Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-12, 18-22, 24-25 and 34 are pending and being acted upon in this Office Action.
Priority
Applicant’ claim priority to provisional application 63/074,439, filed September 3, 2020, and 63/074,436, filed September 3, 2020 is acknowledged.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on June 26, 2026 has been considered by the examiner and an initialed copy of the IDS is included with this Office Action.
Rejection Withdrawn
The rejection of claim 11 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph is withdrawn in light of the claim amendment.
The rejection of claims 1-4, 7, 10 and 12 under 35 U.S.C. 103 as being unpatentable over Nadkarni (WO2017109619 publication, of record, published June 29, 2017; PTO 892) in view of Geall (US20210095283, of record, claimed earliest priority to 62/613,742, filed January 4, 2018; PTO 892) and Gagnon et al (Current Pharmaceutical Biotechnology 10: 440-446, 2009; PTO 1449) is withdrawn in view of the claim amendment. In particular, none of the references above teach anti-TfR antibody Fab.
The provisional rejection of claims 1-12, 18-22, 24-25 and 34 on the ground of nonstatutory double patenting as being unpatentable over claim 29 of copending Application No. 17/616,870 (hereinafter US20220306685) is withdrawn in light of the claim amendment.
Claim rejections under - 35 U.S.C. 112
The following is a quotation of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), first paragraph:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-12, 18-22, 24-25 and 34 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
The Written Description Guidelines for examination of patent applications indicates, “the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical characteristics and/or other chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show applicant was in possession of the claimed genus.” (see MPEP 2163).
Claim 1 encompasses a method of processing complexes each comprising an antibody covalently linked to any one or more charge-neutral oligonucleotides, wherein the antibody is a Fab fragment of any anti-transferrin receptor antibody, wherein the one or more charge-neutral oligonucleotides are phosphorodiamidate morpholino oligomers (PMOs), and wherein the one or more charge-neutral oligonucleotides are 8 to 30 nucleotides in length, the method comprising: (i) contacting a mixture comprising an organic solvent, the complexes and unlinked charge- neutral oligonucleotides with a mixed-mode resin that comprises positively-charged metal sites and negatively charged ionic sites, under conditions in which the complexes adsorb to the mixed-mode resin, and (ii) eluting the complexes from the mixed-mode resin under conditions in which the complexes dissociate from the mixed-mode resin.
Claim 2 encompasses the method of claim 1, wherein the organic solvent is Dimethylacetamide (DMA), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN), or propylene glycol (PG).
Claim 3 encompasses the method of claim 1, wherein the organic solvent is at 5%-30% (v/v) in the mixture in step (i), optionally wherein the organic solvent is at 15% (v/v) in the mixture in step (i).
Claim 4 encompasses the method of claim 1, wherein the mixture in step (i) further comprises up to 10 mM phosphate ions and/or up to 20 mM chloride ions.
Claim 5 encompasses the method of claim 1, further comprising washing the mixed-mode resin between step (i) and step (ii) with a washing solution comprising an organic solvent, optionally wherein the organic solvent is Dimethylacetamide (DMA), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN), or propylene glycol (PG).
Claim 6 encompasses the method of claim 5, wherein the organic solvent is at 5%-30% (v/v) in the washing solution, optionally wherein the organic solvent is at 15% (v/v) in the washing solution.
Claim 7 encompasses the method of claim 5, wherein the washing solution further comprises up to 10 mM phosphate ions and/or up to 20 mM chloride ions.
Claim 8 encompasses the method of claim 1, wherein step (ii) comprises applying an elution solution to the mixed-mode resin to elute the complexes, wherein the elution solution comprises an organic solvent, optionally wherein the organic solvent is Dimethylacetamide (DMA), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN), or propylene glycol (PG).
Claim 9 encompasses the method of claim 8, wherein the organic solvent is at 10%-30% (v/v) in the elution solution, optionally wherein the organic solvent is at 10% (v/v) in the elution solution.
Claim 10 encompasses the method of claim 8, wherein the elution solution comprises at least 30 mM phosphate ions, optionally wherein the elution solution comprises at least 100 mM phosphate ions.
Claim 11 encompasses the method of claim 8, comprising increasing the concentration of phosphate ions in the elution solution, optionally wherein the concentration of the phosphate ions increases is increased from at least 10 mM to at least 100 mM.
Claim 12 encompasses the method of claim 8, wherein the elution solution has a pH of 7.6- 8.5.
Claim 18 encompasses a method of processing complexes each comprising an antibody covalently linked to any one or more oligonucleotides, wherein the antibody is a Fab fragment of any anti-transferrin receptor antibody, wherein the one or more charge-neutral oligonucleotides are phosphorodiamidate morpholino oligomers (PMOs), and wherein the one or more charge-neutral oligonucleotides are 8 to 30 nucleotides in length, the method comprising:
contacting a mixture with a mixed-mode resin that comprises positively-charged metal sites and negatively charged ionic sites, under conditions in which the complexes adsorb to the mixed-mode resin, wherein the mixture comprises the complexes, unlinked oligonucleotides, and trace amounts of unlinked antibodies that comprise an alkyne group; and
(ii) eluting the complexes from the mixed-mode resin under conditions in which the complexes dissociate from the mixed-mode resin, wherein the mixture in step (i) is produced by a method comprising:
(a) obtaining a first intermediate comprising an oligonucleotide covalently linked to a cleavable linker comprising a valine-citrulline sequence;
(b) linking the first intermediate obtained in step (a) with a compound comprising a bicyclononyne to obtain a second intermediate; and
(c) linking the second intermediate obtained in step (b) to an antibody to obtain the complexes;
wherein the compound comprising the bicyclononyne is present in the reaction of step (c) in an amount that is less than 5% of the starting amount of the compound in step (b), optionally wherein the oligonucleotide is covalently linked to the cleavable linker comprising the valine- citrulline sequence at the 5' end and/or the antibody is linked via a lysine.
Claim 19 encompasses the method of claim 18, wherein the mixture in step (i) has not been subjected to previous purification.
Claim 20 encompasses the method of 18, wherein the mixture of step (i) comprises trace amounts of phosphate ions and/or chloride ions.
Claim 21 encompasses the method of claim 18, further comprising washing the mixed-mode resin between step (i) and step (ii) with a washing solution comprising up to 20 mM phosphate ions and/or up to 30 mM chloride ions, optionally wherein the solution comprises up to 10 mM phosphate ions and/or up to 25 mM chloride ions.
Claim 22 encompasses the method of claim 21, wherein the washing solution has a pH of 5.0-7.6.
Claim 24 encompasses the method of claim 18, wherein step (ii) comprises applying an elution solution comprising at least 30 mM phosphate ions and/or at least 50 mM chloride ions to the mixed-mode resin to elute the complexes, optionally wherein the elution solution comprises at least 100 mM phosphate ions and/or at least 100 mM chloride ions.
Claim 25 encompasses the method of claim 24, wherein the elution solution has a pH of 7.5-8.5.
Claim 34 encompasses a method of processing complexes each comprising an antibody covalently linked to one or more oligonucleotides, wherein the antibody is a Fab fragment of an anti-transferrin receptor antibody, wherein the one or more charge-neutral oligonucleotides are phosphorodiamidate morpholino oligomer (PMOs), and wherein the one or more charge-neutral oligonucleotides are 8 to 30 nucleotides in length, the method comprising:
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The specification defines antibody as follow:
[0051] Antibody: As used herein, the term “antibody” refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., paratope that specifically binds to an antigen. In some embodiments, an antibody is a full-length antibody, e.g., a full-length IgG. In some embodiments, an antibody is a chimeric antibody. In some embodiments, an antibody is a humanized antibody. However, in some embodiments, an antibody is a Fab fragment, a F(ab′)2 fragment, a Fv fragment or a scFv fragment. In some embodiments, an antibody is a nanobody derived from a camelid antibody or a nanobody derived from shark antibody. In some embodiments, an antibody is a diabody. In some embodiments, an antibody comprises a framework having a human germline sequence. In another embodiment, an antibody comprises a heavy chain constant domain selected from the group consisting of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constant domains. In some embodiments, an 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, an antibody comprises a constant domain, e.g., an Fc region. An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of an antibody described herein can be an alpha (a), delta (A), epsilon (E), gamma (γ) or mu (p) heavy chain. In some embodiments, the heavy chain of an antibody described herein can comprise a human alpha (a), delta (A), epsilon (E), gamma (γ) or mu (p) heavy chain. In a particular embodiment, an antibody described herein comprises a human gamma 1 CH1, CH2, and/or CH3 domain. 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, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A et al., (1991) supra. In some embodiments, the 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, an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and/or methylation. In some embodiments, an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecule are conjugated to the antibody via N-glycosylation. O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and/or phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecule are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan. In some embodiments, the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit. In some embodiments, an antibody is a construct that comprises a polypeptide comprising one or more antigen binding fragments of the disclosure covalently linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Example 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). Still further, an antibody may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal poly histidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).
Regarding “charge-neutral oligonucleotide”, the specification defines as follow:
[0088] Charge-neutral oligonucleotide: As used herein, the term “charge-neutral oligonucleotide” refers to oligonucleotide analogs comprising charge-neutral backbones at a physiological pH (e.g., pH 7.35-pH 7.45). Examples of charge-neutral oligonucleotides include, without limitation, phosphorodiamidate morpholino oligomers (PMOs) and peptide nucleic acids (PNA), e.g., as described in Jarver et al., (Nucleic Acid Therapeutics, Vol. 25, No. 2, 2015), incorporated herein by reference.
Regarding “organic solvent”, the specification discloses:
[0089] Organic Solvent: As used herein, the term “organic solvent” refers to a carbon-based substance that is capable of dissolving other substances. By being carbon based, organic solvents have carbon atoms present in the structure of their compound. Non-limiting examples of organic solvents that can be used in accordance with the present disclosure include, without limitation, Dimethylacetamide (DMA), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN), or propylene glycol (PG).
[0126] Organic solvents commonly used in chromatography methods can be used throughout the methods described herein. In some embodiments, the organic solvent used in step (i) of the methods of processing complexes described herein is Dimethylacetamide (DMA), isopropyl alcohol (IPA), dimethyl sulfoxide (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).
Thus, the claims encompass a method of processing complexes each comprising any Fab from any anti-TfR antibody covalently linked to any one or more charge-neutral oligonucleotide via any linker using a mixed-mode resin (hydroxyapatite resin) in any conditions and methods of making said complexes.
Regarding antibody, the specification discloses mouse transferrin receptor antibody. The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 34. The six CDRs are shown in Table 3.
The humanized anti-TfR antibody comprises a heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35 and a light chain variable region comprises the amino acid sequence of SEQ ID NO: 36.
The specification discloses several alternative strategies for the purification of the crude mixture of a complex comprising anti-TfR Fab covalently linked to oligonucleotide, unlinked oligonucleotide, and unlinked anti-TfR Fab from Example 1 were examined, including cation exchange (CEX) and anion exchange (AEX) resins. It was found that none of the alternative strategies was as effective as the approach described here (using ceramic hydroxyapatite resin).
The specification exemplifies Synthesis of a Complex Comprising an Antibody Linked to a Charge-Neutral Oligonucleotide (Conjugation Method 2—Two Step Conjugation)
[0505] A muscle-targeting complex was generated comprising an oligonucleotide (e.g., a charge-neutral oligonucleotide) covalently linked, via a cathepsin cleavable linker, to an anti-transferrin (anti-TfR) receptor Fab antibody. Anti-TfR Fabs can be recombinantly produced (e.g., in CHO cells) and purified. The oligonucleotide used is a phosphorodiamidate morpholino oligomer (PMO) that is 30 nucleotides in length.
[0506] The anti-TfR Fab was diluted with propylene glycol to a final concentration of 40% v/v propylene glycol and incubated with 5-fold molar excess of endo-BCN-PEG3-PFP (endo-bicyclononyne-PEG3-pentafluorophenyl, dissolved in DMSO at a concentration of 20 mg/mL] for 2 hr at room temperature (˜22.5° C.). It was anticipated that labeling should yield 2.0-2.5 moles of BCN per mole of Fab. Post labeling, the reaction product was sterile filtered or depth filtered to remove precipitated BCN. The filtered solution was then assayed for average reactive BCN moieties analytically using LCMS (ThermoFisher MAbPac RP 4 um 2.1×100 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° C.; in-source CID 20 eV; positive polarity; spray voltage 3.5 kV; scan range 1000-3000 m/z).
[0507] Anti-TfR having a degree of labeling (D0L) of >2.3 was taken to the next step of the conjugation, and was purified into 10% isopropanol in PBS at pH 7.2 by tangential flow filtration using a 10 kDa molecular weight cutoff (1.2 bar), with 5 filtrate volumes, to remove free BCN and propylene glycol. Complete removal of BCN and propylene glycol was verified by analytical HPLC-SEC (Waters Xbridge Protein BEH SEC 3.5 um, 7.8×300 mm, 0.3 mL/min, 100 mM PO.sub.4, 100 mM NaCl, 15% v/v acetonitrile pH 7.0). SEC traces of the crude and purified products are shown in FIG. 8. The recovery of BCN-labeled anti-TfR was >90% of starting material. The purified solution was concentrated to 3.5 mg/mL for further conjugation steps.
[0508] In a separate reaction, the oligonucleotide (e.g., a charge-neutral oligonucleotide) was conjugated to a linker molecule. The oligonucleotide was dissolved at 35 mg/mL in anhydrous DMSO at 37° C. The linker molecule (azide-PEG3-Val-Cit-PAB-PNP) was dissolved at 40 mg/mL in anhydrous DMF and was added at 2.7-fold molar excess to the oligonucleotide with 3-fold molar excess of N,N-Diisopropylethylamine (DIPEA). This linker conjugation reaction was allowed to proceed for 2 hours at room temperature (˜22.5° C.). Progress and completion of the reaction was measured using a ninhydrin assay (Kaiser test) prior to quenching the reaction via acetone precipitation.
[0509] Precipitation was conducted by adding 8 volumes of chilled acetone to the product solution, and the precipitate was pelleted by centrifugation at 3500×g at 8° C. for 20 minutes. The pellet was then washed with 3 volumes of acetone to remove remaining free linker and was centrifuged again at 3500× g at 8° C. for 20 minutes. The purified oligonucleotide-linker was then dissolved in 20% v/v acetonitrile in nuclease-free water at a concentration of 30 mg/ml. The concentration and yield were measured by optical density (OD) in 0.1N HCl, demonstrating a yield of greater than 90%. Analytical RP-HPLC was conducted (Waters BEH-C18, 4.6 mm×150 mm, 0.5 mL/min. 5-90% v/v acetonitrile in water, 30 minute run time) on the crude linker/oligonucleotide conjugation reaction product (FIG. 9) and the purified oligonucleotide-linker (FIG. 10) to confirm removal of free linker by the precipitation and washing steps. Confirmatory LCMS of the purified oligonucleotide-linker was also conducted (FIG. 11).
[0510] To conjugate the anti-TfR and the oligonucleotide, BCN-labeled antibody was mixed with 5-fold molar excess of oligonucleotide-PAB-VC-PEG3-azide (FIG. 1A) in a glass bottle overnight at room temperature (˜22.5° C.).
[0511] Completion of the reaction was evaluated by SDS-PAGE (FIG. 12) and analytical SEC analysis (FIG. 13), which demonstrated less than 10% unlinked anti-TfR antibody (DAR0) and a 90% coupling efficiency by densitometry.
Example 8. Conjugation Process for Preparation of a Fab-Oligonucleotide (Charge-Neutral Oligonucleotide) Conjugate
(Conjugation Method 1—Pre-Reaction Conjugation)
[0512] This example describes the preparation of a conjugate composed of an oligonucleotide covalently linked via a val-cit cathepsin cleavable peptide linker to an anti-transferrin receptor (anti-TfR) Fab antibody. Anti-TfR Fabs can be recombinantly produced (e.g., in CHO cells) and purified. The oligonucleotide used is a phosphorodiamidate morpholino oligomer (PMO) that is 30 nucleotides in length. Prior to the conjugation with the Fab, an intermediate containing the oligonucleotide and the linker is generated via a copper-free 3+2 click reaction between the azide group of an oligonucleotide-PAB-VC-PEG3-azide molecule (FIG. 1A) and the strained bicyclononyne moiety on an endo-BCN-PEG4-PFP ester (FIG. 1B) heterobifunctional crosslinker (“the pre-reaction”). Lyophilized oligonucleotide-PAB-VC-PEG3-azide (98.1 mg) was solubilized in a 4 mL glass Wheaton vial in 0.32 mL of MilliQ water. Following solubilization, 0.32 mL of N,N-dimethylacetamide (DMA) was added and the mixture was gently agitated for 5-10 minutes. Prior to continuing, the vial was inspected carefully to ensure the oligonucleotide-PAB-VC-PEG3-azide was completely dissolved and no residue remained on the walls of the glass vial. The concentration of the oligonucleotide-PAB-VC-PEG3-azide stock solution in 1:1 DMA:water was determined with a Nanodrop UV/vis instrument by using aliquots diluted 25-, 50-, and 100-fold in 1:1 DMA:water containing a final concentration of 0.1 M HCl at 265 nm, using an extinction coefficient of 318,050 M.sup.−1 cm.sup.−1. The HCl was added to ensure accuracy of the concentration measurement. The calculated concentration at each dilution was averaged to determine the solution concentration of 10.1 mM.
[0513] A 32.5 mg/mL (53.5 mM) stock solution of endo-BCN-PEG4-PFP ester was prepared by weighing approximately 25 mg of endo-BCN-PEG4-PFP ester oil into a 4 mL glass Wheaton vial. The appropriate volume of DMA was then added to afford the 32.5 mg/mL stock solution.
[0514] The pre-reaction was conducted with the following final solution reaction conditions: 5.87 μM (6.5 μmol) oligonucleotide-PAB-VC-PEG3-azide, 5.34 mM endo-BCN-PEG4-PFP ester (1.1:1.0 mol:mol equivalents) in 60:40 v/v % DMA to 25 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.5 buffer at room temperature. The reaction was set-up in a 4 mL glass Wheaton vial by adding the appropriate amounts of the reactants and stock solutions as indicated in Table 6. The total final volume of the pre-reaction was 1.11 ml.
Example 9. Purification of Anti-TfR Fab-Oligonucleotide Conjugate
[0520] Following synthesis of the anti-TfR Fab-oligonucleotide conjugate described in Example 8, a two-part purification process was conducted. First, free payload was removed by hydroxyapatite (HA) chromatography. The HA eluate was then buffer exchanged into the final formulation. At the 45 mg scale of Fab, the final buffer exchange was performed with a 30 kDa centrifugal filter device. Prior to loading onto the HA column, the crude reaction product from Example 8 (anti-TfR Fab-oligonucleotide conjugate) was diluted and the pH adjusted from 7.5 to 5.7. First, the 7.5 mL of crude conjugate was diluted by addition of 16.5 mL of 15 v/v % DMA in water and the solution was thoroughly mixed. To this mixture, 0.75 mL of 500 mM MES (pH 3.3) was added to adjust the pH down to 5.7.
[0521] Chromatographic purification to remove unreacted oligonucleotide species was performed using a 5 mL Bio Rad CHT Type I (ceramic hydroxyapatite) cartridge on an AKTA Pure chromatography system. Prior to loading the diluted conjugate pool from the reaction mixture preparation step, the CHT cartridge was prepared and equilibrated according to the manufacturer's instructions using 15:85 v/v % of DMA to 10 mM sodium phosphate, pH 5.8 buffer. Following equilibration, the conjugate pool was loaded at a flow rate of 5 mL/min. After loading the conjugate, the column was washed for a minimum of 7 CV with 15:85 v/v % of DMA in 10 mM sodium phosphate buffer (pH 5.8). After completion of the wash, elution was initiated via a step gradient with 100 mM sodium phosphate, pH 7.6 buffer containing DMA at 15:85 v/v % at a flow rate of 5 mL/min. The entire elution peak, identified by monitoring at 260 nm and 280 nm, was collected and pooled.
[0522] Analysis of the flow through during the HA column loading step by SEC (FIG. 16) indicated the presence of little to no Fab-oligonucleotide conjugate in the flow-through. Only peaks due to oligonucleotide payload species were observed, at ˜10.5 and ˜11.3 minutes. Conversely, SEC analysis of the pooled elution peak shows only conjugate species (FIG. 17), with multiple peaks and shoulders due to the size differences of the conjugates with different oligonucleotide (e.g., PMO) payload loadings. No peaks for payload species at 10.5 or 11.3 minutes were observed.
However, the specification does not disclose a representative number of species of Fab from any and all anti-transferrin receptor and one or more oligonucleotides having phosphorodiamidate morpholino oligomers (PMOs) that are charge-neutral and are 8 to 30 nucleotides in length that are used in the claimed methods, nor sufficient relevant identifying characteristics in the form of structure, e.g., amino acid sequences of the heavy and light chain variable regions for any and all anti-TfR antibodies Fab fragment and nucleotide sequences of any and all potential oligonucleotides having phosphorodiamidate morpholino oligomers (PMOs) that are charge-neutral and are 8 to 30 nucleotides in length for treating subjects having rare muscle diseases, including myotonic dystrophy (e.g., myotonic dystrophy type 1), Facioscapulohumeral muscular dystrophy (FSHD), Pompe disease, Centronuclear myopathy, Fibrodysplasia Ossificans Progressiva, Friedreich's ataxia, and Duchenne muscular dystrophy. In some embodiments, depending on the condition to be treated, different oligonucleotides may be used in such complexes. The specification does not describe the structure of oligonucleotides that are share by members of the genus that capable of treating any diseases above.
The disclosure of just anti-TfR Fab covalently linked to phosphorodiamidate morpholino oligomer (PMO) that is 30 nucleotides in length via a linker comprising azido-PEG3-Val-Cit-PAB-PNP
and BCN-PEG4-PFP together as shown in Formula D in claim 34 is not representative of the genus of antibody covalently linked to one or more charge-neutral oligonucleotides or oligonucleotides payload. Clearly, just any charge-neutral oligonucleotides of 8 to 30 nucleotides in length covalently linked to any Fab of any anti-transferrin receptor will not predictably function to modulate expression or activity of any and all genes in muscle cells, e.g., in a subject having or suspected of having a muscle disease, such as rare muscle diseases, including myotonic dystrophy (e.g., myotonic dystrophy type 1), Facioscapulohumeral muscular dystrophy (FSHD), Pompe disease, Centronuclear myopathy, Fibrodysplasia Ossificans Progressiva, Friedreich's ataxia, and Duchenne muscular dystrophy. The specification does not disclose, and the art does not teach, the genus as broadly encompassed in the claims.
Regarding conditions for purifying any and all possible antibody covalently linked to any oligonucleotides via any linkers, Wiener et al (Scientific Reports 10: 1457, 2020; PTO 892) teaches that one bottleneck in the development of antibody-oligonucleotide conjugates is the conjugation reaction between the two molecules. All antibodies and oligonucleotides have to be functionalized first with the respective reactive chemical group. Despite the vast variety of commercially-available crosslinking reagents, the oligonucleotide labeling of antibodies is not robust. The reason for this is multilayered, including problems related to a loss in the specificity of the antibody due to the masking of the antigen binding site, a change in polarity upon the addition of the reactive conjugation group and oligonucleotide, and a lack of purification methods for the removal of excess oligonucleotides, which increases the rate of false positive errors. Another problem is that antibody-oligonucleotide conjugation is a consecutive reaction with a heterogeneous outcome of single-, multiple-, and non-labeled antibodies depending on the reaction conditions. The temperature, time, and molar stoichiometries of the antibody and oligonucleotide make the conjugation reaction a multiparameter optimization problem.
Wiener further teaches that the yield and product types of the click reaction are dependent on the number of factors, including the click conjugation reaction of antibodies with oligonucleotides, the crosslinker used, reaction temperature, duration, oligonucleotide length, and secondary structure and the removal of excess click reagents after antibody and oligonucleotide functionalization, see entire document, abstract, p. 2.
For example, an increased number of click functional group, e.g., DBCO molecules conjugated to the antibody reduces its solubility. Typically crosslinkers are dissolved in organic solvent such as dimethyl sulfoxide (DMSO), see p. 3. Removal of excess click reagents after antibody and oligonucleotide functionalization was essential for increasing the yield of the subsequent click reaction by reducing the occurrence of unwanted reactions with left-over reactants.
Thus, the specification does not disclose a representative number of species of charge-neutral oligonucleotides covalently linked to Fab of anti-TfR antibody comprising any cleavable linker comprising a valine-citrulline sequence to be used for treating muscle disease in the claimed methods. Given the lack of guidance as to the binding specificity of the antibody and sequence specificity of the oligonucleotide, and the delivery of oligonucleotides to targeted tissues, one of skill in the art would conclude that the specification fails to disclose a representative number of species of antibody-oligonucleotide conjugates to describe the genera encompassed by the claimed methods at the time of filing.
Vas-Cath Inc. v. Mahurkar, 19 USPQ2d 1111, makes clear that “applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the written description inquiry, whatever is now claimed.” (See page 1117.) The specification does not “clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed.” (See Vas-Cath at page 1116.).
Adequate written description requires more than a mere statement that it is part of the invention and reference to a potential method for isolating it. See Fiers v. Revel, 25 USPQ2d 1601, 1606 (CAFC 1993) and Amgen Inc. v. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016.
One cannot describe what one has not conceived. See Fiddles v. Baird, 30 USPQ2d 1481, 1483. In Fiddles v. Baird, claims directed to mammalian FGF’s were found unpatentable due to lack of written description for the broad class. The specification provided only the bovine sequence. Thus, the specification fails to describe these DNA sequences.
For genus claims, an adequate written description of a claimed genus requires more than a generic statement of an invention's boundaries. A patent must set forth either a representative number of species falling within the scope of the genus or structural features common to the members of the genus. Kubin, Exparte, 83 USPQ2d 1410 (Bd. Pat. App. & Int. 2007); Ariad Pharms., Inc. v. Eli Lilly& Co., 598 F.3d 1336, 1350 (Fed. Cir. 2010).
Therefore, only (1) a method of processing complexes each comprising a Fab that binds to transferrin receptor covalently linked to one or more phosphorodiamidate morpholino oligonucleotide (PMO) that is 30 nucleotides in length, the method comprises: (1) contacting a mixture comprising an organic solvent, the complexes and unlinked PMOs with a ceramic hydroxyapatite resin that comprises positively-charged metal sites and negatively charged ionic sites, under condition in which the complexes absorb to the resin, (ii) eluting the complexes with a step gradient of 100 mM sodium phosphate, pH 7.6 buffer containing dimethylacetamide (DMA) at 15:85 v/v %, (2) the method above further comprising washing the resin between step (i) and step (ii) with a washing solution comprising 15:85 v/v % of DMA in 10 mM sodium phosphate buffer at pH 5.8, (3) the method above wherein the mixture in step (i) is produced by a method comprising (a) obtaining a first intermediate comprising reacting one or more phosphorodiamidate morpholino oligomer (PMO) that is 30 nucleotides in length with a cleavable linker comprising PAB-VC-PEG3-azide (b) linking the first intermediate comprising PMO-PAB-VC-PEG3-azide molecule obtained in step (a) with a compound comprising endo-bicyclononyne (BCN)-PEG3-pentafluorophenyl ester (endo-BCN-PEG3-PFP) to obtain a second intermediate, and (c) linking the second intermediate in step (b) to an antibody or antigen binding fragment thereof that binds to transferrin receptor to obtain the complexes; wherein the compound comprising BCN is present in the reaction of step (c) in an amount that is less than 5% of the starting amount of the compound in step (b), wherein the PMOs are linked to the cleavable linker comprising the valine-citrulline sequence at the 5’ end of the PMOs and the antibody is linked via a lysine, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 35 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 36, but not the full breadth of the claims meets the written description provision of 35 U.S.C. § 112, first paragraph.
Applicants’ arguments filed June 26, 2026 have been fully considered but are not found persuasive.
Applicants’ position is that claims 1, 18, and 34 have been amended to structurally define the antibody and one or more charge-neutral oligonucleotides. Specifically, the amended claims now recite, in pertinent part, that the antibody is a Fab fragment of an anti-transferrin receptor antibody, wherein the one or more charge-neutral oligonucleotides are phosphorodiamidate morpholino oligomers (PMOs), and wherein the one or more charge-neutral oligonucleotides are 8 to 30 nucleotides in length.
The instant specification provides sufficient written description for each feature currently recited in amended claims. See, e.g., paragraphs [0051] (definition of antibody); [0114] (describing the suitability of the claimed methods for isolating complexes comprising an antibody covalently linked to one or more oligonucleotides, and optional structural features of the antibody, i.e., Fab fragments of anti-transferrin receptor antibodies); section starting at [00180] (additional disclosure of anti-transferring receptor antibodies); [0072] (definition of oligonucleotide); [0088] (definition of charge-neutral oligonucleotide); [00108] (describing PMO oligonucleotides of the relevant lengths); section starting at [00296] (additional disclosure of oligonucleotide size/structure); and Examples 7- 9, which demonstrate of the synthesis, conjugation, and purification of a complex comprising an anti-transferrin (anti-TfR) receptor Fab antibody covalently linked to a PMO oligonucleotide as claimed. Applicant respectfully notes Examples 7-9 exemplify the efficacy of the claimed methods, and submits that recitation of further specific conditions and ranges of buffer constituents would be unduly limiting in the claims. Adequate written description does not require that Applicant describe and exemplify every species within a claimed genus. MPEP § 2163.
Applicant respectfully submits that based upon the teachings of the present Application and knowledge on the art at the time of filing, a person of ordinary skill in the art would have recognized Applicant to be in possession of the complexes subjected to the claimed methods; thus, the claims as presently amended comply with the written description requirement. Accordingly, withdrawal of this rejection is respectfully requested.
In response, the amendment to claims 1, 18, and 34 is acknowledged.
Regarding Fab fragment from any anti-transferrin antibody, the specification discloses just one humanized anti-TfR antibody comprises a heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35 and a light chain variable region comprises the amino acid sequence of SEQ ID NO: 36.
However, the specification does not describe i. Complete structure, e.g., heavy and light chains variable domains, ii. Partial structure, e.g., six CDRs and functional features share by members of the genus that correlated with binding to any and all transferrin receptor. The specification does not describe a representative number of species falling within the scope of the genus or structural features common to the members of the genus so the one of skill in the art can visualize or recognize the member of the genus of the actual claimed Fab.
At the time the invention was made, it was known in the art that antibodies have a large repertoire of distinct structures and that a huge variety of antibodies can be made to bind to a single epitope.
For example, Lloyd et al. taught that hundreds of functional antibody fragments can be isolated from an antibody library that bind to the same antigen wherein these antibodies have distinct heavy and light chain sequences (Lloyd et al. Protein Engineering, Design & Selection 22:159-168, 2009; PTO 892; see, e.g., Discussion).
For example, Piche-Nicholas et al MABS 10(1): 81-94, 2018; PTO 892) teaches altering complementary-determining region (CDRs) by 1-5 mutations significantly alter binding affinity to FcRn in vitro, see entire document, abstract, p. 95, right col, in particular. Engineering CDRs by modify local charge and thus maintain affinity to FcRn at 400 nM or weaker in vitro while retaining antigen binding may have far-reaching implications in the half-life optimization efforts of IgG therapeutics with respect to in vivo pharmacokinetics, see p. 90, in particular. Given that hundreds of unique antibody structures may bind a single antigen, the structure of an antibody cannot be predicted from the structure of the antigen (as held in Amgen), and a single species, or small group of species, cannot define a structure-function relationship so as to be representative of all the antibodies that bind to that antigen (as held in Abbvie).
Regarding one or more charge-neutral oligonucleotides are phosphorodiamidate morpholino oligomers (PMOs), the specification des not describe the structure, e.g., nucleotide sequence of any and all possible one or more charge-neutral oligonucleotides are phosphorodiamidate morpholino oligomers (PMOs), nor describe the structure common to members of the genus. The specification does not disclose a representative number of species of Fab from any and all anti-transferrin receptor covalently linked to one or more oligonucleotides having phosphorodiamidate morpholino oligomers (PMOs) that are charge-neutral and are 8 to 30 nucleotides in length that can be used for treating subjects having rare muscle diseases, including myotonic dystrophy (e.g., myotonic dystrophy type 1), Facioscapulohumeral muscular dystrophy (FSHD), Pompe disease, Centronuclear myopathy, Fibrodysplasia Ossificans Progressiva, Friedreich's ataxia, and Duchenne muscular dystrophy. In some embodiments, depending on the condition to be treated, different oligonucleotides may be used in such complexes. The specification does not describe the structure of oligonucleotides that are share by members of the genus of oligonucleotides that capable of treating any diseases above.
Regarding cleavable linker comprising a valine-citrulline sequence, and compound comprising bicyclononyne (claim 18), the specification discloses just azide-PEG3-Val-Cit-PAB-PNP and endo-BCN-PEG3-PFP having the structures show in claim 34.
However, "[A] sufficient description of a genus ... requires the disclosure of either a representative number of species falling within the scope of the genus or structural features common to the members of the genus so that one of skill in the art can 'visualize or recognize' the members of the genus." Ariad, 598 F.3d at 1350 (quoting Eli Lilly, 119 F.3d at 1568-69). An adequate written description must contain enough information about the actual makeup of the claimed products - "a precise definition, such as structure, formula, chemic name, physical properties of other properties, of species falling with the genus sufficient to distinguish the gene from other materials", which may be present in "functional terminology when the art has established a correlation between structure and function" (Amgen page 1361).
Regarding conditions for purifying any and all possible antibody covalently linked to any oligonucleotides via any linkers, Wiener et al (of record, Scientific Reports 10: 1457, 2020; PTO 892) teaches that one bottleneck in the development of antibody-oligonucleotide conjugates is the conjugation reaction between the two molecules. All antibodies and oligonucleotides have to be functionalized first with the respective reactive chemical group. Despite the vast variety of commercially-available crosslinking reagents, the oligonucleotide labeling of antibodies is not robust. The reason for this is multilayered, including problems related to a loss in the specificity of the antibody due to the masking of the antigen binding site, a change in polarity upon the addition of the reactive conjugation group and oligonucleotide, and a lack of purification methods for the removal of excess oligonucleotides, which increases the rate of false positive errors. Another problem is that antibody-oligonucleotide conjugation is a consecutive reaction with a heterogeneous outcome of single-, multiple-, and non-labeled antibodies depending on the reaction conditions. The temperature, time, and molar stoichiometries of the antibody and oligonucleotide make the conjugation reaction a multiparameter optimization problem.
Wiener further teaches that the yield and product types of the click reaction are dependent on the number of factors, including the click conjugation reaction of antibodies with oligonucleotides, the crosslinker used, reaction temperature, duration, oligonucleotide length, and secondary structure and the removal of excess click reagents after antibody and oligonucleotide functionalization, see entire document, abstract, p. 2.
For example, an increased number of click functional group, e.g., DBCO molecules conjugated to the antibody reduces its solubility. Typically crosslinkers are dissolved in organic solvent such as dimethyl sulfoxide (DMSO), see p. 3. Removal of excess click reagents after antibody and oligonucleotide functionalization was essential for increasing the yield of the subsequent click reaction by reducing the occurrence of unwanted reactions with left-over reactants.
Vas-Cath Inc. v. Mahurkar, 19 USPQ2d 1111, makes clear that “applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the ‘written description’ inquiry, whatever is now claimed.” (see page 1117). The specification does not “clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed.” (see Vas-Cath at page 1116).
Adequate written description requires more than a mere statement that it is part of the invention and reference to a potential method for isolating it. See Fiers v. Revel, 25 USPQ2d 1601, 1606 (CAFC 1993) and Amgen Inc. v. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016.
One cannot describe what one has not conceived. See Fiddles v. Baird, 30 USPQ2d 1481, 1483. In Fiddles v. Baird, claims directed to mammalian FGF’s were found unpatentable due to lack of written description for the broad class. The specification provided only the bovine sequence.
For these reasons, the rejection is maintained.
New Ground of Rejection and Objection Necessitated by Amendment filed June 26, 2026
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1, 4, 5, 7-8 and 10-12 are rejected under 35 U.S.C. 102 (a)(2) as being anticipated by US20220306685 (claimed earliest priority to 62/858,964 filed June 7, 2019; PTO 892).
Claim 1 recites a method of processing complexes each comprising an antibody covalently linked to one or more charge-neutral oligonucleotides, wherein the antibody is a Fab fragment of an anti-transferrin receptor antibody, wherein the one or more charge-neutral oligonucleotides are phosphorodiamidate morpholino oligomers (PMOs), and wherein the one or more charge-neutral oligonucleotides are 8 to 30 nucleotides in length, the method comprising: (i) contacting a mixture comprising an organic solvent, the complexes and unlinked charge- neutral oligonucleotides with a mixed-mode resin that comprises positively-charged metal sites and negatively charged ionic sites, under conditions in which the complexes adsorb to the mixed-mode resin, and(ii) eluting the complexes from the mixed-mode resin under conditions in which the complexes dissociate from the mixed-mode resin.
Claim 4 recites the method of claim 1, wherein the mixture in step (i) further comprises up to 10 mM phosphate ions and/or up to 20 mM chloride ions.
Claim 5 recites the method of claim 1, further comprising washing the mixed-mode resin between step (i) and step (ii) with a washing solution comprising an organic solvent, optionally wherein the organic solvent is Dimethylacetamide (DMA), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN), or propylene glycol (PG).
Claim 7 recites the method of claim 5, wherein the washing solution further comprises up to 10 mM phosphate ions and/or up to 20 mM chloride ions.
Claim 8 recites the method of claim 1, wherein step (ii) comprises applying an elution solution to the mixed-mode resin to elute the complexes, wherein the elution solution comprises an organic solvent, optionally wherein the organic solvent is Dimethylacetamide (DMA), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN), or propylene glycol (PG).
Claim 10 recites the method of claim 8, wherein the elution solution comprises at least 30 mM phosphate ions, optionally wherein the elution solution comprises at least 100 mM phosphate ions.
Claim 11 recites the method of claim 8, comprising increasing the wherein the elution solution comprises a gradually increasing concentration of phosphate ions in the elution solution, optionally wherein the concentration of the phosphate ions increases is increased from at least 10 mM to at least 100 mM.
Claim 12 recites the method of claim 8, wherein the elution solution has a pH of 7.6- 8.5.
Regarding claim 1, Weeden teaches methods of processing (e.g., purifying) complexes (e.g., or plurality of complexes each comprising an antibody, e.g., anti-transferrin receptor antibody (aka anti-TfR) Fab ([0016], [0045]) covalently linked to one or more oligonucleotides are 8 to 30 nucleotides in length, e.g., [0324], wherein the one or more oligonucleotides are phosphorodiamide morpholino oligomers (PMOs, see para. [0017], [0032]) are purified and isolated from unlinked oligonucleotide using an mixed-mode resin that comprises positively-charged metal sites and negatively charged ionic sites, e.g., hydroxyapatite resin (see para. [0475], reference claims 29-51, in particular), the method of isolating (processing) plurality of complexes comprising:
[0025] (i) contacting a mixture comprising the complexes and unlinked oligonucleotides with a mixed-mode resin that comprises positively-charged metal sites and negatively charged ionic sites, under conditions in which the complexes adsorb to the mixed-mode resin, and
[0026] (ii) eluting the complexes from the mixed-mode resin under conditions in which the complexes dissociate from the mixed-mode resin, see reference claim 29.
[0027] 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 hydroxyfluoroapatite resin, a fluoroapatite resin, or a chlorapatite resin, reference claims 30-31.
[0017] In some embodiments, the oligonucleotide is single stranded. In some embodiments, the oligonucleotide is an antisense oligonucleotide, optionally a gapmer or a phosphorodiamidate morpholino oligomer (PMO), see para. [0346].
[0324] Oligonucleotides may be of a variety of different lengths, e.g., depending on the format. In some embodiments, an 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 lengths, etc.
[0016] In some embodiments, the antibody is a full length IgG, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a scFv, or a Fv fragment. In some embodiments, the antibody is an anti-transferrin receptor antibody, see para. [0242] to [0245], [0256].
Regarding claim 4, Weeden teaches:
[0028] In some embodiments, the mixture in step (i) further comprises up to 20 mM phosphate ions and/or up to 30 mM chloride ions, optionally wherein the mixture in step (i) further comprises up to 10 mM phosphate ions and/or up to 25 mM chloride ions. In some embodiments, the unlinked oligonucleotide does not adsorb to the mixed-mode resin in step (i).
Claim 5 is included as organic solvent such as Dimethylacetamide (DMA), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN), or propylene glycol (PG) is optional.
Regarding claim 7, Weeden teaches:
[0029] In some embodiments, the mixture in step (i) further comprises up to 5 mM phosphate ions and/or up to 10 mM chloride ions, optionally wherein the mixture in step (i) further comprises up to 3 mM phosphate ions and/or up to 8 mM chloride ions. In some embodiments, some or all of the unlinked oligonucleotide adsorb to the mixed-mode resin in step (i). In some embodiments, the method further comprises washing the mixed-mode resin between step (i) and step (ii) with a solution comprising up to 20 mM phosphate ions and/or up to 30 mM chloride ions, optionally wherein the solution comprises up to 10 mM phosphate ions and/or up to 25 mM chloride ions.
The reference up to 25 mM chloride ions encompasses the claimed range of 20 mM chloride ions.
[0154] In some embodiments, when some or all of the unlinked oligonucleotides also adsorb to the mix-mode resin, the methods described herein further comprises washing the mixed-mode resin between step (i) and step (ii) with a solution that would dissociate the unlinked oligonucleotides but not the complexes from the mixed-mode resin. In some embodiments, the solution used for washing comprises up to 20 mM phosphate ions and/or up to 30 mM chloride ions, e.g. 20 mM phosphate ion and 30 mM chloride ion. In some embodiments, the solution used for washing comprises up to 20 mM (e.g., up to 20 mM, up to 15 mM, up to 10 mM, or up to 5 mM) phosphate ion. Additionally, in some embodiments, the solution used for washing comprises up to 30 mM (e.g., up to 30 mM, up to 25 mM, up to 20 mM, up to 15 mM, up to 10 mM, or up to 5 mM) chloride ion. In some embodiments, the solution used for washing comprises 5-20 mM (e.g., 5-20 mM, 5-15 mM, 5-10 mM, 10-20 mM, 10-15 mM, or 15-20 mM) phosphate ion and/or 5-30 mM chloride ions (e.g., 5-30 mM, 5-25 mM, 5-20 mM, 5-15 mM, 5-10 mM, 10-30 mM, 10-25 mM, 10-20 mM, 10-15 mM, 15-30 mM, 15-25 mM, 15-20 mM, 20-30 mM, 20-25 mM, or 25-30 mM). In some embodiments, the solution used for washing comprises 20 mM, 15 mM, 10 mM, 5 mM, or 1 mM phosphate ion and/or 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM chloride ion. In some embodiments, the solution used for washing comprises 20 mM phosphate ion and/or 30 mM chloride ion, e.g., 20 mM phosphate ion and 30 mM chloride ion. In some embodiments, the solution used for washing comprises up to 10 mM phosphate ions and/or up to 25 mM chloride ions, e.g., 10 mM phosphate ions and up to 25 mM chloride ions.
Regarding claims 10-11, Weeden teaches:
[0030] In some embodiments, step (ii) comprises applying an elution solution comprising at least 30 mM phosphate ions and/or at least 50 mM chloride ions to the mixed-mode resin to elute the complexes, optionally wherein the elution solution comprises at least 100 mM phosphate ions and/or at least 100 mM chloride ions.
Regarding claim 11, Weeden teaches:
[0030] In some embodiments, step (ii) comprises applying an elution solution comprising at least 30 mM phosphate ions and/or at least 50 mM chloride ions to the mixed-mode resin to elute the complexes, optionally wherein the elution solution comprises at least 100 mM phosphate ions and/or at least 100 mM chloride ions.
Regarding claim 12, Weeden teaches:
[0480] To remove free payload, a 50 ml mixed-mode fluorapatite (FA) column was used. The eluate from the HIC column was diluted 1:3 in nuclease free water. After equilibrating the column with 10 mM sodium phosphate, 10 mM sodium chloride, pH 7.6, the diluted HIC eluate was loaded onto the column. A wash step using 10 mM sodium phosphate, 10 mM sodium chloride, pH 7.6 was performed for 5 CVs. During this wash step, the free payload loaded onto the column eluted. After washing was complete, the mAB conjugates were eluted using a 100 mM sodium phosphate, 100 mM sodium chloride, pH 7.6 (FIGS. 14 and 15).
Claim 8 is included as the organic solvent such as Dimethylacetamide (DMA), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN), or propylene glycol (PG) is optional.
Thus, the reference teachings anticipate the claimed invention.
Claim Objection
Claim 12 is objected to because of the following informality: “7.6-8.5” should have been “7.6 to 8.5”.
Claim 18 is objected to because of the following informalities:
“an antibody” in step (c) should have been “the Fab fragment”.
“antibody” at the last line should have been “Fab fragment”.
Claim 22 is objected to because of the following informality: “5.0-7.6” should have been “5.0 to 7.6”.
Claim 25 is objected to because of the following informality: “7.5-8.5” should have been “7.5 to 8.5”.
Conclusion
Claims 18-22, 24, 25 and 34 are free of prior art.
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to PHUONG HUYNH whose telephone number is (571)272-0846. The examiner can normally be reached on 9:00 a.m. to 6:30 p.m. The examiner can also be reached on alternate alternative Friday from 9:00 a.m. to 5:30 p.m.
If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Misook Yu, can be reached at 571-270-3497. The fax phone number for the organization where this application or proceeding is assigned is 571-272-0839.
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/PHUONG HUYNH/ Primary Examiner, Art Unit 1641
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