent dye is SYBR green.
  • the oligonucleotide primers used for qPCR comprise a forward primer and a reverse primer complementary to universal site 1 and universal site 2.
  • the methods described herein comprise qPCR to quantify more than one binding targets.
  • the method further comprises administering to the oligonucleotide component to be quantified by qPCR a probe complementary to the first identifier sequence.
  • the probe comprises a reporter dye and a quencher dye.
  • the reporter dye is a fluorescent label as described herein. Examples of quencher dyes include, but are not limited to, QSY 35, BHQ-0, Eclipse, BHQ-1, QSY 7, QSY 9, ElleQuencher, Iowa Black, QSY21, BHQ-3, and BHQ-10.
  • the method comprises the use of a plurality of oligonucleotide primer pairs, each oligonucleotide primer pair comprising a forward primer and a reverse primer complementary to universal site 1 and the second identifier sequence.
  • the methods described herein are used to generate an antigen profile.
  • methods for identification, quantification, or a combination thereof are used to generate an antigen profile.
  • an antigen profile is generated for a biological sample from a subject.
  • the subject has or is suspected of having a condition.
  • the condition is cancer.
  • an antigen profile is generated for soluble antigens. In some embodiments, an antigen profile is generated for cellular antigens. Methods of generating antigen profiles for soluble and cellular antigens are illustrated in FIG. 13.
  • methods for determining the soluble antigen profile from a subject comprise: 1) administering a sample from the subject, wherein the sample comprises a plurality of soluble antigens, to a solid support comprising a plurality of immobilized capture antibodies, wherein each of the plurality of immobilized capture antibodies binds to a subset of the plurality of soluble antigens in the sample; 2) removing soluble antigens that did not bind to the plurality of capture antibodies; 3) incubating the bound soluble antigens with an antibody oligonucleotide conjugate library comprising a plurality of antibody oligonucleotides, wherein each of the plurality of antibody oligonucleotides conjugates binds to a soluble antigen; 4) removing antibody oligonucleotide conjugates that did not bind to a soluble antigen immobilized to a capture antibody; 5) cleaving the oligonucleotide component of each of the plurality of immobilized capture antibodies,
  • the sample is blood, saliva, a tissue sample, urine, or sputum.
  • the tissue sample is an abnormal tissue sample (e.g. a biopsy).
  • the soluble antigen profile from a subject is compared to a second soluble antigen profile.
  • the second soluble antigen profile is from the same subject.
  • the second soluble antigen profile is from a different subject.
  • the soluble antigen profile from a subject is compared to a soluble antigen profile with a known concentration.
  • the soluble antigen profile from a subject is compared to a plurality of control soluble antigen profiles.
  • each of the plurality of control soluble antigen profiles has a known concentration about 4 to about 7 orders of magnitude different from the soluble antigen profile from the subject.
  • at least one of the control antigens in the control soluble antigen profile comprises control antigens is a purified soluble antigen or a recombinant soluble antigen.
  • the soluble antigen profile from the subject is compared to a standard curve of a control antigen.
  • the solid support is an ELISA well or a bead. In some embodiments, the solid support is an array. In some embodiments, any suitable solid support is used. In some embodiments, any suitable method is used to immobilize the capture antibody to the solid support.
  • determining the soluble antigen profile comprises targeted amplification.
  • targeted amplification comprises qPCR.
  • determining the antigen profile comprises universal amplification.
  • targeted amplification comprises NGS.
  • methods for determining the cellular antigen profile from a subject comprise: 1) fixation of a plurality of cellular antigens of a sample from the subject; 2) incubating the fixed plurality of cellular antigens with an antibody oligonucleotide conjugate library comprising a plurality of antibody oligonucleotide conjugates, wherein each of the plurality of antibody oligonucleotide conjugates binds to a cellular antigen 3) removing unbound antigen oligonucleotide conjugates that did not bind to a cellular antigen; 4) cleaving the oligonucleotide component of each of the plurality of oligonucleotide conjugates bound to the antigens; 5) isolating the cleaved oligo components; and 6) determining the cellular antigen profile (FIG. 13).
  • the sample is blood, saliva, a tissue sample, urine, or sputum.
  • the tissue sample is an abnormal tissue sample (e.g. a biopsy).
  • the cellular antigen profile from a subject is compared to a second cellular antigen profile.
  • the second cellular antigen profile is from the same subject.
  • the second cellular antigen profile is from a different subject.
  • the cellular antigen profile from a subject is compared to a plurality of control cellular antigen profiles.
  • each of the plurality of cellular antigen profiles has a known concentration about 4 to about 7 orders of magnitude different from the cellular antigen profile from the subject.
  • control antigens in the control cellular antigen profile comprises control antigens is a purified cellular antigen or a recombinant cellular antigen.
  • the cellular antigen profile from the subject is compared to a standard curve of a control antigen.
  • the cellular antigen profile comprises an antigen profile of cell surface antigens, intracellular antigens, intraorganellar antigens, or a combination thereof.
  • fixation comprises fixation, permeabilization, or a combination thereof.
  • fixation comprises use of a fixation agent.
  • the fixation agent is formaldehyde, paraformaldehyde, formalin, glutaraldehyde, a mercuric chloride-based fixative, dimethyl suberimidate (DMS), or a combination thereof.
  • the fixation agent is a precipitating fixative.
  • the precipitating fixative is ethanol, methanol, or acetone.
  • permeabilization comprises use of a permeabilization agent.
  • the permeabilization agent is an organic solvent or a detergent.
  • the organic solvent is methanol or acetone.
  • the detergent is saponin, Triton X-100, or Tween-20.
  • a fixation agent is also a permeabilization agent.
  • determining the cellular antigen profile comprises targeted amplification.
  • targeted amplification comprises qPCR.
  • determining the antigen profile comprises universal amplification.
  • targeted amplification comprises NGS.
  • determining an antigen profile is used to identify a therapeutic target.
  • the therapeutic target is a unique surface protein of an abnormal cell of a subject compared to a normal cell of the subject.
  • the abnormal cell is a tumor cell.
  • the subject is human.
  • the method further comprises use of the surface protein of the abnormal cell as a therapeutic target for a therapeutic compound. An illustration of the identification of a unique surface protein and use of a therapeutic compound to target the unique surface protein is illustrated in FIG. 22.
  • a therapeutic compound comprising: (a) a primary identifier oligonucleotide conjugate comprising (i) a targeting component that binds to a target on a cancer cell, and (ii) first oligonucleotide component that is attached to the targeting component at the end 3 'end of the first
  • the therapeutic compound is a bispecific antibody, an antibody-drug conjugate, an antibody-protein conjugate, and an antibody-fluorophore conjugate.
  • the therapeutic compound is delivered to a specific tissue in the subject. In some embodiments, the specific tissue is a tumor tissue.
  • the subject in need thereof suffers from or is suspected of suffering from cancer or an infectious disease.
  • the first oligonucleotide component of the primary identifier oligonucleotide conjugate is a PNA and the second oligonucleotide component of the secondary therapeutic oligonucleotide conjugate is a PNA.
  • the first oligonucleotide component of the primary identifier oligonucleotide conjugate is a PNA and the second oligonucleotide component of the secondary therapeutic oligonucleotide conjugate is a DNA.
  • oligonucleotide conjugate is a DNA and the second oligonucleotide component of the secondary therapeutic oligonucleotide conjugate is a PNA.
  • the primary identifier oligonucleotide is administered to the subject before the secondary therapeutic oligonucleotide. In some embodiments, the primary identifier oligonucleotide is administered to the subject at the same time as the secondary therapeutic oligonucleotide. In some embodiments, the primary identifier oligonucleotide is hybridized to the secondary therapeutic oligonucleotide in vitro prior to administering to the subject.
  • an aspect of the disclosure includes therapeutic methods for treating a disease or disorder (e.g., a cancer) in a mammalian subject using one or more oligonucleotide conjugates in accordance with an aspect of the disclosure.
  • a disease or disorder e.g., a cancer
  • an oligonucleotide conjugate comprises a targeting component, a linker component, and an oligonucleotide component.
  • an oligonucleotide component comprises an identifier sequence that identifies a binding target of a targeting component.
  • a subject in need of treatment is administered an oligonucleotide conjugate that comprises an oligonucleotide conjugate that comprises an
  • the oligonucleotide component with an identifier sequence that identifies a binding target of the targeting component.
  • the targeting component of the oligonucleotide conjugate binds to the binding target within the subject.
  • a therapeutic secondary oligonucleotide conjugate is administered to the subject.
  • the therapeutic secondary oligonucleotide conjugate comprises an oligonucleotide component that hybridizes with the oligonucleotide component of the first oligonucleotide conjugate, and also comprises a therapeutic moiety.
  • Hybridization between the oligonucleotide components results in localization of the therapeutic secondary oligonucleotide conjugate to the site within the subject where the targeting component of the oligonucleotide conjugate is bound. As such, a therapeutic activity of the therapeutic moiety is exerted at the site of localization, thereby facilitating targeted delivery of the therapeutic effects at the location of the binding target.
  • targeted delivery comprises tissue-specific delivery. In some embodiments, tissue-specific delivery comprises delivery to a tumor tissue.
  • a therapeutic method further comprises administering a pro-drug to the subject.
  • the pro-drug is converted to an active composition in the vicinity of the therapeutic secondary oligonucleotide conjugate, and exerts a therapeutic activity at the site of localization.
  • a therapeutic secondary oligonucleotide conjugate includes an enzyme as a therapeutic moiety, and the methods involve administering a pro-drug to the subject.
  • the pro-drug contacts the enzyme, which is bound to the therapeutic secondary oligonucleotide conjugate, the pro-drug is converted by the enzyme into an active composition and exerts a therapeutic activity at the site of localization.
  • An aspect of the disclosure includes systems and devices thereof configured to carry out the subject methods, e.g., to contact a cell with a plurality of oligonucleotide conjugates, cleave a cleavage component to release a plurality of identifier sequences from the cell, and detect the plurality of identifier sequences to determine the presence of a plurality of surface proteins on the cell.
  • a system includes a sample acquisition component that is configured to accept one or more samples from a user.
  • a sample acquisition system is configured to receive a liquid or fluid sample.
  • a sample acquisition system is configured to receive a solid sample (e.g., a tissue biopsy sample, a histological sample comprising a tissue specimen mounted on a substrate (e.g., a glass slide)). Any suitable sample format can be utilized in the subject methods of analysis.
  • a sample acquisition system is configured to receive a plurality of samples and to process the samples in series or in parallel.
  • a sample acquisition system is configured to receive a plurality of samples in a multi-well plate format, and is configured to analyze a sample from each well of the multi-well plate either in series or in parallel.
  • a system includes a plurality of tubing and pumping components that are configured to transport and/or manipulate one or more fluids (e.g., one or more fluid samples, one or more liquid reagents, etc.).
  • a system includes one or more pipetting components that are configured to aspirate a specified volume of a fluid and to deliver it to a particular location within the system.
  • a system includes one or more flow meters that are configured to control a volume of a sample or a reagent that is delivered to a particular location within the system.
  • a system includes one or more fluid reservoirs that contain a reagent that is used in the subject methods.
  • An aspect of the subject systems include one or more assay components that are configured to perform an analysis on a sample.
  • an assay system includes one or more polymerase chain reaction (PCR) analysis components that are configured to perform one or more PCR assays on a sample.
  • PCR polymerase chain reaction
  • an assay system includes one or more colorimetric analysis components that are configured to perform one or more colorimetric analyses on a sample. Assay components in accordance with an aspect of the disclosure are configured to perform both qualitative and quantitative analyses.
  • an assay component is configured to determine whether an analyte is present in a sample at a concentration that is above or below a target, or threshold, concentration. In one aspect, an assay component is configured to quantitatively determine an amount of an analyte that is present in a sample, e.g., by comparison of a measured assay value to a standard curve generated from known analyte values.
  • a system includes a nucleic acid sequencing component that is configured to determine a sequence of a nucleic acid.
  • a nucleic acid sequencing component is configured to perform high-throughput sequencing of a plurality of nucleic acids.
  • a sequencing component is configured to perform parallel sequencing by synthesis.
  • a system includes one or more data acquisition components that are configured to acquire data from a sample or assay component, or that are configured to analyze a plurality of data.
  • a data acquisition component is configured to carry out a qualitative or quantitative data analysis and to generate a report that displays the result(s) of the analysis.
  • An aspect of the disclosure includes a controller, processor and computer readable medium that are configured or adapted to control or operate one or more components of the subject systems and devices.
  • a system includes a controller that is in
  • a system includes a processor and a computer-readable medium, which may include memory media and/or storage media.
  • Applications and/or operating systems embodied as computer-readable instructions on computer-readable memory can be executed by the processor to provide some or all of the functionalities described herein.
  • a system includes a user interface, such as a graphical user interface (GUI), that is adapted or configured to receive input from a user, and to execute one or more of the methods as described herein.
  • GUI graphical user interface
  • a GUI is configured to display data or information to a user.
  • a system includes a processor that is configured to generate a report that summarizes one or more results of the subject methods.
  • a report can include data generated by one or more aspects of the subject methods.
  • kits that at least include a plurality of oligonucleotide conjugates as described herein, and instructions for how to use the oligonucleotide conjugates to carry out one or more of the methods described herein.
  • a kit includes a number of different oligonucleotide conjugates that ranges from about 2 to about 300, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, or about 300 different oligonucleotide conjugates.
  • a kit includes a plurality of oligonucleotide conjugates that are individually packaged (e.g., that are each present in a separate container).
  • the instructions for using the oligonucleotide conjugates as discussed above are generally recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e.
  • the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium, e.g., a digital storage medium, e.g., a portable flash drive, a CD-ROM, a diskette, etc.
  • the instructions may take any form, including complete instructions for how to use the systems and devices, or as a website address with which instructions posted on the Internet may be accessed.
  • EXAMPLE 1 CONSTRUCTION AND PROPERTIES OF ANTIBODY-OLIGONUCLEOTIDE- CONJUGATES (AOCs)
  • AOC antibody oligonucleotide conjugate
  • each AOC is composed of four functional domains that permit restriction enzyme-mediated cleavage of oligonucleotides from antibodies, universal amplification of cleaved oligonucleotide tags, and AOC-specific quantification (FIG. 1A).
  • the utility of AOCs hinges upon efficient binding to target cell surface molecules and effective restriction enzyme-mediated cleavage of the oligonucleotide tags from antibodies.
  • a variety of AOCs are constructed with varying oligonucleotide lengths and predicted secondary structures. These AOCs are evaluated for their ability to be processed by Apal to release the
  • oligonucleotide from the attached antibody.
  • the degree of cleavage is evaluated by qPCR, SDS- PAGE, HPLC, and ESI-MS.
  • each AOC is tested for its ability to bind live and fixed cells, and for immobilized AOCs on cell surfaces to be processed by Apal for oligonucleotide cleavage.
  • binding of an AOC to its target antigen is largely determined by its association constant (K a ), it may also be affected by repulsive interactions between the similarly negatively charged oligonucleotide component and the cell surface (Gartner ZJ, Bertozzi CR. Programmed assembly of 3 -dimensional microtissues with defined cellular connectivity.
  • Tetrazine Ligation Fast Bioconjugation Based on Inverse-Electron-Demand Diels- Alder Reactivity. Journal of the American Chemical Society. 2008; 130(41): 13518-9; Rieder U, Luedtke NW. Alkene-Tetrazine Ligation for Imaging Cellular DNA. Angewandte Chemie International Edition. 2014;53(35):9168-72; Devaraj NK, Weissleder R, Hilderbrand SA.
  • Tetrazine-Based Cycloadditions Application to Pretargeted Live Cell Imaging. Bioconjugate Chemistry. 2008;19(12):2297-9).
  • This conjugation strategy allows for low concentration biomolecules ( ⁇ 1 uM) to be utilized as well as very short reaction times (- 30 minutes for reaction completion).
  • synthetic oligonucleotides bearing a 3' amino terminus and 5' biotin group are reacted with tra «s-cyclooctene-PEG4-NHS ester in 150 mM sodium phosphate, 150 mM borate pH 8.5 to afford tra «5-cyclooctene-PEG4 modified oligonucleotides at the 3' terminus.
  • Antibodies are modified via covalent linkage of methyltetrazine-PEG4 to lysine residues on the surface of each antibody.
  • the AOCs are initially constructed in solution by mixing modified antibodies and oligonucleotides at a ratio of 1 : 1, each at a concentration of luM, in phosphate buffered saline.
  • each AOC is composed of four different domains. Two universal domains that comprise all AOCs, a unique identifier sequence unique to a particular AOC, and a constant restriction domain used to separate the oligonucleotide component from the antibody.
  • the oligonucleotide is coupled to the antibody through a 3' trans- cyclooctene moiety that reacts with methyltetrazine introduced onto surface lysines on the antibody.
  • Herceptin- AOCs are tested with the SK-BR-3 and MDA-MB-231 breast cancer cell lines, which are known to be positive and negative for HER2 expression, respectively (Axup JY, Bajjuri KM, Ritland M, Hutchins BM, Kim CH, Kazane SA, Haider R, Forsyth JS, Santidrian AF, Stafin K, Lu Y, Tran H, Seller AJ, Biroc SL, Szydlik A, Pinkstaff JK, Tian F, Sinha SC, Felding-Habermann B, Smider VV, Schultz PG.
  • the cells are placed on ice and blocked for non-specific binding for 1 hour with human Fc blocking agent and a random sequence 3' phosphorothioate protected 30-mer oligonucleotide.
  • Labeling of the viable cells is performed for 1 hour on ice with 20 uL of Herceptin- AOCs at 100 nM in PBS/1% BSA.
  • the unbound conjugates are washed away from the cell surface and the biotinylated oligonucleotides are then probed with avidin-HRP.
  • HRP substrate Upon washing away avidin-HRP, HRP substrate is applied and is analyzed colorimetrically in a microplate reader.
  • the limit of detection (LOD) for HER2 present on varying quantities of SK-BR3 cells by the various Herceptin conjugates is determined and compared against negative controls (Herceptin only, oligonucleotides only, PBS only).
  • Effective cleavage of oligonucleotide components from antibodies is determined by the position of the 6 base Apal restriction site within the oligonucleotide component, owing to the effect of flanking nucleotides and potential steric hindrance of the antibody towards the 3' terminus.
  • the effect of restriction site positioning on oligonucleotide cleavage is resolved by engineering a series of AOCs wherein the restriction site begins at various positions between nucleotides 1-20.
  • Apal and a complementary oligonucleotide to the restriction domain is incubated with the cell- bound Herceptin oligonucleotide conjugates at room temperature.
  • the Apal restriction enzyme was selected as it can operate at room temperature, in conditions non-cytotoxic to cells, and because it can act on a double stranded DNA substrate requiring only a single nucleotide spacer from the terminal 5' or 3' ends.
  • the ability of Apal to cleave the Herceptin- AOCs is screened first in vitro and analyzed by SDS-PAGE electrophoresis.
  • Apal requires potassium as well as magnesium for activity, and so optimal concentrations of these ions in tris-buffered saline is determined for maximal activity and minimal cytotoxicity.
  • Cleaved oligonucleotide from the cell surface is analyzed using an avidin-HRP assay as before, as well as qPCR with SYBR Green (Dezfouli M, Vickovic S, Iglesias MJ, Nilsson P, Schwenk JM, Ahmadian A. Magnetic bead assisted labeling of antibodies at nanogram scale. Proteomics. 2014; 14(1): 14-8; Lourenco EV, Roque-Barreira MC. Immunoenzymatic quantitative analysis of antigens expressed on the cell surface (cell-ELISA).
  • a standard curve is generated by analyzing C t values (threshold cycle) of known concentration of oligonucleotide and comparing against the values obtained from performing qPCR on the cleaved
  • tetrazine ligation an efficient bio-orthogonal reaction between tetrazine and trans-cyclooctene that is amenable to very low concentration (uM to nM) of reagents that are stable in aqueous buffer.
  • uM to nM very low concentration
  • the product of tetrazine ligation is permanent, as the release of N 2 prevents the reversibility of the reaction (Blackman ML, Royzen M, Fox JM. Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels- Alder Reactivity. Journal of the American Chemical Society. 2008; 130(41): 13518-9).
  • oligonucleotides are functionalized with tram'-cyclooctene using DEAE magnetic beads.
  • Antibodies are functionalized with tetrazine, such that mostly 1 tetrazine functionality is introduced onto the antibody, and incubated with the immobilized tram'-cyclooctene
  • oligonucleotides to afford an AOC, where that AOC is composed of an antibody conjugated to a single oligonucleotide. All unreacted functionalized oligonucleotides are quenched with Cy3- tetrazine, permitting visualization of the reaction and determination of yield. Synthesized AOCs are purified away from unreacted antibody by incubating AOCs with DEAE magnetic beads. These beads are then washed to remove unmodified antibody, and then the purified AOCs are eluted.
  • the released AOCs may be further purified using magnetic Protein A beads, Protein G beads, Protein L beads, or any other affinity agent(Dezfouli M, Vickovic S, Iglesias MJ, Nilsson P, Schwenk JM, Ahmadian A. Magnetic bead assisted labeling of antibodies at nanogram scale. Proteomics. 2014; 14(1): 14-8). Purification using Protein A beads removes any DNA that was not conjugated in the previous step. Elution of the AOC from the magnetic beads with low pH treatment affords the synthesized AOC in high yield (-70-95%). Alternatively, AOCs may be further purified by using a lOOkD MWCO centrifugal filter to remove unconjugated DNA. Finally, purified AOCs are visualized using SDS-PAGE and analyzed by UV-Vis or ESI-MS to confirm single oligonucleotide modification of antibody.
  • AOCs in diagnostics and biomarker discovery hinges upon the sensitive and reproducible quantification of cell surface antigens in target populations and single cells.
  • the AOC library consists of AOCs targeting HER2 as well as CD receptors that were determined to be highly variable or invariant according to published HT-FACS data in these lines and GLUT1, a glucose transporter universally expressed in all tissues at similar levels, to be used as an internal reference (Younes M, Lechago LV, Somoano JR, Mosharaf M, Lechago J. Wide Expression of the Human Erythrocyte Glucose Transporter Glutl in Human Cancers. Cancer Research. 1996;56(5): 1164-7).
  • Each AOC contains a 5' biotin molecule that is used to recruit avidin-HRP for all cell- ELISAs conducted.
  • the cell-ELISA experiments are used to obtain the relative quantification levels of each of the CD receptors probed with reference to GLUTl .
  • FACS is performed on each of the cell lines for confirmation of literature values of each of the CD receptor expression levels.
  • the oligonucleotide from the AOC immobilized on cells is cleaved.
  • This cleaved oligonucleotide is analyzed by qPCR using AOC specific primers and compared to cell-ELISA data to ensure the data from the cell-ELISA data can be recapitulated using solely the cleaved oligonucleotide tag as a read-out (Dezfouli M, Vickovic S, Iglesias MJ, Nilsson P, Schwenk JM, Ahmadian A. Magnetic bead assisted labeling of antibodies at nanogram scale. Proteomics. 2014;14(1): 14-8; Ullal AV, Peterson V, Agasti SS, Tuang S, Juric D, Castro CM, Weissleder R.
  • next-generation sequencing technologies (Illumina MiSeq or NextSeq).
  • NGS next-generation sequencing
  • the cell surface profile of cell types not previously analyzed Jurkat, Granta, SUDHL-1 will be screened in order to identify receptors that are significantly up-regulated in each of these cell lines.
  • a population of cells is first incubated with a library of AOCs. These AOCs are allowed to bind to the cell surface, and any unbound AOCs are washed away. Cells may be analyzed as a population, or may be sorted into single cells. The oligonucleotides bound to antibodies may then be separated upon incubation with Apal and a complementary oligonucleotide (top portion of FIG. 2). As shown in the bottom portion of FIG. 2, isolation of the cleaved oligonucleotide is followed by sample barcoding and amplification of all cleaved oligonucleotides isolated. Amplification is performed using universal primers where one primer is "barcoded". All amplified products are then ligated with NGS library adapters for next-generation sequencing analysis.
  • oligonucleotides in serum have limited stability (-days) depending on the modifications that are introduced, other oligonucleotide mimetics have extremely high stability (peptide nucleic acid, phosphorothioate, 2'fluoro, morpholino), have already found clinical utility, retain the ability for non-covalent Watson-Crick base pairing, and can be readily synthesized using automated synthesizers (Karkare S, Bhatnagar D. Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino. Applied Microbiology Biotechnology. 2006;71(5): 575-86).
  • antibodies have huge clinical potential due to their high stability, circulation half-life, and ability to target limitless antigens (Chapman AP, Antoniw P, Spitali M, West S, Stephens S, King DJ. Therapeutic antibody fragments with prolonged in vivo half-lives. Nature Biotechnology. 1999; 17(8):780-3).
  • current antibody-drug- conjugates (ADCs) or bispecific antibodies have to be constructed individually or their effectiveness is limited by stoichiometry (Chames P, Van Regenmortel M, Weiss E, Baty D. Therapeutic antibodies: successes, limitations and hopes for the future. British Journal of Pharmacology. 2009; 157(2):220-33).
  • an AOC was constructed not with an oligonucleotide but instead with peptide nucleic acid (PNA), the AOC could potentially be used in therapeutics (Kazane SA, Axup JY, Kim CH, Ciobanu M, Wold ED, Barluenga S, Hutchins BA, Schultz PG, Winssinger N, Smider VV. Self-Assembled Antibody Multimers through Peptide Nucleic Acid Conjugation. Journal of the American Chemical Society. 2013; 135(l):340-6).
  • a library of diagnostic AOCs using oligonucleotides is first used to profile a particular patient biopsy to determine the cell surface marker up-regulated with respect to adjacent normal tissue.
  • This AOC would directly inform the correct treatment, where this treatment would be two PNA conjugates, in which an antibody-PNA conjugate (APC) would bind the up-regulated cell surface marker (i.e., HER2) and also where this first APC is barcoded with a PNA of a particular sequence.
  • This sequence would be complementary to a PNA on a second PNA-conjugate.
  • This secondary APC i.e., anti-CD3 binding
  • This strategy would effectively be a dynamic CAR T-cell therapy (or "smart" bispecific antibody) where a cancer could first be profiled, the up-regulated biomarker determined, and where this obtained information directly informs treatment with a library of APCs to choose from.
  • peptide nucleic acid will be used for conjugation to antibodies or other molecules rather than simple oligonucleotides.
  • a library of antibody-PNA-conjugates all contain PNA of fixed sequence, X.
  • a library of therapeutic PNA conjugates are also constructed, and all contain PNA of complementary fixed sequence, X' (FIG. 3A).
  • a single APC is used to label these cells, and because of the fixed sequence, this single APC is then used to recruit any PNA conjugate to the surface of HER2 positive cells.
  • a single APC can be converted into an antibody-drug-conjugate, an antibody-enzyme conjugate, a bispecific antibody, or a CAR T- Cell mimetic therapy (FIG. 3B).
  • the potential of having any of these therapies associated with any APC would allow for "mix and match" therapeutics where any targeting molecule could be coupled with any therapeutic molecule.
  • the strategy would use the same molecules for both diagnosis and therapeutics.
  • This platform could be used to analyze how a cancer responds to treatment to allow for rational dynamic therapy for the most effective therapeutic to be utilized.
  • preventative treatment to cancer could be provided. Exosomes or circulating-tumor-cells (CTCs) are analyzed through a routine blood sample and preventative treatment is offered before a cancer has the opportunity to become metastatic (Alix-Panabieres C, Pantel K. Challenges in circulating tumour cell research. Nature Reviews Cancer. 2014; 14(9):623-31; Joosse SA, Gorges TM, Pantel K. Biology, detection, and clinical implications of circulating tumor cells.
  • the cell-surface biomarker directs an ssDNA conjugate to bind (left panel) and a DNA-Enzyme-Inhibitor (IDE) construct with a complementary ssDNA strand is recruited (middle panel).
  • IDE DNA-Enzyme-Inhibitor
  • the enzyme is activated at the surface of a living cell, where it can cleave a doxorubicin-based peptidyl prodrug for cell-specific cytotoxicity (right panel).
  • Example 5 SDS-PAGE electrophoresis validation of protein oligonucleotide conjugates
  • Herceptin was functionalized with DBCO-PEG4-NHS ester to afford Herceptin modified with 3 DBCO functional groups.
  • Herceptin was functionalized with azido-DA or azido-nsDA using 1.1 equivalents of either oligonucleotide and reacted for 16 hours at 37 degrees Celsius. The reaction was purified by alEX to afford Herceptin-DA or Herceptin-nsDA. Unconjugated Herceptin (left lane), is shown in Herceptin with a 22-mer ssDNA oligo "DA" (middle lane), and Herceptin with a 22-mer ssDNA oligo "nsDA" (right lane).
  • SK-BR3 (HER2 + ) or MDA-MB-231 (HER2 " ) cells were targeted with Herceptin-DA, Herceptin-DA*, Herceptin-nsDA, or DA (as shown), then labeled with either IDE or goat anti- human HRP to visualize surface-bound conjugates (FIG. 6A).
  • Herceptin species should only be recruited to the cell surface of SK-BR3 cells, not to MDA-MB-213 cells, and only Herceptin- DA should recruit and activate IDE (FIG. 6B).
  • SK-BR3 or MDA-MB-231 cells were targeted with DA, Herceptin-nsDA, or Herceptin-DA conjugates.
  • FIG. 6C After labeling, these cells were incubated with IDE, and, on removal of unbound IDE, a fluorescent substrate reporter was used to visualize and quantify of surface-bound IDE (FIG. 6C). The experiment in FIG. 6C was repeated, except the cells were incubated with goat anti-human HRP to visualize bound Herceptin species (FIG. 6D). Importantly, both Herceptin-nsDA and Herceptin-DA labeled the SK-BR3 cells, demonstrating that the IDE labeling in FIG. 6C was sequence-dependent. The experiment in FIG. 6D was repeated with only SK-BR3 cells, and all of the Herceptin conjugates labeled the cells equally (FIG. 6E).
  • Example 7 Confocal microscopy images of Herceptin-DA recruitment of oligonucleotides to the surface of SK-BR3 (HER2 + ) cells
  • Herceptin-DA is able to recruit DI-(F) to the cell periphery in the HER2 + cell line (SK-BR3), as can be seen from the co-staining of 594-Concanavalin A and DI-(F) (FIG. 7A).
  • Herceptin-nsDA is unable to recruit DI-(F) to the surface of SK-BR3 cells (FIG. 7B).
  • Herceptin-DA is unable to recruit DI- (F) to the cell surface of the HER2 " cell line (MBA-MB-231) (FIG. 7C). Scale bars, 20 urn.
  • Example 8 LC 50 determination for oligonucleotide-Herceptin, IDE, and peptidyl prodrug treatment with SK-BR3 or MDA-MB-231 cells
  • Example 9 Recruitment of IDE selectively to living cells that have surface FR
  • Either KB (FR + ) or A549 (FR " ) cells were targeted with either Folate-DA, Folate- nsDA, or DA, then labeled with either IDE or avidin-HRP to allow for visualization of surface- bound conjugates (FIG. 9A). Only KB cells that present FR on the cell surface will be targeted by folate molecules, and only when Folate-DA is used for targeting will IDE be recruited to the cell surface, allowing for visualization by a fluorescent substrate reporter. For the A549 cells, which do not express the folate receptor, IDE will not be recruited (FIG. 9B). KB or A549 cells were targeted with DA, Folate-nsDA, or Folate-DA conjugates.
  • Example 10 Confocal microscopy of Folate-DA recruitment of oligonucleotides to the surface of FR + cells
  • Folate-DA recruited DI-(F) to the cell periphery in the FR + cell line, KB, as seen from the co-staining of 594-Concanavalin A and DI-(F) (FIG. 10A).
  • Folate-nsDA was unable to recruit DI-(F) to the cell surface of KB cells (FIG. 10B).
  • Cy5- Folate was able to stain the cellular periphery of FR + KB Cells (FIG. IOC).
  • Folate-DA was unable to recruit DI-(F) to the cell periphery of the FR " cell line, A549 (FIG. 10D).
  • Folate- nsDA was unable to recruit DI-(F) to the cell periphery of the FR " cell line, A549 (FIG. 10E). Cy5-Folate was unable to stain the cellular periphery of the FR " cell line, A549 (FIG. 10F).
  • Example 11 HPLC and cellular viability analysis after 48 h of peptidyl prodrug treatment with Folate-oligonucleotide conjugate and IDE
  • FIG. 11 A The sequences of each construct are shown (FIG. 11 A). The ability of the various targeting constructs to label living cells and affect the proteolytic degradation of the peptidyl prodrug (PD) was evaluated, with absorbance at 476 nm being used to monitor doxorubicin- associated molecules. HPLC curves were normalized such that the maximum peak was set at an absorbance of 1 (FIG. 11B). Doxorubicin products from treatment on FR + KB cells. Either, DA, Folate-nsDA, Folate-DA, or media were used for initial targeting of living cells, followed by IDE application.
  • PD peptidyl prodrug
  • L-Dox was incubated with cells without any targeting or labeling treatment to determine L-Dox generated doxorubicin products (FIG. 11C).
  • the Folate-DA targeting and IDE recruitment allowed for significant degradation of PD to L-Dox, AL-Dox, and Dox on KB (FR + ) cells (FIG. 11D).
  • XTT analysis indicated that cytotoxicity was induced only for Folate-DA (FIG. HE).
  • Each oligo is comprised of five functional domains with total length of 70 nucleotides (nt) (FIG. 12A), produced as shown in FIG. 12B.
  • nt nucleotides
  • FIG. 12A Listed form the 3' end: 1) a 3 '-amino terminal spacer sequence7 nt; 2) a first universal sequence serves as a reverse NGS priming site, 18 nt; 4) a first identifier sequence that identifies the binding target of the targeting component, 8 nt; 5) a second universal sequence serves as a forward NGS priming site, 17 nt; 6) and a second identifier sequence that identifies the binding target of the targeting component, 20nt.
  • Oligos are ordered from IDT and PAGE purified to insure greater than 99% accuracy of the oligo synthesis.
  • the first identifier sequences are designed such that 2 bases would have to be miscalled in order to confuse one first identifier sequence for another.
  • the universal and second identifier sequences are designed to be -55% GC rich and devoid of even partial matches to the central barcode.
  • the second identifier sequences are used in combination with first universal sequences for targeted quantification of a specific AOC via qPCR as a library quality check prior to executing an NGS run, or to confirm results thereafter. Additionally, second identifier sequences can be used for the hybridization of complementary oligos with diagnostic moieties.
  • Antibodies are immobilized on magnetic Protein A/G beads. Surface lysine residues on the antibodies are reacted with Methyltetrazine-PEG4-NHS ester (MT-NHS) in 100 mM sodium phosphate, pH 7.5.
  • MT-NHS Methyltetrazine-PEG4-NHS ester
  • single-stranded DNA oligonucleotides bearing a 3' amino terminus group are first immobilized on weak anion exchange superparamagnetic beads (DEAE) and reacted with trans-cyclooctene-PEG4-NHS ester (TCO-NHS) (14) in 50 mM sodium phosphate, 50 mM Borate, pH 8.5 to afford trans-cyclooctene-PEG4 modified oligonucleotides at the 3 ' terminus.
  • the modified antibodies and oligos are then eluted off of their magnetic beads and mixed together at a ratio of 1 : 1 for 1 hour. At this point the reaction solution, a mixture of antibody, AOC, and free DNA, is quenched.
  • AOCs are then dual affinity purified by immobilizing antibodies and washing away free DNA. Antibodies are then eluted and AOCs are purified from unmodified antibodies via immobilization of DNA. Both AOC and unmodified antibodies are recovered in this final step (FIG. 16A).
  • a human serum sample is added to a well pre-coated with a mixture of capture antibodies, which immobilize the proteins of interest. All unbound sample is washed away.
  • a library of detection AOCs are added to the well, where they bind to their target proteins immobilized by the capture antibodies. All unreacted AOCs are washed away and DNA barcodes are universally amplified via PCR and analyzed using NGS (FIG. 13).
  • Example 16 Generation of AOCs with about 1 to 1.2 antibody: oligo ratio.
  • Controlling the number of oligonucleotides per antibody is critical to the function of AOCs, as repulsive interactions between negatively charged oligonucleotide tags and similarly charged molecules in biological samples could affect the binding of an AOC to its target antigen.
  • Conventional conjugation methods routinely produce antibody to target molecule ratios between 1 :4 to 1 :8, hampering the utility of these conjugates for most applications.
  • Methods using engineered cysteine residues, unnatural amino acids, and other custom linkers can routinely achieve a 1 :2 ratio, but remain limited by availability of recombinant antibodies, difficulties in scalable production of proteins with unnatural amino acids, as well as the considerable time and cost constraints inherent to these approaches.
  • a solid-phase chemistry method is for the rapid production of AOCs modified with 1- 1.2 oligos per antibody. This conjugation method does not require antibody engineering, and is robust across host species and antibody isotypes. A density of functionality is also engineered into the oligonucleotide component of the AOCs to permit both targeted and universal amplification via PCR, as well as for the controlled hybridization of fluorescent probes. These features afford flexibility in the application space of the AOCs, including competitive antibody profiling, single-plex qPCR-based protein quantification, and multiplexed proteomics using NGS. A simple workflow (FIG. 15) allows users to rely on common techniques such as ELISA and PCR for the completion of an AOC-based proteomic profiling.
  • Antibodies are immobilized on magnetic Protein A/G beads. Surface lysine residues on the antibodies are reacted with Methyltetrazine-PEG4-NHS ester (MT-NHS) (13) in 100 mM sodium phosphate, pH 7.5.
  • MT-NHS Methyltetrazine-PEG4-NHS ester
  • single-stranded DNA oligonucleotides bearing a 3' amino terminus group are first immobilized on weak anion exchange superparamagnetic beads (DEAE) and reacted with trans-cyclooctene-PEG4-NHS ester (TCO-NHS) (14) in 50 mM sodium phosphate, 50 mM Borate, pH 8.5 to afford trans-cyclooctene-PEG4 modified oligonucleotides at the 3 ' terminus.
  • the modified antibodies and oligos are then eluted off of their magnetic beads and mixed together at a ratio of 1 : 1 for 1 hour. At this point the reaction solution, a mixture of antibody, AOC, and free DNA, is quenched.
  • AOCs are then dual affinity purified by immobilizing antibodies and washing away free DNA. Antibodies are then eluted and AOCs are purified from unmodified antibodies via immobilization of DNA. Both AOC and unmodified antibodies are recovered in this final step. Importantly, this procedure works with any off the shelf antibody, irrespective of host and isotype, and generates AOCs that are primarily singly modified (FIG. 16A). Solid phase functionalization of the antibodies and oligos is critical in controlling not only the number of modifications per antibody, but also the region of the antibody being modified, with analysis of AOCs via gel electrophoresis under reducing conditions showing that it is consistently a single heavy chain that is oligo-modified.
  • Cross-reactivity of commercial and native antibodies is the main limitation of affinity- based multiplexed proteomics.
  • Cross reactivity experiments such as those highlighted in FIGS. 18A-18D are incredibly complex and represent perhaps the greatest bottleneck in the generation of antibody based multiplexed assays.
  • IL-4 was the only problematic AOCs of the 11 tested, with evidence of off-target binding to captured IL-22, IL-33, IFNy and TSLP proteins. Therefore, it was determined that the detection of all of the proteins tested could be multiplexed, with the exception of IL-4.
  • interaction matrix
  • immunoreactivity of AOCs may not be limited to other analytes in a given panel, and it is important to understand binding profile of AOCs across the whole proteome.
  • an antibody qualification pipeline was devised that characterized antibodies via generation of proteome-wide binding data.
  • Example 19 Development of a robust pipeline for the identification of high quality antibodies for use in multiplexed proteomic assays
  • Affinity reagents with low dissociation constants (low rate of dissociation, koff) and high specificity are of critical importance to implementing multiplexed detection.
  • An antibody qualification pipeline was devised to streamline the selection of highly specific, sensitive and multiplex-compatible antibodies for our assays.
  • AOCs are generated from each of the antibodies using the conjugation procedures described and whole proteome binding profiles are generated using the methods represented in FIG. 19.
  • batches of related AOCs are analyzed using comprehensive human proteome microarrays to determine the specificity, relative affinity, and multiplex compatibility of each antibody.
  • AOC sensitivity is determined via qPCR of AOCs reacted with its corresponding purified recombinant target protein in PBS at concentrations spanning 8 orders of magnitude, from 1 nM to 100 aM.
  • AOC-1 a specific AOC, (AOC-1) is labeled via hybridization of a short, 3 ' fluorophore modified oligonucleotide that is complementary to the 20nt antibody specific sequence of the AOC-1 (FIG. 19).
  • AOC-1 a specific AOC, (AOC-1) is labeled via hybridization of a short, 3 ' fluorophore modified oligonucleotide that is complementary to the 20nt antibody specific sequence of the AOC-1 (FIG. 19).
  • Proteome wide binding targets of the AOC-1 are readout, in a matter of minutes, with a fluorescence microarray scanner.

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Abstract

La présente invention décrit des conjugués oligonucléotidiques et leurs utilisations. Les aspects des conjugués oligonucléotidiques de l'invention comprennent un constituant de ciblage, un constituant de liaison, un constituant de clivage, et un constituant oligonucléotidique. L'oligonucléotide comprend une séquence d'identification ou une première et une seconde séquence d'identification, permettant la détection et la quantification d'une cible en utilisant des procédés tels que la séquence de génération suivante et la PCR quantitative. Des procédés de fabrication et d'utilisation des conjugués oligonucléotidiques dans le diagnostic, la prévention et/ou le traitement du cancer et d'autres maladies sont également décrits.
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Publication number Priority date Publication date Assignee Title
CN108103174A (zh) * 2017-12-14 2018-06-01 中源协和基因科技有限公司 用于qPCR精确定量Illumina平台二代测序样本的定量标准品及其复制方法
WO2020056021A1 (fr) * 2018-09-11 2020-03-19 The Johns Hopkins University Système de libération de médicament dépendant de la force pour améliorer la destruction sélective et réduire au minimum les effets indésirables dans le traitement du cancer
EP3903104A4 (fr) * 2018-12-27 2022-09-21 Bio-Rad Laboratories, Inc. Transfert western multiplex séquentiel
WO2021174416A1 (fr) * 2020-03-03 2021-09-10 深圳先进技术研究院 Procédé de détection de cellule tumorale circulante
WO2021211791A1 (fr) * 2020-04-15 2021-10-21 Academia Sinica Conjugués d'oligonucléotides et leur préparation et application
TWI817107B (zh) * 2020-04-15 2023-10-01 中央研究院 寡核苷酸共軛物及其製備與應用
WO2021260061A3 (fr) * 2020-06-24 2022-02-24 Sapreme Technologies B.V. Dérivés de saponine à fenêtre thérapeutique améliorée
WO2021261996A3 (fr) * 2020-06-24 2022-03-10 Sapreme Technologies B.V. Conjugués de saponine à base de nhs
WO2021260054A3 (fr) * 2020-06-24 2022-04-21 Sapreme Technologies B.V. Dérivés de saponine à fenêtre thérapeutique améliorée
WO2022125755A1 (fr) * 2020-12-10 2022-06-16 Thermo Fisher Scientific Inc. Procédé et composition pour une analyse monocellulaire multiplexée et multimodale
WO2024069235A2 (fr) 2022-09-30 2024-04-04 Sixfold Bioscience Ltd. Compositions contenant des oligonucléotides ayant des applications théranostiques

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