Anti-transferrin receptor antibodies and their use

Abstract

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

Description

[Technical Field]

[0001] cross reference This application claims the interests of U.S. Provisional Patent Application No. 62 / 784,181, filed on 21 December 2018, which is incorporated herein by reference in its entirety. [Background technology]

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

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

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

[0005] In certain embodiments, anti-transferrin receptor antibodies comprising a variable heavy chain (VH) region and a variable light chain (VL) region are disclosed herein, wherein the VH region comprises an HCDR1 sequence including 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 including SEQ ID NO: 3. In some embodiments, the VH region comprises an HCDR1 sequence including SEQ ID NO: 1, an HCDR2 sequence including SEQ ID NO: 2, and an HCDR3 sequence including SEQ ID NO: 3. In some embodiments, the VH region comprises an HCDR1 sequence including SEQ ID NO: 1, an HCDR2 sequence including SEQ ID NO: 4, and an HCDR3 sequence including SEQ ID NO: 3. In some embodiments, the VH region comprises an HCDR1 sequence including SEQ ID NO: 1, an HCDR2 sequence including SEQ ID NO: 5, and an HCDR3 sequence including SEQ ID NO: 3. In some embodiments, the VL region includes 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, if present it is F. In some embodiments, the VL region includes the LCDR1 sequence including sequence number 6, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, where X5 is selected from D or E, and X6 is present or absent, if present it is F. In some embodiments, the VL region includes the LCDR1 sequence including sequence number 6, the LCDR2 sequence including sequence number 7, and the LCDR3 sequence including sequence number 8. In some embodiments, the VL region includes the LCDR1 sequence including sequence number 6, the LCDR2 sequence including sequence number 9, and the LCDR3 sequence including sequence number 10. In some embodiments, the VL region includes an LCDR1 sequence containing sequence number 11, an LCDR2 sequence containing sequence number 12, and an LCDR3 sequence containing sequence number 10.In some embodiments, the VH region includes an HCDR1 sequence containing sequence number 1, an HCDR2 sequence containing sequence number 2, and an HCDR3 sequence containing sequence number 3, while the VL region includes an LCDR1 sequence containing sequence number 6, an LCDR2 sequence containing sequence number 7, and an LCDR3 sequence containing sequence number 8. In some embodiments, the VH region includes an HCDR1 sequence containing sequence number 1, an HCDR2 sequence containing sequence number 4, and an HCDR3 sequence containing sequence number 3, while the VL region includes an LCDR1 sequence containing sequence number 6, an LCDR2 sequence containing sequence number 7, and an LCDR3 sequence containing sequence number 8. In some embodiments, the VH region includes an HCDR1 sequence containing sequence number 1, an HCDR2 sequence containing sequence number 5, and an HCDR3 sequence containing sequence number 3, while the VL region includes an LCDR1 sequence containing sequence number 6, an LCDR2 sequence containing sequence number 9, and an LCDR3 sequence containing sequence number 10. In some embodiments, the VH region includes an HCDR1 sequence containing SEQ ID NO: 1, an HCDR2 sequence containing SEQ ID NO: 4, and an HCDR3 sequence containing SEQ ID NO: 3, while the VL region includes an LCDR1 sequence containing SEQ ID NO: 11, an LCDR2 sequence containing SEQ ID NO: 12, and an LCDR3 sequence containing SEQ ID NO: 10. In some embodiments, the VH region includes 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 includes 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 includes a humanized antibody or its conjugated fragment, or a chimeric antibody or its conjugated fragment. In some embodiments, the anti-transferrin receptor antibody includes a polyspecific antibody or its conjugated fragment. In some embodiments, the anti-transferrin receptor antibody includes a bispecific antibody or its conjugated fragment.In some embodiments, the anti-transferrin receptor antibody includes IgG-scFv, nanobody, BiTE, diabody, DART, TandAb, scdiabody, scdiabody-CH3, triplebody, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2.scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, tandem scFv-Fc, or intrabody. In some embodiments, the anti-transferrin receptor antibody includes an IgG1 framework. In some embodiments, the anti-transferrin receptor antibody includes an IgG2 framework. In some embodiments, the IgG2 framework is an IgG2b framework. In some embodiments, the anti-transferrin receptor antibody includes an IgG4 framework. In some embodiments, the anti-transferrin receptor antibody further includes at least one mutation in the Fc region. In some embodiments, at least one mutation modulates effector function. In some embodiments, at least one mutation weakens or eliminates Fc-γ receptor binding. In some embodiments, at least one mutation is located at residue positions D265, N297, K322, L328, or P329, where the residue position is relative to IgG1. In some embodiments, the Fc region includes two or more, three or more, or four or more mutations. In some embodiments, the Fc region includes 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 includes mutations at D265 and N297. In some embodiments, the anti-transferrin receptor antibody includes a heavy chain (HC) sequence selected from SEQ ID NOs: 23-46 and a light chain (LC) sequence selected from SEQ ID NOs: 47-50. In some embodiments, the anti-transferrin receptor antibody specifically binds to the human transferrin receptor (TfR).

[0006] In certain embodiments, anti-transferrin receptor antibody conjugates comprising an anti-transferrin receptor antibody described herein and a payload are disclosed herein. In some embodiments, the payload comprises a small molecule, peptide, protein, or polynucleic acid molecule. In some embodiments, the payload comprises a polynucleic acid molecule. In some embodiments, the polynucleic acid molecule comprises short interfering nucleic acid (siNA), small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), small hairpin RNA (shRNA), antisense oligonucleotide (ASO), PMO, or mRNA. In some embodiments, the payload comprises dsRNA. In some embodiments, the payload comprises antisense oligonucleotide (ASO). In some embodiments, the payload comprises a small molecule, peptide, or protein. In some embodiments, the payload comprises a microtubule disruptor, DNA modifier, or Akt inhibitor. In some embodiments, the payload comprises auristatin or its derivatives, drastatin or its derivatives or analogs, maytansinoids, or pyrrolobenzodiazepines or their derivatives. In some embodiments, the auristatin or its derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin 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 agent. In some embodiments, the immunomodulatory agent 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 an 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 the payload to the 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)) is included, where A comprises an anti-transferrin antibody, B comprises a payload, and X 1 The linker consists of a single bond or a linker, and n is an average value selected from 1 to 12. In some embodiments, the payload is a polynucleic acid molecule. In some embodiments, the polynucleic acid molecule includes a passenger strand and a guide strand. In some embodiments, the guide strand includes at least one modified internucleotide bond, at least one inverted debase moiety, at least one 5'-vinylphosphonate-modified non-natural nucleotide, or a combination thereof. In some embodiments, the at least one 5'-vinylphosphonate-modified non-natural 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 includes modification of a sugar moiety at the 2' position. In some embodiments, the 2'-position modification is selected from nucleotides modified with 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). In some embodiments, the passenger chain comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorodiamidate morpholinooligomer-modified non-natural nucleotides. In some embodiments, the passenger strand is shorter than the guide strand, thereby generating a 5' overhang, a 3' overhang, a single blunt end, or a combination thereof. In some embodiments, the passenger strand is equal in length to the guide strand, thereby generating blunt ends at each end of the polynucleic acid molecule. In some embodiments, the passenger strand is AX1 Conjugates to. In some embodiments, A-X 1 conjugates to the 5' end of the passenger strand. In some embodiments, A-X 1 conjugates to the 3' end of the passenger strand. In some embodiments, the anti-transferrin receptor antibody conjugate is A-X 1 -(B-X 2 -C) n (Formula (II)), wherein A comprises an anti-transferrin receptor antibody, B comprises a polynucleic acid molecule, C consists of a polymer, and X 1 consists of a single bond or a first linker, X 2 consists of 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 conjugates to A-X 1 and X 2 -C. In some embodiments, A-X 1 conjugates to the 5' end of the passenger strand, and X 2 -C conjugates to the 3' end of the passenger strand. In some embodiments, X 2 -C conjugates to the 5' end of the passenger strand, and A-X 1 conjugates to the 3' end of the passenger strand. In some embodiments, X 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 endosome-lysing moiety.

[0007] In certain embodiments, a nucleic acid polymer encoding the anti-transferrin receptor antibody described herein is disclosed herein.

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

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

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

[0011] In certain embodiments, methods for treating a subject's cancer are disclosed herein, the methods comprising the step of administering to a subject an anti-transferrin receptor antibody conjugate or a pharmaceutical composition described herein for treating the subject's cancer. In some embodiments, the cancer is a solid tumor. 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 lymphoma, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, esophageal cancer, prostate cancer, kidney cancer, skin cancer, leukemia, thyroid cancer, liver cancer, or uterine cancer. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is recurrent or refractory cancer.

[0012] In certain embodiments, methods for treating a subject's amyotrophy or myotonic dystrophy are disclosed herein, the methods comprising the step of administering to the subject an anti-transferrin receptor antibody conjugate or a pharmaceutical composition described herein, wherein the polynucleic acid molecule hybridizes to a target sequence of atrogyn, and wherein the polynucleic acid molecule mediates RNA interference to atrogyn, thereby treating the subject's amyotrophy. In some embodiments, the amyotrophy is amyotrophy associated with diabetes or amyotrophy associated with cancer cachexia. In some embodiments, the amyotrophy is associated with insulin deficiency, chronic renal failure, congestive heart failure, chronic respiratory disease, chronic infection, fasting, denervation, sarcopenia, or myotonic dystrophy type 1 (DM1). In some embodiments, the subject's reticulocyte levels do not decrease after administration of the anti-transferrin receptor antibody. In some embodiments, administration of an anti-transferrin receptor antibody conjugate downregulates the levels of SSB siRNA or SSB mRNA in the subject. In some embodiments, the downregulation of SSB siRNA or SSB mRNA occurs within the 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 upregulated genes within 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 forkheadbox transcription factor. In some embodiments, the atrogene comprises the atrogin-1 gene (FBXO32), the MuRF1 gene (TRIM63), FOXO1, FOXO3, or MSTN. In some embodiments, the atrogene comprises DMPK. In some embodiments, the subject is human.

[0014] In certain embodiments, a method for treating a subject's muscular dystrophy is disclosed herein, the method comprising the step of administering to a subject an anti-transferrin receptor antibody conjugate or a pharmaceutical composition described herein, thereby treating the subject's muscular dystrophy. 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 human.

[0015] In certain embodiments, kits 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 are disclosed herein. [Brief explanation of the drawing]

[0016] Various aspects of this disclosure are specifically described in the attached claims. A better understanding of the features and merits of this disclosure can be obtained by referring to the following detailed description of exemplary embodiments in which the principles of this disclosure are utilized, and to the attached drawings below. This patent application file includes at least one drawing in color. A copy of this publication of the patent application containing the color drawing will be provided by the Secretariat after the request and payment of the required fees. [Figure 1] An example of an SSB passenger chain structure is shown. [Figure 2] An exemplary blunt-ended double-stranded structure with 19 complementary bases and one 3' dinucleotide overhang is illustrated. Purified single-stranded siRNAs were doubled to obtain double-stranded siRNAs. [Figure 3A]The in vitro binding of TfR1.IgG2 mAb and TfR1.IgG2 mAb-SSB to recombinant human TfR1 is illustrated. [Figure 3B] This paper illustrates the in vitro binding of TfR1.IgG2 mAb and TfR1.IgG2 mAb-SSB to recombinant cynomolgus monkey TfR1. [Figure 4A] The following illustrates the SSB mRNA levels upon siRNA delivery in Hel92.1.7 cells treated with hTfR1.IgG2 mAb SSB or hTfR1.IgG2 mAb MSTN (negative control) conjugates. [Figure 4B] This paper illustrates SSB mRNA levels during siRNA delivery in immortalized human skeletal muscle cells treated with hTfR1.IgG2 mAb SSB or hTfR1.IgG2 mAb MSTN (negative control) conjugates. [Figure 5A] The levels of SSB mRNA and SSB siRNA in the gastrocnemius muscle of cynomolgus monkeys after administration of hTfR1.IgG2 mAb-SSB conjugate at 30 and 60 mg / kg (n=3) are illustrated. [Figure 5B] The levels of SSB mRNA and SSB siRNA in the quadriceps femoris muscle of cynomolgus monkeys after administration of hTfR1.IgG2 mAb-SSB conjugate at 30 and 60 mg / kg (n=3) are illustrated. [Figure 6] The relative reticulocyte levels of cynomolgus monkeys before and after administration of the hTfR1.IgG2 mAb-SSB conjugate at 30 and 60 mg / kg are illustrated. [Figure 7] The binding constants of exemplary anti-TfR antibodies against cynomolgus monkey CD71 are illustrated. [Figure 8] The binding constants of exemplary anti-TfR antibodies against human CD71 are shown. [Figure 9A] This illustrates the binding of an exemplary anti-TfR antibody to TfR under competitive conditions. [Figure 9B] Figure 9A shows the binding constants of the tested anti-TfR antibodies. [Figure 10A]This demonstrates that the binding of exemplary anti-TfR antibodies to TfR is maintained. [Figure 10B] Figure 10A shows the binding constants of the tested anti-TfR antibody. [Figure 11] This shows the ADCC activity of an exemplary anti-TfR antibody. [Figure 12] This shows that the anti-TfR antibody does not bind to TfR2. [Figure 13A] This shows % SSB mRNA knockdown in HEL92 cells. [Figure 13B] Figure 13A shows the EC50 of the tested anti-TfR antibody. [Figure 14] The ADCC activity of exemplary anti-TfR antibodies is illustrated. [Figure 15] The CDC activity of exemplary anti-TfR antibodies is illustrated. [Figure 16] This shows the uptake of the TfR1.mAb conjugate in primary human skeletal muscle cells (myotubes). [Figure 17] This 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 mutants. [Figure 18] This shows the absolute reticulocyte levels in cynomolgus monkeys before and after administration of TfR1-targeted AOC (single dose on day 1). [Figure 19] This shows the SSB mRNA levels in the muscle of cynomolgus monkeys 21 days after a single dose of TfR1 mAb SSB conjugate (n=3). [Figure 20A] This shows the SSB siRNA levels in cynomolgus monkey tissue 21 days after a single 6 mg / kg administration of the hIgG1 TfR-Val2ii-SSB conjugate (n=2). [Figure 20B] This shows the SSB mRNA levels in the tissues of cynomolgus monkeys 21 days after a single 6 mg / kg administration of the hIgG1 TfR-Val2ii-SSB conjugate (n=2). [Modes for carrying out the invention]

[0017] Transferrin receptors (TfRs) are a family of membrane glycoproteins 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 in various cell types, while TfR2 is specifically expressed in hepatocytes.

[0018] In some cases, abnormal expression of TfR1 is prominent in various cancers. In fact, one study showed elevated TfR1 expression levels 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 indicated that iron uptake is elevated in tumor-initiating cells (Rychtarcikova, et al., “Tumorinitiating cells of breast and prostate origin show the alterations in the expression of genes related to iron metabolism,” Oncotarget. 8:6376-6398 (2017)).

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

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

[0021] In some examples, an 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 containing 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 containing SEQ ID NO: 3.

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

[0023] [Table 1]

[0024] In some embodiments, the VH region includes an HCDR1 sequence containing sequence number 1, an HCDR2 sequence containing sequence number 2, 4, or 5, and an HCDR3 sequence containing sequence number 3. In some examples, the VH region includes an HCDR1 sequence containing sequence number 1, an HCDR2 sequence containing sequence number 2, and an HCDR3 sequence containing sequence number 3. In some examples, the VH region includes an HCDR1 sequence containing sequence number 1, an HCDR2 sequence containing sequence number 4, and an HCDR3 sequence containing sequence number 3. In some examples, the VH region includes an HCDR1 sequence containing sequence number 1, an HCDR2 sequence containing sequence number 5, and an HCDR3 sequence containing sequence number 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 includes sequences LCDR1, LCDR2, and LCDR3 selected from Table 2.

[0027] [Table 2]

[0028] In some examples, the VL region includes the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence containing sequence number 7, 9, or 12, and the LCDR3 sequence containing sequence number 8 or 10, where X3 is selected from N or S.

[0029] In some examples, the VL region includes an LCDR1 sequence containing sequence number 6 or 11, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence containing sequence number 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 includes an LCDR1 sequence containing sequence number 6 or 11, an LCDR2 sequence containing sequence number 7, 9, or 12, and an LCDR3 sequence QHFWGTPLTX6, where X6 may or may not be present, and if present, it is F.

[0031] In some examples, the VL region includes the LCDR1 sequence containing sequence number 6, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, where X5 is selected from D or E, and X6 may or may not be present, and if present, it is F.

[0032] In some examples, the VL region contains the LCDR1 sequence containing sequence number 6, the LCDR2 sequence containing sequence number 7, and the LCDR3 sequence containing sequence number 8.

[0033] In some examples, the VL region contains the LCDR1 sequence containing sequence number 6, the LCDR2 sequence containing sequence number 9, and the LCDR3 sequence containing sequence number 10.

[0034] In some examples, the VL region contains the LCDR1 sequence containing sequence number 11, the LCDR2 sequence containing sequence number 12, and the LCDR3 sequence containing sequence number 10.

[0035] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence including 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 including SEQ ID NO: 3, and the VL region comprising 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 containing 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 containing SEQ ID NO: 3, and the VL region comprising an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence containing SEQ ID NO: 7, 9, or 12, and an LCDR3 sequence containing 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, the VH region comprising an HCDR1 sequence containing 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 containing SEQ ID NO: 3, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 6 or 11, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence containing 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, the VH region comprising an HCDR1 sequence containing 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 containing SEQ ID NO: 3, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 6 or 11, an LCDR2 sequence containing 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, the VH region comprising an HCDR1 sequence containing 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 containing SEQ ID NO: 3, and the VL region comprising an LCDR1 sequence containing 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, an anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing 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 containing SEQ ID NO: 3, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 6, an LCDR2 sequence containing SEQ ID NO: 7, and an LCDR3 sequence containing SEQ ID NO: 8.

[0041] In some examples, an anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing 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 containing SEQ ID NO: 3, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 6, an LCDR2 sequence containing SEQ ID NO: 9, and an LCDR3 sequence containing SEQ ID NO: 10.

[0042] In some examples, an anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing 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 containing SEQ ID NO: 3, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 11, an LCDR2 sequence containing SEQ ID NO: 12, and an LCDR3 sequence containing SEQ ID NO: 10.

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

[0044] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, where the VH region comprises an HCDR1 sequence containing SEQ ID NO: 1, an HCDR2 sequence containing SEQ ID NO: 2, and an HCDR3 sequence containing SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence containing SEQ ID NO: 6 or 11, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence containing SEQ ID NO: 8 or 10, where 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, where the VH region comprises an HCDR1 sequence containing SEQ ID NO: 1, an HCDR2 sequence containing SEQ ID NO: 2, and an HCDR3 sequence containing SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence containing SEQ ID NO: 6 or 11, an LCDR2 sequence containing SEQ ID NO: 7, 9, or 12, and an LCDR3 sequence QHFWGTPLTX6, where X6 may or may not be present, and if present, is F.

[0046] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, where the VH region comprises an HCDR1 sequence containing SEQ ID NO: 1, an HCDR2 sequence containing SEQ ID NO: 2, and an HCDR3 sequence containing SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence containing 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.

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

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

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

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

[0051] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, where the VH region comprises an HCDR1 sequence containing SEQ ID NO: 1, an HCDR2 sequence containing SEQ ID NO: 4, and an HCDR3 sequence containing SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence containing SEQ ID NO: 6 or 11, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence containing SEQ ID NO: 8 or 10, where 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, where the VH region comprises an HCDR1 sequence containing SEQ ID NO: 1, an HCDR2 sequence containing SEQ ID NO: 4, and an HCDR3 sequence containing SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence containing SEQ ID NO: 6 or 11, an LCDR2 sequence containing SEQ ID NO: 7, 9, or 12, and an LCDR3 sequence QHFWGTPLTX6, where X6 may or may not be present, and if present, is F.

[0053] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, where the VH region comprises an HCDR1 sequence containing SEQ ID NO: 1, an HCDR2 sequence containing SEQ ID NO: 4, and an HCDR3 sequence containing SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence containing 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.

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

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

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

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

[0058] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, where the VH region comprises an HCDR1 sequence containing SEQ ID NO: 1, an HCDR2 sequence containing SEQ ID NO: 5, and an HCDR3 sequence containing SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence containing SEQ ID NO: 6 or 11, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence containing SEQ ID NO: 8 or 10, where 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, where the VH region comprises an HCDR1 sequence containing SEQ ID NO: 1, an HCDR2 sequence containing SEQ ID NO: 5, and an HCDR3 sequence containing SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence containing SEQ ID NO: 6 or 11, an LCDR2 sequence containing SEQ ID NO: 7, 9, or 12, and an LCDR3 sequence QHFWGTPLTX6, where X6 may or may not be present, and if present, is F.

[0060] In some examples, the anti-transferrin receptor antibody comprises a VH region and a VL region, where the VH region comprises an HCDR1 sequence containing SEQ ID NO: 1, an HCDR2 sequence containing SEQ ID NO: 5, and an HCDR3 sequence containing SEQ ID NO: 3, and the VL region comprises an LCDR1 sequence containing 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.

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

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

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

[0064] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region, where the sequence of the VH region comprises approximately 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 approximately 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 contains sequences selected from sequence numbers 13-16 (Table 3), and the VL region contains sequences selected from sequence numbers 18-21 (Table 4). The regions highlighted in Tables 3 and 4 represent the sequences of CDR1, CDR2, or CDR3, respectively.

[0066] [Table 3]

[0067] [Table 4]

[0068] In some embodiments, the anti-transferrin receptor antibody includes a VH region and a VL region, as illustrated 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 its binding fragment. In some cases, the anti-transferrin receptor antibody is a humanized antibody or its binding fragment, a chimeric antibody or its binding fragment, a monoclonal antibody or its binding fragment, a multispecific antibody or its binding fragment, or a bispecific antibody or its binding fragment. In some cases, the anti-transferrin receptor antibody is a monovalent Fab', a bivalent Fab2, an 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 its binding fragment, or a chemically modified derivative thereof.

[0071] In some embodiments, the anti-transferrin receptor antibody is a polyspecific antibody. In some cases, the polyspecific antibody comprises two or more target-binding sites, each of which specifically binds to an antigen, and the two or more antigens are distinct. In some cases, the polyspecific antibody comprises target-binding sites that specifically bind to three or more distinct antigens, four or more distinct antigens, or five or more distinct 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 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 an antibody based on the T cell receptor (BEAT), bispecific T cell engager (BiTE), Biclonics, Fab-scFv-Fc, Two-in-one / Dual Action This includes Fab (DAF), FinomAb, scFv-Fc-(Fab) fusion, Dock-aNd-Lock (DNL), Adaptir (formerly SCORPION), tandem diabody (TandAb), dual-affinity-retargeting (DART), and nanobody.

[0073] In some cases, a bispecific antibody is a trifunctional antibody or a bispecific mini-antibody. In other cases, a bispecific antibody is a trifunctional antibody. In some cases, a trifunctional antibody is a full-length monoclonal antibody containing binding sites for two different antigens.

[0074] In some cases, bispecific antibodies are bispecific miniantibodies. In some examples, bispecific miniantibodies include bivalent Fab2, F(ab)'3 fragments, bis-scFv(scFv)2, diabodies, minibodies, triabodies, tetrabodies, or bispecific T cell engagers (BiTEs). In some embodiments, a bispecific T cell engager is a fusion protein containing two single-chain variable fragments (scFvs) in which 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 contains an F(ab)'3 fragment or 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, anti-transferrin receptor antibodies include 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 include an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some examples, the anti-transferrin receptor antibody includes an IgG framework (e.g., IgG1, IgG2, IgG3, or IgG4). In some cases, the anti-transferrin receptor antibody includes an IgG1 framework. In some cases, the anti-transferrin receptor antibody includes an IgG2 (e.g., IgG2a or IgG2b) framework. In some cases, the anti-transferrin receptor antibody includes an IgG2a framework. In some cases, the anti-transferrin receptor antibody includes an IgG2b framework. In some cases, the anti-transferrin receptor antibody includes an IgG3 framework. In some cases, the anti-transferrin receptor antibody includes an IgG4 framework.

[0078] In some cases, anti-transferrin receptor antibodies contain one or more mutations in the framework region, for example, in the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some cases, one or more mutations are intended to stabilize the antibody and / or increase its half-life. In some cases, one or more mutations are intended to modulate Fc receptor interactions, thereby reducing or eliminating the function of Fc effectors such as FcyR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In further cases, one or more mutations are intended to modify glycosylation.

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

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

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

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

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

[0084] In some embodiments, the Fc region includes 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 includes mutations at L233 and L234. In some examples, the Fc region includes mutations at L233 and L234 in combination with mutations at residue positions K321, L327, or P328. In some cases, the Fc region includes mutations at L233, L234, and K321. In some cases, the Fc region includes mutations at L233, L234, and L327. In some cases, the Fc region includes mutations at L233, L234, and K321. In some cases, the Fc region contains mutations at L233, L234, and P328. In some examples, the Fc region contains mutations at D264 and N296. In some cases, equivalent positions to residues L233, L234, D264, N296, K321, L327, or P328 in the framework of IgG1, IgG2, IgG3, or IgG4 are intended. In some cases, mutations to residues corresponding to residues L233, L234, D264, N296, K321, L327, or P328 in Sequence ID No. 23 in the framework of IgG1, IgG2, or IgG4 are also intended.

[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 cases, the Fc region comprises L233A, L234A, and K321G. In some cases, the Fc region comprises L233A, L234A, and L327R. In some cases, the Fc region comprises L233A, L234A, and K321G. In some cases, the Fc region includes L233A, L234A, and P328G. In some examples, the Fc region includes D264A and N296G.

[0086] In some embodiments, the human IgG constant region is, for example, Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al., 2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS, 103(11): 4005-4010, Shields et al., 2001 JBC, 276(9): 6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: 152-164; Alegre The amino acid modifications described in 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) contains a sequence selected from Table 6. In some cases, the light chain (LC) contains a sequence selected from Table 7. Highlighted regions indicate their 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 longer than that of a reference anti-transferrin receptor antibody.

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

[0096] In some cases, the antibody or its binding fragment is recombinantly expressed, and the nucleic acid encoding the antibody or its binding fragment is assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the synthesis of duplicate oligonucleotides containing a portion of the sequence encoding the antibody, annealing and ligation of the oligonucleotides, and subsequent amplification of the ligated oligonucleotides by PCR.

[0097] Alternatively, nucleic acid molecules encoding antibodies can be arbitrarily generated 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 that can hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific to a particular gene sequence.

[0098] In some cases, antibodies or their binding may be generated at will by immunizing animals such as rabbits to produce polyclonal antibodies, or more preferably by producing monoclonal antibodies, as described, for example, 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 an antibody can be optionally obtained by screening a Fab expression library (e.g., Huse et al., 1989, Science 246:1275-1281) for clones of FAb fragments that bind to a specific antigen, or by screening an antibody library (see Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

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

[0100] In some embodiments, techniques described for the production of single-chain antibodies (USPat. 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 for the production of single-chain antibodies. Single-chain antibodies are formed by linking heavy and light chain fragments of the Fv region via amino acid bridges, resulting in single-chain polypeptides. Techniques for assembling 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 itself is introduced into host cells by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In certain embodiments, antibody expression is regulated by a constitutive, inducible, or tissue-specific promoter.

[0102] In some embodiments, various host expression vector systems are used to express the antibodies or their conjugated fragments described herein. Such host expression systems represent not only vehicles that generate and then purify the antibody coding sequence, but also cells that are transfected with the appropriate nucleotide coding sequence, or that express the antibody or its conjugated fragment in insights when transfected. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibodies or their binding fragment coding sequences; yeast (e.g., Saccharomyces picia) transformed with recombinant yeast expression vectors containing antibodies or their binding fragment coding sequences; insect cell lines (e.g., baculovirus) infected with recombinant virus expression vectors containing antibodies or their binding fragment coding sequences; plant cell lines infected with recombinant virus 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 antibodies or their binding fragment coding sequences; or mammalian cell lines (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) having recombinant expression constructs containing the genome of mammalian cells (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter).

[0103] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some cases, cell lines that stably express antibodies are manipulated at will. Rather than using expression vectors containing viral replication origins, host cells are transformed with DNA controlled by appropriate expression regulators (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introduction of exogenous DNA, the cells are manipulated and grown in enriched medium for 1-2 days, then switched to selective medium. The selectable markers in the recombinant plasmid provide resistance to selection, allowing the cell to stably integrate the plasmid into its chromosome, grow and form a foci, which can then be cloned and expanded into a cell line. This method can be advantageously used to manipulate cell lines expressing antibodies or their binding fragments.

[0104] In some cases, but not limited to, many selection systems are used, including 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, which are utilized in TK cells, HGPRT cells, or APRT cells, respectively. Similarly, resistance to antimetabolites is used as a selection criterion for the following genes: dhfr, which gives 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 gives resistance to mycophenolate (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which gives resistance to 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), as well as hygro (Santerre et al., 1984, Gene 30:147), which provides resistance to hygromycin.The commonly known methods in the field of available recombinant DNA technology 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 cases, antibody expression levels increase with vector amplification (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) for consideration). If the marker in the antibody-expressing vector system is amplified, an increase in the level of the inhibitor present in the host cell culture increases the number of copies of the labeled gene. Since the amplified region is related to the antibody's nucleotide sequence, antibody production also increases (Crouse et al., 1983, Mol. Cell Biol. 3:257).

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

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

[0108] In some cases, the payload-to-antibody ratio (drug-to-antibody ratio or DAR ratio) is approximately 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, anti-transferrin receptor antibody conjugates are

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

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

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

[0113] In some embodiments, B includes small molecules, peptides, or proteins.

[0114] In some embodiments, B comprises 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 It conjugates to AX. In some cases, AX 1 It is conjugated to the 5' end of the passenger chain. In some cases, AX 1 It is conjugated to the 3' end of the passenger chain.

[0115] In some cases, anti-transferrin receptor antibody conjugates are

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

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

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

[0119] In some cases, X 1 and X 2 Each of these is an independent non-polymer linker.

[0120] In some cases, equation (II)AX 1 -(BX 2 -C) n The conjugate further contains D, which is the endosomal lysis portion.

[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 natural ligation. In some examples, conjugation is 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, As described in 4116-4125. In some examples, the conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, polynucleic acid molecules are site-specifically or non-specifically conjugated to the binding site via natural ligation chemistry.

[0122] In some cases, B is conjugated to A by a site-specific method utilizing "traceless" coupling technology (PhiloChem). In some cases, the "traceless" coupling technology utilizes the N-terminal 1,2-aminothiol group of the binding site that conjugates with polynucleic acid molecules containing aldehyde groups. (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 cases, B is conjugated to A by a site-specific method utilizing a non-natural amino acid introduced into the binding site. In some cases, the non-natural amino acid includes p-acetylphenylalanine (pAcPhe). In some cases, the keto group of pAcPhe is selectively ligated to the alkoxyamine-derived conjugate site 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-specific method utilizing an enzyme-catalyzed process. In some examples, the site-specific method utilizes SMARTag® technology (Redwood). In some examples, SMARTag® technology involves the production of formylglycine (FGly) residues from cysteine ​​by formylglycine-producing enzyme (FGE) via an oxidation process in the presence of an aldehyde tag, and the subsequent conjugation of FGly to alkylhydrazine-functionalized polynucleic acid molecules 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 cases, the enzyme-catalyzed process involves microbial transglutaminase (mTG). In some cases, B conjugates to A using a microbial transglutaminase-catalyzed process. In some cases, mTG catalyzes the formation of a covalent bond between the amide side chain of glutamine in the recognition sequence and the primary amine of the functionalized polynucleic acid molecule. In some cases, mTG is produced from Streptomyces mobarensis. (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 cases, B is conjugated to A by a method described in PCT International Publication WO2014 / 140317, which utilizes a sequence-specific transpeptidase.

[0127] In some examples, B conjugates A in a manner as described in U.S. Patent Application Publications 2015 / 0105539 and 2015 / 0105540.

[0128] payload Polynucleic acid molecules In some embodiments, the payload is a polynucleic acid molecule. In some examples, 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 examples, the polynucleic acid molecule modulates mRNA splicing, thereby regulating the subsequent production of the encoded protein.

[0129] In some embodiments, the polynucleic acid molecule includes short interfering nucleic acids (siNAs), small interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), or small hairpin RNAs (shRNAs).

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

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

[0132] In further embodiments, the polynucleic acid molecule includes mRNA.

[0133] In some cases, polynucleotide molecules hybridize to target sequences of atrophy-related genes (also called atogenes). Atogenes, or atrophy-related genes, are genes that are upregulated or downregulated in atrophying muscles. In some cases, upregulated atogenes include genes encoding proteins involved in ubiquitin ligases, forkheadbox transcription factors, growth factors, deubiquitinating enzymes, or glucocorticoid-induced atrophy. In some cases, the polynucleotide molecules described herein hybridize to target sequences of ubiquitin ligases (e.g., E3 ubiquitin ligase or mitochondrial ubiquitin ligase). In some cases, the polynucleotide molecules described herein hybridize to target sequences of forkheadbox transcription factors. In some cases, the polynucleotide molecules described herein hybridize to target sequences of growth factors. In some cases, the polynucleotide molecules described herein hybridize to target sequences of deubiquitinating enzymes.

[0134] In some embodiments, the polynucleic acid molecules described herein hybridize to the target sequences 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 sequences 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 polynucleotide molecules described herein hybridize to the target sequence of FBXO30. In some cases, the polynucleotide molecules described herein hybridize to the target sequence of FBXO40. In some cases, the polynucleotide molecules described herein hybridize to the target sequence of NEDD4. In some cases, the polynucleotide molecules described herein hybridize to the target sequence of TRIM32. In some cases, the polynucleotide molecules described herein hybridize to the target sequence of MUL1. In some cases, the polynucleotide molecules described herein hybridize to the target sequence of STUB1. In some cases, the polynucleotide molecules described herein hybridize to the target sequence of FOXO1. In some cases, the polynucleotide molecules described herein hybridize to the target sequence of FOXO3. In some cases, the polynucleotide 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 polynucleotide molecules described herein hybridize to the target sequence of CTSL2. In some cases, the polynucleotide molecules described herein hybridize to the target sequence of TGIF. In some cases, the polynucleotide molecules described herein hybridize to the target sequence of MYOG. In some cases, the polynucleotide molecules described herein hybridize to the target sequence of HDAC2. In some cases, the polynucleotide molecules described herein hybridize to the target sequence of HDAC3. In some cases, the polynucleotide molecules described herein hybridize to the target sequence of MT1L. In some cases, the polynucleotide molecules described herein hybridize to the target sequence of MT1B. In some cases, the polynucleotide molecules described herein hybridize to the target sequence of DMPK.

[0135] In some cases, polynucleic acid molecules hybridize to target regions of misspliced ​​mRNA, resulting in diseases or disorders such as, but not limited to, neuromuscular diseases, genetic diseases, cancer, hereditary disorders, or cardiovascular diseases. In some cases, the neuromuscular disease or disorder may be Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy.

[0136] In some cases, polynucleotide molecules target mutated exons in the DMD gene that cause Duchenne muscular dystrophy. Exemplary exons that mutate 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 cases, polynucleotide molecules hybridize to the target regions of oncogenes. Exemplary oncogenes include, but are not limited to, Abl, AKT-2, ALK, AML1 (or RUNX1), AR, AXL, BCL-2, 3, 6, BRAF, c-MYC, EGFR, ErbB-2 (Her2, Neu), Fms, FOS, GLI1, HPRT1, IL-3, INTS2, JUN, KIT, KS3, K-sam, LBC (AKAP13), LCK, LMO1, and LMO2. Examples include 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, polynucleic acid molecules hybridize to the target regions of KRAS, EGFR, AR, HPRT1, CNNTB1(β-catenin), or β-catenin-related genes.

[0138] In some embodiments, the polynucleic acid molecule includes mRNA. In some cases, mRNA encodes a cytotoxic protein or peptide. Exemplary cytotoxic proteins or peptides include α-pore-forming toxins (e.g., cytolysin A from E. coli), β-pore-forming toxins (e.g., α-hemolysin, PVL-pantonevale leucocidin, aerolysin, Clostridium epsilon toxin, Clostridium perfringens enterotoxin), and two-component toxins (anthrax toxin, edema toxin, Clostridium botulinum C2 toxin, Clostridium spiroform toxin, Clostridium perfringens iota toxin, Clostridium difficile Examples of bacterial cytotoxins include cytotoxic toxins (A and B), prions, parasporins, cholesterol-dependent cytolysins (e.g., pneumolysin), pore-forming toxins (e.g., gramicidin A), cyanotoxins (e.g., microcystin, nodularin), hemotoxins, neurotoxins (e.g., botulinum neurotoxin), cytotoxins, cholera toxin, diphtheria toxin, pseudomonas exotoxin A, tetanus toxin, or immunotoxins (idarubicin, lysine A, CRM9, porkweed antiviral protein, DT).

[0139] In some cases, mRNA encodes cytotoxic peptides, or immune system-related peptides such as cytotoxic T cell or B cell epitopes, to stimulate a specific immune response through the presentation of these epitopes with MHC I complexes, complement system membrane attack complex proteins (MACs), perforins, granzymes, and granulosins.

[0140] In some cases, mRNA encodes apoptosis-inducing proteins or peptides such as apoptosis protease activator-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, polynucleic acid molecules are nucleic acid decoys. In some examples, nucleic acid decoys are mimics of protein-binding nucleic acids, such as RNA-based protein-binding mimics. Exemplary nucleic acid decoys include transactivation region (TAR) decoys and rev response element (RRE) decoys.

[0142] In some cases, the payload is an aptamer. An aptamer is a small oligonucleotide or peptide molecule that binds to a specific target molecule. Exemplary nucleic acid aptamers include DNA aptamers, RNA aptamers, or XNA aptamers, which are RNA and / or DNA aptamers containing one or more non-native nucleotides. Exemplary nucleic acid aptamers include ARC19499 (Archemix Corp.), REG1 (Regado Biosciences), and ARC1905 (Ophthotech).

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

[0144] In some embodiments, nucleotide analogs or artificial nucleotide bases comprise nucleic acids having modifications 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 the alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogens, sulfur, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols, and oxygen. In some examples, the alkyl moiety comprises further modifications. In some examples, the modification comprises azo groups, keto groups, aldehyde groups, carboxyl groups, nitro groups, nitroso groups, nitrile groups, heterocyclic (e.g., imidazole, hydrazino, or hydroxylamino) groups, isocyanate or cyanate groups, or sulfur-containing groups (e.g., sulfoxides, sulfones, sulfides, or disulfides). In some examples, the alkyl moiety comprises further heterosubstitutions. In some examples, the carbon of the heterocyclic group is substituted with nitrogen, oxygen, or sulfur. In some examples, heterocyclic substitutions include, but are not limited to, morpholinos, imidazoles, and pyrrolidinos.

[0145] In some cases, modification at the 2'-hydroxyl group is either 2'-O-methyl modification or 2'-O-methoxyethyl (2'-O-MOE) modification. In some cases, 2'-O-methyl modification adds a methyl group to the 2'-hydroxyl group of the ribose moiety, while 2'O-methoxyethyl modification adds a methoxyethyl group to the 2'-hydroxyl group of the ribose moiety. Typical chemical structures of 2'-O-methyl modified adenosine molecules and 2'O-methoxyethyl modified 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 bonds the amine group to the 2' oxygen. In some examples, this modification neutralizes the overall negative charge derived 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 nature. Exemplary chemical structures of 2'-O-aminopropyl nucleoside phosphoramidites are illustrated below.

[0148] [ka]

[0149] In some examples, modification at the 2' hydroxyl group is locked or cross-linked ribose modification (e.g., locked nucleic acid or LNA), where the oxygen molecule bonded at the 2' carbon is linked to the 4' carbon by a methylene group, thereby forming a 2'-C,4'-C-oxymethylene-linked bicyclic ribonucleotide monomer. Exemplary representations of the chemical structure of LNA are illustrated below. The representation shown on the left highlights the chemical bonding of the LNA monomer. The representation shown on the right shows the locked 3'-endo( 3 E) It emphasizes the structure.

[0150] [ka]

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

[0152] [ka]

[0153] In some embodiments, further modification at the 2'-hydroxyl group includes 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 analog is a modified base, for example, 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 having a modification at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halolysine, 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 thiobases, e.g., 2-thiouridine and 4-thiouridine, and 2-thiocytidine, dihydrouridine, pseudouridine, cuosin, alkaeosin, naphthyl and substituted naphthyl groups, any O-alkylated and N-alkylated purines and pyrimidines, e.g., N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine The modified nucleotides include 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groups, e.g., aminophenol or 2,4,6-trimethoxybenzene, modified cytosine acting as a G-clamp nucleotide, 8-substituted adenine and guanine, 5-substituted uracil and thymine, azapyrimidine, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. The modified nucleotides further include nucleotides modified to a sugar moiety, as well as nucleotides having a non-ribosyl sugar or an analogue thereof. For example, the sugar moiety may be mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocyclic or carbocyclic, or based on these.The term nucleotide further includes those known in the art as universal bases. Examples of universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularin.

[0155] In some embodiments, nucleotide analogs further include morpholino, peptide nucleic acid (PNA), methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, 1',5'-anhydrohexitol nucleic acid (HNA), or combinations thereof. Morphorino or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structures mimic native nucleic acid structures by deviating from normal sugar and phosphate structures. In some examples, a five-membered ribose ring is replaced by a six-membered morpholino ring containing four carbon atoms, one nitrogen atom, and one oxygen atom. In some cases, the ribose monomer is bonded by a phosphorodiamidate group instead of a phosphate group. In some cases, the alteration of the skeleton removes all positive and negative charges, allowing the morpholino neutral molecule to pass through the cell membrane without the help of cell delivery agents, such as those used in charged oligonucleotides.

[0156] [ka]

[0157] In some embodiments, the peptide nucleic acid (PNA) does not contain sugar rings or phosphate bonds, and bases are bound to it, appropriately spaced by oligoglycine-like molecules, thereby removing the skeletal charge.

[0158] [ka]

[0159] In some embodiments, one or more modifications may optionally occur at internucleotide bonds. In some examples, the modified internucleotide bonds may include, but are not limited to, phosphorothioates, phosphorodithioates, methylphosphonates, 5'-alkylenephosphonates, 5'-methylphosphonates, 3'-alkylenephosphonates, borontrifluoridates, 3'-5' or 2'-5' linked boranophosphates and selenophosphates, phosphotriesters, thionoalkylphosphotriesters, hydrogen phosphonate bonds, alkylphosphonates, alkylphosphonothioates, arylphosphonothioates, phosphoroselenoates, phosphorodiselenoates, phosphinates, phosphoramidates, 3'-alkylphosphoramides, and phosphoropiperidines. This includes phosphorothioate, phosphoranilothioate, phosphoranilide, ketone, sulfone, sulfonamide, carbonate, carbamate, methylenehydrazo, methylenedimethylhydrazo, formacetal, thioformacetal, oxime, methyleneimino, methylenemethylimino, thioamide, bonding to a riboacetyl group, aminoethylglycine, silyl, or siloxane bonding, for example saturated or unsaturated, and / or substituted, and / or alkyl or cycloalkyl bonding containing or not containing 1 to 10 carbon heteroatoms, morpholino structures in which a base is directly or indirectly bonded to the aza nitrogen of the skeleton, bonding to amides, or polyamides, and combinations thereof. Phosphothioate antisense oligonucleotides (PS ASOs) are antisense oligonucleotides containing a phosphorothioate bond. Exemplary PS ASOs are illustrated below.

[0160] [ka]

[0161] In some examples, the modifications are methyl or thiol modifications, such as methylphosphonate modifications or thiolphosphonate modifications. Exemplary thiolphosphonate nucleotides (left) and methylphosphonate nucleotides (right) are illustrated below.

[0162] [ka]

[0163] In some examples, the modified nucleotides include, but are not limited to, 2'-fluoroN3-P5'-phosphoramidites, as illustrated below:

[0164] [ka]

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

[0166] [ka]

[0167] In some embodiments, the above-described 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 bonds the amine group to the 2' oxygen. In some examples, this modification neutralizes the overall negative charge derived 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 nature.

[0170] In some cases, 5'-vinylphosphonates are further modified with locked or cross-linked ribose modifications (e.g., locked nucleic acids or LNAs), where the oxygen molecule bonded at the 2' carbon is bonded to the 4' carbon by a methylene group, thereby forming a 2'-C,4'-C-oxymethylene-linked bicyclic ribonucleotide monomer. An exemplary representation of the chemical structure of a 5'-vinylphosphonate-modified LNA is shown below, where X is O or S, B is a heterocyclic base moiety, and J is an internucleotide binding group that attaches to adjacent nucleotides of the polynucleotide.

[0171] [ka]

[0172] In some embodiments, further modification at the 2'-hydroxyl group includes 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 analog is a modified base, for example, 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 having a modification at the 5-position, such as 5-(2-amino)propyluridine, 5-halocytidine, 5-halolysine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methyl Ruguanosine, 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, quosin, alkaeosin, naphthyl and substituted naphthyl groups, any O-alkylated and N-alkylated purines and pyrimidines (e.g., N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine) The nucleotides include 5-oxyacetic acid (pyridine-4-one, or pyridine-2-one), phenyl and modified phenyl groups, e.g., aminophenol or 2,4,6-trimethoxybenzene, modified cytosine acting as a G-clamp nucleotide, 8-substituted adenine and guanine, 5-substituted uracil and thymine, azapyrimidine, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. The 5'-vinylphosphonate modified nucleotides further include these nucleotides modified to the sugar moiety, as well as 5'-vinylphosphonate modified nucleotides having a non-ribosyl sugar or an analogue thereof.For example, the sugar moiety may be, in some cases, mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocyclic or carbocyclic, or based on these. The term nucleotide further includes those known in the art as universal bases. Universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularin.

[0174] In some embodiments, 5'-vinylphosphonate-modified nucleotide analogs further include morpholino, peptide nucleic acid (PNA), methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoroN3-P5' phosphoramidite, or 1',5'-anhydrohexitol nucleic acid (HNA). Morphorino or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structures mimic natural nucleic acid structures but deviate from the normal sugar and phosphate structures. In some examples, a five-membered ribose ring is replaced by a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomer is bonded by a phosphorodiamidate group instead of a phosphate group. In some cases, the skeletal alteration removes all positive and negative charges, allowing the morpholino neutral molecule to cross the cell membrane without the help of cell delivery agents, such as those used in charged oligonucleotides. Non-limiting examples of 5'-vinylphosphonate-modified morpholino oligonucleotides are illustrated below, where X is O or S and B is a heterocyclic base moiety.

[0175] [ka]

[0176] In some embodiments, the 5'-vinylphosphonate-modified morpholino or PMO described above is a PMO containing a positive or cationic charge. In some examples, PMO is PMOplus(Sarepta). PMOplus refers to a phosphorodiamidate morpholino oligomer containing any number of (1-piperazino)phosphinylideneoxy, (1-(4-(omega-guanidino-alkanoyl))-piperazino)phosphinylideneoxy bonds (e.g., as described in PCT International Publication WO2008 / 036127). In some examples, PMO is a PMO as 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 comprising at least one bond or at least one of the disclosed terminal modifications, such as those described in PCT International Publication WO2011 / 150408 and U.S. Patent Application Publication 2012 / 0065169.

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

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

[0180] [ka]

[0181] In some embodiments, the peptide nucleic acid (PNA) does not contain sugar rings or phosphate bonds, and bases are bound to it, appropriately spaced by oligoglycine-like molecules, thereby removing the skeletal charge.

[0182]

Chem.

[0183] In some embodiments, one or more modifications of the 5'-vinylphosphonate-modified oligonucleotide optionally occur at the internucleotide linkage. In some examples, the modified internucleotide linkages include, but are not limited to, phosphorothioate, phosphorodithioate, methylphosphonate, 5'-alkylenephosphonate, 5'-methylphosphonate, 3'-alkylenephosphonate, boron trifluoride, boranophosphate ester of 3'-5' linkage or 2'-5' linkage and selenophosphate, phosphotriester, thionoalkylphosphotriester, hydrogen phosphonate linkage, alkylphosphonate, alkylphosphonothioate, arylphosphonothioate, phosphoroselenoate, phosphorodiselenoate, phosphinate, phosphoramidate, 3'-alkylphosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate, phosphoropiperazidate, phosphoranilothioate, phosphoranilidate, ketone, sulfone, sulfonamide, carbonate, carbamate, methylene hydrazo, methylene dimethyl hydrazo, formal acetal, thioformal acetal, oxime, methylene imino, methylene methyl imino, thioamidate, linkage with riboacetyl group, aminoethyl glycine, silyl or siloxane linkage, for example, saturated or unsaturated, and / or substituted, and / or alkyl or cycloalkyl linkage containing 1 to 10 carbons with or without heteroatoms containing heteroatoms, morpholino structure in which the base is directly or indirectly bonded to the azanitrogen of the backbone, amide, or linkage with polyamide, and combinations thereof.

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

[0185] [Chemical formula]

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

[0187] [Chemical formula]

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

[0189] [Chemical formula]

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

[0191] [Chemical formula]

[0192] In some examples, the modified internucleotide linkage is an amide linkage. Non-limiting examples of amide linkages are shown below.

[0193] [Chemical formula]

[0194] In some examples, 5'-vinylphosphonate-modified nucleotides include modified nucleic acids exemplified, but not limited, as follows.

[0195] In some embodiments, one or more modifications involve a modified phosphate skeleton in which the modification generates a neutral or uncharged skeleton. In some examples, the phosphate skeleton is modified by alkylation, which generates an uncharged or neutral phosphate skeleton. As used herein, alkylation includes methylation, ethylation, and propylation. In some cases, alkyl refers to a linear or branched saturated hydrocarbon group containing 1 to 6 carbon atoms, as used herein in the context of alkylation. In some examples, 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 cases, the modified phosphate is a phosphate group described in U.S. Patent No. 9481905.

[0196] In some embodiments, the further modified phosphate skeleton includes methylphosphonate, ethylphosphonate, methylthiophosphonate, or methoxyphosphonate. In some cases, the modified phosphate is methylphosphonate. In some cases, the modified phosphate is ethylphosphonate. In some cases, the modified phosphate is methylthiophosphonate. In some cases, the modified phosphate is methoxyphosphonate.

[0197] In some embodiments, one or more modifications further include modifications of the ribose moiety, phosphate backbone, and nucleoside, or modifications of nucleotide analogs at the 3' or 5' terminus. For example, the 3' terminus optionally includes a 3' cationic group, or by inverting the nucleoside at the 3' terminus via a 3'-3' bond. In another alternative, the 3' terminus optionally conjugates with an aminoalkyl group, e.g., 3'C5-aminoalkyldT. In an additional alternative, the 3' terminus optionally conjugates with a debasing site, e.g., an aprinic acid or apyrimidine acid site. In some examples, the 5' terminus conjugates with an aminoalkyl group, e.g., a 5'-O-aminoalkyl substituent. In some cases, the 5' terminus conjugates with a debasing site, e.g., an aprinic acid or apyrimidine acid site.

[0198] In some embodiments, the polynucleic acid molecule comprises one or more of the artificial nucleotide analogs described herein. In some examples, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificial nucleotide analogs described herein. In some embodiments, the artificial nucleotide analogs include 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, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, or combinations 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 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'-ON-methylacetamide (2'-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidite, 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 2'-O-methyl-modified nucleotides.In some embodiments, the poly nucleic 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, the poly nucleic 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 poly nucleic acid molecule comprises a plurality of phosphorodiamidate morpholino oligomers or a plurality of peptide nucleic acid modified unnatural nucleotides, and optionally comprises at least one inverted abasic moiety. In some examples, the poly nucleic 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 unnatural nucleotides. In some examples, the poly nucleic acid molecule comprises 100% phosphorodiamidate morpholino oligomer modified unnatural nucleotides.

[0200] In some examples, the poly nucleic 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 unnatural nucleotides. In some examples, the poly nucleic acid molecule comprises 100% peptide nucleic acid modified unnatural nucleotides.

[0201] In some embodiments, the poly nucleic acid molecule comprises one or more nucleotide analogs in which each nucleotide analog is stereochemically isomeric. In such examples, the poly nucleic acid molecule is a chiral molecule. In some cases, the nucleotide analog comprises backbone stereochemistry. In further cases, the nucleotide analog comprises chiral analogs as described in U.S. Patent Nos. 9,982,257, 9,695,211, or 9,605,019.

[0202] In some examples, polynucleic acid molecules contain at least one of the following modification levels: approximately 5% to approximately 100%, approximately 10% to approximately 100%, approximately 20% to approximately 100%, approximately 30% to approximately 100%, approximately 40% to approximately 100%, approximately 50% to approximately 100%, approximately 60% to approximately 100%, approximately 70% to approximately 100%, approximately 80% to approximately 100%, and approximately 90% to approximately 100%.

[0203] In some cases, polynucleic acid molecules contain at least one of the following modifications: approximately 10% to approximately 90%, approximately 20% to approximately 90%, approximately 30% to approximately 90%, approximately 40% to approximately 90%, approximately 50% to approximately 90%, approximately 60% to approximately 90%, approximately 70% to approximately 90%, and approximately 80% to approximately 100%.

[0204] In some cases, polynucleic acid molecules contain at least one of the following modifications: approximately 10% to approximately 80%, approximately 20% to approximately 80%, approximately 30% to approximately 80%, approximately 40% to approximately 80%, approximately 50% to approximately 80%, approximately 60% to approximately 80%, and approximately 70% to approximately 80%.

[0205] In some examples, polynucleic acid molecules contain at least one of the following modifications: approximately 10% to approximately 70%, approximately 20% to approximately 70%, approximately 30% to approximately 70%, approximately 40% to approximately 70%, approximately 50% to approximately 70%, and approximately 60% to approximately 70%.

[0206] In some examples, polynucleic acid molecules contain at least one of the following modifications: approximately 10% to approximately 60%, approximately 20% to approximately 60%, approximately 30% to approximately 60%, approximately 40% to approximately 60%, and approximately 50% to approximately 60%.

[0207] In some cases, polynucleic acid molecules contain at least one of the following modifications: approximately 10% to approximately 50%, approximately 20% to approximately 50%, approximately 30% to approximately 50%, and approximately 40% to approximately 50%.

[0208] In some cases, polynucleic acid molecules contain at least one of the following modifications: approximately 10% to approximately 40%, approximately 20% to approximately 40%, and approximately 30% to approximately 40%.

[0209] In some cases, polynucleic acid molecules contain at least one of approximately 10% to 30% modifications and approximately 20% to 30% modifications.

[0210] In some cases, polynucleotide molecules contain approximately 10% to 20% modification.

[0211] In some cases, polynucleic acid molecules contain approximately 15% to 90%, 20% to 80%, 30% to 70%, or 40% to 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% modification.

[0213] In some embodiments, the polynucleic acid molecule includes 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, polynucleic acid molecules contain 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, approximately 5% to approximately 100% of the polynucleic acid molecule contains the artificial nucleotide analogs described herein. In some examples, approximately 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 molecule contains the artificial nucleotide analogs described herein. In some examples, approximately 5% of the polynucleic acid molecule contains the artificial nucleotide analogs described herein. In some examples, approximately 10% of the polynucleic acid molecule contains the artificial nucleotide analogs described herein. In some examples, approximately 15% of the polynucleic acid molecule contains the artificial nucleotide analogs described herein. In some examples, approximately 20% of the polynucleic acid molecule contains the artificial nucleotide analogs described herein. In some examples, approximately 25% of the polynucleic acid molecule contains the artificial nucleotide analogs described herein. In some examples, approximately 30% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 35% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 40% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 45% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 50% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 55% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 60% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 65% ​​of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 70% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 75% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 80% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 85% of the polynucleic acid molecules contain the artificial nucleotide analogs described herein.In some examples, approximately 90% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 95% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 96% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 97% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 98% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 99% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 100% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some embodiments, the artificial nucleotide analogs include 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, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, or combinations thereof.

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

[0217] In some embodiments, the polynucleic acid molecule is assembled from two distinct polynucleotides, where one polynucleotide comprises a sense strand and the second polynucleotide comprises the antisense strand of the polynucleic acid molecule. In other embodiments, the sense strand is linked to the antisense strand by a linker molecule, which in some examples 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, where the pyrimidine nucleotide in the sense strand comprises a 2'-O-methylpyrimidine nucleotide, and the purine nucleotide in the sense strand comprises a 2'-deoxypurine nucleotide. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, where the pyrimidine nucleotide present in the sense strand comprises a 2'-deoxy-2'-fluoropyrimidine nucleotide, and the purine nucleotide present in the sense strand comprises a 2'-deoxypurine nucleotide.

[0219] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, where the pyrimidine nucleotide, if present in the antisense strand, is a 2'-deoxy-2'-fluoropyrimidine nucleotide, and the purine nucleotide, if present in the antisense strand, is a 2'-O-methylpurine nucleotide.

[0220] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, where the pyrimidine nucleotide, if present in the antisense strand, is a 2'-deoxy-2'-fluoropyrimidine nucleotide, and the purine nucleotide, if present in the antisense strand, comprises a 2'-deoxy-purine nucleotide.

[0221] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, where the sense strand includes terminal capping portions at the 5'-end, 3'-end, or both the 5' and 3' ends of the sense strand. In other embodiments, the terminal capping portions are inverted deoxydecate portions.

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

[0224] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, where the sense strand comprises 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 nucleotide interlinks 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) universally base-modified nucleotides, as well as optionally the 3'-terminus, 5'-terminus, or both 3'- and 5'-terminus of the sense strand. The antisense chain contains cap molecules, and optionally includes about 1 to about 10 or more, in particular about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate nucleotide interbondings, 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) universally base-modified nucleotides, as well as terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the antisense chain. In other embodiments, one or more pyrimidine nucleotides of the sense and / or antisense strands, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoronucleotides, or with or without one or more phosphorothioate nucleotide interbonds, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, and / or with 3'-terminus, 5'-terminus, or both 3'- and 5'-terminus end cap molecules present on the same or different strands.

[0225] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, where the sense strand comprises 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 nucleotide interlinks 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) universally base-modified nucleotides, as well as optionally the 3'-end, 5'-end, or both ends of the sense strand. The antisense chain comprises cap molecules, and the antisense chain comprises 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 nucleotide interbondings, 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) universally base-modified nucleotides, as well as optionally terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the antisense chain. In other embodiments, one or more pyrimidine nucleotides of the sense and / or antisense strands, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoronucleotides, with or without 3'-terminal, 5'-terminal, or both 3'- and 5'-terminal terminal cap molecules present on the same or different strands.

[0226] In some embodiments, a polynucleic acid molecule comprises a sense strand and an antisense strand, where the antisense strand comprises 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 nucleotide interlinks 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) universally base-modified nucleotides, as well as optionally the 3'-terminus, 5'-terminus, or The antisense strand comprises terminal cap molecules at both the 3'- and 5'- ends, and optionally includes terminal cap molecules at the 3'-, 5'-, or both the 3'- and 5'- ends. In other embodiments, one or more sense and / or antisense strands, for example, 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, are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoronucleotides, with or without using 3'-terminus, 5'-terminus, or both 3' and 5'-terminus end cap molecules present on the same or different strands.

[0227] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, where the antisense strand comprises 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 nucleotide interlinks 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) universally base-modified nucleotides, as well as optionally the 3'-terminus, 5'-terminus, or 3'- and 5'-terminus of the sense strand. The antisense chain contains both terminal cap molecules, and the antisense chain has 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 nucleotide interbonds, 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, as well as optionally containing terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the antisense chain. In other embodiments, one or more sense and / or antisense strands, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides, are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoronucleotides, with or without using 3'-terminus, 5'-terminus, or both 3'- and 5'-terminus end cap molecules 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 extended pharmacokinetics. In some embodiments, the double-stranded polynucleic acid molecule comprises a passenger strand (e.g., sense strand) and a guide strand (e.g., antisense strand) with multiple modifications.

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

[0230] In some embodiments, the polynucleic acid molecules described herein are chemically modified short-interference nucleic acid molecules having about 1 to about 25, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate nucleotide interbondings in each chain of the polynucleic acid molecule.

[0231] In another embodiment, the polynucleic acid molecules described herein include 2'-5' nucleotide internucleotide bonds. In some examples, the 2'-5' nucleotide internucleotide bonds are located at the 3'-terminus, 5'-terminus, or both the 3'-terminus and 5'-terminus of one or both sequence strands. In further examples, the 2'-5' nucleotide internucleotide bonds are located at various other positions within one or both sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides containing all the nucleotide internucleotide bonds of pyrimidine nucleotides in one or both strands of the polynucleic acid molecule include 2'-5' nucleotide internucleotide bonds, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides containing all the nucleotide internucleotide bonds of purine nucleotides in one or both strands of the polynucleic acid molecule include 2'-5' nucleotide internucleotide bonds.

[0232] In some cases, the polynucleotide molecule is a polynucleotide having a double, asymmetrical, hairpin-shaped, or asymmetrical hairpin-shaped secondary structure with a self-complementary sense region and an antisense region, where the antisense region contains a nucleotide sequence complementary to the nucleotide sequence of another target nucleic acid molecule or a portion thereof, and the sense region contains a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In other cases, the polynucleotide molecule is a cyclic single-chain polynucleotide having two or more loop structures and a base containing a self-complementary sense region and an antisense region, where the antisense region contains a nucleotide sequence complementary to the nucleotide sequence of a target nucleic acid molecule or a portion thereof, and the sense region contains a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and the cyclic polynucleotide is processed in vivo or in vitro to produce an active polynucleotide molecule capable of mediating RNAi. In further cases, the polynucleic acid molecule further comprises a single-chain polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid molecule or a portion thereof (for example, such a polynucleic acid molecule does not need to be present in the polynucleic acid molecule of the nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), and the single-chain 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-chain polynucleic acid molecule that mediates RNAi activity in a cell or a reconstituted in vitro system, wherein the polynucleic acid molecule comprises a single-chain polynucleotide complementary to a target nucleic acid sequence, and one or more pyrimidine nucleotides present in the polynucleic acid are 2'-deoxy-2'-fluoropyrimidine nucleotides (for example, here all pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides, or alternatively, multiple pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides), and any purine nucleotides present in the polynucleic acid are 2'-deoxypurine nucleotides. The polynucleotides are 2'-deoxyribonucleotides (for example, all purine nucleotides are 2'-deoxypurine nucleotides, or alternatively, multiple purine nucleotides are 2'-deoxypurine nucleotides), and terminal cap modifications are optionally present at the 3'-terminus, 5'-terminus, or both the 3' and 5'-terminus of the antisense sequence, and the polynucleotide molecule optionally further contains about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2'-deoxyribonucleotides at the 3' terminus of the polynucleotide molecule, where the terminal nucleotides further contain one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide bonds, and the polynucleotide molecule optionally further contains terminal phosphate groups such as a 5'-terminal phosphate group.

[0234] In some examples, an asymmetric double helix is ​​a linear polynucleotide molecule comprising an antisense region, a loop region containing nucleotides or non-nucleotides, and a sense region, wherein the sense region contains fewer nucleotides than the antisense region to the extent that it has enough complementary nucleotides to base-pair with the antisense region and form a double helix with a loop. For example, an asymmetric hairpin polynucleotide molecule has an antisense region (e.g., about 19 to about 22 nucleotides) that is long enough to mediate RNAi in a cell or in vitro system and has a loop region containing about 4 to about 8 nucleotides, and a sense region having about 3 to about 18 nucleotides complementary to the antisense region. In some cases, an asymmetric hairpin polynucleotide molecule further includes a chemically modified 5' terminal phosphate group. In further cases, the loop region of an asymmetric hairpin polynucleotide molecule contains nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

[0235] In some embodiments, an asymmetric double helix is ​​a polynucleic acid molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region contains fewer nucleotides than the antisense region to the extent that it has enough complementary nucleotides to form a double helix with the antisense region. For example, an asymmetric double polynucleic acid molecule comprises an antisense region (e.g., about 19 to about 22 nucleotides) that is long enough 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 may have resistance to nucleases such as ribonucleases like RNase H, deoxyribonucleases like DNase, or exonucleases like 5'-3' exonuclease and 3'-5' exonuclease, compared to natural 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), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidite, or combinations thereof, are RNases. These molecules possess resistance to nucleases such as ribonucleases like RNase H, deoxyribonucleases like DNase, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases. In some cases, 2'-O-methyl modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, 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 resistance). In some cases, 2'-O-aminopropyl modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, 2'-deoxy-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease).In some cases, T-deoxy-2'-O-fluoro-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 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). In some cases, 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). In some cases, 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). In some cases, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 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). In some cases, LNA-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, ENA-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, HNA-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease).In some cases, morpholino is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, PNA-modified polynucleic acid molecules are resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, methylphosphonate-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, thiolphosphonate-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, polynucleic acid molecules containing 2'-fluoroN3-P5'-phosphoramidites are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, the 5' conjugate described herein inhibits 5'-3' exonuclease cleavage. In some cases, the 3' conjugate described herein inhibits 3'-5' exonuclease cleavage.

[0237] In some embodiments, one or more of the artificial nucleotide analogs described herein exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. One or more 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), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, or 2'-fluoroN3-P5'-phosphoramidite, exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-methyl-modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-methoxyethyl (2'-O-MOE)-modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-aminopropyl-modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, 2'-deoxy-modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, T-deoxy-2'-fluoro-modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-aminopropyl (2'-O-AP)-modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE)-modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules.In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, LNA modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, ENA modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, PNA modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, HNA-modified polynucleotide molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules. In some cases, morpholino-modified polynucleotide molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules. In some cases, methylphosphonate-modified polynucleotide molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules. In some cases, thiolphosphonate-modified polynucleotide molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules. In some cases, polynucleotide molecules containing 2'-fluoroN3-P5'-phosphoramidite exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules. In some cases, the increased affinity is exemplified by low Kd, high melting temperature (Tm), or a combination thereof.

[0238] In some embodiments, the polynucleic acid molecules described herein are chiral pure (or stereopure) polynucleic acid molecules, or polynucleic acid molecules comprising 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 enantiomers. 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 molecules are those described in U.S. Patent Application Publications 2014 / 194610 and 2015 / 211006, and PCT International Publication WO2015107425.

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

[0240] In further embodiments, the polynucleic acid molecules described herein are modified to increase their stability. In some embodiments, the polynucleic acid molecule is RNA (e.g., siRNA). In some examples, the polynucleic acid molecule is modified 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 by modifications such as 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), or by locked or cross-linked ribose structures (e.g., LNA or ENA). In some cases, polynucleotide molecules are modified with 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, polynucleotide molecules further contain morpholino, PNA, HNA, methylphosphonate nucleotides, thiolphosphonate nucleotides, and / or 2'-fluoroN3-P5'-phosphoramidite to increase their stability. In some examples, polynucleotide molecules are chiral-pure (or stereopure) polynucleotide molecules. In some examples, chiral-pure (or stereopure) polynucleotide molecules are modified to increase their stability. Appropriate modifications of RNA to increase delivery stability will be apparent to those skilled in the art.

[0241] In some cases, universal bases refer to nucleotide base analogs that form base pairs with each of the nearly indistinguishable native DNA / RNA bases. Non-exclusive examples of universal bases include C-phenyl, C-naphthyl, and other aromatic derivatives known in the prior art, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, e.g., 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 disruptors, DNA modifiers, or Akt inhibitors.

[0243] In some embodiments, the payload includes a microtubule disruptor. Exemplary microtubule disruptors include, but are not limited to, 2-methoxyestradiol, auristatin, chalcone, colchicine, combretastatin, cryptophycin, dictiostatin, discodermorid, dolastain, eryuterobin, epothilon, halichondrin, laurimalid, meitansine, noscapinoid, paclitaxel, perolside, fomopsin, podophyllotosin, rhizoxin, spongstatin, taxane, tubulysin, vinca alkaloid, vinorelbine, or their derivatives or analogues.

[0244] In some embodiments, tubulicin is an analog or derivative of tubulicin as described in U.S. Patents 8,580,820 and 8,980,833, and U.S. Patent Publications 20130217638, 20130224228, and 201400363454.

[0245] In some embodiments, maytansin is a maytansinoid. In some embodiments, the maytansinoid is DM1, DM4, or anthamitosin. In some embodiments, the maytansinoid is DM1. In some embodiments, the maytansinoid is DM4. In some embodiments, the maytansinoid is anthamitosin. In some embodiments, the maytansinoid is a derivative or analog of a maytansinoid as described in U.S. Patents 5,208,020, 5,416,064, 7,276,497, and 6,716,821, or U.S. Patent Publications 2013,029,900 and 2013,032,3268.

[0246] In some embodiments, the payload is drastine or a derivative or analog thereof. In some embodiments, drastine is drastine 10 or drastine 15, or a derivative or analog thereof. In some embodiments, the drastine 10 analog is auristatin, sobridotin, simprostatin 1, or simprostatin 3. In some embodiments, the drastine 15 analog is semadotin or tacidotin.

[0247] In some embodiments, the durastin 10 analog is auristatin or an auristatin derivative. In some embodiments, the auristatin or auristatin derivative is auristatin E (AE), auristatin F (AF), auristatin E 5-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, auristatin is an auristatin derivative or analog as described in U.S. Patents 6,884,869, 7659,241, 7498,298, 7964,566, 7750,116, 8288,352, 8703,714, and 8871,720.

[0248] In some embodiments, the payload includes a DNA modifier. In some embodiments, the DNA modifier includes a DNA cleavage agent, a DNA intercalator, a DNA transcription inhibitor, or a DNA crosslinker. In some examples, the DNA cleavage agent includes bleomycin A2, calicheamicin, or its derivatives or analogs. In some examples, the DNA intercalator includes doxorubicin, epirubicin, PNU-159682, duocalmycin, pyrrolobenzodiazepine, oligomycin C, daunorubicin, barurubicin, topotecan, or its derivatives or analogs. In some examples, the DNA transcription inhibitor includes dactinomycin. In some examples, the DNA crosslinker includes mitomycin C.

[0249] In some embodiments, the DNA modifier includes amsacrin, anthracycline, camptothecin, doxorubicin, duocalmycin, enediyne, etoposide, indolinobenzodiazepine, netropsin, teniposide, or their derivatives or analogues.

[0250] In some embodiments, the anthracycline is doxorubicin, daunorubicin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mitramycin, nemorubicin, pixantrone, subarubicin, or barurubicin.

[0251] In some embodiments, the camptothecin analog is topotecan, irinotecan, silatecan, cositecan, exatecan, rhutecan, jaimatecan, belothecan, rubitecan, or SN-38.

[0252] In some embodiments, duocalmycin is duocalmycin A, duocalmycin B1, duocalmycin B2, duocalmycin C1, duocalmycin C2, duocalmycin D, duocalmycin SA, or CC-1065. In some embodiments, the engine is calichemycin, esperamicin, or dynemycin A.

[0253] In some embodiments, the pyrrolobenzodiazepine is anthramycin, abeymycin, thikamycin, DC-81, mazetramycin, neotramycin A, neotramycin B, polotoramycin, protracalcin, cibanomycin (DC-102), cibilomycin, or tomaimycin. In some embodiments, the pyrrolobenzodiazepine is a tomaimycin derivative as described in U.S. Patents 8,404,678 and 8,163,736. In some embodiments, pyrrolobenzodiazepines are those described in U.S. Patents No. 8,426402, 8802667, 8809320, 6562806, 6608192, 7704924, 7067511, US7612062, 7244724, 7528126, 7049311, 8633185, 8501934, and 8697688, as well as U.S. Patent 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. 2014,028,697.

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

[0256] In some embodiments, the payload includes polymerase inhibitors, including, but not limited to, polymerase II inhibitors such as α-amanitin and poly(ADP-ribose) polymerase (PARP) inhibitors. Exemplary PARP inhibitors include, but not limited to, iniparib (BSI 201), talazoparib (BMN-673), olaparib (AZD-2281), olaparib, lucaparib (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-rays. Radiopaque materials are well known to those skilled in the art. Exemplary radiopaque materials include iodides, bromides, or barium salts. Additional radiopaque materials include, but are not limited to, organobismuth derivatives (e.g., U.S. Patent No. 5,939,045), radiopaque polyurethanes (e.g., see U.S. Patent No. 5,346,981), organobismuth composites (e.g., see U.S. Patent No. 5,256,334), and radiopaque barium polymer composites (e.g., see U.S. Patent No. 4,866,132).

[0258] In some examples, the payload includes, for example, a detectable label used in an immunoconjugate, and includes any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. 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 quantitative 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 radiative labeling is, but is 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 cases, the payload is ionizing radiation to the cells. 60 The present invention includes radiosensitizers that enhance the cytotoxic effects of substances (such as those produced by Co or X-ray sources). Many radiosensitizers are known and include, but are not limited to, benzoporphyrin derivative compounds (see, e.g., U.S. Patent No. 5,945,439), 1,2,4-benzotriazine oxide (see, e.g., U.S. Patent No. 5,849,738), compounds containing certain diamines (see, e.g., U.S. Patent No. 5,700,825), BCNT (see, e.g., U.S. Patent No. 5,872,107), radiosensitized nitrobenzoic acid amide derivatives (see, e.g., U.S. Patent No. 4,474,814), various heterocyclic derivatives (see, e.g., U.S. Patent No. 5,064,849), platinum complexes (see, e.g., U.S. Patent No. 4,921,963), and others.

[0261] In some cases, the payload obtains an alpha emitter, i.e., a radioactive isotope that emits alpha particles. Alpha emitters have recently been shown to be effective in treating cancer (see, for example, 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 are not limited to, 213 Bi, 211 Includes At, etc.

[0262] In some cases, the payload includes immunomodulators. Useful immunomodulators include antihormone agents that inhibit the hormonal effects on tumors, and immunosuppressants that suppress cytokine production, downregulate the expression of autoantigens, or mask MHC antigens. Representative antihormone agents include antiestrogens such as tamoxifen, raloxifene, aromatase inhibitory 4(5)-imidazoles, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY 117018, onapunstone, and toremifene, as well as antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, and antiadrenal agents. Examples of immunosuppressants, though not limited to them, include 2-amino-6-aryl-5-substituted pyrimidines, azathioprine, cyclophosphamide, bromocriptine, danazol, dapsone, glutaraldehyde, anti-idiotype antibodies against MHC antigens and MHC fragments, cyclosporine A, steroids such as glucocorticosteroids, streptokinase, or rapamycin.

[0263] In some embodiments, the payload includes a protein or peptide toxin or a fragment thereof. Exemplary enzymatically active toxins or fragments thereof include, but are not limited to, diphtheria toxin A fragment, unbound active fragment of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesin A chain, α-sarcin, certain tung tree proteins, certain diansin proteins, pokeweed proteins (PAP, PAPII, and PAP-S), bitter melon inhibitors, curcin, crotin, saponaria officinalis inhibitors, geronin, mitogillin, restrictosin, phenomycin, enomycin, and trichothecenes.

[0264] In some cases, the payload is an immunomodulator. Exemplary immunomodulators include, but are not limited to, ganciclovies, etanercept, tacrolimus, sirolimus, voclosporine, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and their analogues, xanthines, stem cell growth factors, lymphatoxins, hematopoietic factors, tumor necrosis factor (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., Examples include interferon-alpha, interferon-beta, interferon-gamma, stem cell growth factors known as "S1 factor," erythropoietin, and thrombopoietin, or combinations thereof.

[0265] In some examples, the payload includes cytokines. In some embodiments, the cytokines include IL-2, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, interferons (e.g., IFNα, IFNβ), or TNFα.

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

[0267] In some examples, C is the PEG portion. In some examples, the PEG portion conjugates to the 5' end of the polynucleotide molecule, while the binding portion conjugates to the 3' end. In some examples, the PEG portion conjugates to the 3' end of the polynucleotide molecule, while the binding portion conjugates to the 5' end. In some examples, the PEG portion conjugates to an internal region of the polynucleotide molecule. In some examples, the PEG portion, the binding portion, or a combination thereof conjugates to an internal region of the polynucleotide molecule. In some examples, the conjugation is direct. In some examples, the conjugation is via a natural ligation.

[0268] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some examples, the polydisperse material includes a dispersion distribution of materials of different molecular weights, characterized by an average weight (weight-average) size and degree of dispersion. 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 indicated molecular weight 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 approximately 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, 27 The values ​​are 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 100,000Da.

[0270] In some embodiments, C is a polyalkylene oxide (e.g., PEG), and the values ​​are approximately 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. It has a molecular weight of 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 the values ​​are approximately 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 It has molecular weights 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 approximately 200 Da. In some examples, the molecular weight of C is approximately 300 Da. In some examples, the molecular weight of C is approximately 400 Da. In some examples, the molecular weight of C is approximately 500 Da. In some examples, the molecular weight of C is approximately 600 Da. In some examples, the molecular weight of C is approximately 700 Da. In some cases, the molecular weight of C is approximately 800 Da. In some cases, the molecular weight of C is approximately 900 Da. In some cases, the molecular weight of C is approximately 1000 Da. In some cases, the molecular weight of C is approximately 1100 Da. In some cases, the molecular weight of C is approximately 1200 Da. In some cases, the molecular weight of C is approximately 1300 Da. In some cases, the molecular weight of C is approximately 1400 Da. In some cases, the molecular weight of C is approximately 1450 Da.In some cases, the molecular weight of C is approximately 1500 Da. In some cases, the molecular weight of C is approximately 1600 Da. In some cases, the molecular weight of C is approximately 1700 Da. In some cases, the molecular weight of C is approximately 1800 Da. In some cases, the molecular weight of C is approximately 1900 Da. In some cases, the molecular weight of C is approximately 2000 Da. In some cases, the molecular weight of C is approximately 2100 Da. In some cases, the molecular weight of C is approximately 2200 Da. In some cases, the molecular weight of C is approximately 2300 Da. In some cases, the molecular weight of C is approximately 2400 Da. In some cases, the molecular weight of C is approximately 2500 Da. In some cases, the molecular weight of C is approximately 2600 Da. In some cases, the molecular weight of C is approximately 2700 Da. In some cases, the molecular weight of C is approximately 2800 Da. In some cases, the molecular weight of C is approximately 2900 Da. In some cases, the molecular weight of C is approximately 3000 Da. In some cases, the molecular weight of C is approximately 3250 Da. In some cases, the molecular weight of C is approximately 3350 Da. In some cases, the molecular weight of C is approximately 3500 Da. In some cases, the molecular weight of C is approximately 3750 Da. In some cases, the molecular weight of C is approximately 4000 Da. In some cases, the molecular weight of C is approximately 4250 Da. In some cases, the molecular weight of C is approximately 4500 Da. In some cases, the molecular weight of C is approximately 4600 Da. In some cases, the molecular weight of C is approximately 4750 Da. In some cases, the molecular weight of C is approximately 5000 Da. In some cases, the molecular weight of C is approximately 5500 Da. In some cases, the molecular weight of C is approximately 6000 Da. In some cases, the molecular weight of C is approximately 6500 Da. In some cases, the molecular weight of C is approximately 7000 Da. In some cases, the molecular weight of C is approximately 7500 Da. In some cases, the molecular weight of C is approximately 8000 Da. In some cases, the molecular weight of C is approximately 10,000 Da. In some cases, the molecular weight of C is approximately 12,000 Da. In some cases, the molecular weight of C is approximately 20,000 Da. In some cases, the molecular weight of C is approximately 35,000 Da. In some cases, the molecular weight of C is approximately 40,000 Da. In some cases, the molecular weight of C is approximately 50,000 Da.In some cases, the molecular weight of C is approximately 60,000 Da. In other cases, the molecular weight of C is approximately 100,000 Da.

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

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

[0273] [ka]

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

[0275] In some cases, cMAP is further conjugated to the PEG portion to produce cMAP-PEG copolymers, mPEG-cMAP-PEGm triblock polymers, or cMAP-PEG-cMAP triblock polymers. In some cases, the PEG portion is in the range of approximately 500 Da to approximately 50,000 Da. In some cases, the PEG portion is in the range of approximately 500 Da to approximately 1000 Da, over 1000 Da to approximately 5000 Da, over 5000 Da to approximately 10,000 Da, over 10,000 to approximately 25,000 Da, over 25,000 Da to approximately 50,000 Da, or any combination of two or more of these ranges.

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

[0277] Endosome lysis portion In some embodiments, the anti-transferrin receptor antibody conjugate further comprises an additional conjugation moiety. In some examples, the additional conjugation moiety is an endosomal lysate moiety. In some cases, the endosomal lysate moiety is a cellular compartment release component, a compound that can be released from any of the cellular compartments known in the art, such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other endoplasmic reticulum having cells. In some cases, the endosomal lysate moiety comprises an endosomal lysate polypeptide, an endosomal lysate polymer, an endosomal lysate lipid, or an endosomal lysate small molecule. In some cases, the endosomal lysate moiety comprises an endosomal lysate polypeptide. In other cases, the endosomal lysate moiety comprises an endosomal lysate polymer.

[0278] Endosomal soluble polypeptides In some embodiments, the anti-transferrin receptor antibody conjugate is further conjugated to an endosomolytic polypeptide. In some embodiments, the conjugate of formula (I): A-(X 1 -B) n , or the conjugate of formula (II): A-X 1 -(B-X 2 -C) n 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 amphiphilic polypeptide. In further cases, the endosomolytic polypeptide is a peptidomimetic. In some examples, the endosomolytic polypeptide includes INF, melittin, meucin, or derivatives thereof. In some examples, the endosomolytic polypeptide includes INF or a derivative thereof. In other cases, the endosomolytic polypeptide includes melittin or a derivative thereof. In further cases, the endosomolytic polypeptide includes meucin or a derivative thereof.

[0279] In some examples, INF7 is a 24-residue polypeptide, and this sequence includes CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 51) or GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 52). In some examples, INF7 or a derivative thereof includes the following sequences: 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 cases, melittin is a 26-residue polypeptide, and this sequence includes CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 56) or GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 57). In some examples, melittin includes the polypeptide sequence described in U.S. Patent No. 8,501,930.

[0281] In some cases, mucins are antimicrobial peptides (AMPs) derived from the venom glands of scorpions (Mesobuthus eupeus). In some cases, mucins consist of mucin-13 containing IFGAIAGLLKNIF-NH2 (SEQ ID NO: 58) and mucin-18 containing FFGHLFKLATKIIPSLFQ (SEQ ID NO: 59).

[0282] In some examples, the endosomal lytic polypeptide comprises a polypeptide whose sequence is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence-identical to INF7 or its derivatives, melittin or its derivatives, or mucin or its derivatives. In some examples, the endosomal lytic portion comprises INF7 or its derivatives, melittin or its derivatives, or mucin or its derivatives.

[0283] In some cases, the endosomal lysis region contains sequences as illustrated in Table 8.

[0284] [Table 8-1]

[0285] [Table 8-2]

[0286] In some cases, the endosomal lysis portion contains a Bak BH3 polypeptide that induces apoptosis through antagonism of repressor targets such as Bcl-2 and / or Bcl-xL. In some examples, the endosomal lysis portion contains 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 cases, the endosomal lysating portion contains polypeptides (e.g., cell-permeable polypeptides) as described in PCT International Publication No. WO2013 / 166155 or No. WO2015 / 069587.

[0288] Endosome-soluble polymers In some embodiments, formula (I): A - (X 1 -B) n , or formula (II):AX 1 -(BX 2 -C) n The conjugate further conjugates with the endosomal soluble polymer. As used herein, the endosomal soluble polymer includes linear, branched network, star-shaped, comb-shaped, or ladder-shaped polymers. In some examples, the endosomal soluble polymer is a homopolymer or copolymer containing two or more different types of monomers. In some cases, the endosomal soluble polymer is a polycationic polymer. In other cases, the endosomal soluble polymer is a polyanionic polymer.

[0289] In some cases, polycationic polymers contain monomeric units that are positively charged, neutrally charged, or negatively charged, with a net charge of positive. In other cases, polycationic polymers contain nonpolymeric 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 contain monomer units that are positively charged, neutrally charged, or negatively charged, with a net charge of negative. In other cases, polyanionic polymers contain two or more nonpolymer molecules containing negative charges. Exemplary anionic polymers include p(alkyl acrylate) (e.g., poly(propylacrylic acid) (PPAA)) or poly(N-isopropylacrylamide) (NIPAM). Further examples include L-phenylalanine-poly(L-lysine isophthalamide) polymers described in PP75, 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 endosome-soluble polymers described herein are pH-responsive endosome-soluble polymers. pH-responsive polymers include polymers that increase in size (swell) or disintegrate in response to the pH of the environment. Polyacrylic acid and chitosan are examples of pH-responsive polymers.

[0292] In some cases, the endosome-lysing portion described herein is a membrane-breaking polymer. In some cases, the membrane-breaking polymer includes a cationic polymer, a neutral or hydrophobic polymer, or an anionic polymer. In some cases, the membrane-breaking polymer is a hydrophilic polymer.

[0293] In some examples, the endosome-lysating moieties described herein are membrane-disrupting polymers. Exemplary pH-responsive membrane-disrupting polymers include p(alkylacrylic acid), poly(N-isopropylacrylamide) (NIPAM) copolymers, succinyl p(glycidol), and p(β-malic acid) polymers.

[0294] In some examples, p(alkylacrylic acid) includes poly(propylacrylic acid (polyPAA), poly(methacrylic acid (PMAA), poly(ethylacrylic acid) (PEAA), and poly(propylacrylic acid) (PPAA). In some examples, 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-breaking polymer contains p(butylacrylate-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-breaking polymer contains p(styrene-alt-maleic anhydride). (See Henry, et al., Biomacromolecules 7:2407-2414 (2006))

[0297] In some embodiments, the pH-responsive membrane-breaking polymers include pyridyl disulfide acrylate (PDSA) polymers 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) polymers. (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-breaking polymer includes a soluble polymer having the following base structure:

[0299] [ka]

[0300] In some examples, the endosome-lysating moieties described herein are further conjugated to additional conjugates, such as polymers (e.g., PEG) or modified polymers (e.g., cholesterol-modified polymers).

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

[0302] Endosomolytic lipid In some embodiments, the endosomolytic moiety is a lipid (e.g., a fusogenic lipid). In some embodiments, Formula (I): A-(X 1 -B) n , or Formula (II): A-X 1 -(B-X 2 -C) nThe conjugate further conjugates with endosomal soluble lipids (e.g., fusion lipids). Exemplary fusion 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)methaneamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethaneamine (XTC).

[0303] In some cases, the endosomal lysate portion is a lipid (e.g., a fusion lipid) as described in PCT International Publication No. WO09 / 126,933.

[0304] Endosomal soluble small molecules In some embodiments, the endosome-lysing portion is a small molecule. In some embodiments, formula (I): A-(X 1 -B) n , or formula (II):AX 1 -(BX 2 -C) nThese molecules further conjugate with endosomal soluble small molecules. Suitable exemplary small molecules as endosomal soluble moieties include, but are not limited to, quinine, chloroquine, hydroxychloroquine, amodiaquine (carnoquines), amopyroquine, primaquine, mefloquine, nivaquines, halophanthrin, quinone imines, or combinations thereof. In some examples, the quinoline endosome lysis portion is not limited to, but includes 7-chloro-4-(4-diethylamino-1-methylbutylamino)quinoline (chloroquine), 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline (hydroxychloroquine), 7-fluoro-4-(4-diethylamino-1-methylbutylamino)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, and 7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline. Phosphorus, 7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline, 7-fluoro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline, 4-(1-carboxy-4-diethylamino-1-butylamino)quinoline, 7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline, 7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline, 7-fluoro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline, 4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline, 7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline, 7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline,4-(4-ethyl-(2-hydroxy-ethyl)-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) (Tyl)-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 C-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline, 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, 8-[(4-aminopentyl)amino-6-methoxydihydrochloridequinoline, 1-acetyl-1,2,3,4-tetrahydroquinoline, 8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride, 1-butyryl-1,2,3,4-tetrahydroquinoline, 3-chloro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxyquinoline,Examples include 3-fluoro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline, 4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline, 3,4-dihydro-1-(2H)-quinoline carboxyaldehyde, 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 endosomal lysate portion is Naisbitt et al (1997, J Pharmacol Exp Therapy). It is a small molecule described in U.S. Patent No. 280:884-893 and U.S. Patent No. 5,736,557.

[0305] In some embodiments, the endosomal lysis portion is nigericin or a conjugate thereof, such as a folate-nigericin ester conjugate, a folate-nigericin amide conjugate, or a folate-nigericin carbamate conjugate. In some examples, the endosomal lysis portion 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 either severable or inseverable linkers. In some examples, the linker is severable. In other examples, the linker is inseverable.

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

[0308] In some cases, the non-polymer linker does not contain the polymers described above. In some cases, the non-polymer linker does not contain the polymer contained by polymer portion C. In some cases, the non-polymer linker does not contain polyalkylene oxides (e.g., PEG). In some cases, the non-polymer linker does not contain PEG.

[0309] In some examples, the linker includes homobifunctional linkers. Exemplary homobifunctional linkers include, but are not limited to, Lomant's reagents: dithiobis(succinimidylpropionate) DSP, 3'3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidylcarbonate (DSC), dimethyladipimidate (DMA), dimethylpimerimidate (DMP), dimethylsverimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio (O) Propionamide) butane (DPDPB), bismaleimide hexane (BMH), aryl halide-containing compounds (DFDNB), for example, 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) Contains formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0310] In some embodiments, the linker includes a heterobifunctional linker. Exemplary heterobifunctional linkers include, but are not limited to, amine-reactive and sulfhydryl-crosslinked linkers, e.g., 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 Zyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl(4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl(4-iodoactail)aminobenzoate (sulfo- sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyloxy)succinimidide ester (GMBs), N-(γ-maleimidobutyloxy)sulfosuccinimidide ester (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-nitrophenyliodoacetate (NPIA),Carbonyl-reactive and sulfhydryl-reactive crosslinkers, e.g., 4-(4-N-maleimidophenyl)butyrate hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), amine-reactive and photoreactive crosslinkers, e.g., N-hydroxysuccinimidyl-4 -Azidosalicylic acid (NH-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NH-AsA), sulfosuccinimidyl-(4-azidosalicyamide)hexanoate (sulfo-NH-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicyamide)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidosalicylic acid Dobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxy Succinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamide)-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-nitrophenyldiazopirubate (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive crosslinking linkers, for example,Examples include 1-(ρ-azidosalicylamide)-4-(iodoacetamide)butane (AsIB), N-[4-(ρ-azidosalicylamide)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimide, carbonyl-reactive and photoreactive crosslinkers such as ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive crosslinkers such as 4-(ρ-azidosalicylamide)butylamine (AsBA), and arginine-reactive and photoreactive crosslinkers such as ρ-azidophenylglyoxal (APG).

[0311] In some examples, the linker contains a reactive functional group. In some cases, the reactive functional group contains a nucleophile that is reactive to an electrophile present in the bonding portion. Exemplary electrophiles 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 nucleophiles include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0312] In some embodiments, the linker contains a maleimide group. In some examples, the maleimide group is also called a maleimide spacer. In some examples, the maleimide group further contains caproic acid to form maleimidocaproyl (mc). In some cases, the linker contains maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other examples, the maleimide group contains 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 catalytic action of thiosuccinimide ring hydrolysis, thereby preventing the maleimide from undergoing elimination via the retromichael reaction. In some examples, the self-stabilizing maleimide is the 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 contains a self-stabilizing maleimide. In some examples, the linker is a self-stabilizing maleimide.

[0314] In some embodiments, the linker includes a peptide moiety. In some examples, the peptide moiety includes at least 1, 2, 3, 4, 5, or more than 6 amino acid residues. In some examples, the peptide moiety includes up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In some examples, the peptide moiety includes 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 an incleavable peptide moiety. In some examples, the peptide portion includes 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 contains peptide moieties 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 contains Val-Cit. In some cases, the linker is Val-Cit.

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

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

[0317] In some embodiments, the linker is a self-destructing linker or a self-destructing linker. In some cases, the linker is a self-destructing linker. In other cases, the linker is a self-destructing linker (e.g., a cyclized self-destructing linker). In some examples, the linker includes linkers 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 includes a branched, multifunctional linker moiety. In some examples, the dendritic linker is used to increase the molar ratio of polynucleotide B pairs to binding moiety A. In some examples, the dendritic linker includes a PAMAM dendrimer.

[0319] In some embodiments, the linker is a traceless linker, or a linker that, after cleavage, leaves no linker portion (e.g., an atom or linker group) to the binding portion A, polynucleotide B, polymer C, or endosomal lysate portion D. Exemplary traceless linkers include, but are not limited to, germanium linkers, silicon linkers, sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium linkers, or phenylhydrazide linkers. In some cases, the linker is a traceless aryl-triazene linker, such as the one described in Hejesen, et al., “A traceless aryl-triazene linker for DNA-directed chemistry,” Org Biomol Chem 11(15):2493-2497 (2013). In some cases, 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 cases, the linker is a traceless linker as described in U.S. Patent No. 6,821,783.

[0320] In some embodiments, the linker is based on U.S. Patent Nos. 6,884,869, 7,498,298, 8,288,352, 8,609,105, or 8,697,688, U.S. Patent Publication Nos. 2014 / 0127239, 2013 / 028919, 2014 / 286970, and 2013 / 03 Linkers described in publications 09256, 2015 / 037360, or 2014 / 0294851, or PCT International Publications WO2015057699, WO2014080251, WO2014197854, WO2014145090, or WO2014177042.

[0321] In some embodiments, X 1 and X 2 Each of these is independently a single bond or a nonpolymer linker. In some examples, X1 and X 2 Each of them is an independent single bond. In some cases, X 1 and X 2 Each of these is an independent non-polymer linker.

[0322] In some examples, X 1 It contains single bonds or nonpolymer linkers. In some examples, X 1 It is a single bond. In some examples, X 1 It is a nonpolymer linker. In some examples, the linker is a C1-C6 alkyl group. In some cases, X 1 X1 is a C1-C6 alkyl group, for example, a C5, C4, C3, C2, or C1 alkyl group. In some cases, a C1-C6 alkyl group is an unsubstituted C1-C6 alkyl group. As used in the context of linkers, and especially as used in the context of X1, alkyl means a saturated straight-chain or branched hydrocarbon radical containing up to six carbon atoms. In some examples, X 1 This includes the homobifunctional or heterobifunctional linkers described above. In some cases, X 1 It includes a heterobifunctional linker. In some cases, X 1 This includes sMCC. In other examples, X 1 It contains a heterobifunctional linker optionally conjugated to a C1-C6 alkyl group. In other examples, X 1 This includes sMCCs optionally conjugated to C1-C6 alkyl groups. In further examples, X 1 This does not include the homobifunctional or heterobifunctional linkers described above.

[0323] In some examples, X 2 X is a single bond or linker. In some examples, X 2 It is a single bond. In other cases, X 2 is a linker. In further cases, X 2 is a nonpolymer linker. In some embodiments, X 2 is a C1-C6 alkyl group. In some examples, X2 X is a homobifunctional or heterobifunctional linker as described above. In some examples, X 2 The above is a homobifunctional linker. In some examples, X 2 X is a heterobifunctional linker as described above. In some examples, X 2 This includes a maleimide group, for example, maleimidocaproyl(mc), or the self-stabilizing maleimide group described above. In some examples, X 2 It contains peptide moieties such as Val-Cit. In some examples, X 2 It contains benzoic acid groups such as PABA. In further examples, X 2 It comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In further examples, X 2 It contains an mc group. In further examples, X 2 It contains an mc-val-cit group. In further examples, X 2 It contains a val-cit-PABA group. In further examples, X 2 It contains an mc-val-cit-PABA group.

[0324] How to use In some embodiments, a method for delivering a payload to a target site is described herein by the use of an anti-transferrin receptor antibody described herein. In some examples, the target site is a cell that overexpresses a protein associated with disease or illness. In some examples, the target site is a cell containing improperly processed mRNA encoding a non-functional protein or a protein with reduced expression leading to disease or illness. In some examples, the target site is a tumor site. In further examples, the target site is a site in the brain.

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

[0326] In one embodiment, muscular atrophy refers to a significant loss of muscle strength. Significant loss of muscle strength means a decrease in the strength of the diseased, damaged, or unused muscle tissue of the subject compared to the same muscle tissue of a control subject. In one embodiment, significant loss of muscle strength is 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 means a decrease in the strength of unused muscle tissue compared to the muscle strength of the same muscle tissue of the same subject before the non-use period. In one embodiment, significant loss of muscle strength is a decrease 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 the non-use period.

[0327] In another embodiment, muscular atrophy refers to a significant loss of muscle mass. Significant loss of muscle mass means a decrease in the muscle volume of the diseased, damaged, or unused muscle tissue of the subject compared to the same muscle tissue of a control subject. In one embodiment, 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, significant loss of muscle mass means a decrease in the muscle volume of unused muscle tissue compared to the muscle volume of the same muscle tissue of the same subject before the non-use period. In one embodiment, 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 the non-use period. Muscle volume can be measured voluntarily by evaluating the cross-sectional area of ​​the muscle, for example, by magnetic resonance imaging (e.g., muscle volume / cross-sectional area (CSA) MRI).

[0328] In some embodiments, muscular 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-associated muscular atrophy, or disuse-associated muscular atrophy.

[0329] Cachexia is the acquired, accelerating loss of muscle mass caused by an underlying disease. In some cases, cachexia refers to weight loss that cannot be recovered through nutrition and is commonly associated with underlying diseases such as cancer, COPD, AIDS, and heart failure. When cachexia is seen in patients with advanced cancer, it is called "cancer cachexia." Cancer cachexia affects a large proportion of patients with advanced cancer and is associated with reduced treatment resistance, response to treatment, quality of life, and survival. In some cases, cancer cachexia is defined as a multifactorial syndrome characterized by progressive loss of skeletal muscle mass, with or without loss of body fat mass, which cannot be fully recovered with conventional nutritional support and can lead to progressive dysfunction. In some cases, skeletal muscle loss appears to be the most important event in cancer cachexia. In addition, the classification of cancer cachexia suggests that diagnostic criteria should take into account not only weight loss as a signaling event of the cachexic process, but also the patient's initial reserves, low BMI, or low level of muscle mass.

[0330] Denervation is damage to peripheral motor neurons involving partial or complete blockage of nerve fibers between organs and the central nervous system, resulting in blockage of nerve conduction and motor neuron firing, and consequently, impaired skeletal muscle contractility. This loss of nerve function is localized or generalized by the loss of entire motor neuron units. The inability of skeletal muscles to contract leads to muscular atrophy. In some cases, denervation is associated with, or a consequence of, degenerative, metabolic, or inflammatory neuropathy (e.g., Guillain-Barré syndrome, peripheral neuropathy, or exposure to environmental toxins or drugs). In further cases, denervation is associated with physical injury, such as surgical procedures.

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

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

[0333] Diabetes mellitus (diabetes mellitus, DM) includes type 1 diabetes, type 2 diabetes, type 3 diabetes, type 4 diabetes, bidiabetes, latent autoimmune diabetes (LAD), gestational diabetes, neonatal diabetes mellitus (NDM), juvenile-onset adult-onset diabetes mellitus (MODY), Wolfram syndrome, Alström syndrome, prediabetes, or diabetes insipidus. Type 2 diabetes, also called non-insulin-dependent diabetes mellitus, is the most common type of diabetes, accounting for 95% of all diabetes cases. In some cases, type 2 diabetes is caused by a combination of factors, including insulin resistance due to impaired pancreatic β-cell function, resulting in hypertensive glucose levels. In some cases, elevated glucagon levels stimulate the liver to produce an abnormal amount of unnecessary glucose, which contributes to hypertensive glucose levels. Type 1 diabetes, also called insulin-dependent diabetes mellitus, accounts for approximately 5% to 10% of all diabetes cases. Type 1 diabetes is an autoimmune disease in which T cells attack and destroy insulin-producing β-cells in the pancreas. In some embodiments, type 1 diabetes is caused by genetic and environmental factors. Type 4 diabetes is a type of diabetes that affects approximately 20% of diabetes patients aged 65 and older. 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 caused by insulin resistance in the brain.

[0334] Chronic obstructive pulmonary disease (COPD) is a type of obstructive pulmonary disease characterized by long-term respiratory impairment 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 disorders, or primary liver cancer.

[0336] Congestive heart failure is a condition in which the heart is unable to supply enough blood and oxygen to the body's tissues.

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

[0338] In some embodiments, chronic infections such as AIDS can further cause muscular atrophy.

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

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

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

[0342] In some cases, treatment with glucocorticoids can further cause muscular atrophy. Illustrative glucocorticoids include, but are not limited to, cortisol, dexamethasone, betamethasone, prednisone, methylprednisolone, and prednisolone.

[0343] Disuse-related muscular atrophy results from immobilization of the limbs (e.g., fracture of a limb or joint, or orthopedic surgery such as hip or knee replacement). As used herein, “immobilization” or “immobilized” means partial or complete restriction of movement of a limb, muscle, bone, tendon, joint, or other body part for an extended period (e.g., two, three, four, five, six days, one, two weeks, or longer). In some cases, the period of immobilization includes short or brief periods of unrestricted movement, such as bathing, changing or adjusting external devices. Limb immobilization is carried out by any variety of external devices, including, but not limited to, braces, slings, casts, bandages, and splints (any of which may be made of hard or soft materials, including, but not limited to, cloth, gauze, fiberglass, plastic, plaster, or metal), and any variety of internal devices, including surgically implanted splints, plates, orthoses, etc. In the context of limb immobilization, restrictions on movement include single-joint or multi-joint (e.g., single joints such as the shoulder or hip joint, compound joints such as the radiocarpal joint, and multi-joints such as the knee joint, including, but not limited to, one or more of the following: joints of the hand, shoulder, elbow, wrist, auxiliary joints, sternoclavicular joint, vertebral joints, temporomandibular joint, sacroiliac joint, hip, knee, and ankle), 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 ligaments of the ankle, and tendons and ligaments of the jaw or temporomandibular joint), single Bone or multiple bones (for example, 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), single or multiple muscles (for example, but not limited to, one or more of the following: latissimus dorsi, trapezius, deltoid, pectoralis, biceps, triceps, external oblique, abdominal muscles, gluteus maximus, flexor thigh, quadriceps femoris, gastrocnemius, and diaphragm), one or more limbs, one or more arms and legs), or the entire or a part of the skeletal muscular system (for example, in the case of a full-body cast or a spiked cast).

[0344] Myotonic dystrophy is a multi-systemic neuromuscular disorder 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 extension in the DM protein kinase (DMPK) gene, 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 control of the polyadenylatin site and loss of MBNL protein function lead to downstream accumulation of nuclear lesions, increasing missplicing events and subsequently causing myotonia and other clinical symptoms.

[0345] In some embodiments, methods for treating diseases or disorders characterized by misspliced ​​mRNA are described herein. In some embodiments, the anti-transferrin receptor antibodies described herein deliver polynucleic acid molecules to the site of the misspliced ​​mRNA transcript to induce exon skipping or exon inclusion.

[0346] In some cases, diseases or disorders resulting from improperly spliced ​​or partially spliced ​​mRNA include, but are not limited to, neuromuscular diseases, genetic diseases, cancers, hereditary disorders, or cardiovascular diseases.

[0347] In some cases, genetic diseases or disorders include autosomal dominant disorders, autosomal recessive disorders, X-linked dominant disorders, X-linked recessive disorders, Y-linked disorders, mitochondrial genetic diseases, or multifactorial or polygenetic disorders.

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

[0349] In some cases, improperly spliced ​​or partially spliced ​​mRNA can cause cancer. For example, improperly spliced ​​or partially spliced ​​mRNA can affect cellular processes involved in cancer, including, but not limited to, proliferation, motility, and drug response. Some examples include solid tumors or blood cancers. Some examples of cancers include bladder cancer, lung cancer, brain cancer, melanoma, breast cancer, non-Hodgkin lymphoma, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, esophageal cancer, prostate cancer, kidney cancer, skin cancer, leukemia, thyroid cancer, liver cancer, or uterine cancer.

[0350] In some cases, improperly spliced ​​or partially spliced ​​mRNA can cause neuromuscular disease or disorder. Exemplary neuromuscular diseases include muscular dystrophy 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 involve mutations in the DMD gene, which encodes the protein dystrophin. Facioscapulohumeral muscular dystrophy has been shown to involve mutations in the double homeobox 4 (DUX4) gene.

[0351] In some cases, improperly spliced ​​or partially spliced ​​mRNA causes Duchenne muscular dystrophy. Duchenne muscular dystrophy is caused by mutations in the DMD gene that lead to severe muscle weakness and the loss of functional dystrophin production. In some cases, Duchenne muscular dystrophy is the result of mutations in exons within the DMD gene. In some cases, Duchenne muscular dystrophy is the result of at least one mutation in 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 of the DMD gene. In some cases, Duchenne muscular dystrophy results from at least one mutation in 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 of the DMD gene. In some cases, Duchenne muscular dystrophy results from at least one mutation in exons 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, and 55 of the DMD gene. In some cases, many exons mutate. For example, mutations in exons 48-50 are common in patients with Duchenne muscular dystrophy. In some cases, Duchenne muscular dystrophy is the result of a mutation in exon 51. In some cases, Duchenne muscular dystrophy is the result of a mutation in exon 23. In some cases, the mutation involves the deletion of one or more exons. In some cases, the mutation involves the duplication of one or more exons.In some cases, the mutations are involved in exon point mutations. For example, it has been shown that some patients have a nonsense point mutation 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 administration routes. In some examples, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intra-arterial, intraperitoneal, intrathecal, intracerebral, intraventricular, or intracranial) administration. In other examples, the pharmaceutical compositions described herein are formulated for oral administration. In yet another example, the pharmaceutical compositions described herein are formulated for nasal administration.

[0353] In some embodiments, the pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, rapidly dissolving formulations, tablets, capsules, pills, delayed-release formulations, sustained-release formulations, pulsed-release formulations, multi-particle formulations (e.g., nanoparticle formulations), and mixed formulations of immediate-release and controlled-release.

[0354] In some cases, the pharmaceutical formulation includes multi-particle formulations. In some cases, the pharmaceutical formulation includes nanoparticle formulations. In some cases, the nanoparticles include cMAP, cyclodextrin, or lipids. In some cases, the nanoparticles include solid lipid nanoparticles, polymer nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micelle solutions. Further exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metallic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as those having covalently bonded metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In some examples, nanoparticles are nanoparticles of metals, such as 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 thereof, their alloys, or oxides.

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

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

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

[0358] In some examples, nanoparticle formulations include paramagnetic nanoparticles, superparamagnetic nanoparticles, metallic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as those having covalently bonded metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, or quantum dots. In some examples, the polynucleic acid molecules or binding sites 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 sites described herein are directly or indirectly conjugated to the nanoparticles.

[0359] In some embodiments, the pharmaceutical formulation includes a delivery vector, such as a recombinant vector for the 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 viruses, retroviruses, adenoviruses, or alphaviruses. In some examples, the recombinant vector capable of expressing polynucleic acid molecules results in stable expression in target cells. In further examples, viral vectors that result in transient expression of polynucleic acid molecules are used.

[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, and diluents. 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, and pregelatinized starch. 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, pharmaceutical formulations further include pH adjusters or buffers, such as acids including acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases including sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trishydroxymethylaminomethane; and buffers including citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0362] In some cases, a pharmaceutical formulation contains one or more salts in amounts necessary to bring the osmotic pressure of the composition into an acceptable range. Such salts include sodium, potassium, or ammonium cations and anions of chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite, and suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0363] Treatment regimen In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic purposes. In some embodiments, the pharmaceutical compositions are administered once daily, twice daily, three times daily, or more frequently. The pharmaceutical compositions are administered daily, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month, or more frequently. The pharmaceutical compositions are administered for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, eighteen months, two years, three years, or longer.

[0364] In some embodiments, one or more pharmaceutical compositions are administered simultaneously, sequentially, or at regular time intervals. In some embodiments, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered sequentially. In further cases, one or more pharmaceutical compositions are administered at regular time intervals (for example, the first administration of the first pharmaceutical composition is on day 1, and thereafter, at least 1, 2, 3, 4, 5 days or more before the administration of at least the 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. In some cases, two or more different pharmaceutical compositions are administered sequentially and simultaneously with no time interval between administrations. In other cases, two or more different pharmaceutical compositions are administered sequentially and simultaneously with intervals of approximately 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, and 2 days between administrations.

[0366] If the patient's condition improves, the physician may, at their discretion, continue administering the composition, or alternatively, temporarily reduce or temporarily discontinue the dose of the administered composition for a specific period (i.e., a “drug-free period”). In some cases, the length of the drug-free period may vary between 2 days and 1 year, including, but are not limited to, 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 28, 35, 50, 70, 100, 120, 150, 180, 200, 250, 280, 300, 320, 350, or 365 days. The dose reduction during drug-free periods ranges from 10% to 100%, including, but are not limited to, 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 improves, a maintenance dose is administered as needed. Subsequently, the dose, frequency, or both may be reduced, depending on the symptoms, to a level at which the improved disease, impairment, or illness is maintained.

[0368] In some embodiments, the amount of a given drug corresponding to such a quantity varies depending on factors such as the specific compound, the severity of the disease, and the unique characteristics of the subject or host requiring treatment (e.g., body weight). Nevertheless, it is routinely determined by methods known in the art, according to the specific environment surrounding the case, including, for example, the specific drug being administered, the route of administration, and the subject or host being treated. In some examples, the desired dose is conveniently presented as a single dose, or as divided doses administered simultaneously (or over a short period of time), or as sub-doses administered at appropriate intervals, e.g., two, three, four times or more times per day.

[0369] Due to the large number of variables in each treatment regimen, the ranges mentioned above are merely suggestive, and significant deviations from these recommendations are not uncommon. Such dosages are not limited but depend on many variables, including the activity of the compound used, the disease or illness being treated, the mode of administration, the individual subject's requirements, the severity of the disease or illness being treated, and the physician's judgment.

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

[0371] Kit / Product In certain embodiments, kits and products used in conjunction with one or more compositions and methods described herein are disclosed herein. Such kits include a partitioned carrier, packaging container, or container for housing one or more containers such as vials, tubes, etc., each of which contains one of the other elements used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.

[0372] 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 materials suitable for the selected formulation and the intended mode of administration and treatment.

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

[0374] A kit typically includes a label listing the contents and / or instructions for use, and an accompanying document with instructions for use. A set of instructions is also usually included.

[0375] In one embodiment, the label is on or attached to the container. In one embodiment, the label is on the container if the letters, numbers, or other markings forming the label are affixed, molded, or engraved onto the container itself. The label is attached to the container if it is present, for example, as an accompanying document, within the receptacle or transport device that holds the container. In one embodiment, the label is used to indicate that the contents are to be used for a particular therapeutic purpose. The label also indicates how to use the contents, for example, by the method described herein.

[0376] In certain embodiments, the pharmaceutical composition is presented in a pack or dispenser device containing one or more unit dosage forms comprising the compounds provided herein. The pack includes, for example, metal foil 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 is also accompanied by a notice that accompanies a container in a form specified by a government agency that controls the manufacture, use, or sale of the pharmaceutical, and this notice reflects the government agency's approval regarding the form of the drug for administration to humans or animals. Such a notice is, for example, a label approved by the U.S. Food and Drug Administration with respect to a prescription drug or approved package insert. In one embodiment, a composition comprising the compounds provided herein, formulated on a suitable pharmaceutical carrier, is also prepared, placed in a suitable container, and labeled for the treatment of the indicated disease.

[0377] Specific terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the field to which the claimed subject matter belongs. It will be understood that the above general statements and the following detailed statements are illustrative and descriptive only and not limited to any subject matter. In this application, the use of the singular includes the plural unless otherwise specified. Where used in the specification and appended claims, the singular forms "a," "an," and "the" include the plural referent unless the context explicitly states otherwise. In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "including" is not limited to the other forms such as "include," "includes," and "included."

[0378] As used herein, ranges and quantities can be expressed as "approximately" specific values ​​or ranges. "Approximately" also includes exact quantities. Thus, "approximately 5 μL" also means "approximately 5 μL" and "5 μL". In general, the term "approximately" includes quantities that are expected to be within experimental error.

[0379] The paragraph headings used in this specification are for organizational purposes only and should not be interpreted as limiting the subject matter discussed.

[0380] Antibodies and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. These terms are used synonymously. In some cases, the antigen specificity of immunoglobulins is known.

[0381] The term "antibody" is used in its broadest sense to encompass fully assembled antibodies, antibody fragments capable of binding antigens (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 mentioned above.

[0382] As used herein, the terms “monoclonal antibody” and “mAb” refer to antibodies obtained from a substantially homogeneous population of antibodies; that is, individual antibodies within the population are identical except for possible spontaneous mutations, which may be present in small amounts.

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

[0384] As used herein, the term “variable” refers to the fact that certain portions of the variable domain differ significantly in sequence within the antibody. The variable region confers antigen-binding specificity. However, variability is not uniformly distributed throughout the antibody’s variable domain. Instead, it is concentrated in three regions, both in the light and heavy chain variable domains, called complementarity-determining regions (CDRs) or hypervariable regions. The more highly conserved portions of the variable domain are called framework (FR) regions. The natural heavy and light chain variable domains each contain four FR regions, the majority of which employ a β-pleated sheet configuration, connected by three CDRs forming loop connections, and in some cases, forming part of the β-pleated sheet configuration. The CDRs in each chain are held together in close proximity by the FR regions, and together with the CDRs of other chains, contribute to the formation of the antibody’s antigen-binding site (see Kabat et al. (1991) NIH PubL. No. 91-3242, Vol. I, pages 647-669). The constant domain does not directly participate in antibody binding to antigens, but exhibits various effector functions such as Fc receptor (FcR) binding, antibody involvement in antibody-dependent cell-mediated cytotoxicity, induction of complement-dependent cell-mediated cytotoxicity, and mast cell degranulation.

[0385] As used herein, the term “hypervariable region” refers, as used herein, to an amino acid residue of an antibody responsible for antigen binding. The hypervariable region consists of amino acid residues from the "complementarity-determining region" or "CDR" (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 loop" (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 This includes Lesk, (1987) J.Mol.Biol., 196:901-917). A “framework” or “FR” residue is a variable domain residue other than a hypervariable region residue, as is considered herein.

[0386] An "antibody fragment" is a portion of an intact antibody, preferably containing 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 having a single antigen-binding site, and an "Fc" fragment, whose name reflects its ability to be readily crystallized. Pepsin treatment produces an F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigens.

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

[0388] The Fab fragment consists of a constant domain of the light chain and a first constant domain of the heavy chain (C H1 ) also includes. The Fab fragment contains one or more cysteine ​​in the antibody hinge region, heavy chain C H1 The addition of a few residues at the carboxyl terminus of the domain distinguishes it from the Fab' fragment. Fab'-SH is the heretical nomenclature for Fab' fragments in which the cysteine ​​residue of the constant domain has a free thiol group. The Fab' fragment is generated by reduction of the heavy chain disulfide crosslink of the F(ab')2 fragment. Other chemical couplings of antibody fragments are also known.

[0389] The "light chain" of an antibody (immunoglobulin) from any vertebrate species can be assigned to one of two distinctly different types, called kappa (κ) and lambda (λ), based on the amino acid sequence of the constant region.

[0390] Depending on the amino acid sequence of the heavy chain constant domain, 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 different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional structures of various classes of immunoglobulins are well known. Different isotypes have different effector functions. For example, the human IgG1 and IgG3 isotypes have ADCC (antibody-dependent cell-mediated cytotoxicity) activity.

[0391] In some cases, the CDR of an antibody is (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. See Mol. Biol. 215(1): 175-82; and US Patent No. 7,709,226); or (iii) the ImMunoGeneTics (IMGT) numbering system, such 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) determined according to MacCallum et al, 1996, J. Mol. Biol., 262:732-745. For example, see 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, CDRs within an antibody heavy chain molecule are typically located at amino acid positions 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3), which can optionally contain one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3). As is well known to those skilled in the art, with 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 the FR and / or CDR, 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 within the antibody heavy chain molecule are typically located at amino acid positions 26-31 (CDR1), 52-56 (CDR2), and 95-102 (CDR3), which can optionally contain 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 within the antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3). As is well known to those skilled in the art, with the Chotia numbering system, the actual linear amino acid sequence of the antibody variable domain may contain fewer or additional amino acids due to shortening or lengthening of the FR and / or CDR, 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 part of the heavy chain and / or light chain originates from a specific source or species, while the rest of the heavy chain and / or light chain originates from a different source or species.

[0395] The term "humanized antibody" refers to an antibody whose framework or CDR has been modified to constitute a CDR of an immunoglobulin that has different specificity compared to the CDR of the parental immunoglobulin.

[0396] As used herein, the terms “individual,” “subject,” and “patient” mean any mammal. In some embodiments, the mammal is human. In some embodiments, the mammal is non-human. No term is limited to situations characterized by supervision (e.g., constant or intermittent) of a healthcare worker (e.g., physician, registered nurse, clinical nurse, physician’s assistant, nursing assistant, or hospice staff). [Examples]

[0397] These examples are provided for illustrative purposes only and do not 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 were created for each product. Cell proliferation and protein A titer were monitored in these stable pools from day 8 post-transfection. Once the culture reached a viability of 70%, the threshold was 0.6 × 10⁻⁶ e⁻¹. 6 When the cell / mL was reached, the stable pool was subcultured. When the cell viability exceeded 97%, the highest-productivity pool in the tested pool was used for 600 mL of fed-batch overgrow cultures (FOG) per product, 0.2 × 10 e 6Cells were seeded at a concentration of cells / mL. FOG cultures were fed on days 4 and 8, and collected on day 11 by centrifugation and filtration sterilization. The sterile cell culture supernatant was purified by protein A purification using a 3×5 ml MabSelectSuRE column on an AKTA purifier (running at 10 mL / min). The column was equilibrated with 50 mM sodium phosphate. The cells were washed with 250 mM sodium chloride at pH 7.0, 50 mM sodium phosphate, and 1 M sodium chloride at pH 7.0, and eluted with 10 mM sodium formate at pH 3.5. The eluted fraction was neutralized by diluting 1:2 with 2×PBS, and then the pH was adjusted to 7.4 using diluted NaOH.

[0399] Antibodies were analyzed by SE-HPLC and SDS-PAGE. Replicated samples were analyzed by SE-HPLC using a Zorbax GF-250 9.4mm ID×25cm column (Agilent). An 80 μl aliquot of 1 mg / ml sample was injected and HPLC was performed at 1 ml / min for 15 minutes with 50 mM sodium phosphate, 150 mM sodium chloride, 500 mM L-arginine, and pH 6.0. Small peaks of less than 16.89% were observed in all variants, with retention times of approximately 7.66 minutes, consistent with soluble aggregates. Soluble aggregate concentration was analyzed using Empower v3 software.

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

[0401] [Table 9]

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

[0403] hTfR (100 u) was dissolved in 300 uL of water to obtain a stock concentration of 4.3 uM. The hTfR was then diluted to a maximum concentration of 43 nM and tested using a 3-fold series dilution. hTfR was injected at a rate of 200 u / min over 2 minutes, followed by a 1-hour dissociation phase.

[0404] The reaction data was processed by subtracting it from an internal spot control surface, and further double-referenced by buffer injection.

[0405] Table 10 shows the bond constants obtained at 25°C.

[0406] [Table 10] [Examples]

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

[0408] CDRs of mouse IgG2 antibody against hTfR1 were subcloned into a human IgG2 background and transfected into CHO-K1SP cells. See Example 4 for the sequence. A stable cell pool was selected and seeded in Dynamis medium (GIBCO) in cellbags (Healthcare) at 37°C and 5% CO2 using a Wave Bioreactor (GE Healthcare). Feeding was performed every two days from day 4, with a final culture volume of 8% (25 liters), for a total incubation period of 14 days. The culture supernatant was collected, deep filtered, and purified using Monofinity A Resin (GenScript) at a flow rate of 30 ml / min. The eluted protein buffer was added to PBS, and the molecular weight and purity of the purified protein were analyzed by SDS-PAGE and SEC-HPLC under reducing and non-reducing conditions. The final protein purity exceeded 98%.

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

[0410] SSB siRNA double strands were used as the conjugate for this experiment. The sequence of the 21-mer SSB guide / antisense strand was (5'~3')UUACAUUAAAGUCUGUUGUUU. Single strands were fully assembled on solid phase using standard phosphoramidite chemistry and purified using HPLC. The potency of the double strands was optimized and immunogenicity reduced using base, sugar, and phosphate modifications, which are well described in the field of RNAi. The siRNA passenger strand contained a C6-NH2 conjugation handle at the 5' end. See Figure 1. siRNA double strands were designed as blunt-end double strands with complementary 19 bases and one 3' dinucleotide overhang. The conjugation handle was annexed to the siRNA passenger strand via a phosphate diester on the terminal bases. See Figure 2.

[0411] Step 1: Antibody reduction by TCEP

[0412] The antibody was subjected to a buffer exchange with 25 mM borate buffer (pH 8) containing 1 mM DTPA to a maximum concentration of 10 mg / ml. To this solution, 4 equivalents of TCEP in the same borate buffer were added and incubated at 37°C for 2 hours. The resulting reaction mixture was combined with a solution of BisMal-siRNA (1.25 equivalents) in 10 mM acetate buffer at pH 6.0 at room temperature (RT) and stored overnight at 4°C. Analysis of the reaction mixture by analytical SAX column chromatography revealed the antibody-siRNA conjugate along with the unreacted antibody and siRNA. The reaction mixture was treated with 10EQ 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 using AKTA Pure FPLC with anion exchange chromatography (SAX) method-1. The fraction containing the DAR1 antibody-siRNA conjugate was isolated, concentrated, and buffered with PBS at 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 at 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] Powerful Anion Exchange Chromatography (SAX) Method - 2

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

[0421] Solvent A: 80% 10mM Tris pH8, 20% ethanol; Solvent B: 80% 10mM Tris pH8, 20% ethanol, 1.5M NaCl; Flow rate: 0.75 mL / min

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

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

[0424] [Table 11]

[0425] Analysis data table for the conjugate used in this example: HPLC retention time (RT) in minutes, and purity % based on 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) in 1 ng / μL of PBS (Gibco 14190) and incubated overnight at 4°C. The plates were washed four times with 100 μL of Tris-buffered saline + Tween (20xTBST, Cell Signaling 9997S). 100 μL of Superblock (ThermoFisher PI-37535) was added to each well and incubated at room temperature for 1 hour. The washing process was repeated before adding the sample. The sample was added at a maximum concentration of 10 nM and 50 μL / well. The plate was incubated at room temperature for another hour, and the washing process was repeated. The secondary antibody (Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson Immunoresearch, 109-035-098) was diluted 1:5000 in the added Superblock and 50 μL / well. The plate was incubated at room temperature for 1 hour and washed again. 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 control wavelength of 570 nm was subtracted. The binding constants were determined using GraphPad Prism Specific Binding with Hill Slope.

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

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

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

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

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

[0433] [Table 12]

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

[0435] [Table 13]

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

[0437] [Table 14]

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

[0439] Tissue siRNA enrichment was quantified using a stem-loop qPCR assay. Briefly, 15–50 mg of tissue fragments were homogenized in 500 μL of Trizol using TissueLyser II plate-based homogenizer (Qiagen), and then diluted in TE buffer + 0.1% v / v Triton X-100. siRNA was spiked into homogenized tissue from untreated animals, and a standard curve was generated by serial dilution with 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 sequence-specific stem-loop RT primers. The cDNA obtained from the RT step was used for real-time PCR with TaqMan Fast Advanced Master Mix (Applied Biosystems) with 1.5 μM forward primer, 0.75 μM reverse primer, and 0.2 μM probe. Table 13 shows the sequences of the SSB siRNA antisense strands used for measurement, as well as all primers and probes. 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 a linear equation derived from the standard curve.

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

[0441] To monitor the safety of selected anti-hTfR1 antibodies in primates, hematological and clinical chemistry analyses were performed according to the schedule shown in Table x. With the exception of dose-dependent but transient depletion of reticulocytes (Figure 6), no treatment-related effects were observed in hematological or clinical chemistry parameters for up to 28 days post-administration. The transient downregulation observed in reticulocytes was attributed to a side effect of the TfR1 antibody. We observed that mouse TfR1 antibodies with intact activator function or complement binding 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)). Because primates have a low fraction of reticulocytes expressing high TfR1 levels, reticulocyte depletion was only transient and not as pronounced as in rodents. Importantly, other studies have shown that this activity can be effectively suppressed by mutations that remove the ADCC / CDC activity of the antibody. [Examples]

[0442] Human / cynomolgus monkey cross-reactivity anti-TfR1 antibody generation, characterization, and humanization

[0443] Sixteen variants of the chimeric anti-transferrin 1 mAb were designed using modern in silico antibody humanization and deimmunization programs well documented in the art, and subjected to the NHP test of Example 1. See Table XYZ for the variant sequences. As part of the design, manufacturability was assessed by identifying high-risk post-translational modifications (PTMs), and where feasible, these were removed as part of the humanization activity via amino acid substitution. Immunogenicity risk was also assessed, identifying high-risk epitopes and removing them where feasible. Furthermore, mutations were introduced into the Fc domain of the variants to remove the action factor functions (ADCC and CDC). Subsequently, the sixteen variants were expressed in mammalian cell cultures using techniques well documented in the art and purified using affinity chromatography based on protein A resin. The mAb variants were then fully characterized, and siRNA conjugates were prepared as described below.

[0444] Human and cynomolgus monkey TfR1 ELISA assay

[0445] The goal of these assays was to confirm that 16 variant human anti-TfR1 antibodies bind to TfR1 in both humans and cynomolgus monkeys. The human or cynomolgus monkey 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) in 1 ng / μL of PBS (Gibco 14190) and incubated overnight at 4°C. The plates were washed four times with 100 μL of Tris-buffered saline + Tween (20x TBST, Cell Signaling 9997S). 100 μL of Superblock (ThermoFisher PI-37535) was added to each well and incubated at room temperature for 1 hour. The washing process was repeated before adding the samples. Samples were added at a maximum concentration of 10 nM and 50 μL / well. The plates were incubated at room temperature for another 1 hour, and the washing process was repeated. The secondary antibody (Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson Immunoresearch, 109-035-098) was diluted 1:5000 in 50 μL / well with added Superblock. The plate was incubated at room temperature for 1 hour and washed again. 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, subtracting the control 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 of 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 antibody binds to TfR in the presence of holo-transferrin.

[0450] Antibodies were biotinylated using a 50-fold molar excess of EZ-Link No-Weigh NHS-Biotin (Thermo Scientific A39256) according to the manufacturer's instructions. Half-well high-binding plates (Costar #3690) were coated with 500 ng / mL of generated human holo-transferrin (R&D Systems 2914-HT) overnight in PBS at 4°C. For comparison, plates were directly coated with hTfR. Plates were washed four times with 100 μL of Tris-buffered saline + Tween (20xTBST, Cell Signaling 9997S). 100 μL of Superblock (ThermoFisher PI-37535) was added to each well and incubated at room temperature for 1 hour. After repeating the washing process, hTfR (200 ng / mL in 25 μL) was added to the transferrin plate, or Superblock was added to the hTfR plate and incubated for 30 minutes. To achieve a high concentration of biotinylated antibody, it was diluted to 20 nM and added to the plate in a 3-fold serial dilution. 25 μl / well was added to the 25 μl already present on the plate. The plate was incubated for 1 hour, and the washing process was repeated. Streptavidin-HRP (R&D Systems DY998) was added according to the dilution recommended in the package insert, and the final washing process 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 control 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 maintains binding when TfR is bound to HFE.

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

[0454] FcγRIIIA(CD16a)ELISA

[0455] The goal of this assay was to determine the potential of antibodies for ADCC activity by measuring their binding to the FcγRIIIA(CD16a) genotype V158. A half-well high-binding 96-well plate (Costar #3690) was coated with recombinant CD16a protein (Sino Biological 10389-H27H1) in 2 ng / μL of PBS (Gibco 14190) and incubated overnight at 4°C. The plate was washed four times with 100 μL of Tris-buffered saline + Tween (20xTBST, Cell Signaling 9997S). 100 μL of Superblock (ThermoFisher PI-37535) was added to each well and incubated at room temperature for 1 hour. The washing process was repeated before adding the samples. Samples were added at a maximum concentration of 1 μM and 50 μL / well. The plate was incubated at room temperature for another 1 hour, and the washing process was repeated. The secondary antibody (Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson Immunoresearch, 109-035-098) was diluted 1:5000 in 50 μL / well with added Superblock. The plate was incubated at room temperature for 1 hour and washed again. 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, subtracting the control wavelength of 570 nm. Binding constants were determined using GraphPad Prism Specific Binding with Hill Slope. See Figure 11.

[0456] In vitro efficacy 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). The antibody siRNA conjugate was diluted to the maximum dose of 100 nM. 20 μl of the conjugate was added to the 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 allowed to stand at 37°C and 5% CO2 for 72 hours. The culture medium was removed from the wells, and 150 μl of Trizol (Ambion 15596018) was added. The plates were frozen at -80°C overnight or longer before analysis. RNA was isolated using the Direct-zol 96 RNA Isolation Kit (Zymo Research R2056) according to the manufacturer's instructions. RNA was reverse transcribed using the High Capacity cDNA Kit (Applied Biosystems 4368814) according to the manufacturer's instructions, and qPCR was performed using the Taqman Fast Advanced Master Mix (Applied Biosystems 4444558) with SSB and PPIB Taqman probe sets (ThermoFisher Hs04187362_g1 and Hs00168719_m1). The percentage of mRNA was calculated using the ΔΔCt method, and cells treated with PBS were set to 100% expression.

[0458] DAR1 conjugates containing active siRNA (SSB) or inactive or scrambled siRNA (Scr) were prepared 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 chain on the ribose 2' hydroxyl group. This was introduced during solid-phase synthesis, and although the activity of the guide chain was not inhibited, the uptake assay could be performed. The purity of the conjugates was evaluated by analytical HPLC using ion-exchange chromatography method-2, and the chromatographic retention time and purity are listed in Table 15 below.

[0459] [Table 15]

[0460] Analysis data table for the conjugate used in this example: HPLC retention time (RT) in minutes, and purity % based on chromatographic peak area.

[0461] Antibody-dependent cell-mediated cytotoxicity (ADCC) in PBMCs mediated by TfR1 antibodies and antibody-siRNA conjugates (ASCs)

[0462] Studies in mice and non-human primates (NHPs) have demonstrated that antibodies binding to mouse / cynomolgus monkey TfR cells possessing activator function and / or complement-binding ability selectively deplete TfR-expressing reticulocytes. To confirm whether the mutant cells possessed activator function, an ADCC assay was performed using peripheral blood mononuclear cells (PBMCs) obtained from healthy human donors as effector cells.

[0463] material:

[0464] PBMC from BUYPBMC.COM, lot #2010113378, possessing 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 RPMI 1640 (Life Technologies) serum (complete medium) containing 10% thermally inactivated FBS (56°C for 30 minutes) and 2% L-glutamine.

[0468] hIgG1 mAb mutant

[0469] procedure:

[0470] Thaw the PBMC cells gently in a 37°C water bath while stirring. After thawing, add 1 mL of warm culture medium to the vial drop by drip infusion over 30 seconds to allow the cells to adjust to the environmental change. Gradually add the cells to a 15 or 50 mL conical tube containing 9 mL of warm culture medium. Rinse the first vial containing 1 mL of cell-containing medium to collect cells adhering to the sides. Add the rinse medium to the conical tube. Pellet the cells by centrifugation at 350xg for 8-12 minutes. Discard the supernatant. Gently tap to suspend the cell pellet (avoid excessive shearing force). Add 10 mL of warm culture medium to the conical tube and rinse the cells again. Pellet the cells by centrifugation at 350xg for 8-12 minutes. Discard the supernatant from the second wash. Gently tap to suspend the cell pellet (avoid excessive shearing force). Suspend the cells in 10 mL of warm culture medium if necessary. The cells are incubated overnight at 37°C in a T75 tray to allow them to adapt.

[0471] Target cells HEL-92 are collected and washed twice with cold assay medium to ensure high viability. 4 x 10⁶ cells are placed on ice in a 96-well round-bottom plate. 4 / well, cold 50μl (8x10 5Target cells were seeded in assay medium (RPMI-1640 containing 1% BSA and 100 units / mL of penicillin and streptomycin). Dilutions of the test and control antibodies / ASC (10 μl) (6-fold, starting from 10 μg / ml) were added to plates containing the target cells listed in the table below, and then incubated on ice for 30 minutes to allow opsonization. 10 μl of medium was added to the control well to maintain volume.

[0472] Contrast:

[0473] - Background low control: Modify the spontaneous release from target cells (low control) with a control for the spontaneous LDH release of 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 control with maximum LDH release from target cells is required to determine 100% LDH release. 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] - Measure antibody-independent cytotoxicity (AICC) in wells containing target and effector cells without adding antibodies.

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

[0477] - Modify the spontaneous release of LDH from effector cells using a control of spontaneous LDH release from effector cells. Add various numbers of effector cells to be used in the experimental wells. Adjust the final volume to 100 μL / well with culture medium.

[0478] - The culture medium background control needs to be corrected for any contribution from LDH activity that may be present in the serum containing the culture medium. Add 100 μL of culture medium to three wells (without cells).

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

[0480] 45 minutes before collecting the supernatant, add 10 μL of lysis buffer (10X) to the target cell maximum LDH release control (positive control) and the volume-corrected control. Add 10 μL of PBS to the background low control containing cells, samples, and other controls. After incubation (350 g, 10 min), centrifuge the plate. Transfer 50 μL of supernatant to a 96-well clear flat-bottom plate, add 50 μL of reaction mixture to each sample well, and mix by gently tapping. Incubate the plate at room temperature for 30 minutes, protected from light. Add 50 μL of stop solution to each sample well and mix by gently tapping. Measure the 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] Specific ADCC activity was calculated as follows:

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

[0483] The results are shown in Figure 14.

[0484] Complement-dependent cell-mediated cytotoxicity (ADCC) in rabbit serum mediated by TfR1 antibodies and antibody-siRNA conjugates (ASCs)

[0485] material:

[0486] Freeze-dried rabbit complement. Reconstitute with ice-cold distilled water. Gently stir to ensure all freeze-dried material is thawed. Use within 1 hour of reconstitution. Always keep the reconstituted material on ice. If not fully active at 1 / 2 dilution, discard the aliquot.

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

[0488] Viobility 405 / 452 fixable dye.

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

[0490] hIgG1 mutant

[0491] procedure:

[0492] HEL92.1.7 target cells were collected and washed twice with cold assay medium. 5 × 10⁶ cells were placed in a 96-well round-bottom plate. 4 Cells were seeded in 25 μl of cold assay medium (RPMI-1640 containing 1% BSA and 100 units / mL of penicillin and streptomycin) per well. Dilutions of test and control antibodies (25 μl) (5-fold, starting from 100 ug / ml to a final 50 ug / ml) were added to plates containing 25 μl of target cells, and then incubated on ice for 30 minutes to allow opsonization.

[0493] Contrast:

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

[0495] - Measure antibody-independent cytotoxicity (AICC) in wells containing target and CDC cells without adding antibodies.

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

[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 muscle tubules.

[0501] To monitor ASC uptake into muscle cells, primary human skeletal myoblasts (Thermo Fisher Scientific A11440) were seeded on 24-well collagen plates in 1 mL of DMEM (ATCC 30-2002) supplemented with 10% FBS (Nucleus Biologics FBS1824) and 1x ITS (Thermo Fisher Scientific 41400045). The cells were incubated at 37°C and 5% CO2 until the myoblasts reached confluence. At this point, differentiation into myotubes was induced by incubation for 2 days with 1000 μl of 1 mL DMEM (ATCC30-2002) supplemented with 2% horse serum (ATCC 30-2040) and 1x ITS (Thermo Fisher Scientific 41400045). The culture medium was replaced with 500 μl of differentiation medium, and 50 μl of TfR1.IgG2 mAb-SSB(Cy5) conjugate diluted in PBS was added to final concentrations of 1 and 10 nM. Cells were incubated at 37°C and 5% CO2 for 24 hours, washed three times with 500 μl of PBS, and lysed in 150 μl of T-PER lysis buffer (Thermo Fisher Scientific 78510) using a freeze-thaw cycle. 75 μl of lysed cells were diluted with 75 μl of nuclease-free water, and fluorescence was measured using a TECAN plate reader (Ex 635 nM - Em 675 nM). The results are shown as fluorescence in cells in response to input (Figure 16).

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

[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 on 24-well collagen plates (Thermo Fisher A1142802) in 1 mL of DMEM (ATCC30-2002) supplemented with 10% FBS (Nucleus Biologics FBS1824) and 1xITS (Thermo Fisher Scientific 41400045). The cells were incubated at 37°C and 5% CO2 until the myoblasts reached confluence. At this point, differentiation into myotubes was induced by incubation for 2 days with 1000 μl of 1 mL DMEM (ATCC30-2002) supplemented with 2% horse serum (ATCC 30-2040) and 1xITS (Thermo Fisher Scientific 41400045). The culture medium was refreshed, and 100 μl of TfR1.IgG2 mAb-SSB conjugate diluted in PBS was added. The treated cells were incubated for 72 hours. For sample collection, the medium was removed from the wells, and 150 μl of Trizol (Ambion 15596018) was added. The plates were frozen at -80°C overnight or longer before analysis. RNA was isolated using the Direct-zol 96 RNA kit according to the manufacturer's instructions and quantified spectroscopically. RNA (100=200 ng) was reverse transcribed using the High Capacity cDNA kit (Thermo Fisher #4368813) according to the manufacturer's instructions. mRNA concentration was quantified using TaqMan qPCR with appropriately designed primers and probes. PPIB (housekeeping gene) was used as an internal RNA loading control. The ΔΔCt method was used to calculate the mRNA percentage, and PBS-treated cells were set to 100% expression. All SSB siRNA conjugates tested downregulated SSBs by 50% with similar efficacy (Figure 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] Exemplary SSB siRNA knockdown of anti-TfR antibody in cynomolgus monkey studies Treatment of cynomolgus monkeys with exemplary anti-TfR antibodies will be tested to determine the percentage of SSB mRNA downregulation in the gastrocnemius muscle. Dosages of 30 mg / kg, 10 mg / kg, and 3 mg / kg will be tested. The activity of the antibody conjugate will be investigated on days 21 and / or 28 post-administration. Safety in cynomolgus monkeys will also be monitored by hematological and clinical chemistry analyses. [Examples]

[0511] The hIgG1 TfR-Var2ii and hIgG1 TfR-Var9ii SSB conjugates do not affect the absolute reticulocyte count in cynomolgus monkeys. hIgG1 TfR-Var2ii and hIgG1 TfR-Var9ii are humanized IgG1 antibodies that target hTfR1 and contain mutations in the hinge region of the IgG1 heavy chain, designed to remove the activator function (LALA+L328R). In contrast to chimeric hIgG2 TfR1 antibodies, administration of SSB conjugates of hIgG1 TfR-Var2ii and hIgG1 TfR-Var9ii to cynomolgus monkeys did not decrease reticulocyte counts. This result is consistent with other studies demonstrating that the depletion of immature reticulocytes by TfR1-targeted antibodies can be effectively suppressed by mutations that remove the ADCC / CDC activity of the antibody (WO2014 / 189973A2).

[0512] method:

[0513] On day 1, cynomolgus monkeys (males, 2-3 years old, body weight 2-3 kg) were given an intravenous (IV) infusion over 30 minutes (+ / - 3 minutes). As shown in Figure 18, blood samples were collected from the peripheral veins of conscious, restrained subjects at different time points after administration. [Examples]

[0514] SSB conjugates of hIgG1 TfR-Var2ii Ab and hIgG1 TfR-Var9ii Ab downregulate SSB RNA levels in cynomolgus monkey muscle. Compared to pre-administration SSB mRNA levels, a single dose of 1 or 6 mg / kg (siRNA) of hIgG1 TfR-Var2ii or hIgG1 TfR-Var9ii SSB conjugate downregulated SSB mRNA levels in the gastrocnemius and quadriceps femoris muscles by up to 72% at day 21 post-administration (Figure 19). The activity of the humanized antibody was 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 the 6 mg / kg AOC dose).

[0515] method:

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

[0517] AOC-mediated SSB knockdown, rather than siRNA delivery, is specific to muscle. Twenty-one days after a single dose of 6 mg / kg of hIgG1 TfR-Var2ii-SSB, SSB siRNA concentrations of 10–100 nM were observed in most tissues. The highest siRNA concentrations were in the liver and adrenal glands (≤1000 nM), while the lowest was in the brain (2 nM). Skeletal muscle siRNA concentrations ranged from 3–20 nM. Despite relatively low siRNA exposure, a decrease of over 50% in SSB mRNA levels was observed only in skeletal muscle and the heart. These results demonstrate that the delivery of oligonucleotide payloads by TfR1-targeted antibodies is specific to muscle and is driven by cell-specific transport pathways rather than siRNA exposure.

[0518] method:

[0519] On day 1, cynomolgus monkeys (males, 2-3 years old, body weight 2-3 kg) were given intravenous (IV) injection over 30 minutes (+ / - 3 minutes). On day 21 after administration, muscle biopsies were taken from sedated animals via 6 mm puncture. All other tissue samples were collected within 30 minutes of autopsy. The tissue samples were processed and SSB mRNA values ​​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 the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) with sequence-specific stem-loop RT primers. The cDNA obtained from the RT step was then used for real-time PCR, and the Ct values ​​were converted to plasma or tissue concentrations using a linear equation obtained from the standard curve.

[0520] While preferred embodiments of the Disclosure have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided only as examples. Numerous variations, alterations, and substitutions are currently conceivable by those skilled in the art without departing from the Disclosure. It should be understood that various alternatives to the embodiments of the Disclosure described herein may be used in the practice of the Disclosure. The following claims define the scope of the Disclosure, and the methods and structures within the claims, and their equivalents, are intended to be included in the Disclosure.

Claims

1. An anti-transferrin receptor antibody comprising a variable heavy chain (VH) region and a variable light chain (VL) region, (i) The VH region includes an HCDR1 sequence containing sequence number 1, an HCDR2 sequence containing sequence number 4, and an HCDR3 sequence containing sequence number 3, and the VL region includes an LCDR1 sequence containing sequence number 6, an LCDR2 sequence containing sequence number 7, and an LCDR3 sequence containing sequence number 8, (ii) The VH region includes an HCDR1 sequence containing sequence number 1, an HCDR2 sequence containing sequence number 2, and an HCDR3 sequence containing sequence number 3, and the VL region includes an LCDR1 sequence containing sequence number 6, an LCDR2 sequence containing sequence number 9, and an LCDR3 sequence containing sequence number 10, or (iii) The VH region includes an HCDR1 sequence containing sequence number 1, an HCDR2 sequence containing sequence number 5, and an HCDR3 sequence containing sequence number 3, and the VL region includes an LCDR1 sequence containing sequence number 11, an LCDR2 sequence containing sequence number 12, and an LCDR3 sequence containing sequence number 10, Anti-transferrin receptor antibodies, including those mentioned above.

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

3. An antibody or antigen-binding fragment that binds to a transferrin receptor, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, (i) The VH region is the sequence of sequence number 14, and the VL region is the sequence of sequence number 18. (ii) The VH region is an array of sequence number 13, and the VL region is an array of sequence number 20, or (iii) The VH region is the sequence of sequence number 15, and the VL region is the sequence of sequence number 21. An antibody or its antigen-binding fragment, including an antibody.

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

5. The antibody or its antigen-binding fragment according to claim 3, wherein the antigen-binding fragment is selected from IgG-scFv, BiTE, diabody, DART, TandAb, sc diabody, sc diabody-CH3, triplebody, 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. The antibody or its antigen-binding fragment according to claim 5, wherein the antigen-binding fragment is selected from BiTE, diabody, DART, TandAb, scdiabody, scdiabody-CH3, triplebody, 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, and scFv-KIH.

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

8. The anti-transferrin receptor antibody according to claim 1, further comprising at least one mutation in the Fc region, wherein the at least one mutation modulates effector function.

9. The anti-transferrin receptor antibody according to claim 8, wherein the effector function includes weakening or removing Fc-γ receptor binding.

10. The anti-transferrin receptor antibody according to claim 8, wherein the at least one mutation is located at residue position D265, N297, K322, L328, or P329, where the residue position is relative to IgG1.

11. The anti-transferrin receptor antibody according to claim 8, wherein the Fc region contains mutations at L233 and L234, where the residues correspond to positions 233 and 234 of SEQ ID NO:

23.

12. The anti-transferrin receptor antibody according to claim 8, wherein the Fc region is based on the IgG1 Fc region or the IgG2b Fc region and includes mutations at D265 and N297.

13. The anti-transferrin receptor antibody according to claim 1, wherein the anti-transferrin receptor antibody specifically binds to the human transferrin receptor (TfR).

14. A pharmaceutical composition comprising an anti-transferrin receptor antibody according to claim 1, or an antibody or antigen-binding fragment thereof according to claim 3, and a pharmaceutically acceptable excipient.

15. An anti-transferrin receptor antibody conjugate comprising the following formula (I) (Chemical Formula 1), 【Chemistry 1】 During the ceremony, A comprises an anti-transferrin receptor antibody or its antigen-binding fragment, wherein the anti-transferrin receptor antibody comprises a variable heavy chain (VH) region and a variable light chain (VL) region, where, (i) The VH region includes an HCDR1 sequence containing sequence number 1, an HCDR2 sequence containing sequence number 4, and an HCDR3 sequence containing sequence number 3, and the VL region includes an LCDR1 sequence containing sequence number 6, an LCDR2 sequence containing sequence number 7, and an LCDR3 sequence containing sequence number 8, (ii) The VH region includes an HCDR1 sequence containing sequence number 1, an HCDR2 sequence containing sequence number 2, and an HCDR3 sequence containing sequence number 3, and the VL region includes an LCDR1 sequence containing sequence number 6, an LCDR2 sequence containing sequence number 9, and an LCDR3 sequence containing sequence number 10, or (iii) The VH region includes an HCDR1 sequence containing sequence number 1, an HCDR2 sequence containing sequence number 5, and an HCDR3 sequence containing sequence number 3, and the VL region includes an LCDR1 sequence containing sequence number 11, an LCDR2 sequence containing sequence number 12, and an LCDR3 sequence containing sequence number 10, Includes, B contains polynucleic acid molecules, X １ An anti-transferrin receptor antibody conjugate, consisting of a single bond or linker, where n is an average value selected from 1 to 12.

16. An anti-transferrin receptor antibody conjugate comprising the following formula (I) (Chemical Formula 2), 【Chemistry 2】 During the ceremony, A comprises an anti-transferrin receptor antibody or its antigen-binding fragment, wherein the anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, where, (i) The VH region is the sequence of sequence number 14, and the VL region is the sequence of sequence number 18. (ii) The VH region is an array of sequence number 13, and the VL region is an array of sequence number 20, or (iii) The VH region is the sequence of sequence number 15, and the VL region is the sequence of sequence number 21. B contains polynucleotide molecules, X １ It consists of a single bond or a linker, and, n is an average value selected from 1 to 12, representing an anti-transferrin receptor antibody conjugate.

17. The anti-transferrin receptor antibody conjugate according to claim 15 or 16, wherein the antigen-binding fragment is selected from IgG-scFv, BiTE, diabody, DART, TandAb, scdiabody, scdiabody-CH3, triplebody, 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, scdiabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabody.

18. The anti-transferrin receptor antibody conjugate according to claim 15 or 16, wherein the polynucleic acid molecule is selected from short interfering nucleic acids (siRNA), 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 anti-transferrin receptor antibody conjugate according to claim 15 or 16, wherein the polynucleic acid molecule is double-stranded RNA (dsRNA).

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

21. The anti-transferrin receptor antibody conjugate according to claim 15 or 16, wherein the polynucleic acid molecule comprises a passenger chain and a guide chain.

22. The anti-transferrin receptor antibody conjugate according to claim 21, wherein the guide chain comprises at least one modified internucleotide bond, at least one inverted debase moiety, at least one 5'-vinylphosphonate modified non-natural nucleotide, or a combination thereof.

23. The anti-transferrin receptor antibody conjugate according to claim 15 or 16, wherein the polynucleic acid molecule further comprises modification of a sugar moiety at the 2' position, the modification at the 2' position being selected from nucleotides modified with 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).

24. The anti-transferrin receptor antibody conjugate according to claim 21, wherein the passenger chain 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. The anti-transferrin receptor antibody conjugate according to claim 21, wherein A-X1 is conjugated to the 5' end or the 3' end of the passenger chain.

26. The anti-transferrin receptor antibody conjugate according to claim 15 or 16, wherein the polynucleic acid molecule hybridizes to a target sequence of a gene, and the polynucleic acid molecule mediates RNA interference to the gene.

27. The anti-transferrin receptor antibody conjugate according to claim 26, wherein the gene comprises an upregulated gene within 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, forkheadbox transcription factor, atrogin-1 gene (FBXO32), MuRF1 gene (TRIM63), FOXO1, FOXO3, MSTN, DMD, or DMPK.

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

29. A pharmaceutical composition comprising an anti-transferrin receptor antibody conjugate according to claim 15 or 16 and a pharmaceutically acceptable excipient.

Images