Anti-transferrin receptor antibodies and uses thereof

Abstract

To provide use of an anti-transferrin receptor antibody or its antigen-binding fragment in manufacture of formulations for treating diseases.SOLUTION: There are disclosed herein, in certain embodiments, anti-transferrin receptor antibodies, anti-transferrin receptor antibody conjugates, and pharmaceutical compositions which comprise the anti-transferrin receptor antibodies or conjugates. In some embodiments, there are also disclosed herein 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 entirety of which is 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 of preparing and using anti-transferrin receptor antibodies.

[0003] In addition to the existing use of 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.Transferrin receptor is one of the most widely utilized 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 to develop improved anti-transferrin receptor antibodies for pharmaceutical use. Summary of the Invention

[0004] Disclosed herein in certain embodiments 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 (VH) region and a variable light (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 LCDR1 sequence RTSENIYX3NLA, LCDR2 sequence AX4TNLAX5, and 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 LCDR1 sequence comprising SEQ ID NO:6, LCDR2 sequence AATNLAX5, and 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 LCDR1 sequence comprising SEQ ID NO:6, LCDR2 sequence comprising SEQ ID NO:7, and LCDR3 sequence comprising SEQ ID NO:8. In some embodiments, the VL region comprises LCDR1 sequence comprising SEQ ID NO:6, LCDR2 sequence comprising SEQ ID NO:9, and 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 sc diabody, a sc diabody-CH3, a triple body, a minibody, a minibody, a TriBi minibody, 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 sc diabody-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-gamma 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 NO:23-46 and a light chain (LC) sequence selected from SEQ ID NO:47-50. In some embodiments, the anti-transferrin receptor antibody specifically binds to the human transferrin receptor (TfR).

[0006] In certain embodiments, disclosed herein 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, a peptide, a protein, or a 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 small 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 consists of 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), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (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 is 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 is a single bond or a second linker, and n is an average value selected from 1-12. In some embodiments, C is 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 that include 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 pharma- ceutical acceptable excipient. In some embodiments, the pharmaceutical compositions are formulated for systemic administration. In some embodiments, the pharmaceutical compositions are formulated for parenteral administration.

[0010] In certain embodiments, disclosed herein are methods of delivering a payload to a targeted site of interest in a subject, comprising administering to the subject an anti-transferrin receptor antibody conjugate as described herein, or a pharmaceutical composition as described herein, to deliver the payload to the targeted site of interest. In some embodiments, the targeted site of interest is a cell that contains 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, comprising administering to the subject an anti-transferrin receptor antibody conjugate as described herein or a pharmaceutical composition as described herein to treat the cancer in the subject of interest. 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, colon 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 a metastatic cancer. In some embodiments, the cancer is a recurrent or refractory cancer.

[0012] In certain embodiments, disclosed herein are methods of treating muscle atrophy or myotonic dystrophy in a subject, comprising administering to the subject an anti-transferrin receptor antibody conjugate as described herein or a pharmaceutical composition as 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 reticulocyte level of the subject is not decreased after administration of an anti-transferrin receptor antibody. In some embodiments, administering the anti-transferrin receptor antibody conjugate downregulates the level of SSB siRNA or SSB mRNA in the 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 a human.

[0014] In certain embodiments, disclosed herein are methods of treating muscular dystrophy in a subject, comprising administering to the subject an anti-transferrin receptor antibody conjugate as described herein or a pharmaceutical composition as 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 description 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, in which: 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. [Diagram 2] The structure of an exemplary blunt-ended duplex with 19 complementary bases and a single 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 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 following 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 of cynomolgus monkeys following administration of hTfR1.IgG2 mAb-SSB conjugate at 30 and 60 mg / kg (n=3). [Figure 6] 1 illustrates 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 an exemplary anti-TfR antibody to TfR in a competitive setting. [Figure 9B] FIG. 9A shows the binding constants of the tested anti-TfR antibodies. [Figure 10A]1 shows that binding of an exemplary anti-TfR antibody to TfR is maintained. [Figure 10B] FIG. 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] FIG. 13A shows the EC50 of the tested anti-TfR antibodies. [Figure 14] 1 illustrates the ADCC activity of exemplary anti-TfR antibodies. [Figure 15] 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). [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 dose 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 dose of hIgG1 TfR-Val2ii-SSB conjugate (n=2). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 to facilitate iron uptake into cells. There are two subtypes of TfRs: transferrin receptor 1 (TfR1 or CD71) and transferrin receptor 2 (TfR2). TfR1 is ubiquitously expressed on various types of cells, whereas TfR2 is specifically expressed on hepatocytes.

[0018] In some cases, abnormal expression of TfR1 is prominent in various cancers. Indeed, one study showed that the expression level of TfR1 is elevated in breast cancer cells (Pizzamiglio, et al. “Expression of iron-related proteins differentiate 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)). Furthermore, another study has noted 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] Disclosed herein in some embodiments are anti-transferrin receptor antibodies, anti-transferrin receptor antibody conjugates, and pharmaceutical compositions comprising same. Disclosed herein in further embodiments are methods utilizing anti-transferrin receptor antibodies for the delivery of a payload, and methods of treating a disease or condition 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 antibodies specifically bind to the transferrin receptor (TfR). In some examples, the anti-transferrin receptor antibodies specifically bind to human transferrin receptor (TfR). In some cases, the anti-transferrin receptor antibodies specifically bind to transferrin receptor 1 (TfR1) (or CD71). In some cases, the anti-transferrin receptor antibodies specifically bind 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, the VH region comprising 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, and, 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, where 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, where 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, where 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 a LCDR1 sequence comprising SEQ ID NO:6, a LCDR2 sequence comprising SEQ ID NO:7, and a LCDR3 sequence comprising SEQ ID NO:8.

[0033] In some examples, the VL region comprises a LCDR1 sequence comprising SEQ ID NO:6, a LCDR2 sequence comprising SEQ ID NO:9, and a 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 an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence AX4TNLAX5, and an 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.

[0036] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising 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 comprising 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, 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 an 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, and if present, is F.

[0040] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising 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 comprising 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, the VH region comprising 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 comprising 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, the VH region comprising 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 comprising 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 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.

[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, where 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, 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.

[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 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.

[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, where 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, 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.

[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 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.

[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, where 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, 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.

[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 respective CDR1, CDR2, or CDR3 sequences.

[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. In some cases, 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. In some cases, 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) fusions, 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 trifunctional antibody or a bispecific miniantibody. In some instances, a bispecific antibody is a trifunctional antibody. In some instances, a trifunctional antibody 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, a bis-scFv (scFv)2, a diabody, a minibody, a triabody, a tetrabody, or a 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)'3 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 antibodies comprise an IgG framework (e.g., IgG1, IgG2, IgG3, or IgG4). In some cases, the anti-transferrin receptor antibodies comprise an IgG1 framework. In some cases, the anti-transferrin receptor antibodies comprise an IgG2 (e.g., IgG2a or IgG2b) framework. In some cases, the anti-transferrin receptor antibodies comprise an IgG2a framework. In some cases, the anti-transferrin receptor antibodies comprise an IgG2b framework. In some cases, the anti-transferrin receptor antibodies comprise an IgG3 framework. In some cases, the anti-transferrin receptor antibodies comprise an IgG4 framework.

[0078] In some cases, the anti-transferrin receptor antibody comprises one or more mutations in the 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 are to stabilize the antibody and / or increase half-life. In some examples, the one or more mutations are to modulate Fc receptor interaction and reduce or eliminate 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 are to modify glycosylation.

[0079] In some embodiments, the one or more mutations are located in the Fc region. In some examples, the Fc region comprises a mutation at residue position L234, L235, or a combination thereof. In some examples, the mutation comprises L234 and L235. In some examples, the mutation comprises 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 a mutation or combination of mutations at residue positions L235, L236, D265, N297, K322, L328, or P329. In some examples, the Fc region comprises a mutation at L235 and L236. In some examples, the Fc region comprises a mutation at L235 and L236 in combination with a mutation at residue position K322, L328, or P329. In some cases, the Fc region comprises a mutation at L235, L236, and K322. In some cases, the Fc region comprises a mutation at L235, L236, and L328. In some cases, the Fc region comprises a mutation at L235, L236, and P329. In some cases, the Fc region comprises a mutation 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 cases, the Fc region comprises mutations at L233, L234, and K321. In some cases, the Fc region comprises mutations at L233, L234, and L327. In some cases, 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 the IgG constant regions described in, e.g., 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., 2007 Cancer Res, 67(18): 8882-8890; 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 positions 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 described herein (e.g., antibodies and binding fragments thereof) 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 then 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 expressing 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 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 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) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity are used. 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 (US 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 by 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 the antibody nucleotide sequence or the antibody nucleotide sequence is introduced into a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the antibody. In certain embodiments, expression of the antibody is regulated by a constitutive, inducible, or tissue-specific promoter.

[0102] In some embodiments, a variety of 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 sequences, but also cells which, when transfected with the appropriate nucleotide coding sequences, express the antibody or binding fragment thereof in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody or binding fragment coding sequence, yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the antibody or binding fragment coding sequence, insect cell systems (e.g., baculovirus) infected with recombinant viral expression vectors containing the antibody or binding fragment 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 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 promoters) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter).

[0103] For long-term and 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 that contain 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 selectable markers. After introduction of the foreign DNA, cells are engineered to grow in rich medium for 1-2 days and then switched to selective medium. The selectable marker on the recombinant plasmid confers resistance to selection, allowing the 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 binding fragments thereof.

[0104] In some examples, a number of selection systems are used, including, but not limited to, the 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); 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 which may 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 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 conjugates 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 further examples, 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 instances, 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 instances, the DAR ratio of B to A is about 1. In some instances, the DAR ratio of B to A is about 2. In some instances, the DAR ratio of B to A is about 3. In some instances, the DAR ratio of B to A is about 4. In some instances, the DAR ratio of B to A is about 6. In some instances, the DAR ratio of B to A is about 8. In some instances, the DAR ratio of B to A is about 10. In some instances, the DAR ratio of B to A is about 12. In some instances, the DAR ratio of B to A is about 16.

[0112] In some examples, the DAR ratio of the polynucleic acid molecule (B) to the 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 the polynucleic acid molecule (B) to the anti-transferrin receptor antibody A is about 1. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the anti-transferrin receptor antibody A is about 2. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the anti-transferrin receptor antibody A is about 3. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the anti-transferrin receptor antibody A is about 4. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the anti-transferrin receptor antibody A is about 5. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the anti-transferrin receptor antibody A is about 6. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the anti-transferrin receptor antibody A is about 7. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the anti-transferrin receptor antibody A is about 8. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the anti-transferrin receptor antibody A is about 9. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the anti-transferrin receptor antibody A is about 10. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the anti-transferrin receptor antibody A is about 11. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the anti-transferrin receptor antibody A is about 12. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the anti-transferrin receptor antibody A is about 13. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the 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, a peptide, or a 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 is 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 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 the methods described in 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 instances, B is conjugated to A in a site-directed manner utilizing "traceless" coupling technology (PhiloChem). In some instances, the "traceless" coupling technology utilizes an N-terminal 1,2-aminothiol group of the linking moiety that is conjugated to a polynucleic acid molecule that contains 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 by 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 utilizing an enzyme-catalyzed process. In some examples, the site-directed method utilizes SMARTag™ technology (Redwood). In some examples, SMARTag™ technology involves the generation of formylglycine (FGly) residues 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 instances, the enzyme-catalyzed process includes microbial transglutaminase (mTG). In some instances, B is conjugated to A utilizing a microbial transglutaminase-catalyzed process. In some instances, 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 instances, mTG is produced from 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 expression suppression (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 small 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, i.e., an atrophy-associated gene, is a gene that is up-regulated or down-regulated in atrophying muscle. In some examples, the up-regulated atrophy gene includes a gene encoding a protein involved in a ubiquitin ligase, a forkhead box transcription factor, a growth factor, a deubiquitinase, or a 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 the target sequence of FBXO30. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of FBXO40. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of NEDD4. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of TRIM32. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of MUL1. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of STUB1. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of FOXO1. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of FOXO3. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of MSTN. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of USP14. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of USP19. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of DDIT4.In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of CTSL2. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of TGIF. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of MYOG. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of HDAC2. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of HDAC3. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of MT1L. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of MT1B. In some cases, the polynucleic acid molecules described herein hybridize to the target sequence of DMPK.

[0135] In some examples, the polynucleic acid molecule hybridizes the target region of the mis-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 a cancer gene. Exemplary cancer genes 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, 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 KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or a β-catenin-related gene.

[0138] In some embodiments, the polynucleic acid molecule comprises an 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 (Bacillus anthracis toxin, Bacillus subtilis toxin, Clostridium botulinum C2 toxin, Clostridium spiroforme toxin, Clostridium perfringens iota toxin, Clostridium difficile toxin, Bacillus subtilis ... Examples of toxins include bacterial cytotoxins such as leukocyte 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 neurotoxins), 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 a nucleic acid decoy.In some examples, the nucleic acid decoy is a mimic of protein-binding nucleic acid, such as RNA-based protein-binding mimic.Exemplary nucleic acid decoys include 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, the polynucleic acid molecule comprises natural or synthetic or artificial nucleotide analogs or bases. In some cases, the polynucleic acid molecule comprises a combination of DNA, RNA, and / or nucleotide analogs. In some examples, the synthetic or artificial nucleotide analogs or bases comprise modifications in one or more of the ribose moiety, the phosphate moiety, the nucleoside moiety, or a combination 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 comprises H, OR, R, halo, SH, SR, NH2, NHR, NR2, 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 comprises 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, the 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, the 2'-O-methyl modification adds a methyl group to the 2' hydroxyl group of the ribose moiety, while the 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 the 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 an 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 connectivity 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 acid (ENA), such as 2'-4'-ethylene bridged nucleic acid, which locks the sugar structure into a C3'-endo sugar puckering conformation. ENA is part of the bridged nucleic acid class of modified nucleic acid, which also includes LNA. Exemplary chemical structures of ENA and bridged nucleic acid 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 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 Deazanucleotides such as -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-alkylated and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine These include 5-oxyacetic 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 modified on the sugar moiety, as well as nucleotides having non-ribosyl sugars or analogs thereof. For example, the sugar moiety is or is based on mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles, in some cases.The term nucleotide further includes those 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 analogue further comprises morpholino, peptide nucleic acid (PNA), methyl phosphonate nucleotide, thiol phosphonate nucleotide, 2'-fluoro N3-P5'-phosphoramidite, 1',5'-anhydrohexitol nucleic acid (HNA), or a combination thereof. Morpholino or phosphorodiamidate morpholino oligos (PMOs) comprise synthetic molecules whose structure mimics natural nucleic acid structures by deviations from 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 phosphorodiamidate groups instead of phosphate groups. In some cases, backbone alterations remove all positive and negative charges, allowing the morpholino neutral molecule to cross cell membranes without the aid of a cell delivery agent 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 the backbone charge.

[0158] [ka]

[0159] In some embodiments, one or more modifications are optionally made at the internucleotide bond. In some examples, the modified internucleotide bond is, but is not limited to, phosphorothioate, phosphorodithioate, methylphosphonate, 5'-alkylenephosphonate, 5'-methylphosphonate, 3'-alkylenephosphonate, borontrifluoridates, 3'-5' or 2'-5' boranophosphate and selenophosphate, phosphotriester, thionoalkylphosphotriester, hydrogen phosphonate bond, alkylphosphonate, alkylphosphonothioate, arylphosphonothioate, phosphoroselenoate, phosphorodiselenoate, phosphinate, phosphoramidate, 3'-alkylphosphoramidate, phosphoropiperazide ... Phosphorothioate, phosphoroanilothioate, phosphoroanilidate, ketone, sulfone, sulfonamide, carbonate, carbamate, methylenehydrazo, methylenedimethylhydrazo, formacetal, thioformacetal, oxime, methyleneimino, methylenemethylimino, thioamidate, bond with riboacetyl group, aminoethylglycine, silyl, or siloxane bond, alkyl or cycloalkyl bond with or without heteroatom, such as saturated or unsaturated and / or substituted and / or heteroatom containing 1-10 carbons, morpholino structure, amide, or polyamide bond in which base is directly or indirectly bonded to aza nitrogen of backbone, and combinations thereof. Phosphorothioate antisense oligonucleotide (PS ASO) is an antisense oligonucleotide containing phosphorothioate bond. Exemplary PS ASO is 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, where 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 an 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 links 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-methyluridine, 5 ... uridine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methoxyuridine, deazanucleotides (such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, or 6-azothymidine), 5-methyl-2-thiouridine, other thiobases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queousine, archaeosine, naphthyl and substituted naphthyl groups, any O-alkylated and N-alkylated purines and pyrimidines (e.g., N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5'-oxyacetic 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 further includes those 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.

[0174] In some embodiments, the 5'-vinylphosphonate modified nucleotide analogue further comprises a morpholino, a peptide nucleic acid (PNA), a methyl phosphonate nucleotide, a thiol phosphonate nucleotide, a 2'-fluoro N3-P5' phosphoramidite, or a 1',5'-anhydrohexitol nucleic acid (HNA). Morpholino or phosphorodiamidate morpholino oligos (PMOs) comprise synthetic molecules whose structure mimics the natural nucleic acid structure but deviates from the normal sugar and phosphate structure. 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, the ribose monomers are linked by a phosphorodiamidate group instead of a phosphate group. In some cases, the backbone alteration removes all positive and negative charges, allowing the morpholino neutral molecule to cross cell membranes without the aid of a cell delivery agent as 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 that contains a positive or cationic charge. In some examples, the PMO is PMOplus (Sarepta). PMOplus refers to phosphorodiamidate morpholino oligomers that contain 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 that includes 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 the backbone charge.

[0182] [ka]

[0183] In some embodiments, one or more modifications of the 5'-vinylphosphonate modified oligonucleotides are optionally made 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, boranophosphate and selenophosphate at 3'-5' or 2'-5' bonds, phosphotriester, thionoalkylphosphotriester, hydrogen phosphonate bond, alkylphosphonate, alkylphosphonothioate, arylphosphonothioate, phosphoroselenoate, phosphorodiselenoate, phosphinate, phosphoramidate, 3'-alkylphosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate, phosphoropyridine ... 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, e.g., 1-10 carbons with or without heteroatoms, morpholino structures in which the base is directly or indirectly attached 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 or thiolphosphonate modification. Exemplary thiolphosphonate nucleotides (left), phosphorodithioate (middle), 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 examples, 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 examples, 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 1-6 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 a methylphosphonate, an ethylphosphonate, a methylthiophosphonate, or a methoxyphosphonate. In some embodiments, the modified phosphate is a methylphosphonate. In some embodiments, the modified phosphate is an ethylphosphonate. In some embodiments, the modified phosphate is a methylthiophosphonate. In some embodiments, the modified phosphate is a methoxyphosphonate.

[0197] In some embodiments, the one or more modifications further include modifications of the ribose moiety, the phosphate backbone, and the nucleoside, or the nucleotide analogue 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 amino alkyl group, e.g., a 3'C5-amino alkyl dT. In an additional alternative, the 3' end is optionally conjugated with an abasic site, e.g., an apurinic or apyrimidinic site. In some instances, the 5' end is conjugated with an amino alkyl group, e.g., a 5'-O-amino alkyl substituent. In some cases, the 5' end is conjugated with an abasic site, e.g., an apurinic or apyrimidinic 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 nucleotides, thiolphosphonate nucleotides, 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), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or , 2'-ON-methylacetamide (2'-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate 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, the 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-naturally occurring nucleotides. In some examples, the polynucleic acid molecule comprises 100% peptide nucleic acid modified non-naturally occurring nucleotides.

[0201] In some embodiments, the polynucleic acid molecule comprises one or more nucleotide analogs, each nucleotide analog being stereochemically isomeric.In such an example, the polynucleic acid molecule is a chiral molecule.In some cases, the nucleotide analog comprises backbone stereochemistry.In further cases, the nucleotide analog comprises 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% modification, about 10% to about 100% modification, about 20% to about 100% modification, about 30% to about 100% modification, about 40% to about 100% modification, about 50% to about 100% modification, about 60% to about 100% modification, about 70% to about 100% modification, about 80% to about 100% modification, and about 90% to about 100% modification.

[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, a polynucleic acid molecule contains 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 comprise 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 comprise an artificial nucleotide analog described herein. In some examples, about 5% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 10% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 15% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 20% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 25% of the polynucleic acid molecules comprise 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 molecule comprises 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 nucleotides, thiolphosphonate nucleotides, 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 modifications comprise 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 analogue described herein. In some embodiments, the polynucleic acid molecule comprises about 11 modifications, wherein the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the polynucleic acid molecule comprises about 12 modifications, wherein the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the polynucleic acid molecule comprises about 13 modifications, wherein the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the polynucleic acid molecule comprises about 14 modifications, wherein the modifications comprise an artificial nucleotide analogue described herein. In some embodiments, the polynucleic acid molecule comprises about 15 modifications, wherein the modifications comprise an artificial nucleotide analogue 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, a polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides, when present in said antisense strand, are 2'-deoxy-2'-fluoro pyrimidine nucleotides, and the purine nucleotides, when present in said antisense strand, are 2'-O-methyl purine nucleotides.

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

[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 examples, the phosphate backbone modification is phosphorothioate. In some cases, the passenger strand comprises more phosphorothioate modifications than the guide strand. In other cases, the guide strand comprises more phosphorothioate modifications than the passenger strand. In further cases, the passenger strand comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate modifications. In further cases, 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 a terminal capsid 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 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 strand 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 terminal 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 from 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 a terminal 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 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, a polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense 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 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, 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 strand 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, the 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 at 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 an end cap molecule at the 3'-end, 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, 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., a sense strand) and a guide strand (e.g., an antisense strand) that contain multiple modifications.

[0229] In some embodiments, a 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 from 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 contain 2'-5' internucleotide linkages. In some examples, the 2'-5' internucleotide linkages are at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 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, contain 2'-5' internucleotide linkages, 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, contain 2'-5' internucleotide linkages.

[0232] In some cases, the polynucleic acid molecule is a polynucleotide having a double, asymmetric double, hairpin, or asymmetric hairpin secondary structure with self-complementary sense and antisense regions, where the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence of another target nucleic acid molecule or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.In other cases, the polynucleic acid molecule is a circular single-stranded polynucleotide having two or more loop structures and a base comprising self-complementary sense and antisense regions, where the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid molecule or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and the circular polynucleotide is processed in vivo or in vitro to generate an active polynucleic acid molecule capable of mediating RNAi. In further cases, the polynucleic acid molecule further comprises a single-stranded polynucleotide having a nucleotide sequence complementary to a nucleotide sequence 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, where the polynucleic acid molecule comprises a single-stranded polynucleotide having complementarity to a target nucleic acid sequence, and where one or more pyrimidine nucleotides present in the polynucleic acid are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., where all of the pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides, or alternatively, where a plurality of the 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, where 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, the 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 comprises fewer nucleotides than the antisense region to the extent that the sense region has sufficient complementary nucleotides to base-pair with the antisense region and form a looped duplex. For example, the asymmetric hairpin polynucleic acid molecule comprises an antisense region (e.g., about 19 to about 22 nucleotides) having a length sufficient to mediate RNAi in a cell or in vitro system and 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 including a sense region and an antisense region, where 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 includes an antisense region (e.g., about 19 to about 22 nucleotides) having a length sufficient 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, e.g., ribonucleases such as RNase H, deoxyribonucleases such as DNases, or exonucleases such as 5'-3' exonuclease and 3'-5' exonuclease, as 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 capable of inhibiting RNase activity. 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' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'O-methoxyethyl (2'-O-MOE) 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 modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease 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 resistant to nucleases (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 a comparable naturally occurring polynucleic acid molecule. In some examples, a 2'-O-methyl modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent naturally occurring polynucleic acid molecule. In some examples, a 2'-O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent naturally occurring polynucleic acid molecule. In some examples, a 2'-O-aminopropyl modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent naturally occurring polynucleic acid molecule. In some examples, a 2'-deoxy modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent naturally occurring polynucleic acid molecule. In some examples, a T-deoxy-2'-fluoro modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent naturally occurring polynucleic acid molecule. In some examples, a 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent naturally occurring polynucleic acid molecule. 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, a 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, a TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, a 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, an LNA modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, an ENA modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, a PNA modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, the HNA modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to the equivalent natural polynucleic acid molecule. In some examples, the morpholino modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to the equivalent natural polynucleic acid molecule. In some examples, the methylphosphonate nucleotide modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to the equivalent natural polynucleic acid molecule. In some examples, the thiol phosphonate nucleotide modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to the equivalent natural polynucleic acid molecule. In some examples, the polynucleic acid molecule comprising 2'-fluoro N3-P5'-phosphoramidite has increased binding affinity to its mRNA target compared to the equivalent natural polynucleic acid molecule. 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 that contain a single enantiomer. In some examples, the polynucleic acid molecules contain L-nucleotides. In some examples, the polynucleic acid molecules contain D-nucleotides. In some examples, the polynucleic acid molecule composition contains 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its enantiomer. In some cases, the polynucleic acid molecule composition contains 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture. In some examples, the polynucleic acid molecule is a polynucleic acid molecule 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 aptamer-conjugated moieties. In some examples, the aptamer-conjugated moieties are DNA aptamer-conjugated moieties. In some examples, the aptamer-conjugated moieties are Alphamer (Centauri Therapeutics), which includes an aptamer moiety that recognizes a specific cell surface target and a moiety that displays a specific epitope for binding to circulating antibodies. In some examples, the polynucleic acid molecules described herein are further modified to include aptamer-conjugated moieties as described in U.S. Patent Nos. 8,604,184, 8,591,910, and 7,850,975.

[0240] In further embodiments, the polynucleic acid molecule described herein is modified to increase its stability. In some embodiments, the polynucleic acid molecule is RNA (e.g., siRNA). In some examples, the polynucleic acid molecule is modified by 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 by 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), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modification, or by a locked or bridged ribose structure (e.g., LNA or ENA). In some cases, the polynucleic acid molecule is modified by 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, the polynucleic acid molecule further comprises morpholino, PNA, HNA, methyl phosphonate nucleotide, thiol phosphonate nucleotide, and / or 2'-fluoro N3-P5'-phosphoramidite to increase its stability. In some cases, the polynucleic acid molecule is a chirally pure (or stereopure) polynucleic acid molecule. In some cases, the chirally pure (or stereopure) polynucleic acid molecule is modified to increase its stability. The appropriate modification of RNA to increase the stability of delivery will be clear to those skilled in the art.

[0241] In some cases, universal base refers to nucleotide base analogs that form base pairs with each of the natural DNA / RNA bases that are almost indistinguishable.Non-limiting examples of universal bases 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, phomopsin, podophyllotocin, rhizoxin, spongiostatin, taxane, tubulysin, vinca alkaloid, vinorelbine, or derivatives or analogs 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 maytansine 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 Publication Nos. 2013029900 and 20130323268.

[0246] In some embodiments, the payload is dolastine or a derivative or analog thereof. In some embodiments, the dolastine is dolastine 10 or dolastine 15, or a derivative or analog thereof. In some embodiments, the dolastine 10 analog is auristatin, sobridotin, simprostatin 1, or simprostatin 3. In some embodiments, the dolastine 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 an 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 as 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, pyrrolobenzodiazepines, 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, dimatecan, 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, abeimicin, ticamycin, DC-81, mazethramycin, neothramycin A, neothramycin B, polothramycin, prothracalcin, 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, pyrrolobenzodiazepines are 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, US7,612,062, 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 alpha-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 examples, the payload includes a "radiopaque" label, e.g., a label visualized using X-ray. 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 (see, e.g., U.S. Pat. No. 5,939,045), radiopaque polyurethanes (see, e.g., U.S. Pat. No. 5,346,981), organobismuth complexes (see, e.g., U.S. Pat. No. 5,256,334), radiopaque barium polymer complexes (see, 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 detectable labels used, for example, in immunoconjugates. 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 Radiation sensitizers include radiosensitizers that enhance the cytotoxic effects of radioactive materials (such as those generated by Co or X-ray sources, etc.). 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), compounds containing certain diamines (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 will be 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 Includes At etc.

[0262] In some examples, the payload comprises an immunomodulator. Useful immunomodulators include anti-hormonal 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 anti-hormonal agents include, for example, antiestrogens such as tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxyphene, keoxyphene, 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, a-sarcin, certain aleurone forsythia proteins, certain dianthine 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 glucosamine, 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., Interferon alpha, beta, gamma), stem cell growth factor, also 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 crosslinked networks of two- or three-dimensional 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, polyolefins, polyamides, polycyanoacrylates, polyimides, polyethylene terephthalates (PET, PETG), polyethylene terephthalates (PETE), polytetramethylene glycols (PTG), or polyurethanes, and mixtures thereof. As used herein, a mixture refers to the use of various polymers within the same compound, as in the context of block copolymers. In some cases, a block copolymer is a polymer in which at least one portion of the polymer is constructed from a monomer 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 is conjugated to an internal site 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 natural ligation.

[0268] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some examples, polydisperse materials contain a disperse distribution of materials of different molecular weights, characterized by average weight (weight average) size and dispersity. In some examples, monodisperse PEG contains molecules of one size. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the molecular weight given 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, 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, 290 The 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, the molecular weight of C is about 200 Da. In some examples, the molecular weight of C is about 300 Da. In some examples, the molecular weight of C is about 400 Da. In some examples, the molecular weight of C is about 500 Da. In some examples, the molecular weight of C is about 600 Da. In some examples, the molecular weight of C is 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 that contains more than one repeating ethylene oxide unit. In some examples, the discrete PEG (dPEG) contains 2-60, 2-50, or 2-48 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, dPEG contains about 22 or more repeating ethylene oxide units.In some examples, dPEG comprises about 24 or more repeating ethylene oxide units. In some examples, dPEG comprises about 26 or more repeating ethylene oxide units. In some examples, dPEG comprises about 28 or more repeating ethylene oxide units. In some examples, dPEG comprises about 30 or more repeating ethylene oxide units. In some examples, dPEG comprises about 35 or more repeating ethylene oxide units. In some examples, dPEG comprises about 40 or more repeating ethylene oxide units. In some examples, dPEG comprises about 42 or more repeating ethylene oxide units. In some examples, dPEG comprises about 48 or more repeating ethylene oxide units. In some examples, dPEG comprises about 50 or more repeating ethylene oxide units. In some cases, dPEG is synthesized stepwise from pure (e.g., about 95%, 98%, 99%, or 99.5%) starting materials as a single molecular weight compound. In some cases, the dPEG has a specific molecular weight rather than an average molecular weight. In some cases, the dPEG described herein is Quanta Biodesign, LMD's dPEG.

[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 produce 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, for example, an endosome, a lysosome, an endoplasmic reticulum (ER), a Golgi apparatus, a microtubule, a peroxisome, or other endoplasmic reticulum with a cell. 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. 1 -B) n Or, the formula (II): AX 1 -(BX 2 -C) n The conjugate of 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 comprises CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO:51) or GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO:52). In some examples, INF7 or a derivative thereof comprises 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 identity 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-permeable polypeptide) as 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, the formula (II): AX 1 -(BX 2 -C) n The conjugate of is further conjugated with an endosomolytic polymer. As used herein, an endosomolytic polymer includes a linear, branched network, star, comb, or ladder polymer. In some examples, the endosomolytic polymer is a homopolymer or copolymer that includes two or more different types of monomers. In some cases, the endosomolytic polymer is a polycationic polymer. In other cases, the endosomolytic polymer is a polyanionic polymer.

[0289] In some instances, polycationic polymers include monomeric units that are positively charged, neutrally charged, or negatively charged, with a net positive charge. In other instances, polycationic polymers include non-polymeric molecules that contain 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 include monomeric units that are positively charged, neutrally charged, or negatively charged, and the net charge is negative. In other cases, polyanionic polymers include non-polymeric molecules that contain 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 polymer described herein is a pH-responsive endosomolytic polymer. 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 examples, the endosomolytic moieties described herein are membrane disruptive polymers. In some cases, the membrane disruptive polymers include cationic polymers, neutral or hydrophobic polymers, or anionic polymers. In some examples, 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-1 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 detergent (e.g., Triton X-100). In some examples, the endosomolytic moieties described herein comprise a polymer (e.g., a poly(amidoamine)) conjugated to a detergent (e.g., Triton X-100). In some examples, the endosomolytic moieties described herein comprise 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, the formula (II): AX 1 -(BX 2 -C) nThe conjugate is further conjugated to an endosomolytic lipid (eg, 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, the 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), amphopiroquine, primaquine, mefloquine, nivaquines, halofantrine, quinoneimines, or combinations thereof. In some examples, the quinoline endosomolytic moiety may include, but is 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-diethyl-amino-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-diethyl-amino-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)-1-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,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 a quinolinyl derivative or analog as described by 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 an ester conjugate of folate-nigericin, an amide conjugate of folate-nigericin, or a carbamate conjugate of folate-nigericin. 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 include repeating units of a monomer produced by a polymerization process. Exemplary non-polymeric linkers include, but are not limited to, C1-C6 alkyl groups (e.g., C5, C4, C3, C2, or C1 alkyl groups), homobifunctional cross-linkers, heterobifunctional cross-linkers, peptide linkers, traceless linkers, self-immolative linkers, maleimide-based linkers, or combinations thereof. In some cases, the non-polymeric linker includes C1-C6 alkyl groups (e.g., C5, C4, C3, C2, or C1 alkyl groups), homobifunctional cross-linkers, heterobifunctional cross-linkers, peptide linkers, traceless linkers, self-immolative linkers, maleimide-based linkers, or combinations thereof. In further cases, the non-polymeric linker does not include more than two of the same type of linker, 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 that is included by the 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'-pyridyl dithio) (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 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)toluamide]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 (M2C2H), 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)hexanoate (sulfo-NH-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), 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 Succinimidyl (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.1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidocarbonyl-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 includes 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), as 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 elimination via a retro-Michael reaction. 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 up to 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 cases, the linker comprises Val-Cit. In some cases, 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 a mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In further cases, the linker comprises a 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 (e.g., 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 does not leave a linker moiety (e.g., an atom or a linker group) to the binding moiety A, the polynucleotide B, the polymer C, or the endosomolytic moiety D after cleavage. 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 may be any of the compounds described in U.S. Pat. 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, 2013 / 03 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 cases, X 1 comprises a single bond or a non-polymeric linker. In some examples, X 1 is a single bond. In some cases, X 1 is a non-polymeric linker. In some examples, the linker is a C1-C6 alkyl group. 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 the linker, and in particular as used in the context of X1, alkyl means a saturated linear or branched hydrocarbon radical containing up to 6 carbon atoms. In some examples, X 1 comprises a homobifunctional linker or a heterobifunctional linker as described above. 1 comprises a heterobifunctional linker. 1 In another embodiment, X comprises sMCC. 1 In another example, X comprises a heterobifunctional linker optionally conjugated to a C1-C6 alkyl group. 1 In a further example, X comprises sMCC optionally conjugated to a C1-C6 alkyl group. 1 does not include the homobifunctional or heterobifunctional linkers described above.

[0323] In some cases, 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, such as 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 In further examples, X 2 In a further example, X may include a combination of maleimide groups, peptide moieties, and / or benzoic acid groups. 2 In a further example, X 2 In a further example, X 2 In a further example, X 2 contains the mc-val-cit-PABA group.

[0324] How to use In some embodiments, methods are described herein for delivering payloads 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, the present invention provides a method for treating a disease or disorder characterized by overexpressed protein.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 diseased, damaged, or unused muscle tissue of a subject, compared to the same muscle tissue of a control subject. In one embodiment, 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 of a control subject. In another embodiment, significant loss of muscle strength refers to a decrease in strength of unused muscle tissue, compared to the muscle strength of the same muscle tissue of the same subject before a period of non-use. In one embodiment, 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 of 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 of diseased, damaged, or unused muscle tissue of a subject, compared to the same muscle tissue of a control subject. In an embodiment, the 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 of a control subject. In another embodiment, the significant loss of muscle mass refers to a decrease in muscle volume of unused muscle tissue, compared to the muscle volume of the same muscle tissue of the same subject before a period of non-use. In an embodiment, the 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 of the same subject before a period of non-use. 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 atrophy includes 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 caused by an underlying disease. In some instances, cachexia refers to the loss of body weight that cannot be restored by nutrition and is commonly associated with an underlying disease, such as cancer, COPD, AIDS, heart failure, etc. When cachexia is seen 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, response to therapy, quality of life, and reduced 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 fat mass, which cannot be fully restored by conventional nutritional support and may lead to progressive functional impairment. In some instances, the loss of skeletal muscle appears to be the most important event in cancer cachexia. In addition, the classification of cancer cachexia suggests that the diagnostic criteria should not only be that weight loss is a signal event of the cachectic process, but also that the patient's initial reserves, low BMI, or low levels of muscularity must be taken into account.

[0330] Denervation is damage to peripheral motor neurons with partial or complete blockage of nerve fibers between organs and the central nervous system, causing blockage of nerve conduction and motor neuron firing, which in turn impedes skeletal muscle contractability. This loss of nerve function may be localized or generalized with loss of entire motor neuron units. The inability of skeletal muscles to contract causes muscle atrophy. In some instances, denervation is associated with or as a result of degenerative, metabolic, or inflammatory neuropathies (e.g., Guillain-Barre 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 that describes diseases of the muscles. 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-Dreifuss, 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 voluntary muscles of the body. 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 with 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, or diabetes insipidus. Type 2 diabetes, also called 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 beta cell function in the pancreas, resulting in hypertensive glucose levels. In some cases, increased glucagon levels stimulate the liver to produce abnormal amounts of unnecessary glucose, which causes hypertensive glucose levels. Type 1 diabetes, also called insulin-dependent diabetes, comprises 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 about 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 characterised 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 diseases, 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, a chronic infection such as AIDS further causes 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 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 muscular atrophy occurs as a result of limbs being immobilized (e.g., due to limb or joint fractures, or orthopedic surgery such as hip or knee replacement surgery). 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, 1, 2, or more days). 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 is performed 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 constraint of movement may be at a single or multiple joints (e.g., single joints such as the shoulder or hip, compound joints such as the radiocarpal joint, and compound joints 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 ..., 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 tendon or liga 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, femoral 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 a 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 the control of polyadenylatin sites and loss of MBNL protein function cause downstream accumulation of nuclear foci and increased mis-splicing events, subsequently resulting in myotonia and other clinical symptoms.

[0345] In some embodiments, the present invention describes a method for treating diseases or disorders characterized by mis-spliced ​​mRNA.In some embodiments, the anti-transferrin receptor antibody described herein delivers polynucleic acid molecules 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 due to improperly spliced ​​or partially spliced ​​mRNAs. 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, there 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 disease or disorder. 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 mutation. Becker muscular dystrophy and Duchenne muscular dystrophy have been shown to be involved in mutations in the DMD gene, which encodes the protein dystrophin. Facioscapulohumeral muscular dystrophy has been shown to be involved in mutations in the double homeobox 4 (DUX4) gene.

[0351] In some instances, improperly spliced ​​or partially spliced ​​mRNA causes Duchenne muscular dystrophy, which is caused by a mutation in the DMD gene that causes severe muscle weakness and abolishes the production of functional dystrophin. In some instances, Duchenne muscular dystrophy is the result of a mutation in an exon 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, pulsatile 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 a solid lipid nanoparticle, a polymeric nanoparticle, a self-emulsifying nanoparticle, a liposome, a microemulsion, or a micellar solution. 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 include a core or a core and a shell, such as a core-shell nanoparticle.

[0356] In some examples, the nanoparticles are further coated with molecules for binding 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, histones, 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 less than about 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.

[0358] In some examples, the nanoparticle formulation comprises 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, or quantum dots.In some examples, the polynucleic acid molecules or binding moieties described herein are directly or indirectly conjugated to the nanoparticles.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 nanoparticles.

[0359] In some embodiments, the pharmaceutical preparation comprises a delivery vector, for example a recombinant vector for delivery of polynucleic acid molecules to cells. 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 polynucleic acid molecules provides stable expression in target cells. In further examples, viral vectors are used that provide transient expression of polynucleic acid molecules.

[0360] In some embodiments, the pharmaceutical formulation includes a carrier or carrier material selected based on its 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, taurocholic acid, 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 in an acceptable range.

[0362] In some examples, 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, with suitable salts including sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0363] Treatment regimen In some embodiments, the pharmaceutical composition described herein is administered for therapeutic use. In some embodiments, the pharmaceutical composition is administered once a day, twice a day, three times a day, or more. The pharmaceutical composition is administered every day, 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 composition is 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 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 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 administered simultaneously. In some examples, two or more different pharmaceutical compositions are administered simultaneously at the same time. In some cases, two or more different pharmaceutical compositions are administered simultaneously in sequence, without any time gap between administrations. In other cases, two or more different pharmaceutical compositions are administered simultaneously in sequence, with about 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, 2 days between administrations.

[0366] If the patient's condition improves, at the physician's discretion, administration of the composition may continue; alternatively, the dose of the composition being administered may be temporarily reduced or temporarily suspended for a period of time (i.e., a "drug holiday"). In some instances, the length of the drug holiday may vary between 2 days and 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 from 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 or frequency of administration, or both, 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 in need of treatment, but will nevertheless be routinely determined in a manner 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, for example, as 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 merely 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 physician.

[0370] In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, determining 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 the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between the LD50 and the ED50. Compounds that exhibit high therapeutic indices are preferred. The 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 is preferably located within a range of circulating concentrations that includes the ED50 with minimal toxicity. Dosages vary within this range depending on the dosage form used and the route of administration utilized.

[0371] Kits / Products In certain embodiments, kits and products are disclosed herein 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 different elements used in the methods described herein.Suitable containers include, for example, bottles, vials, syringes, and test tubes.In one embodiment, the container is formed from 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 include 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] The kit typically includes 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, the label is on the container if the letters, numbers, or other indicia forming the label are affixed, molded, or engraved on the container itself. The label is associated with the container if it is present in the 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, as 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 the compounds 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 comprises 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 is, for example, a label approved by the U.S. Food and Drug Administration for prescription drugs or an approved package insert. In one embodiment, compositions comprising the compounds provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for the 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 to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to be limiting of any subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used within 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 term "including" is non-limiting, as are other forms such as "include," "includes," and "included."

[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 amounts 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] "Antibody" and "immunoglobulin" (Ig) 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')2, Fv, single chain antibodies, diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, humanized antibodies, etc.), and recombinant peptides including those described above.

[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 usually heterotetrameric glycoproteins of about 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 constant domain of the light chain 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 widely 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 areas 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 and are connected by three CDRs that form loop connections, and in some cases form 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 is comprised of 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 Institute of Health, Bethesda, Md.) and / or amino acid residues 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 (13) 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 crystallize easily. 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 In this structure, 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 include heavy chain C that contains 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 domain. Fab'-SH is the designation 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 which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional structures of the 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, e.g., as described 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 typically reside at amino acid positions 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3), which may 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 typically reside 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 may 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 typically occur at amino acid positions 26-31 (CDR1), 52-56 (CDR2), and 95-102 (CDR3), which may 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 typically occur 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 may 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 worker (e.g., a physician, registered nurse, bedside nurse, physician assistant, nursing assistant, or hospice worker). EXAMPLES

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

[0398] Production and characterization of humanized anti-TfR antibodies Nucleic acids encoding exemplary anti-TfR antibodies were stably transfected into CHOK1SV GSKO cells, and three stable pools per product were generated. The stable pools were monitored for cell growth and Protein A titer from day 8 post-transfection onwards. Cultures were cultured once at 70% viability and a threshold of 0.6×10e 6 Stable pools were passaged when they reached 0.2 × 10e cells / mL. When cell viability was >97%, the highest producing pool tested was used to culture 600 mL of fed-batch overgrow cultures (FOG) per product. 6FOG cultures were fed on days 4 and 8 and harvested on day 11 by centrifugation and filter sterilization. Sterile cell culture supernatants were purified by Protein A purification using 3 x 5 ml MabSelectSuRE columns in parallel on an AKTA purifier (running at 10 mL / min). 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. Eluted fractions were neutralized by diluting 1:2 with 2 x PBS, 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). 80 μl aliquots of 1 mg / ml samples were injected and run at 1 ml / min for 15 min in sodium phosphate 50 mM, sodium chloride 150 mM, L-arginine 500 mM, pH 6.0. All variants showed a small peak less than 16.89% with a retention time of approximately 7.66 min, consistent with soluble aggregates. Soluble aggregate concentration was 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 GLM sensor chip coated with Protein A for mAb capture. The running buffer contained 10 mM HEPES, 150 mM NaCl, pH 7.4, 0.05% tween-20, 0.2 mg / ml BSA. Data was collected at 25°C. Based on the resulting stock concentrations, all mAbs were diluted to 2 ug / ml in running buffer. Each was then captured on the Protein A surface for 40 seconds.

[0403] hTfR (100ug) was dissolved in 300uL of water to give a stock concentration of 4.3uM. hTfR was dilted to 43nM as the highest concentration and tested in 3-fold serial dilutions. hTfR was injected at 200ul / 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-referenced with buffer injections.

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

[0406] [Table 10] EXAMPLES

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

[0408] The CDRs of a mouse IgG2 antibody against hTfR1 were subcloned into a human IgG2 background and transfected into CHO-K1SP cells. For sequences, see Example 4. 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). Feeding of 8% of the final culture volume (25 liters) was performed every 2 days from day 4 for a total of 14 days of incubation. Culture supernatants were harvested, depth filtered, and purified using Monofinity A Resin (GenScript) at a flow rate of 30 ml / min. The eluted proteins were buffered in PBS and the molecular weight and purity of the purified proteins were analyzed by SDS-PAGE under reducing and non-reducing conditions, and by 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 SSB siRNA duplex was used as the conjugate used in this experiment. The sequence of the 21-mer SSB guide / antisense strand was (5'-3')UUACAUUAAAGUCUGUUGUUU. The single strands were fully assembled on the solid phase using standard phosphoramidite chemistry and purified using HPLC. Base, sugar, and phosphate modifications well described in the field of RNAi 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 concentration of up to 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 showed 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 all 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, ProPacTM SAX-10, Bio LCTM, 4 x 250mm

[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 conjugates 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 times (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 hTfR1-IgG2 mAb siRNA conjugates 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. Plates were washed four times with 100 μL Tris-buffered saline + Tween (20xTBST, Cell Signaling 9997S). 100 μL Superblock (ThermoFisher PI-37535) was added to each well and incubated at room temperature for 1 hour. The washing step was repeated before adding samples. Samples were added at a concentration of up to 10 nM, 50 μL / well. Plates were incubated at room temperature for another hour and the washing step was repeated. Secondary antibodies (Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson Immunoresearch, 109-035-098) were 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 reference wavelength of 570 nm was subtracted. Binding constants were determined using GraphPad Prism Specific Binding with Hill Slope.

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

[0429] The ability of 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 RPMI1640 containing 10% fetal bovine serum (Nucleus Biologics FBS1824). Cells were diluted to 100,000 / mL and 100 μL was added to each well of the plate. Antibody conjugates were diluted to a maximum concentration of 100 nM. As a negative control, 20 μL of conjugate or PBS was added to multiple wells of a 96-well plate and the treated cells were left at 37° C. and 5% CO2 for 72 hours.

[0430] Immortalized human skeletal muscle cells (Institute of Myology, Paris) were seeded in 500 μl skeletal muscle cell growth medium (PromoCell C-23260) on 24-well collagen plates (Thermo Fisher A1142802) and incubated at 37°C in 5% CO2 until myoblasts were confluent. At this point, myotube differentiation was induced by incubation for 4 days in 500 μl differentiation medium (DMEM (Gibco 10566-016) supplemented with insulin 10 ug / ml and gentamicin 50 μg / ml). 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 for at least overnight before analysis. RNA was isolated and quantified spectrophotometrically using the Direct-zol 96 RNA kit according to the manufacturer's instructions. RNA (100 = 200ng) 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 % 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) (Figure 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 years old, male, and weighed 2-3 kg. Cynomolgus monkeys received 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 taken from a peripheral vein in conscious, restrained animals, or from the gastrocnemius and quadriceps muscles, respectively, in sedated animals at different times as outlined in Table 12.

[0433] [Table 12]

[0434] A stem-loop qPCR assay was used to determine the plasma concentration of hIgG2 TfR1.mAb-siSSB conjugate. 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 plasma from untreated animals and then serially diluting in TE buffer + 0.1% v / v Triton X-100. The antisense strand of the siRNA was reverse transcribed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) with 25 nM of sequence-specific stem-loop RT primer. The cDNA obtained from the RT step was utilized 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 for 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 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 that of the mouse anti-transferrin mAb conjugates tested in mice.

[0437] [Table 14]

[0438] To assess siRNA concentration and activity of the conjugates in muscle, muscle biopsies (gastrocnemius and quadriceps) were obtained according to the schedule shown in Table 12. Muscle biopsies were taken by 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 tissues and quantified spectrophotometrically using the Direct-zol 96 RNA kit according to the manufacturer's instructions. RNA (100=200ng) 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 % of mRNA was calculated using the method, and the SSB mRNA concentration in the same animal before treatment or the SSB concentration in animals treated with PBS was set to 100% expression.

[0439] Quantification of tissue siRNA enrichment was determined using a stem-loop qPCR assay. Briefly, 15-50 mg tissue pieces were homogenized in 500 uL 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 then serially diluting in TE buffer + 0.1% v / v Triton X-100. The antisense strand of the siRNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) with 25 nM of sequence-specific stem-loop RT primer. cDNA obtained from the RT step was utilized 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 SSB siRNA antisense strand and all primers and probes used for measurement are shown in Table 13. Quantitative PCR reaction was performed using standard cycle conditions in QuantStudio 7 Flex Real-Time PCR System (Life Technologies). Ct value was converted to plasma or tissue concentration using the linear equation obtained from the standard curve.

[0440] Treatment of cynomolgus monkeys with the conjugate resulted in downregulation of SSB mRNA up to 62% in gastrocnemius muscle and up to 75% in quadriceps muscle (Figure 5A and Figure 5B). 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 treatment. These results demonstrate that the selected TfR1 antibody can effectively deliver siRNAs to monkey muscle tissues and that the activity of the transferrin receptor-targeted AOC translates between species.

[0441] To monitor the safety of selected anti-hTfR1 antibodies in primates, hematology and clinical chemistry were analyzed according to the schedule shown in Table x. 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 up to 28 days after dosing. The transient downregulation observed in reticulocytes was considered a side effect of the TfR1 antibody. We observed that mouse TfR1 antibodies 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 depletion of reticulocytes was only transient and was not as obvious as 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. EXAMPLES

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

[0443] Modern in silico antibody humanization and deimmunization programs well described in the art were used to design 16 variants of the chimeric anti-transferrin 1 mAb that was NHP tested 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 as part of the humanized activity via amino acid substitutions. We also attempted to assess immunogenicity risk, identifying high-risk epitopes and, where feasible, removing them. We further introduced mutations in the Fc domain of the variants to remove effector functions (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 made as described below.

[0444] Human and cynomolgus 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 TfR1. Human or cynomolgus transferrin receptor ELISA assay protocols are described below.

[0446] 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. Plates were washed 4 times with 100 μL Tris-buffered saline + Tween (20xTBST, Cell Signaling 9997S). 100 μL Superblock (ThermoFisher PI-37535) was added to each well and incubated for 1 hour at room temperature. The washing step was repeated before adding samples. Samples were added at concentrations up to 10 nM, 50 μL / well. Plates were incubated for an additional hour at room temperature and the washing step was repeated. Secondary antibody (Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson Immunoresearch, 109-035-098) was diluted 1:5000 in Superblock and 50 μL / well added. Plates were 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), 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 subtracted from the reference wavelength of 570 nm. 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 blockade ELISA assay

[0449] The goal of this assay was to confirm that the 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 500ng / 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 4 times with 100μL Tris-buffered saline + Tween (20xTBST, Cell Signaling 9997S). 100μL Superblock (ThermoFisher PI-37535) was added to each well and incubated for 1 hour at room temperature. The washing step was repeated, and then hTfR (200ng / mL in 25μl) 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 to obtain a high concentration and added to the plate in 3-fold serial dilutions. 25 μl / well was added to the 25 μl already on the plate. Plates were incubated for 1 hour and the washing step was repeated. Streptavidin-HRP (R&D Systems DY998) was added according to the dilution recommended in the package insert and a final washing step was performed. Binding was measured by adding 50 μL of 1-Step™ Ultra TMB-ELISA (ThermoFisher, 34028), incubated for 5 minutes, 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 and the reference wavelength of 570 nm 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 is 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 TfR binding in the presence of transferrin, but substituting the cofactor HFE (hereditary hemochromatosis protein, mybiosource.com, MBS953891) for transferrin, see Figure 10A-B.

[0454] FcγRIIIA (CD16a) ELISA

[0455] The goal of this assay was to determine potential ADCC activity of antibodies by measuring binding to FcγRIIIA (CD16a) genotype V158. Half-well high-binding 96-well plates (Costar #3690) were coated with recombinant CD16a protein (Sino Biological 10389-H27H1) at 2ng / μL in PBS (Gibco 14190) and incubated overnight at 4°C. Plates were washed 4 times with 100μL Tris-buffered saline + Tween (20xTBST, Cell Signaling 9997S). 100μL Superblock (ThermoFisher PI-37535) was added to each well and incubated for 1 hour at room temperature. The washing step was repeated before adding samples. Samples were added at a concentration of up to 1μM, 50μL / well. Plates were incubated for an additional hour at room temperature and the washing step was repeated. Secondary antibody (Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson Immunoresearch, 109-035-098) was diluted 1:5000 in Superblock and 50 μL / well added. Plates were 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), 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 subtracted from the control wavelength of 570 nm. 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 that TfR mAb conjugates are capable of siRNA delivery and gene-specific downregulation. mAb variants were conjugated to active siRNA (SSB) or scrambled control (Scr). HEL92.1.7 cell line (ATCC® TIB-180™) was cultured in RPMI1640 (Gibco A10491) containing 10% fetal bovine serum (Nucleus Biologics FBS1824). Antibody siRNA conjugates were 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. Cells were left 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 with Taqman Fast Advanced Master Mix (Applied Biosystems 4444558) with SSB and PPIB Taqman probe sets (ThermoFisher Hs04187362_g1 and Hs00168719_m1). % mRNA 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 the incorporation assay 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 purity are listed in Table 15 below.

[0459] [Table 15]

[0460] Analytical data table for the conjugates used in this example: HPLC retention times (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 non-human primates (NHPs) have demonstrated that antibodies that bind to mouse / cynomolgus TfR with effector function and / or complement fixation selectively deplete TfR-expressing reticulocytes.To determine whether the variants had effector function, ADCC assays were performed using peripheral blood mononuclear cells (PBMCs) from healthy human donors as effector cells.

[0463] material:

[0464] PBMC 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 for 30 seconds to allow the cells to adjust to the environmental changes. Slowly 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. Flip gently to resuspend the cell pellet (avoid excessive shear forces). 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. Flip gently to resuspend the cell pellet (avoid excessive shear forces). Resuspend the cells in 10 mL of warm culture medium if necessary. Acclimate the cells by incubating overnight at 37° C. in a T75.

[0471] Harvest target cells HEL-92 and wash 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 (8x10 5) Target cells are plated in assay medium (RPMI-1640 with 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, then incubated on ice for 30 minutes to allow opsonization. 10 μl of medium is added to control wells, maintaining the volume.

[0472] Control:

[0473] - Background low control: Correct the spontaneous release from target cells (low control) with the spontaneous LDH release control of the 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 targets and effector cells without the addition of antibody.

[0476] The following two controls are used to monitor the assay conditions but are not necessary for the ADCC calculation.

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

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

[0479] After incubation on ice for 30 min, 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. Centrifuge the plate after incubation (350 g, 10 minutes). Transfer 50 μL of supernatant to a 96-well clear flat-bottom plate and add 50 μL of reaction mixture to each sample well and tap gently to mix. Incubate the plate for 30 minutes at room temperature protected from light. Add 50 μL of stop solution to each sample well and tap gently to mix. Measure absorbance at 490 nm and 680 nm. To determine LDH activity, subtract the absorbance at 680 nm (background signal from the instrument) 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. Mix 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 4 Cells were plated per well in cold 25 μl assay medium (RPMI-1640 with 1% BSA and 100 units / mL penicillin and streptomycin). Dilutions (5-fold, starting at 100 μg / ml for a final 50 μg / ml) of test and control antibodies (25 μl) were added to the plates containing 25 μl of target cells, then incubated on ice for 30 minutes to allow opsonization.

[0493] Control:

[0494] - Background low control: Correct the spontaneous release from target cells (low control) with the target cell spontaneous LDH release control. 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 targets and CDC cells without the addition of antibody.

[0496] After 30 min incubation, 50 μL of complement was added to each well and the plates were incubated for an additional 60 min at 37°C, except for the medium and low control (with 50 μL of medium). At the end of the incubation (350 g, 10 min), 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 min at room temperature protected from light. The 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 were confluent. At this point, differentiation into myotubes was induced by incubating for 2 days in 1000 μl of 1 mL DMEM (ATCC30-2002) supplemented with 2% horse serum (ATCC 30-2040) and 1xITS (Thermo Fisher Scientific 41400045). The medium was replaced with 500ul differentiation medium and 50ul of TfR1.IgG2 mAb-SSB(Cy5) conjugate diluted in PBS was added to a final concentration of 1 and 10nM. Cells were incubated at 37°C and 5% CO2 for 24 hours, washed 3 times with 500ul PBS and lysed in 150ul of T-PER lysis buffer (Thermo Fisher Scientific 78510) using freeze-thaw cycles. 75ul of lysed cells were diluted with 75ul of nuclease-free water and fluorescence was determined using a TECAN plate reader (Ex 635nM-Em 675nM). 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 1xITS (Thermo Fisher Scientific 41400045). Cells were incubated at 37°C and 5% CO2 until myoblasts were confluent. At this point, differentiation into myotubes was induced by incubating for 2 days in 1000 μl of 1 mL DMEM (ATCC30-2002) supplemented with 2% horse serum (ATCC 30-2040) and 1xITS (Thermo Fisher Scientific 41400045). 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. The medium was removed from the wells for harvesting and 150 μl of Trizol (Ambion 15596018) was added. Plates were frozen at -80°C for at least overnight before analysis. RNA was isolated using the Direct-zol 96 RNA kit according to the manufacturer's instructions and quantified spectrophotometrically. RNA (100=200ng) 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. % of 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). EXAMPLES

[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] EXAMPLES

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

[0511] SSB conjugates of hIgG1 TfR-Var2ii and hIgG1 TfR-Var9ii have no effect on 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 the effector function (LALA+L328R). In contrast to chimeric hIgG2 TfR1 antibodies, reticulocyte values ​​are not reduced after administration of SSB conjugates of hIgG1 TfR-Var2ii and hIgG1 TfR-Var9ii to cynomolgus monkeys. 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 antibody (WO2014 / 189973A2).

[0512] method:

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

[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 gastrocnemius and quadriceps by up to 72% at day 21 post-treatment (Figure 19). The activity of the humanized antibodies is similar to that of the parental chimeric IgG2 TfR1 antibody. No significant downregulation of SSB was observed with unconjugated TfR-Var2ii Ab administered at 60 mg / kg (equivalent to an AOC dose of 6 mg / kg).

[0515] method:

[0516] On day 1, cynomolgus monkeys (male, 2-3 years old, BW 2-3 kg) received a 30 min (+ / - 3 min) intravenous (IV) infusion. On days -10 and +21 post-dose, muscle biopsies (gastrocnemius and quadriceps) were taken from sedated animals via 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 tissues 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 concentrations were quantified using TaqMan qPCR with appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control. The % of mRNA was calculated using the ΔΔCt method, and the SSB mRNA concentration of the same animals before treatment or the SSB concentration of animals treated with PBS was set to 100% expression. EXAMPLES

[0517] AOC-mediated SSB knockdown, but not siRNA delivery, is muscle specific. After a single dose of 6 mg / kg hIgG1 TfR-Var2ii-SSB, 21 days later, we observed SSB siRNA concentrations of 10-100 nM in most tissues. The highest siRNA levels 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 the 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 oligonucleotide payloads by TfR1-targeting antibodies is muscle-specific and directed by cell-specific transport pathways rather than siRNA exposure.

[0518] method:

[0519] On day 1, cynomolgus monkeys (male, 2-3 years old, BW 2-3 kg) were given 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. cDNA obtained from the RT step was then utilized for real-time PCR, and Ct values ​​were converted to plasma or tissue concentrations using the linear equation obtained from the standard curve.

[0520] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent 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 implementing the present disclosure. It is intended that the following claims define the scope of the present disclosure, and that methods and structures within the scope of the claims, and equivalents thereof, are covered by the present disclosure.

Claims

1. 1. Use of an anti-transferrin receptor antibody, or antigen-binding fragment thereof, in the manufacture of a medicament for treating a disease in a subject, said treatment comprising administering said anti-transferrin receptor antibody, or antigen-binding fragment thereof, wherein said anti-transferrin receptor antibody, or antigen-binding fragment thereof, comprises a variable heavy (VH) region and a variable light (VL) region; (a) 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; (b) 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; or (c) 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; 2. Use of an anti-transferrin receptor antibody or antigen-binding fragment thereof, comprising:

2. The use of claim 1 , wherein the anti-transferrin receptor antibody or antigen-binding fragment thereof comprises a multispecific antibody or binding fragment thereof.

3. 1. Use of an antibody or antigen-binding fragment thereof that binds to a transferrin receptor for the manufacture of a medicament for treating a disease in a subject, said treatment comprising administering said antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) region and a variable light chain (VL) region, (a) the VH region comprises the sequence of SEQ ID NO: 14, and the VL region comprises the sequence of SEQ ID NO: 18; (b) the VH region comprises the sequence of SEQ ID NO: 13, and the VL region comprises the sequence of SEQ ID NO: 20, or (c) the VH region comprises the sequence of SEQ ID NO: 15, and the VL region comprises the sequence of SEQ ID NO: 21; Use of an antibody or antigen-binding fragment thereof having the formula:

4. The use according to claim 3 , wherein the antibody or antigen-binding fragment thereof comprises a humanized antibody or antigen-binding fragment thereof, or a chimeric antibody or antigen-binding fragment thereof.

5. 4. The use of claim 3, wherein the antigen-binding fragment is selected from IgG-scFv, BiTE, diabody, DART, TandAb, sc diabody, sc diabody-CH3, triple body, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, Fab', F(ab')2, F(ab')3, F(ab')2-scFv2, scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabody.

6. 6. The use of claim 5, wherein the antigen-binding fragment is selected from a BiTE, a diabody, a DART, a TandAb, a sc diabody, a sc diabody-CH3, a triple body, a scFv-CH3 KIH, a Fab-scFv-Fc KIH, a Fab-scFv, a scFv-CH-CL-scFv, a Fab', a F(ab')2, a F(ab')3, a F(ab')2-scFv2, a scFv, and a scFv-KIH.

7. 2. The use of claim 1, wherein the anti-transferrin receptor antibody comprises at least one of an IgG1 framework, an IgG2 framework, an IgG2b framework, or an IgG4 framework.

8. The use of claim 1, wherein the anti-transferrin receptor antibody further comprises at least one mutation in the Fc region, wherein the at least one mutation modulates an effector function.

9. The use according to claim 8, wherein the effector function comprises weakening or eliminating Fc-γ receptor binding.

10. 9. The use of claim 8, wherein the at least one mutation is at residue position D265, N297, K322, L328, or P329, wherein the residue positions are relative to IgG1.

11. The use of claim 8, wherein the Fc region comprises mutations at L233 and L234, wherein the residues correspond to positions 233 and 234 of SEQ ID NO:

23.

12. The use according to claim 8, wherein the Fc region is based on an IgG1 Fc region or an IgG2b Fc region and comprises mutations at D265 and N297.

13. the anti-transferrin receptor antibody comprises a heavy chain (HC) sequence and a light chain (LC) sequence; (a) the HC comprises a sequence selected from SEQ ID NOs: 29-34, and the LC comprises the sequence of SEQ ID NO: 47; (b) the HC comprises a sequence selected from SEQ ID NOs: 23-28, and the LC comprises the sequence of SEQ ID NO: 49; or (c) the HC comprises a sequence selected from SEQ ID NOs: 35-40, and the LC comprises a sequence of SEQ ID NO: 50; 2. The use according to claim 1.

14. The use of claim 1 , wherein the anti-transferrin receptor antibody specifically binds to the human transferrin receptor (TfR).

15. 1. Use of an anti-transferrin receptor antibody conjugate in the manufacture of a formulation for treating a disease in a subject, the formulation comprising: 【Chemistry 1】 During the ceremony, A comprises an anti-transferrin receptor antibody or antigen-binding fragment thereof, said anti-transferrin receptor antibody or antigen-binding fragment thereof comprising a variable heavy (VH) region and a variable light (VL) region; (a) 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; (b) 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; or (c) the VH region has a HCDR1 sequence comprising SEQ ID NO:1, a HCDR2 sequence comprising SEQ ID NO:5, and a HCDR3 sequence comprising SEQ ID NO:3, and the VL region has a LCDR1 sequence comprising SEQ ID NO:11, a LCDR2 sequence comprising SEQ ID NO:12, and a LCDR3 sequence comprising SEQ ID NO:10; B comprises a polynucleic acid molecule, X １ consists of a single bond or a linker, and Use of an anti-transferrin receptor antibody conjugate, wherein n is an average value selected from 1-12.

16. 1. Use of an anti-transferrin receptor antibody conjugate in the manufacture of a formulation for treating a disease in a subject, the formulation comprising: 【Chemistry 2】 During the ceremony, A comprises an anti-transferrin receptor antibody or antigen-binding fragment thereof, said anti-transferrin receptor antibody or antigen-binding fragment thereof comprising a variable heavy (VH) region and a variable light (VL) region; (a) the VH region comprises the sequence of SEQ ID NO: 14, and the VL region comprises the sequence of SEQ ID NO: 18; (b) the VH region comprises the sequence of SEQ ID NO: 13, and the VL region comprises the sequence of SEQ ID NO: 20, or (c) the VH region comprises the sequence of SEQ ID NO: 15, and the VL region has the sequence of SEQ ID NO: 21; B comprises a polynucleic acid molecule, X １ consists of a single bond or a linker, and Use of an anti-transferrin receptor antibody conjugate, wherein n is an average value selected from 1-12.

17. 17. The use according to claim 15 or 16, wherein the antigen-binding fragment is selected from IgG-scFv, BiTE, diabody, DART, TandAb, sc diabody, sc diabody-CH3, triple body, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, Fab', F(ab')2, F(ab')3, F(ab')2-scFv2, scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabody.

18. 17. The use according to claim 15 or 16, wherein the polynucleic acid molecule is selected from short interfering nucleic acid (siNA), small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), small hairpin RNA (shRNA), antisense oligonucleotide (ASO), phosphorodiamidate morpholino oligo (PMO), and mRNA.

19. The use according to claim 15 or 16, wherein the polynucleic acid molecule is double-stranded RNA (dsRNA).

20. The use according to claim 15 or 16, wherein the polynucleic acid molecule is a single-stranded antisense oligonucleotide (ASO).

21. The use according to claim 15 or 16, wherein the polynucleic acid molecule comprises a passenger strand and a guide strand.

22. The use of claim 21, wherein the guide strand comprises at least one modified internucleotide bond, at least one inverted abasic moiety, at least one 5'-vinylphosphonate modified non-natural nucleotide, or a combination thereof.

23. 17. The use according to claim 15 or 16, wherein the polynucleic acid molecule further comprises a modification of the sugar moiety at the 2'-position, the modification at the 2'-position being selected from 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamide (2'-O-NMA) modified nucleotides.

24. The use of claim 21, 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.

25. A-X １ The use according to claim 21 , wherein said passenger strand is conjugated to the 5′ end of said passenger strand or to the 3′ end of said passenger strand.

26. The use according to claim 15 or 16, wherein said polynucleic acid molecule hybridizes to a target sequence of a gene and said polynucleic acid molecule mediates RNA interference against said gene.

27. 27. The use of claim 26, wherein the genes comprise upregulated genes 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, E3 ligase, forkhead box transcription factor, atrogin-1 gene (FBXO32), MuRF1 gene (TRIM63), FOXO1, FOXO3, MSTN, DMD, or DMPK.

28. 27. The use of claim 26, wherein the RNA interference is preferentially mediated in muscle when the anti-transferrin receptor antibody conjugate is administered to a subject.

29. 2. The use according to claim 1, wherein the disease is muscle atrophy or myotonic dystrophy.

30. 30. The use according to claim 29, wherein the muscle atrophy is diabetes-related muscle atrophy or cancer cachexia-related muscle atrophy.

31. 30. The use of claim 29, 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).

32. 30. The use according to claim 29, wherein the myotonic dystrophy is DM1.

33. The use of claim 1, wherein the subject's reticulocyte levels are not decreased following administration of the anti-transferrin receptor antibody or antigen-binding fragment thereof.

34. 2. The use of claim 1, wherein administration of an anti-transferrin receptor antibody or antigen-binding fragment thereof conjugate downregulates levels of SSB siRNA or SSB mRNA in the subject.

35. The use of claim 34, wherein the downregulation of SSB siRNA or SSB mRNA is in muscle.

36. 36. The use according to claim 35, wherein the muscle is a skeletal muscle.

37. 36. The use according to claim 35, wherein the muscle is cardiac muscle.

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