Anti-transferrin receptor antibodies and uses thereof

Abstract

SOLUTION: Disclosed herein, in certain embodiments, are anti-transferrin receptor antibodies, anti-transferrin receptor antibody conjugates, and pharmaceutical compositions which comprise the anti-transferrin receptor antibodies or conjugates. In some embodiments, also disclosed herein are methods of delivering a payload utilizing an anti-transferrin receptor antibody described herein, and methods of treatment with use of an anti-transferrin receptor antibody described herein.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 784,181, filed December 21, 2018, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] The present invention is in the field of pharmaceutical formulations, and in particular relates to antibodies. The present invention provides anti-transferrin receptor antibodies and methods for preparing and using anti-transferrin receptor antibodies.

[0003] In addition to their existing use in diagnostic methods, antibodies have been shown to be useful as therapeutic agents. For example, immunotherapy, or the use of antibodies for therapeutic purposes, has been utilized in recent years to treat cancer and other disorders. The transferrin receptor is one of the most widely used target receptors in developing targeted cancer diagnostic or therapeutic agents. This type II transmembrane glycoprotein is responsible for intracellular iron transport and is found at low levels on the surface of many normal cell types. There is a need for the development of improved anti-transferrin receptor antibodies for pharmaceutical use. Summary of the Invention

[0004] In certain embodiments, disclosed herein are anti-transferrin receptor antibodies, anti-transferrin receptor antibody conjugates, and pharmaceutical compositions comprising the anti-transferrin receptor antibodies or conjugates. Also disclosed herein, in some embodiments, are methods of delivering a payload utilizing the anti-transferrin receptor antibodies described herein, and methods of treatment using the anti-transferrin receptor antibodies described herein.

[0005] In certain embodiments, disclosed herein are anti-transferrin receptor antibodies comprising a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence of EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO:3. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:2, and an HCDR3 sequence comprising SEQ ID NO:3. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:4, and an HCDR3 sequence comprising SEQ ID NO:3. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:5, and an HCDR3 sequence comprising SEQ ID NO:3. In some embodiments, the VL region comprises the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence AX4TNLAX5, and the LCDR3 sequence QHFWGTPLTX6, where X3 is selected from N or S, X4 is selected from A or G, X5 is selected from D or E, and X6 is present or absent and, if present, is F. In some embodiments, the VL region comprises the LCDR1 sequence comprising SEQ ID NO:6, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, where X5 is selected from D or E and X6 is present or absent and, if present, is F. In some embodiments, the VL region comprises the LCDR1 sequence comprising SEQ ID NO:6, the LCDR2 sequence comprising SEQ ID NO:7, and the LCDR3 sequence comprising SEQ ID NO:8. In some embodiments, the VL region comprises the LCDR1 sequence comprising SEQ ID NO:6, the LCDR2 sequence comprising SEQ ID NO:9, and the LCDR3 sequence comprising SEQ ID NO:10. In some embodiments, the VL region comprises an LCDR1 sequence comprising SEQ ID NO:11, an LCDR2 sequence comprising SEQ ID NO:12, and an LCDR3 sequence comprising SEQ ID NO:10.In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 2, and an HCDR3 sequence comprising SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6, an LCDR2 sequence comprising SEQ ID NO: 7, and an LCDR3 sequence comprising SEQ ID NO: 8. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 4, and an HCDR3 sequence comprising SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6, an LCDR2 sequence comprising SEQ ID NO: 7, and an LCDR3 sequence comprising SEQ ID NO: 8. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 5, and an HCDR3 sequence comprising SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6, an LCDR2 sequence comprising SEQ ID NO: 9, and an LCDR3 sequence comprising SEQ ID NO: 10. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:4, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:11, an LCDR2 sequence comprising SEQ ID NO:12, and an LCDR3 sequence comprising SEQ ID NO:10. In some embodiments, the VH region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs:13-16. In some embodiments, the VL region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs:18-21. In some embodiments, the anti-transferrin receptor antibody comprises a humanized antibody or binding fragment thereof, or a chimeric antibody or binding fragment thereof. In some embodiments, the anti-transferrin receptor antibody comprises a multispecific antibody or binding fragment thereof. In some embodiments, the anti-transferrin receptor antibody comprises a bispecific antibody or binding fragment thereof.In some embodiments, the anti-transferrin receptor antibody comprises an IgG-scFv, a nanobody, a BiTE, a diabody, a DART, a TandAb, a scdiabody, a scdiabody-CH3, a triplebody, a miniantibody, a minibody, a TriBiminibody, a scFv-CH3 KIH, a Fab-scFv-Fc KIH, a Fab-scFv, a scFv-CH-CL-scFv, a F(ab')2, a F(ab')2-scFv2.scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scdiabody-Fc, a diabody-Fc, a tandem scFv-Fc, or an intrabody. In some embodiments, the anti-transferrin receptor antibody comprises an IgG1 framework. In some embodiments, the anti-transferrin receptor antibody comprises an IgG2 framework. In some embodiments, the IgG2 framework is an IgG2b framework. In some embodiments, the anti-transferrin receptor antibody comprises an IgG4 framework. In some embodiments, the anti-transferrin receptor antibody further comprises at least one mutation in the Fc region. In some embodiments, the at least one mutation modulates effector function. In some embodiments, the at least one mutation weakens or eliminates Fc-γ receptor binding. In some embodiments, the at least one mutation is at residue position D265, N297, K322, L328, or P329, where the residue positions are relative to IgG1. In some embodiments, the Fc region comprises two or more, three or more, or four or more mutations. In some embodiments, the Fc region comprises mutations at L233 and L234, where the residues correspond to positions 233 and 234 of SEQ ID NO: 23. In some embodiments, the Fc region comprises mutations at D265 and N297. In some embodiments, the anti-transferrin receptor antibody comprises a heavy chain (HC) sequence selected from SEQ ID NOs: 23-46 and a light chain (LC) sequence selected from SEQ ID NOs: 47-50. In some embodiments, the anti-transferrin receptor antibody specifically binds to the human transferrin receptor (TfR).

[0006] Disclosed herein, in certain embodiments, is an anti-transferrin receptor antibody conjugate comprising an anti-transferrin receptor antibody described herein and a payload. In some embodiments, the payload comprises a small molecule, peptide, protein, or polynucleic acid molecule. In some embodiments, the payload comprises a polynucleic acid molecule. In some embodiments, the polynucleic acid molecule comprises a short interfering nucleic acid (siNA), a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a PMO, or an mRNA. In some embodiments, the payload comprises a dsRNA. In some embodiments, the payload comprises an antisense oligonucleotide (ASO). In some embodiments, the payload comprises a small molecule, a peptide, or a protein. In some embodiments, the payload comprises a microtubule-disrupting agent, a DNA-modifying agent, or an Akt inhibitor. In some embodiments, the payload comprises an auristatin or a derivative thereof, a dolastatin or a derivative or analog thereof, a maytansinoid, or a pyrrolobenzodiazepine or a derivative thereof. In some embodiments, the auristatin or derivative thereof is monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF). In some embodiments, the maytansinoid is DM1 or DM4. In some embodiments, the pyrrolobenzodiazepine is a pyrrolobenzodiazepine dimer. In some embodiments, the payload comprises an immunomodulator or immunomodulatory drug. In some embodiments, the immunomodulatory drug comprises a cytokine. In some embodiments, the payload comprises a protein or peptide toxin or a fragment thereof. In some embodiments, the payload is conjugated to the anti-transferrin receptor antibody via a linker. In some embodiments, the anti-transferrin receptor antibody is further conjugated to two or more payloads. In some embodiments, the ratio of payload to anti-transferrin receptor antibody is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, or 12:1.In some embodiments, the anti-transferrin receptor antibody conjugate is A-(X. 1 -B) n (Formula (I)), wherein A comprises an anti-transferrin antibody, B comprises a payload, and X 1 wherein n is a single bond or a linker, and n is an average value selected from 1-12. In some embodiments, the payload is a polynucleic acid molecule. In some embodiments, the polynucleic acid molecule comprises a passenger strand and a guide strand. In some embodiments, the guide strand comprises at least one modified internucleotide linkage, at least one inverted abasic moiety, at least one 5'-vinylphosphonate modified unnatural nucleotide, or a combination thereof. In some embodiments, the at least one 5'-vinylphosphonate modified unnatural nucleotide is located about 1, 2, 3, 4, or 5 bases away from the 5' end of the guide strand. In some embodiments, the polynucleic acid molecule further comprises a modification of the sugar moiety at the 2' position. In some embodiments, the modification at the 2'-position is selected from 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2-O-NMA) modified nucleotides. In some embodiments, the passenger strand comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorodiamidate morpholino oligomer-modified non-natural nucleotides. In some embodiments, the passenger strand is shorter in length than the guide strand, thereby generating a 5' overhang, a 3' overhang, a blunt end at one end, or a combination thereof. In some embodiments, the passenger strand is equal in length to the guide strand, thereby generating a blunt end at each end of the polynucleic acid molecule. In some embodiments, the passenger strand is AX1 In some embodiments, AX 1 is conjugated to the 5' end of the passenger strand. 1 is conjugated to the 3' end of the passenger strand. In some embodiments, the anti-transferrin receptor antibody conjugate comprises AX 1 -(BX 2 -C) n (Formula (II)), wherein A comprises an anti-transferrin receptor antibody, B comprises a polynucleic acid molecule, C comprises a polymer, and X 1 is a single bond or a first linker, and X 2 In some embodiments, C is a polyethylene glycol. In some embodiments, the polynucleic acid molecule comprises a passenger strand and a guide strand. In some embodiments, the passenger strand is AX 1 and X 2 In some embodiments, AX is conjugated to -C. 1 is conjugated to the 5' end of the passenger strand, and X 2 -C is conjugated to the 3' end of the passenger strand. 2 -C is conjugated to the 5' end of the passenger strand, and AX 1 is conjugated to the 3' end of the passenger strand. 1 and X 2 are each independently a non-polymeric linker. In some embodiments, the anti-transferrin receptor antibody conjugate further comprises D. In some embodiments, D is an endosomolytic moiety.

[0007] In certain embodiments, disclosed herein are nucleic acid polymers that encode the anti-transferrin receptor antibodies described herein.

[0008] In certain embodiments, disclosed herein are vectors comprising a nucleic acid polymer encoding an anti-transferrin receptor antibody described herein.

[0009] In certain embodiments, pharmaceutical compositions are disclosed herein, the pharmaceutical compositions comprising an anti-transferrin receptor antibody described herein or an anti-transferrin receptor antibody conjugate described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

[0010] In certain embodiments, disclosed herein are methods for delivering a payload to a targeted site of interest in a subject, the methods comprising administering to the subject an anti-transferrin receptor antibody conjugate described herein or a pharmaceutical composition described herein to deliver the payload to the targeted site of interest. In some embodiments, the targeted site of interest is a cell containing an overexpressed causative protein. In some embodiments, the targeted site of interest is a tumor site. In some embodiments, the targeted site of interest is a site in the brain.

[0011] In certain embodiments, disclosed herein are methods of treating cancer in a subject, the methods comprising administering to the subject an anti-transferrin receptor antibody conjugate described herein or a pharmaceutical composition described herein to treat the cancer in the subject. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is bladder cancer, lung cancer, brain cancer, melanoma, breast cancer, non-Hodgkin's lymphoma, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, esophageal cancer, prostate cancer, kidney cancer, skin cancer, leukemia, thyroid cancer, liver cancer, or uterine cancer. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is relapsed or refractory cancer.

[0012] In certain embodiments, disclosed herein are methods for treating muscle atrophy or myotonic dystrophy in a subject, the methods comprising administering to the subject an anti-transferrin receptor antibody conjugate described herein or a pharmaceutical composition described herein, wherein the polynucleic acid molecule hybridizes to a target sequence of an atrogene, and wherein the polynucleic acid molecule mediates RNA interference against the atrogene, thereby treating the muscle atrophy in the subject. In some embodiments, the muscle atrophy is diabetes-related muscle atrophy or cancer cachexia-related muscle atrophy. In some embodiments, the muscle atrophy is associated with insulin deficiency, chronic renal failure, congestive heart failure, chronic respiratory disease, chronic infection, fasting, denervation, sarcopenia, or myotonic dystrophy type 1 (DM1). In some embodiments, the subject's reticulocyte levels are not reduced after administration of the anti-transferrin receptor antibody. In some embodiments, administration of the anti-transferrin receptor antibody conjugate downregulates the level of SSB siRNA or SSB mRNA in a subject. In some embodiments, the downregulation of SSB siRNA or SSB mRNA is in muscle. In some embodiments, the muscle is skeletal muscle. In some embodiments, the muscle is cardiac muscle.

[0013] In some embodiments, the myotonic dystrophy is DM1. In some embodiments, the atrogene comprises an upregulated gene in the IGF1-Akt-FoxO pathway, the glucocorticoid-GR pathway, the PGC1α-FoxO pathway, the TNFα-NFκB pathway, or the myostatin-ActRIIb-Smad2 / 3 pathway. In some embodiments, the atrogene encodes an E3 ligase. In some embodiments, the atrogene encodes a forkhead box transcription factor. In some embodiments, the atrogene comprises the atrogin-1 gene (FBXO32), the MuRF1 gene (TRIM63), FOXO1, FOXO3, or MSTN. In some embodiments, the atrogene comprises DMPK. In some embodiments, the subject is human.

[0014] In certain embodiments, disclosed herein are methods for treating muscular dystrophy in a subject, the methods comprising administering to the subject an anti-transferrin receptor antibody conjugate described herein or a pharmaceutical composition described herein, thereby treating the muscular dystrophy in the subject. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy. In some embodiments, the subject is a human.

[0015] Disclosed herein, in certain embodiments, is a kit comprising an anti-transferrin receptor antibody described herein, an anti-transferrin receptor antibody conjugate described herein, a nucleic acid polymer described herein, a vector described herein, or a pharmaceutical composition described herein. [Brief explanation of the drawings]

[0016] Various aspects of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the disclosure are utilized, and the accompanying drawings. The patent application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1] 1 illustrates the structure of an exemplary SSB passenger strand. [Figure 2] The structure of an exemplary blunt-ended duplex with 19 complementary bases and one 3' dinucleotide overhang is illustrated. The purified single strand was duplexed to obtain a double-stranded siRNA. [Figure 3A]1 illustrates the in vitro binding of TfR1.IgG2 mAb and TfR1.IgG2 mAb-SSB to recombinant human TfR1. [Figure 3B] 1 illustrates the in vitro binding of TfR1.IgG2 mAb and TfR1.IgG2 mAb-SSB to recombinant cynomolgus monkey TfR1. [Figure 4A] 1 illustrates SSB mRNA levels upon siRNA delivery in He192.1.7 cells treated with hTfR1.IgG2 mAb SSB or hTfR1.IgG2 mAb MSTN (negative control) conjugates. [Figure 4B] 1 illustrates SSB mRNA levels upon siRNA delivery in immortalized human skeletal muscle cells treated with hTfR1.IgG2 mAb SSB or hTfR1.IgG2 mAb MSTN (negative control) conjugates. [Figure 5A] 1 illustrates the levels of SSB mRNA and SSB siRNA in the gastrocnemius muscle of cynomolgus monkeys after administration of hTfR1.IgG2 mAb-SSB conjugate at 30 and 60 mg / kg (n=3). [Figure 5B] 1 illustrates the levels of SSB mRNA and SSB siRNA in the quadriceps muscle of cynomolgus monkeys after administration of hTfR1.IgG2 mAb-SSB conjugate at 30 and 60 mg / kg (n=3). [Figure 6] 1 illustrates the relative reticulocyte levels in cynomolgus monkeys before and after administration of hTfR1.IgG2 mAb-SSB conjugate at 30 and 60 mg / kg. [Figure 7] 1 illustrates the binding constants of exemplary anti-TfR antibodies to cynomolgus monkey CD71. [Figure 8] 1 illustrates the binding constants of exemplary anti-TfR antibodies to human CD71. [Figure 9A] 1 illustrates the binding of exemplary anti-TfR antibodies to TfR in a competitive setting. [Figure 9B] Figure 9A shows the binding constants of the tested anti-TfR antibodies. [Figure 10A]1 shows that binding of exemplary anti-TfR antibodies to TfR is maintained. [Figure 10B] Figure 10A shows the binding constants of the tested anti-TfR antibodies. [Figure 11] 1 shows the ADCC activity of exemplary anti-TfR antibodies. [Figure 12] 1 shows that anti-TfR antibodies do not bind to TfR2. [Figure 13A] % SSB mRNA knockdown in HEL92 cells is shown. [Figure 13B] Figure 13A shows the EC50 of the tested anti-TfR antibodies. [Figure 14] 1 illustrates the ADCC activity of exemplary anti-TfR antibodies. [Figure 15] 1 illustrates the CDC activity of exemplary anti-TfR antibodies. [Figure 16] 1 shows uptake of TfR1.mAb conjugates in primary human skeletal muscle cells (myotubes). [Figure 17] 1 shows SSB mRNA levels in primary human skeletal muscle cells treated with SSB or Scramble siRNA conjugates of TfR1.hIgG2 mAb or TfR1.hIgG1 mAb variants. [Figure 18] Absolute reticulocyte levels in cynomolgus monkeys before and after administration of TfR1-targeted AOC (single dose on day 1) are shown. [Figure 19] Shown are SSB mRNA levels in muscle of cynomolgus monkeys 21 days after a single administration of TfR1 mAb SSB conjugate (n=3). [Figure 20A] Shown are SSB siRNA levels in tissues of cynomolgus monkeys 21 days after a single 6 mg / kg administration of hIgG1 TfR-Val2ii-SSB conjugate (n=2). [Figure 20B] Shown are SSB mRNA levels in tissues of cynomolgus monkeys 21 days after a single 6 mg / kg administration of hIgG1 TfR-Val2ii-SSB conjugate (n=2). DETAILED DESCRIPTION OF THE INVENTION

[0017] Transferrin receptors (TfRs) comprise a family of membrane glycoproteins and are encoded by the gene TFRC. TfRs are involved in iron metabolism by interacting with the iron-transferrin complex, facilitating iron uptake into cells. There are two subtypes of TfR: transferrin receptor 1 (TfR1 or CD71) and transferrin receptor 2 (TfR2). TfR1 is ubiquitously expressed on various cell types, whereas TfR2 is specifically expressed in hepatocytes.

[0018] In some cases, abnormal expression of TfR1 is prominent in various cancers. Indeed, one study showed that TfR1 expression levels are elevated in breast cancer cells (Pizzamiglio, et al. “Expression of iron-related proteins differentiates non-cancerous and cancerous breast tumors,” Int J Mol Sci. 2017;18). Another study showed that TFR1 is overexpressed in brain cancer (Rosager, et al., “Transferrin receptor-1 and ferritin heavy and light chains in astrocytic brain tumors: Expression and prognostic value,” PLoS One 12:e0182954 (2017)). Another study showed that iron uptake is elevated in tumor-initiating cells (Rychtarcikova, et al., “Tumorinitiating cells of breast and prostate origin show the alterations in the expression of genes related to iron metabolism,” Oncotarget. 8:6376-6398 (2017)).

[0019] In some embodiments, disclosed herein are anti-transferrin receptor antibodies, anti-transferrin receptor antibody conjugates, and pharmaceutical compositions comprising same. In further embodiments, disclosed herein are methods utilizing anti-transferrin receptor antibodies for the delivery of a payload, and methods for treating a disease or disorder by utilizing the presence of the transferrin receptor for targeted delivery.

[0020] Anti-transferrin receptor antibody In certain embodiments, anti-transferrin receptor antibodies are disclosed herein. In some examples, the anti-transferrin receptor antibody specifically binds to the transferrin receptor (TfR). In some examples, the anti-transferrin receptor antibody specifically binds to human transferrin receptor (TfR). In some cases, the anti-transferrin receptor antibody specifically binds to transferrin receptor 1 (TfR1) (or CD71). In some cases, the anti-transferrin receptor antibody specifically binds to human transferrin receptor 1 (TfR1) (or human CD71).

[0021] In some examples, the anti-transferrin receptor antibody comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO:3.

[0022] In some embodiments, the VH region of the anti-transferrin antibody comprises an HCDR1, HCDR2, and HCDR3 sequence selected from Table 1.

[0023] [Table 1]

[0024] In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:2, 4, or 5, and an HCDR3 sequence comprising SEQ ID NO:3. In some examples, the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:2, and an HCDR3 sequence comprising SEQ ID NO:3. In some examples, the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:4, and an HCDR3 sequence comprising SEQ ID NO:3. In some examples, the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:5, and an HCDR3 sequence comprising SEQ ID NO:3.

[0025] In some embodiments, the VL region of the anti-transferrin receptor antibody comprises the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence AX4TNLAX5, and the LCDR3 sequence QHFWGTPLTX6, where X3 is selected from N or S, X4 is selected from A or G, X5 is selected from D or E, and X6 is present or absent, or, if present, is F.

[0026] In some embodiments, the VL region of the anti-transferrin receptor antibody comprises an LCDR1, LCDR2, and LCDR3 sequence selected from Table 2.

[0027] [Table 2]

[0028] In some examples, the VL region comprises an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence comprising SEQ ID NO:7, 9, or 12, and an LCDR3 sequence comprising SEQ ID NO:8 or 10, wherein X3 is selected from N or S.

[0029] In some examples, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6 or 11, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO: 8 or 10, wherein X4 is selected from A or G and X5 is selected from D or E.

[0030] In some examples, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6 or 11, an LCDR2 sequence comprising SEQ ID NO: 7, 9, or 12, and an LCDR3 sequence QHFWGTPLTX6, where X6 is present or absent and, if present, is F.

[0031] In some examples, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6, an LCDR2 sequence AATNLAX5, and an LCDR3 sequence QHFWGTPLTX6, wherein X5 is selected from D or E, and X6 is present or absent, and if present, is F.

[0032] In some examples, the VL region comprises an LCDR1 sequence comprising SEQ ID NO:6, an LCDR2 sequence comprising SEQ ID NO:7, and an LCDR3 sequence comprising SEQ ID NO:8.

[0033] In some examples, the VL region comprises an LCDR1 sequence comprising SEQ ID NO:6, an LCDR2 sequence comprising SEQ ID NO:9, and an LCDR3 sequence comprising SEQ ID NO:10.

[0034] In some examples, the VL region comprises an LCDR1 sequence comprising SEQ ID NO:11, an LCDR2 sequence comprising SEQ ID NO:12, and an LCDR3 sequence comprising SEQ ID NO:10.

[0035] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence of EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO: 3; and the VL region comprises the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence AX4TNLAX5, and the LCDR3 sequence QHFWGTPLTX6, where X3 is selected from N or S, X4 is selected from A or G, X5 is selected from D or E, and X6 is present or absent, or if present, is F.

[0036] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence of EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO: 3; and the VL region comprises the LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence comprising SEQ ID NO: 7, 9, or 12, and an LCDR3 sequence comprising SEQ ID NO: 8 or 10, where X3 is selected from N or S.

[0037] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence of EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO: 3; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6 or 11, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO: 8 or 10, where X4 is selected from A or G and X5 is selected from D or E.

[0038] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence of EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO: 3; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6 or 11, an LCDR2 sequence comprising SEQ ID NO: 7, 9, or 12, and the LCDR3 sequence QHFWGTPLTX6, where X6 is present or absent, and, if present, is F.

[0039] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence of EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO: 3; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6, an LCDR2 sequence AATNLAX5, and an LCDR3 sequence QHFWGTPLTX6, where X5 is selected from D or E, and X6 is present or absent, or if present, is F.

[0040] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence of EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO: 3; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6, an LCDR2 sequence comprising SEQ ID NO: 7, and an LCDR3 sequence comprising SEQ ID NO: 8.

[0041] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence of EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO: 3; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6, an LCDR2 sequence comprising SEQ ID NO: 9, and an LCDR3 sequence comprising SEQ ID NO: 10.

[0042] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence of EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO: 3; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 11, an LCDR2 sequence comprising SEQ ID NO: 12, and an LCDR3 sequence comprising SEQ ID NO: 10.

[0043] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:2, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises the LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence comprising SEQ ID NO:7, 9, or 12, and an LCDR3 sequence comprising SEQ ID NO:8 or 10, wherein X3 is selected from N or S.

[0044] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:2, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:6 or 11, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO:8 or 10, wherein X4 is selected from A or G and X5 is selected from D or E.

[0045] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:2, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:6 or 11, an LCDR2 sequence comprising SEQ ID NO:7, 9, or 12, and the LCDR3 sequence QHFWGTPLTX6, wherein X6 is present or absent and, if present, is F.

[0046] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:2, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:6, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, wherein X5 is selected from D or E, and X6 is present or absent, and if present is F.

[0047] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:2, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:6, an LCDR2 sequence comprising SEQ ID NO:7, and an LCDR3 sequence comprising SEQ ID NO:8.

[0048] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:2, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:6, an LCDR2 sequence comprising SEQ ID NO:9, and an LCDR3 sequence comprising SEQ ID NO:10.

[0049] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:2, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:11, an LCDR2 sequence comprising SEQ ID NO:12, and an LCDR3 sequence comprising SEQ ID NO:10.

[0050] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:4, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises the LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence comprising SEQ ID NO:7, 9, or 12, and an LCDR3 sequence comprising SEQ ID NO:8 or 10, wherein X3 is selected from N or S.

[0051] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 4, and an HCDR3 sequence comprising SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6 or 11, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO: 8 or 10, wherein X4 is selected from A or G and X5 is selected from D or E.

[0052] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:4, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:6 or 11, an LCDR2 sequence comprising SEQ ID NO:7, 9, or 12, and the LCDR3 sequence QHFWGTPLTX6, wherein X6 is present or absent, and, if present, is F.

[0053] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:4, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:6, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, wherein X5 is selected from D or E, and X6 is present or absent, and if present is F.

[0054] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:4, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:6, an LCDR2 sequence comprising SEQ ID NO:7, and an LCDR3 sequence comprising SEQ ID NO:8.

[0055] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:4, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:6, an LCDR2 sequence comprising SEQ ID NO:9, and an LCDR3 sequence comprising SEQ ID NO:10.

[0056] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:4, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:11, an LCDR2 sequence comprising SEQ ID NO:12, and an LCDR3 sequence comprising SEQ ID NO:10.

[0057] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:5, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises the LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence comprising SEQ ID NO:7, 9, or 12, and an LCDR3 sequence comprising SEQ ID NO:8 or 10, wherein X3 is selected from N or S.

[0058] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 5, and an HCDR3 sequence comprising SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6 or 11, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO: 8 or 10, wherein X4 is selected from A or G and X5 is selected from D or E.

[0059] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 5, and an HCDR3 sequence comprising SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6 or 11, an LCDR2 sequence comprising SEQ ID NO: 7, 9, or 12, and the LCDR3 sequence QHFWGTPLTX6, wherein X6 is present or absent, and, if present, is F.

[0060] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:5, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:6, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, wherein X5 is selected from D or E, and X6 is present or absent, and if present is F.

[0061] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:5, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:6, an LCDR2 sequence comprising SEQ ID NO:7, and an LCDR3 sequence comprising SEQ ID NO:8.

[0062] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:5, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:6, an LCDR2 sequence comprising SEQ ID NO:9, and an LCDR3 sequence comprising SEQ ID NO:10.

[0063] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1, an HCDR2 sequence comprising SEQ ID NO:5, and an HCDR3 sequence comprising SEQ ID NO:3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO:11, an LCDR2 sequence comprising SEQ ID NO:12, and an LCDR3 sequence comprising SEQ ID NO:10.

[0064] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the sequence of the VH region comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 13-16, and the sequence of the VL region comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 18-21.

[0065] In some embodiments, the VH region comprises a sequence selected from SEQ ID NOs: 13-16 (Table 3), and the VL region comprises a sequence selected from SEQ ID NOs: 18-21 (Table 4). The highlighted regions in Tables 3 and 4 represent the sequences of the respective CDR1, CDR2, or CDR3.

[0066] [Table 3]

[0067] [Table 4]

[0068] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region as exemplified in Table 5.

[0069] [Table 5]

[0070] In some embodiments, the anti-transferrin receptor antibody is a full-length antibody. In other embodiments, the anti-transferrin receptor antibody is a binding fragment thereof. Optionally, the anti-transferrin receptor antibody is a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a multispecific antibody or binding fragment thereof, or a bispecific antibody or binding fragment thereof. Optionally, the anti-transferrin receptor antibody is a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or binding fragment thereof, or a chemically modified derivative thereof.

[0071] In some embodiments, the anti-transferrin receptor antibody is a multispecific antibody. In some cases, the multispecific antibody comprises two or more target binding sites, where each of the two or more target binding sites specifically binds to an antigen, and the two or more antigens are different. In some cases, the multispecific antibody comprises target binding sites that specifically bind to three or more different antigens, four or more different antigens, or five or more different antigens.

[0072] In some embodiments, the anti-transferrin receptor antibody is a bispecific antibody. In some cases, the bispecific antibody or binding fragment is a bispecific antibody, such as Knobs-into-Holes (KiH), Asymmetric Re-engineering Technology-immunoglobulin (ART-Ig), Triomab quadroma, Bispecific Monoclonal Antibody (BiMAb, BsmAb, BsAb, bsMab, BS-Mab, or Bi-MAb), FcΔAdp, XmAb, Azymetric, Bispecific Binding by Antibody Based on T Cell Receptor (BEAT), Bispecific T Cell Engager (BiTE), Biclonics, Fab-scFv-Fc, Two-in-one / Dual Action These include Fab (DAF), FinomAb, scFv-Fc-(Fab) fusion, Dock-aNd-Lock (DNL), Adaptir (formerly SCORPION), tandem diabodies (TandAb), Dual-affinity-ReTargeting (DART), and nanobodies.

[0073] In some instances, a bispecific antibody is a triabody or a bispecific miniantibody. In some instances, a bispecific antibody is a triabody. In some instances, a triabody is a full-length monoclonal antibody that contains binding sites for two different antigens.

[0074] In some cases, the bispecific antibody is a bispecific miniantibody. In some examples, the bispecific miniantibody comprises a bivalent Fab2, F(ab)'3 fragment, bis-scFv (scFv)2, diabody, minibody, triabody, tetrabody, or bispecific T cell engager (BiTE). In some embodiments, the bispecific T cell engager is a fusion protein comprising two single-chain variable fragments (scFvs), where the two scFvs target epitopes of two different antigens.

[0075] In some examples, the anti-transferrin receptor antibody is a trispecific antibody. In some examples, the trispecific antibody comprises a F(ab)' fragment or a triabody. In some embodiments, the anti-transferrin receptor antibody is a trispecific antibody as described in Dimas, et al., "Development of a trispecific antibody designed to simultaneously and efficiently target three different antigens on tumor cells," Mol. Pharmaceutics, 12(9):3490-3501 (2015).

[0076] In some examples, the anti-transferrin receptor antibody comprises the antibody format illustrated in Figure 2 of Brinkmann and Kontermann, "The making of bispecific antibodies," MABS 9(2): 182-212 (2017).

[0077] In some embodiments, the anti-transferrin receptor antibodies described herein comprise an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some examples, the anti-transferrin receptor antibody comprises an IgG framework (e.g., IgG1, IgG2, IgG3, or IgG4). In some cases, the anti-transferrin receptor antibody comprises an IgG1 framework. In some cases, the anti-transferrin receptor antibody comprises an IgG2 (e.g., IgG2a or IgG2b) framework. In some cases, the anti-transferrin receptor antibody comprises an IgG2a framework. In some cases, the anti-transferrin receptor antibody comprises an IgG2b framework. In some cases, the anti-transferrin receptor antibody comprises an IgG3 framework. In some cases, the anti-transferrin receptor antibody comprises an IgG4 framework.

[0078] In some cases, the anti-transferrin receptor antibody comprises one or more mutations in a framework region, e.g., in the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some examples, the one or more mutations stabilize the antibody and / or increase half-life. In some examples, the one or more mutations modulate Fc receptor interactions, reducing or eliminating Fc effector function, such as FcyR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In further examples, the one or more mutations modify glycosylation.

[0079] In some embodiments, one or more mutations are located in the Fc region. In some examples, the Fc region includes mutations at residue positions L234, L235, or a combination thereof. In some examples, the mutations include L234 and L235. In some examples, the mutations include L234A and L235A. In some cases, the residue positions are relative to IgG1.

[0080] In some examples, the Fc region comprises mutations at residue positions L234, L235, D265, N297, K322, L328, or P329, or a combination thereof. In some examples, the mutations comprise L234 and L235 in combination with mutations at residue positions K322, L328, or P329. In some cases, the Fc region comprises mutations at L234, L235, and K322. In some cases, the Fc region comprises mutations at L234, L235, and L328. In some cases, the Fc region comprises mutations at L234, L235, and P329. In some cases, the Fc region comprises mutations at D265 and N297. In some cases, the residue positions are relative to IgG1.

[0081] In some examples, the Fc region comprises L234A, L235A, D265A, N297G, K322G, L328R, or P329G, or a combination thereof. In some examples, the Fc region comprises L234A and L235A in combination with K322G, L328R, or P329G. In some cases, the Fc region comprises L234A, L235A, and K322G. In some cases, the Fc region comprises L234A, L235A, and L328R. In some cases, the Fc region comprises L234A, L235A, and P329G. In some cases, the Fc region comprises D265A and N297G. In some cases, the residue positions are relative to IgG1.

[0082] In some examples, the Fc region comprises mutations at residue positions L235, L236, D265, N297, K322, L328, or P329, or a combination of mutations. In some examples, the Fc region comprises mutations at L235 and L236. In some examples, the Fc region comprises mutations at L235 and L236 in combination with mutations at residue positions K322, L328, or P329. In some cases, the Fc region comprises mutations at L235, L236, and K322. In some cases, the Fc region comprises mutations at L235, L236, and L328. In some cases, the Fc region comprises mutations at L235, L236, and P329. In some cases, the Fc region comprises mutations at D265 and N297. In some cases, the residue positions are relative to IgG2b.

[0083] In some embodiments, the Fc region comprises L235A, L236A, D265A, N297G, K322G, L328R, or P329G, or a combination thereof. In some examples, the Fc region comprises L235A and L236A. In some examples, the Fc region comprises L235A and L236A in combination with K322G, L328R, or P329G. In some cases, the Fc region comprises L235A, L236A, and K322G. In some cases, the Fc region comprises L235A, L236A, and L328R. In some cases, the Fc region comprises L235A, L236A, and P329G. In some cases, the Fc region comprises D265A and N297G. In some cases, the residue positions are relative to IgG2b.

[0084] In some embodiments, the Fc region comprises mutations at residue positions L233, L234, D264, N296, K321, L327, or P328, where the residues correspond to positions 233, 234, 264, 296, 321, 327, and 328 of SEQ ID NO: 23. In some examples, the Fc region comprises mutations at L233 and L234. In some examples, the Fc region comprises mutations at L233 and L234 in combination with mutations at residue positions K321, L327, or P328. In some instances, the Fc region comprises mutations at L233, L234, and K321. In some instances, the Fc region comprises mutations at L233, L234, and L327. In some instances, the Fc region comprises mutations at L233, L234, and K321. In some instances, the Fc region comprises mutations at L233, L234, and P328. In some instances, the Fc region comprises mutations at D264 and N296. In some instances, equivalent positions to residues L233, L234, D264, N296, K321, L327, or P328 in the framework of IgG1, IgG2, IgG3, or IgG4 are contemplated. In some instances, mutations to residues corresponding to residues L233, L234, D264, N296, K321, L327, or P328 of SEQ ID NO: 23 in the framework of IgG1, IgG2, or IgG4 are also contemplated.

[0085] In some embodiments, the Fc region comprises L233A, L234A, D264A, N296G, K321G, L327R, or P328G, where the residues correspond to positions 233, 234, 264, 296, 321, 327, and 328 of SEQ ID NO:23. In some examples, the Fc region comprises L233A and L234A. In some examples, the Fc region comprises L233A and L234A in combination with K321G, L327R, or P328G. In some instances, the Fc region comprises L233A, L234A, and K321G. In some instances, the Fc region comprises L233A, L234A, and L327R. In some instances, the Fc region comprises L233A, L234A, and K321G. In some instances, the Fc region comprises L233A, L234A, and P328G. In some examples, the Fc region comprises D264A and N296G.

[0086] In some embodiments, the human IgG constant region is selected from the group consisting of, for example, Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al., 2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS, 103(11): 4005-4010, Shields et al., 2001 JBC, 276(9): 6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: 152-164; Alegre et al, 1992 J Immunol, 148: 3461-3468; Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1): 1-11, are used to modify antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0087] In some embodiments, the anti-transferrin receptor antibodies described herein are full-length antibodies comprising a heavy chain (HC) and a light chain (LC). In some cases, the heavy chain (HC) comprises a sequence selected from Table 6. In some cases, the light chain (LC) comprises a sequence selected from Table 7. The highlighted regions indicate the respective CDRs.

[0088] [Table 6-1]

[0089] [Table 6-2]

[0090] [Table 6-3]

[0091] [Table 6-4]

[0092] [Table 6-5]

[0093] [Table 7]

[0094] In some embodiments, the anti-transferrin receptor antibodies described herein have an improved serum half-life compared to a reference anti-transferrin receptor antibody. In some examples, the improved serum half-life is at least 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or more, longer than the reference anti-transferrin receptor antibody.

[0095] Production of antibodies or binding fragments thereof In some embodiments, the polypeptides (e.g., antibodies and binding fragments thereof) described herein are produced using any method known in the art to aid in the synthesis of polypeptides (e.g., antibodies), inter alia, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.

[0096] In some examples, antibodies or binding fragments thereof are recombinantly expressed, and nucleic acids encoding the antibodies or binding fragments thereof are assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves synthesis of overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligation of the oligonucleotides, and subsequent amplification of the ligated oligonucleotides by PCR.

[0097] Alternatively, nucleic acid molecules encoding antibodies are optionally produced from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell that expresses immunoglobulins) by PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence.

[0098] In some instances, the antibody or binding fragment thereof is optionally produced by immunizing an animal such as a rabbit to produce polyclonal antibodies, or more preferably, by producing monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497), or by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Alternatively, clones encoding at least the Fab portion of the antibody are optionally obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) or antibody libraries (see Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937) for clones of FAb fragments that bind to a specific antigen.

[0099] In some embodiments, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) are used by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region, e.g., a humanized antibody.

[0100] In some embodiments, techniques described for the production of single-chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted to produce single-chain antibodies. Single-chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single-chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

[0101] In some embodiments, an expression vector containing an antibody nucleotide sequence or the antibody nucleotide sequence is introduced into host cells by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In certain embodiments, antibody expression is regulated by a constitutive, inducible, or tissue-specific promoter.

[0102] In some embodiments, various host-expression vector systems are utilized to express the antibodies or binding fragments thereof described herein. Such host-expression systems represent not only vehicles for producing and subsequently purifying the antibody coding sequence, but also cells that are transfected with the appropriate nucleotide coding sequences or that, when transfected, express the antibody or binding fragment in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody or binding fragment thereof coding sequence; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the antibody or binding fragment thereof coding sequence; insect cell systems (e.g., baculovirus) infected with recombinant viral expression vectors containing the antibody or binding fragment thereof coding sequence; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody or binding fragment thereof coding sequence; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter).

[0103] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some instances, cell lines that stably express antibodies are optionally engineered. Rather than using expression vectors containing viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, cells are engineered to grow in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker on the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to form foci that can be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express antibodies or their binding fragments.

[0104] In some examples, a number of selection systems are used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes utilized in tk cells, hgprt cells, or aprt cells, respectively. Similarly, antimetabolite resistance has been used as a selection criterion for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); and neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932, and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215), and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods generally known in the art of recombinant DNA technology that can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

[0105] In some instances, antibody expression levels are increased by vector amplification (for a review, see Bebbington and Hentschel, *The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning*, Vol. 3 (Academic Press, New York, 1987)). If the marker in the antibody expression vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Because the amplified region is related to the antibody nucleotide sequence, antibody production will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0106] In some examples, any method known in the art for purification or analysis of antibodies or antibody conjugates is used, for example, by chromatography (e.g., ion exchange, affinity, especially affinity to specific antigens followed by Protein A, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for purification of proteins. Exemplary chromatographic methods include, but are not limited to, strong anion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and fast protein liquid chromatography.

[0107] Anti-transferrin receptor antibody conjugate In some embodiments, the anti-transferrin receptor antibody is further conjugated to a payload. In some examples, the payload comprises a small molecule. In other examples, the payload comprises a protein or peptide. In a further example, the payload comprises a polynucleic acid molecule.

[0108] In some examples, the ratio of payload to anti-transferrin receptor antibody (drug-to-antibody ratio or DAR ratio) is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, or 16:1.

[0109] In some cases, the anti-transferrin receptor antibody conjugate comprises:

[0110] [ka] Including, During the ceremony, A comprises an anti-transferrin receptor antibody, B contains the payload, X 1 consists of a single bond or a linker, and n is the average value selected from 1-12.

[0111] In some examples, the DAR ratio of B to A (anti-transferrin receptor antibody) is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some cases, the DAR ratio of B to A is about 1. In some cases, the DAR ratio of B to A is about 2. In some cases, the DAR ratio of B to A is about 3. In some cases, the DAR ratio of B to A is about 4. In some cases, the DAR ratio of B to A is about 6. In some cases, the DAR ratio of B to A is about 8. In some cases, the DAR ratio of B to A is about 10. In some cases, the DAR ratio of B to A is about 12. In some cases, the DAR ratio of B to A is about 16.

[0112] In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 1. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 2. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 3. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 4. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 5. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 6. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 7. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 8. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 9. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 10. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 11. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 12. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 13. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 14. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 15. In some examples, the DAR ratio of polynucleic acid molecule (B) to anti-transferrin receptor antibody A is about 16.

[0113] In some embodiments, B comprises a small molecule, peptide, or protein.

[0114] In some embodiments, B comprises a polynucleic acid molecule. Optionally, the polynucleic acid molecule comprises a passenger strand and a guide strand. Optionally, the passenger strand is AX 1 In some cases, AX 1 is conjugated to the 5' end of the passenger strand. 1 is conjugated to the 3' end of the passenger strand.

[0115] In some cases, the anti-transferrin receptor antibody conjugate comprises:

[0116] [ka] Including, During the ceremony, A comprises an anti-transferrin receptor antibody, B contains the payload, C is a polymer; X 1 is a single bond or a first linker, X 2 consists of a single bond or a second linker, and n is the average value selected from 1-12.

[0117] In some cases, C is polyethylene glycol.

[0118] In some cases, B is a polynucleic acid molecule. In some cases, the polynucleic acid molecule comprises a passenger strand and a guide strand. In some cases, the passenger strand is AX 1 and X 2 In some embodiments, AX is conjugated to -C. 1 is conjugated to the 5' end of the passenger strand, and X 2 -C is conjugated to the 3' end of the passenger strand. 2 -C is conjugated to the 5' end of the passenger strand, and AX 1 is conjugated to the 3' end of the passenger strand.

[0119] In some cases, X 1 and X 2 are each independently a non-polymeric linker.

[0120] In some cases, the compound of formula (II)AX 1 -(BX 2 -C) n The conjugate of further comprises D, an endosomolytic moiety.

[0121] Conjugation Chemistry In some embodiments, B is conjugated to A by a chemical ligation process. In some examples, B is conjugated to A by native ligation. In some examples, conjugation can be performed using methods such as those described by Dawson, et al. "Synthesis of proteins by native chemical ligation," Science 1994, 266, 776-779; Dawson, et al. "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J. Am. Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology," Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu, et al. "Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol," Angew. Chem. Int. Ed. 2006, 45, 4116-4125. In some examples, the conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, the polynucleic acid molecule is site-specifically or non-specifically conjugated to the binding moiety via native ligation chemistry.

[0122] In some examples, B is conjugated to A in a site-directed manner using "traceless" coupling technology (PhiloChem). In some examples, the "traceless" coupling technology utilizes an N-terminal 1,2-aminothiol group of the linking moiety to conjugate with a polynucleic acid molecule containing an aldehyde group. (See Casi et al., "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery," JACS 134(13): 5887-5892 (2012)).

[0123] In some examples, B is conjugated to A via a site-directed method utilizing an unnatural amino acid introduced into the linking moiety. In some examples, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some examples, the keto group of pAcPhe is selectively linked to an alkoxy-amine derived conjugate moiety to form an oxime bond. (See Axup et al., "Synthesis of site-specific antibody-drug conjugates using unnatural amino acids," PNAS 109(40): 16101-16106 (2012)).

[0124] In some examples, B is conjugated to A by a site-directed method that utilizes an enzyme-catalyzed process. In some examples, the site-directed method utilizes SMARTag™ technology (Redwood). In some examples, SMARTag™ technology involves the generation of a formylglycine (FGly) residue from cysteine by formylglycine generating enzyme (FGE) via an oxidation process in the presence of an aldehyde tag, and the subsequent conjugation of FGly to an alkylhydrazine-functionalized polynucleic acid molecule via hydrazino-Pictet-Spengler (HIPS) ligation. (See Wu et al., “Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,” PNAS 106(9): 3000-3005 (2009); Agarwal, et al., “A Pictet-Spengler ligation for protein chemical modification,” PNAS 110(1):46-51 (2013))

[0125] In some examples, the enzyme-catalyzed process includes microbial transglutaminase (mTG). In some cases, B is conjugated to A using a microbial transglutaminase-catalyzed process. In some examples, mTG catalyzes the formation of a covalent bond between the amide side chain of a glutamine in the recognition sequence and a primary amine of a functionalized polynucleic acid molecule. In some examples, mTG is produced by Streptomyces mobaraensis. (See Strop et al., “Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” Chemistry and Biology 20(2) 161-167 (2013)).

[0126] In some examples, B is conjugated to A by the methods described in PCT International Publication No. WO2014 / 140317, which utilize a sequence-specific transpeptidase.

[0127] In some examples, B is conjugated to A by methods such as those described in U.S. Patent Application Publication Nos. 2015 / 0105539 and 2015 / 0105540.

[0128] payload Polynucleic acid molecule In some embodiments, the payload is a polynucleic acid molecule. In some instances, the polynucleic acid molecule is involved in gene therapy, such as RNA interference (RNAi) or gene silencing (e.g., antisense oligonucleotide) therapy. In some instances, the polynucleic acid molecule regulates the splicing of mRNA, thereby regulating the subsequent production of the encoded protein.

[0129] In some embodiments, the polynucleic acid molecule comprises a short interfering nucleic acid (siNA), a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a microRNA (miRNA), or a short hairpin RNA (shRNA).

[0130] In other embodiments, the polynucleic acid molecule comprises an antisense oligonucleotide.

[0131] In some embodiments, the polynucleic acid molecule comprises a PMO.

[0132] In a further embodiment, the polynucleic acid molecule comprises mRNA.

[0133] In some examples, the polynucleic acid molecule hybridizes to the target sequence of an atrophy-associated gene (also called an atrophy gene). An atrophy-associated gene is a gene that is up- or down-regulated in atrophying muscles. In some examples, up-regulated atrogenes include genes encoding proteins involved in ubiquitin ligases, forkhead box transcription factors, growth factors, deubiquitinases, or glucocorticoid-induced atrophy. In some examples, the polynucleic acid molecule described herein hybridizes to the target sequence of a ubiquitin ligase (e.g., E3 ubiquitin ligase or mitochondrial ubiquitin ligase). In some examples, the polynucleic acid molecule described herein hybridizes to the target sequence of a forkhead box transcription factor. In some examples, the polynucleic acid molecule described herein hybridizes to the target sequence of a growth factor. In some examples, the polynucleic acid molecule described herein hybridizes to the target sequence of a deubiquitinase.

[0134] In some embodiments, the polynucleic acid molecules described herein hybridize to the target sequence of FBXO32, TRIM63, TRAF6, FBXO30, FBXO40, NEDD4, TRIM32, MUL1, STUB1, FOXO1, FOXO3, MSTN, USP14, USP19, DDIT4, CTSL2, TGIF, MYOG, HDAC2, HDAC3, MT1L, MT1B, or DMPK. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of FBXO32, TRIM63, FOXO1, FOXO3, or MSTN. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of FBXO32. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of TRIM63. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of TRAF6. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence for FBXO30. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence for FBXO40. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence for NEDD4. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence for TRIM32. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence for MUL1. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence for STUB1. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence for FOXO1. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence for FOXO3. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence for MSTN. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence for USP14. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence for USP19. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence for DDIT4.In some cases, the polynucleic acid molecules described herein hybridize to a target sequence of CTSL2. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence of TGIF. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence of MYOG. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence of HDAC2. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence of HDAC3. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence of MT1L. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence of MT1B. In some cases, the polynucleic acid molecules described herein hybridize to a target sequence of DMPK.

[0135] In some examples, the polynucleic acid molecule hybridizes to the target region of an incorrectly spliced mRNA that results in a disease or disorder, such as, but not limited to, a neuromuscular disease, a genetic disease, cancer, a genetic disease, or a cardiovascular disease. In some cases, the neuromuscular disease or disorder is Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy.

[0136] In some examples, the polynucleic acid molecule targets an exon that is mutated in the DMD gene that causes Duchenne muscular dystrophy. Exemplary exons mutated in the DMD gene causing Duchenne muscular dystrophy include, but are not limited to, exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78.

[0137] In some examples, the polynucleic acid molecule hybridizes to a target region of an oncogene. Exemplary oncogenes include, but are not limited to, Abl, AKT-2, ALK, AML1 (or RUNX1), AR, AXL, BCL-2, 3, 6, BRAF, c-MYC, EGFR, ErbB-2 (Her2, Neu), Fms, FOS, GLI1, HPRT1, IL-3, INTS2, JUN, KIT, KS3, K-sam, LBC (AKAP13), LCK, LMO1, and LMO2. , LYL1, MAS1, MDM2, MET, MLL (KMT2A), MOS, MYB, MYH11 / CBFB, NOTCH1 (TAN1), NTRK1 (TRK), OST (SLC51B), PAX5, PIM1, PRAD-1, RAF, RAR / PML, HRAS, KRAS, NRAS, REL / NRG, RET, ROS, SKI, SRC, TIAM1, or TSC2. In some cases, the polynucleic acid molecule hybridizes to a target region of a KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or β-catenin-related gene.

[0138] In some embodiments, the polynucleic acid molecule comprises mRNA. In some cases, the mRNA encodes a cytotoxic protein or peptide. Exemplary cytotoxic proteins or peptides include alpha-pore-forming toxins (e.g., cytolysin A from Escherichia coli), beta-pore-forming toxins (e.g., alpha-hemolysin, PVL-Panton-Valentine leukocidin, aerolysin, Clostridium epsilon toxin, Clostridium perfringens enterotoxin), bicomponent toxins (e.g., Bacillus anthracis toxin, Clostridium botulinum C2 toxin, Clostridium spiroforme toxin, Clostridium perfringens iota toxin, Clostridium difficile toxin), and the like. Examples of such toxins include bacterial cytotoxins such as cytolethal toxins (A and B), prions, parasporins, cholesterol-dependent cytolysins (e.g., pneumolysin), pore-forming toxins (e.g., gramicidin A), cyanotoxins (e.g., microcystin, nodularin), hemotoxins, neurotoxins (e.g., botulinum neurotoxin), cytotoxins, cholera toxin, diphtheria toxin, Pseudomonas exotoxin A, tetanus toxin, or immunotoxins (idarubicin, ricin A, CRM9, pokeweed antiviral protein, DT).

[0139] In some instances, the mRNA encodes a peptide associated with the immune system, such as a cytotoxic peptide or a cytotoxic T cell or B cell epitope, to stimulate a specific immune response through presentation of the epitope to the MHC I complex, membrane attack complex proteins (MAC) of the complement system, perforin, granzymes, and granulysin.

[0140] In some cases, the mRNA encodes apoptosis-inducing proteins or peptides such as apoptotic protease activating factor-1 (Apaf-1), cytochrome-c, caspase initiator proteins (CASP2, CASP8, CASP9, CASP10), apoptosis-inducing factor (AIF), p53, p73, p63, Bcl-2, Bax, granzyme B, poly ADP-ribose polymerase (PARP), and P21-activated kinase 2 (PAK2).

[0141] In some embodiments, the polynucleic acid molecule is nucleic acid decoy.In some examples, nucleic acid decoy is the mimic of protein-binding nucleic acid, such as RNA-based protein-binding mimic.Exemplary nucleic acid decoy includes transactivation region (TAR) decoy and rev response element (RRE) decoy.

[0142] In some examples, the payload is an aptamer. Aptamers are small oligonucleotide or peptide molecules that bind to specific target molecules. Exemplary nucleic acid aptamers include DNA aptamers, RNA aptamers, or XNA aptamers, which are RNA and / or DNA aptamers that contain one or more non-natural nucleotides. Exemplary nucleic acid aptamers include ARC19499 (Archemix Corp.), REG1 (Regado Biosciences), and ARC1905 (Ophthotech).

[0143] In some embodiments, polynucleic acid molecules comprise natural, synthetic, or artificial nucleotide analogs or bases. In some cases, polynucleic acid molecules comprise a combination of DNA, RNA, and / or nucleotide analogs. In some instances, synthetic or artificial nucleotide analogs or bases comprise modifications in one or more of the ribose moiety, phosphate moiety, nucleoside moiety, or combinations thereof.

[0144] In some embodiments, the nucleotide analog or artificial nucleotide base comprises a nucleic acid having a modification at the 2' hydroxyl group of the ribose moiety. In some examples, the modification includes H, OR, R, halo, SH, SR, NH, NHR, NR, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogen, sulfur, thiol, thioether, thioester, amine (primary, secondary, or tertiary), amide, ether, ester, alcohol, and oxygen. In some examples, the alkyl moiety further comprises a modification. In some examples, the modification includes an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic (e.g., imidazole, hydrazino, or hydroxylamino) group, an isocyanate group, or a cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some examples, the alkyl moiety further comprises a heterosubstitution. In some examples, a carbon of the heterocyclic group is replaced by nitrogen, oxygen, or sulfur. In some examples, heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidino.

[0145] In some cases, the modification at the 2' hydroxyl group is a 2'-O-methyl modification or a 2'-O-methoxyethyl (2'-O-MOE) modification. In some cases, a 2'-O-methyl modification adds a methyl group to the 2' hydroxyl group of the ribose moiety, while a 2'O-methoxyethyl modification adds a methoxyethyl group to the 2' hydroxyl group of the ribose moiety. Exemplary chemical structures of a 2'-O-methyl modification of an adenosine molecule and a 2'O-methoxyethyl modification of a uridine are illustrated below.

[0146] [ka]

[0147] In some embodiments, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification, in which an extended amine group containing a propyl linker attaches the amine group to the 2' oxygen. In some instances, this modification neutralizes the overall negative charge from the phosphate of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar, thereby improving its cellular uptake properties due to its zwitterionic properties. An exemplary chemical structure of a 2'-O-aminopropyl nucleoside phosphoramidite is illustrated below.

[0148] [ka]

[0149] In some instances, the modification at the 2' hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA), in which the oxygen molecule attached at the 2' carbon is linked to the 4' carbon by a methylene group, thereby forming a 2'-C,4'-C-oxy-methylene linked bicyclic ribonucleotide monomer. An exemplary representation of the chemical structure of an LNA is illustrated below. The representation shown on the left highlights the chemical bond nature of the LNA monomer. The representation shown on the right shows the locked 3'-endo( 3 E) emphasizes structure.

[0150] [ka]

[0151] In some embodiments, the modification at the 2' hydroxyl group includes ethylene nucleic acids (ENAs), such as 2'-4'-ethylene bridged nucleic acids, which lock the sugar structure into a C3'-endo sugar puckering conformation. ENAs are part of the bridged nucleic acid class of modified nucleic acids, which also includes LNAs. Exemplary chemical structures of ENAs and bridged nucleic acids are illustrated below.

[0152] [ka]

[0153] In some embodiments, further modifications at the 2' hydroxyl group include 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0154] In some embodiments, the nucleotide analogs contain modified bases, such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine, and other nucleotides with modifications at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2 ... Deazanucleotides such as 6-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methoxyuridine, 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine, and 2-thiocytidine, dihydrouridine, pseudouridine, queosine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine Modified nucleotides include 5-hydroxyacetic acid, pyridin-4-one, pyridin-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracil and thymine, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. Modified nucleotides further include nucleotides with modifications to the sugar moiety, as well as nucleotides with non-ribosyl sugars or their analogs. For example, the sugar moiety may be, or be based on, mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles.The term nucleotide also includes what are known in the art as universal bases. By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0155] In some embodiments, the nucleotide analog further comprises morpholino, peptide nucleic acid (PNA), methyl phosphonate nucleotide, thiol phosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, 1',5'-anhydrohexitol nucleic acid (HNA), or a combination thereof. Morpholino or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structure mimics natural nucleic acid structures by deviating from the normal sugar and phosphate structures. In some instances, the five-membered ribose ring is replaced with a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some instances, ribose monomers are linked by phosphorodiamidate groups instead of phosphate groups. In some instances, backbone modifications remove all positive and negative charges, allowing the morpholino neutral molecules to cross cell membranes without the aid of cellular delivery agents, as used with charged oligonucleotides.

[0156] [ka]

[0157] In some embodiments, peptide nucleic acids (PNAs) contain no sugar rings or phosphate linkages, and the bases are linked and appropriately spaced by oligoglycine-like molecules, thereby eliminating backbone charge.

[0158] [ka]

[0159] In some embodiments, one or more modifications optionally occur at the internucleotide bond. In some examples, the modified internucleotide bond includes, but is not limited to, phosphorothioates, phosphorodithioates, methyl phosphonates, 5'-alkylene phosphonates, 5'-methyl phosphonates, 3'-alkylene phosphonates, boron trifluorides, 3'-5' or 2'-5' linked boranophosphates and selenophosphates, phosphotriesters, thionoalkylphosphotriesters, hydrogen phosphonate bonds, alkyl phosphonates, alkyl phosphonothioates, aryl phosphonothioates, phosphoroselenoates, phosphorodiselenoates, phosphinates, phosphoramidates, 3'-alkylphosphoramidates, phosphoropiperazides, and the like. Phosphorothioate antisense oligonucleotides include esters, phosphoroanilothioates, phosphoroanilidates, ketones, sulfones, sulfonamides, carbonates, carbamates, methylenehydrazoates, methylenedimethylhydrazoates, formacetals, thioformacetals, oximes, methyleneiminos, methylenemethyliminos, thioamidates, bonds with riboacetyl groups, aminoethylglycine, silyl, or siloxane bonds, alkyl or cycloalkyl bonds, such as saturated or unsaturated and / or substituted and / or heteroatom-containing 1-10 carbon atoms with or without heteroatoms, morpholino structures in which the base is directly or indirectly bound to the aza nitrogen of the backbone, amide, or polyamide bonds, and combinations thereof. Phosphorothioate antisense oligonucleotides (PS ASOs) are antisense oligonucleotides containing phosphorothioate bonds. Exemplary PS ASOs are illustrated below.

[0160] [ka]

[0161] In some examples, the modification is a methyl or thiol modification, such as a methyl phosphonate modification or a thiol phosphonate modification. Exemplary thiol phosphonate nucleotides (left) and methyl phosphonate nucleotides (right) are illustrated below.

[0162] [ka]

[0163] In some examples, modified nucleotides include, but are not limited to, 2'-fluoro N3-P5'-phosphoramidites, exemplified as follows:

[0164] [ka]

[0165] In some examples, modified nucleotides include, but are not limited to, hexitol nucleic acids (or 1',5'-anhydrohexitol nucleic acids (HNA)), exemplified as follows:

[0166] [ka]

[0167] In some embodiments, the nucleotide analog or artificial nucleotide base comprises a 5'-vinylphosphonate-modified nucleotide nucleic acid having a modification at the 5' hydroxyl group of the ribose moiety. In some embodiments, the 5'-vinylphosphonate-modified nucleotide is selected from the nucleotides provided below, wherein X is O or S, and B is a heterocyclic base moiety.

[0168] [ka]

[0169] In some embodiments, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification, in which an extended amine group containing a propyl linker attaches the amine group to the 2' oxygen. In some instances, this modification neutralizes the overall negative charge from the phosphate of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar, thereby improving its cellular uptake properties due to its zwitterionic properties.

[0170] In some instances, the 5'-vinylphosphonate is further modified with a locked or bridged ribose modification (e.g., locked nucleic acid or LNA), in which the oxygen molecule attached at the 2' carbon is linked to the 4' carbon by a methylene group, thereby forming a 2'-C,4'-C-oxy-methylene-linked bicyclic ribonucleotide monomer. An exemplary representation of the chemical structure of a 5'-vinylphosphonate-modified LNA is illustrated below, where X is O or S, B is a heterocyclic base moiety, and J is an internucleotide linking group that connects adjacent nucleotides of a polynucleotide.

[0171] [ka]

[0172] In some embodiments, further modifications at the 2' hydroxyl group include 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0173] In some embodiments, the nucleotide analogs include modified bases, such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine, and other nucleotides with modifications at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2 ... guanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methoxyuridine, deazanucleotides (such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, or 6-azocthymidine), 5-methyl-2-thiouridine, other thiobases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queusine, archaeosine, naphthyl and substituted naphthyl groups, any O-alkylated and N-alkylated purines and pyrimidines (e.g., N-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5'-hydroxyacetic acid, pyridin-4-one, or pyridin-2-one), phenyl and modified phenyl groups, such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. 5'-Vinylphosphonate-modified nucleotides further include those nucleotides modified on the sugar moiety, as well as 5'-vinylphosphonate-modified nucleotides having non-ribosyl sugars or analogs thereof.For example, the sugar moiety may optionally be or be based on mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles. The term nucleotide also includes what are known in the art as universal bases. Exemplary universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0174] In some embodiments, the 5'-vinylphosphonate-modified nucleotide analog further comprises a morpholino, peptide nucleic acid (PNA), methyl phosphonate nucleotide, thiol phosphonate nucleotide, 2'-fluoroN3-P5' phosphoramidite, or 1',5'-anhydrohexitol nucleic acid (HNA). Morpholino or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structure mimics that of natural nucleic acids but deviates from the normal sugar and phosphate structures. In some instances, the five-membered ribose ring is replaced with a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, ribose monomers are linked by a phosphorodiamidate group instead of a phosphate group. In some cases, backbone modification removes all positive and negative charges, allowing the neutral morpholino molecule to cross cell membranes without the aid of a cellular delivery agent, as is used with charged oligonucleotides. Non-limiting examples of 5'-vinylphosphonate modified morpholino oligonucleotides are illustrated below, where X is O or S, and B is a heterocyclic base moiety.

[0175] [ka]

[0176] In some embodiments, the 5'-vinylphosphonate-modified morpholino or PMO described above is a PMO containing a positive or cationic charge. In some examples, the PMO is PMOplus (Sarepta). PMOplus refers to a phosphorodiamidate morpholino oligomer containing any number of (1-piperazino)phosphinylideneoxy, (1-(4-(omega-guanidino-alkanoyl))-piperazino)phosphinylideneoxy bonds (such as those described in PCT International Publication No. WO2008 / 036127). In some examples, the PMO is a PMO described in U.S. Patent No. 7,943,762.

[0177] In some embodiments, the morpholino or PMO described above is PMO-X (Sarepta). In some cases, PMO-X refers to a phosphorodiamidate morpholino oligomer containing at least one bond or at least one of the disclosed terminal modifications, such as those described in PCT International Publication No. WO2011 / 150408 and U.S. Application Publication No. 2012 / 0065169.

[0178] In some embodiments, the morpholino or PMO described above is a PMO as described in Table 5 of U.S. Application Publication No. 2014 / 0296321.

[0179] An exemplary representation of the chemical structure of a 5'-vinylphosphonate modified nucleic acid is illustrated below, where X is O or S, B is a heterocyclic base moiety, and J is an internucleotide linkage.

[0180] [ka]

[0181] In some embodiments, peptide nucleic acids (PNAs) contain no sugar rings or phosphate linkages, and the bases are linked and appropriately spaced by oligoglycine-like molecules, thereby eliminating backbone charge.

[0182] [ka]

[0183] In some embodiments, one or more modifications of the 5'-vinylphosphonate modified oligonucleotide are optionally at the internucleotide bond. In some examples, the modified internucleotide bond may be, but is not limited to, phosphorothioate, phosphorodithioate, methylphosphonate, 5'-alkylenephosphonate, 5'-methylphosphonate, 3'-alkylenephosphonate, boron trifluoride, 3'-5' or 2'-5' linked boranophosphate and selenophosphate, phosphotriester, thionoalkylphosphotriester, hydrogen phosphonate bond, alkylphosphonate, alkylphosphonothioate, arylphosphonothioate, phosphoroselenoate, phosphorodiselenoate, phosphinate, phosphoramidate, 3'-alkylphosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate, phosphoropyridine, phospho ... These include perazidates, phosphoroanilothioates, phosphoroanilidates, ketones, sulfones, sulfonamides, carbonates, carbamates, methylenehydrazo, methylenedimethylhydrazo, formacetals, thioformacetals, oximes, methyleneimino, methylenemethylimino, thioamidates, bonds with riboacetyl groups, aminoethylglycines, silyl or siloxane bonds, alkyl or cycloalkyl bonds, e.g., saturated or unsaturated and / or substituted and / or containing heteroatoms, with or without heteroatoms, from 1 to 10 carbons, morpholino structures in which the base is bonded directly or indirectly to the aza nitrogen of the backbone, amides, or polyamides, and combinations thereof.

[0184] In some examples, the modification is a methyl or thiol modification, such as a methylphosphonate modification or a thiolphosphonate modification. Exemplary thiolphosphonate nucleotides (left), phosphorodithioates (center), and methylphosphonate nucleotides (right) are illustrated below.

[0185] [ka]

[0186] In some examples, 5'-vinylphosphonate modified nucleotides include, but are not limited to, phosphoramidites exemplified as follows:

[0187] [ka]

[0188] In some instances, the modified internucleotide linkage is a phosphorodiamidate linkage. Non-limiting examples of phosphorodiamidate linkages with morpholino systems are shown below.

[0189] [ka]

[0190] In some instances, the modified internucleotide linkage is a methylphosphonate linkage. Non-limiting examples of methylphosphonate linkages are shown below:

[0191] [ka]

[0192] In some instances, the modified internucleotide bond is an amide bond. Non-limiting examples of amide bonds are shown below:

[0193] [ka]

[0194] In some examples, 5'-vinylphosphonate modified nucleotides include, but are not limited to, the modified nucleic acids exemplified below.

[0195] In some embodiments, the one or more modifications include a modified phosphate backbone, where the modification produces a neutral or uncharged backbone. In some instances, the phosphate backbone is modified by alkylation, which produces an uncharged or neutral phosphate backbone. As used herein, alkylation includes methylation, ethylation, and propylation. In some instances, alkyl group, as used herein in the context of alkylation, refers to a linear or branched saturated hydrocarbon group containing 1 to 6 carbon atoms. In some instances, exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethylbutyl groups. In some instances, the modified phosphate is a phosphate group described in U.S. Pat. No. 9,481,905.

[0196] In some embodiments, the further modified phosphate backbone comprises methyl phosphonate, ethyl phosphonate, methylthiophosphonate, or methoxyphosphonate. In some embodiments, the modified phosphate is methyl phosphonate. In some embodiments, the modified phosphate is ethyl phosphonate. In some embodiments, the modified phosphate is methylthiophosphonate. In some embodiments, the modified phosphate is methoxyphosphonate.

[0197] In some embodiments, the one or more modifications further include modifications of the ribose moiety, the phosphate backbone, and the nucleoside, or modifications of the nucleotide analog at the 3' or 5' end. For example, the 3' end optionally includes a 3' cationic group, or by inverting the nucleoside at the 3' end with a 3'-3' bond. In another alternative, the 3' end is optionally conjugated with an aminoalkyl group, such as a 3'C5-aminoalkyl dT. In a further alternative, the 3' end is optionally conjugated with an abasic site, such as an apurinic or apyrimidinic acid site. In some examples, the 5' end is conjugated with an aminoalkyl group, such as a 5'-O-aminoalkyl substituent. In some cases, the 5' end is conjugated with an abasic site, such as an apurinic or apyrimidinic acid site.

[0198] In some embodiments, the polynucleic acid molecule comprises one or more of the artificial nucleotide analogs described herein. In some examples, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificial nucleotide analogs described herein. In some embodiments, the artificial nucleotide analog comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoro N3-P5'-phosphoramidite, or a combination thereof. In some embodiments, the polynucleic acid molecule is 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or , 2'-ON-methylacetamide (2'-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methyl phosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof. In some embodiments, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more artificial nucleotide analogs selected from 2'-O-methyl modified nucleotides.In some embodiments, a polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides. In some examples, a polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more thiolphosphonate nucleotides.

[0199] In some embodiments, a polynucleic acid molecule comprises a plurality of phosphorodiamidate morpholino oligomers or a plurality of peptide nucleic acid modified non-natural nucleotides, and optionally comprises at least one inverted abasic moiety. In some examples, the polynucleic acid molecule comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorodiamidate morpholino oligomer modified non-natural nucleotides. In some examples, the polynucleic acid molecule comprises 100% phosphorodiamidate morpholino oligomer modified non-natural nucleotides.

[0200] In some examples, the polynucleic acid molecule comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more peptide nucleic acid modified non-natural nucleotides. In some examples, the polynucleic acid molecule comprises 100% peptide nucleic acid modified non-natural nucleotides.

[0201] In some embodiments, the polynucleic acid molecule comprises one or more nucleotide analogs, each of which is a stereochemically isomeric form. In such instances, the polynucleic acid molecule is a chiral molecule. In some cases, the nucleotide analog comprises a backbone stereochemistry. In further cases, the nucleotide analog comprises a chiral analog as described in U.S. Patent No. 9,982,257, 9,695,211, or 9,605,019.

[0202] In some examples, the polynucleic acid molecule comprises at least one of about 5% to about 100% modifications, about 10% to about 100% modifications, about 20% to about 100% modifications, about 30% to about 100% modifications, about 40% to about 100% modifications, about 50% to about 100% modifications, about 60% to about 100% modifications, about 70% to about 100% modifications, about 80% to about 100% modifications, and about 90% to about 100% modifications.

[0203] In some cases, the polynucleic acid molecule comprises at least one of about 10% to about 90% modifications, about 20% to about 90% modifications, about 30% to about 90% modifications, about 40% to about 90% modifications, about 50% to about 90% modifications, about 60% to about 90% modifications, about 70% to about 90% modifications, and about 80% to about 100% modifications.

[0204] In some cases, the polynucleic acid molecule comprises at least one of about 10% to about 80% modifications, about 20% to about 80% modifications, about 30% to about 80% modifications, about 40% to about 80% modifications, about 50% to about 80% modifications, about 60% to about 80% modifications, and about 70% to about 80% modifications.

[0205] In some examples, the polynucleic acid molecule comprises at least one of about 10% to about 70% modifications, about 20% to about 70% modifications, about 30% to about 70% modifications, about 40% to about 70% modifications, about 50% to about 70% modifications, and about 60% to about 70% modifications.

[0206] In some examples, the polynucleic acid molecule comprises at least one of about 10% to about 60% modifications, about 20% to about 60% modifications, about 30% to about 60% modifications, about 40% to about 60% modifications, and about 50% to about 60% modifications.

[0207] In some cases, the polynucleic acid molecule comprises at least one of about 10% to about 50% modifications, about 20% to about 50% modifications, about 30% to about 50% modifications, and about 40% to about 50% modifications.

[0208] In some cases, the polynucleic acid molecule comprises at least one of about 10% to about 40% modifications, about 20% to about 40% modifications, and about 30% to about 40% modifications.

[0209] In some cases, the polynucleic acid molecule comprises at least one of about 10% to about 30% modifications and about 20% to about 30% modifications.

[0210] In some cases, the polynucleic acid molecule contains about 10% to about 20% modifications.

[0211] In some cases, the polynucleic acid molecule contains from about 15% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% modifications.

[0212] In further cases, the polynucleic acid molecule contains at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modifications.

[0213] In some embodiments, the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, or more modifications.

[0214] In some examples, the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, or more modified nucleotides.

[0215] In some examples, about 5 to about 100% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 5% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 10% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 15% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 20% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 25% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 30% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 35% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 40% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 45% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 50% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 55% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 60% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 65% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 70% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 75% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 80% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 85% of the polynucleic acid molecules comprise the artificial nucleotide analogs described herein.In some examples, about 90% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 95% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 96% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 97% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 98% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 99% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 100% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some embodiments, the artificial nucleotide analog comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoro N3-P5'-phosphoramidite, or a combination thereof.

[0216] In some embodiments, the polynucleic acid molecule comprises from about 1 to about 25 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 1 modification, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 2 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 3 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 4 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 5 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 6 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 7 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 8 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 9 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 10 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 11 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 12 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 13 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 14 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 15 modifications, wherein the modifications comprise an artificial nucleotide analog described herein.In some embodiments, the polynucleic acid molecule comprises about 16 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 17 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 18 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 19 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 20 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 21 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 22 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 23 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 24 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 25 modifications, wherein the modifications comprise artificial nucleotide analogs described herein.

[0217] In some embodiments, the polynucleic acid molecule is assembled from two separate polynucleotides, where one polynucleotide comprises the sense strand and the second polynucleotide comprises the antisense strand of the polynucleic acid molecule. In other embodiments, the sense strand is connected to the antisense strand by a linker molecule, which in some instances is a polynucleotide linker or a non-nucleotide linker.

[0218] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides in the sense strand comprise 2'-O-methylpyrimidine nucleotides and the purine nucleotides in the sense strand comprise 2'-deoxypurine nucleotides. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides present in the sense strand comprise 2'-deoxy-2'-fluoropyrimidine nucleotides and the purine nucleotides present in the sense strand comprise 2'-deoxypurine nucleotides.

[0219] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein pyrimidine nucleotides, when present in the antisense strand, are 2'-deoxy-2'-fluoro pyrimidine nucleotides, and purine nucleotides, when present in the antisense strand, are 2'-O-methyl purine nucleotides.

[0220] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein pyrimidine nucleotides, when present in the antisense strand, are 2'-deoxy-2'-fluoro pyrimidine nucleotides, and purine nucleotides, when present in the antisense strand, comprise 2'-deoxy-purine nucleotides.

[0221] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a terminal cap moiety at the 5'-end, the 3'-end, or both the 5' and 3'-ends of the sense strand, hi other embodiments, the terminal cap moiety is an inverted deoxy abasic moiety.

[0222] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises a phosphate backbone modification at the 3' end of the antisense strand. In some instances, the phosphate backbone modification is phosphorothioate. In some instances, the passenger strand comprises more phosphorothioate modifications than the guide strand. In other instances, the guide strand comprises more phosphorothioate modifications than the passenger strand. In further instances, the passenger strand comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate modifications. In further instances, the guide strand comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate modifications.

[0223] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises a glyceryl modification at the 3' end of the antisense strand.

[0224] In some embodiments, a polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand has one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally terminal capsids at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the sense strand. The antisense strand comprises about 1 to about 10 or more, particularly about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally an end-cap molecule at the 3'-end, 5'-end, or both the 3'- and 5'-ends of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, or with one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages, and / or with or without end cap molecules at the 3'-terminus, the 5'-terminus, or both the 3'- and 5'-terminus, present on the same or different strands.

[0225] In some embodiments, a polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand has about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the sense strand. and the antisense strand comprises from about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally terminal cap molecules at the 3'-end, 5'-end, or both the 3'- and 5'-ends of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strand are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus, present on the same or different strands.

[0226] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand contains one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally at the 3'-terminus, 5'-terminus, or 6'-terminus of the sense strand. and the antisense strand comprises from about 1 to about 10 or more, particularly about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally an end-cap molecule at the 3'-end, the 5'-end, or both the 3'- and 5'-end of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages, and / or terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus, present on the same or different strands.

[0227] In some embodiments, a polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand has from about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the sense strand. and the antisense strand comprises from about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally, end-cap molecules at the 3'-end, 5'-end, or both the 3'- and 5'-ends of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strand are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without about 1 to about 5, e.g., about 1, 2, 3, 4, 5, or more, phosphorothioate internucleotide linkages, and / or terminal cap molecules at the 3'-terminus, the 5'-terminus, or both the 3'- and 5'-terminus, present on the same or different strands.

[0228] In some embodiments, the polynucleic acid molecule is a double-stranded polynucleic acid molecule having one or more of the following properties: high hepatocyte stability, reduced overall charge, reduced hepatocyte uptake, or enhanced pharmacokinetics. In some embodiments, the double-stranded polynucleic acid molecule comprises a passenger strand (e.g., sense strand) and a guide strand (e.g., antisense strand) that contain multiple modifications.

[0229] In some embodiments, the double-stranded polynucleic acid molecule comprises a guide strand (e.g., antisense strand) having one or more of the modifications described above, and a passenger strand (e.g., sense strand) having a plurality of phosphorodiamidate morpholino oligomers or a plurality of peptide nucleic acid modified non-natural nucleotides.

[0230] In some embodiments, the polynucleic acid molecules described herein are chemically modified short interfering nucleic acid molecules having about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages in each strand of the polynucleic acid molecule.

[0231] In another embodiment, the polynucleic acid molecules described herein comprise 2'-5' internucleotide linkages. In some examples, the 2'-5' internucleotide linkages are at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus of one or both strands of the sequence. In further examples, the 2'-5' internucleotide linkages are present at various other locations within one or both strands of the sequence, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, including all internucleotide linkages of pyrimidine nucleotides in one or both strands of the polynucleic acid molecule, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, including all internucleotide linkages of purine nucleotides in one or both strands of the polynucleic acid molecule, comprise 2'-5' internucleotide linkages.

[0232] In some cases, the polynucleic acid molecule is a polynucleotide with a double, asymmetric double, hairpin, or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises the nucleotide sequence complementary to the nucleotide sequence of another target nucleic acid molecule or a part thereof, and the sense region has the nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof.In other cases, the polynucleic acid molecule is a circular single-stranded polynucleotide with a base comprising two or more loop structures and self-complementary sense and antisense regions, wherein the antisense region comprises the nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid molecule or a part thereof, and the sense region has the nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof, and the circular polynucleotide is processed in vivo or in vitro to generate an active polynucleic acid molecule that can mediate RNAi. In further cases, the polynucleic acid molecule further comprises a single-stranded polynucleotide having a nucleotide sequence complementary to that of a target nucleic acid molecule or a portion thereof (e.g., such a polynucleic acid molecule need not be present within the polynucleic acid molecule of a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), and the single-stranded polynucleotide further comprises a terminal phosphate group such as a 5'-phosphate (see, e.g., Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568) or a 5',3'-diphosphate.

[0233] In some embodiments, the polynucleic acid molecule is a single-stranded polynucleic acid molecule that mediates RNAi activity in a cell or a reconstituted in vitro system, wherein the polynucleic acid molecule comprises a single-stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the polynucleic acid are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides, or alternatively, wherein a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and any purine nucleotides present in the polynucleic acid are 2'-deoxy purine nucleotides. and a terminal cap modification is optionally present at the 3'-terminus, the 5'-terminus, or both the 3'- and 5'-terminus of the antisense sequence; the polynucleic acid molecule optionally further comprises about one to about four (e.g., about 1, 2, 3, or 4) terminal 2'-deoxyribonucleotides at the 3'-terminus of the polynucleic acid molecule, wherein the terminal nucleotides further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages; and the polynucleic acid molecule optionally further comprises a terminal phosphate group, such as a 5'-terminal phosphate group.

[0234] In some instances, an asymmetric duplex is a linear polynucleic acid molecule comprising an antisense region, a loop portion comprising nucleotides or non-nucleotides, and a sense region, where the sense region contains fewer nucleotides than the antisense region, sufficient to base-pair with the antisense region and form a looped duplex. For example, an asymmetric hairpin polynucleic acid molecule has a length sufficient to mediate RNAi in a cell or in vitro system and comprises an antisense region (e.g., about 19 to about 22 nucleotides) having a loop region comprising about 4 to about 8 nucleotides, and a sense region having about 3 to about 18 nucleotides complementary to the antisense region. In some instances, the asymmetric hairpin polynucleic acid molecule further comprises a chemically modified 5'-terminal phosphate group. In further instances, the loop portion of the asymmetric hairpin polynucleic acid molecule comprises nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

[0235] In some embodiments, an asymmetric duplex is a polynucleic acid molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region contains fewer nucleotides than the antisense region, but enough complementary nucleotides to base-pair with the antisense region to form a duplex. For example, an asymmetric duplex polynucleic acid molecule comprises an antisense region (e.g., about 19 to about 22 nucleotides) of sufficient length to mediate RNAi in a cell or in vitro system, and a sense region having about 3 to about 18 nucleotides complementary to the antisense region.

[0236] In some cases, one or more of the artificial nucleotide analogs described herein are resistant to nucleases, such as ribonucleases such as RNase H, deoxyribonucleases such as DNases, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases, compared to naturally occurring polynucleic acid molecules. In some examples, artificial nucleotide analogs, including 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof, are RNase inhibitors. Resistant to nucleases, such as ribonucleases such as RNase H, deoxyribonucleases such as DNase, or exonucleases, such as 5'-3' exonucleases and 3'-5' exonucleases. In some examples, 2'-O-methyl modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonucleases, or 3'-5' exonucleases resistant). In some examples, 2'O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonucleases, or 3'-5' exonucleases resistant). In some examples, 2'-O-aminopropyl modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonucleases, or 3'-5' exonucleases resistant). In some examples, the 2'-deoxy modified polynucleic acid molecule is nuclease resistant (eg, RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant).In some examples, T-deoxy-2'-O-fluoro modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, the LNA-modified polynucleic acid molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some examples, the ENA-modified polynucleic acid molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some examples, the HNA-modified polynucleic acid molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant).In some examples, the morpholino is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some examples, the PNA-modified polynucleic acid molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some examples, the methylphosphonate nucleotide-modified polynucleic acid molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some examples, the thiolphosphonate nucleotide-modified polynucleic acid molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some examples, polynucleic acid molecules comprising 2'-fluoro N3-P5'-phosphoramidites are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some examples, the 5' conjugates described herein inhibit 5'-3' exonuclease cleavage. In some examples, the 3' conjugates described herein inhibit 3'-5' exonuclease cleavage.

[0237] In some embodiments, one or more of the artificial nucleotide analogs described herein have increased binding affinity for their mRNA target compared to a comparable naturally occurring polynucleic acid molecule. One or more of the artificial nucleotide analogs, including 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2'-fluoro N3-P5'-phosphoramidites, have increased binding affinity for their mRNA targets compared to the equivalent naturally occurring polynucleic acid molecule. In some examples, 2'-O-methyl modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable naturally occurring polynucleic acid molecules. In some examples, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable naturally occurring polynucleic acid molecules. In some examples, 2'-O-aminopropyl modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable naturally occurring polynucleic acid molecules. In some examples, 2'-deoxy modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable naturally occurring polynucleic acid molecules. In some examples, T-deoxy-2'-fluoro modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable naturally occurring polynucleic acid molecules. In some examples, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable naturally occurring polynucleic acid molecules. In some instances, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules.In some examples, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some examples, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some examples, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some examples, LNA modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some examples, ENA modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some examples, PNA modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some examples, HNA-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, morpholino-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, methylphosphonate nucleotide-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, thiolphosphonate nucleotide-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, polynucleic acid molecules comprising 2'-fluoroN3-P5'-phosphoramidites have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, the increased affinity is exemplified by a lower Kd, a higher melting temperature (Tm), or a combination thereof.

[0238] In some embodiments, the polynucleic acid molecules described herein are chirally pure (or stereopure) polynucleic acid molecules or polynucleic acid molecules comprising a single enantiomer. In some examples, the polynucleic acid molecules comprise L-nucleotides. In some examples, the polynucleic acid molecules comprise D-nucleotides. In some examples, the polynucleic acid molecule composition comprises 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its enantiomer. In some cases, the polynucleic acid molecule composition comprises 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture. In some examples, the polynucleic acid molecules are polynucleic acid molecules described in U.S. Patent Application Publication Nos. 2014 / 194610 and 2015 / 211006 and PCT International Publication No. WO2015107425.

[0239] In some embodiments, the polynucleic acid molecules described herein are further modified to include an aptamer-conjugated moiety. In some examples, the aptamer-conjugated moiety is a DNA aptamer-conjugated moiety. In some examples, the aptamer-conjugated moiety is Alphamer (Centauri Therapeutics), which includes an aptamer portion that recognizes a specific cell surface target and a portion that displays a specific epitope for binding to circulating antibodies. In some examples, the polynucleic acid molecules described herein are further modified to include an aptamer-conjugated moiety as described in U.S. Patent Nos. 8,604,184, 8,591,910, and 7,850,975.

[0240] In further embodiments, the polynucleic acid molecules described herein are modified to increase their stability. In some embodiments, the polynucleic acid molecule is RNA (e.g., siRNA). In some examples, the polynucleic acid molecule is modified with one or more of the modifications described above to increase its stability. In some cases, the polynucleic acid molecule is modified at the 2' hydroxyl position, such as with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modification, or with a locked or bridged ribose structure (e.g., LNA or ENA). In some cases, the polynucleic acid molecule is modified with 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, the polynucleic acid molecule further comprises morpholino, PNA, HNA, methylphosphonate nucleotide, thiolphosphonate nucleotide, and / or 2'-fluoroN3-P5'-phosphoramidite to increase its stability. In some cases, the polynucleic acid molecule is a chiral pure (or stereopure) polynucleic acid molecule. In some cases, the chiral pure (or stereopure) polynucleic acid molecule is modified to increase its stability. Suitable modifications of RNA to increase the stability of delivery will be apparent to those skilled in the art.

[0241] In some cases, universal base refers to the nucleotide base analogue that forms base pairs with each of the natural DNA / RNA bases that are almost indistinguishable.Non-limiting examples of universal base include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamide, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole and 6-nitroindole, as known in the prior art (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0242] Small molecules, proteins, and peptides In some embodiments, the payload is a small molecule. In some examples, the small molecule is a cytotoxic payload. Exemplary cytotoxic payloads include, but are not limited to, microtubule-disrupting agents, DNA-modifying agents, or Akt inhibitors.

[0243] In some embodiments, the payload comprises a microtubule-disrupting agent. Exemplary microtubule-disrupting agents include, but are not limited to, 2-methoxyestradiol, auristatin, chalcone, colchicine, combretastatin, cryptophycin, dictyostatin, discodermolide, dolastain, eleutherobin, epothilone, halichondrin, laulimalide, maytansine, noscapinoid, paclitaxel, peloruside, fomopsin, podophyllotocin, rhizoxin, spongistatin, taxane, tubulysin, vinca alkaloid, vinorelbine, or a derivative or analog thereof.

[0244] In some embodiments, the tubulysin is an analog or derivative of tubulysin as described in U.S. Patent Nos. 8,580,820 and 8,980,833, and U.S. Application Publication Nos. 20130217638, 20130224228, and 201400363454.

[0245] In some embodiments, the maytansinoid is a maytansinoid. In some embodiments, the maytansinoid is DM1, DM4, or ansamitocin. In some embodiments, the maytansinoid is DM1. In some embodiments, the maytansinoid is DM4. In some embodiments, the maytansinoid is ansamitocin. In some embodiments, the maytansinoid is a maytansinoid derivative or analog as described in U.S. Patent Nos. 5,208,020, 5,416,064, 7,276,497, and 6,716,821, or U.S. Patent Application Publication Nos. 2013029900 and 20130323268.

[0246] In some embodiments, the payload is dolastin or a derivative or analog thereof. In some embodiments, the dolastin is dolastin 10 or dolastin 15, or a derivative or analog thereof. In some embodiments, the dolastin 10 analog is auristatin, soblidotin, simprostatin 1, or simprostatin 3. In some embodiments, the dolastin 15 analog is cemadotin or tacidotin.

[0247] In some embodiments, the dolastin 10 analog is an auristatin or an auristatin derivative. In some embodiments, the auristatin or auristatin derivative is auristatin E (AE), auristatin F (AF), auristatin E5-benzoylvalerate (AEVB), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), or monomethyl auristatin D (MMAD), auristatin PE, or auristatin PYE. In some embodiments, the auristatin derivative is monomethyl auristatin E (MMAE). In some embodiments, the auristatin derivative is monomethyl auristatin F (MMAF). In some embodiments, the auristatin is an auristatin derivative or analog such as those described in U.S. Patent Nos. 6,884,869, 7,659,241, 7,498,298, 7,964,566, 7,750,116, 8,288,352, 8,703,714, and 8,871,720.

[0248] In some embodiments, the payload comprises a DNA modifying agent. In some embodiments, the DNA modifying agent comprises a DNA cleaving agent, a DNA intercalator, a DNA transcription inhibitor, or a DNA cross-linking agent. In some examples, the DNA cleaving agent comprises bleomycin A2, calicheamicin, or a derivative or analog thereof. In some examples, the DNA intercalator comprises doxorubicin, epirubicin, PNU-159682, duocarmycin, pyrrolobenzodiazepine, oligomycin C, daunorubicin, valrubicin, topotecan, or a derivative or analog thereof. In some examples, the DNA transcription inhibitor comprises dactinomycin. In some examples, the DNA cross-linking agent comprises mitomycin C.

[0249] In some embodiments, the DNA modifying agent comprises amsacrine, anthracycline, camptothecin, doxorubicin, duocarmycin, enediyne, etoposide, indolinobenzodiazepine, netropsin, teniposide, or a derivative or analog thereof.

[0250] In some embodiments, the anthracycline is doxorubicin, daunorubicin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mithramycin, nemorubicin, pixantrone, sabarubicin, or valrubicin.

[0251] In some embodiments, the camptothecin analog is topotecan, irinotecan, ciratecan, cositecan, exatecan, lutotecan, jamatecan, belotecan, rubitecan, or SN-38.

[0252] In some embodiments, the duocarmycin is duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, or CC- 1065. In some embodiments, the enediyne is calicheamicin, esperamicin, or dynemicin A.

[0253] In some embodiments, the pyrrolobenzodiazepine is anthramycin, abeymycin, ticamycin, DC-81, mazethramycin, neothramycin A, neothramycin B, polothramycin, prothracarcin, sivanomycin (DC-102), sibiromycin, or tomaymycin. In some embodiments, the pyrrolobenzodiazepine is a tomaymycin derivative as described in U.S. Patent Nos. 8,404,678 and 8,163,736. In some embodiments, the pyrrolobenzodiazepine is as described in U.S. Patent Nos. 8,426,402, 8,802,667, 8,809,320, 6,562,806, 6,608,192, 7,704,924, 7,067,511, US 7,612,062, 7,244,724, 7,528,126, 7,049,311, 8,633,185, 8,501,934, and 8,697,688, and U.S. Application Publication No. US20140294868.

[0254] In some embodiments, the pyrrolobenzodiazepine is a pyrrolobenzodiazepine dimer. In some embodiments, the PBD dimer is a symmetric dimer. Examples of symmetric PBD dimers include, but are not limited to, SJG-136 (SG-2000), ZC-423 (SG2285), SJG-720, SJG-738, ZC-207 (SG2202), and DSB-120 (Table 2). In some embodiments, the PBD dimer is an asymmetric dimer. Examples of asymmetric PBD dimers include, but are not limited to, SJG-136 derivatives as described in U.S. Patent Nos. 8,697,688 and 9,242,013, and U.S. Patent Publication No. 20140286970.

[0255] In some embodiments, the payload comprises an Akt inhibitor. In some cases, the Akt inhibitor comprises ipatasertib (GDC-0068) or a derivative thereof.

[0256] In some embodiments, the payload comprises a polymerase inhibitor, including, but not limited to, a polymerase II inhibitor such as α-amanitin, and a poly(ADP-ribose) polymerase (PARP) inhibitor. Exemplary PARP inhibitors include, but are not limited to, iniparib (BSI 201), talazoparib (BMN-673), olaparib (AZD-2281), olaparib, rucaparib (AG014699, PF-01367338), veliparib (ABT-888), CEP 9722, MK 4827, BGB-290, or 3-aminobenzamide.

[0257] In some embodiments, the payload is a contrast agent. In some instances, the payload comprises a "radiopaque" label, e.g., a label visualized using X-rays. Radiopaque materials are well known to those skilled in the art. Exemplary radiopaque materials include iodides, bromides, or barium salts. Additional radiopaque materials include, but are not limited to, organobismuth derivatives (e.g., U.S. Pat. No. 5,939,045), radiopaque polyurethanes (e.g., U.S. Pat. No. 5,346,981), organobismuth complexes (e.g., U.S. Pat. No. 5,256,334), radiopaque barium polymer complexes (e.g., U.S. Pat. No. 4,866,132), and the like.

[0258] In some examples, the payload includes any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means, including, for example, a detectable label used in an immunoconjugate. Useful labels include magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, etc.), radioisotopes (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA), and colorimetric labels, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, nanoparticles, quantum dots, etc.

[0259] In some embodiments, suitable radiolabels include, but are not limited to: 99 Tc, 203 Pb, 67 Ga, 68 Ga, 72 As, 111 In, 113m In, 97 Ru, 62 Cu, 64 Cu,52 Fe, 52m Mn, 51 Cr, 186 Re, 188 Re, 77 As, 90 Y, 67 Cu, 169 Er, 121 Sn, 127 Te, 142 Pr, 143 Pr, 198 Au, 199 Au, 161 Tb, 109 Pd, 165 Dy, 149 Pm, 151 Pm, 153 Sm, 157 Gd, 159 Gd, 166 Ho, 172 Tm, 169 Yb, 175 Yb, 177 Lu, 105 Rh, and 111 Contains Ag.

[0260] In some instances, the payload is a ionizing radiation ( 60

[0013] Radiosensitizers include radiosensitizers that enhance the cytotoxic effects of radioactive materials (such as those generated by Co or X-ray sources). Many radiosensitizers are known, including, but not limited to, benzoporphyrin derivative compounds (see, e.g., U.S. Pat. No. 5,945,439), 1,2,4-benzotriazine oxides (see, e.g., U.S. Pat. No. 5,849,738), certain diamine-containing compounds (see, e.g., U.S. Pat. No. 5,700,825), BCNT (see, e.g., U.S. Pat. No. 5,872,107), radiosensitizing nitrobenzoic acid amide derivatives (see, e.g., U.S. Pat. No. 4,474,814), various heterocyclic derivatives (see, e.g., U.S. Pat. No. 5,064,849), platinum complexes (see, e.g., U.S. Pat. No. 4,921,963), and the like.

[0261] In some instances, the payload is an alpha emitter, i.e., a radioisotope that emits alpha particles. Alpha emitters have recently been shown to be effective in the treatment of cancer (see, e.g., McDevitt et al. (2001) Science 294:1537-1540; Ballangrud et al. (2001) Cancer Res. 61: 2008-2014; Borchardt et al. (2003) Cancer Res. 63:5084-50). Suitable alpha emitters include, but are not limited to: 213 Bi, 211 Including At etc.

[0262] In some examples, the payload comprises an immunomodulator. Useful immunomodulators include antihormonal agents that inhibit the action of hormones on tumors, and immunosuppressants that suppress cytokine production, downregulate the expression of self-antigens, or mask MHC antigens. Representative antihormonal agents include antiestrogens such as tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapunstone, and toremifene, antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, and antiadrenal agents. Immunosuppressants include, but are not limited to, 2-amino-6-aryl-5-substituted pyrimidines, azathioprine, cyclophosphamide, bromocriptine, danazol, dapsone, glutaraldehyde, anti-idiotypic antibodies against MHC antigens and MHC fragments, cyclosporin A, steroids such as glucocorticosteroids, streptokinase, or rapamycin.

[0263] In some embodiments, the payload comprises a protein or peptide toxin or fragment thereof. Exemplary enzymatically active toxins or fragments thereof include, but are not limited to, diphtheria toxin A fragment, nonbinding active fragment of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, certain Jatropha curcas proteins, certain dianthus proteins, pokeweed proteins (PAP, PAPII, and PAP-S), bitter melon inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogillin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0264] In some examples, the payload is an immunomodulatory agent. Exemplary immunomodulatory agents include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporin, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and analogs thereof, xanthines, stem cell growth factors, lymphotoxins, hematopoietic factors, tumor necrosis factors (TNF) (e.g., TNFα), interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony-stimulating factors (e.g., granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF)), interferons (e.g., ribozymes ... Interferon alpha, interferon beta, interferon gamma), stem cell growth factor termed "S1 factor," erythropoietin, and thrombopoietin, or a combination thereof.

[0265] In some examples, the payload comprises a cytokine, hi some embodiments, the cytokine comprises IL-2, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, an interferon (e.g., IFNα, IFNβ), or TNFα.

[0266] polymer In some embodiments, the anti-transferrin receptor antibody conjugates described herein further comprise a polymer (polymer moiety C). In some examples, the polymer moiety is a natural or synthetic polymer consisting of long chains of branched or unbranched monomers and / or two- or three-dimensional crosslinked networks of monomers. In some examples, the polymer moiety comprises a polysaccharide, lignin, rubber, or polyalkylene oxide (e.g., polyethylene glycol). In some examples, at least one polymer moiety includes, but is not limited to, alpha-dihydroxyl polyethylene glycol, omega-dihydroxyl polyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactide acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin, polyamide, polycyanoacrylate, polyimide, polyethylene terephthalate (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethane, and mixtures thereof. As used herein, a mixture refers to the use of different polymers within the same compound, as in connection with block copolymers. In some cases, a block copolymer is a polymer in which at least one portion of the polymer is constructed from monomers of another polymer. In some examples, the polymer portion comprises a polyalkylene oxide. In some examples, the polymer portion comprises PEG. In some examples, the polymer portion comprises polyethyleneimide (PEI) or hydroxyethyl starch (HES).

[0267] In some examples, C is a PEG moiety. In some examples, the PEG moiety is conjugated to the 5' end of the polynucleic acid molecule, while the linking moiety is conjugated to the 3' end of the polynucleic acid molecule. In some examples, the PEG moiety is conjugated to the 3' end of the polynucleic acid molecule, while the linking moiety is conjugated to the 5' end of the polynucleic acid molecule. In some examples, the PEG moiety, the linking moiety, or a combination thereof, is conjugated to an internal site of the polynucleic acid molecule. In some examples, the conjugation is direct conjugation. In some examples, the conjugation is via native ligation.

[0268] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some instances, a polydisperse material comprises a dispersed distribution of materials of different molecular weights, characterized by average weight (weight-average) size and dispersity. In some instances, a monodisperse PEG comprises molecules of one size. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the molecular weight indicated represents the average molecular weight of the polyalkylene oxide, e.g., PEG, molecules.

[0269] In some embodiments, the molecular weight of the polyalkylene oxide (e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 83 00, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

[0270] In some embodiments, C is a polyalkylene oxide (e.g., PEG) and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, , 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG and is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 850 In some examples, C has a molecular weight of 0, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some examples, C has a molecular weight of about 200 Da. In some examples, C has a molecular weight of about 300 Da. In some examples, C has a molecular weight of about 400 Da. In some examples, C has a molecular weight of about 500 Da. In some examples, C has a molecular weight of about 600 Da. In some examples, C has a molecular weight of about 700 Da. In some examples, the molecular weight of C is about 800 Da. In some examples, the molecular weight of C is about 900 Da. In some examples, the molecular weight of C is about 1000 Da. In some examples, the molecular weight of C is about 1100 Da. In some examples, the molecular weight of C is about 1200 Da. In some examples, the molecular weight of C is about 1300 Da. In some examples, the molecular weight of C is about 1400 Da. In some examples, the molecular weight of C is about 1450 Da.In some examples, the molecular weight of C is about 1500 Da. In some examples, the molecular weight of C is about 1600 Da. In some examples, the molecular weight of C is about 1700 Da. In some examples, the molecular weight of C is about 1800 Da. In some examples, the molecular weight of C is about 1900 Da. In some examples, the molecular weight of C is about 2000 Da. In some examples, the molecular weight of C is about 2100 Da. In some examples, the molecular weight of C is about 2200 Da. In some examples, the molecular weight of C is about 2300 Da. In some examples, the molecular weight of C is about 2400 Da. In some examples, the molecular weight of C is about 2500 Da. In some examples, the molecular weight of C is about 2600 Da. In some examples, the molecular weight of C is about 2700 Da. In some examples, the molecular weight of C is about 2800 Da. In some examples, the molecular weight of C is about 2900 Da. In some examples, the molecular weight of C is about 3000 Da. In some examples, the molecular weight of C is about 3250 Da. In some examples, the molecular weight of C is about 3350 Da. In some examples, the molecular weight of C is about 3500 Da. In some examples, the molecular weight of C is about 3750 Da. In some examples, the molecular weight of C is about 4000 Da. In some examples, the molecular weight of C is about 4250 Da. In some examples, the molecular weight of C is about 4500 Da. In some examples, the molecular weight of C is about 4600 Da. In some examples, the molecular weight of C is about 4750 Da. In some examples, the molecular weight of C is about 5000 Da. In some examples, the molecular weight of C is about 5500 Da. In some examples, the molecular weight of C is about 6000 Da. In some examples, the molecular weight of C is about 6500 Da. In some examples, the molecular weight of C is about 7000 Da. In some examples, the molecular weight of C is about 7500 Da. In some examples, the molecular weight of C is about 8000 Da. In some examples, the molecular weight of C is about 10,000 Da. In some examples, the molecular weight of C is about 12,000 Da. In some examples, the molecular weight of C is about 20,000 Da. In some examples, the molecular weight of C is about 35,000 Da. In some examples, the molecular weight of C is about 40,000 Da. In some examples, the molecular weight of C is about 50,000 Da.In some examples, the molecular weight of C is about 60,000 Da. In some examples, the molecular weight of C is about 100,000 Da.

[0271] In some embodiments, the polyalkylene oxide (e.g., PEG) is a discrete PEG, which is a polymeric PEG containing more than one repeating ethylene oxide unit. In some examples, the discrete PEG (dPEG) contains 2 to 60, 2 to 50, or 2 to 48 repeating ethylene oxide units. In some examples, the dPEG contains about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50, or more repeating ethylene oxide units. In some examples, the dPEG contains about 2 or more repeating ethylene oxide units. In some examples, the dPEG contains about 3 or more repeating ethylene oxide units. In some examples, the dPEG contains about 4 or more repeating ethylene oxide units. In some examples, the dPEG contains about 5 or more repeating ethylene oxide units. In some examples, dPEG contains about 6 or more repeating ethylene oxide units. In some examples, dPEG contains about 7 or more repeating ethylene oxide units. In some examples, dPEG contains about 8 or more repeating ethylene oxide units. In some examples, dPEG contains about 9 or more repeating ethylene oxide units. In some examples, dPEG contains about 10 or more repeating ethylene oxide units. In some examples, dPEG contains about 11 or more repeating ethylene oxide units. In some examples, dPEG contains about 12 or more repeating ethylene oxide units. In some examples, dPEG contains about 13 or more repeating ethylene oxide units. In some examples, dPEG contains about 14 or more repeating ethylene oxide units. In some examples, dPEG contains about 15 or more repeating ethylene oxide units. In some examples, dPEG contains about 16 or more repeating ethylene oxide units. In some examples, dPEG contains about 17 or more repeating ethylene oxide units. In some examples, dPEG contains about 18 or more repeating ethylene oxide units. In some examples, dPEG contains about 19 or more repeating ethylene oxide units. In some examples, dPEG contains about 20 or more repeating ethylene oxide units. In some instances, the dPEG contains about 22 or more repeating ethylene oxide units.In some examples, dPEG contains about 24 or more repeating ethylene oxide units. In some examples, dPEG contains about 26 or more repeating ethylene oxide units. In some examples, dPEG contains about 28 or more repeating ethylene oxide units. In some examples, dPEG contains about 30 or more repeating ethylene oxide units. In some examples, dPEG contains about 35 or more repeating ethylene oxide units. In some examples, dPEG contains about 40 or more repeating ethylene oxide units. In some examples, dPEG contains about 42 or more repeating ethylene oxide units. In some examples, dPEG contains about 48 or more repeating ethylene oxide units. In some examples, dPEG contains about 50 or more repeating ethylene oxide units. In some examples, dPEG is synthesized stepwise as a single molecular weight compound from pure (e.g., about 95%, 98%, 99%, or 99.5%) starting materials. In some examples, the dPEG has a specific molecular weight rather than an average molecular weight. In some examples, the dPEG described herein is dPEG from Quanta Biodesign, LMD.

[0272] In some embodiments, the polymer portion C comprises a cationic mucic acid-based polymer (cMAP). In some examples, the cMAP comprises one or more subunits of at least one repeating subunit, the subunit structure being represented as formula (III):

[0273] [ka]

[0274] wherein m, at each occurrence, is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4-6, or 5, and n, at each occurrence, is independently 1, 2, 3, 4, or 5. In some embodiments, m and n are, for example, about 10.

[0275] In some examples, the cMAP is further conjugated to a PEG moiety to form a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some examples, the PEG moiety ranges from about 500 Da to about 50,000 Da. In some examples, the PEG moiety ranges from about 500 Da to about 1000 Da, greater than 1000 Da to about 5000 Da, greater than 5000 Da to about 10,000 Da, greater than 10,000 to about 25,000 Da, greater than 25,000 Da to about 50,000 Da, or any combination of two or more of these ranges.

[0276] In some instances, C is a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some instances, C is a cMAP-PEG copolymer. In other instances, C is an mPEG-cMAP-PEGm triblock polymer. In further instances, C is a cMAP-PEG-cMAP triblock polymer.

[0277] Endosomolytic part In some embodiments, the anti-transferrin receptor antibody conjugate further comprises an additional conjugated moiety. In some examples, the additional conjugated moiety is an endosomolytic moiety. In some cases, the endosomolytic moiety is a compound that can be released from any of the cellular compartments known in the art, such as a cellular compartment-releasing component, e.g., an endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other endoplasmic reticulum containing cells. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide, an endosomolytic polymer, an endosomolytic lipid, or an endosomolytic small molecule. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide. In other cases, the endosomolytic moiety comprises an endosomolytic polymer.

[0278] Endosomolytic Polypeptides In some embodiments, the anti-transferrin receptor antibody conjugate is further conjugated to an endosomolytic polypeptide. In some embodiments, the anti-transferrin receptor antibody conjugate is further conjugated to an endosomolytic polypeptide. 1 -B) n or formula (II): AX 1 -(BX 2 -C) n The conjugate is further conjugated to an endosomolytic polypeptide. In some cases, the endosomolytic polypeptide is a pH-dependent membrane active peptide. In some cases, the endosomolytic polypeptide is an amphipathic polypeptide. In further cases, the endosomolytic polypeptide is a peptidomimetic. In some examples, the endosomolytic polypeptide comprises INF, melittin, mucin, or their respective derivatives. In some examples, the endosomolytic polypeptide comprises INF or a derivative thereof. In other cases, the endosomolytic polypeptide comprises melittin or a derivative thereof. In further cases, the endosomolytic polypeptide comprises mucin or a derivative thereof.

[0279] In some examples, INF7 is a 24-residue polypeptide, and the sequence includes CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 51) or GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 52). In some examples, INF7 or a derivative thereof includes the following sequence: GLFEAIEGFIENGWEGMIWDYGSGSCG (SEQ ID NO: 53), GLFEAIEGFIENGWEGMIDG WYG-(PEG)6-NH2 (SEQ ID NO: 54), or GLFEAIEGFIENGWEGMIWDYG-SGSC-K(GalNAc)2 (SEQ ID NO: 55).

[0280] In some instances, melittin is a 26-residue polypeptide, and the sequence comprises CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 56) or GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 57). In some instances, melittin comprises a polypeptide sequence described in U.S. Patent No. 8,501,930.

[0281] In some instances, the mucin is an antimicrobial peptide (AMP) derived from the venom gland of the scorpion (Mesobuthus eupeus). In some instances, the mucin is composed of mucin-13, which comprises the sequence IFGAIAGLLKNIF-NH2 (SEQ ID NO: 58), and mucin-18, which comprises the sequence FFGHLFKLATKIIPSLFQ (SEQ ID NO: 59).

[0282] In some examples, the endosomolytic polypeptide comprises a polypeptide whose sequence is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identical to INF7 or a derivative thereof, melittin or a derivative thereof, or a mucin or a derivative thereof, hi some examples, the endosomolytic moiety comprises INF7 or a derivative thereof, melittin or a derivative thereof, or a mucin or a derivative thereof.

[0283] In some examples, the endosomolytic moiety comprises a sequence as illustrated in Table 8.

[0284] [Table 8-1]

[0285] [Table 8-2]

[0286] In some cases, the endosomolytic moiety comprises a Bak BH3 polypeptide that induces apoptosis through antagonism of inhibitory targets such as Bcl-2 and / or Bcl-xL. In some examples, the endosomolytic moiety comprises a Bak BH3 polypeptide described in Albarran, et al., "Efficient intracellular delivery of a pro-apoptotic peptide with a pH-responsive carrier," Reactive & Functional Polymers 71: 261-265 (2011).

[0287] In some examples, the endosomolytic moiety comprises a polypeptide (e.g., a cell-penetrating polypeptide) such as those described in PCT International Publication No. WO2013 / 166155 or WO2015 / 069587.

[0288] Endosomolytic polymers In some embodiments, the compound of formula (I): A-(X 1 -B) n or formula (II): AX 1 -(BX 2 -C) n The conjugate is further conjugated to an endosomolytic polymer. As used herein, endosomolytic polymers include linear, branched network, star, comb, or ladder polymers. In some instances, the endosomolytic polymer is a homopolymer or copolymer containing two or more different types of monomers. In some instances, the endosomolytic polymer is a polycationic polymer. In other instances, the endosomolytic polymer is a polyanionic polymer.

[0289] In some instances, polycationic polymers contain positively, neutrally, or negatively charged monomer units, resulting in a net positive charge. In other instances, polycationic polymers contain non-polymeric molecules containing two or more positive charges. Exemplary cationic polymers include, but are not limited to, poly(L-lysine) (PLL), poly(L-arginine) (PLA), polyethyleneimine (PEI), poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), or N,N-diethylaminoethyl methacrylate (DEAEMA).

[0290] In some cases, polyanionic polymers contain positively charged, neutrally charged, or negatively charged monomer units, resulting in a net negative charge. In other cases, polyanionic polymers contain non-polymeric molecules containing two or more negative charges. Exemplary anionic polymers include p(alkyl acrylates) (e.g., poly(propylacrylic acid) (PPAA)) or poly(N-isopropylacrylamide) (NIPAM). Further examples include PP75, the L-phenylalanine-poly(L-lysine isophthalamide) polymer described in Khormaee, et al., "Edosomolytic anionic polymer for the cytoplasmic delivery of siRNAs in localized in vivo applications," Advanced Functional Materials 23: 565-574 (2013).

[0291] In some embodiments, the endosomolytic polymers described herein are pH-responsive endosomolytic polymers. pH-responsive polymers include polymers that increase in size (swell) or collapse depending on the pH of the environment. Polyacrylic acid and chitosan are examples of pH-responsive polymers.

[0292] In some instances, the endosomolytic moieties described herein are membrane-disruptive polymers. In some instances, the membrane-disruptive polymers include cationic polymers, neutral or hydrophobic polymers, or anionic polymers. In some instances, the membrane-disruptive polymers are hydrophilic polymers.

[0293] In some examples, the endosomolytic moiety described herein is a membrane-disrupting polymer. Exemplary pH-responsive membrane-disrupting polymers include p(alkylacrylic acid), poly(N-isopropylacrylamide) (NIPAM) copolymers, succinylated p(glycidol), and p(β-malic acid) polymers.

[0294] In some examples, the p(alkylacrylic acid) includes poly(propylacrylic acid) (polyPAA), poly(methacrylic acid) (PMAA), poly(ethylacrylic acid) (PEAA), and poly(propylacrylic acid) (PPAA). In some examples, the p(alkylacrylic acid) includes the p(alkylacrylic acid) described in Jones, et al., Biochemistry Journal 372: 65-75 (2003).

[0295] In some embodiments, the pH-responsive membrane-disruptive polymer comprises p(butyl acrylate-co-methacrylic acid). (See Bulmus, et al., Journal of Controlled Release 93: 105-120 2003, and Yessine, et al., Biochimica et Biophysica Acta 1613: 28-38 (2003)).

[0296] In some embodiments, the pH-responsive membrane-disruptive polymer comprises p(styrene-alt-maleic anhydride). (See Henry, et al., Biomacromolecules 7:2407-2414 (2006))

[0297] In some embodiments, the pH-responsive membrane-disruptive polymer comprises a pyridyl disulfide acrylate (PDSA) polymer, such as poly(MAA-co-PDSA), poly(EAA-co-PDSA), poly(PAA-co-PDSA), poly(MAA-co-BA-co-PDSA), poly(EAA-co-BA-co-PDSA), or poly(PAA-co-BA-co-PDSA) polymer. (See El-Sayed, et al., "Rational design of composition and activity correlations for pH-responsive and glutathione-reactive polymer therapeutics," Journal of Controlled Release 104: 417-427 (2005), or Flanary et al., "Antigen delivery with poly(propylacrylic acid) conjugation enhanced MHC-I presentation and T-cell activation," Bioconjugate Chem. 20: 241-248 (2009)).

[0298] In some embodiments, the pH-responsive membrane-disruptive polymer comprises a soluble polymer comprising the following base structure:

[0299] [ka]

[0300] In some examples, the endosomolytic moieties described herein are further conjugated to an additional conjugate, for example, a polymer (e.g., PEG), or a modified polymer (e.g., a cholesterol-modified polymer).

[0301] In some examples, the additional conjugate comprises a surfactant (e.g., Triton X-100). In some examples, the endosomolytic moiety described herein comprises a polymer (e.g., poly(amidoamine)) conjugated to a surfactant (e.g., Triton X-100). In some examples, the endosomolytic moiety described herein comprises a poly(amidoamine)-Triton X-100 conjugate (Duncan, et al., "A polymer-Triton X-100 conjugate capable of pH-dependent red blood cell lysis: a model system illustrating the possibility of drug delivery within acidic intracellular compartments," Journal of Drug Targeting 2: 341-347 (1994)).

[0302] Endosomolytic lipids In some embodiments, the endosomolytic moiety is a lipid (e.g., a fusogenic lipid). In some embodiments, the endosomolytic moiety is a lipid having formula (I): A-(X 1 -B) n or formula (II): AX 1 -(BX 2 -C) nThe conjugate is further conjugated to an endosomolytic lipid (e.g., a fusogenic lipid). Exemplary fusogenic lipids include 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (XTC).

[0303] In some examples, the endosomolytic moiety is a lipid (eg, a fusogenic lipid) described in PCT International Publication No. WO 09 / 126,933.

[0304] Endosomolytic small molecules In some embodiments, the endosomolytic moiety is a small molecule. In some embodiments, the endosomolytic moiety is a small molecule. 1 -B) n or formula (II): AX 1 -(BX 2 -C) nis further conjugated to an endosomolytic small molecule. Exemplary small molecules suitable as endosomolytic moieties include, but are not limited to, quinine, chloroquine, hydroxychloroquine, amodiaquine (carnoquines), ampiroquine, primaquine, mefloquine, nivaquines, halofantrine, quinoneimine, or combinations thereof. In some examples, quinoline endosomolytic moieties include, but are not limited to, 7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline (chloroquine), 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline (hydroxychloroquine), 7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline, 4-(4-diethylamino-1-methylbutylamino)quinoline, 7-hydroxy-4-(4-diethylamino-1-methylbutylamino)quinoline, 7-chloro-4-(4-diethylamino-1-butylamino)quinoline (desmethylchloroquine), 7-fluoro-4-(4-diethylamino-1-butylamino)quinoline, 4-(4-diethylamino-1-butylamino)quinoline, 7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline, phosphorus, 7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline, 7-fluoro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline, 4-(1-carboxy-4-diethylamino-1-butylamino)quinoline, 7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline, 7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline, 7-fluoro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline, 4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline, 7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline, 7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline,4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino-)quinoline, 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline, hydroxychloroquine phosphate, 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline (desmethylhydroxychloroquine), 7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline, 4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline, 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline, 7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline, 4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline, 7-hydroxy-4-(1-carboxy 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline, 7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline, 4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline, 7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline amino-1-methylbutylamino)quinoline, 8-[(4-aminopentyl)amino-6-methoxydihydrochloridequinoline, 1-acetyl-1,2,3,4-tetrahydroquinoline, 8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride, 1-butyryl-1,2,3,4-tetrahydroquinoline, 3-chloro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxyquinoline,Examples include 3-fluoro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline, 4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline, 3,4-dihydro-1-(2H)-quinolinecarboxaldehyde, 1,1'-pentamethylenequinolinium iodide, 8-quinolinol sulfate, and their amino, aldehyde, carboxylic acid, hydroxyl, halogen, keto, sulfhydryl, and vinyl derivatives or analogs. In some examples, the endosomolytic moiety is described in Naisbitt et al. (1997, J Pharmacol Exp Therapy 280:884-893) and U.S. Pat. No. 5,736,557.

[0305] In some embodiments, the endosomolytic moiety is nigericin or a conjugate thereof, such as a folate-nigericin ester conjugate, a folate-nigericin amide conjugate, or a folate-nigericin carbamate conjugate. In some examples, the endosomolytic moiety is nigericin, as described in Rangasamy, et. al., "New mechanism for release of endosomal contents: osmotic lysis via nigericin-mediated K / H exchange," Bioconjugate Chem. 29:1047-1059 (2018).

[0306] Linker In some embodiments, the linkers described herein are cleavable or non-cleavable linkers. In some examples, the linker is a cleavable linker. In other examples, the linker is a non-cleavable linker.

[0307] In some cases, the linker is a non-polymeric linker. A non-polymeric linker refers to a linker that does not contain repeating units of a monomer produced by a polymerization process. Exemplary non-polymeric linkers include, but are not limited to, a C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or C1 alkyl group), a homobifunctional cross-linker, a heterobifunctional cross-linker, a peptide linker, a traceless linker, a self-immolative linker, a maleimide-based linker, or a combination thereof. In some cases, the non-polymeric linker includes a C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or C1 alkyl group), a homobifunctional cross-linker, a heterobifunctional cross-linker, a peptide linker, a traceless linker, a self-immolative linker, a maleimide-based linker, or a combination thereof. In further cases, the non-polymeric linker does not include more than two linkers of the same type, for example, more than two homobifunctional cross-linkers or more than two peptide linkers. In further instances, the non-polymeric linker optionally includes one or more reactive functional groups.

[0308] In some examples, the non-polymeric linker does not include a polymer as described above. In some examples, the non-polymeric linker does not include a polymer encompassed by polymer moiety C. In some cases, the non-polymeric linker does not include a polyalkylene oxide (e.g., PEG). In some cases, the non-polymeric linker does not include PEG.

[0309] In some examples, the linker comprises a homobifunctional linker. Exemplary homobifunctional linkers include, but are not limited to, Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3',3'-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)bis ... (e) propionamido) butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl] disulfide (BASE D), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0310] In some embodiments, the linker comprises a heterobifunctional linker. Exemplary heterobifunctional linkers include, but are not limited to, amine-reactive and sulfhydryl-crosslinking linkers, such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoactyl)aminobenzoate (sulfo- sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyloxy)succinimide esters (GMBs), N-(γ-maleimidobutyloxy)sulfosuccinimide esters (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), Succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl-4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA),Carbonyl-reactive and sulfhydryl-reactive cross-linkers, such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (MCH), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive and photoreactive cross-linkers, such as N-hydroxysuccinimidyl-4 -azidosalicylic acid (NH-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NH-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NH-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidosalicylic acid (sulfo-NH-AsA), sulfosuccinimidyl-4-azidosalicylamido dobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxy Succinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3′-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), ρ-nitrophenyl diazopyruvate (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive cross-linkers, e.g.Examples of cross-linkers include 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidecarbonyl-reactive and photoreactive cross-linkers such as ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive cross-linkers such as 4-(ρ-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive cross-linkers such as ρ-azidophenylglyoxal (APG).

[0311] In some examples, the linker comprises a reactive functional group. In some cases, the reactive functional group comprises a nucleophilic group that is reactive to an electrophilic group present in the linking moiety. Exemplary electrophilic groups include carbonyl groups such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0312] In some embodiments, the linker comprises a maleimide group. In some examples, the maleimide group is also referred to as a maleimide spacer. In some examples, the maleimide group further comprises caproic acid to form maleimidocaproyl (mc). In some cases, the linker comprises maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other examples, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), described above.

[0313] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some examples, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby preventing the maleimide from undergoing retro-Michael elimination. In some examples, the self-stabilizing maleimide is a maleimide group described in Lyon, et al., "Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol. 32(10):1059-1062 (2014). In some examples, the linker comprises a self-stabilizing maleimide. In some examples, the linker is a self-stabilizing maleimide.

[0314] In some embodiments, the linker comprises a peptide moiety. In some examples, the peptide moiety comprises at least 1, 2, 3, 4, 5, or more than 6 amino acid residues. In some examples, the peptide moiety comprises at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In some examples, the peptide moiety comprises about 2, about 3, about 4, about 5, or about 6 amino acid residues. In some examples, the peptide moiety is a cleavable peptide moiety (e.g., enzymatically or chemically). In some examples, the peptide moiety is a non-cleavable peptide moiety. In some examples, the peptide moiety comprises Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some examples, the linker comprises a peptide moiety such as Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some instances, the linker comprises Val-Cit. In some instances, the linker is Val-Cit.

[0315] In some embodiments, the linker comprises a benzoic acid group or a derivative thereof. In some examples, the benzoic acid group or a derivative thereof comprises para-aminobenzoic acid (PABA). In some examples, the benzoic acid group or a derivative thereof comprises gamma-aminobutyric acid (GABA).

[0316] In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and / or a benzoic acid group, in any combination. In some embodiments, the linker comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In some examples, the maleimide group is maleimidocaproyl (mc). In some examples, the peptide group is val-cit. In some examples, the benzoic acid group is PABA. In some examples, the linker comprises an mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In further cases, the linker comprises an mc-val-cit-PABA group.

[0317] In some embodiments, the linker is a self-immolative linker or a self-eliminating linker.In some cases, the linker is a self-immolative linker.In other cases, the linker is a self-eliminating linker (for example, a cyclized self-eliminating linker).In some examples, the linker comprises the linker described in U.S. Patent No. 9,089,614 or PCT International Publication No. WO2015038426.

[0318] In some embodiments, the linker is a dendritic linker. In some examples, the dendritic linker comprises a branched multifunctional linker moiety. In some examples, the dendritic linker is used to increase the molar ratio of polynucleotide B to binding moiety A. In some examples, the dendritic linker comprises a PAMAM dendrimer.

[0319] In some embodiments, the linker is a traceless linker, or a linker that, after cleavage, does not leave a linker moiety (e.g., an atom or linker group) to the binding moiety A, the polynucleotide B, the polymer C, or the endosomolytic moiety D. Exemplary traceless linkers include, but are not limited to, a germanium linker, a silicon linker, a sulfur linker, a selenium linker, a nitrogen linker, a phosphorus linker, a boron linker, a chromium linker, or a phenylhydrazide linker. In some cases, the linker is a traceless aryl-triazene linker as described in Hejesen, et al., "A traceless aryl-triazene linker for DNA-directed chemistry," Org Biomol Chem 11(15):2493-2497 (2013). In some examples, the linker is a traceless linker as described in Blaney, et al., "Traceless solid-phase organic synthesis," Chem. Rev. 102:2607-2024 (2002). In some examples, the linker is a traceless linker as described in U.S. Patent No. 6,821,783.

[0320] In some embodiments, the linker is a polymerizable compound as described in U.S. Patent Nos. 6,884,869, 7,498,298, 8,288,352, 8,609,105, or 8,697,688; U.S. Patent Publication Nos. 2014 / 0127239; 2013 / 028919; 2014 / 286970; 09256, 2015 / 037360, or 2014 / 0294851, or PCT International Publication Nos. WO2015057699, WO2014080251, WO2014197854, WO2014145090, or WO2014177042.

[0321] In some embodiments, X 1 and X 2 are each independently a single bond or a non-polymeric linker.1 and X 2 are each independently a single bond. 1 and X 2 are each independently a non-polymeric linker.

[0322] In some instances, X 1 comprises a single bond or a non-polymeric linker. In some examples, X 1 is a single bond. In some instances, X 1 is a non-polymeric linker. In some examples, the linker is a C1-C6 alkyl group. In some cases, X 1 is a C1-C6 alkyl group, such as a C5, C4, C3, C2, or C1 alkyl group. In some cases, the C1-C6 alkyl group is an unsubstituted C1-C6 alkyl group. As used in the context of linkers, particularly in the context of X1, alkyl refers to a saturated, straight- or branched-chain hydrocarbon radical containing up to 6 carbon atoms. In some instances, X 1 comprises a homobifunctional linker or heterobifunctional linker as described above. 1 comprises a heterobifunctional linker. 1 In another example, X 1 comprises a heterobifunctional linker optionally conjugated to a C1-C6 alkyl group. 1 comprises sMCC optionally conjugated to a C1-C6 alkyl group. 1 does not include the homobifunctional or heterobifunctional linkers described above.

[0323] In some instances, X 2 is a single bond or a linker. In some examples, X 2 is a single bond. In other cases, X 2 is a linker. In a further case, X 2 is a non-polymeric linker. In some embodiments, X 2 is a C1-C6 alkyl group. In some examples, X2 is a homobifunctional linker or heterobifunctional linker as described above. In some examples, X 2 is a homobifunctional linker as described above. In some examples, X 2 is a heterobifunctional linker as described above. In some examples, X 2 comprises a maleimide group, e.g., maleimidocaproyl (mc), or a self-stabilizing maleimide group as described above. In some examples, X 2 contains a peptide moiety such as Val-Cit. In some instances, X 2 contains a benzoic acid group, such as PABA. 2 In a further example, X may contain a combination of maleimide groups, peptide moieties, and / or benzoic acid groups. 2 contains mc groups. 2 contains a mc-val-cit group. 2 In a further example, X 2 contains the mc-val-cit-PABA group.

[0324] How to use In some embodiments, described herein are methods for delivering a payload to a targeted target site by use of the anti-transferrin receptor antibodies described herein. In some examples, the targeted target site is a cell that overexpresses a causative protein associated with a disease or condition. In some examples, the targeted target site is a cell that contains improperly processed mRNA that encodes a non-functional protein or a protein with reduced expression that leads to a disease or condition. In some examples, the targeted target site is a tumor site. In a further example, the targeted target site is a site in the brain.

[0325] In some embodiments, methods for treating diseases or disorders characterized by overexpressed proteins are described herein.In some examples, the disease or disorder is muscle atrophy.In some examples, the disease or disorder is myotonic dystrophy.

[0326] In one embodiment, muscle atrophy refers to a significant loss of muscle strength. Significant loss of muscle strength refers to a decrease in the strength of a subject's diseased, damaged, or unused muscle tissue compared to the same muscle tissue in a control subject. In some embodiments, significant loss of muscle strength refers to a decrease in strength of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more compared to the same muscle tissue in a control subject. In another embodiment, significant loss of muscle strength refers to a decrease in the strength of unused muscle tissue compared to the muscle strength of the same muscle tissue in the same subject before a period of non-use. In some embodiments, significant loss of muscle strength refers to a decrease in strength of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more compared to the muscle strength of the same muscle tissue in the same subject before a period of non-use.

[0327] In another embodiment, muscle atrophy refers to a significant loss of muscle mass. Significant loss of muscle mass refers to a decrease in muscle volume in a subject's diseased, injured, or disused muscle tissue compared to the same muscle tissue in a control subject. In some embodiments, a significant loss of muscle volume is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more compared to the same muscle tissue in a control subject. In another embodiment, a significant loss of muscle mass refers to a decrease in muscle volume in disused muscle tissue compared to the muscle volume of the same muscle tissue in the same subject before a period of disuse. In some embodiments, a significant loss of muscle tissue is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more compared to the muscle volume of the same muscle tissue in the same subject before a period of disuse. Muscle volume is optionally measured by assessing muscle cross-sectional area, such as by magnetic resonance imaging (eg, by muscle volume / cross-sectional area (CSA) MRI method).

[0328] In some embodiments, the muscle wasting disease comprises or is associated with cachexia, denervation, myopathy, motor neuron disease, diabetes, chronic obstructive pulmonary disease, liver disease, congestive heart failure, chronic renal failure, chronic infection, sepsis, fasting, sarcopenia, glucocorticoid-related muscle atrophy, or disuse-related muscle atrophy.

[0329] Cachexia is the acquired accelerated loss of muscle mass caused by an underlying disease. In some instances, cachexia refers to weight loss that cannot be restored through nutrition and is commonly associated with underlying diseases such as cancer, COPD, AIDS, and heart failure. When cachexia is observed in patients with terminal cancer, it is called "cancer cachexia." Cancer cachexia affects a large proportion of patients with advanced cancer and is associated with treatment resistance, reduced response to therapy, quality of life, and survival. In some instances, cancer cachexia is defined as a multifactorial syndrome characterized by the ongoing loss of skeletal muscle mass, with or without loss of body fat, which cannot be fully restored with conventional nutritional support and can lead to progressive functional impairment. In some cases, skeletal muscle loss appears to be the most important event in cancer cachexia. In addition, the classification of cancer cachexia suggests that diagnostic criteria should not only be based on weight loss being a signal event of the cachectic process, but also on the patient's initial reserves, low BMI, or low levels of muscularity.

[0330] Denervation is damage to peripheral motor neurons involving partial or complete blockage of nerve fibers between organs and the central nervous system, causing blockage of nerve conduction and motor neuron firing, resulting in impaired skeletal muscle contractility. This loss of nerve function can be localized or generalized, with loss of entire motor neuron units. The inability of skeletal muscles to contract results in muscle atrophy. In some instances, denervation is associated with or results from degenerative, metabolic, or inflammatory neuropathies (e.g., Guillain-Barré syndrome, peripheral neuropathy, or exposure to environmental toxins or drugs). In further instances, denervation is associated with physical injury, e.g., surgery.

[0331] Myopathy is a general term describing muscle disorders. In some instances, myopathy includes myotonia, congenital myopathies such as nemaline myopathy, multicore / minicore myopathy, and myotubular (centronuclear) myopathy, mitochondrial myopathy, familial periodic paralysis, inflammatory myopathies such as metabolic myopathies caused by glycogen or lipid storage diseases, dermatomyositis, polymyositis, inclusion body myositis, myositis ossificans, rhabdomyolysis, and myoglobinuria. In some instances, myopathy is caused by muscular dystrophy syndromes such as Duchenne, Becker, myotonic, facioscapulohumeral, Emery-Dreyfus, oculopharyngeal, scapulohumeral, limb-girdle, Fukuyama, congenital muscular dystrophies, or genetic distal myopathies. In some instances, the myopathy is caused by myotonic dystrophy (e.g., myotonic dystrophy type 1 or DM1). In some instances, the myopathy is caused by DM1.

[0332] Motor neuron diseases (MNDs) encompass neurological disorders that affect motor neurons, the cells that control the body's voluntary muscles. Exemplary motor neuron diseases include, but are not limited to, adult motor neuron disease, infantile spinal muscular atrophy, amyotrophic lateral sclerosis, juvenile spinal muscular atrophy, autoimmune motor neuropathy due to multifocal conduction block, paralysis due to stroke or spinal cord injury, or skeletal immobilization due to trauma.

[0333] Diabetes mellitus (DM) includes type 1 diabetes, type 2 diabetes, type 3 diabetes, type 4 diabetes, double diabetes, latent autoimmune diabetes (LAD), gestational diabetes, neonatal diabetes mellitus (NDM), maturity-onset diabetes of the young (MODY), Wolfram syndrome, Alström syndrome, prediabetes, and diabetes insipidus. Type 2 diabetes, also known as non-insulin-dependent diabetes, is the most common type of diabetes, accounting for 95% of all diabetes cases. In some instances, type 2 diabetes is caused by a combination of factors, including insulin resistance due to impaired pancreatic beta cell function, resulting in hypertensive glucose levels. In some cases, increased glucagon levels stimulate the liver to produce abnormal amounts of unnecessary glucose, which contributes to hypertensive glucose levels. Type 1 diabetes, also known as insulin-dependent diabetes, accounts for approximately 5% to 10% of all diabetes cases. Type 1 diabetes is an autoimmune disease in which T cells attack and destroy insulin-producing beta cells in the pancreas. In some embodiments, type 1 diabetes is caused by genetic and environmental factors. Type 4 diabetes is a type of diabetes that affects approximately 20% of diabetic patients over the age of 65. In some embodiments, type 4 diabetes is characterized by age-related insulin resistance. Type 3 diabetes is used as a term for Alzheimer's disease, which is caused by insulin resistance in the brain.

[0334] Chronic obstructive pulmonary disease (COPD) is a type of obstructive lung disease characterized by long-term breathing problems and poor airflow. Chronic bronchitis and emphysema are two different types of COPD.

[0335] Liver disease (or liver disease) includes fibrosis, cirrhosis, hepatitis, alcoholic liver disease, fatty liver, genetic disease, or primary liver cancer.

[0336] Congestive heart failure is a condition in which the heart cannot pump enough blood and oxygen to the body's tissues.

[0337] Chronic renal failure or chronic kidney disease is a condition characterized by the gradual loss of kidney function over time.

[0338] In some embodiments, chronic infections such as AIDS also cause muscle wasting.

[0339] Sepsis is an immune response to infection that leads to tissue damage, organ dysfunction, and / or death.

[0340] Fasting is the voluntary abstinence or reduction of some or all food, drink, or both for a specified period of time.

[0341] Sarcopenia is a continuing process of muscle atrophy during normal aging, characterized by a gradual loss of muscle mass and strength over months to years, which, as used herein, refers to an aging process that is not affected or accelerated by the presence of disorders and diseases that promote skeletal muscle neurodegeneration.

[0342] In some instances, treatment with glucocorticoids further causes muscle atrophy. Exemplary glucocorticoids include, but are not limited to, cortisol, dexamethasone, betamethasone, prednisone, methylprednisolone, and prednisolone.

[0343] Disuse-related muscle atrophy occurs as a result of limb immobilization (e.g., due to limb or joint fracture or orthopedic surgery such as hip or knee replacement). As used herein, "immobilization" or "immobilized" refers to partial or complete restriction of movement of a limb, muscle, bone, tendon, joint, or other body part for an extended period of time (e.g., 2, 3, 4, 5, 6 days, 1, 2 weeks, or more). In some instances, the period of immobilization includes short or brief periods of unrestrained movement, such as bathing, changing an external device, or adjusting an external device. Limb immobilization can be achieved by any of a variety of external devices, including, but not limited to, braces, slings, casts, bandages, and splints (any of which may be optionally constructed of hard or soft materials, including, but not limited to, cloth, gauze, fiberglass, plastic, plaster, or metal), and any of a variety of internal devices, including surgically implanted splints, plates, braces, and the like. In the context of limb immobilization, the restriction of movement may be at a single or multiple joints (e.g., a single joint such as the shoulder or hip, a compound joint such as the radiocarpal joint, and a compound joint such as the knee, including but not limited to one or more of the following: hand joints, shoulder joints, elbow joints, wrist joints, accessory joints, sternoclavicular joints, vertebral joints, temporomandibular joints, sacroiliac joints, hip joints, knee joints, and foot joints), a single tendon or ligament or multiple tendons or ligaments (e.g., including but not limited to one or more of the following: anterior cruciate ligament, posterior cruciate ligament, rotator cuff tendons, medial collateral ligaments of the elbow and knee, flexor tendons of the hand, lateral ligament of the ankle, and tendons and ligaments of the jaw or temporomandibular joint), a single It may include a bone or bones (e.g., including but not limited to, one or more of the following: skull, mandible, clavicle, ribs, radius, ulna, humerus, pelvis, sacrum, femur, patella, phalanges, carpals, metacarpals, tarsals, metatarsals, fibula, tibia, scapula, and vertebrae), a muscle or muscles (e.g., including but not limited to, one or more of the following: latissimus dorsi, trapezius, deltoid, pectoralis, biceps, triceps, external oblique, abdominal muscles, gluteus maximus, thigh flexors, quadriceps, gastrocnemius, and diaphragm), a limb or limbs, one or more of an arm and a leg, or the entire skeletal muscular system or a portion thereof (e.g., in the case of a full body cast or spica cast).

[0344] Myotonic dystrophy is a multisystemic neuromuscular disease that includes two major types: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is caused by a dominantly inherited "CTG" repeat expansion in the gene DM protein kinase (DMPK), which, when transcribed into mRNA, forms a hairpin that binds with high affinity to the Muscleblind-like (MBNL) family of proteins. MBNL proteins are involved in post-transcriptional splicing, and loss of polyadenylation site regulation and MBNL protein function leads to the accumulation of downstream nuclear foci, increased mis-splicing events, and subsequent myotonicity and other clinical symptoms.

[0345] In some embodiments, methods for treating diseases or disorders characterized by mis-spliced mRNA are described herein. In some embodiments, the anti-transferrin receptor antibody described herein delivers a polynucleic acid molecule to the site of mis-spliced mRNA transcript to induce exon skipping or exon inclusion.

[0346] In some examples, diseases or disorders resulting from improperly spliced or partially spliced mRNA include, but are not limited to, neuromuscular diseases, genetic diseases, cancer, inherited diseases, or cardiovascular diseases.

[0347] In some examples, the genetic disease or disorder comprises an autosomal dominant disorder, an autosomal recessive disorder, an X-linked dominant disorder, an X-linked recessive disorder, a Y-linked disorder, a mitochondrial genetic disorder, or a multifactorial or polygenic disorder.

[0348] In some instances, cardiovascular diseases such as hypercholesterolemia are caused by improperly spliced or partially spliced mRNA. In hypercholesterolemia, a single nucleotide polymorphism in exon 12 of the low-density lipoprotein receptor (LDLR) has been shown to promote exon skipping.

[0349] In some instances, improperly spliced or partially spliced mRNA causes cancer. For example, improperly spliced or partially spliced mRNA affects cellular processes involved in cancer, including, but not limited to, proliferation, motility, and drug response. In some instances, the cancer is a solid cancer or a blood cancer. In some instances, the cancer is bladder cancer, lung cancer, brain cancer, melanoma, breast cancer, non-Hodgkin's lymphoma, cervical cancer, ovarian cancer, colon cancer, pancreatic cancer, esophageal cancer, prostate cancer, kidney cancer, skin cancer, leukemia, thyroid cancer, liver cancer, or uterine cancer.

[0350] In some cases, improperly spliced or partially spliced mRNA causes neuromuscular diseases or disorders. Exemplary neuromuscular diseases include muscular dystrophies such as Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy. In some cases, muscular dystrophy is genetic. In some cases, muscular dystrophy is caused by spontaneous mutations. Becker muscular dystrophy and Duchenne muscular dystrophy have been shown to be associated with mutations in the DMD gene, which encodes the protein dystrophin. Facioscapulohumeral muscular dystrophy has been shown to be associated with mutations in the double homeobox 4 (DUX4) gene.

[0351] In some instances, improperly spliced or partially spliced mRNA causes Duchenne muscular dystrophy, which causes severe muscle weakness and is caused by a mutation in the DMD gene that abolishes the production of functional dystrophin. In some instances, Duchenne muscular dystrophy is the result of an exon mutation in the DMD gene. In some instances, Duchenne muscular dystrophy is the result of a mutation in at least one of exons 1, 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, and 79 in the DMD gene. In some instances, Duchenne muscular dystrophy is the result of a mutation in at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 in the DMD gene. In some instances, Duchenne muscular dystrophy is the result of a mutation in at least one of exons 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, and 55 in the DMD gene. In some instances, multiple exons are mutated. For example, mutations in exons 48-50 are common in patients with Duchenne muscular dystrophy. In some instances, Duchenne muscular dystrophy is the result of a mutation in exon 51. In some instances, Duchenne muscular dystrophy is the result of a mutation in exon 23. In some instances, the mutation comprises a deletion of one or more exons. In some instances, the mutation comprises a duplication of one or more exons.In some cases, the mutations involve exonic point mutations, for example, some patients have been shown to harbor nonsense point mutations in exon 51 of the DMD gene.

[0352] Pharmaceutical preparations In some embodiments, the pharmaceutical formulations described herein are administered to a subject by multiple routes of administration, including, but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal routes of administration. In some examples, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, intracerebral, intraventricular, or intracranial) administration. In other examples, the pharmaceutical compositions described herein are formulated for oral administration. In yet other examples, the pharmaceutical compositions described herein are formulated for nasal administration.

[0353] In some embodiments, pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolve formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsed release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and combined immediate and controlled release formulations.

[0354] In some examples, the pharmaceutical formulation comprises a multiparticulate formulation. In some examples, the pharmaceutical formulation comprises a nanoparticle formulation. In some examples, the nanoparticle comprises cMAP, cyclodextrin, or lipid. In some cases, the nanoparticle comprises solid lipid nanoparticles, polymeric nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micellar solutions. Further exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as those with covalently bound metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In some examples, the nanoparticles are metal nanoparticles, such as nanoparticles of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium, lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium, potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, and combinations, alloys, or oxides thereof.

[0355] In some examples, the nanoparticles comprise a core or a core and a shell, such as core-shell nanoparticles.

[0356] In some examples, the nanoparticles are further coated with molecules for attachment of functional elements (e.g., to one or more of the polynucleic acid molecules or binding moieties described herein). In some examples, the coating comprises chondroitin sulfate, dextran sulfate, carboxymethyldextran, alginic acid, pectin, carrageenan, fucoidan, agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronic acid, glucosamine, galactosamine, chitin (or chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine, histone, protamine, ovalbumin, or dextrin or cyclodextrin. In some examples, the nanoparticles comprise graphene-coated nanoparticles.

[0357] In some cases, the nanoparticles have at least one dimension that is less than about 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.

[0358] In some examples, the nanoparticle formulation comprises a paramagnetic nanoparticle, a superparamagnetic nanoparticle, a metal nanoparticle, a fullerene-like material, an inorganic nanotube, a dendrimer (such as one with a covalently bound metal chelate), a nanofiber, a nanohorn, a nanoonion, a nanorod, a nanorope, or a quantum dot. In some examples, the polynucleic acid molecule or binding moiety described herein is directly or indirectly conjugated to the nanoparticle. In some examples, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more polynucleic acid molecules or binding moieties described herein are directly or indirectly conjugated to the nanoparticle.

[0359] In some embodiments, the pharmaceutical preparation comprises a delivery vector, for example, a recombinant vector for delivering a polynucleic acid molecule to a cell. In some examples, the recombinant vector is a DNA plasmid. In other examples, the recombinant vector is a viral vector. Exemplary viral vectors include vectors derived from adeno-associated virus, retrovirus, adenovirus, or alphavirus. In some examples, the recombinant vector capable of expressing a polynucleic acid molecule provides stable expression in target cells. In further examples, viral vectors are used that provide transient expression of a polynucleic acid molecule.

[0360] In some embodiments, pharmaceutical formulations include a carrier or carrier material selected based on compatibility with the compositions disclosed herein and the release profile characteristics of the desired dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, etc. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholate, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, etc. See, for example, Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980, and Pharmaceutical Dosage Forms and See Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0361] In some examples, the pharmaceutical formulation further comprises a pH adjusting or buffering agent, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trishydroxymethylaminomethane, and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0362] In some instances, the pharmaceutical formulation includes one or more salts in an amount necessary to bring the osmolality of the composition into an acceptable range. Such salts include sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0363] Treatment regimen In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic use. In some embodiments, the pharmaceutical compositions are administered once a day, twice a day, three times a day, or more. The pharmaceutical compositions are administered daily, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month, or more. The pharmaceutical compositions are administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

[0364] In some embodiments, one or more pharmaceutical compositions are administered simultaneously, sequentially, or at regular time intervals. In some embodiments, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered sequentially. In further cases, one or more pharmaceutical compositions are administered at regular time intervals (e.g., a first administration of a first pharmaceutical composition is on day 1, followed by at least 1, 2, 3, 4, 5, or more days before administration of at least a second pharmaceutical composition).

[0365] In some embodiments, two or more different pharmaceutical compositions are co-administered. In some instances, two or more different pharmaceutical compositions are co-administered simultaneously. In some cases, two or more different pharmaceutical compositions are co-administered sequentially without any time gap between administrations. In other cases, two or more different pharmaceutical compositions are co-administered sequentially with about 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, or 2 days gap between administrations.

[0366] If the patient's condition improves, at the physician's discretion, administration of the composition may continue; alternatively, the administered dose of the composition may be temporarily reduced or temporarily discontinued for a specified period of time (i.e., a "drug holiday"). In some examples, the length of the drug holiday may vary from 2 days to 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dose reductions for drug holidays can be 10% to 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0367] Once the patient's condition has improved, a maintenance dose is administered if necessary, after which the dosage and / or frequency of administration can be reduced, depending on the symptoms, to a level at which the improved disease, disorder, or condition is maintained.

[0368] In some embodiments, the amount of a given agent corresponding to such an amount will vary depending on factors such as the particular compound, the severity of the disease, the identity (e.g., body weight) of the subject or host requiring treatment, but is nevertheless routinely determined by techniques known in the art according to the particular circumstances surrounding the case, including, for example, the particular agent being administered, the route of administration, and the subject or host being treated. In some instances, the desired dose is conveniently presented as a single dose, or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, e.g., two, three, four or more sub-doses per day.

[0369] Because of the large number of variables associated with any particular treatment regimen, the foregoing ranges are only suggestive, and significant deviations from these recommendations are not uncommon. Such dosages will vary depending on many variables, including, but not limited to, the activity of the compound being used, the disease or condition being treated, the mode of administration, the requirements of the particular subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0370] In some embodiments, the toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Compounds that exhibit high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are used to formulate a range of dosages for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage will vary within this range depending on the dosage form used and the route of administration utilized.

[0371] Kits / Products In certain embodiments, the present specification discloses kits and products that can be used with one or more compositions and methods described herein. Such kits include a carrier, packaging, or container that is partitioned to accommodate one or more containers, such as vials, tubes, etc., each of which contains one of the components used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the container is made of various materials, such as glass or plastic.

[0372] The products provided herein include packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.

[0373] For example, a container may contain an anti-transferrin receptor antibody and, optionally, one or more target nucleic acid molecules described herein. Such kits optionally include an identifying description or label or instructions for use in the methods described herein.

[0374] Kits typically include a label listing the contents and / or instructions for use and a package insert with instructions for use. A set of instructions is also usually included.

[0375] In one embodiment, the label is on or associated with the container. In one embodiment, a label is on a container if letters, numbers, or other indicia forming the label are affixed, molded, or engraved into the container itself. A label is associated with a container if it is present in a receptacle or carrier that holds the container, for example, as a package insert. In one embodiment, the label is used to indicate that the contents are to be used for a particular therapeutic application. The label also indicates how to use the contents, for example, in the methods described herein.

[0376] In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device containing one or more unit dosage forms comprising a compound provided herein. The pack comprises, for example, metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser also carries a notice attached to the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the drug form for administration to humans or animals. Such notice may be, for example, labeling approved by the U.S. Food and Drug Administration for prescription drugs or approved package inserts. In one embodiment, compositions comprising a compound provided herein formulated with a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated disease.

[0377] Specific Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of any subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the word "including," as well as other forms such as "include," "includes," and "included," is non-limiting.

[0378] As used herein, ranges and amounts can be expressed as "about" a particular value or range. "About" includes the exact amount. Thus, "about 5 μL" also means "about 5 μL" and "5 μL." In general, the term "about" includes an amount that would be expected to be within experimental error.

[0379] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0380] "Antibodies" and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. The terms are used interchangeably. In some instances, the antigen specificity of an immunoglobulin is known.

[0381] The term "antibody" is used in the broadest sense and includes fully assembled antibodies, antibody fragments capable of binding antigen (e.g., Fab, F(ab'), Fv, single-chain antibodies, diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, humanized antibodies, etc.), and recombinant peptides including the foregoing.

[0382] As used herein, the terms "monoclonal antibody" and "mAb" refer to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0383] "Native antibodies" and "native immunoglobulins" are typically heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end, with the light-chain constant domain aligned with the first constant domain of the heavy chain and the light-chain variable domain aligned with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.

[0384] As used herein, the term "variable" refers to the fact that certain portions of the variable domains differ significantly in sequence among antibodies. The variable regions confer antigen-binding specificity. However, variability is not uniformly distributed throughout the variable domains of antibodies. The variability is concentrated in three regions called complementarity-determining regions (CDRs) or hypervariable regions, both in the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework (FR) regions. Natural heavy- and light-chain variable domains each contain four FR regions, most of which adopt a β-pleated sheet configuration, connected by three CDRs that form loops connecting, and in some cases, forming part of, the β-pleated sheet configuration. The CDRs in each chain are held together in close proximity by the FR regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al. (1991) NIH PubL. No. 91-3242, Vol. I, pages 647-669). The constant domains are not directly involved in binding an antibody to an antigen, but exhibit various effector functions, such as Fc receptor (FcR) binding, participation of the antibody in antibody-dependent cellular cytotoxicity, induction of complement-dependent cytotoxicity, and mast cell degranulation.

[0385] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs" (i.e., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) and / or from the "hypervariable loops" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Clothia and Lesk, (1987) J. Mol. Biol., 196:901-917). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as considered herein.

[0386] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab, F(ab')2, and Fv fragments, diabodies, linear antibodies (Zapata et al. (1995) Protein Eng. 10:1057-1062), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and an "Fc" fragment, the name of which reflects its ability to readily crystallize. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0387] "Fv" is a small antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. H -V L It is in this structure that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv, which contains only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

[0388] The Fab fragment contains the constant domain of the light chain and the first constant domain of the heavy chain (C H1 Fab fragments also contain heavy chain C fragments containing one or more cysteines from the antibody hinge region. H1 It differs from Fab' fragments by the addition of a few residues at the carboxy terminus of the domains. Fab'-SH is the designation used herein for Fab' in which the cysteine residues of the constant domains bear a free thiol group. Fab' fragments are produced by reduction of the heavy chain disulfide bridges of the F(ab')2 fragment. Other chemical couplings of antibody fragments are also known.

[0389] The "light chains" of antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0390] Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, IgM, and IgY. Some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of various classes of immunoglobulins are well known. Different isotypes have different effector functions. For example, human IgG1 and IgG3 isotypes have ADCC (antibody-dependent cell-mediated cytotoxicity) activity.

[0391] In some examples, the CDRs of the antibody are numbered according to (i) the Kabat numbering system (Kabat et al. (197) Ann. NY Acad. Sci. 190:382-391 and, Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), or (ii) the Chothia numbering scheme (e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948; Chothia et al., 1992, J. Mol. Biol., 227:799-817; Tramontano A et al., 1990, J. Mol. Biol. 215(1): 175-82; and US Patent No. 7,709,226); or (iii) the ImMunoGeneTics (IMGT) numbering system, as described, for example, in Lefranc, M.-P., 1999, The Immunologist, 7: 132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212 (“IMGT CDRs”); or (iv) as determined according to MacCallum et al., 1996, J. Mol. Biol., 262:732-745. See, e.g., Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

[0392] With respect to the Kabat numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acid positions 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3), which can optionally include one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme). Using the Kabat numbering system, the CDRs in an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3). As is well known to those skilled in the art, using the Kabat numbering system, the actual linear amino acid sequence of an antibody variable domain can contain fewer or additional amino acids due to shortening or lengthening of FRs and / or CDRs, and therefore the Kabat number of an amino acid is not necessarily the same as its linear amino acid number.

[0393] With respect to the Chotia numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acid positions 26-31 (CDR1), 52-56 (CDR2), and 95-102 (CDR3), which can optionally include one or two additional amino acids after 31 (referred to as 31A and 31B in the Chotia numbering scheme). Using the Chotia numbering system, the CDRs in an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3). As is well known to those skilled in the art, using the Chotia numbering system, the actual linear amino acid sequence of an antibody variable domain can contain fewer or additional amino acids due to shortening or lengthening of FRs and / or CDRs, and therefore the Chotia number of an amino acid is not necessarily the same as its linear amino acid number.

[0394] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0395] The term "humanized antibody" refers to an antibody in which the framework or CDRs have been modified to constitute an immunoglobulin CDR with different specificity compared to the CDRs of the parent immunoglobulin.

[0396] As used herein, the terms "individual," "subject," and "patient" refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms are limited to situations characterized by the supervision (e.g., full-time or intermittent) of a health care professional (e.g., a physician, registered nurse, bedside nurse, physician assistant, nursing assistant, or hospice worker). [Example]

[0397] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein. [Example]

[0398] Production and characterization of humanized anti-TfR antibodies Nucleic acids encoding exemplary anti-TfR antibodies were stably transfected into CHOK1SV GSKO cells, generating three stable pools per product. The stable pools were monitored for cell growth and Protein A titer from day 8 onwards post-transfection. Once the cultures reached a viability threshold of 70% and a titer of 0.6 x 10e 6 Stable pools were passaged when they reached a cell viability of >97%. The highest producing pool tested was used to grow 600 mL fed-batch overgrow cultures (FOG) at 0.2 x 10e cells / mL per product. 6Cells were plated at 1000 cells / mL. FOG cultures were fed on days 4 and 8 and harvested on day 11 by centrifugation and filter sterilization. Sterile cell culture supernatant was purified by Protein A purification using 3 x 5 ml MabSelectSuRE columns in parallel on an AKTA purifier (running at 10 mL / min). The columns were equilibrated with 50 mM sodium phosphate, 250 mM sodium chloride, pH 7.0, washed with 50 mM sodium phosphate and 1 M sodium chloride, pH 7.0, and eluted with 10 mM sodium formate, pH 3.5. The eluted fractions were neutralized by diluting 1:2 with 2 x PBS, and then the pH was adjusted to 7.4 with diluted NaOH.

[0399] Antibodies were analyzed by SE-HPLC and SDS-PAGE. Duplicate samples were analyzed by SE-HPLC using a Zorbax GF-250 9.4 mm ID x 25 cm column (Agilent). An 80 μl aliquot of 1 mg / ml sample was injected and run at 1 ml / min for 15 minutes in 50 mM sodium phosphate, 150 mM sodium chloride, 500 mM L-arginine, pH 6.0. All variants showed a small peak (<16.89%) with a retention time of approximately 7.66 minutes, consistent with soluble aggregates. Soluble aggregate concentrations were analyzed using Empower v3 software.

[0400] Table 9 illustrates the construct design and HPLC analysis of the tested anti-TfR antibodies.

[0401] [Table 9]

[0402] The binding kinetics of nine exemplary humanized anti-TfR antibodies and the parent chimeric antibody were characterized. Tests were performed on a BioRad ProteOn XPR36 optical biosensor using a Protein A-coated GLM sensor chip for mAb capture. The running buffer contained 10 mM HEPES, 150 mM NaCl, pH 7.4, 0.05% Tween-20, and 0.2 mg / ml BSA. Data were collected at 25°C. Based on the resulting stock concentrations, all mAbs were diluted to 2 μg / ml in running buffer. Each was then captured on the Protein A surface for 40 seconds.

[0403] hTfR (100 μg) was dissolved in 300 μL of water to give a stock concentration of 4.3 μM. hTfR was diluted to 43 nM as the highest concentration and tested in 3-fold serial dilutions. hTfR was injected at 200 μl / min over 2 minutes, followed by a 1-hour dissociation phase.

[0404] Response data were processed by subtracting data from an internal spot control surface and additionally double-referencing with buffer injections.

[0405] Table 10 shows the binding constants determined at 25°C.

[0406] [Table 10] [Example]

[0407] In vivo gene downregulation using hIgG2 TfR1 chimeric antibody-siRNA (SSB) conjugates

[0408] The CDR of a mouse IgG2 antibody against hTfR1 was subcloned into a human IgG2 background and transfected into CHO-K1SP cells. (See Example 4 for sequences.) Stable cell pools were selected and seeded in Dynamis medium (GIBCO) in cellbags (Healthcare) at 37°C and 5% CO2 using a Wave Bioreactor (GE Healthcare). Starting on day 4, cells were fed at 8% of the final culture volume (25 liters) every two days for a total of 14 days of incubation. The culture supernatant was harvested, depth filtered, and purified using Monofinity A Resin (GenScript) at a flow rate of 30 ml / min. The eluted protein buffer was placed in PBS, and the molecular weight and purity of the purified protein were analyzed by SDS-PAGE under reducing and non-reducing conditions and SEC-HPLC. The final protein purity was greater than 98%.

[0409] Conjugation of TfR1-IgG2 mAb chimera with SSB siRNA using a bis-maleimide (BisMal) linker

[0410] The conjugate used in this experiment was an SSB siRNA duplex. The sequence of the 21-mer SSB guide / antisense strand was (5'-3') UUACAUUAAAGUCUGUUGUUU. The single strands were fully assembled on a solid phase using standard phosphoramidite chemistry and purified using HPLC. Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize duplex potency and reduce immunogenicity. The siRNA passenger strand contained a C6-NH2 conjugation handle at the 5' end (see Figure 1). The siRNA duplex was designed as a blunt-end duplex with 19 bases of complementarity and one 3' dinucleotide overhang. The conjugation handle was connected to the siRNA passenger strand via a phosphodiester on the terminal base (see Figure 2).

[0411] Step 1: Antibody reduction with TCEP

[0412] The antibody was buffer exchanged into 25 mM borate buffer (pH 8) containing 1 mM DTPA to a maximum concentration of 10 mg / ml. To this solution, 4 equivalents of TCEP in the same borate buffer were added and incubated at 37°C for 2 hours. The resulting reaction mixture was combined with a solution of BisMal-siRNA (1.25 equivalents) in 10 mM acetate buffer, pH 6.0, at room temperature (RT) and stored at 4°C overnight. Analysis of the reaction mixture by analytical SAX column chromatography revealed the antibody-siRNA conjugate along with unreacted antibody and siRNA. The reaction mixture was treated with 10 eq of N-ethylmaleimide (10 mg / mL in DMSO) to cap any remaining free cysteine residues.

[0413] Step 2: Purification

[0414] The crude reaction mixture was purified by AKTA Pure FPLC using anion exchange chromatography (SAX) method 1. The fraction containing the DAR1 antibody-siRNA conjugate was isolated, concentrated, and buffer exchanged with PBS, pH 7.4.

[0415] Anion Exchange Chromatography Method (SAX)-1

[0416] Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5mm ID x 15cm, 13μm

[0417] Solvent A: 20 mM Tris buffer, pH 8.0; Solvent B: 20 mM Tris, 1.5 M NaCl, pH 8.0; Flow rate: 6.0 mL / min.

[0418] gradient: a) %A %B column volume b) 100 0 1 c)811 9 0.5 d) 50 50 13 e) 40 60 0.5 f)0 100 0.5 g)100 0 2

[0419] Strong Anion Exchange Chromatography (SAX) Method-2

[0420] Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4 x 250 mm

[0421] Solvent A: 80% 10 mM Tris pH 8, 20% ethanol, Solvent B: 80% 10 mM Tris pH 8, 20% ethanol, 1.5 M NaCl, Flow rate: 0.75 mL / min:

[0422] gradient: a) Time %A %B b).0 90 10 c) 3.00 90 10 d) 11.00 40 60 e)14.00 40 60 f)15.00 20 80 g) 16.00 90 10 h)20.00 90 10

[0423] The purity of the conjugate was assessed by analytical HPLC using SAX method-2 (Table 11).

[0424] [Table 11]

[0425] Analytical data table for the conjugates used in this example: HPLC retention time (RT) in minutes, and % purity by chromatographic peak area.

[0426] In vitro activity of hTfR1-IgG2 mAb siRNA DAR1 conjugate

[0427] The binding ability of the hTfR1-IgG2 mAb siRNA conjugate to human and cynomolgus monkey TfR1 was evaluated using an ELISA assay. Half-well high-binding 96-well plates (Costar #3690) were coated with recombinant human transferrin receptor protein (Sino Biological 11020-H07H) or recombinant cynomolgus monkey transferrin receptor protein (Sino Biological 90253-C07H) at 1 ng / μL in PBS (Gibco 14190) and incubated overnight at 4°C. The plates were washed four times with 100 μL of Tris-buffered saline + Tween (20xTBST, Cell Signaling 9997S). 100 μL of Superblock (ThermoFisher PI-37535) was added to each well and incubated for 1 hour at room temperature. The wash step was repeated before adding samples. Samples were added at a maximum concentration of 10 nM, 50 μL / well. The plate was incubated for another hour at room temperature, and the wash step was repeated. The secondary antibody (Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson Immunoresearch, 109-035-098) was diluted 1:5000 in the added Superblock and 50 μL / well. The plate was incubated for 1 hour at room temperature and washed once more. Binding was measured by adding 50 μL of 1-Step™ Ultra TMB-ELISA (ThermoFisher, 34028), followed by a 5-minute incubation and the reaction was stopped by adding 25 μL of Stop Solution 2N sulfuric acid (R&D Systems DY994). Absorbance was measured at 450 nm, with the 570 nm reference wavelength subtracted. Binding constants were determined using GraphPad Prism Specific Binding with Hill Slope.

[0428] The unconjugated and conjugated hTfR1.IgG2 mAb antibodies bind to recombinant human and cynomolgus TfR1 with similar affinity (FIGS. 3A-B).

[0429] The ability of the TfR1.IgG2 mAb-SSB conjugate to downregulate SSB expression was monitored in HEL92.1.7 and human skeletal muscle cells. HEL92.1.7 cells (ATCC® TIB-180™) were cultured in RPMI 1640 containing 10% fetal bovine serum (Nucleus Biologics FBS1824). Cells were diluted to 100,000 / mL, and 100 μL was added to each well of a plate. The antibody conjugate was diluted to a maximum concentration of 100 nM. As a negative control, 20 μL of the conjugate or PBS was added to multiple wells of a 96-well plate, and the treated cells were incubated at 37°C and 5% CO2 for 72 hours.

[0430] Immortalized human skeletal muscle cells (Institute of Myology, Paris) were seeded in 500 μl of skeletal muscle cell growth medium (PromoCell C-23260) onto 24-well collagen plates (Thermo Fisher A1142802) and incubated at 37°C in 5% CO2 until myoblasts reached confluence. At this point, myotube differentiation was induced by incubation in 500 μl of differentiation medium (DMEM (Gibco 10566-016) supplemented with 10 μg / ml insulin and 50 μg / ml gentamicin) for 4 days. The medium was refreshed, and 50 μl of TfR1.IgG2 mAb-SSB conjugate diluted in PBS was added. Treated cells were incubated for 72 hours. To harvest and analyze either cell type, the medium was removed from the wells and 150 μL of Trizol (Ambion 15596018) was added. Plates were frozen at -80°C overnight or longer before analysis. RNA was isolated and quantified spectrophotometrically using the Direct-zol 96 RNA kit according to the manufacturer's instructions. RNA (100 = 200 ng) was reverse transcribed using the High Capacity cDNA kit (Thermo Fisher #4368813) according to the manufacturer's instructions. mRNA concentrations were quantified using TaqMan qPCR with appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control. ΔΔC t The percentage of mRNA was calculated using the method, and cells treated with PBS were set to 100% expression. In these experiments, the TfR1.IgG2 mAb-SSB conjugate downregulated SSB by up to 60%, while SSB was downregulated by up to 25% in cells treated with the TfR1.IgG2 mAb-MSTN conjugate (negative control) (Figures 4A-B).

[0431] Activity and safety of hTfR1-IgG2 mAb SSB siRNA conjugate in cynomolgus monkeys

[0432] The PK, PD, and safety characteristics of the hIgG2 TfR1.mAb-siSSB conjugate were evaluated in cynomolgus monkeys. Cynomolgus monkeys were 2-3 year old males weighing 2-3 kg. Cynomolgus monkeys were administered 30 mg / kg or 60 mg / kg (mAb concentration) of the conjugate or PBS via a 30-minute (+ / - 3 minute) intravenous (IV) infusion. Blood samples and muscle biopsies were collected from a peripheral vein in conscious, restrained animals or from the gastrocnemius and quadriceps muscles in sedated animals at different times as outlined in Table 12.

[0433] [Table 12]

[0434] The plasma concentration of the hIgG2 TfR1.mAb-siSSB conjugate was determined using a stem-loop qPCR assay. Briefly, plasma samples were diluted directly in TE buffer + 0.1% v / v Triton X-100. A standard curve was generated by spiking siRNA into the plasma of untreated animals and then serially diluting with TE buffer + 0.1% v / v Triton X-100. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) with 25 nM of a sequence-specific stem-loop RT primer. The cDNA obtained from the RT step was used for real-time PCR using TaqMan Fast Advanced Master Mix (Applied Biosystems) with 1.5 μM forward primer, 0.75 μM reverse primer, and 0.2 μM probe. The sequence of the SSB siRNA antisense strand, as well as all primers and probes used in the measurements, are listed in Table 13. Quantitative PCR reactions were performed using standard cycling conditions in a QuantStudio 7 Flex Real-Time PCR System (Life Technologies). Ct values were converted to plasma or tissue concentrations using the linear equation derived from the standard curve.

[0435] [Table 13]

[0436] The clearance and half-life of the conjugates are shown in Table 14. The PK properties of these conjugates were similar to those of the mouse anti-transferrin mAb conjugates tested in mice.

[0437] [Table 14]

[0438] To assess siRNA concentration and intramuscular conjugate activity, muscle biopsies (gastrocnemius and quadriceps) were obtained according to the schedule shown in Table 12. Muscle biopsies were collected via a 6 mm puncture, weighed, and flash-frozen in liquid nitrogen. Frozen tissue samples were homogenized in 1 ml of cold TRIZOl (supplier). To determine mRNA knockdown, total RNA was subtracted from the tissue and quantified spectrophotometrically using the Direct-zol 96 RNA kit according to the manufacturer's instructions. RNA (100 = 200 ng) was reverse transcribed using the High Capacity cDNA kit (Thermo Fisher #4368813) according to the manufacturer's instructions. SSB mRNA concentration was quantified using TaqMan qPCR with appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control. ΔΔC t The % mRNA was calculated using the method, and the SSB mRNA concentration of the same animal before treatment or the SSB concentration of PBS-treated animals was set to 100% expression.

[0439] Tissue siRNA enrichment was quantified using a stem-loop qPCR assay. Briefly, 15–50 mg of tissue fragments were homogenized in 500 μL of Trizol using a TissueLyser II plate-based homogenizer (Qiagen) and then diluted in TE buffer + 0.1% v / v Triton X-100. A standard curve was generated by spiking siRNA into homogenized tissue from untreated animals and serially diluting it in TE buffer + 0.1% v / v Triton X-100. The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) with 25 nM of a sequence-specific stem-loop RT primer. cDNA obtained from the RT step was used for real-time PCR using TaqMan Fast Advanced Master Mix (Applied Biosystems) with 1.5 μM forward primer, 0.75 μM reverse primer, and 0.2 μM probe. The sequence of the SSB siRNA antisense strand and all primers and probes used in the measurements are shown in Table 13. Quantitative PCR reactions were performed using standard cycle conditions in a QuantStudio 7 Flex Real-Time PCR System (Life Technologies). Ct values were converted to plasma or tissue concentrations using the linear equation obtained from the standard curve.

[0440] Treatment of cynomolgus monkeys with the conjugate resulted in up to 62% downregulation of SSB mRNA in the gastrocnemius muscle and up to 75% in the quadriceps muscle (Figures 5A and 5B). The siRNA concentrations in these tissues were dose-dependent, ranging from 0.6 to 1.9 nM and 2.0 to 6.5 nM at doses of 30 mg / kg and 60 mg / kg, respectively. Conjugate activity and siRNA concentrations in tissues were similar when examined 21 or 28 days after administration. These results demonstrate that the selected TfR1 antibody can effectively deliver siRNAs to monkey muscle tissue and that the activity of the transferrin receptor-targeting AOC translates across species.

[0441] To monitor the safety of selected anti-hTfR1 antibodies in primates, hematology and clinical chemistry analyses were performed according to the schedule shown in Table 1. With the exception of a dose-dependent but transient depletion of reticulocytes (Figure 6), no treatment-related effects were observed on the hematological or clinical chemistry parameters for up to 28 days after administration. The observed transient downregulation of reticulocytes was considered a side effect of the TfR1 antibody. We observed that a murine TfR1 antibody with intact agonist function or complement fixation ability severely depleted TfR-expressing reticulocytes (Daniels-Wells, et al., “Transferrin receptor 1: a target for antibody-mediated cancer therapy,” Immunotherapy 8(9):991–994 (2016)). Due to the low fraction of reticulocytes expressing high TfR1 levels in primates, the reticulocyte depletion was only transient and less pronounced than in rodents. Importantly, other studies have shown that it is possible to successfully suppress the ADCC / CDC activity of antibodies with mutations that eliminate this activity. [Example]

[0442] Generation, characterization, and humanization of a human / cynomolgus monkey cross-reactive anti-TfR1 antibody

[0443] Using modern in silico antibody humanization and deimmunization programs well described in the art, we designed 16 variants of the chimeric anti-transferrin 1 mAb tested in NHP in Example 1. See Table XYZ for variant sequences. As part of the design, we attempted to assess manufacturability by identifying high-risk post-translational modifications (PTMs) and, where feasible, removing them via amino acid substitutions as part of the humanized activity. We also attempted to assess immunogenicity risk, identifying high-risk epitopes and, where feasible, removing them. Further mutations were introduced into the Fc domain of the variants to remove effector function (ADCC and CDC). The 16 variants were then expressed in mammalian cell culture using techniques well described in the art and purified using protein A resin-based affinity chromatography. The mAb variants were then fully characterized, and siRNA conjugates were generated as described below.

[0444] Human and cynomolgus monkey TfR1 ELISA assays

[0445] The goal of these assays was to confirm that the 16 mutant human anti-TfR1 antibodies bind to both human and cynomolgus monkey TfR1. The human or cynomolgus monkey transferrin receptor ELISA assay protocols are described below.

[0446] Half a well of a high-binding 96-well plate (Costar #3690) was coated with recombinant human transferrin receptor protein (Sino Biological 11020-H07H) or recombinant cynomolgus monkey transferrin receptor protein (Sino Biological 90253-C07H) at 1 ng / μL in PBS (Gibco 14190) and incubated overnight at 4°C. The plate was washed four times with 100 μL of Tris-buffered saline + Tween (20xTBST, Cell Signaling 9997S). 100 μL of Superblock (ThermoFisher PI-37535) was added to each well and incubated for 1 hour at room temperature. The washing step was repeated before the addition of samples. Samples were added at concentrations up to 10 nM, 50 μL / well. The plate was incubated for an additional hour at room temperature, and the washing step was repeated. The secondary antibody (Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson Immunoresearch, 109-035-098) was diluted 1:5000 in the added Superblock and 50 μL / well. Plates were incubated at room temperature for 1 hour and washed once more. Binding was measured by adding 50 μL of 1-Step™ Ultra TMB-ELISA (ThermoFisher, 34028), followed by a 5-minute incubation and the addition of 25 μL of Stop Solution 2N sulfuric acid (R&D Systems DY994). Absorbance was measured at 450 nm and the 570 nm reference wavelength was subtracted. Binding constants were determined using GraphPad Prism Specific Binding with Hill Slope.

[0447] Figures 7 and 8 illustrate the binding results for cynomolgus monkey CD71 and human CD71, respectively.

[0448] Tf-TfR blocking ELISA assay

[0449] The goal of this assay was to confirm that TfR antibodies bind to TfR in the presence of holo-transferrin.

[0450] Antibodies were biotinylated using a 50-fold molar excess of EZ-Link No weigh NHS-Biotin (Thermo Scientific A39256) according to the manufacturer's instructions. Half-well high-binding plates (Costar #3690) were coated with 500 ng / ml purified human holo-transferrin (R&D Systems 2914-HT) in PBS overnight at 4°C. For comparison, plates were directly coated with hTfR. Plates were washed four times with 100 μL of Tris-buffered saline + Tween (20xTBST, Cell Signaling 9997S). 100 μL of Superblock (ThermoFisher PI-37535) was added to each well and incubated for 1 hour at room temperature. After repeating the wash step, hTfR (200 ng / mL in 25 μL) was added to the transferrin plate, or Superblock was added to the hTfR plate, and incubated for 30 minutes. Biotinylated antibodies were diluted to 20 nM for higher concentrations and added to the plate in 3-fold serial dilutions. 25 μl / well was added to the 25 μl already in the plate. The plate was incubated for 1 hour, and the wash step was repeated. Streptavidin-HRP (R&D Systems DY998) was added according to the dilution recommended in the package insert, followed by a final wash step. Binding was measured by adding 50 μL of 1-Step™ Ultra TMB-ELISA (ThermoFisher, 34028), followed by a 5-minute incubation and the addition of 25 μL of Stop Solution 2N sulfuric acid (R&D Systems DY994). Absorbance was measured at 450 nm, and the 570 nm reference wavelength was subtracted. Binding constants were determined using GraphPad Prism Specific Binding with Hill Slope. The change in antibody binding constant in the presence and absence of transferrin was considered for a commercially available antibody (AF2474, R&D Systems) known to have an epitope overlapping with transferrin (see Figures 9A-9B).

[0451] HFE-TfR binding ELISA assay

[0452] The goal of this assay was to confirm that the TfR antibody maintained binding when TfR bound to HFE.

[0453] This assay was performed following the same method as for TfR binding in the presence of transferrin, but using the cofactor HFE (hereditary hemochromatosis protein, mybiosource.com, MBS953891) instead of transferrin. See Figure 10A-B.

[0454] FcγRIIIA (CD16a) ELISA

[0455] The goal of this assay was to identify potential antibody ADCC activity by measuring binding to FcγRIIIA (CD16a) genotype V158. Half of a high-binding 96-well plate (Costar #3690) was coated with recombinant CD16a protein (Sino Biological 10389-H27H1) at 2 ng / μL in PBS (Gibco 14190) and incubated overnight at 4°C. The plate was washed four times with 100 μL of Tris-buffered saline + Tween (20xTBST, Cell Signaling 9997S). 100 μL of Superblock (ThermoFisher PI-37535) was added to each well and incubated for 1 hour at room temperature. The washing step was repeated before adding the samples. Samples were added at a concentration of up to 1 μM, 50 μL / well. The plate was incubated for an additional hour at room temperature, and the washing step was repeated. The secondary antibody (Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson Immunoresearch, 109-035-098) was diluted 1:5000 in the added Superblock and 50 μL / well. Plates were incubated at room temperature for 1 hour and washed once more. Binding was measured by adding 50 μL of 1-Step™ Ultra TMB-ELISA (ThermoFisher, 34028), incubated for 5 minutes, and stopped by adding 25 μL of Stop Solution 2N sulfuric acid (R&D Systems DY994). Absorbance was measured at 450 nm and the 570 nm reference wavelength was subtracted. Binding constants were determined using GraphPad Prism Specific Binding with Hill Slope. See Figure 11.

[0456] In vitro potency assay in HEL92.1.7 cells

[0457] The goal of this assay was to demonstrate the ability of TfR mAb conjugates to deliver siRNA and achieve gene-specific downregulation. The mAb variants were conjugated to active siRNA (SSB) or a scrambled control (Scr). The HEL92.1.7 cell line (ATCC® TIB-180™) was cultured in RPMI1640 (Gibco A10491) containing 10% fetal bovine serum (Nucleus Biologics FBS1824). The antibody-siRNA conjugate was diluted to a maximum dose of 100 nM. 20 μl of conjugate was added to wells of a 96-well plate. 20 μl of PBS was added to some wells as an additional negative control. Cells were diluted to 100,000 / mL, and 100 μL was added to each well of the plate. The cells were incubated at 37°C and 5% CO2 for 72 hours. Media was removed from wells and 150 μl of Trizol (Ambion 15596018) was added. Plates were frozen at -80°C overnight or longer before analysis. RNA was isolated using the Direct-zol 96 RNA Isolation Kit (Zymo Research R2056) according to the manufacturer's instructions. RNA was reverse transcribed using the High Capacity cDNA Kit (Applied Biosystems 4368814) according to the manufacturer's instructions, and qPCR was performed using Taqman Fast Advanced Master Mix (Applied Biosystems 4444558) with the SSB and PPIB Taqman probe sets (ThermoFisher Hs04187362_g1 and Hs00168719_m1). The % mRNA expression was calculated using the ΔΔCt method, with PBS-treated cells set to 100% expression.

[0458] DAR1 containing active siRNA (SSB) or inactive or scrambled siRNA (Scr) was generated and characterized as described in Example 2. In these conjugates, the SSB siRNA contained a Cy5 fluorescent tag conjugated at position 11 of the passenger strand on the ribose 2' hydroxyl. This was introduced during solid-phase synthesis and did not inhibit the activity of the guide strand but allowed for incorporation assays to be performed. The purity of the conjugates was assessed by analytical HPLC using ion-exchange chromatography method-2, and the chromatographic retention times and purities are listed in Table 15 below.

[0459] [Table 15]

[0460] Analytical data table for the conjugates used in this example: HPLC retention time (RT) in minutes, and % purity by chromatographic peak area.

[0461] Antibody-dependent cellular cytotoxicity (ADCC) mediated by TfR1 antibody and antibody-siRNA conjugate (ASC) in PBMCs

[0462] Studies in mice and nonhuman primates (NHPs) have demonstrated that antibodies that bind to mouse / cynomolgus TfR with agonist and / or complement fixation capabilities selectively deplete TfR-expressing reticulocytes. To confirm whether the variants had agonist function, ADCC assays were performed using peripheral blood mononuclear cells (PBMCs) from healthy human donors as effector cells.

[0463] material:

[0464] PBMCs from BUYPBMC.COM, lot#2010113378, with strong ADCC activity.

[0465] Target cells: HEL-92.1.7 (ATCC, #TIB-180), HEL (#JCRB0062)

[0466] Cytotoxicity LDH Kit, Pierce (ThermoFisher), #88953

[0467] Tissue culture medium with serum (complete medium) RPMI 1640 (Life Technologies) containing 10% heat-inactivated FBS (56°C for 30 min) and 2% L-glutamine

[0468] hIgG1 mAb variants

[0469] procedure:

[0470] Thaw PBMC cells in a 37°C water bath with gentle agitation. After thawing, add 1 mL of warm culture medium to the vial drop-wise over a 30-second period to allow the cells to adjust to the environmental change. Gradually add the cells to a 15- or 50-mL conical tube containing 9 mL of warm culture medium. Rinse the first vial with 1 mL of cell-containing medium to recover any cells attached to the sides. Add the rinse medium to the conical tube. Pellet the cells by centrifugation at 350 x g for 8-12 minutes. Discard the supernatant. Gently tap to resuspend the cell pellet (avoid excessive shearing). Rinse the cells again by adding 10 mL of warm culture medium to the conical tube. Pellet the cells by centrifugation at 350 x g for 8-12 minutes. Discard the supernatant from the second wash. Gently tap to resuspend the cell pellet (avoid excessive shearing). If necessary, resuspend the cells in 10 mL of warm culture medium. The cells are allowed to acclimate by overnight incubation at 37°C in a T75.

[0471] Harvest target cells HEL-92 and wash them twice with cold assay medium to ensure high viability. 4 x 10 cells were cultured on ice in a 96-well round-bottom plate. 4 Add 50 μl (8 x 10 mL) of cold 5) Target cells are plated in assay medium (RPMI-1640 containing 1% BSA and 100 units / mL penicillin and streptomycin). Dilutions (6-fold, starting at 10 μg / mL) of test and control antibody / ASC (10 μl) are added to the plates containing target cells as described in the table below, followed by incubation on ice for 30 minutes to allow opsonization. 10 μl of medium is added to control wells to maintain the volume.

[0472] Control:

[0473] - Background low control: Correct the spontaneous release from target cells (low control) with the control for spontaneous LDH release from target cells. Add the same number of target cells used in the experimental wells. Adjust the final volume to 100 μL / well with culture medium.

[0474] - Positive High Control: A target cell maximum LDH release control is required for calculations to determine 100% release of LDH. Add the same number of target cells used in the experimental wells. The final volume should be 100 μL / well (add 10 μL of 10X lysis buffer in step 5).

[0475] - Antibody-independent cellular cytotoxicity (AICC) is measured in wells containing target and effector cells without the addition of antibody.

[0476] The following two controls are used to monitor assay conditions but are not required for ADCC calculations.

[0477] - Correct the spontaneous release of LDH from effector cells with the effector cell spontaneous LDH release control. Add various numbers of effector cells to experimental wells. Adjust the final volume to 100 μL / well with culture medium.

[0478] - Culture medium background controls are required to correct for contributions from LDH activity that may be present in serum containing culture medium. 100 μL of culture medium is added to triplicate wells (without cells).

[0479] After 30 min of incubation on ice, 8 × 10 cells were cultured in 50 μl of warm assay medium (RPMI-1640 containing 1% BSA and 100 units / mL penicillin and streptomycin). 5 PBMC effector cells are added to each well to give a 20:1 ratio of effector:target cells and the plates are incubated at 37° C. for a further 4 hours.

[0480] 45 minutes before harvesting the supernatant, add 10 μL of lysis buffer (10X) to the target cell maximum LDH release control (positive control) and the volume correction control. Add 10 μL of PBS to the background low control containing cells, samples, and other controls. After incubation (350 g, 10 minutes), centrifuge the plate. Transfer 50 μL of supernatant to a 96-well clear flat-bottom plate, add 50 μL of reaction mixture to each sample well, and mix by tapping gently. Incubate the plate for 30 minutes at room temperature, protected from light. Add 50 μL of stop solution to each sample well and mix by tapping gently. Measure the absorbance at 490 nm and 680 nm. To determine LDH activity, subtract the absorbance at 680 nm (instrument background signal) from the absorbance at 490 nm.

[0481] The specific ADCC activity was calculated as follows.

[0482] %ADCC = 100 × ((A 490 (Sample)-A 490 (AICC)) / (A 490 (High control)-A 490 (Low control)

[0483] The results are shown in Figure 14.

[0484] Complement-dependent cytotoxicity (ADCC) mediated by TfR1 antibody and antibody-siRNA conjugate (ASC) in rabbit serum

[0485] material:

[0486] Lyophilized rabbit complement. Reconstitute with ice-cold distilled water. Stir gently to ensure all lyophilized material is dissolved. Use within 1 hour of reconstitution. Keep reconstituted material on ice at all times. Discard aliquot if not fully active at 1 / 2 dilution.

[0487] Target cell: HEL-92.1.7 (ATCC, #TIB-180)

[0488] Viobility 405 / 452 fixable dye.

[0489] Tissue culture medium with serum (complete medium) RPMI 1640 (Life Technologies) containing 10% heat-inactivated FBS (56°C for 30 min) and 2% L-glutamine

[0490] hIgG1 variants

[0491] procedure:

[0492] HEL92.1.7 target cells were harvested and washed twice with cold assay medium. 5 × 10 cells were plated in a 96-well round-bottom plate. 4 Cells were plated per well in 25 μl cold assay medium (RPMI-1640 containing 1% BSA and 100 units / mL penicillin and streptomycin). Dilutions (5-fold, starting from 100 μg / mL for a final concentration of 50 μg / mL) of test and control antibodies (25 μl) were added to the plates containing 25 μl of target cells, followed by incubation on ice for 30 minutes to allow opsonization.

[0493] Control:

[0494] - Low background control: Correct the spontaneous release from target cells (low control) with the control of spontaneous LDH release from target cells. Add the same number of target cells used in the experimental wells. Adjust the final volume to 100 μL / well with culture medium.

[0495] - Antibody-independent cellular cytotoxicity (AICC) is measured in wells containing target and CDC cells without the addition of antibody.

[0496] After 30 minutes of incubation, 50 μL of complement was added to each well, and the plates were incubated for an additional 60 minutes at 37°C, excluding the medium and low control (with 50 μL of medium). At the end of the incubation (350 g, 10 minutes), the plates were centrifuged. Diluted Viobility 405 / 452 dye (0.5 μL dye in 100 μL staining buffer) was added. The plates were incubated for 15 minutes at room temperature, protected from light. Cells were washed and fixed. Flow analysis was performed to measure dead cells.

[0497] Specific CDC activity is calculated as follows:

[0498] %CDC = % dead cells in sample - % dead cells in control

[0499] The results are shown in Figure 15.

[0500] In vitro uptake of human anti-TfR1 IgG1 siRNA conjugate (ASC) into human skeletal myotubes

[0501] To monitor ASC incorporation into muscle cells, primary human skeletal myoblasts (Thermo Fisher Scientific A11440) were seeded onto 24-well collagen plates in 1 mL of DMEM (ATCC 30-2002) supplemented with 10% FBS (Nucleus Biologics FBS1824) and 1xITS (Thermo Fisher Scientific 41400045). Cells were incubated at 37°C and 5% CO2 until myoblasts reached confluence. At this point, differentiation into myotubes was induced by incubating the cells in 1 mL of DMEM (ATCC30-2002) supplemented with 2% horse serum (ATCC 30-2040) and 1xITS (Thermo Fisher Scientific 41400045) for 2 days. The medium was replaced with 500 μl of differentiation medium, and 50 μl of TfR1.IgG2 mAb-SSB(Cy5) conjugate diluted in PBS was added to a final concentration of 1 and 10 nM. Cells were incubated at 37°C and 5% CO2 for 24 hours, washed three times with 500 μl of PBS, and lysed in 150 μl of T-PER lysis buffer (Thermo Fisher Scientific 78510) using freeze-thaw cycles. 75 μl of lysed cells were diluted with 75 μl of nuclease-free water, and fluorescence was determined using a TECAN plate reader (Ex 635 nM - Em 675 nM). Results are shown as fluorescence in cells relative to input (Figure 16).

[0502] In vitro gene downregulation mediated by human anti-TfR1 IgG1 siRNA conjugate (ASC) in human skeletal myotubes

[0503] To monitor the ability of the TfR1.mAb-SSB conjugate to downregulate SSB mRNA levels, primary human skeletal myoblasts (Thermo Fisher Scientific A11440) were seeded onto 24-well collagen plates (Thermo Fisher Scientific A1142802) in 1 mL of DMEM (ATCC30-2002) supplemented with 10% FBS (Nucleus Biologics FBS1824) and 1x ITS (Thermo Fisher Scientific 41400045). Cells were incubated at 37°C and 5% CO2 until myoblasts reached confluence. At this point, differentiation into myotubes was induced by incubating the cells in 1 mL of DMEM (ATCC30-2002) supplemented with 2% horse serum (ATCC 30-2040) and 1x ITS (Thermo Fisher Scientific 41400045) for 2 days. The medium was refreshed, and 100 μl of TfR1.IgG2 mAb-SSB conjugate diluted in PBS was added. Treated cells were incubated for 72 hours. For harvesting, the medium was removed from the wells, and 150 μl of Trizol (Ambion 15596018) was added. Plates were frozen at -80°C overnight or longer before analysis. RNA was isolated using the Direct-zol 96 RNA Kit according to the manufacturer's instructions and quantified spectrophotometrically. RNA (100 = 200 ng) was reverse transcribed using the High Capacity cDNA Kit (Thermo Fisher #4368813) according to the manufacturer's instructions. mRNA concentrations were quantified using TaqMan qPCR with appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control. The % mRNA was calculated using the ΔΔCt method, with PBS-treated cells set to 100% expression. All SSB siRNA conjugates tested downregulated SSB by 50% with similar potency (FIG. 17). [Example]

[0504] hTfR1 heavy chain: 461aa

[0505] NruI-Kozak sequence - artificial signal peptide - hTfR1 mAb HC variable region - human IgG2 constant region (P01859) - stop codon - PmlI

[0506] [ka]

[0507] hTfR1 light chain: 233aa

[0508] AscI-Kozak sequence - artificial signal peptide - hTfR1 mAb LC variable region - human Ig kappa constant region (P01859) - stop codon - FseI

[0509] [ka] [Example]

[0510] SSB siRNA knockdown of an exemplary anti-TfR antibody in a cynomolgus monkey study Treatment of cynomolgus monkeys with exemplary anti-TfR antibodies will be tested to determine the rate of SSB mRNA downregulation in the gastrocnemius muscle. Doses of 30 mg / kg, 10 mg / kg, and 3 mg / kg will be tested. Activity of the antibody conjugate will be investigated 21 and / or 28 days after administration. Safety in the cynomolgus monkeys will also be monitored by hematology and clinical chemistry analysis. [Example]

[0511] SSB conjugates of hIgG1 TfR-Var2ii and hIgG1 TfR-Var9ii do not affect absolute reticulocyte values in cynomolgus monkeys. hIgG1 TfR-Var2ii and hIgG1 TfR-Var9ii are humanized IgG1 antibodies targeting hTfR1, containing mutations in the hinge region of the IgG1 heavy chain designed to remove agonist function (LALA+L328R). In contrast to chimeric hIgG2 TfR1 antibodies, administration of SSB conjugates of hIgG1 TfR-Var2ii and hIgG1 TfR-Var9ii to cynomolgus monkeys did not reduce reticulocyte levels. This result is consistent with studies by others demonstrating that depletion of immature reticulocytes by TfR1-targeting antibodies can be successfully suppressed by mutations that remove the ADCC / CDC activity of the antibodies (WO2014 / 189973A2).

[0512] method:

[0513] On day 1, cynomolgus monkeys (male, 2-3 years old, BW 2-3 kg) were administered a 30-minute (+ / - 3 minute) intravenous (IV) infusion. Blood samples were collected from a peripheral vein in conscious, restrained animals at different time points after administration, as shown in Figure 18. [Example]

[0514] SSB conjugates of hIgG1 TfR-Var2ii Ab and hIgG1 TfR-Var9ii Ab down-regulate SSB RNA levels in muscle of cynomolgus monkeys. Compared to pre-treatment SSB mRNA levels, a single dose of 1 or 6 mg / kg (siRNA) of hIgG1 TfR-Var2ii or hIgG1 TfR-Var9ii SSB conjugate downregulated SSB mRNA levels in the gastrocnemius and quadriceps muscles by up to 72% at 21 days post-treatment (Figure 19). The activity of the humanized antibodies is similar to that of the parent chimeric IgG2 TfR1 antibody. Unconjugated TfR-Var2ii Ab administered at 60 mg / kg (equivalent to an AOC dose of 6 mg / kg) did not significantly downregulate SSB.

[0515] method:

[0516] On day 1, cynomolgus monkeys (male, 2-3 years old, body weight 2-3 kg) received a 30-minute (+ / - 3-minute) intravenous (IV) infusion. On days -10 and +21 post-injection, muscle biopsies (gastrocnemius and quadriceps) were collected from sedated animals via a 6 mm puncture, weighed, and flash-frozen in liquid nitrogen. Frozen tissue samples were homogenized in 1 ml of cold TRIZOl (Thermo Fisher #15596026). To determine mRNA knockdown, total RNA was subtracted from the tissue and quantified spectrophotometrically using the Direct-zol 96 RNA kit according to the manufacturer's instructions. RNA (100 = 200 ng) was reverse transcribed using the High Capacity cDNA kit (Thermo Fisher #4368813) according to the manufacturer's instructions. SSB mRNA levels were quantified using TaqMan qPCR with appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control. The % mRNA was calculated using the ΔΔCt method, and the SSB mRNA concentration in the same animals before treatment or the SSB concentration in PBS-treated animals was set to 100% expression. [Example]

[0517] AOC-mediated, but not siRNA-delivered, SSB knockdown is muscle-specific. Following a single 6 mg / kg hIgG1 TfR-Var2ii-SSB administration, 21 days later, SSB siRNA concentrations of 10–100 nM were observed in most tissues. The highest siRNA concentrations were in the liver and adrenal gland (<1000 nM), and the lowest in the brain (2 nM). siRNA concentrations in skeletal muscle ranged from 3–20 nM. Despite relatively low siRNA exposure, we observed a >50% reduction in SSB mRNA levels only in skeletal muscle and heart. These results demonstrate that delivery of the oligonucleotide payload by the TfR1-targeting antibody is muscle-specific and is driven by cell-specific transport pathways rather than siRNA exposure.

[0518] method:

[0519] On day 1, cynomolgus monkeys (male, 2-3 years old, 2-3 kg body weight) received a 30-minute (+ / - 3-minute) intravenous (IV) infusion. On day 21 post-dose, muscle biopsies were taken from sedated animals via a 6 mm puncture. All other tissue samples were collected within 30 minutes after necropsy. Tissue samples were processed, and SSB mRNA levels were determined as described above (Figure 20B). Tissue SSB siRNA concentrations were determined using the stem-loop qPCR assay described in the Methods section (Figure 20A). The antisense strand of the siRNA was reverse transcribed using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) with a sequence-specific stem-loop RT primer. The cDNA obtained from the RT step was then used for real-time PCR, and Ct values were converted to plasma or tissue concentrations using the linear equation derived from the standard curve.

[0520] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It will be understood that various alternatives to the embodiments of the disclosure described herein may be utilized in practicing the present disclosure. It is intended that the following claims define the scope of the disclosure, and that methods and structures within the scope of the claims and equivalents thereof be covered thereby.

Claims

1. 1. An anti-transferrin receptor antibody comprising a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1 and an HCDR2 sequence comprising EINPIX １ GRSNYAX ２ KFQG and X １ is selected from N or Q, and X ２ An anti-transferrin receptor antibody comprising an HCDR2 sequence in which is selected from Q or E, and an HCDR3 sequence comprising SEQ ID NO:

3.

2. 2. The anti-transferrin receptor antibody of claim 1, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 2, and an HCDR3 sequence comprising SEQ ID NO:

3.

3. 2. The anti-transferrin receptor antibody of claim 1, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 4, and an HCDR3 sequence comprising SEQ ID NO:

3.

4. 2. The anti-transferrin receptor antibody of claim 1, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 5, and an HCDR3 sequence comprising SEQ ID NO:

3.

5. The VL region has the LCDR1 sequence RTSENIYX ３ NLA, LCDR2 sequence AX ４ TNLAX ５ , and LCDR3 sequence QHFWGTPLTX ６ where X ３ is selected from N or S, and X ４ is selected from A or G, and X ５ is selected from D or E, and X ６ The anti-transferrin receptor antibody of any one of claims 1 to 4, wherein is present or absent, and if present is F.

6. The VL region has an LCDR1 sequence containing SEQ ID NO: 6, an LCDR2 sequence containing AATNLAX ５ , and LCDR3 sequence QHFWGTPLTX ６ where X ５ is selected from D or E, and X ６ The anti-transferrin receptor antibody of any one of claims 1 to 5, wherein is present or absent, and if present is F.

7. The anti-transferrin receptor antibody of any one of claims 1 to 6, wherein the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6, an LCDR2 sequence comprising SEQ ID NO: 7, and an LCDR3 sequence comprising SEQ ID NO:

8.

8. The anti-transferrin receptor antibody of any one of claims 1 to 5, wherein the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6, an LCDR2 sequence comprising SEQ ID NO: 9, and an LCDR3 sequence comprising SEQ ID NO:

10.

9. The anti-transferrin receptor antibody of any one of claims 1 to 5, wherein the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 11, an LCDR2 sequence comprising SEQ ID NO: 12, and an LCDR3 sequence comprising SEQ ID NO:

10.

10. 10. The anti-transferrin receptor antibody of any one of claims 1 to 9, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 2, and an HCDR3 sequence comprising SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6, an LCDR2 sequence comprising SEQ ID NO: 7, and an LCDR3 sequence comprising SEQ ID NO:

8.

11. 10. The anti-transferrin receptor antibody of any one of claims 1 to 9, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 4, and an HCDR3 sequence comprising SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6, an LCDR2 sequence comprising SEQ ID NO: 7, and an LCDR3 sequence comprising SEQ ID NO:

8.

12. 10. The anti-transferrin receptor antibody of any one of claims 1 to 9, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 5, and an HCDR3 sequence comprising SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 6, an LCDR2 sequence comprising SEQ ID NO: 9, and an LCDR3 sequence comprising SEQ ID NO:

10.

13. 10. The anti-transferrin receptor antibody of any one of claims 1 to 9, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 4, and an HCDR3 sequence comprising SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 11, an LCDR2 sequence comprising SEQ ID NO: 12, and an LCDR3 sequence comprising SEQ ID NO:

10.

14. The anti-transferrin receptor antibody of any one of claims 1-13, wherein the VH region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 13-16.

15. The anti-transferrin receptor antibody of any one of claims 1-14, wherein the VL region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 18-21.

16. The anti-transferrin receptor antibody of any one of claims 1-15, wherein the anti-transferrin receptor antibody comprises a humanized antibody or binding fragment thereof, or a chimeric antibody or binding fragment thereof.

17. The anti-transferrin receptor antibody of any one of claims 1-16, wherein the anti-transferrin receptor antibody comprises a multispecific antibody or binding fragment thereof.

18. The anti-transferrin receptor antibody of any one of claims 1-17, wherein the anti-transferrin receptor antibody comprises a bispecific antibody or binding fragment thereof.

19. 19. The anti-transferrin receptor antibody of any one of claims 1-18, wherein the anti-transferrin receptor antibody comprises an IgG-scFv, a nanobody, a BiTE, a diabody, a DART, a TandAb, an sc diabody, an sc diabody-CH3, a triplebody, a miniantibody, a minibody, a TriBi minibody, an scFv-CH3 KIH, a Fab-scFv-Fc KIH, a Fab-scFv, an scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, an scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, an sc diabody-Fc, a diabody-Fc, a tandem scFv-Fc, or an intrabody.

20. The anti-transferrin receptor antibody of any one of claims 1-19, wherein the anti-transferrin receptor antibody comprises an IgG1 framework.

21. The anti-transferrin receptor antibody of any one of claims 1-19, wherein the anti-transferrin receptor antibody comprises an IgG2 framework.

22. 22. The anti-transferrin receptor antibody of claim 21, wherein the IgG2 framework is an IgG2b framework.

23. The anti-transferrin receptor antibody of any one of claims 1-19, wherein the anti-transferrin receptor antibody comprises an IgG4 framework.

24. The anti-transferrin receptor antibody of any one of claims 1-23, wherein the anti-transferrin receptor antibody further comprises at least one mutation in the Fc region.

25. 25. The anti-transferrin receptor antibody of claim 24, wherein at least one mutation modulates an effector function.

26. 26. The anti-transferrin receptor antibody of claim 24 or 25, wherein at least one mutation weakens or eliminates Fc-γ receptor binding.

27. 27. The anti-transferrin receptor antibody of any one of claims 24-26, wherein at least one mutation is at residue position D265, N297, K322, L328, or P329, wherein the residue positions are relative to IgG1.

28. 28. The anti-transferrin receptor antibody of any one of claims 24-27, wherein the Fc region comprises two or more, three or more, or four or more mutations.

29. 29. The anti-transferrin receptor antibody of any one of claims 24-28, wherein the Fc region comprises mutations at L233 and L234, wherein the residues correspond to positions 233 and 234 of SEQ ID NO:

23.

30. The anti-transferrin receptor antibody of any one of claims 24-28, wherein the Fc region comprises mutations at D265 and N297.

31. The anti-transferrin receptor antibody of any one of claims 1-30, wherein the anti-transferrin receptor antibody comprises a heavy chain (HC) sequence selected from SEQ ID NOs: 23-46 and a light chain (LC) sequence selected from SEQ ID NOs: 47-50.

32. The anti-transferrin receptor antibody of any one of claims 1-31, wherein the anti-transferrin receptor antibody specifically binds to human transferrin receptor (TfR).

33. 33. An anti-transferrin receptor antibody conjugate comprising the anti-transferrin receptor antibody of claim 1-32 and a payload.

34. 34. The anti-transferrin receptor antibody conjugate of claim 33, wherein the payload comprises a small molecule, peptide, protein, or polynucleic acid molecule.

35. 34. The anti-transferrin receptor antibody conjugate of claim 33, wherein the payload comprises a polynucleic acid molecule.

36. 36. The anti-transferrin receptor antibody conjugate of claim 35, wherein the polynucleic acid molecule comprises a short interfering nucleic acid (siNA), a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a PMO, or an mRNA.

37. 37. The anti-transferrin receptor antibody conjugate of any one of claims 34-36, wherein the payload comprises dsRNA.

38. 37. The anti-transferrin receptor antibody conjugate of any one of claims 34-36, wherein the payload comprises an antisense oligonucleotide (ASO).

39. 34. The anti-transferrin receptor antibody conjugate of claim 33, wherein the payload comprises a small molecule, peptide, or protein.

40. 40. The anti-transferrin receptor antibody conjugate of claim 39, wherein the payload comprises a microtubule disrupting agent, a DNA modifying agent, or an Akt inhibitor.

41. 40. The anti-transferrin receptor antibody conjugate of claim 39, wherein the payload comprises an auristatin or a derivative thereof, a dolastatin or a derivative or analog thereof, a maytansinoid, or a pyrrolobenzodiazepine or a derivative thereof.

42. 42. The anti-transferrin receptor antibody conjugate of claim 41, wherein the auristatin or derivative thereof is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

43. 42. The anti-transferrin receptor antibody conjugate of claim 41, wherein the maytansinoid is DM1 or DM4.

44. 42. The anti-transferrin receptor antibody conjugate of claim 41, wherein the pyrrolobenzodiazepine is a pyrrolobenzodiazepine dimer.

45. 40. The anti-transferrin receptor antibody conjugate of claim 39, wherein the payload comprises an immunomodulator or immunomodulatory drug.

46. 46. The anti-transferrin receptor antibody conjugate of claim 45, wherein the immunomodulator comprises a cytokine.

47. 40. The anti-transferrin receptor antibody conjugate of claim 39, wherein the payload comprises a protein or peptide toxin or a fragment thereof.

48. 48. The anti-transferrin receptor antibody conjugate of any one of claims 33-47, wherein the payload is conjugated to the anti-transferrin receptor antibody via a linker.

49. 49. The anti-transferrin receptor antibody conjugate of any one of claims 33-48, wherein the anti-transferrin receptor antibody is further conjugated to two or more payloads.

50. 50. The anti-transferrin receptor antibody conjugate of any one of claims 33-49, wherein the ratio of payload to anti-transferrin receptor antibody is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, or 12:

1.

51. The anti-transferrin receptor antibody conjugate is 【Chemical 1】 Including, During the ceremony, A comprises an anti-transferrin antibody, B contains the payload, X １ consists of a single bond or a linker, and 51. The anti-transferrin receptor antibody conjugate of any one of claims 33-50, wherein n is an average value selected from 1-12.

52. 52. The anti-transferrin receptor antibody conjugate of claim 51, wherein the payload is a polynucleic acid molecule.

53. 53. The anti-transferrin receptor antibody conjugate of claim 52, wherein the polynucleic acid molecule comprises a passenger strand and a guide strand.

54. 54. The anti-transferrin receptor antibody conjugate of claim 53, wherein the guide strand comprises at least one modified internucleotide linkage, at least one inverted abasic moiety, at least one 5'-vinylphosphonate modified unnatural nucleotide, or a combination thereof.

55. 55. The anti-transferrin receptor antibody conjugate of claim 54, wherein the at least one 5'-vinylphosphonate modified unnatural nucleotide is positioned about 1, 2, 3, 4, or 5 bases away from the 5' end of the guide strand.

56. 56. The anti-transferrin receptor antibody conjugate of any one of claims 51-55, wherein the polynucleic acid molecule further comprises a modification of the sugar moiety at the 2' position.

57. 57. The anti-transferrin receptor antibody conjugate of claim 56, wherein the modification at the 2'-position is selected from 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamide (2-O-NMA) modified nucleotides.

58. 54. The anti-transferrin receptor antibody conjugate of claim 53, wherein the passenger strand comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorodiamidate morpholino oligomer-modified non-natural nucleotides.

59. 59. The anti-transferrin receptor antibody conjugate of any one of claims 52-58, wherein the passenger strand is shorter in length than the guide strand, thereby generating a 5' overhang, a 3' overhang, a blunt end at one end, or a combination thereof.

60. 59. The anti-transferrin receptor antibody conjugate of any one of claims 52-58, wherein the passenger strand is equal in length to the guide strand, thereby generating blunt ends at each end of the polynucleic acid molecule.

61. Passenger chain is A-X １ 61. The anti-transferrin receptor antibody conjugate of any one of claims 52-60, conjugated to

62. A-X １ is conjugated to the 5' end of the passenger strand.

63. A-X １ is conjugated to the 3' end of the passenger strand.

64. The anti-transferrin receptor antibody conjugate is 【Chemistry 2】 Including, During the ceremony, A comprises an anti-transferrin receptor antibody, B comprises a polynucleic acid molecule; C comprises a polymer; X １ consists of a single bond or a first linker, X ２ consists of a single bond or a second linker, and 51. The anti-transferrin receptor antibody conjugate of any one of claims 33-50, wherein n is an average value selected from 1-12.

65. 65. The anti-transferrin receptor antibody conjugate of claim 64, wherein C is polyethylene glycol.

66. 65. The anti-transferrin receptor antibody conjugate of claim 64, wherein the polynucleic acid molecule comprises a passenger strand and a guide strand.

67. Passenger chain is A-X １ and X ２ 67. The anti-transferrin receptor antibody conjugate of claim 66, conjugated to -C.

68. A-X １ is conjugated to the 5' end of the passenger strand, and X ２ 68. The anti-transferrin receptor antibody conjugate of claim 67, wherein -C is conjugated to the 3' end of the passenger strand.

69. X ２ -C is conjugated to the 5' end of the passenger strand, and A-X １ is conjugated to the 3' end of the passenger strand.

70. X １ and X ２ and each independently is a non-polymeric linker.

71. 65. The anti-transferrin receptor antibody conjugate of claim 64, further comprising D.

72. 72. The anti-transferrin receptor antibody conjugate of claim 71, wherein D is an endosomolytic moiety.

73. A nucleic acid polymer encoding the anti-transferrin receptor antibody of claims 1-32.

74. A vector comprising the nucleic acid polymer of claim 73.

75. an anti-transferrin receptor antibody according to any one of claims 1 to 32 or an anti-transferrin receptor antibody conjugate according to any one of claims 33 to 72, and pharmaceutically acceptable excipients, A pharmaceutical composition comprising:

76. 76. The pharmaceutical composition of claim 75, wherein the pharmaceutical composition is formulated for systemic administration.

77. 77. The pharmaceutical composition of claim 75 or 76, wherein the pharmaceutical composition is formulated for parenteral administration.

78. 1. A method of delivering a payload to a target site of interest in a subject, comprising: The method comprises:

80. A method comprising administering to a subject an anti-transferrin receptor antibody conjugate of any one of claims 33-72 or a pharmaceutical composition of any one of claims 75-77 to deliver a payload to a target site of interest.

79. 79. The method of claim 78, wherein the targeted site of interest is a cell containing the overexpressed causative protein.

80. 79. The method of claim 78, wherein the targeted site is a tumor site.

81. 79. The method of claim 78, wherein the targeted site of interest is a site in the brain.

82. 1. A method of treating cancer in a subject, comprising: The method comprises:

80. A method for treating cancer in a subject, comprising administering to the subject an anti-transferrin receptor antibody conjugate of any one of claims 33-72 or a pharmaceutical composition of any one of claims 75-77.

83. 83. The method of claim 82, wherein the cancer is a solid tumor.

84. 83. The method of claim 82, wherein the cancer is a hematological malignancy.

85. 85. The method of any one of claims 82-84, wherein the cancer is bladder cancer, lung cancer, brain cancer, melanoma, breast cancer, non-Hodgkin's lymphoma, cervical cancer, ovarian cancer, colon cancer, pancreatic cancer, esophageal cancer, prostate cancer, kidney cancer, skin cancer, leukemia, thyroid cancer, liver cancer, or uterine cancer.

86. 86. The method of any one of claims 82-85, wherein the cancer is a metastatic cancer.

87. 86. The method of any one of claims 82-85, wherein the cancer is a recurrent or refractory cancer.

88. 1. A method of treating muscle atrophy or myotonic dystrophy in a subject, comprising: The method comprises:

10. A method comprising administering to a subject an anti-transferrin receptor antibody conjugate of claims 33-72 or a pharmaceutical composition of claims 75-77, wherein the polynucleic acid molecule hybridizes to a target sequence of an atrogene, and the polynucleic acid molecule mediates RNA interference against the atrogene, thereby treating muscle wasting in the subject.

89. 89. The method of claim 88, wherein the muscle atrophy is diabetes-related muscle atrophy or cancer cachexia-related muscle atrophy.

90. 89. The method of claim 88, wherein the muscle atrophy is associated with insulin deficiency, chronic renal failure, congestive heart failure, chronic respiratory disease, chronic infection, fasting, denervation, sarcopenia, or myotonic dystrophy type 1 (DM1).

91. 89. The method of claim 88, wherein the myotonic dystrophy is DM1.

92. 89. The method of claim 88, wherein the subject's reticulocyte levels do not decrease after administration of the anti-transferrin receptor antibody.

93. 89. The method of claim 88, wherein administration of the anti-transferrin receptor antibody conjugate downregulates SSB siRNA or SSB mRNA levels in the subject.

94. 94. The method of claim 93, wherein the downregulation of SSB siRNA or SSB mRNA is in muscle.

95. 95. The method of claim 94, wherein the muscle is a skeletal muscle.

96. 95. The method of claim 94, wherein the muscle is cardiac muscle.

97. 89. The method of claim 88, wherein the atrogene comprises an upregulated gene in the IGF1-Akt-FoxO pathway, the glucocorticoid-GR pathway, the PGC1α-FoxO pathway, the TNFα-NFκB pathway, or the myostatin-ActRIIb-Smad2 / 3 pathway.

98. 89. The method of claim 88, wherein the atrogene encodes an E3 ligase.

99. 89. The method of claim 88, wherein the atrogene encodes a forkhead box transcription factor.

100. 89. The method of claim 88, wherein the atrogene comprises the atrogin-1 gene (FBXO32), the MuRF1 gene (TRIM63), FOXO1, FOXO3, or MSTN.

101. 89. The method of claim 88, wherein the atrogene comprises DMPK.

102. 1. A method of treating muscular dystrophy in a subject, comprising: The method comprises:

80. A method comprising administering to a subject an anti-transferrin receptor antibody conjugate of claims 33-72 or a pharmaceutical composition of claims 75-77, thereby treating muscular dystrophy in the subject.

103. 103. The method of claim 102, wherein the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy.

104. 103. The method of claim 102, wherein the muscular dystrophy is Duchenne muscular dystrophy.

105. The method of any one of claims 78-104, wherein the subject is a human.

106. 78. A kit comprising an anti-transferrin receptor antibody of any one of claims 1-32, an anti-transferrin receptor antibody conjugate of any one of claims 33-72, a nucleic acid polymer of claim 73, a vector of claim 74, or a pharmaceutical composition of any one of claims 75-77.

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