Compositions and methods to discover targeted oligonucleotide conjugates

Abstract

A method of selecting a therapeutic oligonucleotide for use in a therapeutic oligonucleotide targeting ligand drug conjugate, the method encompassing: linking one or more p19 polypeptides to a targeting ligand and contacting a therapeutic oligonucleotide to the targeted-p19 polypeptide to form a targeted-p19-oligonucleotide complex.

Description

Attorney Docket No: JBI6815WOPCT1 COMPOSITIONS AND METHODS TO DISCOVER TARGETED OLIGONUCLEOTIDE CONJUGATES BACKGROUND

[0001] Nucleic acid therapeutics offer the potential to regulate the expression of any disease-causing gene or pathway, enabling treatment of many diseases that are considered “undruggable” because they are not amenable to small- or large-molecule targeting. Small- interfering RNA (siRNA) is a promising modality that harnesses the natural cellular defense mechanism of RNA interference (RNAi) in which small, double-stranded RNA molecules bind to complementary mRNA sequences and target them for degradation to shut down expression of target genes. Although unmodified siRNAs are sensitive to nuclease degradation, incorporation of chemical modifications throughout both strands of the siRNA have improved the metabolic stability dramatically. Currently, the difficulty of delivering the large and highly anionic siRNA molecules to target cells and across the cell membrane remains the largest barrier to their wider application as therapeutics.

[0002] Proteins such as centyrins, monoclonal antibodies (mAbs) and antibody fragments as well as peptides and small molecules have all been explored as targeting ligands for targeted siRNA delivery. In the typical process for developing an RNAi (e.g., siRNA) therapeutic, candidates are screened for in vitro activity independently of the delivery technology. Initial screening of siRNA sequences and optimization of backbone chemistries are commonly achieved by electroporation or transfection of target cells. However, siRNAs identified by either method may not be optimal for delivery via the endocytic pathway due to the fundamental differences in how the siRNAs are internalized. Whereas electroporation and transfection directly disrupt the cell / endosomal membrane, siRNAs entering cells through the endosomal pathway are believed to linger for an extended period in the lysosome and only a small percentage is released to the cytosol. As a result, potent siRNAs selected from these initial screens must be re-evaluated as conjugates to assess how well they can withstand the lysosomal environment (among other potential differences). Additionally, while there is a significant area of research devoted toAttorney Docket No: JBI6815WOPCT1 the exploration of new backbone and ribose modifications, the impact of these modifications specifically on conjugate potency and endosomal escape has not been well studied. A tool that can identify optimal siRNAs for ligand-mediated delivery will streamline the siRNA conjugate discovery process by eliminating the need to generate and purify conjugates with each candidate siRNA. Furthermore, as siRNA targeting continues to expand beyond the liver, it will be imperative to develop methods for screening siRNAs in cell types that are difficult to transfect using conventional methods. SUMMARY

[0003] Here an optimized targeted oligonucleotide carrier is described based on the double-stranded RNA binding protein p19 from Carnation Italian Ringspot Virus. p19 is a 19 kDa protein that binds with nanomolar affinity to small dsRNAs and acts as a natural RNA silencing suppressor in plant tombosviruses. It was hypothesized that fusion of p19 to a high-affinity antibody that binds a highly expressed internalizing receptor would enable delivery of bound siRNA into cells. Because the mAb-p19 / oligonucleotide complex can be internalized by receptor-mediated endocytosis, the potency of oligonucleotide delivered via this method is more predictive than if the oligonucleotides were screened by traditional methods.

[0004] As the field of oligonucleotide conjugates continues to expand to targeting cells beyond hepatocytes, it will become increasingly important to have alternative methods for screening candidate oligonucleotides that can be utilized in all cell types including those that are considered difficult-to-transfect such as suspension or primary cells. Although electroporation is commonly used to screen oligonucleotide activity in cells that are recalcitrant to transfection, oligonucleotides delivered by this method are transported directly across the cell membrane and therefore cannot accurately predict the potency of a given oligonucleotide as a conjugate. In contrast, screening oligonucleotide activity with a p19 fusion not only avoids this potential pitfall because the p19 / oligonucleotide complex is internalized by receptor mediated endocytosis, but also can be utilized with any cell type by selection of the appropriate targeting ligand.Attorney Docket No: JBI6815WOPCT1

[0005] The functional delivery of oligonucleotides to cells was demonstrated with a trastuzumab-p19 fusion with the complexes eliciting potent mRNA knockdown. Knockdown achieved with mAb-p19 delivery was observed to correlate well with knockdown from the corresponding mAb-siRNA conjugates, indicating that the mAb-p19 fusions / conjugates is a powerful screening tool in the therapeutic oligonucleotide discovery process. The conjugation of a standalone p19 dimer to a targeting protein was also demonstrated using established site-specific conjugation. The modularity of this system enables not only the ability to pair p19 with different targeting ligands but also the simultaneous screening of internalizing ligands and candidate oligonucleotides. Whereas previously the discovery process has relied on screening internalizing ligands with a proof- of-concept oligonucleotides and independently selecting potent oligonucleotides from a transfection or electroporation screen, the p19 conjugation method enables them to be screened in combination. Targeting ligands and oligonucleotides can then be selected with the knowledge of how they will perform when they are paired together.

[0006] We describe throughout a novel fusion protein or protein conjugate between the viral dsRNA binding protein p19 and an antibody. This fusion functionally delivers siRNA to cells and mimics a mAb-siRNA conjugate in that the formation of the p19 / siRNA complex acts as a surrogate for the chemical conjugation step, and the siRNA enters the cell by receptor-mediated endocytosis. There is no requirement for a purification step prior to treating cells, so a mAb-p19 can easily be incorporated into a high-throughput screening workflow. It was demonstrated that siRNA delivered as a complex with a mAb-p19 is predictive of the potency of the equivalent mAb-siRNA conjugate, and as a result can be a useful tool for discovery of therapeutic siRNAs with the potential to improve upon current approaches and enable faster screening and optimization of siRNAs for extrahepatic delivery. A single p19 fusion can bind siRNAs with a wide range of nucleotide sequences and chemical modification patterns, so this tool can have applications for both selecting sequences and in optimizing the siRNA modification chemistry. Screening for mRNA knockdown with a p19 / siRNA complex thus enables consideration of the endo / lysosomal trafficking when selecting the most potent siRNAs to take forward as conjugates (Figure 15). Overall, targeted p19 fusion proteins can have significant potential as a tool that can help to advance the next generation of siRNA therapeutics.Attorney Docket No: JBI6815WOPCT1

[0007] Described throughout is a method of selecting an oligonucleotide for use in an oligonucleotide conjugate, the method comprising: linking one or more p19 polypeptides to a targeting ligand to form a targeted p19 polypeptide; and contacting an oligonucleotide to the targeted p19 polypeptide to form a targeted-p19-oligonucleotide complex.

[0008] In some embodiments, the oligonucleotide comprises a length of about 16 to about 25 nucleotides.

[0009] In some embodiments, the oligonucleotide is an siRNA, an miRNA, or an antisense RNA.

[0010] In some embodiments, the oligonucleotide is contacted to the targeted p19 polypeptide in a ratio of about 10:1 to about 2:5. In a specific embodiment, the oligonucleotide is contacted to the targeted-p19 polypeptide in a ratio of about 2:1.

[0011] In some embodiments, a cell expressing a target of the oligonucleotide is contacted to the targeted-p19-oligonucleotide polypeptide.

[0012] In some embodiments, the target of the targeting ligand comprises a surface ligand.

[0013] In some embodiments, the method produces a targeted-p19-oligonucleotide polypeptide at a concentration of about 0.01 nM to about 100 nM.

[0014] In some embodiments, the cell is contacted with the targeted-p19-siRNA polypeptide for about 24 to about 96 hours.

[0015] In some embodiments, a level of the target of the oligonucleotide is measured.

[0016] In some embodiments, the level of the target of interest is measured by qRT-PCR.

[0017] In some embodiments, the level of the target of interest in the cell contacted with the targeted-p19-oligonucleotide fusion polypeptide is compared with a control, and optionally wherein the control expresses the target of interest or is treated with an inoperable targeted-p19- oligonucleotide, and optionally wherein a modulation in the level of the target of interest in the cell contacted with the targeted-p19-oligonucleotide fusion polypeptide as compared to the control indicates that the oligonucleotide is capable of modulating the target of interest when fused to the targeting ligand.

[0018] In some embodiments, an optimal oligonucleotide is selected based on the modulation of the level of the target of interest in the cell contacted with the targeted-p19- oliognucleotide fusion polypeptide.Attorney Docket No: JBI6815WOPCT1

[0019] In some embodiments, the targeting ligand is selected from the group consisting of an antibody, an antibody fragment, a miniprotein, or a peptide.

[0020] In some embodiments, the p19 polypeptide is covalently linked to a heavy chain or a light chain of the antibody.

[0021] In some embodiments, p19 polypeptide is covalently linked to the heavy chain of the antibody.

[0022] In some embodiments, the p19 polypeptide and the antibody are linked by a click chemistry method or a conjugation method.

[0023] In some embodiments, the p19 polypeptide and the antibody are linked by a linker sequence.

[0024] In some embodiments, the linker sequence comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO: 21. In a specific embodiment, the linker sequence comprises the amino acid sequence of SEQ ID NO: 21.

[0025] In some embodiments, the p19 polypeptide is a mutant p19 polypeptide.

[0026] In some embodiments, the mutant p19 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 15 or SEQ ID NO: 16. In a specific embodiment, the mutant p19 polypeptide comprises the amino acid sequence of SEQ ID NO: 16.

[0027] In some embodiments, the p19 polypeptide comprises a Δ1-5, a N15K, a G16R, a C134S, and / or a C160A mutation.

[0028] In some embodiments, the oligonucleotide is a therapeutic oligonucleotide.

[0029] In some embodiments, the targeting ligand binds to HER2 or TfR.

[0030] Described throughout is a p19 polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 15 or SEQ ID NO 16.

[0031] In some embodiment, the p19 polypeptide comprises the amino acid sequence of SEQ ID NO: 16, SEQ ID NO:18, or SEQ ID NO: 19.

[0032] In some embodiment, the antibody is linked to the p19 polypeptide.

[0033] In some embodiment, an oligonucleotide is complexed with the p19 polypeptide. In some embodiment, the oligonucleotide is complexed with the p19 polypeptide.Attorney Docket No: JBI6815WOPCT1

[0034] Described throughout is a polynucleotide encoding any one of the p19 polypeptides comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 15 or SEQ ID NO 16.

[0035] Described throughout is a vector comprising the isolated polynucleotide encoding any one of the p19 polypeptides.

[0036] Described throughout is a host cell comprising the vector encoding any of the p19 polypeptides.

[0037] Described throughout is a kit for selecting an siRNA for use in an siRNA targeting ligand drug conjugate, the kit comprising one or more of the p19 polypeptides and instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 shows the design of mAb-p19 fusion proteins and optimization of linker. p19 (dark grey) was fused as a monomer or covalently linked dimer to either the light chain or heavy chain of trastuzumab (light grey). The composition of the linker connecting the two proteins (Linker 1, dashed lines) was varied to determine the impact on fusion protein expression and solubility.

[0039] Figure 2 shows analytical SEC and reduced SDS-PAGE illustrating how the introduction of a deletion mutation in p19 prevents proteolytic cleavage. (A) SEC and (B) reduced SDS-PAGE analysis of fusion construct 7. Partially and fully cleaved protein are visible in addition to the intact fusion. (C) SEC and (D) reduced SDS-PAGE analysis of construct 10, which has a ∆1-5 deletion at the N-terminus of the p19 dimer. This construct elutes as a homogenous peak in aSEC. Although there are minor impurities present in both constructs, the band at ~50 kDa that corresponds to the cleaved heavy chain is no longer visible in the SDS-PAGE of 10.

[0040] Figure 3 shows how Trastuzumab-p19 binds CTNNb1 siRNA and induces mRNA knockdown. (A) aSEC analysis of Tmab-p19, with and without 1 molar equivalent CTNNb1 siRNA_1. (B-C) CTNNb1 siRNA binding to Tmab-p19 was analyzed by EMSA. The native gel was stained with (B) SYBR Green and (C) SYPRO Ruby to illustrate how the siRNA mobility is altered when it is bound to p19. The lower band can be attributed toAttorney Docket No: JBI6815WOPCT1 the protein with 2 siRNAs because it is more negatively charged and therefore migrates faster, and the higher of the two bands arises from the protein with 1 siRNA bound. Trastuzumab-p19 alone does not migrate beyond the well. The band for the un- complexed siRNA is no longer visible above 0.75 equivalents trastuzumab-p19, indicating that all siRNA is bound to the protein. (D-F) HCC1954 cells were treated with complexes consisting of CTNNb1 siRNA and 0-2.5 molar equivalents trastuzumab-p19 for 72 h, and CTNNB1 mRNA expression was detected by qRT-PCR. The (D) overall knockdown and (E) IC50 are dependent on the ratio of trastuzumab-p19:siRNA. (F) mRNA knockdown is most comparable to the trastuzumab-CTNNb1 conjugate at a stoichiometric ratio.

[0041] Figure 4 shows CTNNb1 gene expression measured by qRT-PCR in HCC1954 cells treated with (A) trastuzumab-siRNA conjugates, (B) trastuzumab-p19 / siRNA complexes, and (C) siRNA delivered via lipofectamine transfection. siRNAs have the same sequence but different chemical modifications, as indicated in Table 2. The Spearman correlation was calculated for (D) knockdown and (E) IC50.

[0042] Figure 5 shows siRNA binding and delivery with a trastuzumab-p19 conjugate generated by click chemistry. (A) Schematic shows the generation of Trastuzumab-p19 conjugates from a standalone p19 dimer and azide-labeled mAb using sortase transpeptidation and click chemistry. (B) Tmab-azide-p19 is purified with an average DOL of ~1.8-1.9. (C) Tmab-azide-p19 was confirmed to bind CTNNb1 siRNA by EMSA. CTNNb1 siRNA was incubated with 0-2.5 equiv Tmab-p19 and analyzed by non-reducing native PAGE. The gel was stained with SYBR Green (left) and SYPRO Ruby (right). (D) HCC1954 cells were treated Tmab-p19 / CTNNb1 siRNA complexes, the unconjugated p19 / CTNNb1 siRNA complex, and a covalent Tmab-CTNNb1 conjugate. CTNNb1 mRNA expression was quantified by qRT-PCR.

[0043] Figure 6 shows an aSEC analysis of Trastuzumab-p19 fusion proteins 1-10.

[0044] Figure 7 shows a LC-MS analysis of fusion proteins 3-8. The presence of cleaved heavy chain or light chain is visible in most samples. Note that for fusions 5 and 8 the cleaved species was purified away from the intact species by SEC prior to LC-MS analysis.

[0045] Figure 8 shows how mAb siRNA conjugates were generated by site-specific transglutaminase labeling following by strain-promoted azide alkyne cycloaddition.Attorney Docket No: JBI6815WOPCT1

[0046] Figure 9 shows in vitro silencing from CTNNb1 siRNA delivered as a complex with trastuzumab-p19. (A) HCC1954 cells were treated with CTNNb1 siRNA_1, trastuzumab-p197, and mRNA knockdown was compared with a covalent trastuzumab- CTNNb1 siRNA_1 conjugate. Neither the CTNNb1 siRNA nor trastuzumab-p19 alone result in mRNA knockdown below 100 nM. (B) % CTNNb1 expression at 100 nM siRNA for different ratios of trastuzumab-p19:siRNA, compared to a covalent trastuzumab- CTNNb1 conjugate (DOL = 2).

[0047] Figure 10 shows a visual representation of selected siRNA chemical modification patterns.

[0048] Figure 11 shows an EMSA with CTNNb1 siRNA panel. Each siRNA was incubated with 0-1.0 equivalents trastuzumab-p19 and analyzed by non-reducing native PAGE. The gels were stained with SYBR green (left panels) and SYPRO ruby (right panels) to illustrate how the siRNA mobility is altered when it is bound to p19.

[0049] Figure 12 show the characterization of p19-LPETG-His6. (A) SDS-PAGE of the purified 2xp19-LPETG-His6 reveals highly pure protein at the correct molecular weight. (B) The molecular weight of 2xp19-LPETG-His6 was confirmed by LCMS. (C) aSEC confirms that 2xp19-LPETG-His6is monodisperse in solution. (D) CTNNb1 binding to 2xp19-LPETG-His6 was analyzed by EMSA. CTNNb1 siRNA_1 was incubated with 0-4 equivalents of 2xp19-LPETG-His6 and analyzed by non-reducing native PAGE. The gels were stained with SYBR green (left panel) and SYPRO ruby (right panel)..

[0050] Figure 13 shows that p19-LPETG-His6 was labeled with DBCO via sortase transpeptidation. (A) Deconvoluted LCMS trace of p19-LPETG-His6. (B) 86% labeling with DBCO is observed after 1 h reaction with SrtA and GGG-cysteic acid-DBCO. The mass difference corresponds to loss of the His tag and addition of GGG-cysteic acid- DBCO (C) Purified 2xp19-DBCO. A small amount of hydrolyzed 2xp19 is visible but all unreacted 2xp19-LPETG-His6 has been removed.

[0051] Figure 14 shows (A) SDS-PAGE analysis of the click reaction between Trastuzumab-Azide and 2xp19-DBCO. (B) SDS-PAGE of the purified Trastuzumab-Az- 2xp19 conjugate. Unreacted 2xp19-DBCO was removed by Protein A chromatography.

[0052] Figure 15 is a graphical abstract of the internalization of the mAb-p19 / siRNA complex by receptor-mediated endocytosis.Attorney Docket No: JBI6815WOPCT1

[0053] Figure 16 shows siRNA delivery with trastuzumab-p19 heavy chain and light chain fusions. HCC1954 cells were treated with CTNNb1 siRNA delivered as a DOL=2 trastuzumab-siRNA conjugate and stoichiometric complexes with Tmab-p19 constructs 4 and 7. CTNNb1 mRNA expression was quantified at 72 hours by qRT-PCR. \

[0054] Figure 17 shows siRNA delivery with BBBB175-p19. (A) bcr10 siRNA binding to BBBB175-p19 was analyzed by EMSA. bcr10 siRNA was incubated with 0-2 equivalents of BBBB175-p19 and analyzed by non-reducing native PAGE. The gels were stained with SYBR green (left panel) and SYPRO ruby (right panel).. (B) TF1 cells were treated a covalent BBBB175-bcr10 siRNA conjugate and a BBBB175-p19 / bcr10 siRNA complexes and BCL2 mRNA expression was quantified by qRT-PCR at 72 h. DETAILED DESCRIPTION

[0055] The disclosed compositions and methods can be understood more readily by reference to the following detailed description. The disclosed compositions and methods are not limited to the specific methods described and / or shown herein, and the terminology used herein is for the purpose of exemplifying and describing particular embodiments and is not intended to be limiting of the claimed methods.

[0056] All patents, published patent applications, and publications cited herein are incorporated by reference in their entireties.

[0057] When a list is presented in the disclosure, unless stated otherwise, it is understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” or “A, B, and / or C” is to be interpreted as including the embodiments “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” “A, B, or C,” “A and B,” “A and C,” “B and C,” and “A, B, and C.”

[0058] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0059] The transitional terms “comprising,” “consisting essentially of,” and “consisting of” are intended to connote their generally accepted meanings in the patent vernacular; that is, (i) “comprising,” which is synonymous with “including,” “containing,” orAttorney Docket No: JBI6815WOPCT1 “characterized by,” is inclusive or open-ended, and does not exclude additional, unrecited elements or method steps; (ii) “consisting of” excludes any element, step, or ingredient not specified in the claim; and (iii) “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of” and “consisting essentially of.”

[0060] “About” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of a particular assay, result, or embodiment, “about” means within one standard deviation per the practice in the art, or a range of up to 10%, whichever is larger.

[0061] The term “antibody” or “antibodies” or the like are meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antigen binding fragments, multispecific antibodies, such as bispecific, trispecific, tetraspecific etc., dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity. Typical “full length antibodies” are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g., IgM). Each HC is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CH1, hinge, CH2 and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and the VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Immunoglobulins can be assigned to five major classes: IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgA1, IgA2,Attorney Docket No: JBI6815WOPCT1 IgG1, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0062] “Decrease,” “lower,” “lessen,” “reduce,” or “abate” and similar phrases refers generally to the ability of a test molecule to mediate a reduced response (i.e., downstream effect) when compared to the response mediated by a control or a vehicle. Decrease can be a statistically significant difference in the measured response between the test molecule and the control (or the vehicle), or a decrease in the measured response, such as a decrease of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 30 fold or more, such as 500, 600, 700, 800, 900 or 1000 fold or more (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.).

[0063] “Encode” or “encoding” and similar phrases refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0064] “Enhance,” “promote,” “increase,” “expand” or “improve” and similar phrases refers generally to the ability of a test molecule to mediate a greater response (i.e., downstream effect) when compared to the response mediated by a control or a vehicle. Enhance can be a statistically significant difference in the measured response between the test molecule and control (or vehicle), or an increase in the measured response, such as an increase of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 30 fold or more, such as 500, 600, 700, 800, 900 or 1000 fold or more (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.).

[0065] “Expansion” or the like refers to the outcome of cell division and cell death.Attorney Docket No: JBI6815WOPCT1

[0066] “Express” and “expression” and similar phrases refers the to the well-known transcription and translation occurring in cells or in vitro. The expression product, e.g., the protein, is thus expressed by the cell or in vitro and can be an intracellular, extracellular or a transmembrane protein.

[0067] “Expression vector” or the like refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0068] “Isolated” or the like refers to a homogenous population of molecules (such as synthetic polynucleotides or polypeptides) which have been substantially separated and / or purified away from other components of the system the molecules are produced in, such as a recombinant cell, as well as a protein that has been subjected to at least one purification or isolation step. “Substantially free” or the like refers to a molecule that does not contain other cellular material and / or chemicals and encompasses molecules that are isolated to a higher purity, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0069] “Modulate” or the like refers to the ability of a molecule to mediate a response (e.g., an upstream or downstream effect) when compared to the response mediated by a control or a vehicle.

[0070] “Operatively linked” and similar phrases, when used in reference to nucleic acids or amino acids, refers to the operational linkage of nucleic acid sequences or amino acid sequence, respectively, placed in functional relationships with each other. For example, an operatively linked promoter, enhancer elements, open reading frame, 5' and 3' UTR, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA) and in some instances to the production of a polypeptide (i.e., expression of the open reading frame). “Operatively linked peptide” refers to a peptide in which theAttorney Docket No: JBI6815WOPCT1 functional domains of the peptide are placed with appropriate distance or orientation from each other to impart the intended function of each domain.

[0071] “Covalently linked” or the like means that the payload is attached to the antibody via at least one covalent linkage. The linkage can be direct, i.e., without a linker, or indirect, i.e., via a linker.

[0072] “Linker” or the like refers to a moiety that joins two molecules. The linkers can be, for example, a single covalent bond, a substituted or unsubstituted alkyl moiety, a substituted or unsubstituted heteroalkyl moiety, a polyethylene glycol (PEG) linker, a peptide linker, a sugar-based linker, a cleavable linker, such as a disulfide linkage or a protease cleavage site such as valine- citrulline-PAB, or any one of the sequences listed in Table 1.

[0073] “Polynucleotide” or “nucleic acid” and similar phrases refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide. Polynucleotide may be a DNA or an RNA molecule.

[0074] “Proliferation” in a biological context refers to an increase in cell division, either symmetric or asymmetric division of cells.

[0075] “Protein” or “polypeptide” or the like are used interchangeably and refers to a molecule that comprises at least two amino acids linked by a peptide bond. A protein can be a monomer, or can be protein complex of two or more subunits, the subunits being identical or distinct. Protein can be a heterologous fusion protein, a glycoprotein, or a protein modified by post-translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation or biotinylation. Protein can be recombinantly expressed.

[0076] “Recombinant” refers to polynucleotides, polypeptides, vectors, viruses, and other macromolecules that are prepared, expressed, created or isolated by recombinant means.

[0077] The terms “specifically binds,” “specific binding,” “specifically binding”, or “binds” or the like refer to a proteinaceous molecule binding to an antigen or an epitope within the antigen with greater affinity than for other antigens. In some cases, the proteinaceous molecule binds to the antigen or the epitope within the antigen with anAttorney Docket No: JBI6815WOPCT1 equilibrium dissociation constant (KD) of about 1x10-7M or less, for example about 5x10-8M or less, about 1x10-8M or less, about 1x10-9M or less, about 1x10-10M or less, about 1x10-11M or less, or about 1x10-12M or less, typically with a KD that is at least one hundred fold less than its KDfor binding to a non-specific antigen (e.g., BSA, casein).

[0078] The term “transduction” and the like refers to the introduction of a foreign nucleic acid into a cell using a viral vector.

[0079] The term “variant,” “mutant”, or “altered” or the like refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications, for example, one or more substitutions, insertions or deletions.

[0080] Examples of point mutations in one region of p19 are referred to as follows: N15K and G16R. Δ1-5 refers to the deletion of amino acids 1 to 5 in the N-terminus region of p19.

[0081] The disclosure describes a method of selecting a oligonucleotide for use in a oligonucleotide antibody conjugate. In some examples, the oligonucleotides can be a therapeutic oligonucleotide. The method can comprise linking one or more p19 polypeptides to an antibody. The p19 polypeptide can be a polypeptide having an amino acid sequence of at least 95% identity to SEQ ID NO: 21 or SEQ ID NO 22. Also described are polynucleotides encoding the foregoing or vectors, host cells and methods of making and using the foregoing.

[0082] Variants of the isolated p19 polypeptide are also described. For example, variants can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 amino acid substitutions in the isolated p19 polypeptide as long as they retain or have improved functional properties when compared to the parent polypeptide. In some embodiments, the sequence identity can be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the isolated p19 polypeptide disclosed throughout. In some embodiments, the variation is in the framework regions. In some embodiments, variants are generated by conservative substitutions.

[0083] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions ×100), taking into account the number of gaps, and theAttorney Docket No: JBI6815WOPCT1 length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0084] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput Appl Biosci 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http_ / / _www_gcg_com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0085] “Conservative modifications” and the like refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid modifications. Conservative modifications include amino acid substitutions, additions, and deletions. Conservative amino acid substitutions are those in which the amino acid is replaced with an amino acid residue having a similar side chain. The families of amino acid residues having similar side chains are well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amide (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine) and sulfur- containing side chains (e.g., cysteine, methionine). Furthermore, any native residue in the polypeptide can also be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55- 67; Sasaki et al., (1988) Adv Biophys 35:1-24). Amino acid substitutions to the antibodies can be made by known methods for example by PCR mutagenesis (US Pat. No. 4,683,195). Alternatively, libraries of variants can be generated for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acidsAttorney Docket No: JBI6815WOPCT1 (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting variants can be tested for their characteristics using assays described herein.

[0086] “Oligonucleotide” and the like refers to a polynucleotide formed from a plurality of linked nucleotide units (e.g., ribonucleotides, deoxyribonucleotides, or both). Such oligonucleotides can be obtained from existing nucleic acid sources or can be produced by synthetic methods. In some embodiments, the oligonucleotides each have from about 16 to about 25 nucleotide. In some embodiments, the oligonucleotides each have from 16 to 19, 17 to 20, 18 to 21, 19 to 22, 20 to 23, or 21 to 25 nucleotides. According to particular embodiments, internucleotide linkages for the oligonucleotides include, but are not limited to, phosphodiester linkages, phosphothioate linkages, and mixtures thereof.

[0087] “Conjugate” and the like refers to a protein or peptide covalently linked to one or more heterologous molecule(s). Examples of a protein or peptide that can be covalently linked to the heterologous molecule(s) include, but are not limited to, a therapeutic peptide or protein, an antibody or a fragment thereof. Examples of the heterologous molecule(s) that can be covalently linked to the protein or peptide include, but are not limited to, one or more small molecule compounds, a label, a linker, an oligonucleotide, etc.

[0088] “Ligand” and the like refers to a molecule, or chemical entity, that interacts with or binds to a target of interest, a subunit of target of interest, a domain of a target of interest, a target structural motif of a target of interest, or a fragment of a target of interest. Conjugation to a targeting ligand

[0089] Conjugation described throughout can be performed by a click reaction. For example, the antibody can be conjugated to a first click reaction partner. The antibody conjugate can then be further reacted with a second click reaction partner that comprises the isolated p19 polypeptide to obtain an antibody-p19 polypeptide.

[0090] As used herein, the term “click chemistry” refers to a chemical philosophy describing chemistry tailored to generate covalent bonds quickly and reliably by joining small units comprising reactive groups together (see e.g., Kolb, et al., Angew Chem IntEd Engl. 2001 Jun 1;40(11): 2004-2021). Click chemistry does not refer to a specific reaction, but to a concept including, but not limited to, reactions that mimic reactions found inAttorney Docket No: JBI6815WOPCT1 nature. In some embodiments, click chemistry reactions are modular, wide in scope, give high chemical yields, generate inoffensive byproducts, are stereospecific, exhibit a large thermodynamic driving force to favor a reaction with a single reaction product, and / or can be carried out under physiological conditions. In some embodiments, a click chemistry reaction exhibits high atom economy, can be carried out under simple reaction conditions, uses readily available starting materials and reagents, uses no toxic solvents or uses a solvent that is benign or easily removed, such as water, and / or provides simple product isolation by non-chromatographic methods, such as crystallization or distillation. In certain embodiments, the click chemistry reaction is a Huisgen cycloaddition or the 1,3-dipolar cycloaddition between an azide (-N3) and an alkyne, or an alkyne moiety, to form a 1 ,2,4- triazole linker.

[0091] As used herein, the term “click reaction partner” or “click chemistry handle” and similar phrases refers to a reactant or a reactive group that can partake in a click chemistry reaction. A click reaction partner can be a moiety that is rarely found in naturally-occurring biomolecules and is chemically inert towards biomolecules, but, e.g., when reacted with an azide- reactive or alkyne-reactive group, the reaction can take place efficiently under biologically relevant conditions, for example, in cell culture conditions, such as in the absence of excess heat or harsh reactants. In general, click chemistry reactions require at least two molecules comprising click reaction partners that can react with each other. Such click reaction partners that are reactive with each other are sometimes referred to herein as click chemistry handle pairs, or click chemistry pairs. In some embodiments, the click reaction partners are an azide and a strained alkyne, e.g., a cyclooctyne, or any other alkyne. In other embodiments, the click reaction partners are reactive dienes and suitable tetrazine dienophiles. For example, trans-cyclooctene, norbornene, or biscyclononene can be paired with a suitable tetrazine dienophile as a click reaction pair. In yet other embodiments, tetrazoles can act as latent sources of nitrile imines, which can pair with unactivated alkenes in the presence of ultraviolet light to create a click reaction pair, termed a “photo- click” reaction pair. In other embodiments, the click reaction partners are a cysteine and a maleimide. For example, the cysteine from a peptide (e.g., GGGC (SEQ ID NO: 44) can be reacted with a maleimide that is associated with a chelating agent (e.g., NOTA). Other suitable click chemistry handles are known to those of skill in the art (see,Attorney Docket No: JBI6815WOPCT1 e.g., Spicer et al., “Selective chemical protein modification.” Nature Communications. 2014; 5:4740). In other embodiments, the click reaction partners are Staudinger ligation components, such as phosphine and azide. In other embodiments, the click reaction partners are Diels-Alder reaction components, such as dienes, such as tetrazine, and alkenes, such as trans- cyclooctene (TCO) or norbornene. According to preferred embodiments, one of the first and second click reaction partners comprises an alkyne group, and the other click reaction partner comprises an azide.

[0092] Conditions for carrying out click chemistry reactions are known, and any known conditions for carrying out click chemistry reactions in view of the present disclosure can be used. Examples of conditions include, but are not limited to, incubating the antibody and the isolated p19 polypeptide at a ratio of 1:1 to 1:1000 at a pH of 4 to 10 and a temperature of 20°C to 70°C. A specific example of conditions include incubating the antibody and the isolated p19 polypeptide at a ratio of 1:4 at a pH of 7.2 and a temperature of 22°C.

[0093] In some embodiments, the condition can include an antibody to p19 polypeptide ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In a specific embodiment, the ratio is 1:4.

[0094] In some embodiments, the conditions can include a pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10. In a specific embodiment, the pH is about 7.2. In some embodiments, the incubation occurs at a temperature of about 20°C, 30°C, 40°C, 50°C, 60°C, or 70°C. In some embodiments, the temperature can be room temperature. In a specific embodiment, the temperature is about 20°C to 22°C. Polynucleotides, host cells and vectors

[0095] Also disclosed is an isolated polynucleotide encoding any one of the p19 polypeptides presented throughout.

[0096] Also disclosed is an isolated polynucleotide encoding any one of p19 polypeptides or fragments thereof.

[0097] Also disclosed is an isolated polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO: 15 or SEQ ID NO: 16.Attorney Docket No: JBI6815WOPCT1

[0098] Also disclosed is an isolated or purified nucleic acid comprising a polynucleotide which is complementary to the polynucleotides encoding the p19 polypeptides disclosed throughout or polynucleotides which hybridize under stringent conditions to the polynucleotides encoding the p19 polypeptides disclosed throughout.

[0099] The polynucleotide sequences can be operably linked to one or more regulatory elements, such as a promoter or enhancer, that allow expression of the nucleotide sequence in the intended host cell. The polynucleotide can be a cDNA. The promoter bay be a strong, weak, tissue-specific, inducible, or developmental-specific promoter. Exemplary promoters that can be used are hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, human muscle creatine, and others. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use with the described embodiments. Such viral promoters include Cytomegalovirus (CMV) immediate early promoter, the early and late promoters of SV40, the Mouse Mammary Tumor Virus (MMTV) promoter, the long terminal repeats (LTRs) of Maloney leukemia virus, Human Immunodeficiency Virus (HIV), Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV), and other retroviruses, and the thymidine kinase promoter of Herpes Simplex Virus. Inducible promoters such as the metallothionein promoter, tetracycline-inducible promoter, doxycycline-inducible promoter, promoters that contain one or more interferon-stimulated response elements (ISRE) such as protein kinase R 2',5'-oligoadenylate synthetases, Mx genes, ADAR1, and the like can also be used.

[0100] Disclosed is also a vector comprising the polynucleotide as described throughout. Disclosed is also an expression vector comprising the polynucleotide as described throughout. Such vectors can be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon-based vectors, or any other vector suitable for introduction of the synthetic polynucleotide into a given organism or genetic background by any means. Polynucleotides encoding the p19 polypeptides disclosed throughout can be operably linked to control sequences in the expression vector(s) that ensure the expression of the p19 polypeptides. Such regulatory elements can include a transcriptional promoter, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. Expression vectors can also include one orAttorney Docket No: JBI6815WOPCT1 more nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5' or 3' flanking nontranscribed sequences, 5' or 3' nontranslated sequences (such as necessary ribosome binding sites), a polyadenylation site, splice donor and acceptor sites, or transcriptional termination sequences. An origin of replication that confers the ability to replicate in a host can also be incorporated.

[0101] The expression vectors can comprise naturally-occurring or non-naturally- occurring internucleotide linkages, or both types of linkages. The non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.

[0102] Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the p19 polypeptides disclosed throughout encoded by the incorporated polynucleotides. The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells can be provided by viral sources. Exemplary vectors can be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

[0103] Vectors disclosed throughout can also contain one or more Internal Ribosome Entry Site(s) (IRES). Inclusion of an IRES sequence into fusion vectors can be beneficial for enhancing expression of some proteins. In some embodiments, the vector system will include one or more polyadenylation sites (e.g., SV40), which can be upstream or downstream of any one of the aforementioned nucleic acid sequences. Vector components can be contiguously linked or arranged in a manner that provides optimal spacing for expressing the gene products (i.e., by the introduction of “spacer” nucleotides between the ORFs) or positioned in another way. Regulatory elements, such as the IRES motif, can also be arranged to provide optimal spacing for expression.

[0104] Vectors disclosed throughout can be circular or linear. They can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColE1, SV40, 2μ plasmid, λ, bovine papilloma virus, and the like.Attorney Docket No: JBI6815WOPCT1

[0105] The recombinant expression vectors can be designed for either transient expression, for stable expression, or for both. Also, the recombinant expression vectors can be made for constitutive expression or for inducible expression.

[0106] The vectors can also comprise selection markers, which are well known in the art. Selection markers include positive and negative selection marker. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Exemplary marker genes include antibiotic resistance genes (e.g., neomycin resistance gene, a hygromycin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, a penicillin resistance gene, histidinol resistance gene, histidinol x resistance gene), glutamine synthase genes, HSV- TK, HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). A nucleic acid sequence encoding a selection marker or the cloning site can be upstream or downstream of a nucleic acid sequence encoding a polypeptide of interest or cloning site.

[0107] Exemplary vectors that can be used are Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia), pEE6.4 (Lonza) and pEE12.4 (Lonza). Additional vectors include the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene) can be used. Exemplary plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Exemplary animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The expression vector can be a viral vector, e.g., a retroviral vector, e.g., a gamma retroviral vector.

[0108] Embodiments disclosed throughout further provide host cells comprising any one of the recombinant expression vectors described herein. “Host cell” refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended toAttorney Docket No: JBI6815WOPCT1 refer not only to the particular subject cell but to the progeny of such a cell, and also to a stable cell line generated from the particular subject cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny can not be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Such host cells can be eukaryotic cells, prokaryotic cells, plant cells or archeal cells. Escherichia coli, bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species are examples of prokaryotic host cells. Other microbes, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells can be of mammalian, insect, avian or other animal origins. Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL- 61) or DG44.

[0109] The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance, DH5α E.coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating the recombinant expression vector, the host cell can be a prokaryotic cell, e.g., a DH5α cell. For purposes of producing a p19 polypeptide, or protein, the host cell can be a mammalian cell. The host cell can be a human cell.

[0110] Also provided are a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising the host cell comprising any one of the recombinant expression vectors described, in addition to at least one other cell, e.g., a host cell (), which does not comprise any one of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, a macrophage, an erythrocyte, a neutrophil, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly host cells (e.g., consisting essentially of) comprising the recombinant expression vector. The population also can be a clonal population of cells, in which all cells of the population are clones of a single hostAttorney Docket No: JBI6815WOPCT1 cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

[0111] Disclosed is also a method of producing the p19 polypeptides as disclosed throughout comprising culturing the host cell in conditions the p19 polypeptides are expressed, and recovering the p19 polypeptides produced by the host cell. Methods of making proteins and purifying them are known. Once synthesized (either chemically or recombinantly), the p19 polypeptides can be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, and the like (see generally Scopes, Protein Purification (Springer- Verlag, N.Y., (1982)). A subject protein can be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99%, or more, pure, e.g., free from contaminants such as cell debris, macromolecules, etc. other than the subject protein.

[0112] The polynucleotides encoding the p19 polypeptides disclosed throughout can be incorporated into vectors using standard molecular biology methods. Host cell transformation, culture, antibody expression and purification are done using known methods. Kits

[0113] Disclosed is also a kit comprising the mutant p19 polypeptides.

[0114] The kit can be used for screening uses and as diagnostic kits.

[0115] The kit can be used to selecting an siRNA for use in an siRNA antibody drug conjugate.

[0116] In some embodiments, the kit comprises the mutant p19 polypeptides disclosed throughout and reagents for detecting the target of interest. The kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, an antibody to aAttorney Docket No: JBI6815WOPCT1 label or therapeutic agent, or a radioprotective composition; devices or other materials for preparing the antibody for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

[0117] In some embodiments, the kit comprises the mutant p19 polypeptide in a container and instructions for use of the kit.

[0118] In some embodiments, the mutant p19 polypeptide in the kit is labeled.

[0119] In some embodiments, the kit comprises the mutant p19 polypeptide comprising the sequences of SEQ ID NO 15 or SEQ ID NO 16. EMBODIMENTS

[0120] This invention provides the following non-limiting embodiments. 1. A method of selecting an oligonucleotide for use in an oligonucleotide conjugate, the method comprising: a. linking one or more p19 polypeptides to a targeting ligand to form a targeted p19 polypeptide; and b. contacting an oligonucleotide to the targeted p19 polypeptide to form a targeted-p19-oligonucleotide complex. 2. The method of embodiment 1, wherein the oligonucleotide comprises a length of about 16 to about 25 nucleotides. 3. The method of embodiments 1 or 2, wherein the oligonucleotide is an siRNA, an miRNA, or an antisense RNA. 4. The method of any one of embodiments 1 to 3, wherein the oligonucleotide is contacted to the targeted p19 polypeptide in a ratio of about 10:1 to about 2:5. 5. The method of any one of embodiments 1 to 4, wherein the oligonucleotide is contacted to the targeted-p19 polypeptide in a ratio of about 2:1. 6. The method of any one of embodiments 1 to 5, wherein a cell expressing a target of the oligonucleotide is contacted to the targeted-p19-oligonucleotide polypeptide. 7. The method of any of embodiments 1 to 6, when the target of the targeting ligand comprises a surface ligand.Attorney Docket No: JBI6815WOPCT1 8. The method of any one of embodiments 1 to 7, wherein the method produces a targeted-p19-oligonucleotide polypeptide at a concentration of about 0.01 nM to about 100 nM. 9. The method of any one of embodiments 1 to 8, wherein the cell is contacted with the targeted-p19-siRNA polypeptide for about 24 to about 96 hours. 10. The method of any one of embodiments 6 to 9, wherein a level of the target of the oligonucleotide is measured. 11. The method of any one of embodiments 6 to 10, wherein the level of the target of interest is measured by qRT-PCR. 12. The method of any one of embodiments 6 to 11, wherein the level of the target of interest in the cell contacted with the targeted-p19-oligonucleotide fusion polypeptide is compared with a control, and optionally wherein the control expresses the target of interest or is treated with an inoperable targeted-p19- oligonucleotide, and optionally wherein a modulation in the level of the target of interest in the cell contacted with the targeted-p19- oligonucleotide fusion polypeptide as compared to the control indicates that the oligonucleotide is capable of modulating the target of interest when fused to the targeting ligand. 13. The method of embodiment 12, wherein an optimal oligonucleotide is selected based on the modulation of the level of the target of interest in the cell contacted with the targeted-p19-oliognucleotide fusion polypeptide. 14. The method of any one of embodiments 1 to 13, wherein the targeting ligand is selected from the group consisting of an antibody, an antibody fragment, a miniprotein, or a peptide. 15. The method of any one of embodiments 1 to 14, wherein the p19 polypeptide is covalently linked to a heavy chain or a light chain of the antibody. 16. The method of any one of embodiments 1 to 15, wherein the p19 polypeptide is covalently linked to the heavy chain of the antibody. 17. The method of any one of embodiments 1 to 16, wherein the p19 polypeptide and the antibody are linked by a click chemistry method or a conjugation method. 18. The method of any one of embodiments 1 to 17, wherein the p19 polypeptide and the antibody are linked by a linker sequence.Attorney Docket No: JBI6815WOPCT1 19. The method of embodiment 18, wherein the linker sequence comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO: 21. 20. The method of embodiments 18 or 19, wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 21. 21. The method of any one of embodiments 1 to 20, wherein the p19 polypeptide is a mutant p19 polypeptide. 22. The method of embodiment 21, wherein the mutant p19 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 15 or SEQ ID NO: 16. 23. The method of embodiments 21 or 22, wherein the mutant p19 polypeptide comprises the amino acid sequence of SEQ ID NO: 16. 24. The method of embodiment 21, wherein the p19 polypeptide comprises a Δ1-5, a N15K, a G16R, a C134S, and / or a C160A mutation. 25. The method of any one of embodiments 1 to 24, wherein the oligonucleotide is a therapeutic oligonucleotide. 26. The method of any one of embodiments 1 to 25, wherein the targeting ligand binds to HER2 or TfR. 27. An p19 polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 15 or SEQ ID NO 16. 28. The p19 polypeptide of embodiment 27, comprising the amino acid sequence of SEQ ID NO: 16, SEQ ID NO:18, or SEQ ID NO: 19. 29. A polynucleotide encoding any one of the p19 polypeptides of embodiment 27. 30. A vector comprising the isolated polynucleotide of embodiment 29. 31. A host cell comprising the vector of embodiment 30. 32. A kit for selecting an siRNA for use in an siRNA targeting ligand drug conjugate, the kit comprising one or more of the p19 polypeptides of claim 27 and instructions for use. 33. The p19 polypeptide of embodiment 27, further comprising an antibody, wherein the antibody is linked to the p19 polypeptide. 34. The p19 polypeptide of embodiment 33, further comprising an oligonucleotide wherein the oligonucleotide is complexed with the p19 polypeptide.Attorney Docket No: JBI6815WOPCT1 35. The p19 polypeptide of embodiment 27, further comprising an oligonucleotide, wherein the oligonucleotide is complexed with the p19 polypeptide. EXAMPLES

[0121] The following examples are provided to supplement the prior disclosure and to provide a better understanding of the subject matter described herein. These examples should not be considered to limit the described subject matter. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be apparent to persons skilled in the art and are to be included within, and can be made without departing from, the true scope of the invention. Example 1: Design and production of antibody-p19 fusions

[0122] To develop p19 into a targeted siRNA carrier, a series of fusion proteins were designed consisting of p19 and the HER2 targeting antibody trastuzumab. The p19 construct incorporates the following mutations: C134S and C160A to prevent unwanted crosslinking, and N15K and G16R to increase siRNA affinity which was correlated with more potent silencing. p19 was fused as a monomer or as a covalently linked dimer to the C-terminus of either the heavy or light chain of trastuzumab (Figure 1 and Table 1). Fusions 1-2 have one p19 monomer per chain and are theoretically capable of binding 1 equivalent siRNA per mAb-p19. However, because the p19 domains form homodimers even in the absence of siRNA, there is a risk that these constructs could be prone to oligomerization and aggregation. As an alternative approach, a previously described construct was built upon in which two p19 monomers are connected by a flexible (GGGGS)2 (SEQ ID NO: 20) linker (Linker 2). Covalently linking the p19 monomers has been shown to improve both the thermostability of the p19 protein and the siRNA binding affinity. Fusions 3-8 have one fused dimer per chain and are therefore capable of binding up to 2 equivalents of siRNA per mAb-p19. To explore the effect of the length and composition of the linker between the mAb and p19 (Linker 1) on the properties of theAttorney Docket No: JBI6815WOPCT1 fusion proteins, constructs were designed with three different linker compositions: (GGGGS)2 (SEQ ID NO: 20), (GGGGS)3 (SEQ ID NO: 19), and (AP)5 (SEQ ID NO: 21). All trastuzumab-p19 fusions were expressed in ExpiCHO-S cells. Protein expression was confirmed by Protein A-HPLC using a PA-ID Sensor Cartridge (ThermoFisher, 2100100). The column was equilibrated with 1 x PBS (pH ~7.4), and the flow rate was maintained at 1.0 ml / min. A gradient HPLC was performed with the following conditions: BUFFERS USED Solvent A PBS (NaCl-137mM, KCl-2.7Mm, Na2HPO4-8mM, KH2PO42mM;pH 7.4) Solvent B 80 mM acetic acid; pH 3.0 Time Solvent A Solvent B (mins) (%) (%) 0 100 0 2.00 100 0 2.01 0 100 4.00 0 100 4.01 100 0 5 100 0

[0123] Batches were harvested when the cell viability dropped to ~70%. All proteins were purified by Protein A affinity chromatography on a HiTrap mAbselect PrismA column (Cytiva, Marlborough, MA, 1754800) and size-exclusion chromatography on a Superdex200 HiLoad 26 / 600 column (Cytiva, 28989336). An additional cation exchange step was performed after the size exclusion step for fusion 9, on a HiTrap SP column (Cytiva, 17072910) with an increasing salt gradient (0-1M NaCl) in 10 mM sodium acetate pH 5. The final protein was buffer exchanged into 1xDPBS by gel filtration on a Sephadex G25 column (Cytiva, 17003301). Protein purity was assessed by SDS-PAGE and Size Exclusion-HPLC. For SDS-PAGE, 3 µg total protein was analyzed on a 4-20% Stain Free gel (BioRad). Analytical SEC analysis was performed on an Agilent HPLC 1200 Series equipped with a Tosoh TSK gel G3000SWxl column (Tosoh Bioscience #08541) equilibrated in 1xDPBS + 0.2 M L-Arginine monohydrochloride + 0.01% sodium azide.Attorney Docket No: JBI6815WOPCT1

[0124] The fusions with the more flexible Gly-Ser Linker 1 had expression titers >2-fold higher than those with the more rigid Ala-Pro Linker 1 (Table 1). aSEC analysis of the purified fusion proteins revealed that while the location of p19 (heavy chain vs light chain) did not have much of an impact on the properties of the fusion protein, the number of p19 monomers per chain and the composition of the linker did (Figure 6 and Table 1). expression titer Yield Final protein Number Chain Linker monomer / dimer (mg / L) (mg / L) % Purity SEQ ID 1* LC monomer 241.1 21.525 91% NO: 19expression titers, and yields were determined by aSEC.

[0125] Fusion constructs 4 and 7 showed less visible precipitation and evidence of aggregation than the constructs with the shorter 10 aa Linker 1, suggesting that the greater physical separation between the mAb and p19 domains can increase stability. Fusions 1 and 2, that have 1 p19 monomer per chain, also showed a tendency to aggregate, leading to significant precipitation.

[0126] All constructs, regardless of the linker or location of p19, showed contamination with a lower molecular weight species that corresponds to the heavy chain or light chain without p19 (Figures 2A-B and 6). It was predicted that this contamination could be from proteolytic cleavage of the p19 domain. Samples were analyzed by LC-MS to determine the mass difference between the cleaved and intact species. LC-MS was performed on anAttorney Docket No: JBI6815WOPCT1 Agilent Model G6224 MS-TOF Mass Spectrometer. The instrument was operated in positive electrospray ionization mode and scanned from m / z 600 to 4000 for fragments or 2000 to 6000 m / z for intact mass. Instrument settings included: capillary voltage 3500V; fragmentor 175V; skimmer 65V; gas temperature 325 °C; drying gas flow 5.0 L / min; nebulizer pressure 30 psig; acquisition mode range 100-7000 with 0.42 scan rate.

[0127] Based on the mass difference between the cleaved and intact, the cleavage was predicted to be occurring between residues 3 and 4 of the N-terminal p19 domain (Figure 7). To engineer a more homogenous siRNA carrier and prevent loss of the p19 domain, a deletion mutation was introduced to the N-terminal p19 in constructs 4 and 7, which were selected as the starting point for further optimization because they initially had the highest yields of soluble protein. Because the N-terminus of p19 is not involved in RNA binding, it was proposed that the first 5 residues of p19 could be deleted and the site of proteolysis removed without impacting protein function. Constructs 9 and 10, which incorporate this ∆1-5 mutation, are monodisperse and stable in solution and no longer show evidence of p19 cleavage (Figures 2C-D, 6). These fusions could be purified to >90% homogeneity, which also greatly improved the overall yield of purified protein compared to the initial unoptimized versions. Although the light chain and heavy chain fusions behaved similarly, the heavy chain fusion 10 was chosen for use in subsequent experiments and is referred to going forward as trastuzumab-p19. Example 2: Generation of Trastuzumab-p19 conjugates by click chemistry.

[0128] While construction of the mAb-p19 molecules as genetic fusions has advantages, the time required to design, optimize, and produce new fusion proteins for a target could potentially be a drawback. As an alternative, a conjugation strategy was used to attach p19 to existing mAbs and targeting ligands of interest using established site-specific methods. In one such example, a stand-alone p19 dimer was generated with a C-terminal LPETG sortase recognition sequence, which can be labeled with a biorthogonal click chemistry handle by sortase transpeptidation. The p19 monomers in this construct are connected by the same Linker 2 (SEQ ID NO: 20) described for the fusion proteins. This construct also contains a C-terminal 6xHistidine tag (SEQ ID NO: 45) for affinity purification. TheAttorney Docket No: JBI6815WOPCT1 sequence for 2xp19-LEPTG-His6was codon-optimized for expression in E. coli and expressed from the pJ401 vector (ATUM Bio, Newark CA) in E. coli BL21 competent cells (NEB, Ipswich MA, C2530H). Cells were grown in 1 L cultures of TB medium (Growcells, Irvine CA, MBLE-3270) at 37 °C with shaking at 200 rpm until the OD600 reached ~0.5. Expression was induced by addition of 1 mM IPTG (VWR), and the culture was further incubated ON at 20 °C. Cells were harvested by centrifugation at 6,000xg for 15 minutes at 4 °C. Cells were lysed using BugBuster HT Protein Extraction solution (Millipore Sigma) following the manufacturer’s instructions. Cell lysate was centrifuged at 20,000xg for 30 min at 4 °C. The protein was purified by Ni-NTA affinity chromatography, followed by anion exchange chromatography and size-exclusion chromatography. Anion exchange was performed on a 5 mL HiTrap Q HP column (Cytiva, 17115301) with an increasing salt gradient (0.01-1 M NaCl) in 50 mM Tris pH 7.5. SEC was performed using a HiLoad 16 / 600 Superdex200 column (Cytiva, 28989335) in 50 mM Tris pH 7.5 p19-LPETG-His6could be isolated in good yield (~12 mg / L cell culture) and >99% purity as determined by aSEC and SDS-APGE (Figure 12A-C). Generation of p19-DBCO:

[0129] 2xp19-LPETG-His6was conjugated to DBCO via sortase reaction (Figure 13). 2xp19-LPETG-His6 was incubated with 25 equivalents of Gly3-cysteic acid-DBCO and a 1:50 ratio of SrtA:p19 for 1-2 h at room temperature in buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, and 10 mM CaCl2. Cleavage of the His-tag from 2xp19 and addition of the Gly3-cysteic acid-DBCO peptide was confirmed by LC-MS. 2xp19-DBCO was separated from the cleaved His-tag and SrtA by Ni-NTA chromatography and excess peptide was removed by buffer exchange on a Zeba desalting column (ThermoFisher, 89892) followed by diafiltration in a 10 kDa MWCO centrifugal filter device (Millipore Sigma, Burlington MA, UFC901024).

[0130] Generation of Trastuzumab-p19 conjugates:

[0131] The resulting p19-DBCO were then conjugated to an azide-labeled antibody (Figure 5A). Trastuzumab-azide was prepared by site-specific conjugation with microbial transglutaminase. Trastuzumab was deglycosylated overnight with Rapid PNGaseF (NEB)Attorney Docket No: JBI6815WOPCT1 at 37 °C. The deglycosylated mAb was incubated at 37 °C with 100 equivalents of 3- azidopropylamine (Click Chemistry Tools) and 20% w / v Activa TI transglutaminase. The resulting trastuzumab-azide was purified by Protein A affinity chromatography on a mAbselect SuRe column (Cytiva, 11003495). To prepare the trastuzumab-p19 conjugate, trastuzumab-azide was incubated ON at room temperature (~22 °C) with 4 equivalents p19-DBCO. The addition of p19 was confirmed by SDS-PAGE and intact LCMS \. Conjugates were purified by gel filtration chromatography on a Superdex 200 Increase 10 / 300 GL column equilibrated in 1xDPBS. Fractions containing conjugate were pooled and concentrated using a 100 kDa MWCO centrifugal filter device (Millipore Sigma, UFC903008) and quantified by UV absorption. Purified Trastuzumab-Az-p19 conjugates had a degree of labeling of ~1.8-1.9 as determined from the SDS-PAGE (Figures 5B and 14). Example 3: Trastuzumab-p19 bind chemically modified siRNA

[0132] The ideal targeted p19 is capable of binding siRNA sequences with modifications at all positions of the sugar-phosphate backbone to enable successful delivery of the full range of potential therapeutic siRNAs. Previous work characterizing siRNA delivery with p19 was done with unmodified siRNA or with siRNA that was modified with 2’-OMe at selected positions only. For this study, binding was analyzed between trastuzumab-p19 and a fully modified β-catenin (CTNNb1) siRNA sequence that is stabilized with a previously described pattern of 2’-F, 2’-OMe, and phosphorothioate modifications (Table 2, CTNNb1 siRNA_1). CTNNb1 was targeted due to the availability of highly potent siRNAs that target this broadly expressed gene. SEQ CTNNb1 siRNAs Strand Sequence CTNNb1 siRNA_1 Sense u•a•cuguUgfGAUugauucga•a•aantisense VPu•U•ucgAaUCaaucCaAcagua•g•c 23 CTNNb1 Sense u•a•cuguuggauugauucga•a•a 24 Fully 2’-OMe siRNA_2OMe antisense VPu•u•ucgaaucaauccaacagua•g•c 25Attorney Docket No: JBI6815WOPCT1 CTNNb1 siRNA_2F Sense U•A•CUGUUGGAUUGAUUCGA•A•A 26 Fully 2’-F antisense VPu•U•UCGAAUCAAUCCAACAGUA•G•C27CTNNb1 Alternating 2’-OMe / 2’- Sense u•a•CuGuUgGaUuGaUuCgA•a•A 28 siRNA_2OMe / 2F F antisense VPu•U•uCgAaUcAaUcCaAcAgUa•G•c 29 CTNNb1 siRNA_2 Sense c•u•GuuGGAuuGAuucGAA•A 30 Chemistry variant 2 antisense VPu•U•uCgAaUcAaUcCaAcAg•u•u 31 siRNA_1 with 2’-F CTNNb1 siRNA_1d Sense u•a•cugudTgdGdAdTugauucga•a•a 32 replaced with DNA bases antisense VPu•dT•ucgdAadTdCaaucdCadAcagua•g•c 33 CTNNb1 siRNA_3 Sense (invAb) •c•uguUgGAUugauucga•a•a 34 Chemistry variant 3 antisense VPu•U•ucgAaUCaaucCaAcag•u•u 35 (invAb) •c•uguugGAUugauucgaa•a• CTNNb1 siRNA_4 Sense 36 Chemistry variant 4 (invAb) antisense VPu•U•ucgAauCaaucCaAcag•u•a37 CTNNb1 siRNA_5 Sense c•u•GuugGAUugAuucgA•a•a 38 Chemistry variant 5 antisense VPu•U•ucgAaucaaucCaAcag•u•a 39 CTNNb1 siRNA_1a Sense u•a•cuguUgfGAUugauucga•a•a 40 siRNA_1 w / o 5’-P antisense u•U•ucgAaUCaaucCaAcagua•g•c 41 siRNA_1 w / standard 5’- CTNNb1 siRNA_1b Sense u•a•cuguUgfGAUugauucga•a•a 42 P antisense Pu•U•ucgAaUCaaucCaAcagua•g•c 43 Table 2. Oligonucleotide sequences and chemistry modifications. Chemistry modification: •, phosphorothioate (PS) linkage; lower case nucleotides, 2’-O-methyl (OMe); upper case nucleotides in italics, 2’-deoxy-2’fluoro (F); VP, 5’-(E)-vinylphosphonate

[0133] Complex formation was first analyzed by SEC, and the protein and siRNA components were distinguished by their differing λmax values (280 for protein, 260 for siRNA) (Figure 3A). Analytical SEC was performed using a Superdex2005 / 150 GL column (Cytiva, 28990945) equilibrated in 0.2 mM sodium phosphate pH 6.8. Samples were prepared with 10 µg protein. The siRNA co-eluted with the trastuzumab-p19 protein in a single homogenous peak, which is slightly shifted to a shorter retention time compared to the peak for trastuzumab-p19 protein by itself, suggesting a higher apparent MW. ThisAttorney Docket No: JBI6815WOPCT1 shift is accompanied by an increase in the absorbance at 260 nm as well as an overall increase in peak intensity, confirming that the siRNA is bound to the protein. The peak for the complex is distinct from that of the free siRNA, which elutes at a later retention time of 6.5 min (data not shown).

[0134] To further understand the siRNA binding capacity of trastuzumab-p19, the complex formation was also analyzed by a fluorescence based electrophoretic mobility shift assay (EMSA) (Figure 3B-C). Samples containing 0.25-0.5 µM CTNNb1 siRNA and the desired equivalents of trastuzumab-p19 were prepared in a total volume of 15 µL in 1xdPBS. The complexes were incubated on ice for 20 minutes and then combined with 3 µL of 6X EMSA Gel Loading Dye (ThermoFisher). Samples were loaded onto a 4-20% non- denaturing gel (BioRad) and run at 75 V for 120 minutes in 1X Tris-Glycine Running Buffer. Gels were stained with SYBR Green, imaged, then stained with SYPRO Ruby according to the manufacturer’s protocol. Gels were imaged using the built-in SYBRTMGreen and SYPROTMRuby protocols on a Bio-Rad Gel Doc XR+ gel documentation system.

[0135] At lower equivalents of trastuzumab-p19 most of the siRNA is in the unbound form, but as the concentration of trastuzumab-p19 is increased relative to the concentration of siRNA, shifted siRNA bands emerge that can be attributed to the mAb-p19 / siRNA complex (Figure 3B). The bands for the protein components migrate with the RNA components and provides further evidence that they have formed a complex (Figure 3C). At higher protein equivalents, a mixture of complexes is formed with distinct bands visible for the protein likely corresponding to 0, 1 or 2 siRNA molecules bound. These data establish that fusing p19 to an antibody does not interfere with siRNA binding and that trastuzumab-p19 can bind fully chemically modified siRNA.

[0136] Purified p19-LPETG-His6 was also confirmed to bind CTNNb1 siRNA by EMSA (Figure 12D), as was the trastuzumab-Az-p19 conjugate (Figure 5C).

[0137] Example 4. mRNA knockdown from targeted p19 delivery

[0138] To evaluate whether trastuzumab-p19 can be used to deliver functional siRNA to cells, the CTNNb1 siRNA_1 described above was complexed with trastuzumab-p19 in different ratios and applied to HER2 expressing HCC1954 cells. The trastuzumab-p19Attorney Docket No: JBI6815WOPCT1 mediated knockdown was compared with knockdown from a potent trastuzumab-CTNNb1 siRNA covalent conjugate, which was generated by site-specific transglutaminase labeling followed by strain promoted click chemistry with a DBCO-modified siRNA and has a degree of labeling (DOL) of 2 (Figure 8 and Figure 16). Treatment with the trastuzumab- p19 / siRNA complexes for 72 h resulted in dose-dependent reduction in CTNNb1 mRNA expression at all ratios (Figure 3D-E), and neither the siRNA nor trastuzumab-p19 alone reduced the mRNA levels at concentrations at or below 100 nM, indicating that trastuzumab-p19 is responsible for siRNA uptake (Figure 9A).

[0139] The extent of silencing was highly dependent on the ratio of trastuzumab-p19 to siRNA. It was observed that potency increased concomitantly with trastuzumab-p19 concentration up to one stoichiometric equivalent (1 trastuzumab:2 siRNA) at which point both the maximum level of knockdown and IC50 values were comparable to the direct trasutzumab-CTNNB1 conjugate (Figures 3D-E and 9B). In these samples the siRNA binding sites on trastuzumab-p19 are presumably saturated, maximizing the amount of siRNA that is taken up by the cells. An attenuation in the knockdown was observed as the trastuzumab-p19 concentration is increased above a stoichiometric ratio. For all subsequent assays, a stoichiometric amount of siRNA and trastuzumab-p19 was used to maximize the knockdown (Figure 3F).

[0140] Treating HCC1954 cells with the conjugated trastuzumab-Az-p19 complexed with CTNNb1 siRNA resulted in mRNA knockdown that was comparable to the knockdown obtained from the trastuzumab-p19 fusion proteins (Figure 5D). These results indicate that site-specific conjugation of p19 to a mAb is a viable alternative to a genetic fusion protein for siRNA delivery. These results also demonstrate that no silencing occurs with the unconjugated p19 / siRNA complex, indicating that the mAb is required for cell uptake and that there is no toxicity associated with the standalone p19 protein. Overall, this protocol establishes a method for converting targeting ligands from commercially available “off- the-shelf” mAbs or other proteins and peptides that can be modified with a click chemistry handle, into a cell-targeting p19 without the need to express a new genetic fusion protein.

[0141] mAb and mAb-p19-mediated siRNA uptake:Attorney Docket No: JBI6815WOPCT1 HCC1954 cells (ATCC) were cultured in RPMI 1640 (Gibco, 72400-146) + 10% FBS (Gibco, 16140-063). The cells were maintained at 37°C in a humidified incubator with 5% CO2 for the intended treatment time. HCC1954 cells were plated in 96-well tissue culture plates (~5,000 cells per well) for 24 h and then treated in duplicate with mAb- siRNA conjugates or mAb-p19 / siRNA complexes for 72 h. Cells were lysed and the RNA was isolated using the mRNA catcher PLUS kit (ThermoFisher). mRNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied BioSystems) in a ProFlex ThermoCycler (Applied Biosystems) according to the manufacturer’s instructions. cDNA was stored at -20 °C until analysis. qPCR was performed using TaqMan Fast Advanced 2x Master Mix (Applied Biosystems) with TaqMan Gene Expression Assay Primer / Probes (Applied Biosystems) for CTNNb1 and GAPDH on a ViiA 7 Real-Time PCR System (Applied Biosystems). Target gene CT values were normalized by subtracting the GAPDH CT values to obtain a deltaCT. The average of untreated deltaCT values was then subtracted from the sample deltaCT. Relative mRNA level was then determined using the equation, % Gene expression = 100 × 2(−deltadeltaCT). All data were analyzed in GraphPad Prism and presented as mean ± standard error of the mean (SEM). Dose-response curves were all performed in duplicate with an experimental n=2 (in some cases n=3). Concentrations were log-transformed and data were fit to three parameter sigmoidal curves. Example 5: mRNA knockdown from mAb-p19 delivery predicts mAb-siRNA conjugate potency

[0142] After demonstrating that siRNA activity was comparable for one highly optimized siRNA sequence, it was sought to determine how well trastuzumab-p19 can predict conjugate performance for a small panel of siRNAs that was designed to capture a range of potencies. A set of CTNNb1 siRNAs were designed with different patterns of chemical modifications to the sugar-phosphate backbone, which it was anticipated would have a large impact on the activity of the siRNA (Table 2, Figure 10). All siRNAs have the same nucleotide sequence but vary in the placement of 2’-OH and 2’-F ribose modifications in both strands, and includes modification patterns that are known to perform poorly such as 2_OMe and 2_F (38) as well as those with more optimized 2’-OMe and 2’-F contents. TheAttorney Docket No: JBI6815WOPCT1 identity of the 5’phosphonate was varied on the antisense strand (natural 5’-phosphate vs. stabilized 5’-(E)-vinyl phosphonate), which has a known effect on siRNA stability and knockdown. It was confirmed by EMSA that all chemistry variants bind to p19, suggesting that these chemical modifications do not significantly impact the ability to bind to p19 (Figure 11). The siRNAs were then applied to HCC1954 cells both as equimolar complexes with trastuzumab-p19 and as covalent trastuzumab-CTNNb1 conjugates, and mRNA knockdown at 72 h was measured by qRT-PCR as described in example 4 (Figure 4). mRNA knockdown from these targeted delivery methods was also compared to mRNA knockdown from siRNA delivered via lipofectamine transfection.

[0143] Preparation of siRNA conjugates: To prepare trastuzumab-siRNA conjugates, trastuzumab-azide was incubated ON at 37 °C with 5 equivalents CTNNb1 duplex siRNA modified with a 5’-DBCO on the sense strand (Axolabs, Kulmbach Germany). The extent of conjugation was estimated from reduced and denatured SDS-PAGE. and was estimated to be between 1.89-1.93 for all conjugates. Conjugates were purified by gel filtration on a Superdex 200 Increase 10 / 300 column (Cytiva, 28990944) equilibrated in 1xDPBS. Fractions containing conjugate were pooled and concentrated in 50 kDa MWCO centrifugal filter device (Millipore Sigma). Concentrations were determined by A260 using the extinction coefficient of the conjugate, adjusted for the degree of labeling. Transfection: Cells were plated in 96-well tissue culture plates (~5,000 cells per well) and incubated overnight at 37°C in a humidified incubator with 5% CO2. Unlabeled siRNAs were prepared with equal volume Lipofectamine RNAiMAX transfection reagent (Invitrogen) in Opti-MEM (Gibco, 31985062) and incubated for 20 minutes before adding to cells in duplicate. Following sample addition, plates were incubated for 48 h at 37°C in a humidified incubator with 5% CO2. mRNA expression was measured as described above.

[0144] The trastuzumab-CTNNb1 conjugates exhibited a wide range of potencies, ranging from almost no knockdown at all with CTNNb1 siRNA_2OMe, to ~75% knockdown with IC50 values of <1 nM for the more optimized chemistry variants (Figure 4A). A similar dose-dependent reduction was observed in the CTNNb1 expression when the same siRNAsAttorney Docket No: JBI6815WOPCT1 were applied to cells as complexes with trastuzumab-p19 (Figure 4B). Although the siRNAs overall were slightly less potent when delivered as complexes with trastuzumab- p19 than as covalent conjugates, both the overall knockdown and the IC50 values were well correlated between the two delivery methods indicating that mAb-p19 delivery can be used to as a predictor of conjugate potency (Figure 4D-E).

[0145] The advantages of screening siRNAs with mAb-p19 are evident when comparing the mRNA knockdown from the targeted delivery methods with lipofectamine transfection (Figure 4C). While siRNA delivery with the mAb-p19 reflected the potency of the corresponding conjugates, transfection resulted in extremely potent knockdown at 48 hr from almost all sequences, including ones that were shown to have much lower activity when delivered as a conjugate such as variants 3 and 6. With the exception of CTNNb1 siRNA_2OMe, there was very low correlation between transfection and conjugate potency for any one of the siRNAs tested. These data demonstrate how a p19 fusion can aid in the optimization of modification chemistry during the siRNA conjugate discovery process. Example 6: siRNA delivery with a transferrin receptor targeting mAb-p19

[0146] Targeted p19 fusions can be used to deliver siRNA to any cell provided that the appropriate targeting ligand is selected. Transferrin receptor (TfR) is a ubiquitous cell surface protein that can be used for delivery to multiple tissue types. A fusion of p19 and the TfR targeting mAb BBBB175 was generated for delivery of siRNA to TfR expressing cells.

[0147] Generation of B175-p19 fusions: p19 was fused to the heavy chain of BBBB175 by a GGGGSGGGGSGGGGS linker [SEQ NO. 19] to generate BBBB175-p19. BBBB175- p19 was expressed in Expi293 cells. The expression was confirmed by PA-HPLC as described above and batches were harvested when the cell viability dropped to ~70%. BBBB175-p19 was purified by Protein A affinity chromatography on a HiTrap mAbselect PrismA column (Cytiva, Marlborough, MA, 1754800) and size-exclusion chromatography on a Superdex200 HiLoad 26 / 600 column equilibrated in 1xdPBS pH 7.4 (Cytiva, 28989336). Protein purity was assessed by SDS-PAGE and Size Exclusion-HPLC as described for the trastuzumab fusions.Attorney Docket No: JBI6815WOPCT1

[0148] siRNA binding to BBBB175-p19 was assessed by EMSA. 0.5 µM BCL2 siRNA was incubated with 0.5-2 equivalents of BBBB175-p19. Samples were run on a native non- reducing gel for 2 hr @ 75V, then stained first with SYBR Green and then overnight with SYPRO Ruby according to the manufacturer’s protocol. A clear difference can be seen between the BBBB175-p19 with 1 or 2 equivalents of siRNA bound vs BBBB175-p19 without any siRNA, which does not migrate from the well (Figure 17A).

[0149] BCL2 mRNA knockdown was measured in the acute myeloid leukemia (AML) cell line TF1. Cells were initially plated at 10000 cells per well in 100µL of RPMI1640 media + 10% FBS with 2 ng / µL of the growth factor GM-CSF. Following seeding, the plates were then incubated at 37°C, 5% CO2, 90% RH. Complexes were prepared with 5 µM BCL2 siRNA and 0.5 molar equivalents of BBBB175-p19 and incubated for ~30 min at RT before diluting into RPMI1640 media + 10% FBS with 2 ng / mL GM-CSF to the desired concentration.

[0150] TF1 cells were treated in duplicate with the BBBB175-p19 / siRNA complex for 72 h. BBBB175-bcl2 conjugate (prepared essentially as described above) was included as a positive control and BCL2 siRNA and b175-p19 protein were included as negative controls. mRNA knockdown was assessed by qPCR as described above using TaqMan Gene Expression Assay Primer / Probes (Applied Biosystems) for BCL2 and GAPDH (Figure 17B). The BCL2 knockdown from siRNA delivered as a complex with BBBB175- p19 was comparable to the knockdown from the covalent BBBB175-BCL2 siRNA conjugate. SEQUENCE LISTING: SEQ ID NO: 1 Tmab-p19 [wt HC] HC: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNAttorney Docket No: JBI6815WOPCT1 KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK SEQ ID NO: 2 Tmab-p191 LC: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGG SGGGGSQGNDTREQAKRERWDGGSGGITSPFKLPDESPSWTEWRLYNDETNSNQ DNPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGSWTGDSVNYAASRFLGANQ VGCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELLQLTPVEVESNVSRGAPEGI ETFKKESE SEQ ID NO: 3 Tmab-p192 HC: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGGGGGSGGGGSGGGGSQGNDTREQAKRERWDGGSGGITSPFKLPDE SPSWTEWRLYNDETNSNQDNPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGS WTGDSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELL QLTPVEVESNVSRGAPEGIETFKKESE SEQ ID NO: 4 Tmab-p192 [WT LC] LC: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPAttorney Docket No: JBI6815WOPCT1 SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 5 Tmab-p193 [WT HC] HC: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID NO: 6 Tmab-p193 LC: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGG SMERAIQGNDTREQAKRERWDGGSGGITSPFKLPDESPSWTEWRLYNDETNSNQD NPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGSWTGDSVNYAASRFLGANQV GCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELLQLTPVEVESNVSRGAPEGIET FKKESEGGGGSGGGGSMERAIQGNDTREQAKRERWDGGSGGITSPFKLPDESPSW TEWRLYNDETNSNQDNPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGSWTG DSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELLQLT PVEVESNVSRGAPEGIETFKKESE SEQ ID NO: 7 Tmab-p194 LC: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGAttorney Docket No: JBI6815WOPCT1 SGGGGSMERAIQGNDTREQAKRERWDGGSGGITSPFKLPDESPSWTEWRLYNDET NSNQDNPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGSWTGDSVNYAASRFL GANQVGCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELLQLTPVEVESNVSRG APEGIETFKKESEGGGGSGGGGSMERAIQGNDTREQAKRERWDGGSGGITSPFKLP DESPSWTEWRLYNDETNSNQDNPLGFKESWGFGKVVFKRYLRYDRTEASLHRVL GSWTGDSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQE LLQLTPVEVESNVSRGAPEGIETFKKESE SEQ ID NO: 8 Tmab-p195 LC: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAPAPAPAPAP MERAIQGNDTREQAKRERWDGGSGGITSPFKLPDESPSWTEWRLYNDETNSNQD NPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGSWTGDSVNYAASRFLGANQV GCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELLQLTPVEVESNVSRGAPEGIET FKKESEGGGGSGGGGSMERAIQGNDTREQAKRERWDGGSGGITSPFKLPDESPSW TEWRLYNDETNSNQDNPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGSWTG DSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELLQLT PVEVESNVSRGAPEGIETFKKESE SEQ ID NO: 9 Tmab-p196 HC: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGGGGGSGGGGSMERAIQGNDTREQAKRERWDGGSGGITSPFKLPDE SPSWTEWRLYNDETNSNQDNPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGS WTGDSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELL QLTPVEVESNVSRGAPEGIETFKKESEGGGGSGGGGSMERAIQGNDTREQAKRER WDGGSGGITSPFKLPDESPSWTEWRLYNDETNSNQDNPLGFKESWGFGKVVFKR YLRYDRTEASLHRVLGSWTGDSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGS RTLQHLSEMAIRSKQELLQLTPVEVESNVSRGAPEGIETFKKESEAttorney Docket No: JBI6815WOPCT1 SEQ ID NO: 10 Tmab-p196 [WT LC] LC: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 11 Tmab-p197 HC: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGGGGGSGGGGSGGGGSMERAIQGNDTREQAKRERWDGGSGGITSPF KLPDESPSWTEWRLYNDETNSNQDNPLGFKESWGFGKVVFKRYLRYDRTEASLH RVLGSWTGDSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGSRTLQHLSEMAIRS KQELLQLTPVEVESNVSRGAPEGIETFKKESEGGGGSGGGGSMERAIQGNDTREQ AKRERWDGGSGGITSPFKLPDESPSWTEWRLYNDETNSNQDNPLGFKESWGFGK VVFKRYLRYDRTEASLHRVLGSWTGDSVNYAASRFLGANQVGCTYSIRFRGVSV TISGGSRTLQHLSEMAIRSKQELLQLTPVEVESNVSRGAPEGIETFKKESE SEQ ID NO: 12 Tmab-p198 HC: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYAttorney Docket No: JBI6815WOPCT1 TQKSLSLSPGAPAPAPAPAPMERAIQGNDTREQAKRERWDGGSGGITSPFKLPDES PSWTEWRLYNDETNSNQDNPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGS WTGDSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELL QLTPVEVESNVSRGAPEGIETFKKESEGGGGSGGGGSMERAIQGNDTREQAKRER WDGGSGGITSPFKLPDESPSWTEWRLYNDETNSNQDNPLGFKESWGFGKVVFKR YLRYDRTEASLHRVLGSWTGDSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGS RTLQHLSEMAIRSKQELLQLTPVEVESNVSRGAPEGIETFKKESE SEQ ID NO: 13 Tmab-p199 LC: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGG SGGGGSQGNDTREQAKRERWDGGSGGITSPFKLPDESPSWTEWRLYNDETNSNQ DNPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGSWTGDSVNYAASRFLGANQ VGCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELLQLTPVEVESNVSRGAPEGI ETFKKESEGGGGSGGGGSMERAIQGNDTREQAKRERWDGGSGGITSPFKLPDESP SWTEWRLYNDETNSNQDNPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGSW TGDSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELLQ LTPVEVESNVSRGAPEGIETFKKESE SEQ ID NO: 14 Tmab-p1910 HC: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGGGGGSGGGGSGGGGSQGNDTREQAKRERWDGGSGGITSPFKLPDE SPSWTEWRLYNDETNSNQDNPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGS WTGDSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELL QLTPVEVESNVSRGAPEGIETFKKESEGGGGSGGGGSMERAIQGNDTREQAKRER WDGGSGGITSPFKLPDESPSWTEWRLYNDETNSNQDNPLGFKESWGFGKVVFKR YLRYDRTEASLHRVLGSWTGDSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGS RTLQHLSEMAIRSKQELLQLTPVEVESNVSRGAPEGIETFKKESEAttorney Docket No: JBI6815WOPCT1 SEQ ID NO: 15 mut p19 MERAIQGNDTREQAKRERWDGGSGGITSPFKLPDESPSWTEWRLYNDETNSNQD NPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGSWTGDSVNYAASRFLGANQV GCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELLQLTPVEVESNVSRGAPEGIET FKKESE SEQ ID NO: 16 2xp19-LPETGG-His6: MERAIQGNDTREQAKRERWDGGSGGITSPFKLPDESPSWTEWRLYNDETNSNQD NPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGSWTGDSVNYAASRFLGANQV GCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELLQLTPVEVESNVSRGAPEGIET FKKESEGGGGSGGGGSMERAIQGNDTREQAKRERWDGGSGGITSPFKLPDESPSW TEWRLYNDETNSNQDNPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGSWTG DSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELLQLT PVEVESNVSRGAPEGIETFKKESEGSGSGSLPETGGHHHHHH SEQ ID NO: 17 BBBB175-p19 D1-5 HC EVQLVQSGAE VKKPGESLKI SCKGSGYSFT SYWIGWVRQM PGKGLEWMGI IDPSDSYTRY SPSFQGQVTI SADKSISTAYLQWSSLKASD TAMYYCARMY KGRGHLFDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSQGNDTREQA KRERWDGGSGGITSPFKLPDESPSWTEWRLYNDETNSNQDNPLGFKESWGFGKV VFKRYLRYDRTEASLHRVLGSWTGDSVNYAASRFLGANQVGCTYSIRFRGVSVTI SGGSRTLQHLSEMAIRSKQELLQLTPVEVESNVSRGAPEGIETFKKESEGGGGSGG GGSMERAIQGNDTREQAKRERWDGGSGGITSPFKLPDESPSWTEWRLYNDETNSN QDNPLGFKESWGFGKVVFKRYLRYDRTEASLHRVLGSWTGDSVNYAASRFLGAN QVGCTYSIRFRGVSVTISGGSRTLQHLSEMAIRSKQELLQLTPVEVESNVSRGAPEG IETFKKESE SEQ ID NO: 18 BBBB175-p19 D1-5 LCAttorney Docket No: JBI6815WOPCT1 EIVLTQSPAT LSLSPGERAT LSCRASQSVS KALAWYQQKP GQAPRLLIYA ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ YYRAPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC SEQ ID NO: 19 GGGGSGGGGSGGGGS SEQ ID NO: 20 GGGGSGGGGS SEQ ID NO: 21 APAPAPAPAP SEQ ID NO: 22 CTNNb1 siRNA_1 sense u•a•cuguUgfGAUugauucga•a•a SEQ ID NO: 23 CTNNb1 siRNA_1 antisense VPu•U•ucgAaUCaaucCaAcagua•g•c SEQ ID NO: 24 CTNNb1 siRNA_2OMe sense u•a•cuguuggauugauucga•a•a SEQ ID NO: 25 CTNNb1 siRNA_2OMe antisense VPu•u•ucgaaucaauccaacagua•g•c SEQ ID NO: 26 CTNNb1 siRNA_2F sense U•A•CUGUUGGAUUGAUUCGA•A•A SEQ ID NO: 27 CTNNb1 siRNA_2F antisense VPu•U•UCGAAUCAAUCCAACAGUA•G•C SEQ ID NO: 28 CTNNb1 siRNA_2OMe / 2F sense u•a•CuGuUgGaUuGaUuCgA•a•A SEQ ID NO: 29 CTNNb1 siRNA_2OMe / 2F antisenseAttorney Docket No: JBI6815WOPCT1 VPu•U•uCgAaUcAaUcCaAcAgUa•G•c SEQ ID NO: 30 CTNNb1 siRNA_2 sense c•u•GuuGGAuuGAuucGAA•A SEQ ID NO: 31 CTNNb1 siRNA_2 antisense VPu•U•uCgAaUcAaUcCaAcAg•u•u SEQ ID NO: 32 CTNNb1 siRNA_1d sense u•a•cugudTgdGdAdTugauucga•a•a SEQ ID NO: 33 CTNNb1 siRNA_1d antisense VPu•dT•ucgdAadTdCaaucdCadAcagua•g•c SEQ ID NO: 34 CTNNb1 siRNA_3 sense (invAb) •c•uguUgGAUugauucga•a•a SEQ ID NO: 35 CTNNb1 siRNA_3 antisense VPu•U•ucgAaUCaaucCaAcag•u•u SEQ ID NO: 36 CTNNb1 siRNA_4 sense (invAb) •c•uguugGAUugauucgaa•a• (invAb) SEQ ID NO: 37 CTNNb1 siRNA_4 antisense VPu•U•ucgAauCaaucCaAcag•u•a SEQ ID NO: 38 CTNNb1 siRNA_5 sense c•u•GuugGAUugAuucgA•a•a SEQ ID NO: 39 CTNNb1 siRNA_5 antisense VPu•U•ucgAaucaaucCaAcag•u•a SEQ ID NO: 40 CTNNb1 siRNA_1a sense u•a•cuguUgfGAUugauucga•a•aAttorney Docket No: JBI6815WOPCT1 SEQ ID NO: 41 CTNNb1 siRNA_1a antisense u•U•ucgAaUCaaucCaAcagua•g•c SEQ ID NO: 42 CTNNb1 siRNA_1b sense u•a•cuguUgfGAUugauucga•a•a SEQ ID NO: 43 CTNNb1 siRNA_1b antisense Pu•U•ucgAaUCaaucCaAcagua•g•c

Claims

Attorney Docket No: JBI6815WOPCT1 CLAIMS We claim:

1. A method of selecting an oligonucleotide for use in an oligonucleotide conjugate, the method comprising: a. linking one or more p19 polypeptides to a targeting ligand to form a targeted p19 polypeptide; and b. contacting an oligonucleotide to the targeted p19 polypeptide to form a targeted-p19-oligonucleotide complex.

2. The method of claim 1, wherein the oligonucleotide comprises a length of about 16 to about 25 nucleotides.

3. The method of claim 1 or 2, wherein the oligonucleotide is an siRNA, an miRNA, or an antisense RNA.

4. The method of any one of claims 1 to 3, wherein the oligonucleotide is contacted to the targeted p19 polypeptide in a ratio of about 10:1 to about 2:

5.

5. The method of any one of claims 1 to 4, wherein the oligonucleotide is contacted to the targeted-p19 polypeptide in a ratio of about 2:

1.

6. The method of any one of claims 1 to 5, wherein a cell expressing a target of the oligonucleotide is contacted to the targeted-p19-oligonucleotide polypeptide.

7. The method of any of claims 1 to 6, when the target of the targeting ligand comprises a surface ligand.

8. The method of any one of claims 1 to 7, wherein the method produces a targeted- p19-oligonucleotide polypeptide at a concentration of about 0.01 nM to about 100 nM.

9. The method of any one of claims 1 to 8, wherein the cell is contacted with the targeted-p19-siRNA polypeptide for about 24 to about 96 hours.

10. The method of any one of claims 6 to 9, wherein a level of the target of the oligonucleotide is measured.

11. The method of any one of claims 6 to 10, wherein the level of the target of interest is measured by qRT-PCR.Attorney Docket No: JBI6815WOPCT1 12. The method of any one of claims 6 to 11, wherein the level of the target of interest in the cell contacted with the targeted-p19-oligonucleotide fusion polypeptide is compared with a control, and optionally wherein the control expresses the target of interest or is treated with an inoperable targeted-p19- oligonucleotide, and optionally wherein a modulation in the level of the target of interest in the cell contacted with the targeted-p19- oligonucleotide fusion polypeptide as compared to the control indicates that the oligonucleotide is capable of modulating the target of interest when fused to the targeting ligand.

13. The method of claim 12, wherein an optimal oligonucleotide is selected based on the modulation of the level of the target of interest in the cell contacted with the targeted- p19-oliognucleotide fusion polypeptide.

14. The method of any one of claim 1 to 13, wherein the targeting ligand is selected from the group consisting of an antibody, an antibody fragment, a miniprotein, or a peptide.

15. The method of any one of claims 1 to 14, wherein the p19 polypeptide is covalently linked to a heavy chain or a light chain of the antibody.

16. The method of any one of claims 1 to 15, wherein the p19 polypeptide is covalently linked to the heavy chain of the antibody.

17. The method of any one of claims 1 to 16, wherein the p19 polypeptide and the antibody are linked by a click chemistry method or a conjugation method.

18. The method of any one of claims 1 to 17, wherein the p19 polypeptide and the antibody are linked by a linker sequence.

19. The method of claim 18, wherein the linker sequence comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:

21.

20. The method of claim 18 or 19, wherein the linker sequence comprises the amino acid sequence of SEQ ID NO:

21.

21. The method of any one of claims 1 to 20, wherein the p19 polypeptide is a mutant p19 polypeptide.

22. The method of claim 21, wherein the mutant p19 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 15 or SEQ ID NO: 16.Attorney Docket No: JBI6815WOPCT1 23. The method of claims 21 or 22, wherein the mutant p19 polypeptide comprises the amino acid sequence of SEQ ID NO:

16.

24. The method of claim 21, wherein the p19 polypeptide comprises a Δ1-5, a N15K, a G16R, a C134S, and / or a C160A mutation.

25. The method of any one of claims 1 to 24, wherein the oligonucleotide is a therapeutic oligonucleotide.

26. The method of any one of claims 1 to 25, wherein the targeting ligand binds to HER2 or TfR.

27. An p19 polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 15 or SEQ ID NO 16.

28. The p19 polypeptide of claim 27, comprising the amino acid sequence of SEQ ID NO: 16, SEQ ID NO:18, or SEQ ID NO:

19.

29. A polynucleotide encoding any one of the p19 polypeptides of claim 27.

30. A vector comprising the isolated polynucleotide of claim 29.

31. A host cell comprising the vector of claim 30.

32. A kit for selecting an siRNA for use in an siRNA targeting ligand drug conjugate, the kit comprising one or more of the p19 polypeptides of claim 27 and instructions for use.

33. The p19 polypeptide of claim 27, further comprising an antibody, wherein the antibody is linked to the p19 polypeptide.

34. The p19 polypeptide of claim 33, further comprising an oligonucleotide wherein the oligonucleotide is complexed with the p19 polypeptide.

35. The p19 polypeptide of claim 27, further comprising an oligonucleotide, wherein the oligonucleotide is complexed with the p19 polypeptide.

Images