OLIGONUCLEOTIDE CONJUGATES AND USES THEREOF

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 62/329, 138, filed April 28, 2016, and U.S. Provisional Application No. 62/329,143, filed April 28, 2016, both of which are incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 27, 2017, is named 52184-702_601_SL.txt and is 68,942 bytes in size.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with the support of the United States government under Contract number GM067170 by the National Institute of General Medical Sciences.

BACKGROUND

[0004] Malignant tumors (cancers) 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.

[0005] In attempts to discover effective cellular targets for cancer therapy, researchers have sought to identify 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.

[0006] Every cancer is unique, with a subset of mutations that give rise to a tumorigenic phenotype. As such, the cell surfaces of cancerous cells are highly variable, making treatment of cancer highly dependent on the ability of targeted therapeutic agents to bind tumorigenic tissue. The ability to probe cancerous cells with all currently available targeted therapies (antibody- drug-conjugates, CAR T-cells, or bispecific antibodies) and to quickly determine which treatment comprising these targeted therapies is able to bind cancerous cells would be highly desirable. In addition, 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. In addition, 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.

SUMMARY

[0007] 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.

[0008] 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. In one aspect, the targeting component comprises a polymer. In one aspect, the polymer comprises an amino acid, a nucleic acid, or a polysaccharide. In one aspect, 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). In one aspect, 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. In one aspect, the linker component comprises a polypeptide. In one aspect, the linker component does not comprise a polypeptide. In one aspect, 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. In one aspect, the cleavage component comprises a light- cleavable cleavage component. In one aspect, the cleavage component comprises a chemically- cleavable cleavage component. In one aspect, the cleavage component comprises an

oligonucleotide. In one aspect, the oligonucleotide of the cleavable component comprises a restriction site. In one aspect, the restriction site comprises a number of nucleic acid residues ranging from about 6 to about 12. In one aspect, the length of the oligonucleotide component comprises a number of nucleic acid residues ranging from about 16 to about 120.

[0009] In one aspect, 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. In one aspect, the targeting component binds to a protein, a lipid, a carbohydrate, or a small molecule. In one aspect, the small molecule is a folate molecule. In one aspect, the targeting component binds to a protein. In one aspect, the protein is a lectin or a cytokine. In one aspect, the protein is an intracellular protein, an extracellular protein, or a cell surface protein. In one aspect, the protein is a cell surface protein. In one aspect, the cell surface protein is HER2, FOLR1, NCAM1 (CD56), CD274 (PD-L2), CD278 (ICOS), CD9, CD 104 or CD119. In one aspect, the first and the second universal sequences are different. In one aspect, the first and the second universal sequences are the same. In one aspect, the length of the first universal sequence comprises a number of nucleic acid residues ranging from about 12 to about 60. In one aspect, the length of the second universal sequence comprises a number of nucleic acid residues ranging from about 12 to about 60. In one aspect, the length of the identifier sequence comprises a number of nucleic acid residues ranging from about 5 to about 15. In one aspect, the length of the identifier sequence comprises about 8 nucleic acid residues. In one aspect, 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. In one aspect, the cleavage component is located at the 3' end of the oligonucleotide component. In one aspect, the cleavage component is linked to the targeting component via the linker component.

[0010] In one aspect, 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.

[0011] In one aspect, 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.

[0012] In one aspect, 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. [0013] In one aspect, 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.

[0014] In one aspect, 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.

[0015] In one aspect, 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.

[0016] In one aspect, 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.

[0017] In one aspect, 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.

[0018] 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

oligonucleotide conjugate, wherein 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. In one aspect, the sample comprises a cell. In one aspect, the binding target is a surface protein on the cell. In one aspect, a second identifier sequence is linked to the first identifier sequence after the first identifier sequence has been released from the oligonucleotide conjugate. In one aspect, the surface protein is HER2, FOLR1, NCAM1 (CD56), CD274 (PD-L2), CD278 (ICOS), CD9, CD104 or CD119. In one aspect, the cleaving agent comprises a restriction enzyme. In one aspect, the cleaving agent is a light beam. In one aspect, detecting the first identifier sequence comprises sequencing at least a portion of the first identifier sequence. In one aspect, a method comprises detecting the second identifier sequence. In one aspect, detecting the second identifier sequence comprises sequencing at least a portion of the second identifier sequence. In one aspect, the sequencing comprises high-throughput sequencing. In one aspect, the sequencing comprises parallel sequencing by synthesis. In one aspect, the detection is qualitative. In one aspect, the detection is quantitative.

[0019] 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. In one aspect, the sample comprises a cell. In one aspect, the binding target is a surface protein on the cell. In one aspect, a second identifier sequence is linked to each of the first identifier sequences after step b). In one aspect, detecting the first identifier sequence comprises sequencing at least a portion of the first identifier sequence. In one aspect, a method comprises detecting the second identifier sequence. In one aspect, detecting the second identifier sequence comprises sequencing at least a portion of the second identifier sequence. In one aspect, the sequencing comprises high-throughput sequencing. In one aspect, the sequencing comprises parallel sequencing by synthesis. In one aspect, the detection is qualitative. In one aspect, the detection is quantitative.

[0020] 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 plurality of identifier sequences to determine the presence of the plurality of binding targets in the sample. In one aspect, the sample comprises a cell. In one aspect, the binding target is a surface protein on the cell. [0021] 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

oligonucleotide conjugate, wherein the second oligonucleotide conjugate comprises a detectable moiety; and an oligonucleotide component, wherein at least a portion of the oligonucleotide component of the second oligonucleotide conjugate is complementary to at least a portion of the oligonucleotide component of the first oligonucleotide conjugate; and b) detecting the detectable moiety on the second oligonucleotide conjugate to diagnose the subject for cancer. In one aspect, the targeting component comprises a polymer. In one aspect, the polymer comprises an amino acid, a nucleic acid, or a polysaccharide. In one aspect, 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). In one aspect, 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. In one aspect, the linker component comprises a polypeptide. In one aspect, the linker component does not comprise a polypeptide. In one aspect, 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. In one aspect, 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

oligonucleotide conjugate comprises a number of nucleic acid residues ranging from about 16 to about 120. In one aspect, the target on the cancer cell is a cell surface protein. In one aspect, the cell surface protein is HER2, FOLRl, NCAMl (CD56), CD274 (PD-L2), CD278 (ICOS), CD9, CD 104 or CD119. In one aspect, the targeting component of the first oligonucleotide conjugate comprises a protein linked to the oligonucleotide component by a strain-promoted-azide- alkylene (SPAAC) linker. In one aspect, the targeting component of the first oligonucleotide conjugate comprises a protein linked to the oligonucleotide component by a tetrazine ligation linker. In one aspect, the detectable moiety comprises a fluorophore.

[0022] 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. In one aspect, 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. In one aspect, a kit comprises a cleaving agent that cleaves the cleavage component.

[0023] 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. In one aspect, the functionalized oligonucleotide is separated from the first solid support and is reacted with the functionalized targeting component while the functionalized targeting component is

immobilized on the second solid support. In one aspect, 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. In one aspect, both the functionalized oligonucleotide and the functionalized targeting component are separated from the first and second solid supports before being reacted with one another. In one aspect, 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. In one aspect, 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. In one aspect, 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. In one aspect, the oligonucleotide is non-covalently immobilized on the first solid support. In one aspect, the targeting component is non-covalently immobilized on the second solid support. In one aspect, the first solid support comprises a cationic affinity resin. In one aspect, the cationic affinity resin comprises diethylaminoethanol (DEAE) beads. In one aspect, the second solid support comprises an affinity resin. In one aspect, 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. In one aspect, a method comprises attaching a moiety to the targeting component. In one aspect, the moiety is a detectable moiety. In one aspect, the moiety is a therapeutic moiety. In one aspect, isolating the oligonucleotide conjugate comprises contacting the oligonucleotide conjugate with a magnetic bead.

[0024] 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. In one aspect, the targeting component comprises a polymer. In one aspect, the polymer comprises an amino acid, a nucleic acid, and a polysaccharide. In one aspect, 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). In one aspect, 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. In one aspect, the linker component comprises a polypeptide. In one aspect, the linker component does not comprise a polypeptide. In one aspect, 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. In one aspect, 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 therapeutic secondary oligonucleotide conjugate comprises a number of nucleic acid residues ranging from about 16 to about 120. In one aspect, the target on the cancer cell is a cell surface protein. In one aspect, the cell surface protein is HER2, FOLR1, NCAM1 (CD56), CD274 (PD-L2), CD278 (ICOS), CD9, CD104 or CD119. In one aspect, the targeting component of the first oligonucleotide conjugate comprises a protein linked to the oligonucleotide component by a strain-promoted-azide-alkylene (SPAAC) linker. In one aspect, the targeting component of the first oligonucleotide conjugate comprises a protein linked to the oligonucleotide component by a tetrazine ligation linker. In one aspect, 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. In one aspect, the therapeutic moiety comprises an enzyme. In one aspect, the method further comprises administering a prodrug to the subject, and wherein the prodrug is activated by the enzyme. In one aspect, the surface protein is HER2, FOLR1, NCAM1 (CD56), CD274 (PD-L2), CD278 (ICOS), CD9, CD 104 or CD119. In one aspect, 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 conjugates; and d) detecting the first identifier sequences to determine the presence of the one or more surface proteins on the cell. In one aspect, a second identifier sequence is linked to the first identifier sequence after the first identifier sequence has been released from the first oligonucleotide conjugate. In one aspect, detecting the first identifier sequence comprises sequencing at least a portion of the first identifier sequence. In one aspect, a method comprises detecting the second identifier sequence. In one aspect, detecting the second identifier sequence comprises sequencing at least a portion of the second identifier sequence. In one aspect, the sequencing comprises high-throughput sequencing. In one aspect, the sequencing comprises parallel sequencing by synthesis. In one aspect, the detection is qualitative. In one aspect, the detection is quantitative.

[0025] 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 conjugate.

[0026] Disclosed herein, in certain embodiments, are 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. In some embodiments, the first identifier sequence identifies the targeting component. In some embodiments, the 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. In some embodiments, the second identifier sequence is hybridized to a secondary oligonucleotide conjugate comprising a second oligonucleotide component that is attached to a diagnostic moiety. In some embodiments, 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. In some embodiments, the oligonucleotide component is attached to the targeting component through a linker component. In some embodiments, 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. In some embodiments, the linker component is attached to the oligonucleotide component through a cleavage component. In some embodiments, the cleavage component comprises a light-cleavable cleavage component. In some embodiments, 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. In some embodiments, 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.

[0027] Disclosed herein, in certain embodiments, are methods for detecting the presence of a plurality of target antigens in a biological sample comprising: (a) contacting the biological sample with a plurality of oligonucleotide conjugates wherein 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 to detect the oligonucleotide components that are bound to target antigens thereby detecting the presence of a plurality target antigens. In some

embodiments, 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. In some embodiments, 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. In some embodiments, the second identifier sequence is hybridized to a secondary oligonucleotide conjugate comprising a second oligonucleotide component that is attached to a diagnostic moiety. In some embodiments, 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. In some embodiments, the oligonucleotide component is attached to the targeting component through a linker component. In some embodiments, wherein 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. In some embodiments, the linker component is attached to the oligonucleotide component through a cleavage component. In some embodiments, the cleavage component comprises a light-cleavable cleavage component. In some embodiments, 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. In some embodiments, 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.

[0028] Disclosed herein, in certain embodiments, are therapeutic methods for treating a disease in a subject in need thereof comprising administering to the subject: (a) 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

oligonucleotide conjugate comprising (i) a second oligonucleotide component that hybridizes to the first oligonucleotide component of the primary identifier oligonucleotide conjugate, and (ii) a therapeutic moiety that is attached to the 3' end of the second oligonucleotide conjugate. In some embodiments, the disease is cancer or an infectious disease. In some embodiments, 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. In some embodiments, the therapeutic moiety is a protein. In some

embodiments, 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. In some embodiments, 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). In some embodiments, the oligonucleotide component of the primary identifier oligonucleotide, the secondary therapeutic oligonucleotide, or a combination thereof is a PNA.

[0029] Disclosed herein, in certain embodiments, are 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. In some embodiments, the cell is a cancer cell or an infectious disease cell.

[0030] Disclosed herein, in certain embodiments, are 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. In some embodiments, the cell is a cancer cell or an infectious disease cell.

[0031] Disclosed herein, in certain embodiments, are 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 therapeutic enzyme that is attached to the 3' end of the second oligonucleotide conjugate. In some embodiments, the cell is a cancer cell or an infectious disease cell.

[0032] Disclosed herein, in certain embodiments, are 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. In some embodiments, the cell is a cancer cell or an infectious disease cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 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.

[0034] FIG. 2 depicts various steps in an example diagnostic method that is carried out using the subject oligonucleotide conjugates.

[0035] 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 with a complementary oligonucleotide binding to the first

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.

[0036] FIG. 4 depicts various steps in an example therapeutic method that is carried out using the subject oligonucleotide conjugates.

[0037] FIG. 5 depicts the results of a molecular weight analysis carried out on various different conjugated and unconjugated molecules.

[0038] 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

oligonucleotide conjugate using goat-anti-human HRP to visualize the bound oligonucleotide conjugate. For each pair of bars, the first bar corresponds to SK-BR3 and the second bar corresponds to MD A-MB-231. 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.

[0039] 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). 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.

[0040] 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 MDA-MB-231 cells as a function of concentration for different Herceptin oligonucleotide conjugates and peptidyl prodrug molecules.

[0041] 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

quantification of surface-bound oligonucleotide conjugate using an IDE label on different cells. 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- )·

[0042] 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. 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.

[0043] 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.

[0044] 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

oligonucleotide conjugate shown in FIG. 12A.

[0045] FIG. 13 illustrates examples of the use of oligonucleotide conjugate molecules for both soluble antigen profiling and cellular antigen profiling.

[0046] 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.

[0047] FIG. 15 illustrates a soluble protein detection method.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] FIG. 19 illustrates whole proteome antibody characterization.

[0052] 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.

[0053] 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.

[0054] 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.

DETAILED DESCRIPTION

[0055] 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.

[0056] It is to be understood that the methods and composition described herein are not limited to particular aspects described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the methods and compositions described herein will be limited only by the appended claims.

[0057] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the methods and compositions described herein are not entitled to antedate such publication by virtue of the methods and compositions described herein are Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0058] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the methods and compositions described herein are. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0059] The methods and compositions described herein can employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987, and periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et al., ed., 1994); "A

Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A

Laboratory Manual" (Barbas et al., 2001).

DEFINITIONS

[0060] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. The below terms are discussed to illustrate meanings of the terms as used in this specification, in addition to the understanding of these terms by those of skill in the art. As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0061] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating un-recited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods and compositions described herein are. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods and compositions described herein, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions described herein.

[0062] The terms "polynucleotide", "nucleic acid", "nucleotide" and "oligonucleotide" are used interchangeably herein. These terms refer 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. The following are non-limiting examples of 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. If present, 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.

[0063] The term "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.

[0064] The term "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. For example, a targeting component and an oligonucleotide component can be linked to one another by a linker component.

[0065] The term "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.

[0066] The term "artificial" is used herein to refer to a molecule, component, or composition that does not occur in nature.

[0067] The term "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.

[0068] The term "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). Each L chain has at the N-terminus a variable domain (V _L ) followed by one constant domain (C _L ). The 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). Particular amino acid residues are believed to form an interface between the L chain and H chain variable domains. The pairing of a V _H and V _L together forms a single antigen- binding site.

[0069] The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (C _H ), 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 α, δ, ε, γ, and μ, respectively. The γ 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.

[0070] The "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.

[0071] The term "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.

However, the variability is not evenly distributed across the 110-amino acid span of most variable domains. Instead, the V regions 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. The variable domains of native H and L chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-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).

[0072] 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.

[0073] "Immunoglobulin fragments" comprise a portion of an intact immunoglobulin, preferably the antigen binding or variable region of the intact immunoglobulin. Examples of 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

immunoglobulins formed from immunoglobulin fragments. In some aspects, an immunoglobulin fragment comprises an antigen binding site of the intact immunoglobulin and thus retains the ability to bind antigen.

[0074] In the case of IgG, papain digestion of immunoglobulins produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. 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). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an 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.

[0075] 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.

[0076] "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. In a single-chain Fv (scFv) species, 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.

However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although typically at a lower affinity than the entire binding site.

[0077] "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. Preferably, 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. For a review of 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.

[0078] Unless stated otherwise, the term "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.

[0079] The term "native" with reference to a 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. The term "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. [0080] The term "polypeptide" is used herein in the broadest sense and includes peptide sequences. The term "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.

[0081] 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. Monoclonal

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. For example, 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).

[0082] 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).

[0083] "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 (recipient antibody) 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. In some instances, Fv framework region (FR) residues of the human immunoglobulin are also replaced by corresponding non-human residues.

Furthermore, 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. In general, 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

immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. (1986) Nature 321 :522-525; Riechmann et al. (1988) Nature 332:323- 329; and Presta (1992) Curr. Op. Struct. Biol. 2:593-596.

[0084] The term "human immunoglobulin", as used herein, is intended to include

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. However, the term "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.

[0085] 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. In some aspects, 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

homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Ordinarily, an isolated immunoglobulin or oligonucleotide conjugate will be prepared by at least one purification step.

[0086] The term "specific binding" or "specifically binds to" or is "specific for" 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). Specific binding can be measured, for example, by determining binding of a binding moiety, or an immunoglobulin, to a target molecule compared to binding to a control molecule. For example, 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,

alternatively at least about 40 nM, alternatively at least about 30 nM, alternatively at least about 20 nM, alternatively at least about 10 nM, alternatively at least about 8 nM, alternatively at least about 6 nM, alternatively at least about 4 nM, alternatively at least about 2 nM, alternatively at least about 1 nM, or greater. In certain instances, 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.

[0087] "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). For example, 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.

[0088] As used herein, the "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

Healthcare, Piscataway, N.J.) at 25°C with immobilized antigen CM5 chips.

[0089] The terms "conjugate," "conjugated," and "conjugation" refer 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.

[0090] The term "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. The term "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. The term "fusion" is used herein to refer to the combination of amino acid sequences of different origin.

[0091] The term "epitope" includes any molecular determinant capable of specific binding to a targeting component (e.g., an immunoglobulin). In one aspect, 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.

[0092] The term "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.

[0093] The term "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. The term antigen includes regions known as antigenic determinants or epitopes, as defined above.

[0094] 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) and three light chain variable domain CDRs (CDRL1, CDRL2 and CDRL3). The extent of CDR and framework regions (FRs) is determined by comparison to a compiled database of amino acid sequences in which those regions have been defined according to variability among the sequences and/or structural information from

antibody/antigen complexes. Also included within the scope of the disclosure are 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. Furthermore, certain antibodies might have non-CDR-associated binding sites for an antigen. Such binding sites are specifically included within the present definition.

[0095] A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, 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. Generally, "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.

[0096] 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.

[0097] "Treating" or "treatment" 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. For example, 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.

[0098] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions described herein belong. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions described herein, representative illustrative methods and materials are now described. OLIGONUCLEOTIDE CONJUGATES

[0099] 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.

Targeting Components

[00100] 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.

[00101] In one aspect, 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 . In one aspect, a light chain comprises a kappa light chain. In one aspect, a light chain comprises a lambda light chain.

[00102] In one aspect, each heavy chain includes a variable domain, designated as V _¾ as well as a constant domain designated as C _H 1, followed by one or more heavy chain Fc region domains. In one aspect, 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.

[00103] In one aspect, an immunoglobulin molecule can contain a native polypeptide sequence that occurs in nature. In one aspect, an immunoglobulin molecule can contain a non-naturally occurring polypeptide sequence (i.e., an artificial sequence that does not occur in nature).

[00104] The organization of the variable and constant domains along the light chain generally proceeds from the N-terminus to the C-terminus as V _L -C _L . Similarly, the 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.

[00105] 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. In one aspect, 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.

[00106] In one aspect, a targeting component is an enzyme that specifically binds to a binding target. In one aspect, a targeting component is a small molecule that specifically binds to, or is specifically bound by, a binding target. Examples of small molecules that specifically bind to or are bound by a binding target (e.g., a cell surface binding target) include, but are not limited to, folate. In one aspect, a targeting component is a lectin that specifically binds to, or is specifically bound by, a binding target. In one aspect, a targeting component is a carbohydrate that specifically binds to, or is specifically bound by, a binding target.

[00107] In one aspect, 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). In one aspect, 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).

[00108] In one aspect, a polymeric targeting component comprises a peptide nucleic acid (PNA). 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. In one aspect, a PNA is a thioester peptide nucleic acid (tPNA).

[00109] In one aspect, a polymeric targeting component comprises a polynucleotide that includes a locked nucleic acid (LNA). 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.

[00110] In one aspect, 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.

[00111] In one aspect, a polymeric targeting component comprises a polynucleotide that includes a phosphonoacetate (PACE) phosphoramidite. 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.

[00112] In one aspect, 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).

[00113] In one aspect, 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. For example, in one aspect, a moiety suitable for derivatization of a targeting component (e.g., an antibody) 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

homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. 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. In general, 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.

Binding Targets

[00114] As described above, 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. In one aspect, a binding target can be a protein. In one aspect, a binding target can be a soluble protein (e.g., an extra-cellular protein). Examples of soluble proteins that find use as binding targets in accordance with an aspect of the disclosure include, but are not limited to: IFN-γ, IL-1RA, IL-la, IL-Ιβ, IL-2, IL-21, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL- 12p70, IL-12/IL-23p40, IL-13, IL-15, IL-17A, IL-22, IL-32, IL-33, IL-34, MCP-1, MIF, TNF-a, TSLP, C3a, C5a, CCL17, CTLA-4, CCL28, CCL5, CXCL10, CXCL5, Factor H, FGF2, G- CSF, GM-CSF, Granulysin, LAP, Latent TGF-β, NGF, Nogo-B, Prolactin, Syt 7, TGF-βΙ, TNF- β, TNFSF13, TREM-1 and VEGF.

[00115] In one aspect, a binding target can be a cell surface protein. Examples of 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.

[00116] In one aspect, a binding target is a cell surface protein that is a tumor-associated antigen. One of skill in the art will realize that 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, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GRP, IGFl, IGF2, IL12A, ILIA, IL1B, IL2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2, EGF, FGF10, FGF18, FGF2, FGF4, FGF7, IGFl, IGF1R, IL2, VEGF, BCL2, CD 164,

CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRHl, IGFBP6, ILIA, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, E2F1, EGFR (ERBB1), HER3 (ERBB3), HER4 (ERBB4), ENOl, ESR1, ESR2, IGFBP3, IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL, TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRHl, IGFl, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NRIDI, NR1H3, NR1I3, NR2F6, NR4A3, ESR1, ESR2, NROB l, NR0B2, NR1D2, NR1H2, NR1H4, NR1I2, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2, NR5A1, NR5A2, NR6A1, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOCl, BRCA2, CHGA, CHGB, CLU, COL1A1,

COL6A1, EGF, ERK8, FGF1, FGF10, FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRHl, IGFl, IGF2, IGFBP3, IGFBP6, IL12A, ILIA, IL IB, IL2, IL24, INHA, INSL3, INSL4, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9, MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TFMP3, CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18, CDH19, CDH20, CDH7, CDH8, CDH9, ROB02, CD44, ILK, ITGA1, APC, CD164, COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1, CDH12, CLDN3, CLN3, CYB5, CYC1, DAB21P, DES, DNCL1, ELAC2, EN02, EN03, FASN, FLJ12584, FLJ25530, GAGEBl, GAGECl, GGTl, GSTPl, HIPl, HUMCYT2A, IL29, K6HF, KAIl, KRT2A, MIB l, PARTI, PATE, PC A3, PIAS2, PIK3CG, PPID, PR1, PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPMl, TPM2, TRPC6, ANGPTl, ANGPT2, ANPEP, ECGFl, EREG, FGFl, FGF2, FIGF, FLT1, JAG1, KDR, LAMA5, NRP1, NRP2, PGF, PLXDC1, STABl, VEGF, VEGFC, ANGPTL3, BAI1, COL4A3, IL8, LAMA5, NRP1, NRP2, STABl, ANGPTL4, PECAM1, PF4, PROK2, SERPINFl, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6, CXCL9, IFNA1, IFNBl, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2, EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS 1, THBS2, BAD, BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNEl, CCNE2, CDH1 (E-cadherin), CDKN1B (p27Kipl), CDKN2A (pl6INK4a), COL6A1, CTNNBl (b-catenin), CTSB (cathepsin B), ESRl, ESR2, F3 (TF), FOSLl (FRA-1), GAT A3, GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130), ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki- 67), NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA), PTEN, SERPINB5 (maspin), SERPINEl (PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1), TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A (topoisomerase Iia), TP53, AZGP1 (zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wapl/Cipl), CLDN7 (claudin-7), CLU (clusterin), FGFl, FLRT1 (fibronectin), GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4 integrin), KLF5 (GC Box BP), KRT19 (Keratin 19), KRTHB6 (hair-specific type II keratin),

MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin), PTGS2 (COX-2), RAC2

(p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Sprl), THBS1, THBS2, THBS4, and TNFAIP2 (B94).

[00117] In one aspect, a binding target can be an intracellular protein. Examples of 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.

[00118] As reviewed above, in one aspect, 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.

[00119] 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. 6,914,128); anti-IL-18 antibody (US 2005/0147610 Al), anti-C5, anti-CBL, anti-CD147, anti-gpl20, anti-VLA4, anti-CDl la, anti-CD18, anti-VEGF, anti-CD40L, anti-Id, anti-ICAM-1, anti-CXCL13, anti-CD2, anti-EGFR, anti-TGF-beta 2, anti- E-selectin, anti -Fact VII, anti-Her2/neu, anti-F gp, anti-CDl l/18, anti-CD14, anti-ICAM-3, anti- CD80, anti-CD4, anti-CD3, anti-CD23, anti-beta2-integrin, anti-alpha4beta7, anti-CD52, anti- HLA DR, anti-CD22, anti-CD20, anti-MIF, anti-CD64 (FcR), anti-TCR alpha beta, anti-CD2, anti-Hep B, anti-CA 125, anti-EpCAM, anti-gpl20, anti-CMV, anti-gpllbllla, anti-IgE, anti- CD25, anti-CD33, anti-HLA, anti-VNRintegrin, anti-IL-1 alpha, anti -IL- lb eta, anti-IL-1 receptor, anti-IL-2 receptor, anti-IL-4, anti-IL4 receptor, anti-IL5, anti-IL-5 receptor, anti-IL-6, anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13 receptor, anti-IL-17, and anti-IL-23 (see Presta LG. 2005 Selection, design, and engineering of therapeutic antibodies J Allergy Clin Immunol. 116:731-6 and Clark, M., "Antibodies for Therapeutic Applications," Department of Pathology, Cambridge University, UK, 15 Oct. 2000, published online at M. Clark's home page at the website for the Department of Pathology, Cambridge University).

[00120] 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. Such 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.

5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769 (PCT/US2003/040426, entitled "Immunoglobulin Variants and Uses Thereof), trastuzumab (HERCEPTIN®, Genentech) (see for example U.S. Pat. No. 5,677, 171), a humanized anti-Her2/neu antibody approved to treat breast cancer; pertuzumab (rhuMab-2C4, OMNITARG®), currently being developed by Genentech; an anti-Her2 antibody described in U.S. Pat. No. 4,753,894; 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- EGFR™ (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;

Kettleborough et al., 1991, Protein Eng. 4(7):773-83); ICR62 (Institute of Cancer Research) (PCT WO 95/20045; Modjtahedi et al., 1993, J. Cell Biophys. 1993, 22(1-3): 129-46;

Modjtahedi et al., 1993, Br J Cancer. 1993, 67(2):247-53; Modjtahedi et al, 1996, Br J Cancer, 73(2):228-35; Modjtahedi et al, 2003, Int J Cancer, 105(2):273-80); TheraCFM hR3 (YM Biosciences, Canada and Centro de Immunologia Molecular, Cuba (U.S. Pat. No. 5,891,996; U.S. Pat. No. 6,506,883; Mateo et al, 1997, Immunotechnology, 3(1):71-81); mAb-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102 (KS Biomedix); MRl-1 (IVAX, National Cancer Institute) (PCT WO 0162931A2); and SCIOO (Scancell) (PCT WO 01/88138); alemtuzumab

(CAMPATH®, Millennium), a humanized monoclonal antibody currently approved for treatment of B-cell chronic lymphocytic leukemia; 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

Celltech/Wyeth, alefacept (AMEVIVE®), an anti-LFA-3 Fc fusion developed by Biogen), abciximab (REOPRO®), developed by Centocor/Lilly, basiliximab (SFMULECT®), developed by Novartis, palivizumab (SYNAGIS®), developed by Medimmune, infliximab

(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) and alpha-4-beta-7 antibody being developed by Biogen, VLA-1 mAb, an anti-VLA-1 integrin antibody being developed by Biogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody being developed by Biogen, CAT- 152, an anti-TGF-P2 antibody being developed by Cambridge Antibody Technology, J695, an anti-IL-12 antibody being developed by Cambridge Antibody Technology and Abbott, CAT- 192, an anti-TGFpi antibody being developed by Cambridge Antibody Technology and Genzyme, CAT-213, an anti-Eotaxinl antibody being developed by Cambridge Antibody Technology, LYMPHOSTAT-B® an anti-Blys antibody being developed by Cambridge

Antibody Technology and Human Genome Sciences Inc., TRAIL-RlmAb, an anti-TRAIL-Rl antibody being developed by Cambridge Antibody Technology and Human Genome Sciences, Inc., AVASTIN® bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed by Genentech, an anti-HER receptor family antibody being developed by Genentech, Anti-Tissue Factor (ATF), an anti-Tissue Factor antibody being developed by Genentech, XOLAIR®

(Omalizumab), an anti-IgE antibody being developed by Genentech, RAPTIVA® (Efalizumab), an anti-CDl la antibody being developed by Genentech and Xoma, MLN-02 Antibody (formerly LDP-02), being developed by Genentech and Millennium Pharmaceuticals, HUMAX CD4®, an anti-CD4 antibody being developed by Genmab, HUMAX™-IL15, an anti-IL15 antibody being developed by Genmab and Amgen, HUMAX™-Inflam, being developed by Genmab and Medarex, HUMAX™-Cancer, an anti-Heparanase I antibody being developed by Genmab and Medarex and Oxford GlycoSciences, HUMAX™-Lymphoma, being developed by Genmab and Amgen, HUMAX™-TAC, being developed by Genmab, IDEC-131, and anti-CD40L antibody being developed by IDEC Pharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibody being developed by IDEC Pharmaceuticals, IDEC-114, an anti-CD80 antibody being developed by IDEC Pharmaceuticals, IDEC-152, an anti-CD23 being developed by IDEC Pharmaceuticals, anti-macrophage migration factor (MIF) antibodies being developed by IDEC Pharmaceuticals, BEC2, an anti -idiotypic antibody being developed by Imclone, FMC-1C11, an anti-KDR antibody being developed by Imclone, DC101, an anti-flk-1 antibody being developed by Imclone, anti-VE cadherin antibodies being developed by Imclone, CEA-CIDE® (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibody being developed by Immunomedics,

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

Centocor/J&J, MOR101 and MOR102, anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies being developed by MorphoSys, MOR201, an anti -fibroblast growth factor receptor 3 (FGFR-3) antibody being developed by MorphoSys, 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 5β1 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® (Omalizumab) a humanized anti-IgE antibody developed by Genentech and Novartis, and MLN01, an anti-Beta2 integrin antibody being developed by Xoma. All of the above-cited references in this paragraph are expressly incorporated herein by reference in their entirety.

[00121] Non-protein binding targets are also contemplated, and include, for example, lectins, cytokines, lipids, carbohydrates, and small molecules (e.g., folate).

Linker Components

[00122] 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

component. Non-limiting examples of 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-dioxopyrrolidin-l-yl 4-(pyridin-2- yldisulfanyl) butanoate forming linker moiety 4-mercaptobutanoic acid ("SPDB"), N- Succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"), ethyl eneoxy -CH ₂ CH ₂ 0- as one or more repeating units ("EO" or "PEO"). In one aspect, a linker can be a cleavable linker that is configured decouple a targeting component form an oligonucleotide component under cleavage- promoting conditions. For example, an acid-labile linker (e.g., a hydrazine linker), protease- sensitive (e.g., a peptidase-sensitive) linker, photolabile linker, dimethyl linker or disulfide- containing linker (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020; incorporated herein by reference) can be used.

[00123] In one aspect, a linker component can comprise a polypeptide. Examples of 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).

[00124] In one aspect, a linker comprises a chemical composition that does not include amino acid residues (e.g., does not comprise a polypeptide).

[00125] In one aspect, a linker comprises a tetrazine ligation linker. A non-limiting example of a tetrazine ligation linker is provided in formula (I), below:

(I)

[00126] In one aspect, a linker comprises a strain-promoted-azide-alkylene (SPAAC) linker (also referred to herein as a dibenzylcycootyne (DBCO) linker). A non-limiting example of an SPAAC linker is provided in formula (II), below:

(Π) [00127] In one aspect, a linker comprises a maleimide linker, which comprises the chemical formula H ₂ C ₂ (CO) ₂ H. In one aspect, a linker comprises a succinimide linker, which comprises the chemical formula C ₄ H ₅ N0 ₂ . In one aspect, a linker comprises a hydrazone linker, which comprises the chemical formula In one aspect, a linker comprises a hydrazine linker, which comprises the chemical formula H ₂ N H ₂ .

[00128] In one aspect, a linker comprises a tyrosine linker. A non-limiting example of a tyrosine linker is provided in formula (III), below:

(III)

[00129] In one aspect, a linker comprises a chemoenzymatic linker wherein a primary amine reacts with an LLQG acceptor site on a modified polypeptide or protein. A non-limiting example of a chemoenzymatic linker is provided in formula (IV), below:

(IV)

Cleavage Components

[00130] 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. Examples of 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.

[00131] In one aspect, a cleavage component comprises a polypeptide sequence. In one aspect, 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. In one aspect, 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); Examples of 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).

[00132] In one aspect, a cleavage component comprises a polynucleotide sequence. In one aspect, 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. In one aspect, 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.

[00133] In one aspect, a restriction endonuclease is functional at room temperature and in conditions that are non-cytotoxic to cells. In one aspect, 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).

Oligonucleotide Components

[00134] An aspect of the disclosure includes oligonucleotide components that comprise a plurality of nucleic acid residues. In one aspect, 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.

[00135] In one aspect, 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.. For example, in one aspect, 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. In some embodiments, the identifier sequence is a single stranded oligonucleotide. In some embodiments, the identifier sequence is a single stranded DNA or a single stranded PNA In some embodiments, the identifier sequence identifies the targeting component. In one aspect, an 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, an identifier sequence is 8 nucleic acids in length. In one aspect, the identifier sequence comprises at least one trinucleotide repeat (FIG. 21A). In one aspect, the identifier sequence does not comprise a trinucleotide repeat. In one aspect, the identifier sequence has a CG content >= 0.25% and <= 0.55%. Examples of identifier sequences include, but are not limited to, SEQ ID NOS: 1-96 and SEQ ID NOS: 205-300, as shown in Table 1.

[00136] In another aspect, 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. For example, in one aspect, 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. In some embodiments, the first identifier sequence is a single stranded

oligonucleotide. In some embodiments, 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. In one aspect, the first identifier sequence has a CG content >= 0.25% and <= 0.55%. In some embodiments, the second identifier sequence is a single stranded oligonucleotide. In some embodiments, the second identifier sequence is a single stranded DNA or a single stranded PNA In some embodiments, the second identifier sequence identifies the targeting component. In one aspect, 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. In one aspect, a second identifier sequence is 20 nucleic acids in length. In one aspect, the second identifier sequence comprises at least one trinucleotide repeat (FIG. 21A). In one aspect, the second identifier sequence does not comprise a trinucleotide repeat. In one aspect, the second identifier sequence has a CG content >= 0.25% and <= 0.55%. Examples of 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. Examples of second identifier sequences include, but are not limited to, SEQ ID NOS: 301-396, as shown in Table 2.

[00137] In some embodiments, 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.

Identifier Sequence SEQ ID NO:

CATGGTGT 31

CATATAGT 32

C ATT AG C A 33

CTGGTATT 34

CTGAAGAT 35

CTCCACTT 36

CTCATGAA 37

CTCTTTTT 38

CTAAGCGA 39

CTTTGCAA 40

CTTTTGTC 41

AGCCTTGA 42

AG CT AC A A 43

AGATCCGT 44

AGTGGTTT 45

AGTAGATT 46

AGTAATGT 47

AGTATCGG 48

ACGTGAAT 49

ACATGCGA 50

ACATATGT 51

ACTCGTAT 52

ACTCATCC 53

ACTTTGAC 54

AACGTACC 55

A AC A AG GT 56

AAAAGATG 57

AAATGCGC 58

AAATTACC 59

AATCTAAC 60

AATTGGGT 61

AATTATGG 62

ATGCCAAC 63

ATGATGCA 64

ATCCGAAT 65

ATCCGTTT 66

ATCCAGAC 67

ATCTCTCT 68

ATAGCGTT 69

ATACGTAC 70

ATAACGAC 71

ATTGCCCT 72

ATTTCTCT 73

TGGGTACA 74

TGCACTTC 75

TGATCAAG 76 Identifier Sequence SEQ ID NO:

TGTATGAA 77

TGTTAAGT 78

TCAGAACG 79

TCTGTCAA 80

TCTTATCG 81

TAGCAAAG 82

TAACACCA 83

TAAAGAGC 84

TATGACGG 85

TATGAACC 86

TATGATGG 87

TATCAGTA 88

TTGGACGT 89

TTGCCAAA 90

TTGCTATA 91

TTCTATCT 92

TTCTTGTT 93

TTTGAGAT 94

1 1 1 1 1 1 95

1 1 1 1 CAGC 96

GGCGTATT 205

GGCCAATT 206

GGCAATGT 207

GGCATATC 208

GGCTTGAT 209

GGAGTTAA 210

GGACAGAA 211

GG AC AC AT 212

GG AC AT AC 213

GGACTTCA 214

GGAACTAG 215

G GAT AT A A 216

GGATTCAC 217

GGATTACT 218

GGTGAATC 219

GGTGATGA 220

GGTCAACT 221

GGTCTGTT 222

GGTAGAGT 223

GGTTGAGA 224

GCGAGAAT 225

GCGAATCA 226

GCCAGTAT 227

GCCACAAT 228

G CC ATTGT 229

GCAGAGTA 230 Identifier Sequence SEQ ID NO:

GCAGATAG 231

GCAACCTA 232

GCAACATT 233

GCAATAGA 234

GCAATTGC 235

GCATGCTA 236

GCATGACA 237

GCATCCAA 238

GCATCTAG 239

GCATATGT 240

GCTGTAAC 241

GCTGTTCT 242

GCTCATTC 243

GCTAGATG 244

GCTAACTC 245

GCTATCAT 246

GCTTCTAC 247

GAGCGATT 248

GAGCAGAA 249

GAGACGAA 250

GAGACACA 251

GAGTAAGC 252

GAGTAATT 253

GAGTTATG 254

GACGAATC 255

GACCATAC 256

GACAGTCT 257

GACACCAA 258

GACACCTT 259

GACACTGT 260

GACTGGTT 261

GACTGTGT 262

GACTCTTG 263

GACTTGTA 264

GACTTCGT 265

GACTTCAC 266

GAAGGACT 267

GAAGACCA 268

GAACTGGT 269

G A ACT ACT 270

GAATCGGA 271

GAATCGAC 272

GAATCCTG 273

GAATAACG 274

GATGGAGT 275

GATGAGAC 276 Identifier Sequence SEQ ID NO:

GATGTCAC 277

GATCGTCT 278

GATCATAT 279

GATCTAGG 280

GATAGGTA 281

GATAGTGG 282

GATACCAG 283

GATACATG 284

GATATACT 285

GATTGACC 286

GATTGAAT 287

GATTGTAG 288

GATTGTTA 289

GATTCGGT 290

GATTAGTT 291

GATTACAA 292

GTGGCATA 293

GTGGAGTA 294

GTGGATAG 295

GTGGTGTT 296

GTGAGTGA 297

GTGAAGAC 298

GTGAACGA 299

GTGTCGTT 300

Table 2: Example second identifier sequences.

Identifier Sequence SEQ ID NO:

AGAAGTTAAGGCAGCGGCGA 301

TAACTTACCACTCAGCCGCC 302

ATCGAATTCACGCCGGTCAC 303

TGAATCTTCTCCGCCGACAG 304

TCCTATAAGTCCAG CG GCTC 305

AATCAATCGACGGAGCGACC 306

TTC AACTAAGTG CG G AG CG C 307

CTTAGAACTTGCCGTCGCTC 308

AATCTTGAGTCGGACGCTGC 309

TTCATTATCGTGCGCAGGCC 310

TTAACACGATTCCGGCCACC 311

AATATGGTCTCGGTCGACCG 312

TTGAGATTAGACCGAGCGCC 313

CG ATTACTA AG CGGCGTCTG 314

TTATCGCTATTCCTGCGGCG 315

TTCTATCATGTGCCGCAGGC 316

ATGTATGTTGCGCGTCCGCA 317

TAATCCACTTCTTGGCCGCG 318

TCTCTTATCACCTCGGTCGG 319 TA AG C A ATCTCTG AG G CG CC 320

AAG G AACTAAGTG CCGCCGT 321

TGTATAACGACCGACGTGCG 322

GGATACTATTGGCGGCTCGT 323

TAATGACCATCTCTGCGCGC 324

G GT ACTT ATTG GCGGATGCC 325

TTG ACTTAC A ACC AG CG CG G 326

ACTATTCGAAGTGGCTGGCG 327

AACTGAATAGCGGCGTCGCT 328

TTAGATTCGATGTCCGGCCG 329

TGCAGTATAACCGGTCACGC 330

TTACTACATCTCGCCGGTCG 331

ATTAGAGCATCCGGCGTGAG 332

ACCAGTAATTGAGGCCGCGT 333

ACAAGATAACGAGCACGGCG 334

AAGAACAGAAGTCGGTCGCC 335

TACGTAACATCACCGCTCGG 336

GAAGATTCTTGGCCGGATGG 337

TGTAG AACTACCG CTCTCG C 338

C A AG ATA AC AG CG CG AC AG G 339

CTTAATTGAGGCCAGCGCCT 340

TTAATCCAC ATCCG CG G AG C 341

TAGCAATGATCAGCGCGCAG 342

AT ACTTG AAG CG CTCTCG CG 343

T ATG CTC ATT AG G CTCCG G C 344

TAACTGTAACCTCCGCGACG 345

CTATCATGTTGCCGCCTCAC 346

GAACTAACATGGCCGCAGTC 347

CGTACTATAAGCGGCCGTCA 348

CATAAGTGTAGCGCTTGGCG 349

GGTAATACTAGGCGGTGCGA 350

G G A AC A AT ATG GCGGCCACA 351

ATATGACGAACGACGCGCAC 352

CAGAAGATAAGCGGTCGGTG 353

ACATAATGCTGACGCAGGCG 354

TAGTAGGAATCACGCGCGCT 355

AT ACTG G ATTCG CTCG C ACG 356

CTAACTCATTGCCGCGGTCT 357

GTCTATAGTTGGAGGCTGCC 358

GTCCTTATAAG GCTCTG CG C 359

G G C ATTA ATAG G CG G CG C A A 360

CATTACTAAGGCGACTCGCC 361

TTCGATGTTATGGCGCCGGT 362

TCTGTAATC ACCTG CCGG CT 363

ACGTTATTGTGAGGCGCTGG 364

TCTAATCTAG CCTCCGG CC A 365

ATTAG CTTCTCCG G CG AG CT 366 ATTAAG CCAACCG GCAGTCC 367

CTGTTAACTAG CGTCG CC AC 368

TCTAGTATCACCTCGGCTGC 369

ATCTGAGTATCGCCACGCGT 370

GGAATAAGTAGGCGGCAGGA 371

ATTCCAATACCGTCGTG CCG 372

A ATGTT ATG GCGGCTGCCAG 373

ATCTCCTAATCGCCACCTGC 374

GGAAGATAATGGCGGCCGTT 375

ACATCAAGATGACGCGGCAC 376

ATAACAGCATCGAGGTCCGG 377

ATATACGCATCGACAGCGCG 378

AAGAGACTTAGTGACCGGCG 379

TTGTACATAGAACGGCGCCG 380

TTAGTTACCTTCGGCGGTGC 381

CTA AC ATAG AGCCGCGGCAT 382

ATTGTGATTGCGTGACGCGC 383

ATTATCAGGACCGGCTACGC 384

ATAATTCGGACGAGACGCGG 385

GTATTCGATAGGTGGCACGC 386

GTGAATATGTGGCTGACCGG 387

TAAGATTCAGCTGTGGCCGC 388

CAAG GTTATAG CG CG CTTCC 389

TTCTCTACATTGCGCCGGAC 390

AGATGTACTAGCAGAGGCGC 391

CCATACTTAAGCGGCTACGG 392

TAGTAACTTCCACGCGCCGA 393

C ATG A ATG ATG CGCAGCCAC 394

GAGTATCTATGGCGACCTCC 395

TGTACATCAACCGCTCCTGC 396

[00138] In one aspect, an oligonucleotide component comprises first universal sequence and second universal sequence that are configured for sequencing analysis. In one aspect, 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. In one aspect, 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. In one aspect, 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.

Universal Sequences SEQ ID NO:

ACGCAGAACAAAGACTGC 196

AGGGTCTACCGTCCGT 197

C ATTG GCTGGGTG A ATTC 397

AACG AATCC ACCCG C AG 398

[00139] In one aspect, 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. In one aspect, 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.

[00140] In one aspect, 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. In one aspect, 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.

[00141] In one aspect, 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 of full oligonucleotide component sequences include, but are not limited to, SEQ ID NOS: 100-195 and SEQ ID NOS: 399-446, as shown in Table 4.

Table 4: Example full oligonucleotide component sequences.

Full Oligonucleotide Component Sequence SEQ ID

NO:

CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/

/5BioSG/ AGACTACTCCACAGCTGAGC AACGAATCCACCCGCAG GGTGTTCT 102 CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/

/5BioSG/ GACCCCGATACTTACACGAG AACGAATCCACCCGCAG GCAAAAAC 103 CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/

/5BioSG/ AGTACGCTTACACCCGACAG AACGAATCCACCCGCAG GCTTCGTA 104 CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/

/5BioSG/ CGCCGAAGTATAACCTACGC AACGAATCCACCCGCAG GAGGACTA 105 CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/

AACGAATCCACCCGCAG GACACTGT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 106

AACGAATCCACCCGCAG GACAAGAT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 107

AACGAATCCACCCGCAG GACTTTCT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 108

AACGAATCCACCCGCAG GAACTCTA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 109

AACGAATCCACCCGCAG GATCTTGC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 1 10

AACGAATCCACCCGCAG GATTCCCT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 1 1 1

AACGAATCCACCCGCAG GATTAGCT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 1 12

AACGAATCCACCCGCAG GTGGATAA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 1 13

AACGAATCCACCCGCAG GTCAGACA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 1 14

AACGAATCCACCCGCAG GTCTACAT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 1 15

AACGAATCCACCCGCAG GTACTACA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 1 16

AACGAATCCACCCGCAG GTTCAACA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 1 17

AACGAATCCACCCGCAG GTTATCTG CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 1 18

AACGAATCCACCCGCAG CGTTCTCT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 1 19

AACGAATCCACCCGCAG CCGGAAAA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 120

AACGAATCCACCCGCAG CCAATTTC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 121

AACGAATCCACCCGCAG CCATTTGG CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 122

AACGAATCCACCCGCAG CCATTTAA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 123

AACGAATCCACCCGCAG CAGGTTAT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 124

AACGAATCCACCCGCAG CAGCAACT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 125

AACGAATCCACCCGCAG CACGAAAA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 126

AACGAATCCACCCGCAG CAACGATC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 127

AACGAATCCACCCGCAG CAAAGATG CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 128

AACGAATCCACCCGCAG CAAATGGG CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 129

AACGAATCCACCCGCAG CATGGTGT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 130

AACGAATCCACCCGCAG CATATAGT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 131

AACGAATCCACCCGCAG CATTAGCA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 132

AACGAATCCACCCGCAG CTGGTATT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 133

AACGAATCCACCCGCAG CTGAAGAT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 134

AACGAATCCACCCGCAG CTCCACTT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 135

AACGAATCCACCCGCAG CTCATGAA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 136

AACGAATCCACCCGCAG CTCTTTTT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 137

AACGAATCCACCCGCAG CTAAGCGA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 138

AACGAATCCACCCGCAG CTTTGCAA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 139

AACGAATCCACCCGCAG CTTTTGTC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 140

AACGAATCCACCCGCAG AGCCTTGA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 141

AACGAATCCACCCGCAG AGCTACAA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 142

AACGAATCCACCCGCAG AGATCCGT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 143

AACGAATCCACCCGCAG AGTGGTTT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 144

AACGAATCCACCCGCAG AGTAGATT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 145

AACGAATCCACCCGCAG AGTAATGT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 146 Full Oligonucleotide Component Sequence SEQ ID

NO:

AACGAATCCACCCGCAG AGTATCGG CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 147

AACGAATCCACCCGCAG ACGTGAAT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 148

AACGAATCCACCCGCAG ACATGCGA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 149

AACGAATCCACCCGCAG ACATATGT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 150

AACGAATCCACCCGCAG ACTCGTAT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 151

AACGAATCCACCCGCAG ACTCATCC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 152

AACGAATCCACCCGCAG ACTTTGAC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 153

AACGAATCCACCCGCAG AACGTACC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 154

AACGAATCCACCCGCAG AACAAGGT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 155

AACGAATCCACCCGCAG AAAAGATG CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 156

AACGAATCCACCCGCAG AAATGCGC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 157

AACGAATCCACCCGCAG AAATTACC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 158

AACGAATCCACCCGCAG AATCTAAC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 159

AACGAATCCACCCGCAG AATTGGGT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 160

AACGAATCCACCCGCAG AATTATGG CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 161

AACGAATCCACCCGCAG ATGCCAAC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 162

AACGAATCCACCCGCAG ATGATGCA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 163

AACGAATCCACCCGCAG ATCCGAAT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 164

AACGAATCCACCCGCAG ATCCGTTT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 165

AACGAATCCACCCGCAG ATCCAGAC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 166

AACGAATCCACCCGCAG ATCTCTCT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 167

AACGAATCCACCCGCAG ATAGCGTT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 168

AACGAATCCACCCGCAG ATACGTAC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 169

AACGAATCCACCCGCAG ATAACGAC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 170

AACGAATCCACCCGCAG ATTGCCCT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 171

AACGAATCCACCCGCAG ATTTCTCT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 172

AACGAATCCACCCGCAG TGGGTACA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 173

AACGAATCCACCCGCAG TGCACTTC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 174

AACGAATCCACCCGCAG TGATCAAG CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 175

AACGAATCCACCCGCAG TGTATGAA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 176

AACGAATCCACCCGCAG TGTTAAGT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 177

AACGAATCCACCCGCAG TCAGAACG CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 178

AACGAATCCACCCGCAG TCTGTCAA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 179

AACGAATCCACCCGCAG TCTTATCG CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 180

AACGAATCCACCCGCAG TAGCAAAG CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 181

AACGAATCCACCCGCAG TAACACCA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 182

AACGAATCCACCCGCAG TAAAGAGC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 183

AACGAATCCACCCGCAG TATGACGG CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 184

AACGAATCCACCCGCAG TATGAACC CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 185

AACGAATCCACCCGCAG TATGATGG CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 186

AACGAATCCACCCGCAG TATCAGTA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 187

AACGAATCCACCCGCAG TTGGACGT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 188

AACGAATCCACCCGCAG TTGCCAAA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 189

AACGAATCCACCCGCAG TTGCTATA CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 190

AACGAATCCACCCGCAG TTCTATCT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 191

AACGAATCCACCCGCAG TTCTTGTT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 192

AACGAATCCACCCGCAG TTTGAGAT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 193

AACGAATCCACCCGCAG TTTTCGTT CATTGGCTGGGTTGCTCA ACACACA /3 AmMO/ 194 Full Oligonucleotide Component Sequence SEQ ID

NO:

AACGAATCCACCCGCAG TTTTCAGC CATTGGCTGGGTTGCTCA AC AC AC A /3 AmMO/ 195

/5BioSG/ AGAAGTTAAGGCAGCGGCGA AACGAATCCACCCGCAG GGCGTATT

CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 399

/5BioSG/ TAACTTACCACTCAGCCGCC AACGAATCCACCCGCAG GGCCAATT

CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 400

/5BioSG/ TGAATCTTCTCCGCCGACAG AACGAATCCACCCGCAG GGCATATC

CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 401

/5BioSG/ TCCTATAAGTCCAGCGGCTC AACGAATCCACCCGCAG GGCTTGAT

CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 402

/5BioSG/ AATCAATCGACGGAGCGACC AACGAATCCACCCGCAG GGAGTTAA

CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 403

/5BioSG/ TTCAACTAAGTGCGGAGCGC AACGAATCCACCCGCAG GGACAGAA

CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 404

CTTAGAACTTGCCGTCGCTC AACGAATCCACCCGCAG GGACACAT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 405

AATCTTGAGTCGGACGCTGC AACGAATCCACCCGCAG GGACATAC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 406

TTCATTATCGTGCGCAGGCC AACGAATCCACCCGCAG GGACTTCA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 407

TTAACACGATTCCGGCCACC AACGAATCCACCCGCAG GGAACTAG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 408

AATATGGTCTCGGTCGACCG AACGAATCCACCCGCAG GGATATAA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 409

TTGAGATTAGACCGAGCGCC AACGAATCCACCCGCAG GGATTCAC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 410

CGATTACTAAGCGGCGTCTG AACGAATCCACCCGCAG GG ATT ACT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 411

TTATCGCTATTCCTGCGGCG AACGAATCCACCCGCAG GGTGAATC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 412

TTCTATCATGTGCCGCAGGC AACGAATCCACCCGCAG GGTGATGA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 413

ATGTATGTTGCGCGTCCGCA AACGAATCCACCCGCAG GGTCAACT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 414

T AATCC ACTTCTTG G CCG CG AACGAATCCACCCGCAG GGTCTGTT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 415

TCTCTTATC ACCTCG GTCG G AACGAATCCACCCGCAG GGTAGAGT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 416

TAAGCAATCTCTGAGGCGCC AACGAATCCACCCGCAG GGTTGAGA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 417

AAGGAACTAAGTGCCGCCGT AACGAATCCACCCGCAG GCGAGAAT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 418

TGTATAACGACCGACGTGCG AACGAATCCACCCGCAG GCGAATCA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 419

GGATACTATTGGCGGCTCGT AACGAATCCACCCGCAG GCCAGTAT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 420

TAATGACCATCTCTGCGCGC AACGAATCCACCCGCAG GCCACAAT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 421

G GT ACTTATTG G CG G ATG CC AACGAATCCACCCGCAG GCCATTGT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 422

TTGACTTACAACCAGCGCGG AACGAATCCACCCGCAG GCAGAGTA CATTGGCTGGGTGAATTC 423 Full Oligonucleotide Component Sequence SEQ ID

NO:

ACACACA /3AmMO/

ACTATTCGAAGTGGCTGGCG AACGAATCCACCCGCAG GCAGATAG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 424

AACTGAATAGCGGCGTCGCT AACGAATCCACCCGCAG GCAACCTA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 425

TTAGATTCGATGTCCGGCCG AACGAATCCACCCGCAG GCAACATT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 426

TG C AGTATA ACCG GTC ACG C AACGAATCCACCCGCAG GCAATAGA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 427

TTACTACATCTCGCCGGTCG AACGAATCCACCCGCAG GCAATTGC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 428

ATTAGAGCATCCGGCGTGAG AACGAATCCACCCGCAG GCATGCTA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 429

ACCAGTAATTGAGGCCGCGT AACGAATCCACCCGCAG GCATGACA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 430

ACAAGATAACGAGCACGGCG AACGAATCCACCCGCAG GCATCCAA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 431

AAGAACAGAAGTCGGTCGCC AACGAATCCACCCGCAG GCATCTAG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 432

TACGTAACATCACCGCTCGG AACGAATCCACCCGCAG GCATATGT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 433

GAAGATTCTTGGCCGGATGG AACGAATCCACCCGCAG GCTGTAAC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 434

TGTAGAACTACCGCTCTCGC AACGAATCCACCCGCAG GCTGTTCT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 435

CAAGATAACAGCGCGACAGG AACGAATCCACCCGCAG GCTCATTC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 436

CTTAATTGAGGCCAGCGCCT AACGAATCCACCCGCAG GCTAGATG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 437

TTAATCCACATCCGCGGAGC AACGAATCCACCCGCAG GCTAACTC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 438

TAGCAATGATCAGCGCGCAG AACGAATCCACCCGCAG GCTATCAT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 439

ATACTTGAAGCGCTCTCGCG AACGAATCCACCCGCAG GCTTCTAC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 440

TATGCTCATTAGGCTCCGGC AACGAATCCACCCGCAG GAGCGATT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 441

TAACTGTAACCTCCGCGACG AACGAATCCACCCGCAG GAGCAGAA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 442

CTATC ATGTTG CCG CCTC AC AACGAATCCACCCGCAG GAGACGAA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 443

G A ACTA AC ATG G CCG C AGTC AACGAATCCACCCGCAG GAGACACA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 444

CGTACTATAAGCGGCCGTCA AACGAATCCACCCGCAG GAGTAAGC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 445

C AT AAGTGT AG CG CTTG G CG AACGAATCCACCCGCAG GAGTAATT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 446

GGTAATACTAGGCGGTGCGA AACGAATCCACCCGCAG GAGTTATG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 399

GGAACAATATGGCGGCCACA AACGAATCCACCCGCAG GACGAATC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 400 Full Oligonucleotide Component Sequence SEQ ID

NO:

ATATGACGAACGACGCGCAC AACGAATCCACCCGCAG GACCATAC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 401

CAGAAGATAAGCGGTCGGTG AACGAATCCACCCGCAG GACAGTCT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 402

ACATAATGCTGACGCAGGCG AACGAATCCACCCGCAG GACACCAA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 403

TAGTAGGAATCACGCGCGCT AACGAATCCACCCGCAG GACACCTT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 404

ATACTGGATTCGCTCGCACG AACGAATCCACCCGCAG GACACTGT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 405

CTA ACTC ATTG CCG CG GTCT AACGAATCCACCCGCAG GACTGGTT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 406

GTCT AT AGTTG G AGG CTG CC AACGAATCCACCCGCAG GACTGTGT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 407

GTCCTT ATA AG G CTCTG CG C AACGAATCCACCCGCAG GACTCTTG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 408

GGCATTAATAGGCGGCGCAA AACGAATCCACCCGCAG GACTTGTA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 409

C ATTACTA AG G CG ACTCG CC AACGAATCCACCCGCAG GACTTCGT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 410

TTCGATGTTATGGCGCCGGT AACGAATCCACCCGCAG GACTTCAC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 411

TCTGTAATCACCTGCCGGCT AACGAATCCACCCGCAG GAAGGACT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 412

ACGTTATTGTGAGGCGCTGG AACGAATCCACCCGCAG GAAGACCA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 413

TCTAATCTAGCCTCCGGCCA AACGAATCCACCCGCAG GAACTGGT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 414

ATTAGCTTCTCCGGCGAGCT AACGAATCCACCCGCAG GAACTACT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 415

ATTAAGCCAACCGGCAGTCC AACGAATCCACCCGCAG GAATCGGA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 416

CTGTTAACTAGCGTCGCCAC AACGAATCCACCCGCAG GAATCGAC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 417

TCT AGTATC ACCTCG G CTG C AACGAATCCACCCGCAG GAATCCTG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 418

ATCTGAGTATCGCCACGCGT AACGAATCCACCCGCAG GAATAACG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 419

GGAATAAGTAGGCGGCAGGA AACGAATCCACCCGCAG GATGGAGT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 420

ATTCCAATACCGTCGTGCCG AACGAATCCACCCGCAG GATGAGAC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 421

AATGTTATGGCGGCTGCCAG AACGAATCCACCCGCAG GATGTCAC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 422

ATCTCCTAATCGCCACCTGC AACGAATCCACCCGCAG GATCGTCT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 423

GGAAGATAATGGCGGCCGTT AACGAATCCACCCGCAG GATCATAT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 424

ACATCAAGATGACGCGGCAC AACGAATCCACCCGCAG GATCTAGG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 425 Full Oligonucleotide Component Sequence SEQ ID

NO:

ATAACAGCATCGAGGTCCGG AACGAATCCACCCGCAG GATAGGTA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 426

ATATACGCATCGACAGCGCG AACGAATCCACCCGCAG GATAGTGG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 427

AAGAGACTTAGTGACCGGCG AACGAATCCACCCGCAG GATACCAG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 428

TTGTACATAGAACGGCGCCG AACGAATCCACCCGCAG GATACATG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 429

TTAGTTACCTTCGGCGGTGC AACGAATCCACCCGCAG GAT AT ACT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 430

CTAACATAGAGCCGCGGCAT AACGAATCCACCCGCAG GATTGACC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 431

ATTGTGATTGCGTGACGCGC AACGAATCCACCCGCAG GATTGAAT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 432

ATTATCAGGACCGGCTACGC AACGAATCCACCCGCAG GATTGTAG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 433

ATAATTCGGACGAGACGCGG AACGAATCCACCCGCAG GATTGTTA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 434

GT ATTCG ATAG GTG G C ACG C AACGAATCCACCCGCAG GATTCGGT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 435

GTGAATATGTGGCTGACCGG AACGAATCCACCCGCAG GATTAGTT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 436

TAAGATTCAGCTGTGGCCGC AACGAATCCACCCGCAG GATTACAA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 437

CAAGGTTATAGCGCGCTTCC AACGAATCCACCCGCAG GTG G CAT A CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 438

TTCTCTACATTGCGCCGGAC AACGAATCCACCCGCAG GTGGAGTA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 439

AGATGTACTAGCAGAGGCGC AACGAATCCACCCGCAG GTGGATAG CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 440

CCATACTTAAGCGGCTACGG AACGAATCCACCCGCAG GTGGTGTT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 441

TAGTAACTTCCACGCGCCGA AACGAATCCACCCGCAG GTGAGTGA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 442

CATGAATGATGCGCAGCCAC AACGAATCCACCCGCAG GTGAAGAC CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 443

GAGTATCTATGGCGACCTCC AACGAATCCACCCGCAG GTGAACGA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 444

TGTAC ATC AACCG CTCCTG C AACGAATCCACCCGCAG GTGTCGTT CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 445

ATCATTCCATCGCCAGCGGT AACGAATCCACCCGCAG GTGTCACA CATTGGCTGGGTGAATTC ACACACA /3AmMO/ 446

[00142] In one aspect, 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. For example, in one aspect, a spacer sequence can be placed between a universal sequence and an identifier sequence. In one aspect, 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).

[00143] In some embodiments, 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, a ribonucleic acid (RNA), and a deoxyribonucleic acid (DNA).

Diagnostic Moieties

[00144] Targeting components of the present disclosure can be linked to at least one diagnostic moiety. A "diagnostic moiety" as used herein refers to any moiety that can be detected using techniques that are known in the art, e.g., an assay, an imaging technique, etc. Non-limiting examples 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.

[00145] 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). Non-limiting examples of imaging moieties include paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.

[00146] 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).

[00147] Non-limiting examples of radioactive isotopes include ²¹¹ astatine, ¹⁴ carbon,

51 36 57 58 67 152 67 3 123

chromium, chlorine, cobalt, cobalt, copper, Eu, gallium, hydrogen, iodine,

125 iodine, 131 iodine, 111 indium, 59 iron, 32 phosphorus, 186 rhenium, 188 rhenium, 75 selenium, ³⁵ sulphur, ^99m technicium and/or ⁹⁰ yttrium. 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. Alternatively, 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).

[00148] 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.

[00149] In one aspect, 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. Non- limiting examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase. Non-limiting examples of binding ligands include biotin, avidin and streptavidin compounds.

[00150] Several methods are known in the art for the attachment or conjugation of moiety to a targeting component (e.g., an antibody). Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a

diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and/or tetrachloro-3a-6a-diphenylglycouril-3 attached to the targeting component (U.S. Pat. Nos. 4,472,509 and 4,938,948, which are incorporated by reference herein). 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. In U.S. Pat. No. 4,938,948, 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.

[00151] In one aspect, derivatization of targeting components is accomplished by selectively introducing sulfhydryl groups into the targeting component. For example, in the case of an antibody 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).

[00152] In some embodiments, the diagnostic moiety is biotin (FIG. 17).

[00153] In some embodiments, the diagnostic moiety is displaced by hybridization of an oligonucleotide that is fully complementary to the entire oligonucleotide component of the first oligonucleotide conjugate. In some embodiments, diagnostic moiety is cleaved from the first oligonucleotide conjugate using a nuclease. In some embodiments, the nuclease is a DNase. In some embodiments, the DNase is a double strand specific DNase.

Secondary Oligonucleotide Conjugates

[00154] An aspect of the disclosure includes therapeutic and diagnostic secondary

oligonucleotide conjugates that comprise an oligonucleotide component and a therapeutic or a diagnostic moiety. In one aspect, a secondary oligonucleotide conjugate comprises a linker component that links the oligonucleotide component to the therapeutic or diagnostic moiety. In some embodiments, the second identifier sequence is hybridized to a secondary oligonucleotide conjugate. In some embodiments, the secondary oligonucleotide conjugate comprises a second oligonucleotide component that is attached to a diagnostic moiety.

[00155] In some embodiments, 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, a ribonucleic acid (RNA), and a deoxyribonucleic acid (DNA).

[00156] In one aspect, 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.

Diagnostic Moieties

[00157] As reviewed above, an aspect of the disclosure includes diagnostic secondary oligonucleotide conjugates that comprise a diagnostic moiety. Examples of 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).

Therapeutic Moieties

[00158] As reviewed above, 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,

immunomodulatory activity, or thrombogenic 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

polynucleotides, polypeptides, antibodies, antigen-binding fragments of antibodies, antibody- drug conjugates (ADCs), and cells.

[00159] 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).

[00160] In some embodiments, 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.

[00161] Disclosed herein, in certain embodiments, are 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. In some embodiments, the cell is a cancer cell.

[00162] Disclosed herein, in certain embodiments, are 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. In some embodiments, the cell is a cancer cell. In some embodiments, 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. In some embodiments, the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). In some embodiments, the maytansinoid comprises DM1 (mertansine) or DM4. In some embodiments, the

pyrrolobenzodiazepine is a pyrrolobenzodiazepine dimer. In some embodiments, the taxane is taxol, docetaxel, paclitaxel, larotaxel, tesetaxel, or orataxel. In some embodiments, 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.

[00163] Disclosed herein, in certain embodiments, are 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. In some embodiments, the cell is a cancer cell. In some embodiments, the protein is an enzyme or a toxin. In some embodiments, 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. In some embodiments, 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. In some embodiments, the fusarium is is a fumonisin, a trichothecene, or a zearalenone. In some embodiments, the plant toxin is a ribosome-inactivating protein (RIP). In some embodiments, the RIP is a type 1 RIP or a type 2 RIP. In some embodiments, the type 1 RIP is PAP (pokeweed antiviral protein), gelonin, saporin, curcin, curcin-L, or derivatives thereof. In some embodiments, the saporin is saporin-S6. In some embodiments, the type 2 RIP is ricin, abrin, modeccin, pulchellin, mistletoe lectin I, or volkensin, In some embodiments, the animal toxin is a snake venom or an arthropod venom.. In some embodiments, the snake venom is a venom from a snake in the family Crotalid, Elapid, or Viperid. In some embodiments, the arthropod is a scorpion, a bee, a wasp, a spider, an ant, a centipede, or a caterpillar, In some embodiments, the toxin is an isolated component of an animal toxin. In some embodiments, 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. In some embodiments, 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. coli enterotoxin toxin A subunit, cholera toxin A subunit and Pseudomonas toxin c-terminal, abrin, A/B heat labile toxins, Botulinum toxin, Helix pomatia, Jacalin or Jackfruit, Peanut agglutinin, Sambucus nigra, Tetanus, Ulex, and Viscumin. In some embodiments, the enzyme is a therapeutic enzyme. In some embodiments, the enzyme is an aminopeptidase, an alkaline phosphatase (AP), a glycosidase, a beta galactosidase, horseradish peroxidase (HRP), a penicillin amidase, a β-lactamase, a cytosine deaminase, a nitroreductase, In one aspect, an enzyme works in conjunction with a prodrug by converting the pro-drug to an active composition in the vicinity of the enzyme.

[00164] Disclosed herein, in certain embodiments, are 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. In some embodiments, the cell is a cancer cell. In some embodiments, 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. In some embodiments, the xanthene derivative is fluorescein, rhodamine, Oregon green, eosin, or Texas red. In some embodiments, the fluorophore moiety is a fluorescent label as described herein.

[00165] In one aspect, a therapeutic moiety is a thrombogenic agent. Non-limiting examples of 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.

[00166] In one aspect, a therapeutic moiety is an anti-angiogenic agent. Non-limiting examples of anti-angiogenic agents include drugs that block the pro-angiogenic function of vascular endothelial growth factor (VEGF). Other examples include SU5416, AG3340, endostatin, angiostatin, squalamine, thalidomide, CAI, Neovastat, and 2-methoxyestradiol.

[00167] 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 toluene 2,6-diisocyanate), and bis- active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, 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.

ADDITIONAL ASPECTS

[00168] In one aspect, an oligonucleotide conjugate comprises one or more artificial components. In one aspect, an oligonucleotide conjugate comprises a biopolymer. In one aspect, an oligonucleotide component comprises an antibody (immunoglobulin). In one aspect, an oligonucleotide conjugate comprises an antibody that comprises a variable region. In one aspect, an antibody comprises an antigen-binding site. In one aspect, an oligonucleotide conjugate comprises an intact immunoglobulin. In one aspect, an oligonucleotide conjugate comprises an immunoglobulin fragment. In one aspect, an oligonucleotide conjugate comprises an Fv immunoglobulin fragment. In one aspect, an oligonucleotide conjugate comprises an scFv immunoglobulin fragment. [00169] In one aspect, an oligonucleotide conjugate comprises a native polypeptide. In one aspect, an oligonucleotide conjugate comprises a monoclonal antibody. In one aspect, an oligonucleotide conjugate comprises a chimeric antibody. In one aspect, an oligonucleotide conjugate comprises a humanized antibody. In one aspect, an oligonucleotide conjugate comprises a human antibody. In one aspect, an oligonucleotide conjugate comprises an isolated antibody. In one aspect, an antibody specifically binds to a binding target with a binding affinity. In one aspect, an oligonucleotide conjugate comprises two or more polypeptide components that have been linked by fusion. In one aspect, a targeting component binds to an epitope on a binding target. In one aspect, an antibody binds to an antigen. In one aspect, two or more portions or components of an oligonucleotide conjugate are operably linked to one another.

[00170] In one preferred embodiment, 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. In one preferred embodiment, 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.

[00171] In one preferred embodiment, 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. In one preferred embodiment, 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.

[00172] In one preferred embodiment, 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. In one preferred embodiment, 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.

[00173] In one preferred embodiment, 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. In one preferred embodiment, 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.

[00174] In one preferred embodiment, 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. In one preferred embodiment, 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.

[00175] In one preferred embodiment, 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. In one preferred embodiment, 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.

[00176] In one preferred embodiment, 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. In one preferred embodiment, 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.

[00177] In one preferred embodiment, 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. In one preferred embodiment, 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.

[00178] In one preferred embodiment, 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. In one preferred embodiment, 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.

[00179] In one preferred embodiment, 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. In one preferred embodiment, 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.

Methods of Synthesis

[00180] An aspect of the disclosure includes methods of synthesizing the oligonucleotide conjugates described herein. In one aspect, the subject synthesis methods involve immobilizing one or more components of an oligonucleotide conjugate on a solid support. Non-limiting examples of 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. In one aspect, an affinity resin is a cationic affinity resin that comprises diethylaminoethanol (DEAE) beads.

[00181] In one aspect, a component of an oligonucleotide conjugate is covalently immobilized on a solid support (i.e., covalent bonds are formed between the component of the

oligonucleotide conjugate and the molecules of the solid support). In one aspect, 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). In one aspect, 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. In one aspect, both a first and a second component of an oligonucleotide conjugate are non-covalently immobilized on a first and a second solid support.

[00182] Once immobilized on a solid support (either covalently or non-covalently), a component of an oligonucleotide conjugate can be contacted with one or more reagents that chemically modify the component. For example, in one aspect, an oligonucleotide component is immobilized on a solid support and is contacted with a reagent to create a functionalized oligonucleotide component. In one aspect, 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. In one aspect, 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.

[00183] 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. In one aspect, a functionalized targeting component is contacted with an oligonucleotide component to generate an oligonucleotide conjugate. In one aspect, a functionalized oligonucleotide component is contacted with a targeting component to generate an oligonucleotide conjugate. In one aspect, a functionalized targeting component is contacted with a functionalized oligonucleotide component to generate an oligonucleotide conjugate.

[00184] In one aspect, 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. For example, in one aspect, 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

oligonucleotide conjugate. In one aspect, 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. For example, in one aspect 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. In one aspect, 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.

[00185] In one aspect, 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. For example, in one aspect, 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.

[00186] In one aspect, a subject method of synthesis involves attaching (e.g., conjugating) a moiety to a targeting component of an oligonucleotide conjugate. For example, in one aspect, a method involves attaching a detectable moiety to a targeting component. In one aspect, a method involves attaching a therapeutic moiety to a targeting component. In one aspect, a moiety is attached to a targeting component while the targeting component is immobilized on a solid support. In one aspect, a moiety is attached to a targeting component when the targeting component is separated from a solid support (e.g., in solution).

[00187] In one aspect, 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. In one aspect, 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.

[00188] In one aspect, isolating an oligonucleotide conjugate involves contacting the oligonucleotide conjugate with a magnetic bead. In one aspect, 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.

PHARMACEUTICAL COMPOSITIONS

[00189] For therapeutic uses, oligonucleotide conjugates in accordance with an aspect of the disclosure can be formulated into pharmaceutical compositions. In some embodiments, 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. For example, 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.

[00190] 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.

[00191] Actual dosage levels of the active ingredients in the pharmaceutical 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.

[00192] A composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, a pharmaceutical carrier preferably comprises an isotonic buffered saline solution.

METHODS OF USE

[00193] 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. As such, 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. In some embodiments, use of an oligonucleotide conjugate in a quality assurance assay comprises use of the oligonucleotide conjugate for detecting the presence of a contaminant. In some embodiments, the contaminant is a microbe. In some embodiments, the microbe is a bacterium, fungi, virus, protozoan, or a combination thereof.

Diagnostic Methods

Determination Methods

[00194] 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. As reviewed above, the subject oligonucleotide conjugates comprise a targeting component, a linker component, a cleavage component, and an oligonucleotide component. In one aspect, an oligonucleotide component comprises an identifier sequence that identifies a binding target of a targeting component. In one example of a diagnostic method, 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. In some embodiments, 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. In some embodiments, the cleavage component is not cleaved. If an identifier sequence for a given binding target is found to be present, then 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. In some embodiments, the identifier sequence is a first identifier sequence and the oligonucleotide component further comprises a second identifier sequence.

[00195] The subject diagnostic methods are particularly useful in the identification of cell surface proteins in a sample. For example, in one aspect, a targeting component of an

oligonucleotide conjugate comprises an antibody that binds to a cell surface protein. In some embodiments, 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.

[00196] In one aspect, after a sample has been contacted with a first oligonucleotide conjugate and any unbound first oligonucleotide conjugate has been removed, the 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.

[00197] In one aspect, 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. In this way, the presence of a plurality of binding targets, e.g., a plurality of cell surface proteins on a cancer cell, can be determined. This information can be utilized, e.g., to diagnose specific types of cancer that involve the expression of specific combinations or patterns of biomarkers.

[00198] In one aspect, 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. The results provide information regarding a plurality of biomarkers that are present in the sample. In one aspect, 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

(Illumina MiSeq or NextSeq) (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.

[00199] In one aspect, a primer includes an additional 5' sequence. In one aspect, the 5' sequence comprises an NGS adapter, a sample identifier sequence, or a combination thereof. Table 5: Example primer sequences.

Quantification Methods

[00200] 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. [00201] In one aspect, an oligonucleotide component comprises an identifier sequence that identifies a binding target of a targeting component. In one example of a diagnostic method, 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. In another aspect, an oligonucleotide component comprises a first identifier sequence and a second identifier sequence that identifies a binding target of a targeting component. In one example of a diagnostic method, 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.

[00202] The targeting component of the oligonucleotide conjugate binds to the binding target or targets (if present), and any unbound oligonucleotide conjugates are removed. In some embodiments, 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.

[00203] In some embodiments, 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.

[00204] In some embodiments, the methods described herein comprise qPCR to quantify one binding target. In some embodiments, the method further comprises administering to the oligonucleotide component to be quantified by qPCR an intercalating fluorescent dye prior to beginning qPCR. In some embodiments, the intercalating fluorescent dye is SYBR green. In some embodiments, the oligonucleotide primers used for qPCR comprise a forward primer and a reverse primer complementary to universal site 1 and universal site 2.

[00205] In some embodiments, the methods described herein comprise qPCR to quantify more than one binding targets. In some embodiments, the method further comprises administering to the oligonucleotide component to be quantified by qPCR a probe complementary to the first identifier sequence. In some embodiments, the probe comprises a reporter dye and a quencher dye. In some embodiments, 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. In some embodiments, 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.

Antigen Profiling Methods

[00206] In some embodiments, the methods described herein are used to generate an antigen profile. In some embodiments, methods for identification, quantification, or a combination thereof are used to generate an antigen profile. In some embodiments, an antigen profile is generated for a biological sample from a subject. In some embodiments, the subject has or is suspected of having a condition. In some embodiments, the condition is cancer.

[00207] In some embodiments, 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.

[00208] In some embodiments, 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 oligonucleotide conjugates bound to a soluble antigen; 6) isolating the cleaved oligonucleotide components; and 7) determining the soluble antigen profile (FIG. 13).

[00209] In some embodiments, the sample is blood, saliva, a tissue sample, urine, or sputum. In some embodiments, the tissue sample is an abnormal tissue sample (e.g. a biopsy). In some embodiments, the soluble antigen profile from a subject is compared to a second soluble antigen profile. In some embodiments, the second soluble antigen profile is from the same subject. In some embodiments, the second soluble antigen profile is from a different subject. In some embodiments, the soluble antigen profile from a subject is compared to a soluble antigen profile with a known concentration. In some embodiments, the soluble antigen profile from a subject is compared to a plurality of control soluble antigen profiles. In some embodiments, 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. In some embodiments, 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. In some embodiments, the soluble antigen profile from the subject is compared to a standard curve of a control antigen.

[00210] In some embodiments, 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.

[00211] In some embodiments, determining the soluble antigen profile comprises targeted amplification. In some embodiments, targeted amplification comprises qPCR. In some embodiments, determining the antigen profile comprises universal amplification. In some embodiments, targeted amplification comprises NGS.

[00212] In some embodiments, 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).

[00213] In some embodiments, the sample is blood, saliva, a tissue sample, urine, or sputum. In some embodiments, the tissue sample is an abnormal tissue sample (e.g. a biopsy). In some embodiments, the cellular antigen profile from a subject is compared to a second cellular antigen profile. In some embodiments, the second cellular antigen profile is from the same subject. In some embodiments, the second cellular antigen profile is from a different subject. In some embodiments, the cellular antigen profile from a subject is compared to a plurality of control cellular antigen profiles. In some embodiments, 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. In some embodiments, at least one of the control antigens in the control cellular antigen profile comprises control antigens is a purified cellular antigen or a recombinant cellular antigen. In some embodiments, the cellular antigen profile from the subject is compared to a standard curve of a control antigen.

[00214] In some embodiments, the cellular antigen profile comprises an antigen profile of cell surface antigens, intracellular antigens, intraorganellar antigens, or a combination thereof.

[00215] In some embodiments, fixation comprises fixation, permeabilization, or a combination thereof. In some embodiments, fixation comprises use of a fixation agent. In some embodiments, the fixation agent is formaldehyde, paraformaldehyde, formalin, glutaraldehyde, a mercuric chloride-based fixative, dimethyl suberimidate (DMS), or a combination thereof. In some embodiments, the fixation agent is a precipitating fixative. In some embodiments, the precipitating fixative is ethanol, methanol, or acetone. In some embodiments, permeabilization comprises use of a permeabilization agent. In some embodiments, the permeabilization agent is an organic solvent or a detergent. In some embodiments, the organic solvent is methanol or acetone. In some embodiments, the detergent is saponin, Triton X-100, or Tween-20. In some embodiments, a fixation agent is also a permeabilization agent.

[00216] In some embodiments, determining the cellular antigen profile comprises targeted amplification. In some embodiments, targeted amplification comprises qPCR. In some embodiments, determining the antigen profile comprises universal amplification. In some embodiments, targeted amplification comprises NGS.

[00217] In some embodiments, determining an antigen profile is used to identify a therapeutic target. In some embodiments, the therapeutic target is a unique surface protein of an abnormal cell of a subject compared to a normal cell of the subject. In some embodiments, the abnormal cell is a tumor cell. In some embodiments, the subject is human. In some embodiments, 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.

Therapeutic Methods

[00218] Disclosed herein, in certain embodiments, are methods for delivering a therapeutic compound to a subject in need thereof comprising administering to the subject 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

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 therapeutic moiety that is attached to the 3' end of the second oligonucleotide conjugate. In some embodiments, the therapeutic compound is a bispecific antibody, an antibody-drug conjugate, an antibody-protein conjugate, and an antibody-fluorophore conjugate. In some embodiments, the therapeutic compound is delivered to a specific tissue in the subject. In some embodiments, the specific tissue is a tumor tissue. In some embodiments, the subject in need thereof suffers from or is suspected of suffering from cancer or an infectious disease. [00219] In some embodiments, 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. In some embodiments, 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. In some embodiments, the first oligonucleotide component of the primary identifier

oligonucleotide conjugate is a DNA and the second oligonucleotide component of the secondary therapeutic oligonucleotide conjugate is a PNA.

[00220] In some embodiments, 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.

[00221] 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. In one aspect, an oligonucleotide conjugate comprises a targeting component, a linker component, and an oligonucleotide component. In one aspect, an oligonucleotide component comprises an identifier sequence that identifies a binding target of a targeting component. In one non-limiting example of a therapeutic method, a subject in need of treatment is administered an oligonucleotide conjugate that comprises an

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. Next, 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. . In some embodiments, targeted delivery comprises tissue-specific delivery. In some embodiments, tissue-specific delivery comprises delivery to a tumor tissue.

[00222] In one aspect, 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. For example, in one aspect, a therapeutic secondary oligonucleotide conjugate includes an enzyme as a therapeutic moiety, and the methods involve administering a pro-drug to the subject. When 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.

SYSTEMS AND DEVICES

[00223] 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.

[00224] In one aspect, a system includes a sample acquisition component that is configured to accept one or more samples from a user. In one aspect, a sample acquisition system is configured to receive a liquid or fluid sample. In one aspect, 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. In one aspect, a sample acquisition system is configured to receive a plurality of samples and to process the samples in series or in parallel. For example, in one aspect, 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.

[00225] An aspect of the subject systems include fluid handling components. In one aspect, 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.). In one aspect, 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. In one aspect, 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. In one aspect, a system includes one or more fluid reservoirs that contain a reagent that is used in the subject methods. Reservoirs in accordance with an aspect of the disclosure can be fluidly connected to one or more additional components of the system, which can be configured to deliver a specified volume of a reagent to a particular location within the system. [00226] An aspect of the subject systems include one or more assay components that are configured to perform an analysis on a sample. In one aspect, 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. In one aspect, 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. As such, in one aspect, 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.

[00227] In one aspect, a system includes a nucleic acid sequencing component that is configured to determine a sequence of a nucleic acid. In one aspect, a nucleic acid sequencing component is configured to perform high-throughput sequencing of a plurality of nucleic acids. In one aspect, a sequencing component is configured to perform parallel sequencing by synthesis.

[00228] In one aspect, 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. In one aspect, 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.

[00229] 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. In one aspect, a system includes a controller that is in

communication with one or more components of the systems, as described herein, and is configured to control aspects of the systems and/or execute one or more operations or functions of the subject systems to carry out one or more aspects of the methods described herein. In one aspect, 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.

[00230] In one aspect, 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. In one aspect, a GUI is configured to display data or information to a user. In one aspect, a system includes a processor that is configured to generate a report that summarizes one or more results of the subject methods. In one aspect, a report can include data generated by one or more aspects of the subject methods.

KITS

[00231] Also provided are 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. In one aspect, 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. In one aspect, a kit includes a plurality of oligonucleotide conjugates that are individually packaged (e.g., that are each present in a separate container).

[00232] The instructions for using the oligonucleotide conjugates as discussed above are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, 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.

associated with the packaging or sub-packaging) etc. In one aspect, 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.

[00233] The following examples are provided to aid the understanding of the present disclosure, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the disclosure.

EXAMPLES

EXAMPLE 1: CONSTRUCTION AND PROPERTIES OF ANTIBODY-OLIGONUCLEOTIDE- CONJUGATES (AOCs)

A. Optimizing antibody oligonucleotide conjugate (AOC) architecture for cell-binding, restriction enzyme cleavage and processing of cleaved oligonucleotide tags

[00234] The basic structure of 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. In addition, 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.

[00235] Although the 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.

Proceedings National Academy of Sciences USA. 2009; 106(12):4606-10; Selden NS, Todhunter ME, Jee NY, Liu JS, Broaders KE, Gartner ZJ. Chemically Programmed Cell Adhesion with Membrane- Anchored Oligonucleotides. Journal of the American Chemical Society.

2012; 134(2):765-8). As this repulsive interaction increases as a function of oligonucleotide component length, the relative binding capacity of AOCs ranging in length from 50 to 70 nucleotides is determined. The 3' terminus of the oligonucleotide is conjugated to the antibody using bio-orthogonal chemistry (tetrazine ligation) (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; 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). Specifically, 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. The modification of the 3' terminus is important to minimize oligonucleotide degradation upon incubation with cells as 3' exonucleases confer nearly almost all exonuclease activity in serum (Shaw J-P, Kent K, Bird J, Fishback J, Froehler B. Modified

deoxyoligonucleotides stable to exonuclease degradation in serum. Nucleic Acids Research. 1991; 19(4):747-50). 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.

[00236] For example, as shown in FIG. 1A, 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.

[00237] In order to assess AOC binding to the cell surface, 50-70nt oligonucleotides are conjugated to Herceptin (trastuzamab), a monoclonal antibody targeting HER2. 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. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proceedings of the National Academy of Sciences. 2012; 109(40): 16101-6). For each cell line, adherent cultures grown to 80% confluence in a 10cm dish are trypsinized, counted, and resuspended in 10 mL DMEM at concentration of 5,000 cells/mL. Using a multichannel pipette, 500 cells are plated in each well of a 96-well poly-lysine treated microplate via transfer of 100 μΙ_, of the cell suspension. After 24 hours, 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. 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).

[00238] 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. To determine if the Herceptin-AOCs can be cleaved from the cell surface, 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). Methods Molecular Biology . 2010;588:301-9). 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

oligonucleotide from each cell labeling experiment.

B. Establishing a high-throughput and simplified procedure for the construction of antibody oligonucleotide conjugates (AOCs) bearing a single oligonucleotide tag

[00239] Several methods have been developed for the construction of AOCs, including using heterobifunctional linkers, site-specific oligonucleotide attachment via carbohydrates attached in the Fc region, unnatural amino acids, or bioorthogonal chemistry (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. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proceedings of the National Academy of Sciences. 2012; 109(40): 16101-6; Dennler P, Chiotellis A, Fischer E, Bregeon D, Belmant C, Gauthier L, Lhospice F, Romagne F, Schibli R. Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody- Drug Conjugates. Bioconjugate Chemistry. 2014;25(3):569-78). The utility of heterobifunctional linkers is diminished by the creation of antibody oligomers of unknown composition and the targeting of carbohydrates for functionalization suffers as the technique is not routinely general for all antibody subtypes. From a chemical perspective, the use of unnatural amino acids is ideal, as it can incorporate a single reactive handle into an antibody. In practicality, however, recombinant antibodies are not widely available, poorly scalable, and are difficult to generate (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). Rapid and cost-effective generation of AOC libraries will require the development of methods to produce singly oligonucleotide-modified antibodies with

commercially available antibodies from any source, and with limited amounts (-micrograms). The most attractive approach for large-scale generation of AOCs is 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. Additionally, 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).

[00240] In order to reproducibly generate singly modified antibodies, as shown in FIG. IB, 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.

Example 2: Cell Surface Profiling using AOC Libraries

[00241] The utility of AOCs in diagnostics and biomarker discovery hinges upon the sensitive and reproducible quantification of cell surface antigens in target populations and single cells. To this end, we perform comparative profiling of a small library of 10 AOCs in four cell lines, MDA-MB-231, 22RV1, OVCAR-3, PBMCs, that have been previously analyzed using high- throughput flow cytometry (HT-FACS) as well as the HER2+ SK-BR-3 cell line (Meyer M. Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker

Discovery and Analysis of Cellular Heterogeneity (vol 9, el05602, 2014). Plos One.

2014;9(11)). 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).

[00242] 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 . In addition, 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. Cancer Cell Profiling by Barcoding Allows Multiplexed Protein Analysis in Fine- Needle Aspirates. Science Translational Medicine . 2014;6(219):219ra9). Finally, upon confirmation that the qPCR data is in agreement with the cell-ELISA and FACS data, the cleaved oligonucleotide component is analyzed using next-generation sequencing (NGS) technologies (Illumina MiSeq or NextSeq). Upon confirmation that cell surfaces can be analyzed/profiled using 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. The identification of these up- regulated receptors is confirmed by cell-ELISA, qPCR, and FACS to ensure the analytical method agrees with previously used methods. In order to determine if this technology could have immediate clinical utility, biopsy samples obtained from real cancer patients are analyzed (Scripps Clinic, UCSF, Sanford-Burnham Medical Research Institute), and cells are identified as being normal or cancerous using routine immunohistochemistry analysis, after profiling the cell surface with a library of AOCs. What potential cell surface receptors would be candidate targets for therapeutics for each of these patient samples are then identified (either antibody-drug- conjugate, CAR T-Cell therapy, or bispecific antibodies)( 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. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proceedings of the National Academy of Sciences. 2012; 109(40): 16101-6;

Kochenderfer IN, Rosenberg SA. Treating B-cell cancer with T cells expressing anti-CD 19 chimeric antigen receptors. Nature Reviews Clinical Oncology. 2013; 10(5):267-76; Kim CH, Axup JY, Dubrovska A, Kazane SA, Hutchins BA, Wold ED, Smider VV, Schultz PG.

Synthesis of Bispecific Antibodies using Genetically Encoded Unnatural Amino Acids. Journal of the American Chemical Society. 2012; 134(24):9918-21).

[00243] For example, as shown in FIG. 2, 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.

Example 3: "Mix and Match" Therapeutics

[00244] The information obtained from the cell surface profiling of cancerous cells using AOCs not only has immediate diagnostic application but also therapeutic potential. While

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). In addition, 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). However, 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). If 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).

[00245] In this particular scenario, 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) would be used to recruit immune cells capable of inducing cytotoxicity. 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.

[00246] For example, for therapeutic applications, peptide nucleic acid will be used for conjugation to antibodies or other molecules rather than simple oligonucleotides. A library of antibody-PNA-conjugates (APCs) 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). In the case of HER2 positive cells, 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. In this light, 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.

[00247] Thus, 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. In addition, due to the capability of single-cell analysis using this technology, 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. EMBO Molecular Medicine . 2015;7(1): 1-11; Lin J, Li J, Huang B, Liu J, Chen X, Chen XM, Xu YM, Huang LF, Wang XZ. Exosomes: novel biomarkers for clinical diagnosis. ScientifwWorldJournal . 2015;2015:657086). This rational diagnosis and treatment of cancer has far reaching applications and offers a revolutionary technology in the treatment of not just cancer but other human diseases.

Example 4: General process of ssDNA-directed enzyme recruitment and activation

[00248] As shown in FIG. 4, 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). 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

[00249] As shown in FIG. 5, 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).

Example 6: Recruitment of IDE selectively to cells that have surface HER2

[00250] 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. 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

[00251] Confocal microscopy was used to image Herceptin-DA conjugates and show localization at the cell periphery, using the complementary oligonucleotide DI-(F), where F denotes Dylight 488 (green) (FIGS. 7A-7C). Each conjugate is depicted by the notation, Herceptin-(oligonucleotide name). The sequence nsDA is a 22-nt poly-A oligonucleotide. The cells were stained with a 594-Concanavalin A (red), as well as DAPI (blue). 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

[00252] Various concentrations of Leu-Dox were incubated with cells for 48 h. Various concentrations of peptidyl prodrug were incubated with cells for 48 h after either Herceptin- nsDA or Herceptin-DA labelling, followed by IDE application and removal of unbound IDE. After 48 h, the cells were washed and the medium replaced with fresh media. The cells were incubated for 24 h and viability was determined by performing an XTT assay. Viability of SK- BR3 cells following Herceptin-nsDA or Herceptin-DA targeting, IDE labeling, and peptidyl prodrug application was assessed with Leu-Dox treatment used as a positive control (FIG. 8A). Viability of MDA-MB-231 cells following Herceptin-nsDA or Herceptin-DA targeting, IDE labeling, and peptidyl prodrug application was assessed with Leu-Dox treatment used as a positive control (FIG. 8B).

Example 9: Recruitment of IDE selectively to living cells that have surface FR

[00253] 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. Subsequently, these cells were incubated with IDE to allow for cellular labeling and a fluorescent substrate reporter was used to visualize and quantify the surface-bound IDE (FIG. 9C). The experiment in FIG. 9C was repeated except cells were labeled with avidin-HRP for visualization (FIG. 9D). Both Folate- nsDA and Folate-DA were shown to label KB cells, demonstrating that IDE labeling in FIG. 9C was sequence-dependent.

Example 10: Confocal microscopy of Folate-DA recruitment of oligonucleotides to the surface of FR ⁺ cells

[00254] Confocal microscopy images demonstrating the ability of Folate-DA conjugates to localize at the cellular periphery a complementary oligonucleotide, DI-(F), where F denotes Dylight488 (green). Each conjugate is depicted by the notation Folate-(oligonucleotide name). DI-(F) is fully complementary to the oligonucleotide DA. The cells were stained with a 594- Concanavalin A (red) as well as DAPI (blue). 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

[00255] Cells were targeted with Folate-DA, Folate-nsDA, DA, or media only, then labeled with IDE. 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. 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). After labeling with IDE and application of peptidyl prodrug (PD) with KB cells, XTT analysis indicated that cytotoxicity was induced only for Folate-DA (FIG. HE).

Example 12: DNA Oligonucleotide design

[00256] Each oligo is comprised of five functional domains with total length of 70 nucleotides (nt) (FIG. 12A), produced as shown in FIG. 12B. 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.

[00257] The attachment of the 3' terminus to the antibody is important to minimize

oligonucleotide degradation via 3' exonucleases in serum. In order to clearly distinguish oligos from different AOCs in a multiplexed NGS based quantification, 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.

Example 13: Optimization of Antibody-oligo conjugate (AOC)-synthesis procedures

[00258] Generation of an AOC with a ~1 to 1.2 antibody: oligo ratio is preferred.

[00259] 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. In parallel, 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).

Example 14: Optimization of Antibody-oligo conjugate (AOC)-synthesis procedures

This procedure described in Example 13 works with any off the shelf antibody, irrespective of host and isotype. AOCs that were primarily singly modified were generated (FIG. 16B). Solid phase functionalization of the antibodies and oligos was critical in controlling not only the number of modifications per antibody, but also the region of the antibody being modified. Analysis of AOCs via gel electrophoresis under reducing conditions showed that it was consistently a single heavy chain that was oligo-modified (FIG. 16C).

Example 15: Soluble protein detection using next generation sequencing

[00260] 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.

[00261] 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.

[00262] 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.

Example 17: Antibody conjugation

[00263] 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. In parallel, 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. Gel electrophoresis of AOCs under non-recuing conditions showed major product of singly oligo-modified antibodies across several species and antibody isotypes. (FIG. 16B). The upward shift in the band was the result of DNA conjugation. Gel electrophoresis of AOCs under reducing conditions showed that oligos are attached to a single antibody heavy chain (FIG. 16C)

Example 18: Cross-reactivity of AOCs and unconjugated antibodies

[00264] 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. In this example, it was determined whether or not successful multiplex detection could be carried out for 11 proteins, for which single-plex detection was validated via AOC-ELISA and AOC-qPCR.

[00265] Multiplexing the detection of proteins using antibodies requires rigorous validation to eliminate problems associated with antibody cross-reactivity and interference between reagents. A series of ELISA based experiments were set up to determine whether or not sandwich ELISA pairs could be used together in a single reaction (FIGS. 18A-18D). In these exhaustive interaction matrices, the HRP detection signal comes from a single AOC that is in competition for antigen binding with naked detection antibodies against the captured protein. Along the diagonal in FIG. 18A, the HRP signal comes from on target binding of AOC as shown in FIG. 18B. HRP signal that is detected off the diagonal (FIG. 18C) is due to off target binding as shown for IL4-AOC binding to captured IFNy protein in FIG. 18D. 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. However, interaction matrix

experiments like these are also highly context dependent. Therefore, each time an established assay is expanded, the experiments must again be repeated. More importantly,

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. To address this issue, 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

[00266] 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.

[00267] High-throughput characterization of antibody cross-reactivity has been accomplished using protein arrays such as the Arrayit HuProt™ v2.0 19K Human Proteome Microarrays. These powerful arrays are the most comprehensive human proteome array available, spotted with 19,394 proteins, in duplicate. At ~$1000/array, this method is prohibitively expensive if used only once to characterize each antibody. Notably, AOC technology enables

characterization of binding profiles of multiple antibodies simultaneously.

[00268] 25 antibodies targeting 5 proteins of interest are selected from respected antibody manufactures and according to public antibody validation data provided by the Human Protein Atlas. 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. In a competitive binding reaction, 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.

[00269] Upon adding of a mixture of AOCs to the array, gentle fixation (e.g. 0.5-2% formaldehyde) is applied to "freeze" all binding events for stepwise interrogation with fluorescent DNA probes (FIG. 19). Next, 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. In a final step, the fluorescent probe is specifically displaced from AOC-1 via hybridization of a ssDNA oligo with full complementarity to the 71nt oligonucleotide component of the AOC-1 (FIG. 19; Step 3). To facilitate labeling of the AOC-2, a second fluorescent oligo is simultaneously added during the AOC-1 label displacement step. With a 3 color microarray scanner, whole proteome binding profile data is generated for 30 AOCs in a matter of hours, with only 10 labeling and

displacement wash steps. These data streamline the selection of highly specific, sensitive and multiplex-compatible antibodies.

Example 20: Multiplexed detection of blood proteins

[00270] In order to determine the utility of AOCs in soluble protein quantification, antibody sandwich pairs were obtained for the capture and detection 15 cytokines (CCL17, IL-4, IL-5, IL- 6, JL-7, IL-9, IL-22, IL-33, GMCSF, IFNy, IL-10, IL-12, TSLP, MCP-1, MIF) and a soluble inhibitor of T cell signalling (CTLA4). AOCs were generated for each of the detection antibodies, while capture antibodies were kept in their native form. To determine whether or not antibody function was preserved throughout the synthesis of AOCs, standard curves were generated via ELISA-HRP (FIG. 20A-20C). The limit of detection was determined to be between 62.5 pM to 250 pM for most of these proteins, although IL-10, IL-12, MCP-1, MIF, and CTLA4 could not be detected for undetermined reasons (FIG. 20B). For the 1 1/16 proteins that were detected via ELISA-HRP using AOC detection antibodies, it was then determined whether the same antigens could be detected via AOC-qPCR. These experiments showed that proteins could also be quantified using qPCR, which increased the sensitivity of detection by 1 -2 orders of magnitude over ELISA-HRP (FIG. 20C).

Example 21: Optimization of qPCR

[00271] The current limit of detection as determined via qPCR is in the range of 1 to 0.1 pM for most antigens with a non-optimized workflow. While this level of sensitivity is on par with standard techniques and competing technologies, and would warrant continued product development, it is a reasonable assessment that successful improvement of this sensitivity by at least 2 orders of magnitude is possible in order to achieve low femtomolar sensitivity through optimization of buffer compositions, washing conditions, and rigirous qualification of antibodies.

[00272] To test the performance of the AOC-based multiplexed protein detection method in relevant biological samples, a series of human serum samples spiked with known concentrations (0, InM, ΙΟΟρΜ, ΙΟρΜ, lpM, or lOOfM) of recombinant proteins are set up. Additionally, the performance of the assay in a set of 20 human serum samples that have been analyzed using assays produced by market leader, Luminex are benchmarked. These samples and historical Luminex data are secured through collaborators and potential early adopters at the Blood Systems Research Institute, one Luminex' s single largest academic customers. Target proteins are quantified, in triplicate, via qPCR and next generation sequencing on an Illumina MiniSeq device.

[00273] Although the methods and compositions described herein have been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings described herein that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[00274] Accordingly, the preceding merely illustrates the principles of the methods and compositions described herein. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the methods and compositions described herein and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the methods and compositions and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles and aspects of the methods and compositions described herein as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present methods and compositions described herein, therefore, is not intended to be limited to the exemplary aspects shown and described herein. Rather, the scope and spirit of the methods and compositions is embodied by the appended claims.