CN116457015A - Method for preparing protein-oligonucleotide complex - Google Patents
Method for preparing protein-oligonucleotide complex Download PDFInfo
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- CN116457015A CN116457015A CN202180074252.7A CN202180074252A CN116457015A CN 116457015 A CN116457015 A CN 116457015A CN 202180074252 A CN202180074252 A CN 202180074252A CN 116457015 A CN116457015 A CN 116457015A
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Abstract
Some aspects of the disclosure relate to methods of purifying complexes comprising a protein (e.g., an antibody) covalently linked to a molecular charge (e.g., a charge neutral oligonucleotide, a charge oligonucleotide, or a hydrophobic small molecule) using a mixed mode resin (e.g., a hydroxyapatite resin) comprising positively charged metal sites and negatively charged ion sites. Methods of producing the complexes are also provided.
Description
RELATED APPLICATIONS
The present application claims priority from 35U.S. C. ≡119 (e) to U.S. provisional application sequence No.63/074439 entitled "METHODS OF PREPARING PROTEIN-OLIGONUCLEOTIDE COMPLEXES" filed on 3/9/2020 and U.S. provisional application sequence No.63/074436 entitled "METHODS OF PREPARING PROTEIN-OLIGONUCLEOTIDE COMPLEXES" filed on 3/9/2020; the respective content of which is incorporated herein by reference in its entirety.
Technical Field
The present application relates to methods of purifying complexes (e.g., protein-oligonucleotide conjugates and antibody-drug conjugates).
Reference is made to the sequence listing submitted as a text file through EFS-WEB
The present application contains a sequence listing that has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy created at month 9 of 2021 was named D082470043WO00-SEQ-ZJG and was 57,060 bytes in size.
Background
In recent years, several oligonucleotides (e.g., antisense oligonucleotides) have been developed to combat tissue or cell specific diseases (e.g., muscle specific diseases such as various forms of muscular dystrophy). However, it has proven challenging to deliver these oligonucleotides efficiently to their desired tissues or cells.
Disclosure of Invention
Complexes comprising tissue or cell specific proteins (e.g., antibodies) covalently linked to a therapeutic oligonucleotide provide excellent opportunities for delivery of the therapeutic oligonucleotide. Such therapeutic oligonucleotides include, for example, charge neutral oligonucleotides (e.g., PMO, PNA, etc.) and charged oligonucleotides (e.g., spacer (gapmer), mixtures, siRNA, etc.). However, it is challenging to purify and isolate the complex away from excess proteins and oligonucleotides. In some aspects, the disclosure provides methods of treating a complex comprising a protein covalently linked to an oligonucleotide, which separates unconjugated oligonucleotide and protein (e.g., antibody) from the complex. Methods of producing the complexes are also provided. In some embodiments, the methods of producing the complexes described herein reduce the level of unconnected antibodies comprising alkynyl groups. In some embodiments, the methods of producing the complexes described herein produce only trace amounts of the non-linked antibodies comprising alkynyl groups after the conjugation reaction.
One aspect of the present disclosure relates to a method of treating complexes each comprising an antibody covalently linked to one or more charge neutral oligonucleotides, the method comprising:
(i) Contacting a mixture comprising an organic solvent, the complex, and an unligated charge neutral oligonucleotide with a mixed mode resin comprising positively charged metal sites and negatively charged ion sites under conditions such that the complex adsorbs to the mixed mode resin, and
(ii) Eluting the complex from the mixed mode resin under conditions that dissociate the complex from the mixed mode resin. In some embodiments, the organic solvent is Dimethylacetamide (DMA), isopropanol (IPA), dimethylsulfoxide (dimethyl sulfoxide, DMSO), acetonitrile (ACN), or Propylene Glycol (PG). In some embodiments, the organic solvent is 5% to 30% (v/v) in the mixture of step (i), optionally wherein the organic solvent is 15% (v/v) in the mixture of step (i). In some embodiments, the organic solvent is 30% (v/v) in the mixture of step (i).
In some embodiments, the mixture in step (i) further comprises up to 10mM phosphate ions and/or up to 20mM chloride ions. In some embodiments, the method further comprises washing the mixed mode resin with a washing solution comprising an organic solvent between step (i) and step (ii), optionally wherein the organic solvent is Dimethylacetamide (DMA), isopropanol (IPA), dimethylsulfoxide (DMSO), acetonitrile (ACN), or Propylene Glycol (PG). In some embodiments, the organic solvent is 5% to 30% (v/v) in the wash solution, optionally wherein the organic solvent is 15% (v/v) in the wash solution. In some embodiments, the organic solvent is 30% (v/v) in the wash solution. In some embodiments, the wash solution further comprises up to 10mM phosphate ions and/or up to 20mM chloride ions.
In some embodiments, step (ii) comprises applying an elution solution to the mixed mode resin to elute the complex, wherein the elution solution comprises an organic solvent, optionally wherein the organic solvent is Dimethylacetamide (DMA), isopropanol (IPA), dimethylsulfoxide (DMSO), acetonitrile (ACN), or Propylene Glycol (PG). In some embodiments, the organic solvent is 10% to 30% (v/v) in the elution solution, optionally wherein the organic solvent is 10% (v/v) in the elution solution. In some embodiments, the elution solution comprises at least 30mM phosphate ions, optionally wherein the elution solution comprises at least 100mM phosphate ions. In some embodiments, the elution solution comprises a gradually increasing concentration of phosphate ions, optionally wherein the concentration of phosphate ions increases from at least 10mM to at least 100mM. In some embodiments, the pH of the elution solution is 7.6 to 8.5.
Another aspect of the disclosure relates to a method of producing a complex comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i) An oligonucleotide comprising the following structure was obtained:
(B) Wherein n is 3;
(ii) Obtaining an antibody comprising the structure:
(F) Wherein m is 4; and
(iii) Reacting the oligonucleotide in step (i) with the antibody obtained in step (ii) to obtain the complex.
Another aspect of the disclosure relates to a method of producing a complex comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i) An oligonucleotide comprising the following structure was obtained:
(D) The method comprises the steps of carrying out a first treatment on the surface of the Wherein n is 3 and wherein m is 4;
(ii) Obtaining an antibody; and
(iii) Reacting the oligonucleotide in step (i) with the antibody obtained in step (ii) to obtain the complex.
In some embodiments, the complex comprises the following structure:
(E)
wherein n is 3 and m is 4, and wherein the antibody is linked by lysine.
Another aspect of the disclosure relates to a mixture comprising a complex and an unligated oligonucleotide, the complexes each comprising an antibody covalently linked to one or more oligonucleotides, wherein the mixture is produced by a method comprising:
(i) Obtaining a first intermediate comprising an oligonucleotide covalently linked to a cleavable linker comprising a valine-citrulline sequence;
(ii) Connecting the first intermediate obtained in step (i) with a compound comprising bicyclononene to obtain a second intermediate; and
(iii) Ligating the second intermediate obtained in step (ii) with an antibody to obtain the complex;
wherein the compound comprising a bicyclononene is present in the reaction of step (iii) in an amount less than 5% of the starting amount of the compound in step (ii), optionally wherein the oligonucleotide is covalently attached at the 5' end to the cleavable linker comprising a valine-citrulline sequence and/or the antibody is attached via lysine.
Another aspect of the disclosure relates to a mixture comprising a complex and ii) unligated oligonucleotides, the complexes each comprising an antibody covalently linked to one or more oligonucleotides, wherein the mixture is produced by a method comprising:
(i) Combining one or more oligonucleotides with a linker of formula (a) under reaction conditions that produce a product of formula (B):
(A) Wherein n is 3:
(B) Wherein n is 3;
(ii) Contacting a product of formula (B) with a compound of formula (C) under reaction conditions that produce a product of formula (D):
(C) Wherein m is 4;
(D) Wherein n is 3 and m is 4; and
(iii) Contacting a product of formula (D) with an antibody under reaction conditions that produce a complex of formula (E):
(E) Wherein n is 3 and m is 4;
wherein the compound of formula (C) is present in the reaction of step (iii) in an amount of less than 5% of the starting amount of the compound of formula (C) in the reaction of step (ii).
Another aspect of the present disclosure relates to a method of treating complexes each comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i) Contacting the mixture of either of the two mixtures with a mixed mode resin comprising positively charged metal sites and negatively charged ion sites under conditions such that the complex adsorbs to the mixed mode resin, wherein the mixture comprises a trace amount of non-linked antibodies comprising alkynyl groups; and
(ii) Eluting the complex from the mixed mode resin under conditions that dissociate the complex from the mixed mode resin. In some embodiments, the mixture in step (i) is not pre-purified. In some embodiments, the mixture in step (i) comprises phosphate ions and/or chloride ions in trace amounts. In some embodiments, the method further comprises washing the mixed mode resin with a wash solution comprising up to 20mM phosphate ions and/or up to 30mM chloride ions between step (i) and step (ii), optionally wherein the solution comprises up to 10mM phosphate ions and/or up to 25mM chloride ions. In some embodiments, the pH of the wash solution is from 5.0 to 7.6. In some embodiments, in the washing step, most or all of the unbound oligonucleotides are removed from the mixed-mode resin.
In some embodiments, step (ii) comprises applying an elution solution comprising at least 30mM phosphate ions and/or at least 50mM chloride ions to the mixed mode resin to elute the complex, optionally wherein the elution solution comprises at least 100mM phosphate ions and/or at least 100mM chloride ions. In some embodiments, the pH of the elution solution is from 7.5 to 8.5.
In some embodiments, the antibody is an anti-transferrin receptor antibody. In some embodiments, the oligonucleotide is a charged oligonucleotide. In some embodiments, the oligonucleotide is a negatively charged oligonucleotide. In some embodiments, the oligonucleotide is single stranded. In some embodiments, the oligonucleotide is an antisense oligonucleotide, optionally a spacer. In some embodiments, the oligonucleotide is one strand of a double-stranded oligonucleotide, optionally wherein the double-stranded oligonucleotide is an siRNA, and optionally wherein the one strand is the sense strand of the siRNA. In some embodiments, the oligonucleotide comprises at least one modified internucleotide linkage, optionally wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the oligonucleotide comprises one or more modified nucleotides, optionally wherein the modified nucleotide comprises a 2 '-O-methoxyethyl ribose (MOE), locked nucleic acid (locked nucleic acid, LNA), 2' -fluoro modification, or morpholino modification.
Drawings
Figures 1A to 1C show the chemical structure of molecules involved in the ligation of anti-TfR antibodies to oligonucleotide loads. FIG. 1A shows the structure of the oligonucleotide-PAB-VC-PEG 3-azide. FIG. 1B shows endo-BCN-PEG4-PFP ester. FIG. 1C shows the oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester. BCN: bicyclic nonynes. PFP: a pentafluorophenyl group. PAB: 4-aminobenzoic acid. VC: val-cit. In all fig. 1A to 1C, n is 3 and m is 4.
FIG. 2 shows SDS-PAGE gels of crude Fab-oligonucleotide conjugates produced in reactions containing varying ratios of anti-TfR Fab and oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP esters. The leftmost lane shows the molecular weight ladder and the second lane shows unreacted anti-TfR Fab. Lanes labeled A, B, C, D, E and F show the reaction products. The labels D0, D1, D2 and D3 indicate the reaction products with drug-to-antibody ratios of 0, 1, 2 and 3, respectively.
FIG. 3 shows SDS-PAGE gels of crude Fab-oligonucleotide conjugates produced in reactions containing different ratios of anti-TfR Fab with oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester and different solvent conditions. The leftmost lane shows a molecular weight ladder strap. Lanes labeled G, H, I, J, K, L and M show the reaction products.
FIG. 4 shows the results of RP C18UPLC monitoring the reaction between endo-BCN-PEG4-PFP ester and oligonucleotide-PAB-VC-PEG 4-azide over time. The peak area of endo-BCN-PEG4-PFP ester starting material was measured at 220nm over time and shows the percentage of the original starting material peak at time 0.
Figure 5 shows size exclusion chromatography (size exclusion chromatography, SEC) chromatograms of anti-TfR Fab' -oligonucleotide conjugates after purification measured at 260nm and 280 nm.
FIG. 6 shows SDS-PAGE gels of anti-TfR Fab-oligonucleotide conjugates after purification. The rightmost lane shows a molecular weight ladder strap. The remaining three lanes show the fractions eluted from the purification column. The labels D0, D1, D2 and D3 indicate the reaction products with drug-to-antibody ratios of 0, 1, 2 and 3, respectively.
FIG. 7 is a graph showing the activity of complexes treated using the methods described herein to reduce DMPK mRNA levels in vitro. Complex 1 was prepared by 2-step conjugation: see example 6. Complex 2 was prepared by pre-reaction conjugation: see example 1.
FIG. 8 shows analytical HPLC-SEC traces of crude and purified anti-TfR-BCN reaction products. The curve of the crude product shows a second main peak at about 17.4 minutes, which is absent in the curve of the purified product, indicating that unconjugated BCN was removed. BCN: bicyclic nonynes.
FIG. 9 shows analytical RP-HPLC of crude adaptor-oligonucleotide reaction products showing peaks corresponding to free oligonucleotides and free adaptors in addition to the peaks of the desired oligonucleotide-adaptor products.
FIG. 10 shows analytical RP-HPLC of the purified adaptor-oligonucleotide reaction product by alcohol precipitation. The curve shows a significant decrease in the free oligonucleotide peak and almost disappearance of the free linker peak relative to the curve shown in fig. 9, indicating that free oligonucleotide and linker were removed by the purification process.
FIG. 11 shows LCMS of purified oligonucleotide-linker product showing only one major peak.
FIG. 12 shows SDS-PAGE analysis of anti-TfR-oligonucleotide conjugation reaction products. The left lane shows the molecular weight ladder and the right lane shows the reaction products. DAR0, DAR1, DAR2, DAR3, and DAR4 tags indicate products with drug-to-antibody ratios (DAR) of 0, 1, 2, 3, or 4, respectively.
FIG. 13 shows analytical SEC curves for anti-TfR-oligonucleotide conjugation reaction products.
FIG. 14 shows RP-C18UPLC monitoring of the progress of the pre-reaction at 220nm by the disappearance of endo-BCN-PEG4-PFP ester starting material over time at reaction durations of 5, 35 and 65 minutes. PFP: a pentafluorophenyl group.
FIG. 15 shows SDS-PAGE analysis of crude reaction mixtures after 20 hours conjugation of anti-TfR Fab with oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester. The left lane shows a molecular weight ladder strap. The D0, D1, D2, D3, D4, D5, and D6 tags indicate products with a drug-to-antibody ratio (DAR) of 0, 1, 2, 3, 4, 5, or 6, respectively.
Figure 16 shows Size Exclusion Chromatography (SEC) chromatograms (260 nm) of HA flow-through fractions during chromatographic purification. The chromatogram shows the free (unconjugated) loading substance at 10.5 minutes and 11.3 minutes. Little to no Fab-oligonucleotide conjugate was observed in the loading flow-through (load flow-through) (main peak at about 9.1 min).
Figure 17 shows SEC chromatograms (260 nm) of HA elution peaks during chromatographic purification. The chromatogram shows complete removal of free (unconjugated) oligonucleotide loading species (expected peak at about 10.5 minutes and about 11.3 minutes) and alternatively only Fab-oligonucleotide conjugates (main peak at about 9.1 minutes).
FIG. 18 shows overlapping SEC chromatograms (260 nm) of 24 μg implants of crude conjugation reaction product (4.0 μl at 6 μg/μl; the "crude reaction mixture" curve with two main peaks) and theoretically 24 μg Fab-oligonucleotide conjugates in HA eluate assuming 100% recovery (7.4 μl at 3.24 μg/μl and a volume of 13.9mL; the "HA eluate" curve with only one main peak).
Figures 19A to 19B show SEC chromatograms of final purified anti-TfR-oligonucleotide Fab-oligonucleotide conjugates at 260nm (figure 19A) and 280nm (figure 19B).
FIG. 20 shows SDS-PAGE analysis of purified reaction products in 50mM His (pH 6.0) buffer after conjugation of anti-TfR Fab with the oligonucleotide to form a Fab-oligonucleotide conjugate. The left lane shows the purified Fab-oligonucleotide conjugate reaction product and the right lane shows the molecular weight ladder.
FIG. 21 shows SDS-PAGE analysis of crude reaction products after conjugation of oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester with anti-TfR Fab in different molar ratios. The left lane shows a molecular weight ladder strap. Lanes A, B, C and D show the products of conjugation reactions with 2, 4, 6 or 10 molar equivalents of BCN relative to TfR, respectively. The right lane shows unreacted anti-TfR Fab. The D0, D1, D2, D3, D4, D5, and D6 tags indicate products with a drug-to-antibody ratio (DAR) of 0, 1, 2, 3, 4, 5, or 6, respectively.
FIG. 22 shows SDS-PAGE analysis of crude reaction products after conjugation of oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester with anti-TfR Fab in different molar ratios. The leftmost and rightmost lanes show molecular weight ladder tapes. Lanes I, H, G, F and E show the products of conjugation reactions with 8, 6, 5, 4 or molar equivalents of BCN relative to anti TfR, respectively. The D0, D1, D2, D3, D4, D5, and D6 tags indicate products with a drug-to-antibody ratio (DAR) of 0, 1, 2, 3, 4, 5, or 6, respectively.
FIG. 23 shows the hydrolysis rate of endo-BCN-PEG4-PFP esters. The blue curve results show that the initial hydrolysis rate of endo-BCN-PEG4-PFP ester at 1:1DMA is about 0.9%/hour. The green curve shows the reaction between the model val-cit-PAB-PEG 3-azide loading and endo-BCN-PEG4-PFP ester under the same 1:1DMA reaction conditions. Both curves show absorbance at 220 nm.
Fig. 24A-24C show SEC-UPLC analysis of crude reaction mixture (fig. 24A), sample flow-through (fig. 24B) and combined HA eluate (fig. 24C). The crude reaction mixture contained free oligonucleotide and anti-TfR-oligonucleotide Fab-oligonucleotide conjugates. The sample flow-through contains high DAR material, material with high antibody-oligonucleotide conjugation. The combined HA eluate contains the remaining oligonucleotides and antibody-oligonucleotide conjugates.
Figure 25 shows SEC-UPLC analysis of the final conjugate of buffer exchange. The total conjugation efficiency yield was 62% with residual oligonucleotides shown in the right peak.
Fig. 26A-26B show SEC-UPLC analysis of combined HA eluate (fig. 26A) and sample flow-through (fig. 26B). The sample was maintained at 21℃and the flow rate was 0.25 mL/min. The mobile phase contained 100mM sodium phosphate and 10% MeCN, pH 7.3. The combined HA eluate and sample flow-through shows the presence of free oligonucleotides.
FIG. 27 shows SEC-UPLC of the final antibody fragment-drug conjugate (FDC). The sample was maintained at 21℃and the flow rate was 0.25 mL/min. The mobile phase contained 100mM NaPO and 10% MeCN, pH 7.3. 47.2% of the conjugate was recovered, with the free oligonucleotide shown in the right peak.
FIGS. 28A to 28B show HA purification chromatograms (FIG. 28A) and SDS-PAGE (FIG. 28B) of each solution. The pH of the reaction mixture was adjusted to 5.7 with 500mM MES at pH 3.5.
Fig. 29A to 29C show sample flow-through chromatograms by SEC at different equilibration buffers and loading ratios of 6 mg/mL. FIG. 29A shows sample flow-through in 10mM sodium phosphate equilibration buffer at pH 5.6 at 0% IPA. FIG. 29B shows sample flow-through in 10mM NaPO equilibration buffer at 25% IPA at pH 5.6. FIG. 29C shows sample flow-through in 5mM sodium phosphate equilibration buffer at pH 5.6 at 25% IPA.
Detailed Description
As described herein, the present disclosure provides methods of treating (e.g., producing, purifying) complexes (e.g., complexes comprising a protein (e.g., a muscle targeting agent (e.g., an antibody) covalently linked to a molecular cargo (e.g., an oligonucleotide or small molecule)). In some embodiments, the molecular charge in the complex treatment by the methods described herein is an oligonucleotide (e.g., a charge neutral oligonucleotide (e.g., PMO) or a charged oligonucleotide) or a hydrophobic small molecule. In some embodiments, one or more complexes containing a protein (e.g., an antibody) covalently linked to a molecular load (e.g., a charge neutral oligonucleotide or a charged oligonucleotide) are purified from a mixture comprising the complex and an unconnected (e.g., excess) molecular load (e.g., a charge neutral oligonucleotide or a charged oligonucleotide) using a mixed mode resin (e.g., a hydroxyapatite resin, a ceramic hydroxyapatite resin, a fluoroapatite resin, a chloroapatite resin) comprising positively charged metal sites and negatively charged ion sites, wherein an organic solvent is present in the mobile phase of the mixed mode resin chromatography. The presence of an organic solvent in the mixed-mode chromatography mobile phase reduces non-specific interactions between molecular loadings such as oligonucleotides (e.g., charge neutral oligonucleotides (e.g., PMOs) or charged oligonucleotides) and hydrophobic small molecules) and mixed-mode resins and increases the yield of complexes.
In some embodiments, the purified complexes are particularly useful for delivering molecular cargo (e.g., oligonucleotides) that modulate the expression or activity of a target gene in a muscle cell, for example, in a subject suffering from or suspected of suffering from a muscle disorder. For example, in some embodiments, the complexes may be used to treat subjects with rare muscle disorders including myotonic muscular dystrophy (e.g., type 1 myotonic muscular dystrophy), facial shoulder brachial muscular dystrophy (Facioscapulohumeral muscular dystrophy, FSHD), pompe disease (Pompe disease), central nuclear myopathy, progressive ossifiable fibroplasia, friedreich's ataxia), and duchenne muscular dystrophy (Duchenne muscular dystrophy). In some embodiments, depending on the condition to be treated, different oligonucleotides may be used in such complexes.
Further aspects of the disclosure, including descriptions of defined terms, are provided below.
I. Definition of the definition
And (3) application: as used herein, the term "administering" and variations thereof means providing a complex to a subject in a physiologically and/or pharmacologically useful manner (e.g., to treat a disorder in a subject).
About: as used herein, the term "about" or "approximately," as applied to one or more target values, refers to a value similar to the stated reference value. In certain embodiments, the term "about" or "approximately" refers to a range of values that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) the stated reference value, unless stated otherwise or otherwise apparent from the context (unless such numbers exceed 100% of the possible values).
Antibody: as used herein, the term "antibody" refers to a polypeptide comprising at least one immunoglobulin variable domain or at least one epitope (e.g., paratope) that specifically binds an antigen. In some embodiments, the antibody is a full length antibody, e.g., full length IgG. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a F (ab') 2 fragment, an Fv fragment, or an scFv fragment. In some embodiments, the antibody is a nanobody derived from a camelidae antibody or a nanobody derived from a shark antibody. In some embodiments, the antibody is a diabody. In some embodiments, the antibody comprises a framework with human germline sequences. In another embodiment, the antibody comprises a heavy chain constant domain selected from the group consisting of IgG, igG1, igG2A, igG2B, igG2C, igG3, igG4, igA1, igA2, igD, igM, and IgE constant domains. In some embodiments, the antibody comprises a heavy (H) chain variable region (abbreviated herein as VH) and/or a light (L) chain variable region (abbreviated herein as VL). In some embodiments, the antibody comprises a constant domain, such as an Fc region. Immunoglobulin constant domain refers to a heavy chain or light chain constant domain. The amino acid sequences of the human IgG heavy and light chain constant domains and their functional variations are known. With respect to heavy chains, in some embodiments, the heavy chains of the antibodies described herein may be alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chains. In some embodiments, the heavy chain of an antibody described herein can comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In a particular embodiment, the antibodies described herein comprise human γ1ch1, CH2 and/or CH3 domains. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, such as any known in the art. Non-limiting examples of human constant region sequences have been described in the art, for example, see U.S. Pat. No.5,693,780 and Kabat E A et al, (1991) supra. In some embodiments, a VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, the antibody is modified, e.g., by glycosylation, phosphorylation, SUMO (SUMO) and/or methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody by N-glycosylation, O-glycosylation, C-glycosylation, glycosyl phosphatidyl inositol (GPI anchor attachment), and/or phosphoglycosylation (phosphoglycosylation). In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, an antibody is a construct comprising a polypeptide comprising one or more antigen binding fragments of the present disclosure covalently linked to a linker polypeptide or immunoglobulin constant domain. The linker polypeptide comprises two or more amino acid residues linked by peptide bonds and is used to link one or more antigen binding portions. Examples of linker polypeptides have been reported (see, e.g., holliger, P., et al (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; poljak, R.J., et al (1994) Structure 2:1121-1123). In addition, the antibody may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to make tetrameric scFv molecules (Kipriyanov, S.M., et al (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine residues, labeled peptides and C-terminal polyhistidine tags to make bivalent and biotinylated scFv molecules (Kipriyanov, S.M., et al (1994) mol. Immunol.31:1047-1058).
CDR: as used herein, the term "CDR" refers to a complementarity determining region within an antibody variable sequence. There are three CDRs in each of the variable regions of the heavy and light chains, referred to as CDR1, CDR2 and CDR3, respectively, for each variable region. The term "set of CDRs" as used herein refers to a set of three CDRs capable of binding an antigen that are present within a single variable region. The exact boundaries of these CDRs have been defined differently for different systems. These CDRs may be referred to as Kabat CDR.sub-portions of the CDRs may be designated L1, L2 and L3 or H1, H2 and H3, respectively, wherein "L" and "H" designate the light and heavy chain regions, respectively, which may be referred to as Chothia CDRs, with boundaries overlapping Kabat CDRs Padlan (FASEB J.9:133-139 (1995)) and MacCallum (J Mol Biol 262 (5): 45 (1996)) have described other boundary definitions defining CDRs overlapping Kabat CDRs which may not strictly follow one of the systems described above, but which may still overlap Kabat CDRs in accordance with one of the methods of shortening the residues or even using any of the preferred set of CDRs or the methods of the present invention.
CDR grafted antibody (CDR-grafted antibody): the term "CDR-grafted antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species but wherein the sequences of one or more CDR regions of VH and/or VL are replaced by CDR sequences from another species, e.g., an antibody having murine heavy and light chain variable regions and wherein one or more murine CDRs (e.g., CDR 3) have been replaced by human CDR sequences.
Chimeric antibody: the term "chimeric antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species and constant region sequences from another species, e.g., an antibody having murine heavy and light chain variable regions linked to human constant regions.
Complementary: as used herein, the term "complementary" refers to the ability to precisely pair between two nucleotides or two sets of nucleotides. In particular, complementarity is a term that characterizes the degree to which hydrogen bonding pairing causes binding between two nucleotides or groups of nucleotides. For example, bases at one position of an oligonucleotide are considered complementary to each other if the bases at that position are capable of hydrogen bonding with bases at the corresponding position of the target nucleic acid (e.g., mRNA). Base pairing can include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairing, an adenosine base (a) is complementary to a thymidine base (T) or a uracil base (U), a cytosine base (C) is complementary to a guanosine base (G), and a universal base such as 3-nitropyrrole or 5-nitroindole can hybridize to any A, C, U or T and be considered complementary. Inosine (I) is also known in the art as a universal base and is considered complementary to any A, C, U or T.
Conservative amino acid substitutions: as used herein, "conservative amino acid substitutions" refer to amino acid substitutions that do not alter the relative charge or dimensional characteristics of the protein in which they are made. Variants can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as can be found in references compiling such methods: for example Molecular Cloning: ALaboratory Manual, J.Sambrook, et al, eds., fourth Edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, new York,2012, or Current Protocols in Molecular Biology, F.M. Ausubel, et al, eds., john Wiley & Sons, inc., new York. Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
Covalent attachment: as used herein, the term "covalently linked" refers to a feature in which two or more molecules are linked together by at least one covalent bond. In some embodiments, two molecules may be covalently linked together by a single bond, such as a disulfide bond or disulfide bridge, that serves as a linker between the molecules. However, in some embodiments, two or more molecules may be covalently linked together by a molecule that acts as a linker that links the two or more molecules together by multiple covalent bonds. In some embodiments, the linker may be a cleavable linker. However, in some embodiments, the linker may be a non-cleavable linker.
Cross-reactivity: as used herein and in the context of a targeting agent (e.g., an antibody), the term "cross-reactive" refers to the property of a substance that is capable of specifically binding with similar affinity or avidity to more than one antigen of similar type or class (e.g., antigens of multiple homologs, paralogs or orthologs). For example, in some embodiments, antibodies that are cross-reactive to similar types or classes of human and non-human primate antigens (e.g., human transferrin receptor and non-human primate transfer receptor) are capable of binding with similar affinity or avidity to human and non-human primate antigens. In some embodiments, the antibodies are cross-reactive to human and rodent antigens of similar types or classes. In some embodiments, the antibodies are cross-reactive with a similar type or class of rodent antigens and non-human primate antigens. In some embodiments, the antibodies are cross-reactive with similar types or classes of human, non-human primate, and rodent antigens.
Disease alleles: as used herein, the term "disease allele" refers to any alternative form (e.g., mutant form) of a gene whose allele is associated with a disease and/or contributes directly or indirectly to or contributes to the disease. Disease alleles may include genetic alterations relative to wild-type (non-disease) alleles, including, but not limited to, insertions (e.g., disease-related repeats described below), deletions, missense mutations, nonsense mutations, and splice site mutations. In some embodiments, the disease allele has a loss-of-function mutation. In some embodiments, the disease allele has a function-acquiring mutation. In some embodiments, the disease allele encodes an activating mutation (e.g., encodes a constitutively active protein). In some embodiments, the disease allele is a recessive allele having a recessive phenotype. In some embodiments, the disease allele is a dominant allele having a dominant phenotype.
Disease-related repeat: as used herein, the term "disease-related repeat" refers to a repeat nucleotide sequence at a genomic position, wherein the number of units of the repeat nucleotide sequence is associated with and/or contributes, directly or indirectly, to or causes a genetic disease. Each repeat unit of a disease-related repeat may be 2, 3, 4, 5 or more nucleotides in length. For example, in some embodiments, the disease-related repeat is a dinucleotide repeat. In some embodiments, the disease-related repeat is a trinucleotide repeat. In some embodiments, the disease-related repeat is a tetranucleotide repeat. In some embodiments, the disease-related repeat is a five nucleotide repeat. In some embodiments, the disease-related repeat comprises a CAG repeat, a CTG repeat, a CUG repeat, a CGG repeat, a CCTG repeat, or a nucleotide complement of any thereof. In some embodiments, the disease-related repeat is in a non-coding portion of the gene. However, in some embodiments, the disease-related repeat is in the coding region of the gene. In some embodiments, the disease-related repeat is amplified from a normal state to a length that directly or indirectly contributes to or contributes to the genetic disease. In some embodiments, the disease-related repeat is in RNA (e.g., an RNA transcript). In some embodiments, the disease-related repeat is in DNA (e.g., chromosome, plasmid). In some embodiments, the disease-related repeat is amplified in the chromosome of the germ cell. In some embodiments, the disease-related repeat is amplified in a chromosome of the somatic cell. In some embodiments, the disease-related repeat is amplified to a number of repeat units associated with congenital episodes of the disease. In some embodiments, the disease-related repeat is amplified to a number of repeat units associated with the onset of childhood disease. In some embodiments, the disease-related repeat is amplified to a number of repeat units associated with an adult disease onset.
A frame: as used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region minus the CDRs. Since the exact definition of CDR sequences can be determined by different systems, the meaning of framework sequences accordingly has different interpretations. The six CDRs (CDR-L1, CDR-L2 and CDR-L3 of the light chain and CDR-H1, CDR-H2 and CDR-H3 of the heavy chain) also divide the framework on the light and heavy chains into four sub-regions (FR 1, FR2, FR3 and FR 4) on each chain, with CDR1 located between FR1 and FR2, CDR2 located between FR2 and FR3, and CDR3 located between FR3 and FR 4. In the case where a specific sub-region is not designated as FR1, FR2, FR3 or FR4, the framework regions mentioned by others represent the combined FR within the variable regions of a single native immunoglobulin chain. As used herein, FR represents one of four subregions, and FRs represents two or more of the four subregions constituting the framework region. Human heavy and light chain acceptor sequences are known in the art. In one embodiment, receptor sequences known in the art may be used in the antibodies disclosed herein.
Human antibodies: as used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may comprise amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in CDRs, particularly in CDR 3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.
Humanized antibodies: the term "humanized antibody" refers to an antibody that comprises heavy and light chain variable region sequences from a non-human species (e.g., mouse) but in which at least a portion of the VH and/or VL sequences have been altered to be more "human-like" (i.e., more similar to human germline variable sequences). One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced into non-human VH and VL sequences in place of the corresponding non-human CDR sequences. In one embodiment, humanized anti-transferrin receptor antibodies and antigen binding portions are provided. Such antibodies may be produced by obtaining murine anti-transferrin receptor monoclonal antibodies using conventional hybridoma techniques followed by humanization using in vitro genetic engineering, such as those disclosed in PCT publication No. wo 2005/123126 A2 to kasian et al.
Internalizing cell surface receptors: as used herein, the term "internalized cell surface receptor" refers to a cell surface receptor that is internalized by a cell under an external stimulus (e.g., ligand binding to the receptor). In some embodiments, the internalized cell surface receptor is internalized by endocytosis. In some embodiments, the internalized cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, internalized cell surface receptors are internalized by clathrin-independent pathways, such as phagocytosis, megaloblastic, cell and raft mediated uptake, or constitutive clathrin-independent endocytosis. In some embodiments, the internalized cell surface receptor comprises an intracellular domain, a transmembrane domain, and/or an extracellular domain, which may optionally further comprise a ligand binding domain. In some embodiments, the cell surface receptor is internalized by the cell upon ligand binding. In some embodiments, the ligand may be a muscle targeting protein or a muscle targeting antibody. In some embodiments, the internalized cell surface receptor is a transferrin receptor.
Isolated antibodies: as used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to a transferrin receptor is substantially free of antibodies that specifically bind to antigens other than the transferrin receptor). However, isolated antibodies that specifically bind to the transferrin receptor complex may have cross-reactivity with other antigens (e.g., transferrin receptor molecules from other species). In addition, the isolated antibodies may be substantially free of other cellular material and/or chemicals.
Kabat numbering: the terms "Kabat numbering", "Kabat definition" and "Kabat labeling" are used interchangeably herein. These terms are recognized in the art as referring to the system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen binding portion thereof that are more variable (i.e., hypervariable) than other amino acid residues (Kabat et al (1971) ann.ny Acad, sci.190:382-391 and Kabat, e.a., et al (1991) Sequences of Proteins of Immunological Interest, fifth Edition, u.s.part of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region of CDR1 is amino acids 31 to 35, the hypervariable region of CDR2 is amino acids 50 to 65, and the hypervariable region of CDR3 is amino acids 95 to 102. For the light chain variable region, the hypervariable region of CDR1 is amino acids 24 to 34, the hypervariable region of CDR2 is amino acids 50 to 56, and the hypervariable region of CDR3 is amino acids 89 to 97.
Mixed mode treeAnd (3) grease: as used herein, the term "mixed mode resin" refers to a chromatographic resin or material for purification, separation and/or isolation of biomolecules comprising positively charged metal sites and negatively charged ion sites. In some embodiments, the metal site comprises calcium. In some embodiments, the negatively charged ionic sites comprise phosphate, sulfate, fluoride, or chloride. In some embodiments, the metal site comprises calcium and the negatively charged ion site comprises phosphate, and optionally sulfate, fluoride, or chloride. In some embodiments, the mixed mode resin is an apatite resin. In some embodiments, the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxy fluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin. In some embodiments, the apatite resin comprises a mineral of the formula: ca (Ca) 10 (PO 4 ) 6 (OH) 2 . In some embodiments, the apatite resin comprises a mineral of the formula: ca (Ca) 10 (PO 4 ) 6 F 2 . In some embodiments, the apatite resin comprises a mineral of the formula: ca (Ca) 10 (PO 4 ) 6 Cl 2 。
Molecular loading: as used herein, the term "molecular load" refers to a molecule or substance that plays a role in regulating biological outcome. In some embodiments, the molecular cargo is covalently linked or otherwise associated with the muscle targeting agent. In some embodiments, the molecular cargo is a small molecule, protein, peptide, nucleic acid, or oligonucleotide. In some embodiments, the molecular load is a hydrophobic small molecule. In some embodiments, the molecular charge is an oligonucleotide (e.g., a charge neutral oligonucleotide (e.g., a phosphodiamide morpholino oligomer (phosphorodiamidate morpholino oligomer, PMO)) or a charged oligonucleotide). In some embodiments, the molecular cargo functions to regulate transcription of the DNA sequence, regulate expression of the protein, or regulate activity of the protein. In some embodiments, the molecular cargo is an oligonucleotide, e.g., an oligonucleotide comprising a strand having a region complementary to a target gene.
Muscle disease gene: as used herein, the term "muscle disease gene" refers to a gene having at least one disease allele associated with and/or directly or indirectly contributing to or causing a muscle disease. In some embodiments, the muscle disease is a rare disease, e.g., as defined by the genetic and rare disease information center (Genetic and Rare Diseases Information Center, GARD), which is a program of the national transformation science facilitation center (National Center for Advancing Translational Sciences, NCATS). In some embodiments, the muscle disorder is a rare disorder characterized by affecting less than 200,000 people. In some embodiments, the muscle disorder is a monogenic disorder. In some embodiments, the muscle disease gene is a gene listed in table 1.
Muscle targeting agents: as used herein, the term "muscle targeting agent" refers to a molecule that specifically binds to an antigen expressed on a muscle cell. The antigen in or on the muscle cell may be a membrane protein, such as an integral membrane protein or a peripheral membrane protein. Generally, the muscle targeting agent specifically binds to an antigen on the muscle cell, which aids in internalizing the muscle targeting agent (and any associated molecular load) into the muscle cell. In some embodiments, the muscle targeting agent specifically binds to an internalized cell surface receptor on the muscle and is capable of internalizing into the muscle cell by receptor-mediated internalization. In some embodiments, the muscle targeting agent is a small molecule, protein, peptide, nucleic acid (e.g., aptamer), or antibody. In some embodiments, the muscle targeting agent is covalently linked to the molecular cargo. In some embodiments, the muscle targeting agent is a muscle targeting protein (e.g., an antibody).
Muscle targeting antibodies: as used herein, the term "muscle targeting antibody" refers to a muscle targeting agent that is an antibody that specifically binds to an antigen present in or on a muscle cell. In some embodiments, the muscle targeting antibody specifically binds to an antigen on a muscle cell, which aids in internalizing the muscle targeting antibody (and any associated molecular load) into the muscle cell. In some embodiments, the muscle targeting antibody specifically binds to an internalized cell surface receptor present on a muscle cell. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to a transferrin receptor.
An oligonucleotide: as used herein, the term "oligonucleotide" refers to an oligonucleotide compound of up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), micrornas, spacer polymers, hybrid polymers, phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., cas9 guide RNAs), and the like. The oligonucleotide may be single-stranded or double-stranded. In some embodiments, the oligonucleotides may comprise one or more modified nucleotides (e.g., 2' -O-methyl sugar modification, purine or pyrimidine modification). In some embodiments, the oligonucleotide may comprise one or more modified internucleotide linkages. In some embodiments, the oligonucleotide may comprise one or more phosphorothioate linkages, which may be in an Rp or Sp stereochemical conformation.
Recombinant antibodies: as used herein, the term "recombinant human antibody" is intended to include all human antibodies produced, expressed, produced, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described in more detail in this disclosure), antibodies isolated from recombinant, combinatorial human antibody libraries (Hoogenboom h.r., (1997) TIB tech.15:62-70; azzazy H., andhighsmith w.e., (2002) clin.biochem.35:425-445;Gavilondo J.V., and Larrick j.w. (2002) biotechnology 29:128-145; hoogenboom H., andchares p. (2000) Immunology Today 21:371-378), antibodies isolated from animals transgenic for human immunoglobulin genes (e.g., mice) (see, e.g., taylor, l.d., et. 1992) nucl.20.62:62-62, or any other means involved in the production of antibodies by recombinant means of DNA sequences, 62:62-62:364, or any other means). Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when animals transgenic for human Ig sequences are used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are those sequences that, although derived from and related to human germline VH and VL sequences, may not naturally occur in the human antibody germline repertoire in vivo. One embodiment of the present disclosure provides fully human antibodies capable of binding to human transferrin receptor, which can be produced using techniques well known in the art, such as, but not limited to, using human Ig phage libraries, such as those disclosed in PCT publication No. WO 2005/007699 A2 to Jermus et al.
Complementary region: as used herein, the term "complementary region" refers to a nucleotide sequence, e.g., an oligonucleotide, that is sufficiently complementary to a homologous nucleotide sequence, e.g., a target nucleic acid, such that the two nucleotide sequences are capable of annealing to each other under physiological conditions (e.g., in a cell). In some embodiments, the complementary region is fully complementary to the homologous nucleotide sequence of the target nucleic acid. However, in some embodiments, the complementary region is partially complementary (e.g., at least 80%, 90%, 95%, or 99% complementary) to the homologous nucleotide sequence of the target nucleic acid. In some embodiments, the complementary region comprises 1, 2, 3, or 4 mismatches compared to the homologous nucleotide sequence of the target nucleic acid.
Specific binding: as used herein, the term "specific binding" refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that allows the molecule to be used to distinguish the binding partner from a suitable control in a binding assay or other binding environment. With respect to antibodies, the term "specific binding" refers to the ability of an antibody to bind to a specific antigen with a degree of affinity or avidity that enables the antibody to be used to distinguish the specific antigen from other antigens, e.g., to the extent that allows preferential targeting of certain cells (e.g., myocytes) by binding to an antigen as described herein, as compared to a suitable reference antigen or antigens. In some embodiments, an antibody specifically binds to a target if the KD of the antibody binding to the target is at least about 10-4M, 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M, 10-13M or less. In some embodiments, the antibody specifically binds to a transferrin receptor (e.g., an epitope of the top domain of the transferrin receptor).
The object is: as used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate or rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, e.g., a human patient having or suspected of having a disease. In some embodiments, the subject is a human patient having or suspected of having a muscle disorder (e.g., any of the disorders provided in table 1).
Transferrin receptor: as used herein, the term "transferrin receptor" (also referred to as TFRC, CD71, p90, TFR or TFR 1) refers to an internalized cell surface receptor that binds transferrin to promote uptake of iron by endocytosis. In some embodiments, the transferrin receptor may be of human origin (NCBI gene ID 7037), non-human primate origin (e.g., NCBI gene ID 711568 or NCBI gene ID 102136007), or rodent origin (e.g., NCBI gene ID 22042). In addition, a variety of human transcript variants have been characterized that encode different isoforms of the receptor (e.g., as noted in GenBank RefSeq accession numbers: NP-001121620.1, NP-003225.2, NP-001300894.1, and NP-001300895.1).
An exemplary human transferrin receptor amino acid sequence corresponding to NCBI sequence np_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows:
an exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence np_001244232.1 (transferrin receptor protein 1, rhesus monkey (Macaca mulatta)) is as follows:
an exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence xp_005545315.1 (transferrin receptor protein 1, cynomolgus monkey (Macaca fascicularis)) is as follows:
an exemplary mouse transferrin receptor amino acid sequence corresponding to NCBI sequence np_001344227.1 (transferrin receptor protein 1, mouse (mus musculus)) is as follows:
unconnected molecular load: as used herein, the term "unconjugated molecular cargo" refers to, for example, free molecular cargo (e.g., an oligonucleotide or a small molecule) or excess molecular cargo (e.g., an oligonucleotide or a small molecule) that is present in solution after a conjugation reaction produces a complex comprising a protein linked to the molecular cargo (e.g., an oligonucleotide or a small molecule). In some embodiments, the unconnected molecular payload (e.g., an oligonucleotide or small molecule) is not connected to a protein (e.g., an antibody). In some embodiments, the unconnected molecular payload (e.g., oligonucleotide or small molecule) is not connected to any other moiety. In some embodiments, the unconnected molecular payload (e.g., an oligonucleotide or small molecule) is connected to a functional group, but not to a protein (e.g., an antibody) to form a complex. In some embodiments, the unconnected molecular payload (e.g., an oligonucleotide or small molecule) is connected to the linker or a portion of the linker, but not to the protein (e.g., an antibody) to form a complex.
Unconnected proteins: as used herein, the term "unconjugated protein" refers to free protein or excess protein (e.g., free antibody or excess antibody) present in solution, for example, after conjugation reactions to produce a complex comprising the protein linked to a molecular charge. In some embodiments, the unconnected protein (e.g., antibody) is not chemically modified. In some embodiments, the unligation (e.g., antibody) is chemically modified. In some embodiments, the unconnected (e.g., antibody) is chemically modified to include a functional group, but is not connected to a molecular charge (e.g., oligonucleotide or small molecule). In some embodiments, the functional group is for conjugation to a molecular charge (e.g., by click chemistry). In some embodiments, the functional group is an alkynyl group. In some embodiments, the unconnected (e.g., antibody) comprises an alkynyl.
A complex: as used herein, the term "complex" refers to a conjugate comprising a protein (e.g., an antibody) covalently linked to one or more molecular charges (e.g., a therapeutic agent such as a small molecule or an oligonucleotide). In some embodiments, the protein in the complex comprises a targeting agent (e.g., an antibody). In some embodiments, the targeting agent targets a muscle (e.g., an anti-transferrin receptor antibody).
And (3) treatment: as used herein, the term "treating" includes, but is not limited to, the production (e.g., by conjugation), isolation (e.g., from a reaction mixture), and/or modification of the complexes described herein.
Alkynes: as used herein, the term "alkyne" refers to an unsaturated hydrocarbon that contains at least one carbon-carbon triple bond.
Charged oligonucleotides: as used herein, the term "charged oligonucleotide" refers to an oligonucleotide analog comprising a backbone having a net negative or positive charge at physiological pH (e.g., pH 7.35 to pH 7.45). In some embodiments, the charged oligonucleotide has a net negative charge (referred to herein as a negatively charged oligonucleotide) at physiological pH. In some embodiments, the charged oligonucleotide has a net positive charge (referred to herein as a positively charged oligonucleotide) at physiological pH. In some embodiments, the charged oligonucleotide comprises a phosphodiester backbone having a net negative charge at physiological pH. In some embodiments, the charged oligonucleotide comprises a phosphorothioate backbone having a net negative charge at physiological pH.
Charge neutral oligonucleotides: as used herein, the term "charge neutral oligonucleotide" refers to an oligonucleotide analog comprising a charge neutral backbone at physiological pH (e.g., pH 7.35 to pH 7.45). Some examples of charge neutral oligonucleotides include, but are not limited to, phosphodiamide Morpholino Oligomers (PMO) and peptide nucleic acids (peptide nucleic acid, PNA), e.g., as in As described in et al, (Nucleic Acid Therapeutics, vol.25, no.2,2015), incorporated herein by reference.
Organic solvent: as used herein, the term "organic solvent" refers to a carbon-based material that is capable of dissolving other materials. The organic solvent has carbon atoms in the structure of its compound, since it is carbon-based. Some non-limiting examples of organic solvents that may be used in accordance with the present disclosure include, but are not limited to, dimethylacetamide (DMA), isopropanol (IPA), dimethylsulfoxide (DMSO), acetonitrile (ACN), or Propylene Glycol (PG).
% of (v/v): as used herein, the term "% (v/v)" refers to the% of the volume of one component of the mixture (e.g., organic solvent) in the total volume of the mixture.
Drug-to-antibody Ratio (DAR): as used herein, the term "drug-to-antibody ratio (DAR)" refers to the number of drugs conjugated to an antibody. The number of DARs can vary with the nature of the antibody and drug used and the experimental conditions used for conjugation (ratio of antibody to molecular loading in the starting reaction material, reaction time, nature of solvent and co-solvent, if any). The determined DAR is an average. One example of a method that may be used to determine DAR is described in Dimitrov et al, 2009,Therapeutic Antibodies and Protocols,vol 525,445,Springer Science, which is incorporated herein by reference.
An intermediate: as used herein, the term "intermediate" refers to a molecule that is produced when the process of producing the complex is performed prior to obtaining the complex. In some embodiments, the intermediate comprises an oligonucleotide linked to a linker (e.g., a cleavable linker, e.g., a val-cit linker (i.e., a cleavable linker comprising a valine-citrulline sequence)). In some embodiments, the intermediate comprises an oligonucleotide covalently linked to a linker (e.g., a cleavable linker, e.g., a val-cit linker) that is covalently linked to a compound comprising a bicyclononene. In some embodiments, the intermediate comprises an antibody covalently linked to a compound comprising a bicyclononene.
2' -modified nucleoside: as used herein, the terms "2' -modified nucleoside" and "2' -modified ribonucleoside" are used interchangeably and refer to a nucleoside having a modified sugar moiety at the 2' position. In some embodiments, the 2' -modified nucleoside is a 2' -4' bicyclic nucleoside in which the 2' and 4' positions of the sugar are bridged (e.g., by methylene, ethylene, or (S) -constrained ethyl). In some embodiments, the 2' -modified nucleoside is a non-bicyclic 2' -modified nucleoside, e.g., wherein the 2' position of the sugar moiety is replaced. Some non-limiting examples of 2' -modified nucleosides include: 2' -deoxy, 2' -fluoro (2 ' -F), 2' -O-methyl (2 ' -O-Me), 2' -O-methoxyethyl (2 ' -MOE), 2' -O-aminopropyl (2 ' -O-AP), 2' -O-dimethylaminoethyl (2 ' -O-DMAOE), 2' -O-dimethylaminopropyl (2 ' -O-DMAP), 2' -O-dimethylaminoethoxyethyl (2 ' -O-DMAOOE), 2' -O-N-methylacetamido (2 ' -O-NMA), locked nucleic acids (LNA, methylene bridged nucleic acids), ethylene bridged nucleic acids (ethylene-bridged nucleic acid, ENA), and (S) -constrained ethyl bridged nucleic acids (cEt). In some embodiments, the 2 '-modified nucleosides described herein are high affinity modified nucleotides, and oligonucleotides comprising the 2' -modified nucleotides have increased affinity for a target sequence relative to unmodified oligonucleotides. Some examples of structures of 2' -modified nucleosides are provided below:
Method for treating a complex
In some aspects, the disclosure provides methods of treating (e.g., generating, isolating) complexes comprising a protein (e.g., an antibody) covalently linked to one or more molecular charges (e.g., charge neutral oligonucleotides or charged oligonucleotides).
In some aspects, the disclosure provides methods of producing a complex comprising a protein (e.g., an antibody) covalently linked to one or more oligonucleotides (e.g., charge neutral oligonucleotides or charged oligonucleotides). In some embodiments, the method comprises: (i) The oligonucleotide is covalently linked to a cleavable linker (e.g., val-cit linker) to obtain a first intermediate. In some embodiments, the method further comprises (ii) covalently linking the first intermediate obtained in step (i) to a bicyclononene compound to obtain a second intermediate. In some embodiments, the method further comprises (iii) covalently linking the second intermediate obtained in step (ii) to an antibody to obtain a complex. In some embodiments, the bicyclononene compound is present in the reaction of step (iii) in an amount that is 10% or less or 5% or less (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%) of the starting amount of the bicyclononene compound in step (ii). In some embodiments, a cleavable linker (e.g., val-cit linker) is attached to the 5' end of the oligonucleotide. In some embodiments, a cleavable linker (e.g., val-cit linker) is attached to the oligonucleotide through an additional chemical moiety. In some embodiments, step (iii) results in the attachment of a lysine residue of the antibody to the second intermediate.
In some embodiments, a method of producing a complex comprises (i) covalently linking an oligonucleotide to a cleavable linker (e.g., a val-cit linker) to obtain a first intermediate; (ii) Covalently linking the first intermediate obtained in step (i) with a bicyclononene compound to obtain a second intermediate; and (iii) covalently linking the second intermediate obtained in step (ii) with an antibody to obtain a complex; wherein the bicyclononene compound is present in the reaction of step (iii) in an amount that is 10% or less or 5% or less (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5% or less than 0.1%) of the starting amount of the compound in step (ii). In this method, a cleavable linker (e.g., val-cit linker) is optionally attached to the 5' end of the oligonucleotide. In some embodiments, a cleavable linker (e.g., val-cit linker) is attached to the oligonucleotide through an additional chemical moiety. Furthermore, in some embodiments, step (iii) results in the attachment of a lysine residue of the antibody to the second intermediate.
In some embodiments, the molecular load is a hydrophobic small molecule. In some embodiments, the molecular cargo is an oligonucleotide. In some embodiments, the molecular charge is a charge neutral oligonucleotide. In some embodiments, the molecular charge is a charged oligonucleotide. In some embodiments, the oligonucleotide is a single stranded oligonucleotide (e.g., a charge neutral single stranded oligonucleotide or a charged single stranded oligonucleotide). In some embodiments, the charge neutral strand oligonucleotide is an antisense oligonucleotide. In some embodiments, the charge neutral oligonucleotide is a Phosphodiamide Morpholino Oligomer (PMO). In some embodiments, the charge neutral oligonucleotide is a Peptide Nucleic Acid (PNA). In some embodiments, the charged oligonucleotide is a spacer. In some embodiments, the antibody is covalently linked to the 5' end of a single stranded oligonucleotide (e.g., a spacer or PMO). In some embodiments, the antibody is covalently linked to the 3' end of a single stranded oligonucleotide (e.g., a spacer or PMO). In some embodiments, the antibody is covalently linked to the 5' end of an antisense oligonucleotide (e.g., a spacer or PMO).
In some embodiments, the single stranded oligonucleotide is one strand of a double stranded oligonucleotide. In some embodiments, one strand of a double-stranded oligonucleotide may be covalently conjugated to an antibody, and the complex may be separated using the methods described herein, followed by annealing the other strand of the double-stranded oligonucleotide. In some embodiments, the double-stranded oligonucleotide is an siRNA and the sense strand is covalently attached to the antibody (e.g., at the 3 'end or at the 5' end). In some embodiments, the complex purified using the methods described herein comprises an antibody covalently linked to the 3' end of the sense strand of the siRNA. The antisense strand of the siRNA can be annealed to the sense strand after purification.
In some embodiments, the oligonucleotide comprises at least one modified internucleotide linkage. In some embodiments, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the oligonucleotide comprises one or more modified nucleotides. In some embodiments, the modified nucleotide comprises a 2 '-O-methoxyethyl ribose (MOE), locked Nucleic Acid (LNA), 2' -fluoro modification, or morpholino modification.
In some embodiments, the oligonucleotide is a single stranded oligonucleotide (e.g., an antisense oligonucleotide) containing a modified nucleotide comprising a 2 '-O-methoxyethyl ribose (MOE), locked Nucleic Acid (LNA), 2' -fluoro modification, or morpholino modification. In some embodiments, the antisense oligonucleotide is a spacer containing a modified nucleotide comprising a 2 '-O-methoxyethyl ribose (MOE), locked Nucleic Acid (LNA), or 2' -fluoro modification. In some embodiments, the antisense oligonucleotide is a diamide morpholino oligomer Phosphate (PMO). Antisense oligonucleotides may comprise more than one type of modification described herein, for example with MOE and 2' -fluoro modifications.
In some embodiments, the oligonucleotide is a single stranded oligonucleotide (e.g., one strand of a double stranded RNA (e.g., siRNA) or an antisense oligonucleotide) comprising a modified nucleotide containing a 2 '-O-methoxyethyl ribose (MOE), locked Nucleic Acid (LNA), or 2' -fluoro modification. In some embodiments, the oligonucleotide is the sense strand of an siRNA, which contains a modified nucleotide comprising a 2 '-O-methoxyethyl ribose (MOE), locked Nucleic Acid (LNA), or 2' -fluoro modification. In some embodiments, the oligonucleotide is an antisense strand of an siRNA comprising a modified nucleotide comprising a 2 '-O-methoxyethyl ribose (MOE), locked Nucleic Acid (LNA), or 2' -fluoro modification. The sense and antisense strands of an siRNA can comprise the same type or different types of modifications described herein. One or both strands of the siRNA may comprise more than one type of modification described herein, e.g., with MOE and 2' -fluoro modifications.
In some embodiments, a method of producing a complex comprising an antibody covalently linked to one or more oligonucleotides (e.g., charge neutral oligonucleotides or charged oligonucleotides) comprises:
(i) Covalently linking an oligonucleotide (e.g., a charge neutral oligonucleotide or a charged oligonucleotide) to a linker of formula (a):
Wherein n is 0 to 15 (e.g., 3); to provide a modified oligonucleotide of formula (B):
wherein n is 0 to 15 (e.g., 3);
(ii) Contacting a modified oligonucleotide of formula (B) with a compound of formula (C):
wherein m is 0 to 15 (e.g., 4); to provide a modified oligonucleotide of formula (D):
wherein n is 0 to 15 (e.g., 3), and m is 0 to 15 (e.g., 4); and
(iii) Contacting a modified oligonucleotide of formula (D) with an antibody to provide a complex of formula (E):
wherein n is 0 to 15 (e.g., 3), and m is 0 to 15 (e.g., 4).
In some embodiments, the methods of producing a complex described herein produce a reaction mixture comprising the complex, an unligated oligonucleotide (e.g., a charge neutral oligonucleotide or a charged oligonucleotide), and/or an unligated antibody. In some embodiments, the reaction mixture comprises a complex of formula (E):
wherein n is 0 to 15 (e.g., 3), and m is 0 to 15 (e.g., 4); an unligated oligonucleotide of formula (B):
wherein n is 0 to 15 (e.g., 3); and unconnected antibodies. In some embodiments, the compound of formula (C) is present in the reaction mixture of step (iii) in an amount that is 10% or less or 5% or less (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%) of the starting amount of the compound of formula (C) in the reaction of step (ii), optionally wherein the compound of formula (C) is contacted with the antibody in step (iii) to form an unconnected antibody of formula (F) in the reaction mixture:
Wherein m is 0 to 15 (e.g., 4).
In some embodiments, a method of producing a complex, each comprising an antibody covalently linked to one or more oligonucleotides (e.g., charge neutral oligonucleotides or charged oligonucleotides), comprises covalently linking the oligonucleotides to a linker (e.g., a cleavable linker (e.g., val-cit linker)) to obtain a first intermediate. In some embodiments, the method further comprises covalently linking the antibody to a bicyclononene compound to obtain a second intermediate. In some embodiments, the method further comprises covalently linking the first intermediate to the second intermediate to obtain a complex. In this method, a linker (e.g., val-cit linker) is optionally attached to the 5' end of the oligonucleotide. In some embodiments, a linker (e.g., val-cit linker) is covalently attached to the oligonucleotide through an additional chemical moiety. Furthermore, in some embodiments, step (iii) results in covalent attachment of a lysine residue of the antibody to the second intermediate.
In some embodiments, a method of producing a complex (each comprising an antibody covalently linked to one or more oligonucleotides (e.g., charge neutral oligonucleotides or charged oligonucleotides)) comprises: (i) Covalently ligating the oligonucleotide to a linker (e.g., a cleavable linker (e.g., val-cit linker)) to obtain a first intermediate; (ii) Covalently linking the antibody to a compound comprising a bicyclononene to obtain a second intermediate; and (iii) covalently linking the first intermediate obtained in step (i) with the second intermediate obtained in step (ii) to obtain a complex. In this method, a linker (e.g., val-cit linker) is optionally attached to the 5' end of the oligonucleotide. In some embodiments, a linker (e.g., val-cit linker) is covalently attached to the oligonucleotide through an additional chemical moiety. Furthermore, in some embodiments, step (iii) results in covalent attachment of a lysine residue of the antibody to the second intermediate.
In some embodiments, a method of producing a complex comprising an antibody covalently linked to one or more oligonucleotides (e.g., charge neutral oligonucleotides or charged oligonucleotides) comprises:
(i) Covalently linking the oligonucleotide to a linker of formula (a):
wherein n is 0 to 15 (e.g., 3); to provide a modified oligonucleotide of formula (B):
wherein n is 0 to 15 (e.g., 3);
(ii) Covalently linking an antibody to a compound of formula (C):
wherein m is 0 to 15 (e.g., 4); to provide a modified antibody of formula (F):
wherein n is 0 to 15 (e.g., 3), and m is 0 to 15 (e.g., 4); and
(iii) Contacting a modified oligonucleotide of formula (B) with a modified antibody of formula (F) to provide a complex of formula (E):
wherein n is 0 to 15 (e.g., 3), and m is 0 to 15 (e.g., 4).
In some embodiments, the methods of producing a complex described herein produce a reaction mixture comprising the complex, the unligated oligonucleotide, and/or the unligated antibody. In some embodiments, the reaction mixture comprises a complex of formula (E):
wherein n is 0 to 15 (e.g., 3), and m is 0 to 15 (e.g., 4); an unligated oligonucleotide of formula (B):
wherein n is 0 to 15 (e.g., 3); unconnected antibodies of formula (F)
Wherein m is 0 to 15 (e.g., 4).
In some embodiments, the length of the oligonucleotide (e.g., charge neutral oligonucleotide (e.g., PMO) or charged oligonucleotide) is 10 to 50 (e.g., 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 50, 20 to 40, 20 to 30, 30 to 50, 30 to 40, or 40 to 50) nucleotides. In some embodiments, the oligonucleotide (e.g., a charge neutral oligonucleotide (e.g., PMO) or a charged oligonucleotide) is 15 to 30 (e.g., 15 to 30, 15 to 25, 15 to 20, 20 to 30, 20 to 25, or 25 to 30) nucleotides in length. In some embodiments, the length of the oligonucleotide (e.g., a charge neutral oligonucleotide (e.g., PMO) or charged oligonucleotide) is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the oligonucleotide (e.g., a charge neutral oligonucleotide (e.g., PMO) or a charged oligonucleotide) is 30 nucleotides in length. In some embodiments, the oligonucleotide (e.g., a charge neutral oligonucleotide (e.g., PMO) or a charged oligonucleotide) is covalently linked to the antibody through lysine or cysteine. In some embodiments, an oligonucleotide (e.g., a charge neutral oligonucleotide (e.g., PMO) or a charged oligonucleotide) is covalently linked to an antibody through a linker (e.g., a linker comprising a Val-cit linker). In some embodiments, the linker has formula (G):
Wherein n is 0 to 15 (e.g., 3), and m is 0 to 15 (e.g., 4). In some embodiments, n is 3 and/or (e.g., and) m is 4. In some embodiments, n is 3 and/or (e.g., and) m is 4. In some embodiments, X is NH (e.g., NH from an amine group of lysine), S (e.g., S from a thiol group of cysteine), or O (e.g., O from a hydroxyl group of serine, threonine, or tyrosine) of the antibody.
In some embodiments, the complex has formula (E):
wherein n is 0 to 15 (e.g., 3), and m is 0 to 15 (e.g., 4). In some embodiments, the oligonucleotide is a charged neutral oligonucleotide (e.g., PMO) or a charged oligonucleotide (e.g., spacer).
In some aspects, methods of isolating a complex described herein involve contacting a mixture comprising the complex and an unconnected molecular charge (e.g., a charge neutral oligonucleotide or a charged oligonucleotide) with a mixed mode resin comprising positively charged metal sites and negatively charged ion sites, removing the unconnected molecular charge (e.g., a charge neutral oligonucleotide or a charged oligonucleotide), and eluting the adsorbed complex from the mixed mode resin. In some embodiments, the mixed mode resin is an apatite resin. In some embodiments, the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxy fluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin.
In some embodiments, the mixture comprising the complex and the unconnected molecular payload (e.g., charge neutral oligonucleotide or charged oligonucleotide) subjected to mixed mode chromatography is a reaction mixture of reactions that produce the complex (e.g., if the antibody and molecular payload are by conjugation). In some embodiments, the mixture of complex and unbound molecular charge (e.g., charge neutral oligonucleotide or charged oligonucleotide) subjected to mixed mode chromatography is a reaction mixture that produces a reaction of complex (e.g., if the antibody and molecular charge are by conjugation) that has not been subjected to any prior purification steps prior to mixed mode chromatography. In some embodiments, the average drug-to-antibody ratio (drug to antibody ratio, DAR) of the complexes in the mixture subjected to mixed mode chromatography is at least about 1.0 (e.g., about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, or higher).
In some embodiments, complexes are substantially purified from unconnected molecular payloads (e.g., charge neutral oligonucleotides or charged oligonucleotides) using mixed mode resin chromatography as described herein. In some embodiments, the complex composition after purification using the methods described herein does not comprise any detectable level of unbound oligonucleotides or unbound proteins.
In some embodiments, the methods described herein are suitable for isolating complexes comprising antibodies covalently linked to one or more oligonucleotides. In some embodiments, the antibody may be a full length IgG, fab fragment, fab 'fragment, F (ab') 2 fragment, scFv, or Fv fragment. The specific antibody sequence did not affect the purification outcome. In some embodiments, the antibody is an anti-transferrin receptor antibody (e.g., any of the anti-transferrin receptor antibodies listed in table 2) or any antigen-binding fragment thereof (e.g., a Fab fragment, a Fab 'fragment, a F (ab') 2 fragment, a scFv, or an Fv fragment).
A. Removal of unbound charge-neutral oligonucleotides from complexes using mixed-mode resins
In some embodiments, it is demonstrated herein that the use of a mixed mode resin (e.g., an apatite resin, such as a hydroxyapatite resin) comprising positively charged metal sites and negatively charged ion sites effectively removes unbound molecular loads, particularly charge neutral or hydrophobic molecular loads (e.g., charge neutral oligonucleotides or hydrophobic small molecules), from the complex, and that the use of an organic solvent significantly increases the yield of the complex without unbound molecular loads throughout the purification process. The mixed mode resin purification methods described herein are advantageous compared to other known methods of removing unconnected molecular loads and/or excess salt (desalting). One such known method is Size Exclusion Chromatography (SEC). The mixed mode resin purification process is superior to SEC at least due to its scalability and higher recovery. Recovery of at least 50% of the complexes is achieved using the mixed mode resin method described herein, whereas SEC can only achieve recovery of 20% to 30% of the complexes.
In some aspects, the disclosure provides methods of treating (e.g., isolating) complexes that each comprise an antibody covalently linked to one or more molecular loads. In some embodiments, the molecular load is a small molecule (e.g., a hydrophobic small molecule). In some embodiments, the molecular charge is an oligonucleotide (e.g., a charge neutral oligonucleotide).
In some embodiments, the methods of treating a complex described herein comprise: (i) Contacting a mixture comprising an organic solvent, a complex, and an unconnected molecular charge (e.g., a charge neutral oligonucleotide or a hydrophobic small molecule) with a mixed mode resin comprising positively charged metal sites and negatively charged ion sites under conditions wherein the complex adsorbs to the mixed mode resin, and (ii) eluting the complex from the mixed mode resin under conditions wherein the complex dissociates from the mixed mode resin. In some embodiments, the mixture of step (i) further comprises no more than 30% (e.g., no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, or less than 0.5%) of unconnected antibodies. In some embodiments, the mixture of step (i) further comprises undetectable levels of unconjugated antibodies. In some embodiments, the mixture of step (i) is a reaction mixture that produces a complex. In some embodiments, the mixture of step (i) is a reaction mixture that produces a complex that has not been subjected to any previous purification steps. In some embodiments, the mixture of step (i) comprises a trace amount of an alkynyl-containing unconnected antibody.
In some embodiments, molecular loading (attached or unattached) interacts non-specifically with the mixed mode resin, affecting the yield of the complex. It is shown herein that inclusion of an organic solvent in the mobile phase of mixed mode chromatography effectively reduces/eliminates non-specific interactions between charge neutral oligonucleotides and mixed mode resins. In some embodiments, reducing the non-specific interaction between molecular loads (e.g., charge neutral oligonucleotides or hydrophobic small molecules) results in an increase (e.g., an increase of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, or more) in the yield of the complex as compared to not reducing such non-specific interaction.
Organic solvents commonly used in chromatographic methods can be used in all methods described herein. In some embodiments, the organic solvent used in step (i) of the methods of treating a complex described herein is Dimethylacetamide (DMA), isopropanol (IPA), dimethylsulfoxide (DMSO), acetonitrile (ACN), or Propylene Glycol (PG). In some embodiments, the organic solvent used in step (i) of the methods described herein is Dimethylacetamide (DMA). In some embodiments, the organic solvent used in step (i) of the methods described herein is isopropyl alcohol (IPA). In some embodiments, the organic solvent used in step (i) of the methods described herein is dimethyl sulfoxide (DMSO). In some embodiments, the organic solvent used in step (i) of the methods described herein is Acetonitrile (ACN). In some embodiments, the organic solvent used in step (i) of the methods described herein is Propylene Glycol (PG).
In some embodiments, the organic solvent is 2% to 50% (v/v) in the mixture in step (i). For example, the organic solvent may be 2% to 50% (v/v), 5% to 50% (v/v), 10% to 50% (v/v), 20% to 50% (v/v), 30% to 50% (v/v), 40% to 50% (v/v), 2% to 40% (v/v), 5% to 40% (v/v), 10% to 40% (v/v), 20% to 40% (v/v), 30% to 40% (v/v), 2% to 30% (v/v), 5% to 30% (v/v), 10% to 30% (v/v), 20% to 30% (v/v), 2% to 20% (v/v), 5% to 20% (v/v), 10% to 20% (v/v), 2% to 10% (v/v), 5% to 20% (v/v), 5% to 15% (v/v), 5% to 10% (v/v), 10% to 15% (v/v), 15% to 15% (v/v) or 15% (v) in the mixture in step (i) of the methods described herein. In some embodiments, the organic solvent is 5% to 20% (v/v) in the mixture in step (i). In some embodiments, the organic solvent is 5% (v/v), 6% (v/v), 7% (v/v), 8% (v/v), 9% (v/v), 10% (v/v), 11% (v/v), 12% (v/v), 13% (v/v), 14% (v/v), 15% (v/v), 16% (v/v), 17% (v/v), 18% (v/v), 19% (v/v), or 20% (v/v) in the mixture in step (i) of the methods described herein. In some embodiments, more than 20% (v/v) of an organic solvent may be used in the mixture in step (i) of the methods described herein. In some embodiments, the organic solvent is 15% (v/v) in the mixture in step (i) of the methods described herein. In some embodiments, the organic solvent is 30% (v/v) in the mixture in step (i) of the methods described herein.
In some embodiments, the conditions of complex adsorption in step (i) are achieved by including phosphate ions and/or chloride ions in the mixture in step (i) at a concentration that allows the complex to adsorb to the mixed mode resin. In some embodiments, the pH of the mixture in step (i) is about 5.0 to 8.0 (e.g., about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0). In some embodiments, the pH of the mixture in step (i) is about 5.0 to 6.0 (e.g., about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0). In some embodiments, the mixture in step (i) of the methods described herein does not comprise phosphate ions or chloride ions. In some embodiments, the mixture in step (i) of the methods described herein further comprises up to 10mM phosphate ions. In some embodiments, the mixture in step (i) of the methods described herein further comprises up to 10mM (e.g., up to 10mM or up to 5 mM) phosphate ions, and optionally in some embodiments, the mixture in step (i) of the methods described herein further comprises up to 20mM (e.g., up to 20mM, up to 15mM, up to 10mM or up to 5 mM) chloride ions. In some embodiments, the mixture in step (i) of the methods described herein further comprises 5 to 10mM (e.g., 5, 6, 7, 8, 9, or 10 mM) phosphate ions, and optionally in some embodiments, the mixture in step (i) of the methods described herein further comprises 5 to 20mM (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM) chloride ions. Under these conditions, the unconnected molecular charge (e.g., charge neutral oligonucleotide or hydrophobic small molecule) remains in the flow-through and does not adsorb to the mixed mode resin.
In some embodiments, in step (i), the unconnected molecular charge (e.g., charge neutral oligonucleotide or hydrophobic small molecule) does not adsorb to the mixed mode resin. In some embodiments, in step (i), less than 5% (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5%) of the unconnected molecular load (e.g., charge neutral oligonucleotide or hydrophobic small molecule) is adsorbed to the mixed mode resin. In some embodiments, less than 5% (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5%) of the unconnected molecular load (e.g., charge neutral oligonucleotide or hydrophobic small molecule) non-specifically interacts with the mixed mode resin.
In some embodiments, the mixed-mode resin may be further washed between step (i) and step (ii) under conditions that remove unbound molecular loads (e.g., charge neutral oligonucleotides or hydrophobic small molecules) that are loosely bound but not adsorbed to the mixed-mode resin. In some embodiments, the washing step is performed using a washing solution. In some embodiments, the wash solution comprises an organic solvent. In some embodiments, the organic solvent used in the wash solution is Dimethylacetamide (DMA), isopropanol (IPA), dimethylsulfoxide (DMSO), acetonitrile (ACN), or Propylene Glycol (PG). In some embodiments, the organic solvent used in the wash solution is Dimethylacetamide (DMA). In some embodiments, the organic solvent used in the cleaning solution is isopropyl alcohol (IPA). In some embodiments, the organic solvent used in the wash solution is dimethyl sulfoxide (DMSO). In some embodiments, the organic solvent used in the wash solution is Acetonitrile (ACN). In some embodiments, the organic solvent used in the wash solution is Propylene Glycol (PG).
In some embodiments, the organic solvent is 5% to 20% (v/v) in the wash solution. For example, the organic solvent may be 5% to 20% (v/v), 5% to 15% (v/v), 5% to 10% (v/v), 10% to 20% (v/v), 10% to 15% (v/v), or 15% to 20% (v/v) in the wash solution. In some embodiments, the organic solvent is 5% (v/v), 6% (v/v), 7% (v/v), 8% (v/v), 9% (v/v), 10% (v/v), 11% (v/v), 12% (v/v), 13% (v/v), 14% (v/v), 15% (v/v), 16% (v/v), 17% (v/v), 18% (v/v), 19% (v/v), or 20% (v/v) in the wash solution. In some embodiments, more than 20% (v/v) of the organic solvent may be used in the wash solution. In some embodiments, the organic solvent is 15% (v/v) in the wash solution. In some embodiments, the organic solvent is 30% (v/v) in the wash solution.
In some embodiments, the wash solution comprises phosphate ions and/or chloride at a concentration that removes loosely bound molecular loads (e.g., charge neutral oligonucleotides or hydrophobic small molecules) but does not dissociate the complex from the mixed mode resin. In some embodiments, the pH of the wash solution is from 5.0 to 8.0 (e.g., about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0). In some embodiments, the pH of the wash solution is 5.0 to 6.0 (e.g., 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0). In some embodiments, the wash solution does not contain phosphate ions or chloride ions. In some embodiments, the wash solution further comprises up to 10mM phosphate ions. In some embodiments, the wash solution further comprises up to 10mM (e.g., up to 10mM, or up to 5 mM) phosphate ions, and optionally in some embodiments, the wash solution further comprises up to 20mM (e.g., up to 20mM, up to 15mM, up to 10mM, or up to 5 mM) chloride ions. In some embodiments, the wash solution further comprises 5 to 10mM (e.g., 5, 6, 7, 8, 9, or 10 mM) phosphate ions, and optionally in some embodiments, the wash solution further comprises 5 to 20mM (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM) chloride ions.
In some embodiments, to elute the complex from the mixed mode resin, in step (ii), the mixed mode and bound complex are subjected to conditions that allow the complex to dissociate from the mixed mode resin. In some embodiments, the conditions allowing dissociation of the complex in step (ii) are achieved by applying an elution solution comprising a higher concentration of phosphate ions and/or chloride ions to the mixed mode resin than the concentration of phosphate ions and/or chloride ions in the mixture or wash solution of step (i). In some embodiments, the elution solution comprises a higher concentration of phosphate ions and no chloride ions than the phosphate concentration in the mixture or wash solution of step (i). The eluting step may be accomplished using an eluting solution comprising a single phosphate ion concentration, or using an eluting solution having an increasing phosphate ion concentration gradient.
In some embodiments, the pH of the elution solution is about 6.5 to 8.5 (e.g., about 6.5, 7.0, 7.5, 8.0, or 8.5). In some embodiments, the pH of the elution solution is about 7.6 to 8.5 (e.g., about 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5).
In some embodiments, the eluting solution comprises an organic solvent. In some embodiments, the organic solvent used in the elution solution is Dimethylacetamide (DMA), isopropanol (IPA), dimethylsulfoxide (DMSO), acetonitrile (ACN), or Propylene Glycol (PG). In some embodiments, the organic solvent used in the elution solution is Dimethylacetamide (DMA). In some embodiments, the organic solvent used in the elution solution is isopropyl alcohol (IPA). In some embodiments, the organic solvent used in the elution solution is dimethyl sulfoxide (DMSO). In some embodiments, the organic solvent used in the elution solution is Acetonitrile (ACN). In some embodiments, the organic solvent used in the elution solution is Propylene Glycol (PG).
In some embodiments, the organic solvent is 2% to 50% (v/v) in the eluting solution. For example, the organic solvent may be 2% to 50% (v/v), 5% to 50% (v/v), 10% to 50% (v/v), 20% to 50% (v/v), 30% to 50% (v/v), 40% to 50% (v/v), 2% to 40% (v/v), 5% to 40% (v/v), 10% to 40% (v/v), 20% to 40% (v/v), 30% to 40% (v/v), 2% to 30% (v/v), 5% to 30% (v/v), 10% to 30% (v/v), 20% to 30% (v/v), 2% to 20% (v/v), 5% to 20% (v/v), 10% to 10% (v/v), 5% to 20% (v/v), 5% to 15% (v/v), 5% to 10% (v/v), 10% to 15% (v/v), or 15% to 20% (v/v) in the elution solution. In some embodiments, the organic solvent is 5% to 20% (v/v) in the eluting solution. In some embodiments, the organic solvent is 5% (v/v), 6% (v/v), 7% (v/v), 8% (v/v), 9% (v/v), 10% (v/v), 11% (v/v), 12% (v/v), 13% (v/v), 14% (v/v), 15% (v/v), 16% (v/v), 17% (v/v), 18% (v/v), 19% (v/v), or 20% (v/v) in the elution solution. In some embodiments, more than 20% (v/v) of the organic solvent may be used in the elution solution. In some embodiments, the organic solvent is 15% (v/v) in the elution solution. In some embodiments, the organic solvent is 30% (v/v) in the elution solution.
In some embodiments, the elution solution of step (ii) comprises at least 30mM phosphate ions. In some embodiments, the elution solution of step (ii) comprises at least 30mM (e.g., at least 30mM, at least 40mM, at least 50mM, at least 60mM, at least 70mM, at least 80mM, at least 90mM, at least 100mM, at least 110mM, at least 120mM, at least 130mM, at least 140mM, or at least 150 mM) phosphate ions. In some embodiments, the elution solution of step (ii) comprises at least 100mM phosphate ions. In some embodiments, the elution solution of step (ii) comprises 100mM phosphate ions.
In some embodiments, the elution solution comprises a progressively increasing concentration of phosphate ions. In some embodiments, during step (ii), the concentration of phosphate ions is increased from at least 10mM (e.g., 10mM, 15mM, or 20 mM) to at least 100mM (e.g., 100mM, 150mM, 200mM, 250mM, 300mM, or higher). In some embodiments, during step (ii), the concentration of phosphate ions increases from 10mM to 100mM.
In some embodiments, applying the elution solution to the mixed-mode resin dissociates at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the complex from the mixed-mode resin. In some embodiments, applying the elution solution to the mixed-mode resin dissociates 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the complexes from the mixed-mode resin.
In some embodiments, the elution solution does not contain chloride ions. In some embodiments, the elution solution may further comprise at least 50mM (e.g., at least 50mM, at least 60mM, at least 70mM, at least 80mM, at least 90mM, at least 100mM, at least 110mM, at least 120mM, at least 130mM, at least 140mM, at least 150mM, at least 160mM, at least 170mM, at least 180mM, at least 190mM, or at least 200 mM) of chloride ions.
In some embodiments, the methods described herein further comprise collecting the dissociated complexes. In some embodiments, the methods described herein further comprise preparing the complex in a formulation buffer (formulation buffer) (e.g., by buffer exchange). In some embodiments, buffer exchange may be performed by ultrafiltration/diafiltration (UF/DF) or tangential flow filtration (tangential flow filtration, TFF).
In some aspects, the present disclosure provides methods of treating complexes each comprising an antibody covalently linked to one or more charge-neutral oligonucleotides, the method comprising:
(i) Contacting a mixture comprising 15% (v/v) DMA or IPA, complex, and unligated charge neutral oligonucleotides with a Hydroxyapatite (HA) resin comprising positively charged metal sites and negatively charged ion sites, wherein the pH of the mixture is about 5.7, and optionally further comprising 10mM phosphate ions and/or (e.g., and) 20mM chloride ions;
(ii) Washing the HA resin with a washing solution having a pH of about 5.7 and comprising 15% (v/v) DMA or IPA, and optionally further comprising 10mM phosphate ions and/or (e.g., and) 20mM chloride ions;
(iii) Eluting the complex from the mixed mode resin by applying an elution solution to the HA resin, wherein the elution solution HAs a pH of about 7.6 and comprises:
(a) 15% (v/v) DMA or IPA, and
(a) A phosphate ion concentration gradient of 100mM or in the range of 30mM to 100 mM; and
(iv) The selected complexes are collected.
In some embodiments, purification of the anti-TfR Fab-oligonucleotide conjugate requires a two-part purification process. In some embodiments, the reaction mixture comprising the complex and the unligated Fab and/or oligonucleotide is diluted in nuclease-free water (e.g., 1:3) and the pH is adjusted (e.g., to 5.7) by adding an appropriate buffer (e.g., MES buffer) at an appropriate concentration (e.g., about 50 mM). In some embodiments, a ceramic hydroxyapatite (ceramic hydroxyapatite, HA) column is equilibrated with 10mM sodium phosphate at pH 5.8 using 15:85v/v% organic solvent DMA. In some embodiments, the crude reaction mixture is loaded onto an HA column to remove unconjugated oligonucleotides. In some embodiments, the HA column is washed with 15:85v/v% DMA in 10mM sodium phosphate buffer (pH 5.8). In some embodiments, elution is initiated at a flow rate of 5 mL/min by a step gradient with 100mM sodium phosphate pH 7.6 buffer (containing 15:85v/v% DMA). In some embodiments, the HA eluate is buffer exchanged into the final formulation. In some embodiments, the final purified anti-TfR Fab-oligonucleotide conjugate is analyzed (e.g., by SEC, SDS-PAGE densitometry, and/or BCA).
In some embodiments, the mixtures and solutions used in the methods described herein further comprise phosphate ion and/or chloride ion counter ions. In some embodiments, the counter ion of the phosphate is calcium, sodium, magnesium, potassium, or manganese. In some embodiments, the counter ion used in the methods described herein is sodium. In some embodiments, the phosphate ion source is NaH 2 PO 4 、Na 2 HPO 4 Or Na (or) 3 PO 4 . In some embodiments, the counter ion of chlorine is calcium, sodium, magnesium, potassium, or manganese. In some embodiments, the chloride ion source is NaCl. Those skilled in the art will readily appreciate that many other equivalent salts and ions may be used in the methods described herein.
In some embodiments, the wash solution and/or the eluate solution may further comprise a buffer to maintain a consistent pH. Examples of buffers for use herein include ethylenediamine tetraacetic acid (EDTA), succinate, citrate, aspartic acid, glutamic acid, maleate, dimethylarsinate (cacodate), 2- (N-morpholino) -ethanesulfonic acid (MES), N- (2-acetamido) -2-aminoethanesulfonic Acid (ACES), piperazine-N, N ' -2-ethanesulfonic acid (PIPES), 2- (N-morpholino) -2-hydroxy-propanesulfonic acid (MOPSO), N-bis- (hydroxyethyl) -2-aminoethanesulfonic acid (BES), 3- (N-morpholino) -propanesulfonic acid (MOPS), N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES), 3- (N-tris- (hydroxymethyl) methylamino) -2-hydroxy-propanesulfonic acid (tamso), 3- (N, N-bis [ 2-hydroxyethyl ] amino) -2-hydroxy-propanesulfonic acid (dipes), N- (2-hydroxyethyl) piperazine-N ' - (2-hydroxy-propanesulfonic acid) (hepo), 4- (2-hydroxyethyl) -1-piperazine-N ' - (2-hydroxy-propanesulfonic acid) (hepos), tris (Tricine) and tris- [ N-hydroxymethyl ] glycine (Tricine), N-Bis (2-hydroxyethyl) glycine (Bicine), [ (2-hydroxy-1, 1-Bis (hydroxymethyl) ethyl) amino ] -1-propanesulfonic acid (TAPS), N- (1, 1-dimethyl-2-hydroxyethyl) -3-amino-2-hydroxypropanesulfonic Acid (AMPSO), tris (hydroxymethyl) aminomethane (Tris) and Bis [ 2-hydroxyethyl ] iminotris- [ hydroxymethyl ] methane (Bis-Tris). Other buffer compositions, buffer concentrations, and other components of solutions for use herein will be apparent to those skilled in the art.
Any mixed mode resin comprising positively charged metal sites and negatively charged ion sites may be used in accordance with the present disclosure. In some embodiments, the mixed mode resin used in the methods described herein is an apatite resin. In some embodiments, the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxy fluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin. Apatite resins can include any form of apatite and are typically used as chromatographic solid phases in the isolation and purification of biomolecules (e.g., complexes described herein) using affinity, ion exchange, hydrophobic interactions, or a combination thereof.
In some embodiments, the hydroxyapatite resin is a Bio-Gel HT resin, for example, from Bio-Rad Laboratories, inc (Hercules, ca, USA). In some embodiments, the ceramic hydroxyapatite resin is a Bio-Scale Mini CHT resin, e.g., from Bio-Rad Laboratories, inc. In some embodiments, the apatite resin (e.g., ceramic hydroxyapatite) comprises spherical particles of apatite. In some embodiments, the diameter of the spherical particles of apatite is about 10 microns to about 100 microns, about 25 microns to about 50 microns, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, or about 80 microns. In some embodiments, the apatite resin (e.g., ceramic hydroxyapatite) is type I (medium porosity and high binding capacity) or type II (larger porosity and lower binding capacity). In some embodiments, the apatite particles may be mixed with another separation medium or support for use.
In some embodiments, the mixed-mode resin may be equilibrated prior to contact with the mixture of complex and unconnected molecular charge (e.g., charge neutral oligonucleotide or hydrophobic small molecule). In some embodiments, the mixed mode resin is equilibrated with a wash solution, as described above. In some embodiments, the mixed mode resin is equilibrated to achieve a pH of about 5.0 to 8.0.
In some embodiments, the mixed mode resin is packed into a column (e.g., a vertical column). In some embodiments, the column may be used under pressure, optionally under top-to-bottom or bottom-to-top pressure. In some embodiments, the column may be used without external pressure, such as gravity flow alone. In some embodiments, mixed mode resins are used as the free resin, for example using a batch process. In some embodiments, the batch process may further comprise centrifugation and/or filtration steps after contacting the resin with a mixture of complex and unconnected molecular charges (e.g., charge neutral oligonucleotides or hydrophobic small molecules).
In some embodiments, the complex in the mixture in step (i) of the methods described herein and/or (e.g., and) the complex eluted in step (ii) of the methods described herein comprises an antibody covalently linked to 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) molecular loads (e.g., charge neutral oligonucleotides or hydrophobic small molecules). In some embodiments, at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more) of the complexes in the mixture of step (i) and/or (e.g., and) the complexes eluted in step (ii) of the methods described herein comprise antibodies covalently linked to 1 to 3 (e.g., 1, 2 or 3) molecular loads (e.g., charge neutral oligonucleotides or hydrophobic small molecules). In some embodiments, the complexes in the mixture of step (i) and/or (e.g., and) the complexes eluted in step (ii) of the methods described herein have an average drug-to-antibody ratio (DAR) of at least about 1.5 (e.g., at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, or at least about 2). In some embodiments, the eluate obtained from step (ii) contains undetectable levels of unconnected molecular loadings (e.g., charge neutral oligonucleotides or hydrophobic small molecules). In some embodiments, the eluate obtained from step (ii) contains undetectable levels of unbound antibodies.
B. Removal of unbound charged oligonucleotides relative to complex using mixed mode resin
In some embodiments, it is shown herein that the use of a mixed mode resin (e.g., an apatite resin, such as a hydroxyapatite resin) comprising positively charged metal sites and negatively charged ion sites effectively purifies the complex away from the unligated oligonucleotides. This is largely unexpected because no other purification strategy alternatives are able to remove substantially all of the unligated oligonucleotides from the composition comprising the protein-oligonucleotide complex. Furthermore, the mixed mode resin purification methods described herein are advantageous compared to other known methods of removing unbound oligonucleotides and/or excess salts (desalting). One such known method is Size Exclusion Chromatography (SEC). Mixed mode resin purification is superior to SEC at least because of its scalability and higher recovery. Recovery of at least 90% of the complexes is achieved using the mixed mode resin method described herein, whereas SEC can only achieve recovery of 20% to 30% of the complexes.
In some embodiments, a method of isolating one or more complexes described herein, each comprising an antibody covalently linked to one or more oligonucleotides (e.g., charged oligonucleotides), comprises: (i) Contacting a mixture comprising the complex and the unligated oligonucleotide with a mixed mode resin comprising positively charged metal sites and negatively charged ion sites under conditions that allow the complex to adsorb to the mixed mode resin, and (ii) eluting the complex from the mixed mode resin under conditions that allow the complex to dissociate from the mixed mode resin. In some embodiments, the mixture in step (i) comprises a trace amount of an alkynyl-containing unconnected antibody. In some embodiments, the mixture in step (i) is not subjected to purification prior to contact with the mixed mode resin. In some embodiments, the conditions under which the complex is adsorbed to the mixed-mode resin in step (i) may be adjusted to allow or exclude the adsorption of the unligated oligonucleotides to the mixed-mode resin, as described herein.
In some embodiments, the conditions that cause the complex to adsorb to the mixed-mode resin in step (i) do not allow the unbound oligonucleotides to adsorb to the mixed-mode resin, thus separating the complex from the unbound oligonucleotides. In some embodiments, the conditions are achieved by including phosphate ions and/or chloride ions in the mixture in step (i) in a concentration that allows the complex to adsorb to the mixed-mode resin but the unattached oligonucleotide does not adsorb to the mixed-mode resin. In some embodiments, the mixture comprising the complex and the unligated oligonucleotides further comprises up to 20mM phosphate ions and/or up to 30mM chloride ions. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises up to 20mM (e.g., up to 20mM, up to 15mM, up to 10mM, or up to 5 mM) phosphate ions. In addition, in some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises chloride ions at up to 30mM (e.g., up to 30mM, up to 25mM, up to 20mM, up to 15mM, up to 10mM, or up to 5 mM). In some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises 5 to 20mM (e.g., 5 to 20mM, 5 to 15mM, 5 to 10mM, 10 to 20mM, 10 to 15mM, or 15 to 20 mM) phosphate ions and/or 5 to 30mM chloride ions (e.g., 5 to 30mM, 5 to 25mM, 5 to 20mM, 5 to 15mM, 5 to 10mM, 10 to 30mM, 10 to 25mM, 10 to 20mM, 10 to 15mM, 15 to 30mM, 15 to 25mM, 15 to 20mM, 20 to 30mM, 20 to 25mM, or 25 to 30 mM). In some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises 20mM, 15mM, 10mM, 5mM or 1mM phosphate ions and/or 30mM, 25mM, 20mM, 15mM, 10mM or 5mM chloride ions. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises 20mM phosphate ions and/or 30mM chloride ions, e.g., 20mM phosphate ions and 30mM chloride ions. In some embodiments, the mixture comprising the complex and the unligated oligonucleotides further comprises up to 10mM phosphate ions and/or up to 25mM chloride ions. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises 5 to 10mM phosphate ions and/or 5 to 25mM chloride ions. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises 10mM phosphate ions and/or 25mM chloride ions, e.g., 10mM phosphate ions and 25mM chloride ions. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide comprises a trace amount of phosphate ion. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide comprises a trace amount of chloride ion. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide comprises both phosphate ions and chloride ions in trace amounts. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide does not comprise phosphate ions, does not comprise chloride ions, or does not comprise phosphate ions or chloride ions. Under these conditions, the unligated oligonucleotides remain in the flow-through and are not adsorbed to the mixed mode resin. In some embodiments, the mixed-mode resin may be further washed under these same conditions between step (i) and step (ii) to remove unbound oligonucleotides that are loosely bound but not adsorbed to the mixed-mode resin.
In some embodiments, the conditions that cause the complex to adsorb to the mixed-mode resin in step (i) also allow some or all (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) of the unbound oligonucleotides to adsorb to the mixed-mode resin. In some embodiments, the conditions are achieved by including phosphate ions and/or chloride ions in the mixture of step (i) in a concentration that allows both the complex and the unligated oligonucleotide to adsorb to the mixed-mode resin. In some embodiments, the mixture comprising the complex and the unligated oligonucleotides further comprises up to 5mM phosphate ions and/or up to 10mM chloride ions. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises up to 5mM (e.g., up to 5mM, up to 4mM, up to 3mM, up to 2mM, or up to 1 mM) phosphate ions. In addition, in some embodiments, the mixture comprising the complex and the unbound oligonucleotide further comprises chloride ions up to 10mM (e.g., up to 10mM, up to 9mM, up to 8mM, up to 7mM, up to 6mM, up to 5mM, up to 4mM, up to 3mM, up to 2mM, or up to 1 mM). In some embodiments, the mixture comprising the complex and the unbound oligonucleotide further comprises 1 to 5mM (e.g., 1 to 5mM, 1 to 4mM, 1 to 3mM, 1 to 2mM, 2 to 5mM, 2 to 4mM, 2 to 3mM, 3 to 5mM, 3 to 4mM, or 4 to 5 mM) phosphate ions and/or 1 to 10mM (e.g., 1 to 10mM, 1 to 8mM, 1 to 6mM, 1 to 4mM, 1 to 2mM, 2 to 10mM, 2 to 8mM, 2 to 6mM, 2 to 4mM, 4 to 10mM, 4 to 8mM, 4 to 6mM, 6 to 10mM, 6 to 8mM, or 8 to 10 mM) chloride ions. In some embodiments, the mixture comprising the complex and the unbound oligonucleotide further comprises 5mM, 4mM, 3mM, 2mM or 1mM phosphate ion and/or 10mM, 9mM, 8mM, 7mM, 6mM, 5mM, 4mM, 3mM, 2mM or 1mM chloride ion. In some embodiments, the mixture comprising the complex and the unligated oligonucleotides further comprises up to 3mM phosphate ions and/or up to 8mM chloride ions. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises 1 to 3mM (e.g., 1, 2, or 3 mM) phosphate ions and/or 1 to 8mM (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 mM) chloride ions. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises 3mM phosphate ions and/or 8mM chloride ions, e.g., 3mM phosphate ions and 8mM chloride ions. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide comprises a trace amount of phosphate ion. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide comprises a trace amount of chloride ion. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide comprises both phosphate ions and chloride ions in trace amounts. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide does not comprise phosphate ions, does not comprise chloride ions, or does not comprise phosphate ions or chloride ions. Under these conditions, some of all of the unligated oligonucleotides also adsorbed to the mixed mode resin.
In some embodiments, the pH of the mixture comprising the complex and the unligated oligonucleotide is between 5.0 and 8.0 (e.g., about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0). In some embodiments, the pH of the mixture comprising the complex and the unligated oligonucleotide is from 5.0 to 6.0 or from about 5.0 to 6.0 (e.g., about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0). In some embodiments, the pH of the mixture comprising the complex and the unligated oligonucleotide is 5.7 or about 5.7.
In some embodiments, when some or all of the unbound oligonucleotides are also adsorbed to the mixed-mode resin, the methods described herein further comprise washing the mixed-mode resin with a solution that dissociates the unbound oligonucleotides from the mixed-mode resin but does not dissociate the complexes from the mixed-mode resin between step (i) and step (ii). In some embodiments, the solution for washing comprises up to 20mM phosphate ions and/or up to 30mM chloride ions, e.g., 20mM phosphate ions and 30mM chloride ions. In some embodiments, the solution for washing comprises up to 20mM (e.g., up to 20mM, up to 15mM, up to 10mM, or up to 5 mM) phosphate ions. In addition, in some embodiments, the solution for washing comprises up to 30mM (e.g., up to 30mM, up to 25mM, up to 20mM, up to 15mM, up to 10mM, or up to 5 mM) chloride ions. In some embodiments, the solution for washing comprises 5 to 20mM (e.g., 5 to 20mM, 5 to 15mM, 5 to 10mM, 10 to 20mM, 10 to 15mM, or 15 to 20 mM) phosphate ions and/or 5 to 30mM chloride ions (e.g., 5 to 30mM, 5 to 25mM, 5 to 20mM, 5 to 15mM, 5 to 10mM, 10 to 30mM, 10 to 25mM, 10 to 20mM, 10 to 15mM, 15 to 30mM, 15 to 25mM, 15 to 20mM, 20 to 30mM, 20 to 25mM, or 25 to 30 mM). In some embodiments, the solution for washing comprises 20mM, 15mM, 10mM, 5mM or 1mM phosphate ions and/or 30mM, 25mM, 20mM, 15mM, 10mM or 5mM chloride ions. In some embodiments, the solution for washing comprises 20mM phosphate ions and/or 30mM chloride ions, e.g., 20mM phosphate ions and 30mM chloride ions. In some embodiments, the solution for washing comprises up to 10mM phosphate ions and/or up to 25mM chloride ions, e.g., 10mM phosphate ions and up to 25mM chloride ions.
In some embodiments, when some or all of the unbound oligonucleotides are also adsorbed to the mixed-mode resin, the methods described herein further comprise washing the mixed-mode resin with a series of solutions between step (i) and step (ii) that will dissociate the unbound oligonucleotides (rather than the complex) from the mixed-mode resin. In some embodiments, the first solution for washing comprises 10mM or about 10mM phosphate ions and 10mM or about 10mM chloride ions. In some embodiments, the second solution for washing comprises 15mM or about 15mM phosphate ions and 15mM or about 15mM chloride ions. In some embodiments, the third solution for washing comprises 19mM or about 19mM phosphate ions and 19mM or about 19mM chloride ions. In some embodiments, washing the mixed mode resin between step (i) and step (ii) comprises washing the resin with a first solution comprising 10mM phosphate ions and 10mM chloride ions, a second solution comprising 14.5mM phosphate ions and 14.5mM chloride ions, and a third solution comprising 19mM phosphate ions and 19mM chloride ions.
In some embodiments, the pH of the solution used for washing is from 6.0 to 8.5. In some embodiments, the pH of the solution used for washing is at or about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some embodiments, the pH of the solution used for washing is about 6.5.
In some embodiments, to elute the complex from the mixed mode resin, in step (ii), the mixed mode and bound complex are subjected to conditions that allow the complex to dissociate from the mixed mode resin. In some embodiments, the conditions allowing dissociation of the complex in step (ii) are achieved by applying an elution solution comprising a higher concentration of phosphate ions and/or chloride ions to the mixed mode resin. The eluting step may be accomplished using an eluting solution comprising a single phosphate ion concentration, or using an eluting solution having an increased phosphate ion concentration gradient. The use of an elevated phosphate ion concentration gradient during elution (step (ii)) allows for the separation of complexes with different drug to antibody ratio (DAR) numbers. For example, as the concentration of phosphate ions in the elution solution increases, complexes with lower DAR are eluted first, and complexes with higher DAR are eluted.
In some embodiments, step (ii) comprises applying an elution solution comprising at least 30mM phosphate ions and/or at least 50mM chloride ions to the mixed mode resin to elute the complex. In some embodiments, the elution solution comprises at least 30mM (e.g., at least 30mM, at least 40mM, at least 50mM, at least 60mM, at least 70mM, at least 80mM, at least 90mM, at least 100mM, at least 110mM, at least 120mM, at least 130mM, at least 140mM, or at least 150 mM) phosphate ions. Additionally, in some embodiments, the elution solution comprises at least 50mM (e.g., at least 50mM, at least 60mM, at least 70mM, at least 80mM, at least 90mM, at least 100mM, at least 110mM, at least 120mM, at least 130mM, at least 140mM, at least 150mM, at least 160mM, at least 170mM, at least 180mM, at least 190mM, or at least 200 mM) chloride ions. In some embodiments, the elution solution comprises at least 100mM phosphate ions and/or at least 100mM chloride ions. In some embodiments, the elution solution comprises 100mM phosphate ions and 100mM chloride ions.
In some embodiments, the pH of the elution solution is from 6.0 to 8.5. In some embodiments, the pH of the elution solution is at or about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some embodiments, the pH of the elution solution is about 7.5.
In some embodiments, to isolate complexes comprising anti-TfR Fab covalently linked to charged oligonucleotides, the reaction mixture comprising the complexes and unbound Fab and/or oligonucleotides is diluted in nuclease-free water (e.g., 1:3) and pH adjusted (e.g., to 5.7) by adding an appropriate buffer (e.g., MES buffer) at an appropriate concentration (e.g., about 50 mM). In some embodiments, the diluted reaction mixture is loaded onto a ceramic Hydroxyapatite (HA) column (e.g., resin at a biomolecular concentration of 8 mg/mL). In some embodiments, the column is washed with a wash solution (e.g., 5mM Na2HPO4, 25mM NaCl pH 7.0) to remove unbound oligonucleotides. In some embodiments, the complex comprising an anti-TfR Fab linked to an oligonucleotide is eluted from the HA column in a formulation buffer (e.g., 100mM Na2HPO4, 100mM NaCl,pH 7.6). In some embodiments, the isolated and purified anti-TfR Fab-oligonucleotide conjugate is analyzed (e.g., by SDS-PAGE and/or analytical SEC) to indicate complete removal of the unbound oligonucleotide. In some embodiments, the isolated and purified anti-TfR Fab-oligonucleotide conjugate is analyzed for human TfR1/cyno TfR1 binding and endotoxin levels by ELISA. In some embodiments, the complex may be alternatively purified by cation exchange and anion exchange.
In some embodiments, the mixtures and solutions used in the methods described herein further comprise phosphate ion and/or chloride ion counter ions. In some embodiments, the counter ion of the phosphate is calcium, sodium, magnesium, potassium, or manganese. In some embodiments, the counter ion used in the methods described herein is sodium. In some embodiments, the phosphate ion source is NaH 2 PO 4 、Na 2 HPO 4 Or Na (or) 3 PO 4 . In some embodiments, the counter ion of chlorine is calcium, sodium, magnesium, potassium, or manganese. In some embodiments, the chloride ion source is NaCl. Those skilled in the art will readily appreciate that many other equivalent salts and ions may be used in the methods described herein.
In some embodiments, the wash solution and/or the eluate solution may further comprise a buffer to maintain a consistent pH. In some embodiments, the wash buffer and/or the elution buffer comprises a neutral pH. In some embodiments, the pH of the wash buffer and/or elution buffer is about 6, about 6.5, about 7, about 7.5, about 8, or about 6 to 8. Examples of buffers for use herein include ethylenediamine tetraacetic acid (EDTA), succinate, citrate, aspartic acid, glutamic acid, maleate, dimethylarsinate (cacodate), 2- (N-morpholino) -ethanesulfonic acid (MES), N- (2-acetamido) -2-aminoethanesulfonic Acid (ACES), piperazine-N, N ' -2-ethanesulfonic acid (PIPES), 2- (N-morpholino) -2-hydroxy-propanesulfonic acid (MOPSO), N-bis- (hydroxyethyl) -2-aminoethanesulfonic acid (BES), 3- (N-morpholino) -propanesulfonic acid (MOPS), N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES), 3- (N-tris- (hydroxymethyl) methylamino) -2-hydroxy-propanesulfonic acid (tamso), 3- (N, N-bis [ 2-hydroxyethyl ] amino) -2-hydroxy-propanesulfonic acid (dipes), N- (2-hydroxyethyl) piperazine-N ' - (2-hydroxy-propanesulfonic acid) (hepo), 4- (2-hydroxyethyl) -1-piperazine-N ' - (2-hydroxy-propanesulfonic acid) (hepos), tris (Tricine) and tris- [ N-hydroxymethyl ] glycine (Tricine), N-Bis (2-hydroxyethyl) glycine (Bicine), [ (2-hydroxy-1, 1-Bis (hydroxymethyl) ethyl) amino ] -1-propanesulfonic acid (TAPS), N- (1, 1-dimethyl-2-hydroxyethyl) -3-amino-2-hydroxypropanesulfonic Acid (AMPSO), tris (hydroxymethyl) aminomethane (Tris) and Bis [ 2-hydroxyethyl ] iminotris- [ hydroxymethyl ] methane (Bis-Tris). Other buffer compositions, buffer concentrations, and other components of solutions for use herein will be apparent to those skilled in the art.
Any mixed mode resin comprising positively charged metal sites and negatively charged ion sites may be used in accordance with the present disclosure. In some embodiments, the mixed mode resin used in the methods described herein is an apatite resin. In some embodiments, the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxy fluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin. Apatite resins can include any form of apatite and are typically used as chromatographic solid phases in the separation and purification of biomolecules (e.g., complexes described herein) using affinity, ion exchange, hydrophobic interactions, or a combination thereof.
In some embodiments, the hydroxyapatite resin is a Bio-Gel HT resin, for example, from Bio-Rad Laboratories, inc (Hercules, calif., USA). In some embodiments, the ceramic hydroxyapatite resin is a Bio-Scale Mini CHT resin, such as from Bio-Rad Laboratories, inc. In some embodiments, the apatite resin (e.g., ceramic hydroxyapatite) comprises spherical particles of apatite. In some embodiments, the diameter of the spherical particles of apatite is about 10 microns to about 100 microns, about 25 microns to about 50 microns, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, or about 80 microns. In some embodiments, the apatite resin (e.g., ceramic hydroxyapatite) is type I (medium porosity and high binding capacity) or type II (larger porosity and lower binding capacity). In some embodiments, the apatite particles may be mixed with another separation medium or support for use.
In some embodiments, the mixed-mode resin may be equilibrated prior to contact with the mixture of complex and unligated oligonucleotides. In some embodiments, as described above, a wash solution is used to equilibrate the mixed mode resin. In some embodiments, the mixed mode resin is equilibrated such that the pH of the resin reaches a neutral pH, pH 6 to 8, pH about 6.5, pH about 7.0, pH about 7.5, or pH about 8.0.
In some embodiments, the mixed mode resin is packed into a column (e.g., a vertical column). In some embodiments, the column may be used under pressure, optionally from top to bottom or bottom to top. In some embodiments, the column may be used without external pressure, e.g., gravity flow alone. In some embodiments, mixed mode resins are used as the free resin, for example, using a batch process. In some embodiments, the batch process may further comprise centrifugation and/or filtration steps after contacting the resin with the mixture of complex and unbound oligonucleotides.
In some embodiments, the complex eluted in step (ii) of the mixed mode resin chromatography described herein comprises an antibody covalently linked to 1, 2, or 3 oligonucleotides. In some embodiments, complexes with different numbers of linked oligonucleotides (e.g., 1, 2, or 3) are separated in different elution fractions. In some embodiments, the eluate obtained from step (ii) comprises undetectable levels of unbound oligonucleotides.
C. Composition of purified complexes
The methods described herein can produce a substantially pure complex, wherein the composition of the purified complex does not comprise a detectable amount of unconnected molecular charge (e.g., charge neutral oligonucleotide or charged oligonucleotide) or unconnected antibodies. In some embodiments, the composition of the purified complex comprises at least 9:1, at least 95:5, 96:4, 97:3, 98:2, 99:1, 99.5:0.5, or higher molar or weight ratio of complex to unligated molecular cargo (e.g., charge neutral oligonucleotides or charged oligonucleotides). In some embodiments, the composition of the purified complex comprises at least 9:1, at least 95:5, 96:4, 97:3, 98:2, 99:1, 99.5:0.5, or higher molar or weight ratio of complex to unbound protein.
In some embodiments, the composition of the purified complex does not comprise a detectable level (e.g., a detectable amount) of unconnected protein. In some embodiments, the composition of the purified complex does not comprise a detectable level (e.g., a detectable amount) of unconnected molecular charge (e.g., a charge neutral oligonucleotide or a charged oligonucleotide).
In some embodiments, the composition of the purified complex comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 5%, or less than 0.5% of unconnected protein (e.g., antibody) by mole ratio. In some embodiments, the composition of the purified complex comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 5%, or less than 0.5% of the unconnected molecular load (e.g., charge neutral oligonucleotide or charged oligonucleotide) by mole ratio. In some embodiments, the composition of the purified complex comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 5%, or less than 0.5% of unconnected protein (e.g., antibody) by mole ratio, and comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 5%, or less than 0.5% of unconnected molecular load (e.g., charge neutral oligonucleotide or charged oligonucleotide) by mole ratio.
In some embodiments, the composition of the purified complex comprises at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%) of the complex (i.e., a protein (e.g., an antibody) covalently linked to one or more oligonucleotides) by molar ratio.
In some embodiments, the composition of the purified complex comprises a protein (e.g., an antibody) covalently linked to one oligonucleotide, two oligonucleotides, three oligonucleotides, and/or more oligonucleotides. In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) or more of the complexes in the composition of the purified complexes comprise a protein (e.g., an antibody) (DAR 1) covalently linked to one oligonucleotide. In some embodiments, about 50%, 60%, 70%, 80%, 90%, or 95% of the complexes in the composition of the purified complexes comprise a protein (e.g., an antibody) (DAR 1) covalently linked to one oligonucleotide.
In some embodiments, about 5%, 10%, 15%, 20%, 25%, 30% or more of the complexes in the composition of the purified complexes comprise a protein (e.g., an antibody) (DAR 2) covalently linked to two oligonucleotides. In some embodiments, about 1%, 2%, 3%, 5%, 7%, 10%, 20% or more of the complexes in the composition of the purified complexes comprise a protein (e.g., an antibody) (dar3+) covalently linked to three or more oligonucleotides.
III. Complex
In some aspects, provided herein are complexes comprising a targeting agent (e.g., an antibody) covalently linked to a molecular cargo (e.g., an oligonucleotide). In some embodiments, the complex comprises a muscle targeting antibody covalently linked to an oligonucleotide. The complex may comprise an antibody that specifically binds a single antigenic site or binds at least two antigenic sites that may be present on the same or different antigens. The complexes may be used to modulate the activity or function of at least one gene, protein, and/or nucleic acid. In some embodiments, the molecular load present with the complex is responsible for the modulation of genes, proteins and/or nucleic acids. The molecular cargo may be a small molecule, protein, nucleic acid, oligonucleotide or any molecular entity capable of modulating the activity or function of a gene, protein and/or nucleic acid in a cell. In some embodiments, the molecular cargo is an oligonucleotide that targets a muscle disease allele in a muscle cell.
In some embodiments, the complex comprises a muscle targeting agent, such as an anti-transferrin receptor antibody, covalently linked to a molecular cargo, such as an antisense oligonucleotide targeting a muscle disease allele.
In some embodiments, the complexes may be used to treat a muscle disorder, wherein the molecular load affects the activity of the corresponding gene provided in table 1. For example, depending on the disorder, the molecular load may regulate (e.g., decrease, increase) transcription or expression of a gene, regulate expression of a protein encoded by a gene, or regulate activity of the encoded protein. In some embodiments, the molecular cargo is an oligonucleotide comprising a strand having a region complementary to a target gene provided in table 1.
Table 1-list of muscle diseases and corresponding genes.
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A. Cell targeting agents
Some aspects of the disclosure provide cell targeting agents (e.g., muscle targeting proteins), for example, for delivering oligonucleotides to muscle cells. In some embodiments, such cell-targeting proteins are capable of binding to a particular cell (e.g., by specifically binding to an antigen on the cell) and delivering an associated oligonucleotide to the cell. In some embodiments, the oligonucleotide is bound (e.g., covalently bound) to a cell targeting agent, and the oligonucleotide is internalized (e.g., by endocytosis) into the cell after the cell targeting agent binds to an antigen on the cell.
Some aspects of the present disclosure provide muscle targeting agents, for example, for delivering molecular loads to muscle cells. In some embodiments, such muscle targeting agents are capable of binding to muscle cells, for example, by specifically binding to an antigen on the muscle cells, and delivering an associated molecular load to the muscle cells. In some embodiments, the molecular cargo binds (e.g., covalently binds) to the muscle targeting agent and internalizes into the muscle cell upon binding of the muscle targeting agent to the antigen on the muscle cell, e.g., by endocytosis. Exemplary muscle targeting agents are described in further detail herein, however, it should be understood that the exemplary muscle targeting agents provided herein are not meant to be limiting. It is understood that a variety of types of muscle targeting agents may be used in accordance with the present disclosure, and that any muscle target (e.g., muscle surface protein) may be targeted by any of the types of muscle targeting agents described herein. For example, the muscle targeting agent may include or consist of: small molecules, nucleic acids (e.g., DNA or RNA), peptides (e.g., antibodies), lipids (e.g., microbubbles), or sugar moieties (e.g., polysaccharides).
Some aspects of the present disclosure provide muscle targeting agents that specifically bind to antigens on muscle (e.g., skeletal muscle, smooth muscle, or cardiac muscle). In some embodiments, any of the muscle targeting agents provided herein bind (e.g., specifically bind) to an antigen on skeletal muscle cells, smooth muscle cells, and/or cardiac muscle cells.
By interacting with muscle-specific cell surface recognition elements (e.g., cell membrane proteins), both tissue localization and selective uptake into muscle cells can be achieved. In some embodiments, molecules that are substrates for muscle uptake transporters may be used to deliver molecular loads (e.g., oligonucleotides) into muscle tissue. Binding to the muscle surface recognition element is followed by endocytosis, which may allow even macromolecules (e.g., antibodies) to enter the muscle cells. As another example, an oligonucleotide conjugated to transferrin or an anti-transferrin receptor antibody may be taken up by muscle cells by binding to transferrin receptor and then endocytosed, for example by clathrin mediated endocytosis.
The use of muscle targeting agents can be used to concentrate molecular loads (e.g., oligonucleotides) in the muscle while reducing toxicity associated with effects in other tissues. In some embodiments, the muscle targeting agent concentrates the bound molecular load in the muscle cells as compared to another cell type within the subject. In some embodiments, the muscle targeting agent concentrates the bound molecular load in a muscle cell (e.g., skeletal muscle, smooth muscle, or cardiac muscle cell) in an amount up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold less than in a non-muscle cell (e.g., liver, neuron, blood, or adipocyte). In some embodiments, when the molecular load is delivered to a subject upon binding to a muscle targeting agent, its toxicity in the subject is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or 95%.
In some embodiments, a muscle recognition element (e.g., a muscle cell antigen) may be required in order to achieve muscle selectivity. As one example, the muscle targeting agent may be a small molecule that is a substrate for a muscle-specific uptake transporter. As another example, the muscle targeting agent may be an antibody that enters a muscle cell by transporter mediated endocytosis. As another example, a muscle targeting agent may be a ligand that binds to a cell surface receptor on a muscle cell. It should be appreciated that while the transporter-based approach provides a direct pathway for cell entry, receptor-based targeting may involve stimulated endocytosis to achieve the desired site of action.
Muscle cells encompassed by the present disclosure include, but are not limited to, skeletal muscle cells, smooth muscle cells, cardiac muscle cells, myoblasts, and muscle cells.
i. Muscle targeting antibodies
In some embodiments, the muscle targeting agent is an antibody. Generally, the high specificity of antibodies for their target antigens provides the potential for selective targeting of muscle cells (e.g., skeletal muscle, smooth muscle, and/or cardiomyocytes). This specificity can also limit off-target toxicity. Examples of antibodies capable of targeting muscle cell surface antigens have been reported and are within the scope of the present disclosure. For example, antibodies targeting the surface of muscle cells are described in the following: arahata K., et al, "Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide" Nature 1988;333:861-3; song K.S., et al, "Expression of caveolin-3in skeletal,cardiac,and smooth muscle cells.Caveolin-3is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins" J Biol Chem 1996;271:15160-5; weisbart R.H.et al, "Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb" Mol immunol.2003Mar,39 (13): 78309; the entire contents of each of which are incorporated herein by reference.
a. Anti-transferrin receptor antibodies
Some aspects of the present disclosure are based on the recognition that: substances that bind to transferrin receptor (e.g., anti-transferrin receptor antibodies) are capable of targeting muscle cells. Transferrin receptors are internalized cell surface receptors that transduce ferritin through the cell membrane and are involved in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor binding proteins capable of binding to transferrin receptors. Accordingly, aspects of the present disclosure provide binding proteins (e.g., antibodies) that bind to transferrin receptor. In some embodiments, the binding protein that binds to the transferrin receptor is internalized into the muscle cell along with any bound molecular load (e.g., oligonucleotide). As used herein, an antibody that binds to a transferrin receptor may be referred to as an anti-transferrin receptor antibody. Antibodies that bind (e.g., specifically bind) to a transferrin receptor can be internalized into a cell upon binding to the transferrin receptor, e.g., by receptor-mediated endocytosis.
It will be appreciated that several known methods (e.g., using phage display library design) may be used to generate, synthesize and/or derive anti-transferrin receptor antibodies. Exemplary methods have been characterized in the art and are incorporated by reference (Di ez, P.et al. "High-throughput phase-display screening in array format", enzyme and microbial technology,2015,79,34-41.; christoph M.H. and Stanley, J.R. "Antibody Phage Display: technique and Applications" J Invest Dermatol.2014,134:2.; engleman, edgar (Ed.) "Human Hybridomas and Monoclonal antibodies."1985, springer). In other embodiments, the anti-transferrin antibodies have been previously characterized or disclosed. Antibodies that specifically bind to transferrin receptor are known in the art (see, e.g., U.S. patent No.4,364,934, "Monoclonal antibody to a human early thymocyte antigen and methods for preparing same", U.S. patent No.8,409,573, "Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells", U.S. patent No.9,708,406, "Anti-transferrin receptor antibodies and methods of use", U.S. patent No. 2014, 5, 20, and U.S. patent 9,611,323, "Low affinity blood brain barrier receptor antibodies and uses therefor", U.S. patent No. 2014, 12, 19, WO 2015/098989, "Novel Anti-Transferrin receptor antibody that passes through blood-brin barrer", U.S. patent No. Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody, c.et al, "Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody o 9", "J Biol chem 1982,257:14, 8516-8522", lee et al, "Targeting Rat Anti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse"2000,J Pharmacol.Exp.Ther, 292:1048-1052, etc.).
Any suitable anti-transferrin receptor antibody may be used in the complexes disclosed herein. Examples of anti-transferrin receptor antibodies, including relevant references and binding epitopes, are listed in table 2. In some embodiments, an anti-transferrin receptor antibody comprises complementarity determining regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) of any of the anti-transferrin receptor antibodies provided herein (e.g., an anti-transferrin receptor antibody listed in Table 2).
Table 2-list of anti-transferrin receptor antibody clones, including relevant references and binding epitope information.
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In some embodiments, the muscle targeting agent is an anti-transferrin receptor antibody. In some embodiments, the anti-transferrin receptor antibody specifically binds to a transferrin protein having an amino acid sequence as disclosed herein. In some embodiments, an anti-transferrin receptor antibody may be conjugated toAny extracellular epitope of the transferrin receptor or epitope that begins to be exposed to antibodies, including the apical domain, transferrin binding domain and protease-like domain, specifically binds. In some embodiments, the anti-transferrin receptor antibody binds to an amino acid fragment of a human or non-human primate transferrin receptor (as provided in SEQ ID nos. 1 to 3) in the range of amino acids C89 to F760. In some embodiments, the transferrin receptor antibody is present in an amount of at least about 10 -4 M、10 -5 M、10 -6 M、10 -7 M、10 -8 M、10 -9 M、10 -10 M、10 -11 M、10 -12 M、10 -13 M or less. Anti-transferrin receptor antibodies as used herein may be capable of competing with other anti-transferrin receptor antibodies (e.g., OKT9, 8D 3) for binding, the latter antibody being at 10 -3 M、10 -4 M、10 -5 M、10 -6 M、10 -7 M or less binds to transferrin receptor.
An exemplary human transferrin receptor amino acid sequence corresponding to NCBI sequence np_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows:
an exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence np_001244232.1 (transferrin receptor protein 1, rhesus monkey is as follows:
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an exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence xp_005545315.1 (transferrin receptor protein 1, cynomolgus monkey) is as follows:
an exemplary murine transferrin receptor amino acid sequence corresponding to NCBI sequence np_001344227.1 (transferrin receptor protein 1, mouse) is as follows:
in some embodiments, the anti-transferrin receptor antibody binds to a receptor amino acid segment as follows:
and does not inhibit the binding interactions between transferrin receptor and transferrin and/or human blood pigmentation proteins (also known as HFEs).
Antibodies, antibody fragments, or antigen binding agents may be obtained and/or produced using suitable methods, for example, by using recombinant DNA protocols. In some embodiments, antibodies may also be produced by hybridoma production (see, e.g., kohler, G and Milstein, C. "Continuous cultures of fused cells secreting antibody of predefined specificity" Nature,1975, 256:495-497). The antigen of interest may be used as an immunogen in any form or entity (e.g., recombinant or native form or entity). Hybridomas are screened using standard methods (e.g., ELISA screening) to find at least one hybridoma producing an antibody that targets a particular antigen. Antibodies can also be generated by screening expression libraries of proteins that express the antibodies (e.g., phage display libraries). In some embodiments, phage display library designs may also be used (see, e.g., U.S. Pat. No. 5,223,409, "Directed evolution of novel binding proteins" filed on 3/1/1992, 10/4/1992, "Heterodimeric receptor libraries using phagemids", WO 1991/17271, "Recombinant library screening methods" filed on 5/1/1991, WO 1992/20791, "Methods for producing members of specific binding pairs" filed on 15/5/1992, 28/1992, WO 1992/15679, "Improved epitope displaying phage"). In some embodiments, the antigen of interest may be used to immunize a non-human animal, such as a rodent or goat. In some embodiments, the antibodies are then obtained from a non-human animal, and optionally modified using a variety of methods (e.g., using recombinant DNA techniques). Other examples of antibody production and methods are known in the art (see, e.g., harlow et al, "Antibodies: A Laboratory Manual", cold Spring Harbor Laboratory, 1988).
In some embodiments, the antibody is modified, e.g., by glycosylation, phosphorylation, SUMO, and/or methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody by N-glycosylation, O-glycosylation, C-glycosylation, glycosyl phosphatidyl inositol (GPI anchor attachment), and/or phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, there are about 1 to 10, about 1 to 5, about 5 to 10, about 1 to 4, about 1 to 3, or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by a chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or in a cell, which may optionally lack an enzyme in the N-or O-glycosylation pathway, such as a glycosyltransferase. In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in international patent application publication No. WO2014065661 entitled "Modified antibody, anti-body-conjugate and process for the preparation thereof," published on 5, month 1 of 2014.
Some aspects of the disclosure provide proteins that bind to a transferrin receptor (e.g., an extracellular portion of a transferrin receptor). In some embodiments, a transferrin receptor antibody provided herein specifically binds to a transferrin receptor (e.g., a human transferrin receptor). Transferrin receptors are internalized cell surface receptors that transport transferrin through the cell membrane and are involved in the regulation and homeostasis of intracellular iron levels. In some embodiments, the transferrin receptor antibodies provided herein specifically bind to transferrin receptors from humans, non-human primates, mice, rats, and the like. In some embodiments, a transferrin receptor antibody provided herein binds to a human transferrin receptor. In some embodiments, the transferrin receptor antibodies provided herein specifically bind to human transferrin receptor. In some embodiments, a transferrin receptor antibody provided herein binds to the top domain of a human transferrin receptor. In some embodiments, the transferrin receptor antibodies provided herein specifically bind to the top domain of a human transferrin receptor.
In some embodiments, a transferrin receptor antibody of the disclosure comprises one or more CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences selected from any one of the anti-transferrin receptor antibodies of table 2. In some embodiments, the transferrin receptor antibody comprises a CDR-H1, CDR-H2, and CDR-H3 provided for any one of the anti-transferrin receptor antibodies selected from table 2. In some embodiments, the transferrin receptor antibody comprises a CDR-L1, CDR-L2, and CDR-L3 provided for any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, the transferrin receptor antibody comprises a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 provided for any one of the anti-transferrin receptor antibodies selected from Table 2. The present disclosure also encompasses any nucleic acid sequence encoding a molecule comprising a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 or CDR-L3 provided for any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, the antibody heavy and light chain CDR3 domains may play a particularly important role in the binding specificity/affinity of an antibody for an antigen. Thus, an anti-transferrin receptor antibody of the present disclosure may comprise at least heavy and/or light chain CDR3 of any anti-transferrin receptor antibody selected from table 2.
In some examples, any anti-transferrin receptor antibody of the disclosure has one or more CDRs (e.g., CDR-H or CDR-L) substantially similar to any CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and/or CDR-L3 sequences of one anti-transferrin receptor antibody selected from table 2. In some embodiments, one or more CDRs of an antibody described herein can change one, two, three, four, five, or six amino acid positions along the VH (e.g., CDR-H1, CDR-H2, or CDR-H3) and/or VL (e.g., CDR-L1, CDR-L2, or CDR-L3) regions, so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it was derived is substantially maintained). For example, in some embodiments, the positions of CDRs defining any of the antibodies described herein can be altered by shifting the N-terminal and/or C-terminal boundaries of the CDRs by one, two, three, four, five, or six amino acids relative to the CDR positions of any of the antibodies described herein, so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it was derived is substantially maintained). In another embodiment, one or more CDRs of an antibody described herein can vary (e.g., become shorter or longer) in length along a VH (e.g., CDR-H1, CDR-H2, or CDR-H3) and/or VL (e.g., CDR-L1, CDR-L2, or CDR-L3) by one, two, three, four, five, or more amino acids, so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it was derived is substantially maintained).
Thus, in some embodiments, a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or CDR-H3 described herein can be one, two, three, four, five, or more amino acids shorter than one or more CDRs described herein (e.g., CDRs selected from any anti-transferrin receptor antibody of table 2) so long as immunospecific binding (e.g., binding of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) to a transferrin receptor (e.g., a human transferrin receptor) is maintained relative to the original antibody from which it was derived. In some embodiments, a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or CDR-H3 described herein can be one, two, three, four, five, or more amino acids longer than one or more CDRs described herein (e.g., CDRs selected from any anti-transferrin receptor antibody of table 2) so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it was derived). In some embodiments, the amino moieties of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or CDR-H3 described herein can be extended by one, two, three, four, five, or more amino acids as compared to one or more CDRs described herein (e.g., CDRs of any anti-transferrin receptor antibody selected from table 2) so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it was derived). In some embodiments, the carboxy portion of a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or CDR-H3 described herein can be extended by one, two, three, four, five, or more amino acids as compared to one or more CDRs described herein (e.g., CDRs of any anti-transferrin receptor antibody selected from table 2) so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it was derived). In some embodiments, the amino moieties of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or CDR-H3 described herein can be shortened by one, two, three, four, five, or more amino acids as compared to one or more CDRs described herein (e.g., CDRs of any anti-transferrin receptor antibody selected from table 2) so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it was derived). In some embodiments, the carboxy portion of a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or CDR-H3 described herein can be shortened by one, two, three, four, five, or more amino acids as compared to one or more CDRs described herein (e.g., CDRs of any anti-transferrin receptor antibody selected from table 2) so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it was derived). Any method may be used to determine whether immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained, for example using binding assays and conditions described in the art.
In some examples, any anti-transferrin receptor antibody of the disclosure has one or more CDR (e.g., CDR-H or CDR-L) sequences substantially similar to any anti-transferrin receptor antibody selected from table 2. For example, an antibody may comprise one or more CDR sequences selected from any anti-transferrin receptor antibody of table 2 that comprise up to 5, 4, 3, 2, or 1 amino acid residue variations compared to the corresponding CDR regions of any one of the CDRs provided herein (e.g., CDRs selected from any anti-transferrin receptor antibody of table 2) so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) relative to the binding of the original antibody from which it was derived. In some embodiments, any amino acid variation in any CDR provided herein can be a conservative variation. Conservative variations may be introduced into the CDRs at positions where the residues are unlikely to be involved in interactions with transferrin receptor proteins (e.g., human transferrin receptor proteins), e.g., as determined based on crystal structure. Some aspects of the disclosure provide transferrin receptor antibodies comprising one or more heavy chain Variable (VH) and/or light chain Variable (VL) domains provided herein. In some embodiments, any VH domain provided herein comprises one or more CDR-H sequences provided herein (e.g., CDR-H1, CDR-H2, and CDR-H3), e.g., any CDR-H sequences provided in any anti-transferrin receptor antibody selected from table 2. In some embodiments, any VL domain provided herein comprises one or more CDR-L sequences provided herein (e.g., CDR-L1, CDR-L2, and CDR-L3), e.g., any CDR-L sequences provided in any anti-transferrin receptor antibody selected from table 2.
In some embodiments, the anti-transferrin receptor antibodies of the present disclosure include any antibody comprising a heavy chain variable domain and/or a light chain variable domain of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from table 2). In some embodiments, the anti-transferrin receptor antibodies of the present disclosure include any antibody comprising a variable pair of heavy and light chains of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from table 2).
Some aspects of the disclosure provide anti-transferrin receptor antibodies having heavy chain Variable (VH) and/or light chain Variable (VL) domain amino acid sequences homologous to any of those described herein. In some embodiments, an anti-transferrin receptor antibody comprises a heavy chain variable sequence or a light chain variable sequence having at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity to the heavy chain variable sequence and/or the light chain variable sequence of any anti-transferrin receptor antibody (e.g., any one of the anti-transferrin receptor antibodies selected from table 2). In some embodiments, the cognate heavy chain variable and/or light chain variable amino acid sequences do not change within any CDR sequences provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) can occur in heavy chain variable and/or light chain variable sequences provided herein that do not include any CDR sequences. In some embodiments, any anti-transferrin receptor antibody provided herein comprises a heavy chain variable sequence and a light chain variable sequence comprising a framework region sequence having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to a framework region sequence of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from table 2).
In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., a human transferrin receptor) comprises a light chain variable VL domain comprising any CDR-L domain (CDR-L1, CDR-L2, and CDR-L3) of any anti-transferrin receptor antibody selected from table 2, or a CDR-L domain variant provided herein. In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., a human transferrin receptor) comprises a light chain variable VL domain comprising CDR-L1, CDR-L2, and CDR-L3 of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from table 2). In some embodiments, an anti-transferrin receptor antibody comprises a light chain Variable (VL) region sequence comprising one, two, three, or four framework regions of the light chain variable region sequence of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from table 2). In some embodiments, an anti-transferrin receptor antibody comprises one, two, three, or four framework regions of a light chain variable region sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to one, two, three, or four framework regions of a light chain variable region sequence of any transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from table 2). In some embodiments, the light chain variable framework region derived from the amino acid sequence consists of the amino acid sequence, but there are up to 10 amino acid substitutions, deletions and/or insertions, preferably up to 10 amino acid substitutions. In some embodiments, the light chain variable framework region derived from the amino acid sequence consists of the amino acid sequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues replace amino acids present at similar positions of the corresponding non-human primate or human light chain variable framework region.
In some embodiments, the anti-transferrin receptor antibody that specifically binds to transferrin receptor comprises CDR-L1, CDR-L2, and CDR-L3 of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from table 2). In some embodiments, the antibody further comprises one, two, three, or all four VL framework regions derived from a human or primate VL. The primate or human light chain framework regions of the antibodies selected for use with the light chain CDR sequences described herein can have, for example, at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%) identity to the light chain framework regions of the non-human parent antibody. The amino acid numbering of selected primate or human antibodies in their light chain complementarity determining regions can be the same or substantially the same as the amino acid numbering in the light chain complementarity determining regions of any of the antibodies provided herein (e.g., any anti-transferrin receptor antibody selected from table 2). In some embodiments, the primate or human light chain framework amino acid residues are from a natural primate or human antibody light chain framework region having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% (or more) identity to the light chain framework region of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from table 2). In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three, or all four VL framework regions derived from the human light chain variable kappa subfamily. In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three, or all four VL framework regions derived from the human light chain variable lambda subfamily.
In some embodiments, any of the anti-transferrin receptor antibodies provided herein comprises a light chain variable domain, which further comprises a light chain constant region. In some embodiments, the light chain constant region is a kappa or lambda light chain constant region. In some embodiments, the kappa or lambda light chain constant region is from a mammal, e.g., from a human, monkey, rat, or mouse. In some embodiments, the light chain constant region is a human kappa light chain constant region. In some embodiments, the light chain constant region is a human lambda light chain constant region. It is to be understood that any light chain constant region provided herein can be a variant of any light chain constant region provided herein. In some embodiments, the light chain constant region comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to any light chain constant region of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from table 2).
In some embodiments, the anti-transferrin receptor antibody is any anti-transferrin receptor antibody, e.g., any anti-transferrin receptor antibody selected from table 2.
In some embodiments, the anti-transferrin receptor antibody comprises a VL domain comprising the amino acid sequence of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from table 2), and wherein the constant region comprises the amino acid sequence of a constant region of IgG, igE, igM, igD, igA or IgY immunoglobulin molecule or human IgG, igE, igM, igD, igA or IgY immunoglobulin molecule. In some embodiments, the anti-transferrin receptor antibody comprises any VL domain or VL domain variant, and any VH domain or VH domain variant, wherein the VL and VH domains or variants thereof are from the same antibody clone, and wherein the constant region comprises the amino acid sequence of IgG, igE, igM, igD, igA or IgY immunoglobulin molecules or constant regions of any class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2) or any subclass (e.g., igG2a and IgG2 b) of immunoglobulin molecules. Non-limiting examples of human constant regions are described in the art, for example, see Kabat E a et al, supra (1991).
In some embodiments, antibodies of the present disclosure can bind to a target antigen (e.g., transferrin receptor), such as K, with relatively high affinity D Less than 10 -6 M、10 -7 M、10 -8 M、10 -9 M、10 -10 M、10 -11 M or less. For example, an anti-transferrin receptor antibody can bind to transferrin receptor proteins (e.g., human transferrin receptor) with an affinity of 5pM to 500nM, e.g., 50pM to 100nM, e.g., 500pM to 50 nM. The disclosure also includes antibodies that compete with any of the antibodies described herein for binding to a transferrin receptor protein (e.g., a human transferrin receptor) and have an affinity of 50nM or less (e.g., 20nM or less, 10nM or less, 500pM or less, 50pM or less, or 5pM or less). Any suitable method may be used to test the affinity and binding kinetics of the anti-transferrin receptor antibodies, including but not limited to biosensor technology (e.g., OCTET or BIACORE).
In some embodiments, antibodies of the present disclosure can bind to a target antigen (e.g., transferrin receptor), such as K, with relatively high affinity D Less than 10 -6 M、10 -7 M、10 -8 M、10 -9 M、10 -10 M、10 -11 M or less. For example, an anti-transferrin receptor antibody can bind to transferrin receptor proteins (e.g., human transferrin receptor) with an affinity of 5pM to 500nM, e.g., 50pM to 100nM, e.g., 500pM to 50 nM. The disclosure also includes antibodies that compete with any of the antibodies described herein for binding to a transferrin receptor protein (e.g., a human transferrin receptor) and have an affinity of 50nM or less (e.g., 20nM or less, 10nM or less, 500pM or less, 50pM or less, or 5pM or less). Any suitable method may be used to test the affinity and binding kinetics of the anti-transferrin receptor antibodies, including but not limited to biosensor technology (e.g., OCTET or BIACORE).
In some embodiments, the muscle targeting agent is a transferrin receptor antibody (e.g., an antibody as described in international application publication WO 2016/081643, and variants thereof, incorporated herein by reference).
The heavy and light chain CDRs of antibodies according to the different definition systems are provided in table 3. Different definition systems have been described, such as Kabat definitions, chothia definitions and/or contact definitions. See, e.g., kabat, e.a., et al (1991) Sequences of Proteins of Immunological Interest, fifth Edition, U.S. device of Health and Human Services, NIH Publication No.91-3242,Chothia et al, (1989) Nature 342:877;Chothia,C.et al (1987) j.mol. Biol.196:901-917, al-lazikani et al (1997) j.molecular. Biol.273:927-948, and Almagro, j.mol. Recognit.17:132-143 (2004) see also hgmp.mrc.uk and bioinf.org.uk/abs).
Table 3 heavy and light chain CDRs of mouse transferrin receptor antibodies.
Heavy chain variable domain (VH) and light chain variable domain sequences are also provided:
VH
VL
in some embodiments, the transferrin receptor antibodies of the present disclosure comprise the same CDR-H1, CDR-H2, and CDR-H3 as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 3. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure comprise the same CDR-L1, CDR-L2 and CDR-L3 as the CDR-L1, CDR-L2 and CDR-L3 shown in Table 3.
In some embodiments, a transferrin receptor antibody of the disclosure comprises CDR-H1, CDR-H2, and CDR-H3, which collectively comprise no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variations) as compared to CDR-H1, CDR-H2, and CDR-H3 shown in table 3. By "common" is meant that the total number of amino acid variations in all three heavy chain CDRs is within a defined range. Alternatively or additionally, transferrin receptor antibodies of the present disclosure can comprise CDR-L1, CDR-L2, and CDR-L3, which collectively comprise no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variations) as compared to CDR-L1, CDR-L2, and CDR-L3 shown in table 3.
In some embodiments, a transferrin receptor antibody of the disclosure comprises CDR-H1, CDR-H2, and CDR-H3, at least one of which comprises no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) compared to the corresponding heavy chain CDR shown in table 3. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure can comprise CDR-L1, CDR-L2, and CDR-L3, at least one of which comprises no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) compared to the corresponding light chain CDRs shown in table 3.
In some embodiments, a transferrin receptor antibody of the disclosure comprises CDR-L3 that comprises no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) compared to CDR-L3 shown in table 3. In some embodiments, a transferrin receptor antibody of the disclosure comprises CDR-L3, which comprises 1 amino acid variation compared to CDR-L3 shown in table 3. In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-L3 of QHFAGTPLT (SEQ ID NO:31, according to the Kabat and Chothia definition systems) or QHFAGTPLT (SEQ ID NO:32, according to the Contact definition systems). In some embodiments, the transferrin receptor antibodies of the present disclosure comprise the same CDR-H1, CDR-H2, CDR-H3, CDR-L1 and CDR-L2 as the CDR-H1, CDR-H2 and CDR-H3 shown in Table 3 and comprise a CDR-L3 of QHFAGTPLT (SEQ ID NO:31, according to the Kabat and Chothia definition systems) or QHFAGTPL (SEQ ID NO:32, according to the Contact definition system).
In some embodiments, a transferrin receptor antibody of the disclosure comprises heavy chain CDRs that together have at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with the heavy chain CDRs shown in table 3. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure comprise light chain CDRs which together have at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with the light chain CDRs shown in table 3.
In some embodiments, a transferrin receptor antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID No. 33. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure comprise a VL comprising the amino acid sequence of SEQ ID NO: 34.
In some embodiments, a transferrin receptor antibody of the disclosure comprises a VH comprising NO more than 20 amino acid variations (e.g., NO more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VH set forth in SEQ ID No. 33. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure comprise a VL comprising NO more than 15 amino acid variations (e.g., NO more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VL shown in SEQ ID NO 34.
In some embodiments, a transferrin receptor antibody of the disclosure comprises a VH comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity to the VH set forth in SEQ ID No. 33. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure comprise a VL comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity to the VL shown in SEQ ID No. 34.
In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized antibodies (e.g., humanized variants of the antibodies). In some embodiments, a transferrin receptor antibody of the present disclosure comprises the same CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 as the CDR-H1, CDR-H2 and CDR-H3 shown in Table 3 and comprises a humanized heavy chain variable region and/or a humanized light chain variable region.
Humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some embodiments, fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in either the recipient antibody or the introduced CDR or framework sequences, but are included to further refine and optimize antibody performance. Generally, a humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have a modified Fc region as described in WO 99/58372. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) that are altered relative to the original antibody, also referred to as one or more CDRs derived from one or more CDRs from the original antibody. Humanized antibodies may also be involved in affinity maturation.
In some embodiments, humanization is achieved by grafting CDRs (e.g., as shown in table 3) into IGKV1-NL1 x 01 and IGHV1-3 x 01 human variable domains. In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized variants comprising one or more amino acid substitutions at positions 9, 13, 17, 18, 40, 45 and 70 compared to the VL set forth in SEQ ID NO:34, and/or one or more amino acid substitutions at positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87 and 108 compared to the VH set forth in SEQ ID NO: 33. In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized variants comprising amino acid substitutions at all positions 9, 13, 17, 18, 40, 45 and 70 compared to the VL set forth in SEQ ID NO:34, and/or amino acid substitutions at all positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87 and 108 compared to the VH set forth in SEQ ID NO: 33.
In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized antibodies and comprise residues at positions 43 and 48 of VL as shown in SEQ ID NO. 34. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure are humanized antibodies and comprise residues at positions 48, 67, 69, 71 and 73 of VH as shown in SEQ ID No. 33.
VH and VL amino acid sequences of exemplary humanized antibodies that can be used according to the present disclosure are provided:
humanized VH
Humanized VL
In some embodiments, a transferrin receptor antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID No. 35. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure comprise a VL comprising the amino acid sequence of SEQ ID NO: 36.
In some embodiments, a transferrin receptor antibody of the disclosure comprises a VH comprising NO more than 20 amino acid variations (e.g., NO more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VH set forth in SEQ ID No. 35. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure comprise a VL comprising NO more than 15 amino acid variations (e.g., NO more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VL shown in SEQ ID NO: 36.
In some embodiments, a transferrin receptor antibody of the disclosure comprises a VH comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity to the VH set forth in SEQ ID No. 35. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure comprise a VL comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity to the VL set forth in SEQ ID No. 36.
In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized variants comprising amino acid substitutions at one or more of positions 43 and 48 compared to the VL set forth in SEQ ID NO:34 and/or at one or more of positions 48, 67, 69, 71 and 73 compared to the VH set forth in SEQ ID NO: 33. In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized variants comprising the S43A and/or V48L mutation compared to the VL set forth in SEQ ID NO:34 and/or one or more of the A67V, L69I, V R and K73T mutations compared to the VH set forth in SEQ ID NO: 33.
In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized variants comprising amino acid substitutions at one or more of positions 9, 13, 17, 18, 40, 43, 48, 45, and 70 compared to the VL shown in SEQ ID No. 34, and/or at one or more of positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 48, 66, 67, 69, 71, 73, 75, 81, 83, 87, and 108 compared to the VH shown in SEQ ID No. 33.
In some embodiments, the transferrin receptor antibodies of the present disclosure are chimeric antibodies, which may comprise heavy constant regions and light constant regions from human antibodies. Chimeric antibodies refer to antibodies having a variable region or a portion of a variable region from a first species and a constant region from a second species. Generally, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of antibodies derived from one mammal (e.g., a non-human mammal such as mice, rabbits, and rats), while the constant portions are homologous to sequences in antibodies derived from another mammal (e.g., a human). In some embodiments, amino acid modifications may be made in the variable and/or constant regions.
In some embodiments, the transferrin receptor antibodies described herein are chimeric antibodies, which may comprise heavy and light constant regions from a human antibody. Chimeric antibodies refer to antibodies having a variable region or a portion of a variable region from a first species and a constant region from a second species. Generally, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of antibodies derived from one mammal (e.g., a non-human mammal such as mice, rabbits, and rats), while the constant portions are homologous to sequences in antibodies derived from another mammal (e.g., a human). In some embodiments, amino acid modifications may be made in the variable and/or constant regions.
In some embodiments, the heavy chain of any of the transferrin receptor antibodies described herein can comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region can have any suitable origin, such as human, mouse, rat, or rabbit. In a particular example, the heavy chain constant region is from a human IgG (gamma heavy chain) such as IgG1, igG2 or IgG 4. Exemplary human IgG1 constant regions are given below:
In some embodiments, the light chain of any of the transferrin receptor antibodies described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, CL is a kappa light chain. In other examples, CL is a lambda light chain. In some embodiments, CL is a kappa light chain, the sequence of which is provided below:
other antibody heavy and light chain constant regions are well known in the art, such as those provided in IMGT database (www.imgt.org) or www.vbase2.org/vbstat.
Exemplary heavy and light chain amino acid sequences of the transferrin receptor antibodies are provided below:
heavy chain (VH+human IgG1 constant region)
Light chain (VL + kappa light chain)
Heavy chain (humanized VH+human IgG1 constant region)
Light chain (humanized VL + kappa light chain)
In some embodiments, a transferrin receptor antibody described herein comprises a heavy chain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity to SEQ ID NO: 39. Alternatively or additionally, the transferrin receptor antibodies described herein comprise a light chain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity to SEQ ID No. 40. In some embodiments, the transferrin receptor antibodies described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 39. Alternatively or additionally, the transferrin receptor antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO. 40.
In some embodiments, a transferrin receptor antibody of the disclosure comprises a heavy chain comprising NO more than 20 amino acid variations (e.g., NO more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the heavy chain shown in SEQ ID NO: 39. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure comprise a light chain comprising NO more than 15 amino acid variations (e.g., NO more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the light chain shown in SEQ ID No. 40.
In some embodiments, a transferrin receptor antibody described herein comprises a heavy chain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity to SEQ ID No. 41. Alternatively or additionally, the transferrin receptor antibodies described herein comprise a light chain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity to SEQ ID No. 42. In some embodiments, the transferrin receptor antibodies described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO. 41. Alternatively or additionally, the transferrin receptor antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO. 42.
In some embodiments, a transferrin receptor antibody of the disclosure comprises a heavy chain comprising NO more than 20 amino acid variations (e.g., NO more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the heavy chain of a humanized antibody set forth in SEQ ID NO: 39. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure comprise a light chain comprising NO more than 15 amino acid variations (e.g., NO more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the light chain of the humanized antibody set forth in SEQ ID No. 40.
In some embodiments, the transferrin receptor antibody is an antigen binding fragment (Fab) of an intact antibody (full length antibody). Antigen binding fragments of whole antibodies (full length antibodies) can be prepared by conventional methods. For example, F (ab ') 2 fragments can be produced by pepsin digestion of antibody molecules, and Fab fragments can be produced by reduction of disulfide bonds of F (ab') 2 fragments. Exemplary Fab amino acid sequences of the transferrin receptor antibodies described herein are provided below:
Heavy chain FAB (VH+human IgG1 constant region part)
Heavy chain FAB (humanized VH+human IgG1 constant region part)
The transferrin receptor antibodies described herein can be in any antibody form, including but not limited to, intact (i.e., full length) antibodies, antigen binding fragments thereof (e.g., fab ', F (ab') 2, fv), single chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the transferrin receptor antibodies described herein are scFv. In some embodiments, the transferrin receptor antibodies described herein are scFv-Fab (e.g., scFv fused to a portion of a constant region). In some embodiments, the transferrin receptor antibodies described herein are scFv fused to a constant region (e.g., a human IgG1 constant region shown in SEQ ID NO: 39).
b. Other muscle targeting antibodies
In some embodiments, the muscle targeting antibody is an antibody that specifically binds to hemojuvelin (hemojuvelin), caveolin-3, duchenne muscular dystrophy peptide, myosin Iib, or CD 63. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to a myogenic precursor protein. Exemplary myogenic precursor proteins include, but are not limited to, ABCG2, M-cadherin/cadherin-15, nidogen-1, CD34, foxK1, integrin alpha 7 beta 1, MYF-5, myoD, myogenin, NCAM-1/CD56, pax3, pax7, and Pax9. In some embodiments, the muscle targeting antibody is an antibody that specifically binds skeletal muscle protein. Exemplary skeletal muscle proteins include, but are not limited to, alpha-sarcosins (alpha-sarcogycan), beta-sarcosins, calpain inhibitors, creatine kinase MM/CKMM, eIF5A, enolase 2/neuron-specific enolase, epsilon-sarcosins, FABP3/H-FABP, GDF-8/myosin, GDF-11/GDF-8, integrin alpha 7 beta 1, integrin beta 1/CD29, MCAM/CD146, myoD, myogenin, myosin light chain kinase inhibitors, NCAM-1/CD56, and troponin I. In some embodiments, the muscle targeting antibody is an antibody that specifically binds smooth muscle protein. Exemplary smooth muscle proteins include, but are not limited to, alpha-smooth muscle actin, VE-cadherin, calmodulin binding protein/CALD 1, calmodulin 1, desmin (Desmin), histamine H2R, motilin R/GPR38, transferrin/TAGL, and vimentin. However, it is to be understood that antibodies to other targets are within the scope of the present disclosure, and that the exemplary list of targets provided herein is not intended to be limiting.
c. Antibody characterization/alteration
In some embodiments, conservative mutations may be introduced into an antibody sequence (e.g., CDR or framework sequence) at positions where the residues are unlikely to be involved in an interaction with a target antigen (e.g., transferrin receptor), e.g., as determined based on crystal structure. In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., at residues 231-340 of human IgG 1) and/or the CH3 domain (residues 341-447 of human IgG 1) and/or the hinge region of a muscle-targeting antibody described herein, according to the Kabat numbering system (e.g., EU index in Kabat) to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, fc receptor binding, and/or antigen-dependent cytotoxicity.
In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH 1 domain) such that the number of cysteine residues in the hinge region is altered (e.g., increased or decreased) as described, for example, in U.S. patent No.5,677,425. The number of cysteine residues in the hinge region of the CH1 domain can be altered, for example, to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody or to facilitate linker conjugation.
In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., at residues 231 to 340 of human IgG 1) and/or the CH3 domain (residues 341 to 447 of human IgG 1) and/or the hinge region of a muscle-targeting antibody described herein, according to the Kabat numbering system (e.g., EU index in Kabat) to increase or decrease the affinity of the antibody for Fc receptors (e.g., activated Fc receptors) on the surface of effector cells. Mutations in the Fc region of antibodies that reduce or increase the affinity of the antibody for Fc receptors, and techniques for introducing such mutations into Fc receptors or fragments thereof are known to those of skill in the art. Examples of mutations in the Fc receptor of antibodies that can be made to alter the affinity of the antibody for the Fc receptor are described in the following: such as Smith P et al, (2012) PNAS 109:6181-6186, U.S. Pat. No.6,737,056, and International publication Nos. WO 02/060919, WO 98/23289, and WO 97/34631, which are incorporated herein by reference.
In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into an IgG constant domain or FcRn binding fragment thereof (preferably, fc or hinge-Fc domain fragment) to alter (e.g., reduce or increase) the half-life of an antibody in vivo. See, e.g., international publication Nos. WO 02/060919, WO 98/23289 and WO 97/34631, and U.S. Pat. Nos. 5,869,046, 6,121,022, 6,277,375 and 6,165,745, for mutations that alter (e.g., reduce or increase) the half-life of antibodies in vivo.
In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into an IgG constant domain or FcRn binding fragment thereof (preferably, fc or hinge-Fc domain fragment) to reduce the half-life of an anti-transferrin receptor antibody in vivo. In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into an IgG constant domain or FcRn binding fragment thereof (preferably, fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH 2) domain (residues 231 to 340 of human IgG 1) and/or the third constant (CH 3) domain (residues 341 to 447 of human IgG 1), numbered according to the EU index in Kabat (Kabat E a et al, (1991) supra). In some embodiments, the constant region of IgG1 of the antibodies described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index in Kabat. See U.S. Pat. No.7,658,921, which is incorporated herein by reference. This type of mutant IgG (referred to as a "YTE mutant") has been shown to have a 4-fold increase in half-life compared to the wild-type form of the same antibody (see Dall' Acqua W F et al, (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant domain comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251 to 257, 285 to 290, 308 to 314, 385 to 389, and 428 to 436, numbered according to the EU index in Kabat.
In some embodiments, one, two, or more amino acid substitutions are introduced into the IgG constant domain Fc region to alter one or more effector functions of the anti-transferrin receptor antibody. The affinity-altering effector ligand may be, for example, an Fc receptor or the C1 component of complement. Such methods are described in more detail in U.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation (by point mutation or otherwise) of the constant region domains may reduce Fc receptor binding of circulating antibodies, thereby improving tumor localization. For a description of mutations that delete or inactivate constant domains and thereby improve tumor localization, see, e.g., U.S. Pat. nos. 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of antibodies described herein to remove potential glycosylation sites on the Fc region, which may reduce Fc receptor binding (see, e.g., shields R L et al, (2001) J Biol Chem 276:6591-604).
In some embodiments, one or more amino groups in the constant regions of the muscle-targeting antibodies described herein may be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or eliminated complement dependent cytotoxicity (complement dependent cytotoxicity, CDC). Such a process is described in more detail in U.S. Pat. No.6,194,551 (Idusogie et al). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of an antibody described herein are altered, thereby altering the ability of the antibody to fix complement. Such a process is further described in International publication No. WO 94/29351. In some embodiments, the Fc region of an antibody described herein is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (antibody dependent cellular cytotoxicity, ADCC) and/or to increase the affinity of the antibody for fcγ receptors. Such a method is further described in International publication No. WO 00/42072.
In some embodiments, the heavy and/or light chain variable domain sequences of the antibodies provided herein can be used to generate, for example, CDR grafted, chimeric, humanized or composite human antibodies or antigen binding fragments, as described elsewhere herein. As will be appreciated by one of ordinary skill in the art, any variant (CDR grafted, chimeric, humanized or complexed antibody) derived from any of the antibodies provided herein may be used in the compositions and methods described herein and will retain the ability to specifically bind to a transferrin receptor such that the variant (CDR grafted, chimeric, humanized or complexed antibody) has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to the transferrin receptor relative to the original antibody from which it was derived.
In some embodiments, the antibodies provided herein comprise mutations that confer a desired property to the antibody. For example, to avoid potential complications due to Fab arm exchanges known to occur with native IgG4 mabs, antibodies provided herein may comprise a stable 'Adair' mutation (Angal s., et al, "A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG 4) anti," Mol Immunol 30,105-108; 1993), wherein serine at position 228 (EU numbering, residue 241 according to Kabat numbering) is converted to proline, resulting in an IgG 1-like hinge sequence. Thus, any antibody may comprise a stable 'Adair' mutation.
As provided herein, the antibodies of the present disclosure may optionally comprise a constant region or a portion thereof. For example, a VL domain may be linked at its C-terminus to a light chain constant domain, such as ck or cλ. Similarly, a VH domain or a portion thereof may be linked to all or a portion of a heavy chain, such as IgA, igD, igE, igG and IgM, as well as any isotype subclass. Antibodies can include suitable constant regions (see, e.g., kabat et al Sequences of Proteins of Immunological Interest, no.91-3242,National Institutes of Health Publications,Bethesda,Md (1991)). Thus, antibodies within the scope of the present disclosure may comprise VH and VL domains, or antigen-binding portions thereof, in combination with any suitable constant region.
Muscle targeting peptides
Some aspects of the present disclosure provide muscle targeting peptides as muscle targeting agents. Short peptide sequences (e.g., peptide sequences 5 to 20 amino acids in length) have been described that bind to specific cell types. For example, cell-targeting peptides have been described in the following: vines e., et al, A. "Cell-penetrating and Cell-targeting peptides in drug delivery" Biochim Biophys Acta 2008,1786:126-38; jarver P., et al, "In vivo biodistribution and efficacy of peptide mediated delivery" Trends Pharmacol Sci 2010;31:528-35; samolyova t.i., et al, "Elucidation of Muscle-binding peptides by phage display screening" Muscle Nerve 1999;22:460-6; U.S. patent No.6,329,501, entitled "METHODS AND COMPOSITIONS FOR TARGETING COMPOUNDS TO MUSCLE" to date 11 of 12/2001; and samolyov a.m., et al, "Recognition of cell-specific binding of phage display derived peptides using an acoustic wave sensor," Biomol Eng 2002;18:269-72; the entire contents of each of which are incorporated herein by reference. By designing the peptide to interact with a particular cell surface antigen (e.g., receptor), selectivity for a desired tissue, such as muscle, can be achieved. Skeletal muscle targeting has been studied and is capable of delivering a range of molecular loads. These methods can be highly selective to muscle tissue without many of the practical disadvantages of large antibodies or viral particles. Thus, in some embodiments, the muscle targeting agent is a muscle targeting peptide that is 4 to 50 amino acids in length. In some embodiments, the muscle targeting peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. Any of several methods (e.g., phage display) can be used to produce muscle targeting peptides.
In some embodiments, the muscle targeting peptide may bind to an internalized cell surface receptor, such as a transferrin receptor, that is overexpressed or relatively highly expressed in muscle cells as compared to certain other cells. In some embodiments, the muscle targeting peptide can target (e.g., bind to) a transferrin receptor. In some embodiments, the peptide that targets the transferrin receptor may comprise a fragment of a natural ligand (e.g., transferrin). In some embodiments, the peptide that targets the transferrin RECEPTOR is as described in U.S. Pat. No.6,743,893, "RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR," filed 11/30/2000. In some embodiments, peptides that target transferrin receptor are such as Kawamoto, m.et al, "A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells", "BMC cancer.2011aug 18; 11:359. In some embodiments, the peptide that targets the transferrin receptor is as described in U.S. Pat. No.8,399,653, "TRANSFERRIN/TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY," filed 5.20.2011.
As mentioned above, some examples of muscle targeting peptides have been reported. For example, muscle-specific peptides were identified using phage display libraries presenting surface heptapeptides. As an example, a peptide having the amino acid sequence ASSINLNIA (SEQ ID NO: 6) binds to C2C12 murine myotubes in vitro and to mouse muscle tissue in vivo. Thus, in some embodiments, the muscle targeting agent comprises the amino acid sequence ASSINLNIA (SEQ ID NO: 6). The peptides showed improved specificity for binding to heart and skeletal muscle tissue and reduced binding to liver, kidney and brain following intravenous injection in mice. Additional muscle-specific peptides have been identified using phage display. For example, 12 amino acid peptides were identified by phage display library for muscle targeting in the context of DMD treatment. See Yoshida d., et al, "Targeting of salicylate to skin and muscle following topical injections in rates," Int J Pharm 2002;231:177-84; the entire contents of which are incorporated herein by reference. Here, a 12 amino acid peptide having the sequence SKTFNTHPQSTP (SEQ ID NO: 7) was identified, and the muscle targeting peptide showed improved binding to C2C12 cells relative to the ASSINIA (SEQ ID NO: 6) peptide.
Another method for identifying peptides that are selective for muscle (e.g., skeletal muscle) relative to other cell types includes in vitro selection, which has been described in Ghosh D., et al, "Selection of muscle-binding peptides from context-specific peptide-presenting phage libraries for adenoviral vector targeting" J Virol 2005; 79:13667-72; the entire contents of which are incorporated herein by reference. Nonspecific cell conjugates were selected by pre-incubating random 12-mer peptide phage display libraries with a mixture of non-myocyte types. After several rounds of selection, the 12 amino acid peptide TARGEHKEEELI (SEQ ID NO: 8) appeared most frequently. Thus, in some embodiments, the muscle targeting agent comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 8).
The muscle targeting agent may be an amino acid containing molecule or peptide. The muscle targeting peptide may correspond to a protein sequence that preferentially binds to a protein receptor found in a muscle cell. In some embodiments, the muscle targeting peptide comprises a highly-prone hydrophobic amino acid, such as valine, such that the peptide preferentially targets muscle cells. In some embodiments, the muscle targeting peptide has not been previously characterized or disclosed. These peptides can be contemplated, generated, synthesized, and/or derivatized using any of a number of methods, such as phage display peptide libraries, single-bead single-compound peptide libraries, or site-scanning synthetic peptide combinatorial libraries. Exemplary methods have been characterized in the art and incorporated by reference (Gray, B.P. and Brown, K.C. "Combinatorial Peptide Libraries: mining for Cell-Binding Peptides" Chem Rev.2014,114:2,1020-1081.; samoylova, T.I. and Smith, B.F. "Elucidation of Muscle-Binding Peptides by phage display screening." music Nerve,1999, 22:4.460-6.). In some embodiments, muscle targeting peptides have been previously disclosed (see, e.g., writer M.J.et al. "Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display." J.drug targeting.2004;12:185; cai, D. "BDNF-mediated enhancement of inflammation and injury in the aging heart." Physiol genomics.2006,24:3,191-7.; zhang, L. "Molecular profiling of heart endothelial cells." Circulation,2005,112:11,1601-11.; mcGuire, M.J.et al. "In vitro selection of a peptide with high selectivity for cardiomyocytes in device." J Mol biol.2004,342:1, 171-82.). Exemplary muscle targeting peptides comprise the amino acid sequences of the following groups: CQAQGQLVC (SEQ ID NO: 9), CSERSMNFC (SEQ ID NO: 10), CPKTRRVPC (SEQ ID NO: 11), WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 12), ASSINLIA (SEQ ID NO: 6), CMQHSMRVC (SEQ ID NO: 13) and DDTRHWG (SEQ ID NO: 14). In some embodiments, the muscle targeting peptide may comprise about 2 to 25 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to 10 amino acids, or about 2 to 5 amino acids. Muscle targeting peptides may comprise natural amino acids such as cysteine, alanine, or non-naturally occurring or modified amino acids. Unnatural amino acids include beta-amino acids, homoamino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and others known in the art. In some embodiments, the muscle targeting peptide may be linear; in other embodiments, the muscle targeting peptide may be cyclic, e.g., bicyclic (see, e.g., silvana, m.g. et al mol. Therapy,2018,26:1, 132-147.). The muscle targeting agent may be an aptamer, such as a peptide aptamer, that preferentially targets muscle cells over other cell types.
Muscle targeting receptor ligands
The muscle targeting agent may be a ligand, for example a ligand that binds to a receptor protein. The muscle targeting ligand may be a protein, such as transferrin, which binds to internalized cell surface receptors expressed by muscle cells. Thus, in some embodiments, the muscle targeting agent is transferrin or a derivative thereof that binds to a transferrin receptor. The muscle targeting ligand may alternatively be a small molecule, such as a lipophilic small molecule that preferentially targets muscle cells over other cell types. Some exemplary lipophilic small molecules that can target muscle cells include compounds comprising: cholesterol, cholesteryl, stearic acid, palmitic acid, oleic acid, oleyl, linolene, myristic acid, sterols, dihydrotestosterone, testosterone derivatives, glycerol, alkyl chains, trityl groups and alkoxy acids.
Other muscle targeting agents
One strategy for targeting muscle cells (e.g., skeletal muscle cells) is to use substrates for muscle transporter proteins (e.g., transporter proteins expressed on the myomembrane). In some embodiments, the muscle targeting agent is a substrate for an influx transporter specific for muscle tissue. In some embodiments, the inflow transporter is specific for skeletal muscle tissue. Two major classes of transporters are expressed on skeletal muscle myomembranes: (1) An Adenosine Triphosphate (ATP) -binding cassette (ABC) superfamily that promotes outflow from skeletal muscle tissue and (2) a solute transporter (SLC) superfamily that can promote substrate inflow into skeletal muscle. In some embodiments, the muscle targeting agent is a substrate that binds to the ABC superfamily or the SLC superfamily of transporters. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a natural substrate. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a non-natural substrate, e.g., a synthetic derivative thereof that binds to the ABC or SLC superfamily of transporters.
In some embodiments, the muscle targeting agent is any muscle targeting agent described herein that targets the SLC transporter superfamily (e.g., antibodies, nucleic acids, small molecules, peptides, aptamers, lipids, sugar moieties). In some embodiments, the muscle targeting agent can target a transporter (e.g., can be a substrate). SLC transporters are balanced or use proton or sodium ion gradients generated across the membrane to drive substrate transport. Exemplary SLC transporters with high skeletal muscle expression include, but are not limited to, SATT transporter (ASCT 1; SLC1A 4), GLUT4 transporter (SLC 2A 4), GLUT7 transporter (GLUT 7; SLC2A 7), ATRC2 transporter (CAT-2; SLC7A 2), LAT3 transporter (KIAA 0245; SLC7A 6), PHT1 transporter (PTR 4; SLC15A 4), OATP-J transporter (OATP 5A1; SLC21A 15), OCT3 transporter (EMT; SLC22A 3), OCTN2 transporter (FLJ 46769; SLC22A 5), ENT transporter (ENT 1; SLC29A1 and ENT2; SLC29A 2), PAT2 transporter (SLC 36A 2) and SAT2 transporter (KIAA 1382; SLC38A 2). These transporters may facilitate substrate flow into skeletal muscle, providing opportunities for muscle targeting.
In some embodiments, the muscle targeting agent is a substrate for an equilibrium nucleoside transporter 2 (equilibrative nucleoside transporter, ent 2) transporter. ENT2 has one of the highest mRNA expression in skeletal muscle relative to other transporters. Although human ENT2 (hENT 2) is expressed in most body organs such as brain, heart, placenta, thymus, pancreas, prostate and kidney, it is particularly abundant in skeletal muscle. Human ENT2 promotes its substrate absorption according to its concentration gradient. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleoside bases. The hENT2 transporter has low affinity for all nucleosides (adenosine, guanosine, uridine, thymidine, and cytidine) except inosine. Thus, in some embodiments, the muscle targeting agent is an ENT2 substrate. Exemplary ENT2 substrates include, but are not limited to, inosine, 2',3' -dideoxyinosine, and clofarabine (calofarabine). In some embodiments, any of the muscle targeting agents provided herein are associated with a molecular load (e.g., an oligonucleotide load). In some embodiments, the muscle targeting agent is covalently linked to the molecular cargo. In some embodiments, the muscle targeting agent is non-covalently linked to the molecular cargo.
In some embodiments, the muscle targeting agent is a substrate for an organic cation/carnitine transporter (OCTN 2) that is a sodium ion dependent high affinity carnitine transporter. In some embodiments, the muscle targeting agent is carnitine, mildronate, acetyl carnitine, or any derivative thereof that binds to OCTN 2. In some embodiments, carnitine, mildronate, acetyl carnitine, or derivatives thereof, is covalently linked to a molecular load (e.g., an oligonucleotide load). The muscle targeting agent may be a protein, which is a protein that exists in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein may be a hemojuin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), a protein involved in iron overload and homeostasis. In some embodiments, the hemojuin may be full length or a fragment, or a mutant having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a functional hemojuin protein. In some embodiments, the hemojuvelin mutant may be a soluble fragment, may lack N-terminal signaling, and/or lack a C-terminal anchoring domain. In some embodiments, hemojuvelin may be annotated with GenBank RefSeq accession No. nm_001316767.1, nm_145277.4, nm_202004.3, nm_213652.3, or nm_ 213653.3. It is understood that the hemojuvelin may be of human, non-human primate or rodent origin.
B. Molecular loading
Some aspects of the disclosure provide molecular loading, e.g., for modulating biological outcomes, e.g., transcription of DNA sequences, expression of proteins, or activity of proteins. In some embodiments, the molecular load is covalently linked or otherwise associated with the muscle targeting agent. It should be understood that various types of muscle targeting agents may be used in accordance with the present disclosure. For example, the molecular load may comprise or consist of: oligonucleotides (e.g., antisense oligonucleotides), peptides (e.g., peptides that bind to a disease-associated nucleic acid or protein in a muscle cell), proteins (e.g., proteins that bind to a disease-associated nucleic acid or protein in a muscle cell), or small molecules (e.g., small molecules that modulate the function of a disease-associated nucleic acid or protein in a muscle cell). In some embodiments, such molecular cargo is capable of targeting a muscle cell, for example, by specific binding to a nucleic acid or protein in the muscle cell after delivery to the muscle cell by a relevant muscle targeting agent. In some embodiments, the molecular cargo is an oligonucleotide comprising a strand having a region complementary to a gene provided in table 1. Exemplary molecular loadings are described in further detail herein, however, it is to be understood that the exemplary molecular loadings provided herein are not meant to be limiting.
In some embodiments, at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 10) molecular load (e.g., an oligonucleotide) is covalently linked to a muscle targeting agent. In some embodiments, all molecular loads linked to a muscle targeting agent are the same, e.g., target the same gene. In some embodiments, all of the molecular payloads attached to the muscle targeting agent are different, e.g., the molecular payloads may target different portions of the same target gene, or the molecular payloads may target at least two different target genes. In some embodiments, the muscle targeting agent may be linked to some of the same molecular loads or some of the different molecular loads.
The present disclosure also provides compositions comprising a plurality of complexes, wherein at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the complexes comprise a molecular targeting agent covalently linked to the same number of molecular loads (e.g., oligonucleotides).
i. Oligonucleotides
Any suitable oligonucleotide may be used as the molecular charge, as described herein. In some embodiments, the oligonucleotide may be designed to cause degradation of the mRNA (e.g., the oligonucleotide may be a spacer, siRNA, ribozyme, or aptamer that causes degradation). In some embodiments, the oligonucleotide can be designed to block translation of the mRNA (e.g., the oligonucleotide can be a mixed-mer, siRNA, or aptamer that blocks translation). In some embodiments, the oligonucleotides may be designed to cause degradation of mRNA and block translation. In some embodiments, the oligonucleotide may be a guide nucleic acid (e.g., guide RNA) for guiding the activity of an enzyme (e.g., a gene editing enzyme). Further examples of oligonucleotides are provided herein. It will be appreciated that in some embodiments, oligonucleotides of one format (e.g., antisense oligonucleotides) may be suitably adapted to another format (e.g., siRNA oligonucleotides) by incorporating functional sequences (e.g., antisense strand sequences) from one format into another format.
In some embodiments, the oligonucleotide may comprise a region complementary to a target gene provided in table 1. Further non-limiting examples of selected genes of Table 1 are provided below.
DMPK/DM1
In some embodiments, some examples of oligonucleotides that can be used to target DMPK (e.g., for treating DM 1) are provided in the following: U.S. patent application publication 20100016215A1, published 1/2010, titled Compound And Method For Treating Myotonic Dystrophy; U.S. patent application publication 20130237585A1, published at 7/19 2010, modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression; U.S. patent application publication 20150064181A1, which is published 5/3/5/2015, entitled "Antisense Conjugates For Decreasing Expression Of Dmpk"; U.S. patent application publication 20150238627A1, published on day 27, 8, 2015, entitled "Peptide-Linked Morpholino Antisense Oligonucleotides For Treatment Of Myotonic Dystrophy"; pandey, s.k.et al. "Identification and Characterization of Modified Antisense Oligonucleotides Targeting DMPK in Mice and Nonhuman Primates for the Treatment of Myotonic Dystrophy Type 1"J.of Pharmacol Exp Ther,2015,355:329-340; langlois, M.et al, "Cytoplasmic and Nuclear Retained DMPK mRNAs Are Targets for RNA Interference in Myotonic Dystrophy Cells" J.biological Chemistry,2005,280:17, 16949-16954; jauvin, D.et al, "Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type Mice", mol.Ther: nucleic Acids,2017, 7:465-474; mulders, S.A. et al, "triple-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy" PNAS,2009,106:33, 13915-13920; wheeler, T.M. et al, "Targeting nuclear RNA for in vivo correction of myotonic dystrophy" Nature,2012,488 (7409):111-115; and U.S. patent application publication 20160304877A1, published at 10/20/2016, entitled "Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (Dmpk) Expression," the contents of each of which are incorporated herein by reference in their entirety.
Some examples of oligonucleotides for facilitating DMPK gene editing include U.S. patent application publication 20170088819A1, published 3-2017, titled "Genetic Correction Of Myotonic Dystrophy Type 1"; and international patent application publication WO18002812A1, published on month 4 and 1 of 2018, entitled "Materials And Methods For Treatment Of Myotonic Dystrophy Type 1 (DM 1) And Other Related Disorders", the respective contents of which are incorporated herein by reference in their entirety.
In some embodiments, the oligonucleotide may have a region complementary to a mutant form of DMPK, e.g., a mutant form as reported in: botta A.et al, "The CTG repeat expansion size correlates with the splicing defects observed in muscles from myotonic dystrophy type components," J Med Genet.20088 Oct;45 (10) 639-46; and Machuca-Tzili L.et al, "Clinical and molecular aspects of the myotonic dystrophies: a review," music nerve.2005Jul;32 (1) 1-18; the respective content of which is incorporated herein by reference in its entirety.
In some embodiments, the oligonucleotides provided herein are antisense oligonucleotides that target DMPK. In some embodiments, the targeted oligonucleotide is any antisense oligonucleotide (e.g., a spacer) that targets DMPK, as described in U.S. patent application publication US20160304877A1, published 10/20, 2016, entitled "Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression," incorporated herein by reference. In some embodiments, the DMPK targeting oligonucleotide targets a region of the DMPK gene sequence as shown in Genbank accession No. nm_001081560.2 or as shown in Genbank accession No. ng_ 009784.1.
In some embodiments, the DMPK targeting oligonucleotide comprises a nucleotide sequence comprising a region complementary to a target region of at least 10 consecutive nucleotides (e.g., at least 10, at least 12, at least 14, at least 16 or more consecutive nucleotides) in Genbank accession No. nm_ 001081560.2.
In some embodiments, the DMPK targeting oligonucleotide comprises a spacer motif. "spacer" means a chimeric antisense compound in which an inner region having a plurality of nucleotides supporting cleavage by rnase H is located between outer regions having one or more nucleotides, wherein the nucleotides comprising the inner region are chemically different from the one or more nucleotides comprising the outer region. The inner region may be referred to as a "spacer section" and the outer region may be referred to as a "wing section". In some embodiments, the DMPK targeting oligonucleotide comprises one or more modified nucleotides, and/or one or more modified internucleotide linkages. In some embodiments, the internucleotide linkage is a phosphorothioate linkage. In some embodiments, the oligonucleotide comprises an intact phosphorothioate backbone. In some embodiments, the oligonucleotide is a DNA spacer having a cET terminus (e.g., 3-10-3; cET-DNA-cET). In some embodiments, the DMPK targeting oligonucleotide comprises one or more 6' - (S) -CH 3 Bicyclic nucleotides, one or more β -D-2' -deoxyribonucleotides, and/or one or more 5-methylcytosine nucleotides.
DUX4/FSHD
In some embodiments, examples of oligonucleotides that can be used to target DUX4 (e.g., for treating FSHD) are provided in the following: U.S. patent No. 9,988,628, which is disclosed at 2/2017, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; U.S. patent No. 9,469,851, which was published 10/30 in 2014, entitled "RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX" for example; U.S. patent application publication 20120225034, published 9/6 in 2012, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; PCT patent application publication No. WO 2013/120038, published on 15, 8, 2013, entitled "MORPHOLINO TARGETING DUX FOR TREATING FSHD"; chen et al, "Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics," Molecular Therapy,2016,24:8, 1405-1411; and anseau et al, "Antisense Oligonucleotides Used to Target the DUX4 mRNAas Therapeutic Approaches in Facioscapulohumeral Muscular Dystrophy (FSHD)," Genes,2017,8,93, the respective contents of which have been incorporated herein in their entirety. In some embodiments, the oligonucleotide is an antisense oligonucleotide, morpholino, siRNA, shRNA or other nucleotide that hybridizes to a target DUX4 gene or mRNA.
In some embodiments, for example for treating FSHD, the oligonucleotide may have a region complementary to the hypomethylated compact D4Z4 repeat, as described in: daxinger, et al, "Genetic and Epigenetic Contributors to FSHD,"2015 in Curr Opin Genet Dev; lim J-W, et al, DICER/AGO-dependent epigenetic silencing of D Z4 repeats enhanced by exogenous siRNAsuggests mechanisms and therapies for FSHD Hum Mol genet.20150ep 1;24 4817-4818, each of which is incorporated herein in its entirety.
DNM2/CNM
In some embodiments, examples of oligonucleotides that can be used to target DNM2 (e.g., for treating CNM) are provided in the following: U.S. patent application publication No. 20180142008, which is published 24 days 5 in 2018, entitled "DYNAMIN 2INHIBITOR FOR THE TREATMENT OF DUCHENNE'S MUSCULAR DYSTROPHY", and PCT application publication No. WO 2018/100010A1, which is published 7 days 6 in 2018, entitled "ALLELE-SPECIFIC SILENCING THERAPY FOR DYNAMIN 2-RELATED DISEASES". For example, in some embodiments, the oligonucleotide is an RNAi, an antisense nucleic acid, an siRNA, or a ribozyme that specifically interferes with DNM2 expression. Further examples of oligonucleotides that can be used to target DNM2 are provided in the following: tasfaout, et al, "" Single Intramuscular Injection of AAV-shRNA Reduces DNM2 and Prevents Myotubular Myopathy in Mice, ", 4, 2018, are disclosed in mol. Ther. And Tasfaout, et al," "Antisense oligonucleotide-mediated Dnm2 knockdown prevents and reverts myotubular myopathy in mice," Nature Communications volume 8,Article number:15661 (2017). In some embodiments, the oligonucleotide is a shRNA or morpholino that is effective to target DNM2 mRNA. In some embodiments, the oligonucleotide encodes a wild-type DNM2 that is resistant to miR-133 activity, as described in: todaka, et al, "Overexpression of NF90-NF45 Represses Myogenic MicroRNA Biogenesis, resulting in Development of Skeletal Muscle Atrophy and Centronuclear Muscle Fibers,", month 7 of 2015, is disclosed in mol.cell biol. Further examples of oligonucleotides that can be used to target DNM2 are provided in the following: gibbs, et al, "Two Dynamin-2Genes are Required for Normal Zebrafish Development",2013, in PLoS One, each of which is incorporated herein in its entirety.
In some embodiments, for example for treating CNM, the oligonucleotide may have a region complementary to a mutant in CNM 2 associated with CNM, as described in:et al, "Mutation Spectrum in the Large GTPase Dynamin 2, and Genotype-Phenotype Correlation in Autosomal Dominant Centronuclear Myopathy," as disclosed in 2012 in hum.
Pompe disease
In some embodiments, for example for the treatment of pompe disease, the oligonucleotide-mediated inclusion of exon 2 in GAA disease alleles, such as van der Wal, et al, "GAA Deficiency in Pompe Disease is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells," Mol ter Nucleic acids.2017jun 16;7:101-115, the contents of which are incorporated herein by reference. Thus, in some embodiments, the oligonucleotide may have a region complementary to a GAA disease allele.
In some embodiments, for example for the treatment of pompe disease, oligonucleotides (e.g. RNAi or antisense oligonucleotides) are used to inhibit expression of wild-type GYS1 in muscle cells, as reported, for example, in the following: clayton, et al, ", antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase, 1Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease," published in Mol Ther Nucleic Acids in 2017, or U.S. patent application publication No. 2017182189, published in 29 of 6/2017, entitled "INHIBITING OR DOWNREGULATING GLYCOGEN SYNTHASE BY CREATING PREMATURE STOP CODONS USING ANTISENSE OLIGONUCLEOTIDES," the contents of which are incorporated herein by reference. Thus, in some embodiments, the oligonucleotide may have an antisense strand with a region complementary to the human GYS1 sequence corresponding to RefSeq number nm_002103.4 and/or the mouse GYS1 sequence corresponding to RefSeq number nm_ 030678.3.
ACVR1/FOP
In some embodiments, examples of oligonucleotides that can be used to target ACVR1 (e.g., for treating FOP) are provided in the following: U.S. patent application 2009/0253132, "muted ACVR1 for diagnosis and treatment of Fibrodysplasia Ossificans Progressiva (FOP)", published 10/8/2009; WO 2015/152183, 10/8 of 2015, "Prophylactic agent and therapeutic agent for fibrodysplasia ossificans progressive"; lowery, j.w.et al, "Allele-specific RNA Interference in FOP-Silencing the FOP gene", GENE THERAPY, vol.19,2012, pages 701-702; takahashi, M.et al, "Disease-using animals-specific silencing against the ALK variants, R206H and G356D, in fibrodysplasia ossificans progressiva" Gene Therapy (2012) 19,781-785; shi, s.et al, "anti-sense-Oligonucleotide Mediated Exon Skipping in Activin-Receptor-Like Kinase 2:Inhibiting the Receptor That Is Overactive in Fibrodysplasia Ossificans Progressiva"Plos One,July 2013,Vol 8:7,e69096; U.S. patent application 2017/0159056, published on 8/6/2017, "Antisense oligonucleotides and methods of use thereof"; U.S. Pat. No.8,859,752, "SIRNA-based therapy of Fibrodyplasia Ossificans Progressiva (FOP)", granted on 10/4/2014; WO 2004/094636, published 11/4 2004, "Effective sirna knock-down constructs", the respective content of which is incorporated herein in its entirety.
FXN/Friedel-crafts ataxia
In some embodiments, examples of oligonucleotides that can be used to target FXN and/or otherwise compensate for ataxin deficiency (e.g., for treating friedreich ataxia) are provided in the following: li, L.et al, "Activating frataxin expression by repeat-targeted nucleic acids" Nat.Comm.2016, 7:10606; WO 2016/094374, "Compositions and methods for treatment of friedreich's ataxia", published 6/16 of 2016; WO 2015/020993, 2 months 12 days 2015, published, "RNAi COMPOSITIONS AND METHODS FOR TREATMENT OF FRIEDREICH' S ATAXIA"; WO 2017/186815, published 11/2, 2017, "Antisense oligonucleotides for enhanced expression of frataxin"; WO 2008/018795, 14 d 2, 2008, "Methods and means for treating dna repeat instability associated genetic disorders"; U.S. patent application 2018/0028557, published on 1 month 2 of 2018, "Hybrid oligonucleotides and uses thereof"; WO 2015/023975, 19 months of 2015, publication, "Compositions and methods for modulating RNA"; WO 2015/023939, publication No. 19, 2015, 2, month, "Compositions and methods for modulating expression of frataxin"; U.S. patent application 2017/0281643, published on 10 months 5, 2017, "Compounds and methods for modulating frataxin expression"; li L.et al, "Activating frataxin expression by repeat-targeted nucleic acids" Nature Communications, published under month 2 and 4 of 2016; "Activation of Frataxin Protein Expression by Antisense Oligonucleotides Targeting the Mutant Expanded Repeat" Nucleic Acid Ther.2018Feb;28 (1) 23-33, each of which is incorporated herein in its entirety.
In some embodiments, the oligonucleotide payload is configured (e.g., as a spacer or RNAi oligonucleotide) for inhibiting expression of a native antisense transcript that inhibits FXN expression, e.g., as disclosed in: U.S. Pat. No.9,593,330, 2011, U.S. Pat. No. 6,9, "Treatment of Frataxin (FXN) related diseases by inhibition of natural antisense transcript to FXN," the contents of which are incorporated herein by reference in their entirety.
Some examples of oligonucleotides for facilitating editing of FXN gene include WO 2016/094845, published 6/16, 2016, "Compositions and methods for editing nucleic acids in cells utilizing oligonucleotides"; WO 2015/089354, 18 th month of 2015, "Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders"; WO 2015/139139, 24 th month of 2015, "CRISPR-based methods and products for increasing frataxin levels and uses thereof", and WO 2018/002783, 4 th month of 2018, "Materials and methods for treatment of Friedreich ataxia and other related disorders", the respective contents of which are incorporated herein in their entirety.
Examples of oligonucleotides for promoting FXN gene expression by targeting non-FXN genes (e.g., epigenetic modulators of FXN) include WO 2015/023938, published 19, 2015, 2, and "Epigenetic regulators of frataxin," the contents of which are incorporated herein in their entirety.
In some embodiments, the oligonucleotide may have a region complementary to a sequence as set forth below: FXN gene from human (Gene ID 2395; NC_000009.12) and/or FXN gene from mouse (Gene ID 14297; NC_000085.6). In some embodiments, the oligonucleotide may have a region complementary to a mutated form of FXN, e.g., as reported in: for example Monterini, L.et al. "The Friedreich ataxia GAA triplet repeat: premutation and normal moles." hum.molecular. Genet.,1997, 6:1261-1266; filla, A.et al, "The relationship between trinucleotide (GAA) repeat length and clinical features in Friedreich ataxia." am.J. hum.Genet.1996, 59:554-560; pandolfo, m.friedreich ataxia: the clinical picture j. Neurol 2009,256,3-8, the contents of each of which are incorporated herein by reference in their entirety.
DMD/dystrophy (Dystrophinopath)
Examples of oligonucleotides that can be used to target DMD are provided in the following: U.S. patent application publication US20100130591A1, published 5/27/2010, entitled "MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD"; U.S. Pat. No.8,361,979, entitled "MEANS AND METHOD FOR INDUCING EXON-SKIPPING" issued on 1 month 29 of 2013; U.S. patent application publication 20120059042, published 8/3 in 2012, entitled "METHOD FOR EFFICIENT EXON (44) SKIPPING IN DUCHENNE MUSCULAR DYSTROPHY AND ASSOCIATED MEANS; U.S. patent application publication 20140329881, which was published on month 11 and 6 of 2014, entitled "EXON SKIPPING COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY"; U.S. patent No.8,232,384, entitled "ANTISENSE OLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING AND METHODS OF USE THEREOF" to date 31, 7 in 2012; U.S. patent application publication 20120022134A1, published on 1/26 2012, entitled "METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA; U.S. patent application publication 20120077860, published in 2012, 3 and 29, entitled "ADENO-ASSOCIATED VIRAL VECTOR FOR EXON SKIPPING IN A GENE ENCODING ADISPENSABLE DOMAN PROTEIN"; U.S. patent No.8,324,371, entitled "oligos", granted on 4/12/2012; U.S. patent No.9,078,911, entitled "ANTISENSE OLIGONUCLEOTIDES" to date 14 at 7, 2015; U.S. patent No.9,079,934, entitled "ANTISENSE NUCLEIC ACIDS" to date 14 at 7, 2015; U.S. patent No.9,034,838, entitled "MIR-31IN DUCHENNE MUSCULAR DYSTROPHY THERAPY", issued 5.5 and 19 days 2015; and international patent publication WO2017062862A3, published on date 13, 4, 2017, entitled "OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF"; the respective content of which is incorporated herein in its entirety.
Examples of oligonucleotides for facilitating DMD gene editing include international patent publication WO2018053632A1, published on 29, 3, 2018, entitled "METHODS OF MODIFYING THE DYSTROPHIN GENE AND RESTORING DYSTROPHIN EXPRESSION AND USES THEREOF"; international patent publication WO2017049407A1, published 30/3/2017, entitled "MODIFICATION OF THE DYSTROPHIN GENE AND USES THEREOF"; international patent publication WO2016161380A1, published at 10/6 of 2016, entitled "CRISPR/CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING DUCHENNE MUSCULAR DYSTROPHY AND BECKER MUSCULAR DYSTROPHY"; international patent publication WO2017095967, published in 2017, month 6 and 8, entitled "THERAPEUTIC TARGETS FOR THE CORRECTION OF THE HUMAN DYSTROPHIN GENE BY GENE EDITING AND METHODS OF USE"; international patent publication WO2017072590A1, published on 5 months 4 of 2017, entitled "MATERIALS AND METHODS FOR TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY"; international patent publication WO2018098480A1, published on 5.31.2018, entitled "PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR/CPF1-MEDIATED GENE EDITING"; U.S. patent application publication US20170266320A1, published on month 9 and 21 of 2017, entitled "RNA-Guided Systems for In Vivo Gene Editing"; international patent publication WO2016025469A1, published in 2016, 2, 18, entitled "PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR/CAS9-MEDIATED GENE EDITING"; U.S. patent application publication 2016/0201089, published in 2016 at 7 and 14, entitled "RNA-GUIDED GENE EDITING AND GENE related"; and U.S. patent application publication 2013/0145487, which was published on 6/2013, entitled "MEGANUCLEASE VARIANTS CLEAVING A DNA TARGET SEQUENCE FROM THE DYSTROPHN GENE AND USES THEREOF", the respective content of which is incorporated herein in its entirety. In some embodiments, the oligonucleotide may have a region complementary to the DMD gene sequence of a plurality of species (e.g., selected from human, mouse, and non-human species).
In some embodiments, the oligonucleotide may have a region complementary to a mutant DMD allele, e.g., having at least one mutant DMD allele in any one of exons 1 to 79 of human DMD, which results in frame shifting and incorrect RNA splicing/processing.
MYH 7/hypertrophic cardiomyopathy
Examples of oligonucleotides that can be used as a payload (e.g., for targeting MYH 7) are provided in the following: U.S. patent application publication 20180094262, published on 5/4/2018, entitled Inhibitors of MYH7B and Uses Thereof; U.S. patent application publication 20160348103, which is published in 2016 at 12/1, entitled Oligonucleotides and Methods for Treatment of Cardiomyopathy Using RNA Interference; U.S. patent application publication 20160237430, which is published at 8.18 of 2016, entitled "Allole-specific RNA Silencing for the Treatment of Hypertrophic Cardiomyopathy"; U.S. patent application publication 20160032286, published in 2016, 2, 4, entitled "Inhibitors of MYH7Band Uses Thereof"; U.S. patent application publication 20140187603, published on month 7 and 3 of 2014, entitled "MicroRNA Inhibitors Comprising Locked Nucleotides"; U.S. patent application publication 20140179764, published on month 6 and 26 of 2014, entitled "Dual Targeting of miR-208and miR-499in the Treatment of Cardiac Disorders"; U.S. patent application publication 20120114744, published 5/10 in 2012, entitled "Compositions and Methods to Treat Muscular and Cardiovascular Disorders"; the respective content of which is incorporated herein in its entirety.
In some embodiments, the oligonucleotide may target lncRNA or mRNA, for example, for degradation. In some embodiments, the oligonucleotides may target (e.g., for degradation) a nucleic acid encoding a protein involved in the mismatch repair pathway (e.g., MSH2, mutLalpha, mutSbeta, mutLalpha). Proteins involved in the mismatch repair pathway, wherein mRNA encoding such proteins can be targeted by the oligonucleotides described herein, are described in the following: iyer, r.r.et al, "DNA triplet repeat expansion and mismatch repair" Annu Rev biochem.2015; 84:199-226; schmidt m.h. and Pearson c.e. "Disease-associated repeat instability and mismatch Repair" DNA Repair (Amst): 2016Feb;38:117-26.
a. Oligonucleotide size/sequence
Oligonucleotides may have a variety of different lengths, e.g., depending on format. In some embodiments, the oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, the oligonucleotide is 8 to 50 nucleotides in length, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, 21 to 23 nucleotides in length, and the like.
In some embodiments, when binding of a complementary nucleic acid sequence of an oligonucleotide to a target molecule (e.g., mRNA) interferes with normal function of the target (e.g., mRNA) resulting in lack of activity (e.g., inhibition of translation) or expression (e.g., degradation of the target mRNA), and has a sufficient degree of complementarity to avoid non-specific binding of the sequence to the non-target if, for purposes of the present disclosure, the complementary nucleic acid sequence of the oligonucleotide may specifically hybridize to or be specific for the target nucleic acid: under conditions in which it is desirable to avoid non-specific binding, for example in the case of in vivo assays or therapeutic treatments under physiological conditions, and in the case of in vitro assays, under conditions in which the assay is performed under suitably stringent conditions. Thus, in some embodiments, an oligonucleotide can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to consecutive nucleotides of a target nucleic acid. In some embodiments, the complementary nucleotide sequence need not be 100% complementary to the target nucleic acid to which it is targeted to specifically hybridize or be specific for the target nucleic acid.
In some embodiments, the oligonucleotide comprises a region complementary to the target nucleic acid, the region being 8 to 15, 8 to 30, 8 to 40 or 10 to 50, or 5 to 50 or 5 to 40 nucleotides in length. In some embodiments, the oligonucleotide is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 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, or 50 nucleotides in length to the complementary region of the target nucleic acid. In some embodiments, the complementary region is complementary to at least 8 consecutive nucleotides of the target nucleic acid. In some embodiments, an oligonucleotide may comprise 1, 2, or 3 base mismatches as compared to the contiguous nucleotide portion of the target nucleic acid. In some embodiments, the oligonucleotide may have up to 3 mismatches at 15 bases, or up to 2 mismatches at 10 bases.
In some embodiments, the oligonucleotide is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to a target sequence of any one of the oligonucleotides provided herein. In some embodiments, such target sequences are 100% complementary to the oligonucleotides described herein.
In some embodiments, it is understood that methylation of the nucleobase uracil at the C5 position forms thymine. Thus, in some embodiments, a nucleotide or nucleoside having a C5 methylated uracil (or 5-methyluracil) can be equivalently identified as a thymine nucleotide or nucleoside.
In some embodiments, any one or more thymine bases, T's, of any one of the oligonucleotides provided herein can be independently and optionally uracil bases, U's, and/or any one or more U's of the oligonucleotides provided herein can be independently and optionally T's.
b. Oligonucleotide modification:
the oligonucleotides described herein can be modified, e.g., comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide, and/or combinations thereof. In addition, in some embodiments, the oligonucleotides may exhibit one or more of the following properties: does not mediate alternative splicing; not immunostimulatory; resistance to nucleases; has improved cellular uptake compared to the unmodified oligonucleotide; is nontoxic to cells or mammals; internal excretion of endosomes in cells is improved; minimizing TLR stimulation; or avoid pattern recognition receptors. Any of the modified chemistries or formats of the oligonucleotides described herein may be combined with one another. For example, one, two, three, four, five or more different types of modifications may be included within the same oligonucleotide.
In some embodiments, certain nucleotide modifications may be used that render the oligonucleotides into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide or oligoribonucleotide molecule; these modified oligonucleotides survive longer than the unmodified oligonucleotides intact. Some specific examples of modified oligonucleotides include those containing modified backbones, such as modified internucleoside linkages, e.g., phosphorothioate linkages, phosphotriester linkages, methylphosphonate linkages, short chain alkyl linkages or cycloalkyl intersugar linkages or short chain heteroatom linkages or heterocyclic intersugar linkages. Thus, the oligonucleotides of the present disclosure may be stabilized against nucleolytic degradation, for example, by incorporating modifications (e.g., nucleotide modifications).
In some embodiments, the length of the oligonucleotide may be up to 50 or up to 100 nucleotides, wherein 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45 or more nucleotides of the oligonucleotide are modified nucleotides. The length of the oligonucleotide may be 8 to 30 nucleotides, wherein 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are modified nucleotides. The length of the oligonucleotide may be 8 to 15 nucleotides, wherein 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are modified nucleotides. Optionally, the oligonucleotide may have each nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modified nucleotides. Oligonucleotide modifications are further described herein.
c. Modified nucleotides
In some embodiments, the oligonucleotide comprises a 2' -modified nucleotide, such as 2' -deoxy, 2' -deoxy-2 ' -fluoro, 2' -O-methyl, 2' -O-methoxyethyl (2 ' -O-MOE), 2' -O-aminopropyl (2 ' -O-AP), 2' -O-dimethylaminoethyl (2 ' -O-DMAOE), 2' -O-dimethylaminopropyl (2 ' -O-DMAP), 2' -O-dimethylaminoethoxyethyl (2 ' -O-DMAEOE), or 2' -O-N-methylacetamido (2 ' -O-NMA).
In some embodiments, the oligonucleotide may comprise at least one 2 '-O-methyl-modified nucleotide, and in some embodiments, all nucleotides comprise a 2' -O-methyl modification. In some embodiments, the oligonucleotide comprises a modified nucleotide in which the ribose ring comprises a bridge portion connecting two atoms in the ring, e.g., a 2'-O atom to a 4' -C atom. In some embodiments, the oligonucleotide is "locked", e.g., comprises a modified nucleotide in which the ribose ring is "locked" by a methylene bridge connecting the 2'-O atom and the 4' -C atom. Some examples of LNAs are described in international patent application publication WO/2008/043753, published on month 4 and 17 of 2008, and titled "RNA Antagonist Compounds For The Modulation Of PCSK", the contents of which are incorporated herein by reference in their entirety.
Other modifications that can be used in the oligonucleotides disclosed herein include ethylene bridged nucleic acids (ENA). ENA includes, but is not limited to, 2'-O, 4' -C-ethylene bridged nucleic acids. Some examples of ENAs are provided in the following: international patent publication No. WO 2005/042777, published on month 5 and 12 of 2005, and entitled "APP/ENA anti-sense"; morita et al, nucleic Acid Res., suppl 1:241-242,2001; surono et al, hum. Gene Ther, 15:749-757,2004; koizumi, curr.Opin.mol.Ther.,8:144-149,2006 and Horie et al, nucleic Acids Symp.Ser (Oxf), 49:171-172,2005; the disclosure of which is incorporated herein by reference in its entirety.
In some embodiments, the oligonucleotide may comprise a bridged nucleotide, such as a locked nucleic acid (locked nucleic acid, LNA) nucleotide, a constrained ethyl (constrained ethyl, cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide. In some embodiments, the oligonucleotide comprises a modified nucleotide disclosed in one of the following U.S. patents or patent application publications: us patent 7,399,845, which was granted on month 7 and 15 of 2008, and titled "6-Modified Bicyclic Nucleic Acid Analogs"; us patent 7,741,457, which was granted on month 6 and 22 of 2010, and titled "6-Modified Bicyclic Nucleic Acid Analogs"; us patent 8,022,193, which was granted on day 20, 9, 2011, and entitled "6-Modified Bicyclic Nucleic Acid Analogs"; us patent 7,569,686, which was granted 8/4/2009, and entitled "Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs"; us patent 7,335,765, which was granted at 26/2/2008 and titled "Novel Nucleoside And Oligonucleotide Analogues"; us patent 7,314,923, which was granted on 1 st 2008, and titled "Novel Nucleoside And Oligonucleotide Analogues"; us patent 7,816,333, which was granted on month 10 and 19 of 2010, and titled "Oligonucleotide Analogues And Methods Utilizing The Same" and us publication 2011/0009471, is now us patent 8,957,201, which was granted on month 2 and 17 of 2015, and titled "Oligonucleotide Analogues And Methods Utilizing The Same", each of which is incorporated herein by reference in its entirety for all purposes.
In some embodiments, the oligonucleotide comprises at least one nucleotide modified at the 2 'position of the sugar, preferably a 2' -O-alkyl, 2 '-O-alkyl or 2' -fluoro modified nucleotide. In other preferred embodiments, the RNA modifications include 2 '-fluoro, 2' -amino, and 2 'o-methyl modifications on pyrimidine, abasic residues, or ribose of inverted bases at the 3' end of the RNA.
In some embodiments, the oligonucleotide may have at least one modified nucleotide that results in an increase in Tm of the oligonucleotide of 1 ℃, 2 ℃, 3 ℃, 4 ℃, or 5 ℃ compared to an oligonucleotide without the at least one modified nucleotide. The oligonucleotide may have a plurality of modified nucleotides that result in an overall increase in Tm of the oligonucleotide of 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ or more as compared to the oligonucleotide without the modified nucleotides.
The oligonucleotides may comprise different kinds of alternative nucleotides. For example, the oligonucleotide may comprise alternative deoxyribonucleotides or ribonucleotides and 2' -fluoro-deoxyribonucleotides. The oligonucleotides may comprise alternative deoxyribonucleotides or ribonucleotides and 2' -O-methyl nucleotides. The oligonucleotides may comprise substituted 2 '-fluoro nucleotides and 2' -O-methyl nucleotides. The oligonucleotide may comprise a substitute bridging nucleotide and a 2 '-fluoro or 2' -O-methyl nucleotide.
d. Internucleotide linkages/backbones
In some embodiments, the oligonucleotides may comprise phosphorothioate linkages or other modified internucleotide linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, the oligonucleotides comprise modified internucleotide linkages at the first, second and/or third internucleotide linkages of the 5 'or 3' end of the nucleotide sequence.
Phosphorus-containing linkages that may be used include, but are not limited to: phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methylphosphonates and other alkylphosphonates including 3 'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3' phosphoramidates and aminoalkyl phosphoramidates, phosphorothioate, thiocarbonylalkylphosphonates and borane phosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with inverted polarity, wherein pairs of adjacent nucleoside units link 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U.S. patent no.
In some embodiments, the oligonucleotide may have a heteroatom backbone, such as a methylene (methylimino) or MMI backbone; amide backbone (see De Mesmaeker et al ace. Chem. Res.1995, 28:366-374); morpholino backbone (see Summerton and Weller, U.S. Pat. No.5,034,506); or a Peptide Nucleic Acid (PNA) backbone (in which the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone, the nucleotide being directly or indirectly bound to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al, science 1991,254,1497).
e. Stereospecific oligonucleotides
In some embodiments, the internucleotide phosphorus atoms of the oligonucleotide are chiral, and the properties of the oligonucleotide are adjusted based on the configuration of the chiral phosphorus atoms. In some embodiments, the P-chiral oligonucleotide analogs can be synthesized in a stereocontrolled manner using appropriate methods (e.g., as described in Oka N, wada T, stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorus atoms. Chem Soc Rev. 201mdec; 40 (12): 5829-43). In some embodiments, phosphorothioate-containing oligonucleotides are provided that comprise nucleoside units linked together by substantially all Sp or substantially all Rp phosphorothioate sugar-to-sugar linkages. In some embodiments, such phosphorothioate oligonucleotides with substantially chiral pure intersaccharide linkages are prepared by enzymatic or chemical synthesis, as described, for example, in U.S. patent 5,587,261 issued 12/1996, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the chiral control oligonucleotide provides a selective cleavage pattern for a target nucleic acid. For example, in some embodiments, the chirally controlled oligonucleotides provide single site cleavage within the complementary sequence of the nucleic acid, as described, for example, in U.S. patent application publication 20170037399A1, published on month 2, 2017, entitled "CHIRAL DESIGN," the contents of which are incorporated herein by reference in their entirety.
f. Morpholino compounds
In some embodiments, the oligonucleotide may be a morpholino-based compound. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R.Corey, biochemistry,2002,41 (14), 4503-4510); genesis, volume 30, issue 3,2001; heasman, j., dev.biol.,2002,243,209-214; nasevicius et al, nat. Genet.,2000,26,216-220; lacerra et al, proc.Natl. Acad.Sci.,2000,97,9591-9596; and U.S. Pat. No.5,034,506 issued 7/23/1991. In some embodiments, the morpholino-based oligomeric compound is a diamide morpholino phosphate oligomer (PMO) (e.g., as described in Iverson, curr. Opin. Mol. Ther.,3:235-238,2001; and Wang et al, J. Gene Med.,12:354-364,2010; the disclosures of which are incorporated herein by reference in their entirety).
g. Peptide Nucleic Acid (PNA)
In some embodiments, both the sugar and internucleoside linkages (backbones) of the nucleotide units of the oligonucleotide are replaced with new groups. In some embodiments, the maintenance base unit is used to hybridize to an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is known as a Peptide Nucleic Acid (PNA). In PNA compounds, the sugar-backbone of the oligonucleotide is replaced with an amide-containing backbone (e.g., an aminoethylglycine backbone). The nucleobases are retained and bound directly or indirectly to the aza nitrogen atoms of the backbone amide moiety. Representative publications reporting the preparation of PNA compounds include, but are not limited to, U.S. Pat. nos. 5,539,082;5,714,331; and 5,719,262, each of which is incorporated herein by reference. Further teachings of PNA compounds can be found in Nielsen et al, science,1991,254,1497-1500.
h. Spacer polymers
In some embodiments, the oligonucleotide is a spacer. The spacer oligonucleotide generally has the formula 5'-X-Y-Z-3', wherein X and Z act as flanking regions around spacer Y. In some embodiments, the Y region is a contiguous extension of nucleotides, e.g., a region of at least 6 DNA nucleotides, that is capable of recruiting an rnase (e.g., rnase H). In some embodiments, spacer and target nucleic acid binding, at which point RNase recruits and can then cut the target nucleic acid. In some embodiments, both the 5 'and 3' regions of Y are flanked by X and Z regions comprising high affinity modified nucleotides, e.g., 1 to 6 modified nucleotides. Some examples of modified nucleotides include, but are not limited to, 2'moe or 2' ome or Locked Nucleobase (LNA). In some embodiments, flanking sequences X and Z may be 1 to 20 nucleotides, 1 to 8 nucleotides, or 1 to 5 nucleotides in length. Flanking sequences X and Z may have similar lengths or different lengths. In some embodiments, the spacer segment Y may be a nucleotide sequence of 5 to 20 nucleotides, with a length dimension of 12 nucleotides or 6 to 10 nucleotides.
In some embodiments, the spacer region of the spacer oligonucleotide may comprise, in addition to DNA nucleotides, modified nucleotides known to be acceptable for effective rnase H action, such as C4' -substituted nucleotides, acyclic nucleotides, and nucleotides of arabinose (arabino) configuration. In some embodiments, the spacer comprises one or more unmodified internucleosides. In some embodiments, one or both flanking regions each independently comprise one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five, or more nucleotides. In some embodiments, the spacer region and the two flanking regions each independently comprise a modified internucleoside linkage (e.g., phosphorothioate internucleoside linkage or other linkage) between at least two, at least three, at least four, at least five or more nucleotides.
Spacer polymers can be produced using suitable methods. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of spacer polymers include, but are not limited to, U.S. patent nos. 5,013,830;5,149,797;5,220,007;5,256,775;
5,366,878;5,403,711;5,491,133;5,565,350;5,623,065;5,652,355;5,652,356;5,700,922;5,898,031;7,432,250; and 7,683,036;
U.S. patent publication nos. US 200902286969, US20100197762 and US20110112170; and PCT publication nos. WO 2008049785 and WO2009090182, each of which is incorporated herein by reference in its entirety.
i. Mixed polymer
In some embodiments, the oligonucleotides described herein may be mixed-mer or comprise mixed-mer sequence patterns. In general, a mixed mer is an oligonucleotide comprising both natural and non-natural nucleotides or an oligonucleotide comprising two different types of non-natural nucleotides, typically in an alternative mode. The hybrid polymers generally have higher binding affinities than unmodified oligonucleotides and can be used to specifically bind to target molecules, e.g., to block binding sites on target molecules. Generally, the mixed multimer does not recruit RNase to the target molecule and thus does not promote cleavage of the target molecule. Such oligonucleotides that are incapable of recruiting RNase H have been described, for example, see WO2007/112754 or WO2007/112753.
In some embodiments, the hybrid polymer comprises, or consists of, a repeating pattern of nucleotide analogs and natural nucleotides, or one type of nucleotide analog and a second type of nucleotide analog. However, the hybrid polymer need not comprise a repeating pattern, and may alternatively comprise any arrangement of modified nucleotides and naturally occurring nucleotides, or any arrangement of one modified nucleotide and a second modified nucleotide. The repeat pattern may be, for example, every second or every third nucleotide is a modified nucleotide (e.g., LNA), and the remaining nucleotides are natural nucleotides (e.g., DNA) or 2' substituted nucleotide analogs, such as 2' moes or 2' fluoro analogs, or any other modified nucleotide described herein. It is recognized that a repetitive pattern of modified nucleotides, such as LNA units, may be combined with the modified nucleotides at fixed positions, such as at the 5 'or 3' ends.
In some embodiments, the hybrid polymer does not comprise more than 5, more than 4, more than 3, or more than 2 contiguous regions of natural nucleotides (e.g., DNA nucleotides). In some embodiments, the hybrid polymer comprises at least one region consisting of at least two consecutive modified nucleotides, e.g., at least two consecutive LNAs. In some embodiments, the hybrid polymer comprises at least one region consisting of at least three consecutive modified nucleotide units, e.g., at least three consecutive LNAs.
In some embodiments, the hybrid polymer does not comprise more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2 regions of contiguous nucleotide analogs, such as LNA. In some embodiments, the LNA units may be replaced with other nucleotide analogs, such as those mentioned herein.
The hybrid polymers can be designed to include a mixture of affinity-enhanced modified nucleotides (e.g., LNA nucleotides and 2' -O-methyl nucleotides in a non-limiting example). In some embodiments, the hybrid polymers comprise modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five, or more nucleotides.
Any suitable method may be used to produce the hybrid polymer. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of hybrid polymers include U.S. patent publication nos. US 20060184646, US20090209748, US20090298916, US20110077288, and US20120322851, and U.S. patent No.7687617.
In some embodiments, the hybrid polymer comprises one or more morpholino nucleotides. For example, in some embodiments, the mixed polymers can include morpholino nucleotides mixed (e.g., mixed in an alternating fashion) with one or more other nucleotides (e.g., DNA, RNA nucleotides) or modified nucleotides (e.g., LNA, 2' -O-methyl nucleotides).
In some embodiments, the hybrid polymers can be used for splice correction or exon skipping, e.g., as reported in: touznik a., et al, LNA/DNA mixer-based antisense oligonucleotides correct alternative splicing of the SMN2 gene and restore SMN protein expression in type 1SMA fibroblasts Scientific Reports,volume7,Article number:3672 (2017), chen s.et al, synthesis of a Morpholino Nucleic Acid (MNA) -Uridine Phosphoramidite, and Exon Skipping Using MNA/2' -O-Methyl Mixmer Antisense Oligonucleotide, molecules 2016,21,1582, each of which is incorporated herein by reference.
RNA interference (RNAi)
In some embodiments, the oligonucleotides provided herein may be in the form of small interfering RNAs (small interfering RNAs, sirnas, also referred to as short interfering RNAs or silencing RNAs). siRNA is a class of double stranded RNA molecules, typically about 20 to 25 base pairs in length, that target nucleic acids (e.g., mRNA) for degradation via an RNA interference (RNAi) pathway in a cell. The specificity of an siRNA molecule can be determined by the binding of the antisense strand of the molecule to its target RNA. Although longer siRNAs may also be effective, effective siRNA molecules are typically less than 30 to 35 base pairs in length to prevent triggering of non-specific RNA interference pathways in cells by an interferon response.
After selection of the appropriate target RNA sequence, siRNA molecules comprising nucleotide sequences (i.e., antisense sequences) that are complementary to all or part of the target sequence can be designed and prepared using appropriate methods (see, e.g., PCT publication No. WO 2004/016735; and U.S. patent publications Nos. 2004/007574 and 2008/0081791).
siRNA molecules can be double stranded (i.e., dsRNA molecules comprising an antisense strand and a complementary sense strand) or single stranded (i.e., ssRNA molecules comprising only an antisense strand). The siRNA molecule may comprise a duplex (duplex), asymmetric duplex, hairpin, or asymmetric hairpin secondary structure having a self-complementary sense strand and antisense strand.
Double stranded siRNA may comprise RNA strands of the same length or different lengths. Double stranded siRNA molecules can also be assembled from individual oligonucleotides into a stem-loop structure, wherein the self-complementary sense and antisense regions of the siRNA molecule are linked by: one or more nucleic acid-based or non-nucleic acid-based linkers, and a circular single stranded RNA having two or more loop structures and a stem comprising a self-complementary sense strand and an antisense strand, wherein the circular RNA can be processed in vivo or in vitro to produce an active siRNA molecule capable of mediating RNAi. Thus, small hairpin RNA (shRNA) molecules are also contemplated herein. These molecules contain specific antisense sequences in addition to the reverse complement (sense) sequences, which are typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides single stranded RNA molecules and their reverse complements such that they can be annealed to form dsRNA molecules (optionally with additional processing steps that can result in the addition or removal of one, two, three, or more nucleotides from the 3 'and/or 5' ends of either or both strands). The spacer may be of sufficient length to allow the antisense and sense sequences to anneal and form a duplex structure (or stem) prior to cleavage of the spacer (and optionally, subsequent processing steps that may result in the addition or removal of one, two, three, four or more nucleotides from the 3 'and/or 5' ends of either or both strands). The spacer sequence may be an unrelated nucleotide sequence located between two complementary nucleotide sequence regions that when annealed to a double stranded nucleic acid comprises shRNA.
The total length of the siRNA molecule can vary from about 14 to about 100 nucleotides depending on the type of siRNA molecule designed. Typically, about 14 to about 50 of these nucleotides are complementary to the RNA target sequence, i.e., constitute a specific antisense sequence of the siRNA molecule. For example, when the siRNA is a double-stranded siRNA or a single-stranded siRNA, the length may vary from about 14 to about 50 nucleotides, while when the siRNA is an shRNA or a cyclic molecule, the length may vary from about 40 nucleotides to about 100 nucleotides.
siRNA molecules may comprise a 3' overhang at one end of the molecule, the other end may be blunt-ended or also have an overhang (5 ' or 3 '). When the siRNA molecule comprises overhangs at both ends of the molecule, the length of the overhangs may be the same or different. In one embodiment, the siRNA molecules of the present disclosure comprise a 3' overhang of about 1 to about 3 nucleotides at both ends of the molecule.
k. Micro RNA (miRNA)
In some embodiments, the oligonucleotide may be a microrna (miRNA). Micrornas (referred to as "mirnas") are small non-coding RNAs that belong to a class of regulatory molecules that control gene expression by binding to complementary sites on target RNA transcripts. Generally, mirnas are produced from large RNA precursors, known as primary mirnas (pri-mirnas), which are processed in the nucleus to about 70 nucleotide precursor mirnas, which fold into imperfect stem-loop structures. These precursor mirnas are typically subjected to additional processing steps within the cytoplasm, where mature mirnas of 18 to 25 nucleotides in length are excised from one side of the precursor miRNA hairpin by the rnase III enzyme Dicer.
Mirnas as used herein include primary mirnas, precursor mirnas, mature mirnas, or fragments of variants thereof that retain the biological activity of the mature mirnas. In one embodiment, the miRNA may range in size from 21 nucleotides to 170 nucleotides. In one embodiment, the size of the miRNA is in the range of 70 to 170 nucleotides in length. In another embodiment, mature mirnas of 21 to 25 nucleotides in length may be used.
Aptamer
In some embodiments, the oligonucleotides provided herein may be in the form of an aptamer. In general, an aptamer is any nucleic acid that specifically binds to a target (e.g., small molecule in a cell, protein, nucleic acid) under molecular loading. In some embodiments, the aptamer is a DNA aptamer or an RNA aptamer. In some embodiments, the nucleic acid aptamer is single-stranded DNA or RNA (ssDNA or ssRNA). It is understood that single stranded nucleic acid aptamers may form helical and/or loop structures. The nucleic acid forming the nucleic acid aptamer may comprise natural nucleotides, modified nucleotides, natural nucleotides having a hydrocarbon linker (e.g., alkylene) or polyether linker (e.g., PEG linker) interposed between one or more nucleotides, modified nucleotides having a hydrocarbon or PEG linker interposed between one or more nucleotides, or a combination thereof. Exemplary publications and patents describing aptamers and methods of making aptamers include, for example, lorsch and Szostak,1996; jayasena,1999; U.S. Pat. nos. 5,270,163;5,567,588;5,650,275;5,670,637;5,683,867;5,696,249;5,789,157;5,843,653;5,864,026;5,989,823;6,569,630;8,318,438 and PCT application WO 99/31275, each of which is incorporated herein by reference.
m. ribozyme
In some embodiments, the oligonucleotides provided herein may be in the form of ribozymes. Ribozymes (ribonucleases) are molecules, typically RNA molecules, that are capable of performing a specific biochemical reaction, similar to the action of a protease. Ribozymes are molecules that have catalytic activity, including the ability to cleave at specific phosphodiester linkages in RNA molecules (e.g., mRNA, RNA-containing substrates, lncRNA, and ribozymes themselves) that hybridize thereto.
Ribozymes can take one of several physical structures, one of which is known as "hammerhead". Hammerhead ribozymes consist of a catalytic core comprising 9 conserved bases, a double-stranded stem and loop structure (stem-loop II), and two regions complementary to the catalytic core flanking regions of the target RNA. By forming double-stranded stems I and III, the flanking regions enable specific binding of the ribozyme to the target RNA. Cleavage occurs either in cis (i.e., cleavage of the same RNA molecule containing the hammerhead motif) or in trans (cleavage of RNA substrates other than those containing ribozymes) alongside a particular ribonucleotide triplet by transesterification of the 3',5' -phosphodiester to the 2',3' -cyclic phosphodiester. Without wishing to be bound by theory, it is believed that this catalytic activity requires the presence of specific, highly conserved sequences in the catalytic region of the ribozyme.
Modifications in the ribozyme structure also include substitution of non-nucleotide molecules for or replacement of multiple non-core portions of the molecule. For example, benseler et al (j.am.chem.soc. (1993) 115:8483-8484) discloses hammer-like molecules in which two base pairs of stem II and all four nucleotides of loop II are replaced with non-nucleoside linkers based on hexaethyleneglycol, propyleneglycol, bis (triethyleneglycol) phosphate, tris (propyleneglycol) diphosphate or bis (propyleneglycol) phosphate. Ma et al (biochem (1993) 32:1751-1758;Nucleic Acids Res (1993) 21:2585-2589) replaced the six nucleotide loop of the TAR ribozyme hairpin with a non-nucleotide ethylene glycol-related linker. Thomson et al (Nucleic Acids Res. (1993) 21:5600-5603) replaced loop II with linear non-nucleotide linkers of 13, 17 and 19 atoms in length.
Ribozyme oligonucleotides can be prepared using well-known methods (see, e.g., PCT publication WO9118624, WO9413688, WO9201806, and WO 92/07065; and U.S. Pat. Nos. 5436143 and 5650502), or can be purchased from commercial sources (e.g., US Biochemicals), and if desired, can incorporate nucleotide analogs to increase the resistance of the oligonucleotide to degradation by nucleases in cells. Ribozymes can be synthesized in any known manner, for example, by using commercially available synthesizers such as those produced by Applied Biosystems, inc. Or Milligen. Ribozymes can also be produced in recombinant vectors by conventional means. See Molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory (current edition). Ribozyme RNA sequences can be routinely synthesized, for example, by using RNA polymerase such as T7 or SP6.
n. guide nucleic acid (guide nucleic acid)
In some embodiments, an oligonucleotide is a guide nucleic acid, e.g., a guide RNA (gRNA) molecule. In general, the guide RNA is a short synthetic RNA consisting of: (1) A scaffold sequence that binds to a nucleic acid programmable DNA binding protein (napDNAbp) (e.g., cas 9), and (2) a nucleotide spacer portion that defines a DNA target sequence (e.g., a genomic DNA target) that binds to a gRNA to bring the nucleic acid programmable DNA binding protein into proximity to the DNA target sequence. In some embodiments, napDNAbp is a nucleic acid-programmable protein that forms a complex (e.g., binds or associates) with one or more RNAs that targets the nucleic acid-programmable protein to a target DNA sequence (e.g., a target genomic DNA sequence). In some embodiments, the nucleic acid programmable nuclease when complexed with RNA can be referred to as a nuclease: RNA complex. The guide RNA may be present as a complex of two or more RNAs, or as a single RNA molecule.
The guide RNAs (grnas) present as a single RNA molecule may be referred to as single-guide RNAs (sgrnas), although grnas are also used to refer to guide RNAs that are present as a single molecule or as a complex of two or more molecules. In general, a gRNA that exists as a single RNA species comprises two domains: (1) A domain sharing homology to the target nucleic acid (i.e., directing binding of Cas9 complex to the target); and (2) a domain that binds Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA, and comprises a stem-loop structure. In some embodiments, domain (2) is the same as or homologous to a tracrRNA as provided in jink et al, science 337:816-821 (2012) (the entire contents of which are incorporated herein by reference).
In some embodiments, the gRNA comprises two or more of domains (1) and (2), and may be referred to as amplification gRNA (extended gRNA). For example, as described herein, amplifying the gRNA will bind to two or more Cas9 proteins and bind to the target nucleic acid at two or more different regions. The gRNA comprises a nucleotide sequence complementary to a target site that mediates binding of the nuclease/RNA complex to the target site, providing sequence specificity of the nuclease/RNA complex. In some embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, such as Cas9 (Csn 1) from streptococcus pyogenes (Streptococcus pyogenes) (see, e.g.,
the entire contents of each of which are incorporated herein by reference.
Splice altering oligonucleotides
In some embodiments, an oligonucleotide of the disclosure (e.g., an antisense oligonucleotide comprising morpholino) targets splicing. In some embodiments, the oligonucleotide targets splicing by inducing exon skipping and restoring an in-frame in the gene. As non-limiting examples, the oligonucleotide may induce skipping of an exon encoding a frame shift mutation and/or an exon encoding a premature stop codon. In some embodiments, the oligonucleotide may induce exon skipping by blocking recognition of the splice site by the spliceosome. In some embodiments, exon skipping results in a truncated but functional protein (e.g., a truncated but functional DMD protein as described below) as compared to a reference protein. In some embodiments, the oligonucleotide facilitates inclusion of a particular exon (e.g., exon 7 of the SMN2 gene as described below). In some embodiments, the oligonucleotide may induce inclusion of an exon by targeting a splice site suppression sequence. RNA splicing is involved in muscle diseases including Duchenne Muscular Dystrophy (DMD) and spinal muscular atrophy (spinal muscular atrophy, SMA).
Changes in the gene encoding dystrophin (DMD) (e.g., deletions, point mutations, and duplications) cause DMD. These changes may result in frameshift mutations and/or nonsense mutations. In some embodiments, the oligonucleotides of the disclosure facilitate the skipping of one or more DMD exons (e.g., exon 8, exon 43, exon 44, exon 45, exon 50, exon 51, exon 52, exon 53, and/or exon 55) and produce a functional truncated protein. See, for example, U.S. patent nos. 8,486,907, published on 7, 16, 2013 and U.S.20140275212, published on 18, 9, 2014.
In SMA, there is a loss of functional SMN 1. Although the SMN2 gene is a paralog to SMN1, alternative splicing of the SMN2 gene mainly results in skipping of exon 7 and subsequent generation of a truncated SMN protein that cannot compensate for SMN1 loss. In some embodiments, the oligonucleotides of the disclosure facilitate inclusion of SMN2 exon 7. In some embodiments, the oligonucleotide is an antisense oligonucleotide targeting the SMN2 splice site inhibitory sequence (see, e.g., U.S. patent No. 7,838,657 published 11/23 2010).
p.multimers
In some embodiments, the molecular charge may comprise a multimer (e.g., a concatemer) of 2 or more oligonucleotides linked by a linker. In some embodiments, in this way, the oligonucleotide loading of the complex/conjugate can be increased beyond the available ligation sites on the targeting agent (e.g., available thiol sites on the antibody), or otherwise adjusted to achieve a particular loading capacity. The oligonucleotides in the multimer can be the same or different (e.g., targeting different genes or different loci on the same gene or products thereof).
In some embodiments, the multimer comprises 2 or more oligonucleotides linked together by a cleavable linker. However, in some embodiments, the multimer comprises 2 or more oligonucleotides linked together by a non-cleavable linker. In some embodiments, the multimer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more oligonucleotides linked together. In some embodiments, the multimer comprises 2 to 5, 2 to 10, or 4 to 20 oligonucleotides linked together.
In some embodiments, the multimer comprises 2 or more oligonucleotides that are end-to-end (in a linear arrangement). In some embodiments, a multimer comprises 2 or more oligonucleotides joined end-to-end by an oligonucleotide-based linker (e.g., a poly-dT linker, an abasic linker). In some embodiments, the multimer comprises a 5 'end of one oligonucleotide linked to a 3' end of another oligonucleotide. In some embodiments, the multimer comprises a 3 'end of one oligonucleotide linked to a 3' end of another oligonucleotide. In some embodiments, the multimer comprises a 5 'end of one oligonucleotide linked to a 5' end of another oligonucleotide. Nonetheless, in some embodiments, a multimer may comprise a branching structure comprising multiple oligonucleotides linked together by a branching linker.
Further examples of multimers that can be used in the complexes provided herein are disclosed in the following: for example, U.S. patent application No. 2015/0315588A1, titled Methods of delivering multiple targeting oligonucleotides to a cell using cleavable linkers, which is disclosed on month 11, 5 of 2015; U.S. patent application No. 2015/0247241 A1, titled Multimeric Oligonucleotide Compounds, which is published on month 9, 3 of 2015; U.S. patent application No. US 2011/0158937A1, titled Immunostimulatory Oligonucleotide Multimers, which was published in 2011 at month 6 and 30; and U.S. patent No. 5,693,773, entitled duplex-Forming Antisense Oligonucleotides Having Abasic Linkers Targeting Nucleic Acids Comprising Mixed Sequences Of Purines And Pyrimidines, entitled 12-month 2 1997, the respective contents of which are incorporated herein by reference in their entirety.
C. Joint
The complexes described herein generally comprise a linker that links any of the muscle targeting agents described herein (e.g., anti-TfR antibodies) to the molecular load. The linker comprises at least one covalent bond. In some embodiments, the linker may be a single bond, such as a disulfide bond or a disulfide bridge, that connects the muscle targeting agent (e.g., anti-TfR antibody) to the molecular load. However, in some embodiments, the linker can connect any of the muscle targeting agents described herein (e.g., anti-TfR antibodies) to the molecule through a plurality of covalent bonds. In some embodiments, the linker may be a cleavable linker. However, in some embodiments, the linker may be a non-cleavable linker. The linker is generally stable in vitro and in vivo, and may be stable in certain cellular environments. In addition, typically the linker does not negatively affect the functional properties of the anti-TfR antibody or molecular load. Some examples and methods of linker synthesis are known in the art (see, e.g., kline, t.et al. "Methods to Make Homogenous Antibody Drug conjugates." Pharmaceutical Research,2015,32:11,3480-3493.; jain, n.et al. "Current ADC Linker Chemistry" Pharm res.2015,32:11,3526-3540.; mcCombs, J.R.and Owen, s.c. "Antibody Drug Conjugates: design and Selection of Linker, payload and Conjugation Chemistry" AAPS j.2015,17:2, 339-351.).
The precursor of the linker will typically comprise two different reactive species that allow for attachment to both the anti-TfR antibody and the molecular load. In some embodiments, the two different reactive species may be a nucleophile and/or (e.g., and) an electrophile. In some embodiments, the linker is linked to the muscle targeting agent (e.g., anti-TfR antibody) by conjugation to a lysine residue or a cysteine residue of the anti-TfR antibody. In some embodiments, the linker is linked to the cysteine residue of the muscle targeting agent (e.g., an anti-TfR antibody) through a maleimide-containing linker, wherein optionally the maleimide-containing linker comprises a maleimide caproyl or maleimide methyl cyclohexane-1-carboxylate group. In some embodiments, the linker is attached to the cysteine residue or thiol-functionalized molecular load of the muscle targeting agent (e.g., anti-TfR antibody) through a 3-aryl propionitrile functional group. In some embodiments, the linker is attached to a lysine residue of a muscle targeting agent (e.g., an anti-TfR antibody). In some embodiments, the linker is linked to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) the molecular load via an amide bond, a urethane bond, a hydrazide, a triazole, a thioether, or a disulfide bond.
i. Cutting joint
The cleavable linker may be a protease-sensitive linker, a pH-sensitive linker or a glutathione-sensitive linker. These linkers are generally cleavable only intracellularly, and are preferably stable in the extracellular environment, e.g., the myocyte extracellular.
Protease-sensitive linkers can be cleaved by protease activity. These linkers typically comprise peptide sequences and may be 2 to 10 amino acids, about 2 to 5 amino acids, about 5 to 10 amino acids, about 5 amino acids, about 3 amino acids, or about 2 amino acids in length. In some embodiments, the peptide sequence may comprise a natural amino acid such as cysteine, alanine, or a non-naturally occurring or modified amino acid. Unnatural amino acids include beta-amino acids, homoamino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art. In some embodiments, the protease-sensitive linker comprises a valine-citrulline or an alanine-citrulline sequence. In some embodiments, the protease-sensitive linker can be cleaved by a lysosomal protease (e.g., cathepsin B (cathepsin B)) and/or (e.g., and) an endosomal protease.
pH sensitive linkers are covalent linkages that degrade readily in high or low pH environments. In some embodiments, the pH-sensitive linker may be cleaved at a pH of 4 to 6. In some embodiments, the pH-sensitive linker comprises a hydrazone or a cyclic acetal. In some embodiments, the pH sensitive linker is cleaved in endosomes or lysosomes.
In some embodiments, the glutathione-sensitive linker comprises a disulfide moiety. In some embodiments, the glutathione-sensitive linker is cleaved by disulfide exchange reaction with glutathione species within the cell. In some embodiments, the disulfide moiety further comprises at least one amino acid, such as a cysteine residue.
In some embodiments, the linker is a Val-cit linker (e.g., as described in U.S. Pat. No. 6,214,345, which is incorporated herein by reference). In some embodiments, prior to conjugation, the val-cit linker has the following structure:
in some embodiments, after conjugation, the val-cit linker has the following structure:
in some embodiments, the Val-cit linker is linked to a reactive chemical moiety (e.g., sparc for click chemistry conjugation). In some embodiments, prior to click chemistry conjugation, the val-cit linker attached to the reactive chemical moiety (e.g., sparc for click chemistry conjugation) has the following structure:
Wherein n is any number from 0 to 15. In some embodiments, n is 3.
In some embodiments, the val-cit linker (e.g., via a different chemical moiety) attached to a reactive chemical moiety (e.g., sparc for click chemistry conjugation) is conjugated to a molecular cargo (e.g., an oligonucleotide). In some embodiments, the val-cit linker linked to the reactive chemical moiety (e.g., sparc for click chemistry conjugation) and conjugated to the molecular cargo (e.g., oligonucleotide) has the structure (prior to click chemistry conjugation):
wherein n is any number from 0 to 15. In some embodiments, n is 3.
In some embodiments, following conjugation to a molecular cargo (e.g., an oligonucleotide), the val-cit linker comprises the following structure:
wherein n is any number from 0 to 15, and wherein m is any number from 0 to 15. In some embodiments, n is 3 and m is 4.
Non-cleavable linker
In some embodiments, non-cleavable linkers may be used. Generally, non-cleavable linkers are not readily degraded in a cellular or physiological environment. In some embodiments, the non-cleavable linker comprises an optionally substituted alkyl group, wherein the substitution may include halogen, hydroxy, oxygen species, and other common substitutions. In some embodiments, the linker can comprise an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, a heteroarylene, a peptide sequence comprising at least one unnatural amino acid, a truncated glycan, one or more saccharides that are not enzymatically degradable, an azide, an alkyne-azide, a peptide sequence comprising an LPXT sequence, a thioether, biotin, a biphenyl, a repeat unit of polyethylene glycol or an equivalent compound, an acid ester, an amide, a sulfonamide, and/or (e.g., and) an alkoxy-amine linker. In some embodiments, sortase-mediated ligation will be used to covalently ligate a muscle targeting agent comprising an LPXT sequence (e.g., an anti-TfR antibody) to a polypeptide comprising (G) n Molecular loading of sequences (see, e.g., lift T.sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilisation. Biotechnol Lett.2010,32 (1): 1-10.).
In some embodiments, the linker may comprise a substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O and S; an optionally substituted heterocyclylene group further comprising at least one heteroatom selected from N, O and S; imino, optionally substituted nitrogen species, optionally substituted oxygen species O, optionally substituted sulfur species or poly (alkylene oxide), such as polyethylene oxide or polypropylene oxide.
Linker conjugation
In some embodiments, the linker is linked to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) the molecular load via a phosphate, thioether, ether, carbon-carbon bond, carbamate, or amide bond. In some embodiments, the linker is attached to the oligonucleotide by a phosphate or phosphorothioate group, such as a terminal phosphate of the oligonucleotide backbone. In some embodiments, the linker is linked to the muscle targeting agent (e.g., anti-TfR antibody) through a lysine or cysteine residue present on the muscle targeting agent (e.g., anti-TfR antibody).
In some embodiments, the linker is linked to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) the molecular charge via a cycloaddition reaction between the azide and alkyne to form a triazole, wherein the azide and alkyne can be located on the muscle targeting agent (e.g., anti-TfR antibody), the molecular charge, or the linker. In some embodiments, the alkyne can be a cycloalkyne, such as cyclooctyne. In some embodiments, the alkyne can be a bicyclononene (also known as a bicyclo [6.1.0] nonyne or BCN) or a substituted bicyclononene. In some embodiments, cyclooctane is as described in international patent application publication WO2011136645, published 11/3/2011 under the heading "Fused Cyclooctyne Compounds And Their Use In Metal-free Click Reactions". Both exo-and endo-forms of BCN can be used for ligation conjugation, and BCN in formulas (C), (D), (E), (F), (G) and (H) provided herein can be endo-or exo-BCN.
In some embodiments, the azide may be a sugar or carbohydrate molecule comprising an azide. In some embodiments, the azide may be 6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In some embodiments, the azide-containing sugar or carbohydrate molecule is as described in International patent application publication WO2016170186, which is published 10/27 in 2016 under the heading "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A beta (1, 4) -N-acetylgalactosaminyl transferase". In some embodiments, a cycloaddition reaction is performed between an azide and an alkyne to form a triazole, where the azide and alkyne can be located on a muscle targeting agent (e.g., an anti-TfR antibody), molecular load, or linker, as disclosed in international patent application publication WO2014065661, 5-month 1 of 2014, entitled "Modified antibody, anti-conjugate and process for the preparation thereof"; or International patent application publication WO2016170186, published at 10/27/2016, entitled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β (1, 4) -N-acetylgalactosaminyl transferase".
In some embodiments, the linker further comprises a spacer, such as a polyethylene glycol spacer or an acyl/carbamoyl sulfonamide spacer, such as hydro space TM A spacer. In some embodiments, the spacer is as described in Verkade, J.M.M.et al., "A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, stability, and Therapeutic Index of Antibody-Drug connections", antibodies,2018,7,12.
In some embodiments, the linker is linked to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) the molecular load by a Diels-Alder reaction (Diels-Alder reaction) between the dienophile and the diene/heterodiene, wherein the dienophile and the diene/heterodiene can be located on the muscle targeting agent (e.g., anti-TfR antibody), the molecular load, or the linker. In some embodiments, the linker is linked to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) molecular cargo by other pericyclic reactions (pericyclic reaction), such as an olefinic reaction. In some embodiments, the linker is linked to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) the molecular load by an amide, thioamide, or sulfonamide linkage reaction. In some embodiments, the linker is linked to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) molecular load by a condensation reaction to form an oxime, hydrazone, or semicarbazide group that is present between the linker and the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) molecular load.
In some embodiments, the linker is attached to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) the molecular cargo by a conjugate addition reaction between a nucleophile (e.g., amine or hydroxyl) and an electrophile (e.g., carboxylic acid, carbonate, or aldehyde). In some embodiments, a nucleophile may be present on the linker and an electrophile may be present on the muscle targeting agent (e.g., anti-TfR antibody) or molecular load prior to performing a reaction between the linker and the muscle targeting agent (e.g., anti-TfR antibody) or molecular load. In some embodiments, before the reaction between the linker and the muscle targeting agent (e.g., anti-TfR antibody) or molecular load is performed, an electrophile may be present on the linker and a nucleophile may be present on the muscle targeting agent (e.g., anti-TfR antibody) or molecular load. In some embodiments, the electrophile can be an azide, a pentafluorophenyl, a p-nitrophenyl ester, a silicon center, a carbonyl, a carboxylic acid, an anhydride, an isocyanate, a thioisocyanate, a succinimidyl ester, a sulfosuccinimidyl ester, a maleimide, an alkyl halide, an alkyl pseudohalide, an epoxide, an episulfide, an aziridine, an aryl group, an activated phosphorus center, and/or (e.g., and) an activated sulfur center. In some embodiments, the nucleophile may be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxy, an amino, an alkylamino, an anilino, or a thiol group.
In some embodiments, the val-cit linker attached to a reactive chemical moiety (e.g., sparc for click chemistry conjugation) is conjugated to an anti-TfR antibody by the following structure:
wherein m is any number from 0 to 15. In some embodiments, m is 4.
In some embodiments, the val-cit linker attached to a reactive chemical moiety (e.g., sparc for click chemistry conjugation) is conjugated to an anti-TfR antibody having the structure:
wherein m is any number from 0 to 15. In some embodiments, m is 4.
In some embodiments, the val-cit linker attached to a reactive chemical moiety (e.g., sparc for click chemistry conjugation) and conjugated to an anti-TfR antibody has the following structure:
wherein n is any number from 0 to 15, wherein m is any number from 0 to 15. In some embodiments, n is 3 and/or (e.g., and) m is 4. In some embodiments, the oligonucleotide is covalently linked to a compound comprising the structure of formula (H), thereby forming a complex comprising the structure of formula (E). It is understood that the amide shown adjacent to the anti-TfR 1 antibody in formula (H) is generated from reaction with an amine of the anti-TfR 1 antibody (e.g., lysine epsilon amine).
In some embodiments, the val-cit linker for covalently linking the anti-TfR antibody and the molecular load (e.g., oligonucleotide) comprises the following structure:
wherein n is any number from 0 to 15, wherein m is any number from 0 to 15. In some embodiments, n is 3 and/or (e.g., and) m is 4. In some embodiments, n is 3 and/or (e.g., and) m is 4. In some embodiments, X is NH (e.g., NH from an amine group of lysine), S (e.g., S from a thiol group of cysteine), or O (e.g., O from a hydroxyl group of serine, threonine, or tyrosine) of the antibody. In some embodiments, the method of treating a complex comprises the step of producing a complex.
In some embodiments, a method of producing a complex comprises:
(i) An oligonucleotide comprising the following structure was obtained:
wherein n is 0 to 15 (e.g., 3);
(ii) Obtaining an antibody comprising the structure:
wherein m is 0 to 15 (e.g., 4); and
(iii) Reacting the oligonucleotide of step (i) with the antibody obtained in step (ii) to obtain a complex.
In some embodiments, the method comprises:
(i) An oligonucleotide comprising the following structure was obtained:
wherein n is 0 to 15 (e.g., 3), and wherein m is 0 to 15 (e.g., 4);
(ii) Obtaining an antibody; and
(iii) Reacting the oligonucleotide of step (i) with the antibody obtained in step (ii) to obtain a complex.
In some embodiments, the complex produced using the methods described herein comprises the following structure:
wherein n is 0 to 15 (e.g., 3), and wherein m is 0 to 15 (e.g., 4), and wherein the antibody is covalently linked by lysine.
In some embodiments, the complexes described herein have the following structure:
wherein n is any number from 0 to 10, wherein m is any number from 0 to 10. In some embodiments, n is 3 and m is 4.
In structural formula (I), in some embodiments, L1 is a spacer that is a substituted or unsubstituted aliphatic, a substituted or unsubstituted heteroaliphatic, a substituted or unsubstituted carbocyclic subunit, a substituted or unsubstituted heterocyclic subunit, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene, -O-, -N (R A )-，-S，-C(＝O)-，-C(＝O)O-，-C(＝O)NR A -，-NR A C(＝O)-，-NR A C(＝O)R A -，-C(＝O)R A -，-NR A C(＝O)O-，-NR A C(＝O)N(R A )-，-OC(＝O)-，-OC(＝O)O-，-OC(＝O)N(R A )-，-S(O) 2 NR A -，-NR A S(O) 2 -, or a combination thereof, wherein each R A Independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, L1 is
Wherein L2 is
Wherein a marks the site of direct linkage to the carbamate moiety of formula (I); and b marks the site of covalent attachment (either directly or through another chemical moiety) to the oligonucleotide.
In some embodiments, L1 is:
wherein a marks the site of direct linkage to the carbamate moiety of formula (I); and b marks the site of covalent attachment (either directly or through another chemical moiety) to the oligonucleotide.
In some embodiments, L1 is
In some embodiments, L1 is attached to the 5' phosphate of the oligonucleotide.
In some embodiments, L1 is optional (e.g., need not be present).
It is understood that the amides shown adjacent to the anti-TfR 1 antibody in formula (E), formula (F) and formula (I) result from reaction with the amine of the anti-TfR 1 antibody (e.g., lysine epsilon amine).
D. Some examples of antibody-molecule loading complexes
Also provided herein are some non-limiting examples of complexes comprising any of the muscle targeting agents described herein (e.g., anti-TfR antibodies) covalently linked to any of the molecular payloads (e.g., oligonucleotides) described herein. In some embodiments, an anti-TfR antibody (e.g., any one of the muscle targeting agents provided in table 2 (e.g., an anti-TfR antibody)) is covalently linked to the molecular load (e.g., an oligonucleotide) through a linker. Any of the linkers described herein may be used. In some embodiments, if the molecular charge is an oligonucleotide, the linker is covalently attached to the 5 'end, 3' end, or interior of the oligonucleotide. In some embodiments, the linker is covalently linked to the anti-TfR antibody through a thiol-reactive linkage (e.g., through a cysteine in the anti-TfR antibody). In some embodiments, the linker (e.g., val-cit linker) is covalently attached to the antibody (e.g., anti-TfR antibody described herein) through an n-amine group (e.g., through lysine in the antibody). In some embodiments, the molecular cargo is an oligonucleotide (e.g., an oligonucleotide targeting the atrophy genes (atropy genes) listed in table 1).
One example of a structure of a complex comprising an anti-TfR antibody covalently linked to a molecular load through a Val-cit linker is provided below:
wherein the linker is covalently linked to the antibody by a thiol-reactive linkage (e.g., through a cysteine in the antibody). In some embodiments, the molecular cargo is an oligonucleotide (e.g., an oligonucleotide targeting the atrophy genes listed in table 1).
Another example of a structure of a complex comprising an anti-TfR antibody covalently linked to a molecular load through a Val-cit linker is provided below:
wherein n is a number from 0 to 15, wherein m is a number from 0 to 15, wherein the linker is covalently attached to the antibody via an amine group (e.g., on a lysine residue), and/or (e.g., and) wherein the linker is covalently attached to the oligonucleotide (e.g., at the 5 'end, the 3' end, or internally). In some embodiments, the linker is covalently linked to the antibody via lysine, the linker is covalently linked to the oligonucleotide at the 5' end, n is 3 and m is 4. In some embodiments, the molecular cargo is an oligonucleotide (e.g., an oligonucleotide targeting the atrophy genes listed in table 1).
It is understood that antibodies can be covalently linked to oligonucleotides having different stoichiometries, a property which can be referred to as drug-to-antibody ratio (DAR), where "drug" is an oligonucleotide. In some embodiments, one oligonucleotide is covalently linked to one antibody (dar=1). In some embodiments, two oligonucleotides are covalently linked to one antibody (dar=2). In some embodiments, three oligonucleotides are covalently linked to one antibody (dar=3). In some embodiments, four oligonucleotides are covalently linked to one antibody (dar=4). In some embodiments, a mixture of different complexes is provided, each complex having a different DAR. In some embodiments, the average DAR for the complexes in such mixtures may be in the range of 1 to 3, 1 to 4, 1 to 5, or more. DAR can be increased by conjugating oligonucleotides to different sites on an antibody and/or by conjugating multimers to one or more sites on an antibody. For example, DAR of 2 can be achieved by conjugating a single oligonucleotide to two different sites on an antibody or by conjugating a dimeric oligonucleotide to a single site on an antibody.
In some embodiments, a complex described herein comprises a transferrin receptor antibody (e.g., an antibody as described herein or any variant thereof) covalently linked to an oligonucleotide. In some embodiments, a complex described herein comprises a transferrin receptor antibody (e.g., an antibody as described herein or any variant thereof) covalently linked to an oligonucleotide through a linker (e.g., val-cit linker). In some embodiments, a linker (e.g., val-cit linker) is covalently attached to the 5 'end, 3' end, or interior of the oligonucleotide. In some embodiments, a linker (e.g., val-cit linker) is covalently linked to an antibody (e.g., an antibody as described herein or any variant thereof) through a thiol-reactive linkage (e.g., through a cysteine in the antibody).
In some embodiments, the complexes described herein comprise a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises the same CDR-H1, CDR-H2, and CDR-H3 as the CDR-H1, CDR-H2, and CDR-H3 shown in table 3; and CDR-L1, CDR-L2 and CDR-L3 identical to CDR-L1, CDR-L2 and CDR-L3 shown in Table 3.
In some embodiments, a complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID No. 33 and a VL having the amino acid sequence of SEQ ID No. 34. In some embodiments, a complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID No. 35 and a VL having the amino acid sequence of SEQ ID No. 36.
In some embodiments, the complexes described herein comprise a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 39 and a light chain having the amino acid sequence of SEQ ID No. 40. In some embodiments, the complexes described herein comprise a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO. 41 and a light chain having the amino acid sequence of SEQ ID NO. 42.
In some embodiments, the complexes described herein comprise a transferrin receptor antibody covalently linked to an oligonucleotide through a linker (e.g., val-cit linker), wherein the transferrin receptor antibody comprises CDR-H1, CDR-H2, and CDR-H3 identical to CDR-H1, CDR-H2, and CDR-H3 shown in table 3; and CDR-L1, CDR-L2 and CDR-L3 identical to CDR-L1, CDR-L2 and CDR-L3 shown in Table 3.
In some embodiments, the complexes described herein comprise a transferrin receptor antibody covalently linked to an oligonucleotide through a linker (e.g., val-cit linker), wherein the transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO:33 and a VL having the amino acid sequence of SEQ ID NO: 34. In some embodiments, the complexes described herein comprise a transferrin receptor antibody covalently linked to an oligonucleotide through a linker (e.g., val-cit linker), wherein the transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO:35 and a VL having the amino acid sequence of SEQ ID NO: 36.
In some embodiments, the complexes described herein comprise a transferrin receptor antibody covalently linked to an oligonucleotide through a linker (e.g., val-cit linker), wherein the transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO:39 and a light chain having the amino acid sequence of SEQ ID NO: 40. In some embodiments, the complexes described herein comprise a transferrin receptor antibody covalently linked to an oligonucleotide through a linker (e.g., val-cit linker), wherein the transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 41 and a light chain having the amino acid sequence of SEQ ID No. 42.
In some embodiments, the complexes described herein comprise a transferrin receptor antibody covalently linked to an oligonucleotide through a Val-cit linker, wherein the transferrin receptor antibody comprises the same CDR-H1, CDR-H2, and CDR-H3 as those shown in table 3; and CDR-L1, CDR-L2 and CDR-L3 identical to CDR-L1, CDR-L2 and CDR-L3 shown in Table 3, and wherein said complex comprises the following structure:
wherein the linker Val-cit linker is covalently attached to the 5 'end, the 3' end, or the interior of the oligonucleotide, and wherein the Val-cit linker is covalently attached to the antibody (e.g., an antibody described herein or any variant thereof) through a thiol-reactive linkage (e.g., through a cysteine in the antibody).
In some embodiments, a complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide through a Val-cit linker, wherein the transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO:33 and a VL having the amino acid sequence of SEQ ID NO:34, and wherein the complex comprises the structure:
wherein the linker Val-cit linker is covalently attached to the 5 'end, the 3' end, or the interior of the oligonucleotide, and wherein the Val-cit linker is covalently attached to the antibody (e.g., an antibody described herein or any variant thereof) through a thiol-reactive linkage (e.g., through a cysteine in the antibody).
In some embodiments, a complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide through a Val-cit linker, wherein the transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO:35 and a VL having the amino acid sequence of SEQ ID NO:36, and wherein the complex comprises the structure:
wherein the linker Val-cit linker is covalently attached to the 5 'end, the 3' end, or the interior of the oligonucleotide, and wherein the Val-cit linker is covalently attached to an antibody (e.g., an antibody as described herein or any variant thereof) through a thiol-reactive linkage (e.g., through a cysteine in the antibody).
In some embodiments, a complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide through a Val-cit linker, wherein the transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO:39 and a light chain having the amino acid sequence of SEQ ID NO:40, and wherein the complex comprises the structure:
wherein the linker Val-cit linker is covalently attached to the 5 'end, the 3' end, or the interior of the oligonucleotide, and wherein the Val-cit linker is covalently attached to an antibody (e.g., an antibody as described herein or any variant thereof) through a thiol-reactive linkage (e.g., through a cysteine in the antibody).
In some embodiments, a complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide through a Val-cit linker, wherein the transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO:41 and a light chain having the amino acid sequence of SEQ ID NO:42, and wherein the complex comprises the structure:
wherein the linker Val-cit linker is covalently attached to the 5 'end, the 3' end, or the interior of the oligonucleotide, and wherein the Val-cit linker is covalently attached to an antibody (e.g., an antibody as described herein or any variant thereof) through a thiol-reactive linkage (e.g., through a cysteine in the antibody).
In some embodiments, the complexes described herein comprise a transferrin receptor antibody covalently linked to the 5' end of an oligonucleotide through lysine, wherein the transferrin receptor antibody comprises the same CDR-H1, CDR-H2, and CDR-H3 as set forth in table 3; and CDR-L1, CDR-L2 and CDR-L3 identical to CDR-L1, CDR-L2 and CDR-L3 shown in Table 3, and wherein said complex comprises the following structure:
wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is an oligonucleotide targeting the atrophy genes listed in table 1.
In some embodiments, a complex described herein comprises a transferrin receptor antibody covalently linked to the 5' end of an oligonucleotide through lysine, wherein the transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID No. 33 and a VL having the amino acid sequence of SEQ ID No. 34, and wherein the complex comprises the structure:
wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is an oligonucleotide targeting the atrophy genes listed in table 1. In some embodiments, a complex described herein comprises a transferrin receptor antibody covalently linked to the 5' end of an oligonucleotide via lysine, wherein the transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO:35 and a VL having the amino acid sequence of SEQ ID NO:36, and wherein the complex comprises the structure:
Wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is an oligonucleotide targeting the atrophy genes listed in table 1.
In some embodiments, a complex described herein comprises a transferrin receptor antibody covalently linked to the 5' end of an oligonucleotide through lysine, wherein the transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO:39 and a light chain having the amino acid sequence of SEQ ID NO:40, and wherein the complex comprises the structure:
wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is an oligonucleotide targeting the atrophy genes listed in table 1.
In some embodiments, a complex described herein comprises a transferrin receptor antibody covalently linked to the 5' end of an oligonucleotide through lysine, wherein the transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID No. 41 and a light chain having the amino acid sequence of SEQ ID No. 42, and wherein the complex comprises the structure:
wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is an oligonucleotide targeting the atrophy genes listed in table 1.
It will be appreciated that in some examples of complexes having formula (E), the amide displayed adjacent to the anti-TfR 1 antibody is generated from reaction with an amine (e.g., lysine epsilon amine) of the anti-TfR 1 antibody.
III. preparation
The complexes provided herein may be formulated in any suitable manner. In general, the complexes provided herein are formulated in a manner suitable for pharmaceutical use. For example, the complex may be delivered to a subject using a formulation that minimizes degradation, facilitates delivery and/or uptake, or provides another beneficial property to the complex in the formulation. In some embodiments, provided herein are compositions comprising a complex and a pharmaceutically acceptable carrier. Such compositions may be suitably formulated so that when administered to a subject, either in the immediate environment of administration to the target cells or systemically, a sufficient amount of the complex is able to enter the target muscle cells. In some embodiments, the complex is formulated in a buffer solution such as phosphate buffered saline solution, liposomes, micelle structures, and capsids.
It is to be understood that in some embodiments, the compositions may each comprise one or more components of the complexes provided herein (e.g., muscle targeting agents, linkers, molecular loads, or precursor molecules of any of them).
In some embodiments, the complex is formulated in water or an aqueous solution (e.g., water adjusted with pH). In some embodiments, the complex is formulated in an alkaline buffered aqueous solution (e.g., PBS). In some embodiments, the formulations disclosed herein comprise an excipient. In some embodiments, the excipient imparts improved stability, improved absorption, improved solubility, and/or therapeutic enhancement of the active ingredient to the composition. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, tris base, or sodium hydroxide) or a carrier (e.g., buffer solution, petrolatum (petrolatum), dimethyl sulfoxide, or mineral oil).
In some embodiments, the complex or a component thereof (e.g., an oligonucleotide or antibody) is lyophilized for extended shelf life and then made into a solution prior to use (e.g., administration to a subject). Thus, the excipient in a composition comprising a complex or component thereof described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a disintegration temperature regulator (e.g., dextran, ficoll, or gelatin).
In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Some examples of routes of administration include parenteral administration, e.g., intravenous, intradermal, subcutaneous administration. Typically, the route of administration is intravenous or subcutaneous. In some embodiments, the route of administration is parenteral.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when water-soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier may be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. In some embodiments, the formulation in the composition comprises isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride. Sterile injectable solutions may be prepared by incorporating the required amount of the compound in the selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
In some embodiments, the composition may comprise at least about 0.1% of the complex or component thereof, or more, although the percentage of one or more active ingredients may be from about 1% to about 80% or more by weight or volume of the total composition. Those skilled in the art will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations in preparing such pharmaceutical formulations, and thus a variety of dosages and therapeutic regimens may be desirable.
Methods of use/treatment
Complexes comprising a muscle targeting agent covalently linked to a molecular charge as described herein are effective in treating a muscle disorder (e.g., rare muscle disorder). In some embodiments, the complex is effective in treating a muscle disorder provided in table 1. In some embodiments, the muscle disorder is associated with a disease allele, e.g., a disease allele of a particular muscle disorder may comprise a genetic alteration of a corresponding gene listed in table 1.
In some embodiments, the subject may be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject. In some embodiments, the subject may have a muscle disorder provided in table 1.
One aspect of the present disclosure includes methods involving administering an effective amount of a complex described herein to a subject. In some embodiments, an effective amount of a pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently linked to a molecular payload may be administered to a subject in need of treatment. In some embodiments, the pharmaceutical composition comprising a complex as described herein may be administered by a suitable route, which may include intravenous administration, for example as a bolus or by continuous infusion over a period of time. In some embodiments, intravenous administration may be by intramuscular, intraperitoneal, intracerebroventricular, subcutaneous, intra-articular, intrasynovial, or intrathecal routes. In some embodiments, the pharmaceutical composition may be in solid form, aqueous form, or liquid form. In some embodiments, the aqueous or liquid form may be atomized or lyophilized. In some embodiments, the atomized or lyophilized form can be reconstituted with an aqueous or liquid solution.
Compositions for intravenous administration may comprise a variety of carriers, such as vegetable oils, dimethyl lactamide, dimethyl formamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycols, and the like). For intravenous injection, the water-soluble antibody may be administered by an instillation method by which a pharmaceutical formulation comprising the antibody and physiologically acceptable excipients is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, ringer's solution, or other suitable excipients. An intramuscular formulation, e.g. a sterile formulation in the form of a suitable soluble salt of an antibody, may be dissolved in a pharmaceutically acceptable excipient, e.g. water for injection, 0.9% saline or 5% dextrose solution, and administered.
In some embodiments, the pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently loaded with a molecule is administered by site-specific or local delivery techniques. Some examples of these techniques include implantable reservoir sources of the complex, local delivery catheters, site-specific carriers, direct injection, or direct application.
In some embodiments, a pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently linked to a molecular cargo is administered at an effective concentration to confer therapeutic effect to a subject. As recognized by those of skill in the art, the effective amount will vary depending on the severity of the disease, the unique characteristics of the subject being treated (e.g., age, physical condition, health or weight), the duration of the treatment, the nature of any concurrent treatment, the route of administration, and related factors. These relevant factors are known to those skilled in the art and can be solved by only routine experimentation. In some embodiments, the effective concentration is the maximum dose considered safe for the patient. In some embodiments, the effective concentration will be the lowest possible concentration that provides the greatest efficacy.
Empirical considerations (e.g., the half-life of the complex in the subject) will generally help determine the concentration of the pharmaceutical composition used for treatment. The frequency of administration can be determined and adjusted empirically to maximize therapeutic efficacy.
Generally, for administration of any of the complexes described herein, the initial candidate dose may be about 1 to 100mg/kg or higher, depending on factors such as safety or efficacy. In some embodiments, the treatment will be administered once. In some embodiments, the treatment will be administered daily, every two weeks, weekly, every two months, monthly, or at any time interval that minimizes the safety risk to the subject while providing maximum efficacy. Generally, efficacy and treatment as well as safety risks can be monitored throughout the course of treatment.
The efficacy of the treatment may be assessed using any suitable method. In some embodiments, the efficacy of a treatment may be assessed by evaluating observations of symptoms associated with muscle diseases.
In some embodiments, a pharmaceutical composition comprising a complex described herein comprising a muscle targeting agent covalently to a molecular load is administered to a subject at an effective concentration sufficient to inhibit at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity or expression of a target gene relative to a control (e.g., baseline level of gene expression prior to treatment).
In some embodiments, the pharmaceutical composition may comprise more than one complex comprising a muscle targeting agent covalently linked to a molecular charge. In some embodiments, the pharmaceutical composition may further comprise any other suitable therapeutic agent for treating a subject (e.g., a human subject suffering from a muscle disorder (e.g., a muscle disorder provided in table 1)). In some embodiments, other therapeutic agents may enhance or supplement the efficacy of the complexes described herein. In some embodiments, other therapeutic agents may function to treat symptoms or diseases other than the complexes described herein.
Examples
Example 1 Synthesis of complexes comprising antibodies linked to oligonucleotides (conjugation method 1-pre-reaction conjugation)
A muscle targeting complex was generated comprising an antisense oligonucleotide targeting DMPK covalently linked to an anti-transferrin receptor hIgG1- κfab antibody (anti-TfR Fab) via a cathepsin cleavable linker.
Using 1mL Captureselect TM CH1-XL column (Thermo Fisher, loughborough, UK) anti-TfR Fab was purified from CHO cell culture supernatant. The column was washed with 1 XPBS and then the protein was eluted using 50mM sodium acetate pH 4.0. The protein buffer was then exchanged into 20mM sodium citrate, 150mM NaCl,pH 6.0. HiLoad is then used by preparative SEC TM 16/60Superdex TM The Fab was further purified using a 75pg column (GE Healthcare, little Chalfont, UK) with 20mM sodium citrate, 150mM NaCl,pH 6.0 as the mobile phase. Peak fractions containing monomeric protein were pooled, concentrated and filter sterilized, and then quantified according to a280nm using extinction coefficients (Ec (0.1%)) based on predicted amino acid sequences. Then analyzed by reduced and non-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE), analytical size exclusion chromatography-high performance liquid chromatography (size-exclusion chromatography-high performance liquid chromatography, SEC-HPLC) and Biacore Single Cycle Kinetics (SCK)Purified Fab. anti-TfR Fab buffer was swapped into 50mM HEPES pH 7.5 before continuing the conjugation reaction.
A linker/cargo compound is produced comprising an oligonucleotide (e.g., a charged oligonucleotide) and an azide-valine-citrulline linker. The oligonucleotide (Na+ adduct) was dissolved at 200mg/mL in RNAse-free water. The solution was diluted to 10mg/mL with anhydrous Dimethylformamide (DMF). A 25-fold molar excess of tributylamine was then added to the solution. The linker molecule (azide-PEG 3-Val-Cit-PAB-PNP, dissolved in DMF at 20 mg/mL) was added to the oligonucleotide solution at room temperature (about 25 ℃) in a 2-fold molar excess for 120 minutes. Ninhydrin (Kaiser test) was used to measure the reaction completion and then the reaction was quenched using alcohol precipitation. Alcohol precipitation was accomplished by the addition of 0.1v/v 3M NaCl solution followed by the addition of 3 volumes of isopropanol at-80 ℃. The solution was then thoroughly mixed and then allowed to precipitate at-20 ℃ for 1 hour. The precipitated solution was centrifuged (at 4300 Xg; 8 ℃) for 30 minutes and the solvent was decanted. The precipitate was washed with 80% ethanol (corresponding to the volume of the initial reaction) at-80℃in RNase-free water and centrifuged (at 4300 Xg; 8 ℃) for 20 minutes. The ethanol was then decanted and the precipitate (containing the compound comprising the oligonucleotide and azide-valine-citrulline linker) was dried at 37 ℃ for 10 minutes. The linker/cargo compound comprising the oligonucleotide and azide-valine-citrulline linker was resuspended in 20% acetonitrile in nuclease-free water at a concentration of 20mg/mL.
The pre-reaction is then carried out under the following conditions: 5.87. Mu.M oligonucleotide-PAB-VC-PEG 3-azide (structure shown in FIG. 1A) and 5.34mM endo-BCN-PEG4-PFP ester (1.1:1.0 mol: molar equivalent; structure shown in FIG. 1B) were combined in 60:40v/v% DMA with 25mM 2- (N-morpholino) ethanesulfonic acid (MES) pH 5.5 buffer at room temperature.
Completion of the pre-reaction to give oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester (structure shown in FIG. 1C) was monitored by repeated injections of RP-C18UPLC every 30 minutes (at 5, 35 and 65 minute time points) by observing the disappearance of endo-BCN-PEG4-PFP ester starting material at 220 nm. The reaction was determined to be complete when less than 5% endo-BCN-PEG4-PFP ester remained relative to the 5 minute time point. The crude pre-reaction mixture was immediately subjected to conjugation reaction without any purification. The total pre-reaction time was 90 minutes, defined as the time between the addition of endo-BCN-PEG4-PFP ester to the pre-reaction and the addition of the reaction mixture to the Fab conjugation reaction.
Conjugation of an oligonucleotide (e.g., a charged oligonucleotide) to an anti-TfR Fab involves the formation of an amide bond between the solvent accessible lysine residue of the Fab and the activated PFP ester of the oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester produced in the pre-reaction step.
Conjugation reactions were performed with the following solution reactant parameters: anti-TfR Fab (45 mg,6mg/mL, 125. Mu.M) and oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester at a final concentration of 750. Mu.M (1.0:6.0 mol: mol equivalent of anti-TfR Fab and oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester). The concentration of oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester was assumed to be 100% conversion of BCN to oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester in the pre-reaction. The final reaction mixture consisted of 15:85v/v% DMA with 25mM HEPES pH 7.5 buffer. The reaction was established in a 20mL glass scintillation vial by adding appropriate amounts of reactants and stock solutions. The reaction was carried out at room temperature (about 25 ℃) for 20 hours. The start of the reaction is defined as the time to add the pre-reaction mixture comprising the oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester to the anti-TfR Fab solution. The stop time is defined as the beginning of the pH adjustment prior to subsequent purification. The total duration of the conjugation reaction was 20 hours.
After 20 hours of reaction, the crude conjugate mixture was tested by SDS-PAGE and analyzed by densitometry to determine the drug-to-antibody ratio (DAR) and% unconjugated Fab.
Example 2: purification of complexes comprising muscle targeting antibodies linked to oligonucleotides
The crude reaction product from example 1 was successfully purified using chromatography. The mixture of anti-TfR Fab-oligonucleotide conjugate, unconjugated anti-TfR Fab and unconjugated oligonucleotide was diluted 1:3 in nuclease free water and mixed with 500mM MES bufferThe pH of the compound was adjusted to 5.7. The solution was then loaded onto a ceramic Hydroxyapatite (HA) column (HiLoad-26 mm. Times.40 cm column, CHT) at a biomolecular concentration of 8mg/mL resin TM 40 μm resin; from Biorad, catalog # 732-4324) [ Loading flow: linear 113 cm/hr, 26mm ID-10.0 mL/min volumetric flow, residence time-21.4 min ]]. With 5CV of the washing solution (5 mM Na 2 HPO 4 25mM NaCl pH 7.0) to remove unbound oligonucleotides. After removal of the unbound oligonucleotides, the buffer (100 mM Na 2 HPO 4 100mM NaCl,pH 7.6) the complexes comprising anti-TfR Fab covalently linked to an oligonucleotide are eluted from the HA column.
The isolated and purified anti-TfR Fab-oligonucleotide conjugates were analyzed by SDS-PAGE and analytical SEC to demonstrate complete removal of the unligated oligonucleotides, and human TfR1/cyno TfR1 binding and endotoxin levels were analyzed by ELISA. SEC chromatograms showed that the eluted fraction from the HA resin provided a substantially purified complex comprising anti-TfR Fab covalently linked to an oligonucleotide. In addition, the HA flow-through (e.g., wash fraction) comprises unligated oligonucleotides. These results indicate that the complex can be purified from the unligated oligonucleotide using a hydroxyapatite resin, which is an unexpected purification result that would not otherwise be achievable.
Several alternative strategies for purifying crude mixtures from example 1 comprising complexes, unligated oligonucleotides and unligated anti-TfR Fab containing anti-TfR Fab covalently attached to oligonucleotides were examined, including Cation Exchange (CEX) and Anion Exchange (AEX) resins. None of the alternative strategies were found to be as effective as the method described herein (using ceramic hydroxyapatite resins).
Example 3: effect of BCN to Fab ratio on Fab-oligonucleotide conjugation reactions
To investigate the effect of BCN to anti-TfR ratio from the pre-reaction mixture, a set of reactions was performed according to the general protocol described in example 1.
The pre-reaction between the oligonucleotide-PAB-VC-PEG 3-azide and endo-BCN-PEG4-PFP ester was performed using the following final reaction conditions: 2.43mM oligonucleotide-PAB-VC-PEG 3-azide was reacted with 1.62mM endo-BCN-PEG4-PFP ester (1.5:1 mol: mol ratio) in a 1:1 mixture of DMA and 25mM MES pH 5.5 buffer. The total pre-reaction volume was 0.17mL and the pre-reaction step was allowed to proceed for 4 hours at room temperature.
After the pre-reaction was completed, the crude (i.e., unpurified) pre-reaction mixture containing the oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester was used to establish a series of anti-TfR Fab conjugation reactions. Separate anti-TfR Fab conjugation reactions were performed using 1.0, 1.3, 1.6, 1.9, 2.2 and 2.5 molar equivalents of BCN from the pre-reaction product mixture relative to Fab. All other reaction conditions remained unchanged in the different conjugates. In the conjugation reaction mixture, the Fab concentration was 6mg/mL with 15v/v% DMA in 20mM HEPES pH 7.5 buffer and 1mg total Fab per reaction. The reaction was carried out at room temperature for 20 hours. The final average DAR and% unconjugated Fab (D0) for each conjugate were determined by SDS-PAGE densitometry and the results are shown in table 4. SDS-PAGE gels are shown in FIG. 2.
Table 4.
| Rxn | D0 | Average DAR |
| A | 0.322 | 0.761 |
| B | 0.246 | 0.883 |
| C | 0.156 | 1.055 |
| D | 0.092 | 1.218 |
| E | 0.056 | 1.330 |
| F | 0.053 | 1.431 |
Example 4: effect of BCN to Fab ratio and reaction conditions on Fab-oligonucleotide conjugation reactions
To investigate the effect of BCN to anti-TfR ratio from the pre-reaction mixture, as well as the effect of oligonucleotide and Fab concentrations in the oligonucleotide-Fab conjugation reaction, a set of reactions was performed according to the general protocol described in example 1.
The pre-reaction between the oligonucleotide-PAB-VC-PEG 3-azide and endo-BCN-PEG4-PFP ester was performed using the following reaction conditions: 6.15mM oligonucleotide-PAB-VC-PEG 3-azide and 6.15mM endo-BCN-PEG4-PFP ester (1:1 mol: mol ratio) were reacted in a mixture of DMA and 25mM MES pH 5.5 buffer (60:40 DMA: MES) to a total reaction volume of 0.11mL. The pre-reaction was allowed to proceed for 110 minutes at room temperature.
After the pre-reaction was completed, the crude (i.e., unpurified) pre-reaction mixture containing the oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester was used to establish a series of anti-TfR Fab conjugation reactions. Separate anti-TfR Fab conjugation reactions were performed using 2, 3, 4, 5, 6, 8 and 10 molar equivalents of BCN from the pre-reaction product mixture relative to Fab. The Fab concentration in each reaction was 10mg/mL, with 0.65mg total Fab in each reaction. Except for the DMA content, all other reaction conditions for conjugation remain unchanged in the different conjugation reactions. Reactions using 2, 3, 4, 5 and 6 molar equivalents of BCN were performed in 15v/v% DMA in 50mM HEPES pH 7.5 buffer at 23 to 25 ℃ for 20 hours. Reactions using 8 and 10 molar equivalents of BCN were performed in 20v/v% DMA in 50mM HEPES pH 7.5 buffer at 23 to 25 ℃ for 19 hours. The final average DAR for each conjugate was determined by SDS-PAGE densitometry, the results of which are shown in table 5. SDS-PAGE gels are shown in FIG. 3.
Table 5.
| Rxn | Average DAR |
| G | 1.31 |
| H | 1.63 |
| I | 1.84 |
| J | 1.99 |
| K | 2.38 |
| L | 3.30 |
| M | 3.75 |
Example 5: fab-oligonucleotide conjugation and purification
The pre-reaction between the oligonucleotide-PAB-VC-PEG 3-azide and endo-BCN-PEG4-PFP ester was performed using the following reaction conditions: 2.61mM oligonucleotide-PAB-VC-PEG 3-azide was reacted with 1.74mM endo-BCN-PEG4-PFP ester (1.5:1.0 mol: mol equivalent) in a 1:1 v/v% mixture of DMA and 25mM MES pH 5.5 buffer, with a total reaction volume of 0.44mL. The pre-reaction was allowed to proceed at room temperature for 4.75 hours. The completion of the pre-reaction was monitored by measuring the decrease in peak area of endo-BCN-PEG4-PFP ester starting material by RP C18 UPLC over time (fig. 4).
After the pre-reaction was completed, the crude (i.e., unpurified) pre-reaction mixture containing the oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester was used to establish a series of anti-TfR Fab conjugation reactions. anti-TfR Fab conjugation was performed using 2.2:1 mol:mol equivalent of oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester from the pre-reaction product mixture relative to Fab. The Fab concentration in the conjugation reaction was 6mg/mL and the reaction was performed on a scale of 10mg total Fab. The final reaction mixture consisted of 15v/v% DMA in 20mM HEPES pH 7.5 buffer.
After 19 hours of reaction at room temperature, the crude conjugate product mixture was purified by chromatography to remove free oligonucleotide material. First, after loading onto a 1mL CHT type I hydroxyapatite column, the conjugate was washed with 5 Column Volumes (CV) of 100% buffer a (10 mM sodium phosphate and 10mM sodium chloride, pH 6.5). A second wash was then performed with a 95:5 mixture of 10CV buffer A and buffer B (100 mM sodium phosphate and 100mM sodium chloride, pH 7.5) at a flow rate of 1 mL/min. Finally, a third wash was performed with a 90:10 mixture of 10CV buffer A and buffer B at a flow rate of 1 ml/min. After the washing step, the conjugate was eluted with 100% buffer B at a flow rate of 1 ml/min. This procedure gave 7.4mg (74% yield) of anti-TfR Fab-oligonucleotide conjugate with an average DAR of 1.30. SEC chromatograms and SDS-PAGE gels of the purified anti-TfR Fab-oligonucleotide conjugates are shown in fig. 5 and 6, respectively.
Example 6: synthesis of complexes comprising antibodies linked to oligonucleotides (conjugation method 2-two-step conjugation)
A muscle targeting complex was generated comprising an antisense oligonucleotide covalently linked to an anti-transferrin receptor hIgG 1-kappa antibody (anti-TfR antibody) through a cathepsin cleavable linker.
anti-TfR antibodies were stably expressed in CHO-K1SP cells (Genscript) in 15L fed-batch culture. The supernatant was purified via mabselect sure protein a affinity resin, washed with 20mM phosphate and eluted with 50mM sodium citrate at ph=3.5. The eluate was neutralized with 1.0M NaOH and further refined by Size Exclusion Chromatography (SEC) to DPBS ph=7.4 and concentrated to 10.0mg/mL by Tangential Flow Filtration (TFF).
The purified antibody was site-specifically cleaved (at the sequence of CPAPELLG-GPSVF (SEQ ID NO: 45)) to F (ab') 2 fragments using FragIt solid phase supported IdeS enzyme (1.0 gAb/10mL resin, 120 min at 125rpm in shake flasks at room temperature). The FC domain and any uncleaved IgG were removed on a HiLoad column (26 mm. Times.40 cm, [ loading flow: linear 113 cm/hr, 26mm ID-10.0 mL/min volumetric flow, residence time-21.2 min ]) using CaptureSelect FcXL (thermo scientific) with a binding capacity of 25 mg/mL. The flow-through containing purified F (ab') 2 was collected and verified for any breakthrough by SDS-PAGE (4% to 12% nupage, mes ph=7.0, 160v,40 min) and analytical HPLC-SEC (Zorbax GF-250,9.4mm x 250mm, pbs ph=7.2, 25 ℃). The F (ab ') 2 fragment was reduced to Fab' with 80 molar excess of cysteamine-HCl (ChemImpex#02839) per hinge thiol at 37℃for 90 minutes. Fab 'was then immediately purified by protein L chromatography (GE #17547855, 10 x series) using standard pH gradients (50mM Na3C6H5O7,pH =2.6, gradient from 60% to 100% over 20 column volumes) away from unreduced F (ab') 2 and free cysteamine. anti-TfR Fab may also be recombinantly produced.
anti-TfR Fab was diluted 1:10v/v with acetonitrile (HPLC grade) and combined with 5 molar excess of endo-BCN-PEG3-PFP ((endo) bicyclonine-PEG 3-pentafluorophenyl) (formula (C)); dissolved in DMSO at 20 mg/mL) at room temperature (about 22.5 ℃ C.) for 2 hours. After the reaction, the anti-TfR Fab BCN solution was subjected to sterile filtration or depth filtration to remove precipitated BCN. The average reactive BCN fraction of the filtered solution was then determined using LCMS and azide-a 488 (20 molar excess) for 90 minutes. The Fab incorporating BCN (> 1.3 moles BCN/mole Fab) was purified with 10kDa-TFF (1.2 bar, sartorius VivaFlow 200) at 5 filtrate volumes to remove free BCN. Complete removal of free BCN was verified by analytical HPLC-SEC (Tosoh, TSKgel SuperSW mAb HR,7.8mm×300 mm). Recovery of BCN modified anti-TfR Fab >90% of starting material. The purified Fab incorporating BCN was subjected to the next step.
A linker/cargo compound is produced that comprises an oligonucleotide (e.g., a charged oligonucleotide) and an azide-valine-citrulline linker. The oligonucleotide (Na+ adduct) was dissolved at 200mg/mL in RNAse-free water. The solution was diluted to 10mg/mL with anhydrous Dimethylformamide (DMF). A 25-fold molar excess of tributylamine was then added to the solution. The linker molecule (azide-PEG 3-Val-Cit-PAB-PNP (formula (A)) was added to the oligonucleotide solution in a 2-fold molar excess at about 25℃for 120 minutes, dissolved in DMF at 20mg/mL. Ninhydrin (Kaiser test) was used to measure the reaction completion and then the reaction was quenched using alcohol precipitation. Alcohol precipitation was accomplished by the addition of 0.1v/v 3M NaCl solution followed by the addition of 3 volumes of isopropanol at-80 ℃. The solution was then thoroughly mixed and then allowed to precipitate at-20 ℃ for 1 hour. The precipitated solution was centrifuged (at 4300 Xg; 8 ℃) for 30 minutes and the solvent was decanted. The precipitate was washed with 80% ethanol (corresponding to the volume at which the reaction was started) at-80℃in RNase-free water and centrifuged (4300 Xg; 8 ℃) for 20 minutes. The ethanol was then decanted and the precipitate (containing the compound comprising the oligonucleotide and azide-valine-citrulline linker) was dried at 37 ℃ for 10 minutes. The linker/cargo compound comprising the oligonucleotide and azide-valine-citrulline linker was resuspended in 20% acetonitrile in nuclease-free water at a concentration of 20mg/mL.
The BCN modified anti-TfR Fab was reacted with 2.5 molar equivalents per BCN linker/loading compound comprising an oligonucleotide and an azido valine citrulline linker at room temperature (about 25 ℃) for 2 hours.
Completion of the coupling reaction was assessed by SDS-PAGE and analytical SEC, which showed a coupling efficiency of 78% by densitometry.
The conjugates produced by the conjugation method described in example 1 and the conjugates produced by the conjugation method described in example 6 were tested for biological activity. Both conjugates contained DMPK targeting oligonucleotide (ASO 1) and effectively knocked down DMPK mRNA levels in RD cells (fig. 7).
Example 7: synthesis of complexes comprising antibodies linked to charge neutral oligonucleotides (conjugation method 2-two-step conjugation)
A muscle targeting complex is generated comprising an oligonucleotide (e.g., a charge neutral oligonucleotide) covalently linked to an anti-transferrin (anti-TfR) receptor Fab antibody via a cathepsin cleavable linker. anti-TfR Fab can be recombinantly produced (e.g., in CHO cells) and purified. The oligonucleotides used were diamide Phosphate Morpholino Oligomers (PMO) of 30 nucleotides in length.
anti-TfR Fab was diluted with propylene glycol to a final concentration of 40% v/v propylene glycol and incubated with a 5-fold molar excess of endo-BCN-PEG3-PFP (endo-bicyclonyne-PEG 3-pentafluorophenyl, dissolved in DMSO at a concentration of 20 mg/mL) for 2 hours at room temperature (about 22.5 ℃). The label is expected to produce 2.0 to 2.5 moles of BCN per mole of Fab. After labelling, the reaction product is subjected to sterile filtration or depth filtration to remove precipitated BCN. The average reactive BCN fraction of the filtered solution was then determined using LCMS (ThermoFisher MAbPac RP um 2.1X100 mm, #088647; mobile phase A: 0.1% formic acid in 100% UPLC grade water; mobile phase B: 0.1% formic acid in 100% UPLC grade acetonitrile; flow rate 0.3 mL/min; column temperature 70 ℃, CID 20eV in source; positive polarity; spray voltage 3.5 kv; scan range 1000 to 3000 m/z).
Degree of marking (degree of labeling, DOL)>Anti TfR of 2.3 was used for the next step of conjugation and was purified using a 10kDa molecular weight cut-off (1.2 bar), 5 filtrate volumes, by tangential flow filtration into 10% isopropanol in PBS at pH 7.2 to remove free BCN and propylene glycol. By analytical HPLC-SEC (Waters)Xbridge protein BEH SEC 3.5um, 7.8X105 mM,0.3 mL/min, 100mM PO 4 100mM NaCl,15% v/v acetonitrile pH 7.0) to verify complete removal of BCN and propylene glycol. SEC traces for the crude and purified products are shown in fig. 8. Recovery of BCN-labeled anti-TfR>90% of starting material. The purified solution was concentrated to 3.5mg/mL for further conjugation steps.
In a separate reaction, an oligonucleotide (e.g., a charge neutral oligonucleotide) is conjugated to a linker molecule. The oligonucleotides were dissolved at 35mg/mL in anhydrous DMSO at 37 ℃. The linker molecule (azide-PEG 3-Val-Cit-PAB-PNP) was dissolved in anhydrous DMF at 40mg/mL and added to the oligonucleotide at a 2.7-fold molar excess with a 3-fold molar excess of N, N-Diisopropylethylamine (DIPEA). The linker conjugation reaction was allowed to proceed for 2 hours at room temperature (about 22.5 ℃). The progress and completion of the reaction was measured using ninhydrin assay (Kaiser test) and then quenched by acetone precipitation.
Precipitation was performed by adding 8 volumes of cold acetone to the product solution, and the precipitate was precipitated by centrifugation at 3500×g for 20 minutes at 8 ℃. The precipitate was then washed with 3 volumes of acetone to remove residual free linker and centrifuged again at 3500×g for 20 min at 8 ℃. The purified oligonucleotide-linker was then dissolved in 20% v/v acetonitrile in nuclease-free water at a concentration of 30 mg/mL. Concentration and yield were measured by Optical Density (OD) in 0.1N HCl, indicating a yield of greater than 90%. The crude linker/oligonucleotide conjugation reaction product (FIG. 9) and purified oligonucleotide-linker (FIG. 10) were subjected to analytical RP-HPLC (Waters BEH-C18,4.6 mm. Times.150 mm,0.5 mL/min, 5% to 90% v/v acetonitrile in water, 30 min run time) to confirm removal of free linker by precipitation and washing steps. Verification LCMS of purified oligonucleotide-linkers was also performed (fig. 11).
To conjugate anti-TfR and oligonucleotides, BCN-labeled antibodies were mixed with a 5-fold molar excess of oligonucleotide-PAB-VC-PEG 3-azide (fig. 1A) in a glass vial overnight at room temperature (about 22.5 ℃).
Completion of the reaction was assessed by SDS-PAGE (fig. 12) and analytical SEC analysis (fig. 13), which showed less than 10% of unconjugated anti-TfR antibody (DAR 0) and 90% conjugation efficiency by densitometry.
EXAMPLE 8 conjugation methods for preparing Fab-oligonucleotide (charge neutral oligonucleotide) conjugates
(conjugation method 1-Pre-reaction conjugation)
This example describes the preparation of conjugates composed of oligonucleotides covalently linked to anti-transferrin receptor (anti-TfR) Fab antibodies via val-cit cathepsin cleavable peptide linkers. anti-TfR Fab can be recombinantly produced (e.g., in CHO cells) and purified. The oligonucleotides used were diamide Phosphate Morpholino Oligomers (PMO) of 30 nucleotides in length. Copper-free 3+2 click reaction between the azide group of the oligonucleotide-PAB-VC-PEG 3-azide molecule (fig. 1A) and the strained bicyclononene moiety on the endo-BCN-PEG4-PFP ester (fig. 1B) heterobifunctional crosslinker produced an intermediate comprising the oligonucleotide and linker ("pre-reaction") prior to conjugation to Fab.
Lyophilized oligonucleotide-PAB-VC-PEG 3-azide (98.1 mg) was dissolved in 0.32mL MilliQ water in a 4mL glass Wheaton vial. After dissolution, 0.32mL of N, N-Dimethylacetamide (DMA) was added and the mixture was gently stirred for 5 to 10 minutes. Before proceeding, the vials were carefully checked to ensure that the oligonucleotide-PAB-VC-PEG 3-azide was completely dissolved and no residue remained on the walls of the glass vials. 25-fold, 50-fold and 100-fold aliquots were diluted in water using a 1:1 DMA:1:containing final 0.1M HCl using a Nanodrop UV/vis instrument at 265nm using 318,050M -1 cm -1 To determine the concentration of the stock solution of oligonucleotide-PAB-VC-PEG 3-azide in 1:1DMA: water. HCl is added to ensure the accuracy of the concentration measurement. The calculated concentrations for each dilution were averaged to determine the solution concentration of 10.1 mM.
A32.5 mg/mL (53.5 mM) stock solution of endo-BCN-PEG4-PFP ester was prepared by weighing about 25mg of endo-BCN-PEG4-PFP ester oil into a 4mL glass Wheaton vial. An appropriate volume of DMA was then added to give a stock solution of 32.5 mg/mL.
The pre-reaction is carried out under the following final solution reaction conditions: 5.87. Mu.M (6.5. Mu. Mol) oligonucleotide-PAB-VC-PEG 3-azide, 5.34mM endo-BCN-PEG4-PFP ester (1.1:1.0 mol: mol eq.) in 60:40v/v% DMA with 25mM 2- (N-morpholino) ethanesulfonic acid (MES) pH 5.5 buffer at room temperature. The reaction was performed in a 4mL glass Wheaton vial by adding appropriate amounts of reactants and stock solutions, as shown in table 6. The final total volume of the pre-reaction was 1.11mL.
Table 6.
The completion of the pre-reaction to give the oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester (FIG. 1C) was monitored by repeated injections of RP-C18UPLC every 30 minutes (at 5, 35 and 65 minute time points) by observing the disappearance of endo-BCN-PEG4-PFP ester starting material at 220 nm. The IPC indicated that the reaction was complete at 65 minutes (fig. 14). When less than 5% endo-BCN-PEG4-PFP ester remained relative to the 5 minute time point, the reaction was determined to be complete (table 7). The crude pre-reaction mixture was immediately subjected to conjugation reaction without any purification. The total pre-reaction time was 90 minutes, defined as the time between the addition of endo-BCN-PEG4-PFP ester to the pre-reaction and the addition of the reaction mixture to the Fab conjugation reaction.
TABLE 7 quantification of endo-BCN-PEG4-PFP ester starting materials based on RP-C18 UPLC measurements.
| Time (minutes) | % endo-BCN-PEG4-PFP ester remaining |
| 5 | 100 |
| 35 | 13.6 |
| 65 | 2.3 |
Conjugation of the oligonucleotide to the anti-TfR Fab involved the formation of an amide bond between the solvent accessible lysine residue of the Fab and the activated PFP ester of the oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester produced in the pre-reaction step (fig. 1C).
anti-TfR Fab formulated in 20mM sodium citrate, 100mM sodium chloride was buffer exchanged into 50mM HEPES pH 7.5 prior to setting up the conjugation reaction. anti-TfR Fab (10 mL,10.15 mg/mL) was loaded onto a 50mM HEPES pH 7.5 equilibrated NAP-25 desalting column (4 x 2.5 mL) and eluted with 50mM HEPES pH 7.5 (4 x 3.5 mL). The eluates were combined and concentrated using an Amicon Ultra-15 10kDa centrifugal filtration unit at 4000rcf spin to reduce the volume to 2.86mL. The concentration of anti-TfR Fab obtained in the buffer was 31.75mg/mL by Nanodrop UV/vis measurement.
The conjugation reaction was performed with the following final solution reactant amounts: anti-TfR Fab (45 mg,6mg/mL, 125. Mu.M) and oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester at a final theoretical concentration of 750. Mu.M (6.0:1.0 mol: mol equivalents of oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester vs anti-TfR Fab). The concentration of oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester was assumed to be 100% conversion of BCN to oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester in the pre-reaction. The final reaction mixture consisted of 15:85v/v% DMA with 25mM HEPES pH 7.5 buffer. As shown in table 8, the reaction was established in a 20mL glass scintillation vial by adding appropriate amounts of reactants and stock solutions. The reaction was carried out at room temperature (about 25 ℃) for 20 hours. The start of the reaction is defined as the time to add the pre-reaction mixture comprising the oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester to the anti-TfR Fab solution. The total duration of the conjugation reaction was 20 hours.
Table 8.
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After 20 hours of reaction, the crude conjugate mixture was tested by SDS-PAGE and analyzed by densitometry to determine drug-to-antibody ratio (DAR) and% unconjugated Fab. The results are shown in FIG. 15 and Table 9.
Table 9.
| Substance (B) | Abundance ratio |
| D0 | 0.090 |
| D1 | 0.290 |
| D2 | 0.333 |
| D3 | 0.200 |
| D4 | 0.061 |
| D5 | 0.020 |
| D6 | 0.006 |
| Average DAR | 1.93 |
EXAMPLE 9 purification of anti-TfR Fab-oligonucleotide conjugates
After the synthesis of the anti-TfR Fab-oligonucleotide conjugates described in example 8, a two-part purification procedure was performed. First, the free load was removed by Hydroxyapatite (HA) chromatography. The HA eluate is then buffer exchanged into the final formulation. The final buffer exchange was performed with a 30kDa centrifugation filter unit at 45mg scale of Fab. The crude reaction product from example 8 (anti-TfR Fab-oligonucleotide conjugate) was diluted and the pH was adjusted from 7.5 to 5.7 prior to loading onto the HA column. First, 7.5mL of the crude conjugate was diluted by adding 16.5mL of 15v/v% DMA in water, and the solution was thoroughly mixed. To this mixture was added 0.75mL of 500mM MES (pH 3.3) to adjust the pH down to 5.7.
Chromatographic purification was performed on an AKTA pure chromatography system using a 5mL Bio Rad CHT type I (ceramic hydroxyapatite) cartridge to remove unreacted oligonucleotide material. Prior to loading the diluted conjugate pool from the reaction mixture preparation step, 15:85v/v% DMA with 10mM sodium phosphate pH 5.8 buffer, CHT cassettes were prepared and equilibrated according to manufacturer's instructions. After equilibration, the conjugate pool was loaded at a flow rate of 5 mL/min. After loading the conjugate, 15 in 10mM sodium phosphate buffer (pH 5.8): 85v/v% DMA washed the column a minimum of 7CV. After the washing was completed, the washing was performed with a washing composition comprising 15: elution was initiated by a step gradient at a flow rate of 5 mL/min in 85v/v% DMA 100mM sodium phosphate pH 7.6 buffer. The entire elution peak identified by monitoring at 260nm and 280nm was collected and pooled.
Analysis of the flow-through during the HA column loading step by SEC (fig. 16) indicated the presence of little or no Fab-oligonucleotide conjugate in the flow-through. Peaks due to the oligonucleotide loading substance were only observed at about 10.5 minutes and about 11.3 minutes. In contrast, SEC analysis of pooled elution peaks only showed conjugate species (fig. 17), with multiple peaks and shoulders (shoulder) due to the size differences of the conjugates under loading with different oligonucleotides (e.g., PMOs). No peak of loading substance was observed at 10.5 or 11.3 minutes. The mass balance of the HA purified conjugate relative to Fab was estimated by SEC chromatography. This was achieved by injecting 24 μg of Fab from the crude conjugation reaction product (4 μl injection, at a concentration of 6 μg/mL), and theoretically 24 μg of Fab from the HA eluate pool assuming 100% recovery. The HA eluate pool was 13.9mL, theoretically containing 45mg of Fab at a theoretical concentration of 3.24. Mu.g/. Mu.L. To achieve 24 μg Fab injection, 7.4 μl injection was used. The overlap of these two SEC chromatograms (fig. 18) shows that Fab was almost completely recovered as Fab-oligonucleotide conjugate. The peak height at 9.1 minutes was used to estimate 97% recovery of the reaction product after HA chromatographic purification.
On a reaction scale of 45mg, the HA eluate was buffer exchanged into 50mM His (pH 6.0) using an Amicon Ultra-15 30kDa centrifugal filtration device. First, by rotating the column at 4000rcf, the HA eluate pool was concentrated to about 1.5mL. Buffer exchange was then performed by adding 3mL of 50mM His (pH 6.0) and concentration was performed by centrifugation at 4000rcf until the volume reached about 1.5mL. This procedure was repeated for a total of 5 rounds using a volume of buffer equivalent to 15 volumes to produce the final purified anti-TfR Fab-oligonucleotide conjugate. The resulting purified conjugate (pH 6.0 in 50mM His) was then diluted to a final volume of 3.0mL with an additional 50mM His (pH 6.0).
The final purified anti-TfR Fab-oligonucleotide conjugate was analyzed by SEC, SDS-PAGE densitometry, and BCA. SEC of the final conjugate (shown in fig. 19A and 19B) was almost identical to the corresponding SEC data of the conjugate pool after HA purification, indicating that the purification process did not induce the formation of high molecular weight species. Average DAR, DAR species distribution and percentage of unconjugated Fab were calculated by SDS-PAGE densitometry (SDS-PAGE gel shown in fig. 20), and data analysis was performed with a Image Studio Lite software package from Li-Cor Biosciences (calculation results are shown in table 10). The final average DAR for the purified anti-TfR Fab-oligonucleotide conjugate was 1.96, including 8.1% unconjugated anti-TfR Fab. The protein concentration measured by BCA assay was 10.5mg/mL, indicating a total of 31.4mg of conjugate in the final product with a total process yield of 70%.
Table 10. Average DAR and DAR distribution of purified Fab-oligonucleotide conjugate products.
| Substance (B) | Abundance ratio |
| D0 | 0.081 |
| D1 | 0.324 |
| D2 | 0.312 |
| D3 | 0.168 |
| D4 | 0.071 |
| D5 | 0.032 |
| D6 | 0.011 |
| Average DAR | 1.96 |
EXAMPLE 10 Effect of ratio of BCN to Fab and reaction conditions on Fab-oligonucleotide conjugation reaction
To investigate the effect of BCN to anti-TfR ratio from the pre-reaction mixture, as well as the effect of oligonucleotide and Fab concentration in the oligonucleotide-Fab conjugation reaction, a set of reactions was performed according to the general protocol described in example 8. The oligonucleotide used was a PMO of 30 nucleotides in length.
In the first set of reactions, the pre-reaction between the oligonucleotide PAB-VC-PEG 3-azide and endo-BCN-PEG4-PFP ester was performed using the following conditions: 2.44mM of the oligonucleotide PAB-VC-PEG 3-azide was reacted with 1.63mM endo-BCN-PEG4-PFP ester (1.5:1 mol: mol ratio) in a 1:1 mixture of DMA and 25mM MES pH 5.5 buffer. The total pre-reaction volume was 0.37mL and the pre-reaction step was allowed to proceed at room temperature (about 25 ℃) for 18 hours.
After the pre-reaction was completed, the crude (i.e., unpurified) pre-reaction mixture containing the oligonucleotide PAB-VC-PEG 3-triazole-PEG 4-PFP ester was used to establish a series of anti-TfR Fab conjugation reactions. anti-TfR Fab conjugation reactions were performed using 2, 4, 6 and 10 molar equivalents of BCN from the pre-reaction mixture relative to Fab. All other reaction conditions remained unchanged in the different conjugates. In the conjugation reaction mixture, the Fab concentration was 3mg/mL, with 1mg of total Fab per reaction in 15v/v% DMA in 50mM HEPES pH 7.5 buffer. The conjugation reaction was carried out at 23 to 25 ℃ for 18 hours. The final DAR and DAR species distribution for each conjugate was determined by SDS-PAGE densitometry and the results are shown in table 11. An image of the SDS-PAGE gel is shown in FIG. 21.
Table 11.
In the second set of reactions, the pre-reaction between oligonucleotide-PAB-VC-PEG 3-azide and endo-BCN-PEG4-PFP ester was performed using the following conditions: 6.77mM oligonucleotide-PAB-VC-PEG 3-azide was reacted with 4.84mM endo-BCN-PEG4-PFP ester (1.4:1 mol: mol ratio) in a 1:1 mixture of DMA and 25mM MES pH 5.5 buffer. The total pre-reaction volume was 0.160mL and the pre-reaction step was allowed to proceed for 90 minutes at room temperature (about 25 ℃).
After the pre-reaction was completed, the crude (i.e., unpurified) pre-reaction mixture containing the oligonucleotide PAB-VC-PEG 3-triazole-PEG 4-PFP ester was used to establish a series of anti-TfR Fab conjugation reactions. anti-TfR Fab conjugation reactions were performed using 2, 4, 5, 6 and 8 molar equivalents of BCN from the pre-reaction mixture relative to Fab. All other reaction conditions remained unchanged in the different conjugates. In the conjugation reaction mixture, the Fab concentration was 6mg/mL, with 1mg of total Fab per reaction in 15v/v% DMA in 50mM HEPES pH 7.5 buffer. The conjugation reaction was carried out at 23 to 25 ℃ for 19 hours. The final average DAR for each conjugate was determined by SDS-PAGE densitometry and the results are shown in table 12. An image of the SDS-PAGE gel is shown in FIG. 22.
Table 12.
In a third set of reactions, the pre-reaction between oligonucleotide-PAB-VC-PEG 3-azide and endo-BCN-PEG4-PFP ester was performed using the following conditions: 6.77mM oligonucleotide-PAB-VC-PEG 3-azide was reacted with 4.84mM endo-BCN-PEG4-PFP ester (1.4:1 mol:mol ratio) in a 60:40 mixture of DMA and 25mM MES pH 5.5 buffer. The total pre-reaction volume was 0.160mL and the pre-reaction step was allowed to proceed for 90 minutes at room temperature (about 25 ℃).
After the pre-reaction was completed, the crude (i.e., unpurified) pre-reaction mixture containing the oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester was used to establish a series of anti-TfR Fab conjugation reactions. anti-TfR Fab conjugation reactions were performed using 2, 4, 5, 6, 8 and 10 molar equivalents of BCN from the pre-reaction mixture relative to Fab. All other reaction conditions remained unchanged in the different conjugates. In the conjugation reaction mixture, the Fab concentration was 6mg/mL, with 1mg of total Fab per reaction in 15v/v% DMA in 50mM HEPES pH 7.5 buffer. The conjugation reaction was carried out at room temperature (about 25 ℃) for 19 hours. The final average DAR and DAR0 percentages for each conjugate were determined by SDS-PAGE densitometry and the results are shown in table 13 (reaction J, K, L, M, N, O).
In the fourth set of reactions, the pre-reaction between oligonucleotide-PAB-VC-PEG 3-azide and endo-BCN-PEG4-PFP ester was performed using the following conditions: 6.77mM oligonucleotide-PAB-VC-PEG 3-azide was reacted with 6.45mM endo-BCN-PEG4-PFP ester (1.05:1 mol:mol ratio) in a 60:40 mixture of DMA and 25mM MES pH 5.5 buffer. The total pre-reaction volume was 0.080mL and the pre-reaction step was performed at room temperature (about 25 ℃) for 160 minutes.
After the pre-reaction was completed, the crude (i.e., unpurified) pre-reaction mixture containing the oligonucleotide-PAB-VC-PEG 3-triazole-PEG 4-PFP ester was used to establish a series of anti-TfR Fab conjugation reactions. anti-TfR Fab conjugation reactions were performed using 5, 6 and 7 molar equivalents of BCN from the pre-reaction mixture relative to Fab, based on the concentration of BCN in the pre-reaction. All other reaction conditions remained unchanged in the different conjugates. In the conjugation reaction mixture, the Fab concentration was 6mg/mL or 12mg/mL, with total Fab in each reaction being 0.6mg and 1.2mg, respectively. All reactions were performed in 15v/v% DMA in 25mM HEPES pH 7.5 buffer. The conjugation reaction was carried out at room temperature (about 25 ℃) for about 18 hours. The final average DAR and DAR0 percentages for each conjugate were determined by SDS-PAGE densitometry and the results are shown in table 13 (reaction P, Q, R, S).
Table 13.
EXAMPLE 11 Pre-reaction endo-BCN-PEG4-PFP ester hydrolysis
The simulated pre-reaction test was used to monitor the rate of PFP ester hydrolysis by RP-UPLC. The pre-reaction was performed using the following final reaction conditions: 1.63mM endo-BCN-PEG4-PFP ester in 1:1DMA with 25mM MES pH 5.5 buffer at 20 to 25℃for 20 hours. UPLC chromatographic conditions are provided in section 5.4.3. Samples were injected per hour onto the UPLC to monitor the decrease in endo-BCN-PEG4-PFP ester peak area as a function of time, thereby monitoring the rate of PFP ester hydrolysis of the starting reagent. In addition, the rate of click reaction between endo-BCN-PEG4-PFP ester and oligonucleotide-PAB-VC-PEG 3-azide (the oligonucleotide is PMO) was also evaluated. The pre-reaction was performed using the following conditions: 1.63mM endo-BCN-PEG4-PFP ester, 2.47mM oligonucleotide-PAB-VC-PEG 3-azide linker-load (1:1.5 molar ratio), in 1:1DMA with 25mM MES pH 5.5 buffer, at 20 to 25℃for 20 hours. For this test, the reduction in the peak area of endo-BCN-PEG4-PFP ester in the reaction was monitored by RP-UPLC every 30 minutes, 1 hour or 5 hours, with a total reaction duration of 20 hours. The results are shown in fig. 3.
EXAMPLE 12 initial antibody-oligonucleotide analysis
The efficiency of the reaction mixture in producing high DAR products was evaluated. Initially, the crude reaction mixture, sample flow-through and combined HA eluate were evaluated by SEC-UPLC analysis (fig. 24A to 24C). The crude reaction mixture contained antibody-oligonucleotide conjugates and unconjugated oligonucleotides (fig. 24A). The sample flow-through showed the presence of high DAR species in the presence of unconjugated oligonucleotides (fig. 24B). Unconjugated residual oligonucleotides were present in the pooled HA eluates (fig. 24C). These results were consistent in the final conjugate of buffer exchange (fig. 25), which resulted in an overall yield of 62%. Further analysis of the combined HA eluate and sample flow-through by SEC-UPLC showed that conjugate was lost in mobile phase buffer containing 100mM sodium phosphate and 10% mecn at pH 7.3 (fig. 26A-26B). It was determined that the final antibody fragment-drug conjugate (FDC) mixture under the same reaction conditions contained unconjugated oligonucleotide (potential oligonucleotide dimer) and 47.2% recovered conjugated oligonucleotide (fig. 27). HA purification chromatograms showed low conjugate purification and the presence of unconjugated oligonucleotides (fig. 28A to 28B). Taken together, these results indicate that improved reaction conditions are needed to produce higher concentrations of antibody-oligonucleotide conjugates.
EXAMPLE 13 reaction conditions
To determine the conditions under which high concentrations of antibody-oligonucleotide conjugate were produced, the reaction conditions were varied. However, increasing IPA concentration and decreasing sodium phosphate concentration showed improved retention of unconjugated oligonucleotide flowthrough and FDC during sample application (fig. 30A-30C). Finally, increasing the DMA concentration improved the reaction efficiency, resulting in a higher number of antibody-oligonucleotide conjugates and a lower number of unconjugated oligonucleotides (table 14).
Table 14.
Other embodiments
1. A mixture comprising complexes and unligated oligonucleotides, the complexes each comprising an antibody covalently linked to one or more oligonucleotides, wherein the mixture is produced by a method comprising:
(i) Ligating the oligonucleotide to a val-cit linker to obtain a first intermediate;
(ii) Connecting the first intermediate obtained in step (i) with a compound comprising bicyclononene to obtain a second intermediate; and
(iii) Ligating the second intermediate obtained in step (ii) with an antibody to obtain the complex;
wherein the compound comprising a bicyclononene is present in the reaction of step (iii) in an amount less than 5% of the starting amount of the compound in step (ii), optionally wherein the oligonucleotide is linked at the 5' end to the val-cit linker and/or the antibody is linked via lysine.
2. A mixture comprising complexes and unligated oligonucleotides, the complexes each comprising an antibody covalently linked to one or more oligonucleotides, wherein the mixture is produced by a method comprising:
(i) Ligating the oligonucleotide to a linker of formula (a):
wherein n is 3; to provide an oligonucleotide of formula (B):
wherein n is 3;
(ii) Contacting an oligonucleotide of formula (B) with a compound of formula (C):
wherein m is 4; to provide an oligonucleotide of formula (D):
wherein n is 3 and m is 4; and
(iii) Contacting an oligonucleotide of formula (D) with an antibody to provide a complex of formula (E):
wherein n is 3 and m is 4;
wherein the compound of formula (C) is present in the reaction mixture of step (iii) in an amount of less than 5% of the starting amount of the compound of formula (C) in the reaction of step (ii).
3. A method of treating complexes each comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i) Contacting the mixture of embodiment 1 or embodiment 2 with a mixed mode resin comprising positively charged metal sites and negatively charged ion sites under conditions such that the complex adsorbs to the mixed mode resin, wherein the mixture comprises a trace amount of an alkynyl-containing unconnected antibody; and
(ii) Eluting the complex from the mixed mode resin under conditions that dissociate the complex from the mixed mode resin.
4. The method of embodiment 3, wherein the pH of the mixture in step (i) is from 5.0 to 6.0.
5. The method of embodiment 3 or embodiment 4, wherein the mixture in step (i) is not pre-purified.
6. The method of any of embodiments 3-5, wherein the mixture in step (i) comprises phosphate ions and/or chloride ions in trace amounts.
7. The method of any of embodiments 3-5, wherein the mixture in step (i) does not comprise phosphate ions and/or chloride ions.
8. The method of any one of embodiments 3 to 5, wherein the mixed mode resin is an apatite resin.
9. The method of embodiment 8, wherein the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxy fluorapatite resin, a fluorapatite resin, or a chlorapatite resin.
10. The method of any one of embodiments 3 to 9, wherein in step (i) the unligated oligonucleotides are not adsorbed to the mixed mode resin.
11. The method of any one of embodiments 3 to 9, wherein in step (i), some or all of the unligated oligonucleotides are adsorbed to the mixed mode resin.
12. The method of embodiment 11, further comprising washing the mixed mode resin with a wash solution comprising up to 20mM phosphate ions and/or up to 30mM chloride ions between step (i) and step (ii), optionally wherein the solution comprises up to 10mM phosphate ions and/or up to 25mM chloride ions.
13. The method of embodiment 12, wherein the pH of the wash solution is from 5.0 to 7.6.
14. The method of embodiment 12 or embodiment 13, wherein in the washing step, most or all of the unligated oligonucleotides are removed from the mixed mode resin.
15. The method of any one of embodiments 3 to 14, wherein step (ii) comprises applying an elution solution comprising at least 30mM phosphate ions and/or at least 50mM chloride ions to the mixed mode resin to elute the complex, optionally wherein the elution solution comprises at least 100mM phosphate ions and/or at least 100mM chloride ions.
16. The method of embodiment 15, wherein the pH of the elution solution is from 7.5 to 8.5.
17. The method of any one of embodiments 3 to 16, wherein the antibody is a full length IgG, fab fragment, fab 'fragment, F (ab') 2 fragment, scFv, or Fv fragment.
18. The method of any one of embodiments 3 to 17, wherein the antibody is an anti-transferrin receptor antibody.
19. The method of any one of embodiments 3 to 18, wherein the oligonucleotide is single stranded.
20. The method of embodiment 19, wherein the oligonucleotide is an antisense oligonucleotide, optionally a spacer.
21. The method of embodiment 20, wherein the oligonucleotide is one strand of a double stranded oligonucleotide, optionally wherein the double stranded oligonucleotide is an siRNA, and optionally wherein the one strand is the sense strand of the siRNA.
22. The method of any one of embodiments 3 to 21, wherein the oligonucleotide comprises at least one modified internucleotide linkage, optionally wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.
23. The method of any one of embodiments 3 to 22, wherein the oligonucleotide comprises one or more modified nucleotides, optionally wherein the modified nucleotides comprise 2 '-O-methoxyethyl ribose (MOE), locked Nucleic Acid (LNA), 2' -fluoro modification, or morpholino modification.
24. The method of any one of embodiments 3 to 23, wherein the oligonucleotide is 10 to 50 nucleotides in length, optionally 15 to 25 nucleotides in length.
25. The method of any one of embodiments 3 to 24, wherein the antibody is covalently linked to the 5' of the oligonucleotide.
26. The method of any one of embodiments 3 to 25, wherein the antibody is covalently linked to the 3' of the oligonucleotide.
27. The method of any one of embodiments 3 to 26, wherein the antibody is linked by lysine.
28. The method of any one of embodiments 2 to 27, wherein the eluate obtained from step (ii) comprises undetectable levels of unbound oligonucleotides.
29. A method of treating complexes each comprising an antibody covalently linked to one or more charge-neutral oligonucleotides, the method comprising:
(i) Contacting a mixture comprising an organic solvent, the complex, and an unligated charge neutral oligonucleotide with a mixed mode resin comprising positively charged metal sites and negatively charged ion sites under conditions such that the complex adsorbs to the mixed mode resin, and
(ii) Eluting the complex from the mixed mode resin under conditions that dissociate the complex from the mixed mode resin.
30. The method of embodiment 29, wherein the mixed mode resin is an apatite resin.
31. The method of embodiment 2, wherein the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxy fluorapatite resin, a fluorapatite resin, or a chlorapatite resin.
32. The method of any one of embodiments 29 to 31, wherein the organic solvent is Dimethylacetamide (DMA), isopropanol (IPA), dimethylsulfoxide (DMSO), acetonitrile (ACN), or Propylene Glycol (PG).
33. The method of any one of embodiments 29 to 32, wherein the organic solvent is 5% to 20% (v/v) in the mixture in step (i), optionally wherein the organic solvent is 15% (v/v) in the mixture in step (i).
34. The method of any one of embodiments 29 to 33, wherein the mixture in step (i) does not comprise phosphate ions or chloride ions.
35. The method of any one of embodiments 29 to 34, wherein the mixture in step (i) further comprises up to 10mM phosphate ions and/or up to 20mM chloride ions.
36. The method of any one of embodiments 29 to 35, wherein the pH of the mixture in step (i) is from 5.0 to 6.0.
37. The method of any one of embodiments 29 to 36, wherein in step (i) the unligated charge neutral oligonucleotide is not adsorbed to the mixed mode resin.
38. The method of any one of embodiments 29 to 37, further comprising washing the mixed mode resin with a wash solution comprising an organic solvent between step (i) and step (ii), optionally wherein the organic solvent is Dimethylacetamide (DMA), isopropanol (IPA), dimethylsulfoxide (DMSO), acetonitrile (ACN), or Propylene Glycol (PG).
39. The method of embodiment 38, wherein the organic solvent is 5% to 20% (v/v) in the wash solution, optionally wherein the organic solvent is 15% (v/v) in the wash solution.
40. The method of embodiment 38 or embodiment 39, wherein the wash solution further comprises up to 10mM phosphate ions and/or up to 20mM chloride ions.
41. The method of any one of embodiments 29 to 40, wherein step (ii) comprises applying an elution solution to the mixed mode resin to elute the complex, wherein the elution solution comprises an organic solvent, optionally wherein the organic solvent is Dimethylacetamide (DMA), isopropanol (IPA), dimethylsulfoxide (DMSO), acetonitrile (ACN), or Propylene Glycol (PG).
42. The method of embodiment 41, wherein the organic solvent is 10% to 20% (v/v) in the elution solution, optionally wherein the organic solvent is 10% (v/v) in the elution solution.
43. The method of embodiment 41 or embodiment 42, wherein the elution solution comprises at least 30mM phosphate ions, optionally wherein the elution solution comprises at least 100mM phosphate ions.
44. The method of embodiment 43, wherein the elution solution does not comprise chloride ions.
45. The method of embodiment 41 or embodiment 42, wherein the elution solution comprises a gradually increasing concentration of phosphate ions, optionally wherein the concentration of phosphate ions increases from at least 10mM to at least 100mM.
46. The method of any one of embodiments 41 to 45, wherein the pH of the elution solution is 7.6 to 8.5.
47. The method of any one of embodiments 29 to 46, wherein the antibody is a full length IgG, fab fragment, fab 'fragment, F (ab') 2 fragment, scFv, or Fv fragment.
48. The method of any one of embodiments 29 to 47, wherein the antibody is an anti-transferrin receptor antibody.
49. The method of any one of embodiments 29 to 48, wherein the charge neutral oligonucleotide is single stranded.
50. The method of embodiment 49, wherein the charge neutral oligonucleotide is an antisense oligonucleotide.
51. The method of any one of embodiments 29 to 50, wherein the charge neutral oligonucleotide is a Phosphodiamide Morpholino Oligomer (PMO).
52. The method of any one of embodiments 29 to 51, wherein the charge neutral oligonucleotide is 10 to 50 nucleotides in length, optionally 20 to 30 nucleotides in length.
53. The method of any one of embodiments 29 to 52, wherein the antibody is covalently linked to the 5' of the charge neutral oligonucleotide.
54. The method of any one of embodiments 29 to 53, wherein the antibody is covalently linked to the 3' of the charge neutral oligonucleotide.
55. The method of any one of embodiments 29 to 54, wherein said antibody is covalently linked to said charge neutral oligonucleotide through a linker, optionally a Val-cit linker.
56. The method of embodiment 55, wherein the linker comprises the structure:
wherein n is 3 and m is 4.
57. The method of embodiment 56, wherein the complex comprises the structure:
wherein n is 3 and m is 4, and wherein the antibody is linked by lysine.
58. The method of any one of embodiments 29 to 57, wherein the average drug-to-antibody ratio (DAR) of the complexes in the mixture in step (i) is at least about 1.8.
59. The method of any one of embodiments 29 to 58, wherein the eluate obtained from step (ii) comprises undetectable levels of unbound charge-neutral oligonucleotides.
60. The method of any one of embodiments 29 to 59, wherein the complex is generated by ligating a charge neutral oligonucleotide to an antibody, wherein the charge neutral oligonucleotide comprises the structure:
and wherein the antibody comprises the following structure:
/>
wherein n is 3 and m is 4.
61. The method of any one of embodiments 29 to 59, wherein the complex is generated by ligating a charge neutral oligonucleotide to an antibody, wherein the charge neutral oligonucleotide comprises the structure:
wherein n is 3 and m is 4.
62. A method of producing a complex comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i) An oligonucleotide comprising the following structure was obtained:
wherein n is 3;
(ii) Obtaining an antibody comprising the structure:
wherein m is 4; and
(iii) Reacting the oligonucleotide in step (i) with the antibody obtained in step (ii) to obtain the complex.
63. A method of producing a complex comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i) An oligonucleotide comprising the following structure was obtained:
wherein n is 3 and m is 4;
(ii) Obtaining an antibody; and
(iii) Reacting the oligonucleotide in step (i) with the antibody obtained in step (ii) to obtain the complex.
64. The method of embodiment 62 or embodiment 63, wherein the complex comprises the structure:
wherein n is 3 and m is 4, and wherein the antibody is linked by lysine.
Equivalent and terminology
The disclosure illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of … …" can be replaced with any of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure. Thus, it should be understood that although the present disclosure has been specifically disclosed by some preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure.
In addition, where features or aspects of the present disclosure are described in terms of Markush groups (Markush groups) or other alternative groups, those skilled in the art will recognize that the present disclosure is also thus described in terms of any individual member or subgroup of members of the Markush group or other group.
It is understood that in some embodiments, reference may be made to the sequences shown in the sequence listing in describing the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., RNA counterparts of DNA nucleotides or DNA counterparts of RNA nucleotides) and/or one or more modified nucleotides and/or one or more modified internucleotide linkages and/or one or more other modifications as compared to the specified sequence, while retaining substantially the same or similar complementary properties as the specified sequence.
The use of nouns without quantitative word modifications in the context of describing the invention (especially in the context of the appended claims) will be interpreted as one or more than one unless otherwise indicated herein or clearly contradicted by context. Unless otherwise indicated, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to"). Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Some embodiments of the invention are described herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.
The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.
Sequence listing
<110> Dyne Therapeutics, Inc.
<120> method for preparing protein-oligonucleotide Complex
<130> D0824.70043WO00
<140> not yet allocated
<141> at the same time
<150> US 63/074,439
<151> 2020-09-03
<150> US 63/074,436
<151> 2020-09-03
<160> 45
<170> PatentIn version 3.5
<210> 1
<211> 760
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 1
Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu
1 5 10 15
Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp
20 25 30
Asn Ser His Val Glu Met Lys Leu Ala Val Asp Glu Glu Glu Asn Ala
35 40 45
Asp Asn Asn Thr Lys Ala Asn Val Thr Lys Pro Lys Arg Cys Ser Gly
50 55 60
Ser Ile Cys Tyr Gly Thr Ile Ala Val Ile Val Phe Phe Leu Ile Gly
65 70 75 80
Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr
85 90 95
Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Val Arg Glu Glu Pro
100 105 110
Gly Glu Asp Phe Pro Ala Ala Arg Arg Leu Tyr Trp Asp Asp Leu Lys
115 120 125
Arg Lys Leu Ser Glu Lys Leu Asp Ser Thr Asp Phe Thr Gly Thr Ile
130 135 140
Lys Leu Leu Asn Glu Asn Ser Tyr Val Pro Arg Glu Ala Gly Ser Gln
145 150 155 160
Lys Asp Glu Asn Leu Ala Leu Tyr Val Glu Asn Gln Phe Arg Glu Phe
165 170 175
Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val
180 185 190
Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Arg
195 200 205
Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys
210 215 220
Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys
225 230 235 240
Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile
245 250 255
Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu
260 265 270
Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe
275 280 285
Pro Ile Val Asn Ala Glu Leu Ser Phe Phe Gly His Ala His Leu Gly
290 295 300
Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln
305 310 315 320
Phe Pro Pro Ser Arg Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr
325 330 335
Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp
340 345 350
Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser
355 360 365
Glu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Ile
370 375 380
Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp
385 390 395 400
His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala
405 410 415
Ala Lys Ser Gly Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met
420 425 430
Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile
435 440 445
Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr
450 455 460
Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr
465 470 475 480
Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
485 490 495
Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asn
500 505 510
Val Lys His Pro Val Thr Gly Gln Phe Leu Tyr Gln Asp Ser Asn Trp
515 520 525
Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe
530 535 540
Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp
545 550 555 560
Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu
565 570 575
Ile Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu
580 585 590
Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu Leu Asn
595 600 605
Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp
610 615 620
Leu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser Leu Gln
625 630 635 640
Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu
645 650 655
Thr Thr Asp Phe Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys
660 665 670
Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Leu Ser Pro
675 680 685
Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser
690 695 700
Gly Ser His Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg Lys
705 710 715 720
Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
725 730 735
Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp
740 745 750
Val Trp Asp Ile Asp Asn Glu Phe
755 760
<210> 2
<211> 760
<212> PRT
<213> rhesus monkey (Macaca mulatta)
<400> 2
Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu
1 5 10 15
Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp
20 25 30
Asn Ser His Val Glu Met Lys Leu Gly Val Asp Glu Glu Glu Asn Thr
35 40 45
Asp Asn Asn Thr Lys Pro Asn Gly Thr Lys Pro Lys Arg Cys Gly Gly
50 55 60
Asn Ile Cys Tyr Gly Thr Ile Ala Val Ile Ile Phe Phe Leu Ile Gly
65 70 75 80
Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr
85 90 95
Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Ala Arg Glu Glu Pro
100 105 110
Glu Glu Asp Phe Pro Ala Ala Pro Arg Leu Tyr Trp Asp Asp Leu Lys
115 120 125
Arg Lys Leu Ser Glu Lys Leu Asp Thr Thr Asp Phe Thr Ser Thr Ile
130 135 140
Lys Leu Leu Asn Glu Asn Leu Tyr Val Pro Arg Glu Ala Gly Ser Gln
145 150 155 160
Lys Asp Glu Asn Leu Ala Leu Tyr Ile Glu Asn Gln Phe Arg Glu Phe
165 170 175
Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val
180 185 190
Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Gly
195 200 205
Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys
210 215 220
Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys
225 230 235 240
Lys Asp Phe Glu Asp Leu Asp Ser Pro Val Asn Gly Ser Ile Val Ile
245 250 255
Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu
260 265 270
Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe
275 280 285
Pro Ile Val Lys Ala Asp Leu Ser Phe Phe Gly His Ala His Leu Gly
290 295 300
Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln
305 310 315 320
Phe Pro Pro Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr
325 330 335
Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp
340 345 350
Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Lys Met Val Thr Ser
355 360 365
Glu Asn Lys Ser Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Thr
370 375 380
Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp
385 390 395 400
His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala
405 410 415
Ala Lys Ser Ser Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met
420 425 430
Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile
435 440 445
Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr
450 455 460
Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr
465 470 475 480
Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
485 490 495
Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asp
500 505 510
Val Lys His Pro Val Thr Gly Arg Ser Leu Tyr Gln Asp Ser Asn Trp
515 520 525
Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe
530 535 540
Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp
545 550 555 560
Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu
565 570 575
Val Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu
580 585 590
Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Thr Glu Leu Asn
595 600 605
Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Leu Phe Leu Arg Asp
610 615 620
Leu Asn Gln Tyr Arg Ala Asp Val Lys Glu Met Gly Leu Ser Leu Gln
625 630 635 640
Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu
645 650 655
Thr Thr Asp Phe Arg Asn Ala Glu Lys Arg Asp Lys Phe Val Met Lys
660 665 670
Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr Tyr Phe Leu Ser Pro
675 680 685
Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser
690 695 700
Gly Ser His Thr Leu Ser Ala Leu Leu Glu Ser Leu Lys Leu Arg Arg
705 710 715 720
Gln Asn Asn Ser Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
725 730 735
Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp
740 745 750
Val Trp Asp Ile Asp Asn Glu Phe
755 760
<210> 3
<211> 760
<212> PRT
<213> cynomolgus monkey (Macaca fascicularis)
<400> 3
Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu
1 5 10 15
Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp
20 25 30
Asn Ser His Val Glu Met Lys Leu Gly Val Asp Glu Glu Glu Asn Thr
35 40 45
Asp Asn Asn Thr Lys Ala Asn Gly Thr Lys Pro Lys Arg Cys Gly Gly
50 55 60
Asn Ile Cys Tyr Gly Thr Ile Ala Val Ile Ile Phe Phe Leu Ile Gly
65 70 75 80
Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr
85 90 95
Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Ala Arg Glu Glu Pro
100 105 110
Glu Glu Asp Phe Pro Ala Ala Pro Arg Leu Tyr Trp Asp Asp Leu Lys
115 120 125
Arg Lys Leu Ser Glu Lys Leu Asp Thr Thr Asp Phe Thr Ser Thr Ile
130 135 140
Lys Leu Leu Asn Glu Asn Leu Tyr Val Pro Arg Glu Ala Gly Ser Gln
145 150 155 160
Lys Asp Glu Asn Leu Ala Leu Tyr Ile Glu Asn Gln Phe Arg Glu Phe
165 170 175
Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val
180 185 190
Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Gly
195 200 205
Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys
210 215 220
Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys
225 230 235 240
Lys Asp Phe Glu Asp Leu Asp Ser Pro Val Asn Gly Ser Ile Val Ile
245 250 255
Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu
260 265 270
Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe
275 280 285
Pro Ile Val Lys Ala Asp Leu Ser Phe Phe Gly His Ala His Leu Gly
290 295 300
Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln
305 310 315 320
Phe Pro Pro Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr
325 330 335
Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp
340 345 350
Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Lys Met Val Thr Ser
355 360 365
Glu Asn Lys Ser Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Thr
370 375 380
Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp
385 390 395 400
His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala
405 410 415
Ala Lys Ser Ser Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met
420 425 430
Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile
435 440 445
Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr
450 455 460
Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr
465 470 475 480
Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
485 490 495
Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asp
500 505 510
Val Lys His Pro Val Thr Gly Arg Ser Leu Tyr Gln Asp Ser Asn Trp
515 520 525
Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe
530 535 540
Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp
545 550 555 560
Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu
565 570 575
Val Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu
580 585 590
Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Thr Glu Leu Asn
595 600 605
Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Leu Phe Leu Arg Asp
610 615 620
Leu Asn Gln Tyr Arg Ala Asp Val Lys Glu Met Gly Leu Ser Leu Gln
625 630 635 640
Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu
645 650 655
Thr Thr Asp Phe Arg Asn Ala Glu Lys Arg Asp Lys Phe Val Met Lys
660 665 670
Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr Tyr Phe Leu Ser Pro
675 680 685
Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser
690 695 700
Gly Ser His Thr Leu Ser Ala Leu Leu Glu Ser Leu Lys Leu Arg Arg
705 710 715 720
Gln Asn Asn Ser Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
725 730 735
Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp
740 745 750
Val Trp Asp Ile Asp Asn Glu Phe
755 760
<210> 4
<211> 763
<212> PRT
<213> mice (Mus musculus)
<400> 4
Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu
1 5 10 15
Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp
20 25 30
Asn Ser His Val Glu Met Lys Leu Ala Ala Asp Glu Glu Glu Asn Ala
35 40 45
Asp Asn Asn Met Lys Ala Ser Val Arg Lys Pro Lys Arg Phe Asn Gly
50 55 60
Arg Leu Cys Phe Ala Ala Ile Ala Leu Val Ile Phe Phe Leu Ile Gly
65 70 75 80
Phe Met Ser Gly Tyr Leu Gly Tyr Cys Lys Arg Val Glu Gln Lys Glu
85 90 95
Glu Cys Val Lys Leu Ala Glu Thr Glu Glu Thr Asp Lys Ser Glu Thr
100 105 110
Met Glu Thr Glu Asp Val Pro Thr Ser Ser Arg Leu Tyr Trp Ala Asp
115 120 125
Leu Lys Thr Leu Leu Ser Glu Lys Leu Asn Ser Ile Glu Phe Ala Asp
130 135 140
Thr Ile Lys Gln Leu Ser Gln Asn Thr Tyr Thr Pro Arg Glu Ala Gly
145 150 155 160
Ser Gln Lys Asp Glu Ser Leu Ala Tyr Tyr Ile Glu Asn Gln Phe His
165 170 175
Glu Phe Lys Phe Ser Lys Val Trp Arg Asp Glu His Tyr Val Lys Ile
180 185 190
Gln Val Lys Ser Ser Ile Gly Gln Asn Met Val Thr Ile Val Gln Ser
195 200 205
Asn Gly Asn Leu Asp Pro Val Glu Ser Pro Glu Gly Tyr Val Ala Phe
210 215 220
Ser Lys Pro Thr Glu Val Ser Gly Lys Leu Val His Ala Asn Phe Gly
225 230 235 240
Thr Lys Lys Asp Phe Glu Glu Leu Ser Tyr Ser Val Asn Gly Ser Leu
245 250 255
Val Ile Val Arg Ala Gly Glu Ile Thr Phe Ala Glu Lys Val Ala Asn
260 265 270
Ala Gln Ser Phe Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Lys Asn
275 280 285
Lys Phe Pro Val Val Glu Ala Asp Leu Ala Leu Phe Gly His Ala His
290 295 300
Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His
305 310 315 320
Thr Gln Phe Pro Pro Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val
325 330 335
Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Lys Met Glu
340 345 350
Gly Ser Cys Pro Ala Arg Trp Asn Ile Asp Ser Ser Cys Lys Leu Glu
355 360 365
Leu Ser Gln Asn Gln Asn Val Lys Leu Ile Val Lys Asn Val Leu Lys
370 375 380
Glu Arg Arg Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Tyr Glu Glu
385 390 395 400
Pro Asp Arg Tyr Val Val Val Gly Ala Gln Arg Asp Ala Leu Gly Ala
405 410 415
Gly Val Ala Ala Lys Ser Ser Val Gly Thr Gly Leu Leu Leu Lys Leu
420 425 430
Ala Gln Val Phe Ser Asp Met Ile Ser Lys Asp Gly Phe Arg Pro Ser
435 440 445
Arg Ser Ile Ile Phe Ala Ser Trp Thr Ala Gly Asp Phe Gly Ala Val
450 455 460
Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys
465 470 475 480
Ala Phe Thr Tyr Ile Asn Leu Asp Lys Val Val Leu Gly Thr Ser Asn
485 490 495
Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr Leu Met Gly Lys Ile
500 505 510
Met Gln Asp Val Lys His Pro Val Asp Gly Lys Ser Leu Tyr Arg Asp
515 520 525
Ser Asn Trp Ile Ser Lys Val Glu Lys Leu Ser Phe Asp Asn Ala Ala
530 535 540
Tyr Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe
545 550 555 560
Cys Glu Asp Ala Asp Tyr Pro Tyr Leu Gly Thr Arg Leu Asp Thr Tyr
565 570 575
Glu Ala Leu Thr Gln Lys Val Pro Gln Leu Asn Gln Met Val Arg Thr
580 585 590
Ala Ala Glu Val Ala Gly Gln Leu Ile Ile Lys Leu Thr His Asp Val
595 600 605
Glu Leu Asn Leu Asp Tyr Glu Met Tyr Asn Ser Lys Leu Leu Ser Phe
610 615 620
Met Lys Asp Leu Asn Gln Phe Lys Thr Asp Ile Arg Asp Met Gly Leu
625 630 635 640
Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp Tyr Phe Arg Ala Thr
645 650 655
Ser Arg Leu Thr Thr Asp Phe His Asn Ala Glu Lys Thr Asn Arg Phe
660 665 670
Val Met Arg Glu Ile Asn Asp Arg Ile Met Lys Val Glu Tyr His Phe
675 680 685
Leu Ser Pro Tyr Val Ser Pro Arg Glu Ser Pro Phe Arg His Ile Phe
690 695 700
Trp Gly Ser Gly Ser His Thr Leu Ser Ala Leu Val Glu Asn Leu Lys
705 710 715 720
Leu Arg Gln Lys Asn Ile Thr Ala Phe Asn Glu Thr Leu Phe Arg Asn
725 730 735
Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly Val Ala Asn Ala Leu
740 745 750
Ser Gly Asp Ile Trp Asn Ile Asp Asn Glu Phe
755 760
<210> 5
<211> 197
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 5
Phe Val Lys Ile Gln Val Lys Asp Ser Ala Gln Asn Ser Val Ile Ile
1 5 10 15
Val Asp Lys Asn Gly Arg Leu Val Tyr Leu Val Glu Asn Pro Gly Gly
20 25 30
Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr Gly Lys Leu Val His
35 40 45
Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val
50 55 60
Asn Gly Ser Ile Val Ile Val Arg Ala Gly Lys Ile Thr Phe Ala Glu
65 70 75 80
Lys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr
85 90 95
Met Asp Gln Thr Lys Phe Pro Ile Val Asn Ala Glu Leu Ser Phe Phe
100 105 110
Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro
115 120 125
Ser Phe Asn His Thr Gln Phe Pro Pro Ser Arg Ser Ser Gly Leu Pro
130 135 140
Asn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe
145 150 155 160
Gly Asn Met Glu Gly Asp Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr
165 170 175
Cys Arg Met Val Thr Ser Glu Ser Lys Asn Val Lys Leu Thr Val Ser
180 185 190
Asn Val Leu Lys Glu
195
<210> 6
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 6
Ala Ser Ser Leu Asn Ile Ala
1 5
<210> 7
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 7
Ser Lys Thr Phe Asn Thr His Pro Gln Ser Thr Pro
1 5 10
<210> 8
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 8
Thr Ala Arg Gly Glu His Lys Glu Glu Glu Leu Ile
1 5 10
<210> 9
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 9
Cys Gln Ala Gln Gly Gln Leu Val Cys
1 5
<210> 10
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 10
Cys Ser Glu Arg Ser Met Asn Phe Cys
1 5
<210> 11
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 11
Cys Pro Lys Thr Arg Arg Val Pro Cys
1 5
<210> 12
<211> 20
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 12
Trp Leu Ser Glu Ala Gly Pro Val Val Thr Val Arg Ala Leu Arg Gly
1 5 10 15
Thr Gly Ser Trp
20
<210> 13
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 13
Cys Met Gln His Ser Met Arg Val Cys
1 5
<210> 14
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 14
Asp Asp Thr Arg His Trp Gly
1 5
<210> 15
<400> 15
000
<210> 16
<400> 16
000
<210> 17
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 17
Ser Tyr Trp Met His
1 5
<210> 18
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 18
Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe Lys
1 5 10 15
Ser
<210> 19
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 19
Gly Thr Arg Ala Tyr His Tyr
1 5
<210> 20
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 20
Arg Ala Ser Asp Asn Leu Tyr Ser Asn Leu Ala
1 5 10
<210> 21
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 21
Asp Ala Thr Asn Leu Ala Asp
1 5
<210> 22
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 22
Gln His Phe Trp Gly Thr Pro Leu Thr
1 5
<210> 23
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 23
Gly Tyr Thr Phe Thr Ser Tyr
1 5
<210> 24
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 24
Asn Pro Thr Asn Gly Arg
1 5
<210> 25
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 25
Thr Ser Tyr Trp Met His
1 5
<210> 26
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 26
Trp Ile Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn
1 5 10
<210> 27
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 27
Ala Arg Gly Thr Arg Ala
1 5
<210> 28
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 28
Tyr Ser Asn Leu Ala Trp Tyr
1 5
<210> 29
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 29
Leu Leu Val Tyr Asp Ala Thr Asn Leu Ala
1 5 10
<210> 30
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 30
Gln His Phe Trp Gly Thr Pro Leu
1 5
<210> 31
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 31
Gln His Phe Ala Gly Thr Pro Leu Thr
1 5
<210> 32
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 32
Gln His Phe Ala Gly Thr Pro Leu
1 5
<210> 33
<211> 116
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 33
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe
50 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110
Thr Val Ser Ser
115
<210> 34
<211> 107
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 34
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45
Tyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 35
<211> 116
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 35
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe
50 55 60
Lys Ser Arg Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Met Val
100 105 110
Thr Val Ser Ser
115
<210> 36
<211> 107
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 36
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val
35 40 45
Tyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 37
<211> 330
<212> PRT
<213> Chile person
<400> 37
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 38
<211> 107
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 38
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 39
<211> 446
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 39
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe
50 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 40
<211> 214
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 40
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45
Tyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 41
<211> 446
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 41
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe
50 55 60
Lys Ser Arg Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Met Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 42
<211> 214
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 42
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val
35 40 45
Tyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 43
<211> 226
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 43
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe
50 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro
225
<210> 44
<211> 226
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 44
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe
50 55 60
Lys Ser Arg Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Met Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro
225
<210> 45
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> synthetic
<400> 45
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
1 5 10
Claims (33)
1. A method of treating complexes each comprising an antibody covalently linked to one or more charge-neutral oligonucleotides, the method comprising:
(i) Contacting a mixture comprising an organic solvent, the complex, and an unligated charge neutral oligonucleotide with a mixed mode resin comprising positively charged metal sites and negatively charged ion sites under conditions such that the complex adsorbs to the mixed mode resin, and
(ii) Eluting the complex from the mixed mode resin under conditions that cause the complex to dissociate from the mixed mode resin.
2. The method of any one of claims 1, wherein the organic solvent is Dimethylacetamide (DMA), isopropanol (IPA), dimethylsulfoxide (DMSO), acetonitrile (ACN), or Propylene Glycol (PG).
3. The method of claim 1 or 2, wherein the organic solvent is 5% to 30% (v/v) in the mixture of step (i), optionally wherein the organic solvent is 15% (v/v) in the mixture of step (i).
4. A method according to any one of claims 1 to 3, wherein the mixture of step (i) further comprises up to 10mM phosphate ions and/or up to 20mM chloride ions.
5. The method of any one of claims 1 to 4, further comprising washing the mixed mode resin with a washing solution comprising an organic solvent between step (i) and step (ii), optionally wherein the organic solvent is Dimethylacetamide (DMA), isopropanol (IPA), dimethylsulfoxide (DMSO), acetonitrile (ACN), or Propylene Glycol (PG).
6. The method of claim 5, wherein the organic solvent is 5% to 30% (v/v) in the wash solution, optionally wherein the organic solvent is 15% (v/v) in the wash solution.
7. The method of claim 5 or claim 6, wherein the wash solution further comprises up to 10mM phosphate ions and/or up to 20mM chloride ions.
8. The method of any one of claims 1 to 7, wherein step (ii) comprises applying an elution solution to the mixed mode resin to elute the complex, wherein the elution solution comprises an organic solvent, optionally wherein the organic solvent is Dimethylacetamide (DMA), isopropanol (IPA), dimethylsulfoxide (DMSO), acetonitrile (ACN), or Propylene Glycol (PG).
9. The method of claim 8, wherein the organic solvent is 10% to 30% (v/v) in the elution solution, optionally wherein the organic solvent is 10% (v/v) in the elution solution.
10. The method of claim 8 or claim 9, wherein the elution solution comprises at least 30mM phosphate ions, optionally wherein the elution solution comprises at least 100mM phosphate ions.
11. The method of claim 8 or claim 9, wherein the elution solution comprises a progressively increasing concentration of phosphate ions, optionally wherein the concentration of phosphate ions increases from at least 10mM to at least 100mM.
12. The method of any one of claims 8 to 11, wherein the pH of the elution solution is 7.6 to 8.5.
13. A method of producing a complex comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i) An oligonucleotide comprising the following structure was obtained:
wherein n is 3;
(ii) Obtaining an antibody comprising the structure:
wherein m is 4; and
(iii) Reacting the oligonucleotide of step (i) with the antibody obtained in step (ii) to obtain the complex.
14. A method of producing a complex comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i) An oligonucleotide comprising the following structure was obtained:
(D) The method comprises the steps of carrying out a first treatment on the surface of the Wherein n is 3 and wherein m is 4;
(ii) Obtaining an antibody; and
(iii) Reacting the oligonucleotide of step (i) with the antibody obtained in step (ii) to obtain the complex.
15. The method of claim 14, wherein the complex comprises the structure:
wherein n is 3 and m is 4, and wherein the antibody is linked by lysine.
16. A mixture comprising complexes and unligated oligonucleotides, the complexes each comprising an antibody covalently linked to one or more oligonucleotides, wherein the mixture is produced by a method comprising:
(i) Obtaining a first intermediate comprising an oligonucleotide covalently linked to a cleavable linker comprising a valine-citrulline sequence;
(ii) Connecting the first intermediate obtained in step (i) with a compound comprising bicyclononene to obtain a second intermediate; and
(iii) Ligating the second intermediate obtained in step (ii) with an antibody to obtain the complex;
wherein the compound comprising a bicyclononene is present in the reaction of step (iii) in an amount less than 5% of the starting amount of the compound of step (ii), optionally wherein the oligonucleotide is covalently linked at the 5' end to the cleavable linker comprising a valine-citrulline sequence and/or the antibody is linked via lysine.
17. A mixture comprising complexes and ii) unligated oligonucleotides, the complexes each comprising an antibody covalently linked to one or more oligonucleotides, wherein the mixture is produced by a method comprising:
(i) Combining one or more oligonucleotides with a linker of formula (a) under reaction conditions that produce a product of formula (B):
wherein n is 3;
wherein n is 3;
(ii) Contacting a product of formula (B) with a compound of formula (C) under reaction conditions that produce a product of formula (D):
wherein m is 4:
wherein n is 3 and m is 4; and
(iii) Contacting a product of formula (D) with an antibody under reaction conditions that produce a complex of formula (E):
wherein n is 3 and m is 4;
wherein the compound of formula (C) is present in the reaction of step (iii) in an amount of less than 5% of the starting amount of the compound of formula (C) in the reaction of step (ii).
18. A method of treating complexes each comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i) Contacting the mixture of claim 16 or claim 17 with a mixed mode resin comprising positively charged metal sites and negatively charged ion sites under conditions such that the complex adsorbs to the mixed mode resin, wherein the mixture comprises a trace amount of an alkynyl-containing unconnected antibody; and
(ii) Eluting the complex from the mixed mode resin under conditions that cause the complex to dissociate from the mixed mode resin.
19. The method of claim 18, wherein the mixture of step (i) is not pre-purified.
20.18 or 19, wherein the mixture of step (i) comprises phosphate ions and/or chloride ions in trace amounts.
21. The method of claim 18, further comprising washing the mixed mode resin with a wash solution comprising up to 20mM phosphate ions and/or up to 30mM chloride ions between step (i) and step (ii), optionally wherein the solution comprises up to 10mM phosphate ions and/or up to 25mM chloride ions.
22. The method of claim 21, wherein the pH of the wash solution is from 5.0 to 7.6.
23. The method of claim 21 or claim 22, wherein in the washing step most or all of the unbound oligonucleotides are removed from the mixed-mode resin.
24. The method of any one of claims 18 to 23, wherein step (ii) comprises applying an elution solution comprising at least 30mM phosphate ions and/or at least 50mM chloride ions to the mixed mode resin to elute the complex, optionally wherein the elution solution comprises at least 100mM phosphate ions and/or at least 100mM chloride ions.
25. The method of claim 24, wherein the pH of the elution solution is 7.5 to 8.5.
26. The method of any one of claims 18 to 25, wherein the antibody is an anti-transferrin receptor antibody.
27. The method of any one of claims 18 to 26, wherein the oligonucleotide is a charged oligonucleotide.
28. The method of claim 27, wherein the oligonucleotide is a negatively charged oligonucleotide.
29. The method of claim 27 or claim 28, wherein the oligonucleotide is single stranded.
30. The method of any one of claims 27 to 29, wherein the oligonucleotide is an antisense oligonucleotide, optionally a spacer.
31. The method of claim 30, wherein the oligonucleotide is one strand of a double-stranded oligonucleotide, optionally wherein the double-stranded oligonucleotide is an siRNA, and optionally wherein the one strand is a sense strand of the siRNA.
32. The method of any one of claims 18 to 31, wherein the oligonucleotide comprises at least one modified internucleotide linkage, optionally wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.
33. The method of any one of claims 18 to 32, wherein the oligonucleotide comprises one or more modified nucleotides, optionally wherein the modified nucleotides comprise 2 '-O-methoxyethyl ribose (MOE), locked Nucleic Acid (LNA), 2' -fluoro modification, or morpholino modification.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US202063074436P | 2020-09-03 | 2020-09-03 | |
| US63/074,439 | 2020-09-03 | ||
| US63/074,436 | 2020-09-03 | ||
| PCT/US2021/049141 WO2022051665A1 (en) | 2020-09-03 | 2021-09-03 | Methods of preparing protein-oligonucleotide complexes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116457015A true CN116457015A (en) | 2023-07-18 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202180074252.7A Pending CN116457015A (en) | 2020-09-03 | 2021-09-03 | Method for preparing protein-oligonucleotide complex |
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| CN (1) | CN116457015A (en) |
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