The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 33-37 and 39 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. Claim 32 includes several steps so that it is not clear where in the order of steps the lysing step of claim 33 occurs. Additionally, it appears that the sample contains some type of cellular material, however it is not clear if that is cellular material from a tissue sample, cells that have been cultured/grown in a flask or cellular material in a whole blood sample. For examination purposes, examiner will not limit the sample type and will treat the lysing step as either prior to or immediately following the contacting step. Claim 25 also adds another step without defining which of the steps in either claims 32 or 33 it follows or precedes. Based on claim 38 defining the protease as trypsin and claim 37 defining a step of solubilizing the protein precipitate in a solution comprising trypsin and a chaotropic agent, it appears that the step of claim 35 occurs prior to the contacting step of claim 32.
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)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 32, 35, 38 and 40-41 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Aartsma-Rus (Journal of Neuromuscular Diseases 2019). In the last full paragraph of the right column on page 155, Aartsma-Rus describes a presentation by Hendrik Neubert of an immunoaffinity liquid chromatography (LC)-MS/MS detection system for dystrophin and micro-dystrophin quantification. The method was still in development and was designed to combine high measurement specificity with high sensitivity through enrichment with anti-peptide antibodies. Proteins are extracted from muscle samples with high (5%) SDS buffer followed by precipitation with an organic solvent, then proteins are re-solubilized and digested with trypsin (step of contacting the sample with a protease, forming a protein digest comprising dystrophin peptides). A multiplex peptide immuno-affinity extraction is performed, using antibodies against selected dystrophin peptides (steps of applying the protein digest to an affinity matrix comprising antibodies, or antigen binding fragments thereof, specific for at least one dystrophin peptide and eluting dystrophin peptides bound to the affinity matrix, forming a dystrophin peptide-enriched sample) prior to nanoflow/spray LC-MS (step of performing liquid-chromatography-mass spectrometry (LC/MS) on said dystrophin peptide-enriched sample) akin to previously published methods for other protein analytes (reference 51 is the cited Fan paper (Analytical Chemistry 2016)). Both fresh frozen samples and OCT embedded samples can be used. The protocol takes 1.5 days for sample preparation and 15 minute LC-MS run time per sample. Heavy isotope labelled micro-dystrophin or dystrophin peptides are used for response normalization. At the moment 4 peptides from different regions of the dystrophin protein have been prioritized, but additional peptides can be added if needed. The method currently uses an 11 point standard curve of recombinant micro-dystrophin, the lower limit of qualification is less than 1% of normal dystrophin, while the upper limit is 500%. The coefficient of variation (CV) is typically below 25%, even for the lower ranges of dystrophin levels. The method can measure dystrophin from various species (mouse, human) and detect dystrophin from revertant fibers in DMD patients at low levels (preliminary data were 1.6–5.9%). As before, dystrophin levels of healthy individuals varied. Since this description in Aartsma-Rus teaches the four steps required by claim 32, the claim is anticipated.
With respect to claim 35, the fact that the proteins were extracted followed by precipitation with an organic solvent anticipates claim 35. With respect to claim 38, the fact that the proteins are re-solubilized and digested with trypsin anticipates claim 38. With respect to claim 40, the fact that heavy isotope labelled micro-dystrophin or dystrophin peptides are used for response normalization shows that claim 40 is anticipated. With respect to claim 41, the fact that SEQ. ID NO:242 is the sequence the sequence for human dystrophin and the method described by Aartsma-Rus is for dystrophin and micro-dystrophin shows that at least SEQ. ID NO:242 of claim 41 is anticipated.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 33-34 and 50 are rejected under 35 U.S.C. 103 as being unpatentable over Aartsma-Rus as applied to claim 32 above, further in view of Canessa (Journal of Mass Spectrometry 2019). Aartsma-Rus teaches that the proteins were extracted but does not teach that it occurred through lysis of the sample.
In the paper Canessa described/studied the absolute quantification of dystrophin protein in human muscle biopsies using parallel reaction monitoring (PRM). The need for a reliable and accurate method to quantify dystrophin proteins in human skeletal muscle biopsies has become crucial in order to assess the efficacy of dystrophin replacement therapies in Duchenne muscular dystrophy as well as to gain insight into the relationship between dystrophin levels and disease severity in Becker's muscular dystrophy. Current methods to measure dystrophin such as western blot and immunofluorescence, while straightforward and simple, lack precision and sometimes specificity. They standardized a targeted mass spectrometry method to determine the absolute amount of dystrophin in ng/mg of muscle using full‐length 13C6–Arg– and 13C6,15N2–Lys–labeled dystrophin and parallel reaction monitoring (PRM). The method was found to be reproducible with a limit of quantification as low as 30 pg of dystrophin protein per mg of total muscle proteins. The method was then tested to measure levels of dystrophin in muscle biopsies from a healthy donor and from Duchenne and Becker's muscular dystrophy patients. The paragraph bridging pages 1-2 teaches that over the past few years, promising therapies to restore the missing dystrophin in DMD patients were developed and now are in phase II and III clinical trials. These included the use of antisense oligonucleotide‐based exon skipping to restore the reading frame of the aberrant mRNA splicing in DMD enabling the expression of a truncated but functional dystrophin with the hope to promote a less severe phenotype, similar to that seen with BMD patients. Others have introduced stop codon read through strategy or gene therapy using AAV vector harboring microdystrophin gene. However, the amounts of restored dystrophin using these therapies are not well defined, leading to conflicting conclusions about how much dystrophin is enough to stabilize the sarcolemma and improve patient's outcome. Furthermore, the relationship between dystrophin levels and disease severity in BMD remains unclear. Some BMD patients with low levels of dystrophin do better than other BMD patients with normal levels of dystrophin. Thus, the need of reliable and accurate methods to quantify dystrophin in skeletal muscle tissue is important for use as both a pharmacodynamic biomarker to assess efficacy of dystrophin replacement therapies and as a surrogate biomarker to assess disease severity if correlated with clinical endpoints. Section 2.1 describes the culture of SILAC human myotubes and lysate preparation. After the culturing and harvesting steps were described in the first paragraph of the section, the storage of the human myotubes was as a dry pellet in an Eppendorf tube at −80 °C until lysis. SILAC myotube pellets were lysed by suspending the cells in RIPA buffer w/cOmplete™ Protease Inhibitor Cocktail followed by vigorous vortexing and centrifugation at 14 000 rpm at 4°C for 10 minutes. The supernatant containing SILAC myotube protein extract was collected and protein concentration determined. SILAC myotube protein extract was stored in 200‐μg aliquots at −80 °C until needed. The first full paragraph on page 3 of the paper teaches that the protein pellet was then resuspended in 100 μL of 6M urea to which 5 μL of 200‐mM DTT solution in 25‐mM ammonium bicarbonate was added. Samples were then incubated at 37 °C for 1 hour, and then 20 μL of 200‐mM iodoacetamide solution in 25‐mM ammonium bicarbonate was added followed by incubation for 1 hour at room temperature and in the dark. The excess alkylating iodoacetamide was quenched by adding 20 μL of 200‐mM DTT solution. For in‐solution digestion of the sample by trypsin, 755 μL of 25‐mM ammonium bicarbonate was added followed by adding trypsin solution to a final ratio of enzyme to protein (1:50, w/w). Sample was then incubated overnight at 37 °C using a shaker at 550 rpm. Fifty microliters of 2.5% FA was added to stop reaction. The LC-MS/MS analysis was described in section 2.2.
With respect to claims 33-34, it would have been obvious to one of ordinary skill in the art at the time the application was filed to perform the protein extraction in the method described in Aartsma-Rus with a lysis buffer and in particular the RIPS lysis buffer described by Canessa because of that buffer to extract protein from cells to measure the amount of dystrophin through a LC-MS/MS process taught by Canessa that is similar to that described by Aartsma-Rus so that there is an expectation that the RIPS lysis buffer of Canessa would perform the same function with the cells of Aartsma-Rus. With respect to claim 50, it would have been obvious to one of ordinary skill in the art at the time the application was filed to apply the Aartsma-Rus method on a sample of skeletal muscle obtained from a human diagnosed with Duchenne's muscular dystrophy (DMD) because of the need for such a method as taught by Canessa.
Claims 33, 36-37, 39 and 42-47 are rejected under 35 U.S.C. 103 as being unpatentable over Aartsma-Rus as applied to claims 32 and 35 above, further in view of Fan (Analytical Chemistry 2016). Aartsma-Rus references the Fan paper in conjunction with a multiplex peptide immuno-affinity extraction being performed, using antibodies against selected dystrophin peptides prior to nanoflow/spray LC-MS similar to previously published methods for other protein analytes. Aartsma-Rus does not teach specific dystrophin peptides against which the antibodies are designed specific aspect of the digestion of protein preparation steps.
In the paper Fan describes/teaches the quantitative analysis of human neonatal Fc receptor (FcRn) tissue expression in transgenic mice by online peptide immune-affinity LC-HRMS. Neonatal Fc receptor (FcRn) is the homeostatic receptor responsible for the long half-life of endogenous IgG by protecting it from lysosomal degradation. Understanding systemic FcRn tissue expression is important to predict and design the half-life of therapeutic antibodies and Fc-coupled biotherapeutics. To this end, they measured human FcRn (hFcRn) tissue expression in Tg32, a human FcRn knock-in transgenic mouse model, for which a strong correlation of drug clearance to humans has been demonstrated. Building an hFcRn tissue expression profile in Tg32 was enabled by the development of a tissue preparation procedure composed of bead-based protein extraction and protein precipitation using acetone followed by pellet digestion with trypsin. Digests were then loaded onto an online peptide immuno-affinity flow configuration hyphenated with reversed phase nanoflow chromatography and coupled with high resolution mass spectrometry to quantify hFcRn derived peptides. The workflow allowed bypassing some of the challenges typically associated with membrane protein analysis. They demonstrated acceptable precision and bias for measuring hFcRn in tissue matrices, typically within 20% coefficient of variation and relative error. They also reported hFcRn expression in several Tg32 tissues. They anticipated that establishing a quantitative approach for hFcRn in tissues will enable the systematic measurement of hFcRn concentrations to further increase the accuracy of physiologically based pharmacokinetic (PBPK) models for PK prediction of Fc-containing biotherapeutics. This was anticipated to improve the translation of pharmacokinetic data from preclinical model systems to humans. Page 4241 describes the preparation of a multiplex antipeptide antibody column using antibodies generated in rabbits and ligand affinity purified against the target peptide prior to use. All antipeptide antibodies were mixed and packed into a multiplex antibody column for hFcRn peptide immuno-affinity enrichment. The tissue protein and processing section on page 4241 teaches that approximately 1 g of a 0.9 to 2.0 mm blend of stainless steel beads and one stainless steel cone ball shaped bead (UFO 3.5 mm) were added to 50−100 mg of tissue in 500 μL of cold TER-I buffer with freshly prepared 1× Halt Protease Inhibitor Cocktail. TER-I buffer contains 50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 1 mM NaF, 20 mM Na4P2O7, 0.02% NaN3, and proprietary detergent. Tissues were homogenized using a Bullet Blender in a cold room at 4 °C for 5 min. Homogenates were clarified by centrifugation at 4 °C and 10 000g for 15 min. Tissue debris was collected to perform a second extraction with another 500 μL of cold lysis buffer using the same procedure. Tissue homogenate from the first and second extraction were pooled and adjusted to a final concentration of 50 mg of tissue per mL of lysis buffer. Tissue debris after the second extraction was collected for an extraction efficiency test. A 400 μL aliquot of tissue extracts was precipitated in acetone (1:4, v/v) at −20 °C for 1 h and centrifuged at 4 °C and 10 000g for 15 min. The acetone supernatant was discarded, and another 1 mL of cold acetone was added to wash the protein pellets followed by collection of the final pellet by centrifugation at 4 °C and 10 000g for 15 min. Protein pellets were dried at 37 °C using a shaker for 3 min. A 250 μg amount of TPCK-treated trypsin in 400 μL was used to solubilize tissue lysate precipitates at 37 °C for 16 h. The protein concentration was estimated by measuring the absorbance at 280 nm using a NanoDrop 8000 instrument. Stable isotope labeled (SIL) peptides with sequence extensions to contain trypsin cleavage sites were custom synthesized from New England Peptide with >95% peptide purity and >99% isotopic purity, and the concentration was determined by amino acid analysis. SIL peptide sequences are shown in italics in Figure 1, where an asterisk (*) indicates the labeled amino acids. SIL peptides (500 fmol) were spiked to 40 μL of solubilized tissue lysate. Reduction was carried out at 37 °C for 1 h by adding 10 μL of 50 mM dithiothreitol, followed by alkylation at room temperature, in the dark for 1 h, with the addition of 10 μL of 100 mM iodoacetamide and then by digestion at 37 °C for 16 h with 30 μg of fresh TPCK-treated trypsin with a final volume of 100 μL. The workflow is summarized in Supplementary Figure 1. The rest of the experimental section on pages 4241-4242 describes the immuno-affinity LC-HRMS, the data processing for quantification, the assay quantification and preparation of calibration standards. The final paragraph on page 4246 teaches that the selection of quantification platform (MS or immunoassay) in biological matrices for biomarkers or exogenous proteins such as biotherapeutics and protein toxins has been discussed. This work combines the advantages of both techniques by utilizing antibody enrichment to gain the needed sensitivity and taking advantage of the higher measurement specificity of MS to avoid tissue matrix interferences. There are several advantages of employing multiplexed peptide enrichment coupled with HRMS for protein quantification in tissue matrices including robust quantification performance, improved selectivity/sensitivity, and less matrix interference compared to ligand-binding assays or immunoblotting. In addition, in the context of sample preparation, high tissue extraction efficiency was confirmed by digesting tissue debris after lysate preparation. Peptides released from tissue debris were analyzed and quantified via antipeptide antibody column, which helped assay development in optimizing the extraction procedure and determining extraction efficiency quantitatively. Antipeptide antibody enrichment has been successfully used in previous reports for the quantification of soluble cytokines and membrane receptors by multiple-reaction monitoring in cell lines, biofluids or tissues. The repeated use of the antipeptide antibody column relies on the well-controlled LC conditions for efficient wash of nonspecific binding and quick re-equilibration. It is also worth noting that the amount of antibody immobilized in the column is orders of magnitude higher than that of endogenous hFcRn loaded to the column (mg of antibody versus ng of hFcRn). To understand the lifetime of a column, they suggested performing the column test with digested recombinant protein or SIL peptide, as shown in Figure 2A, in order to determine whether the antipeptide antibody column has any reduction in binding capacities. In this method, the assay sensitivity necessary for detection of endogenous hFcRn concentrations was enabled by immunoaffinity enrichment and the use of high resolution tSIM acquisition for quantification. Following further method development, particularly with respect to understanding and minimizing tissue matrix interference, this assay has the potential to be also applied to hFcRn measurement in human tissues. There are broad clinical applications targeting hFcRn due to its function of promoting half-life extension of albumin or Fc-fusion proteins, and monoclonal antibodies.
With respect to claim 33, it would have been obvio8us to one of ordinary skill in the art at the time the application was filed to use an extraction of protein from tissues using a lysis buffer as taught by Fan because of the desire to perform the method described in Aartsma-Rus on tissues and the ability to recover proteins from tissues using a lysis buffer as taught by Fan. With respect to claims 36, it would have been obvious to one of ordinary skill in the art at the time the application was filed to use acetone as the organic solvent to cause precipitation of protein in the Aartsma-Rus method because Aartsma-Rus teaches Fan as showing a similar process for a different protein extracted from tissue and Fan teaches that acetone precipitate protein from tissue in a similar method. With respect to claims 37 and 39, it would have been obvious to one of ordinary skill in the art at the time the application was filed to modify the method described by Aartsma-Rus by solubilizing the protein precipitate with a solution comprising trypsin and a chaotropic agent and reducing and alkylating of the solubilized protein precipitate as taught by Fan because Aartsma-Rus teaches Fan as showing a similar process for a different protein extracted from tissue and the ability to digest tissue debris after lysate preparation as taught by Fan.
With respect to claims 42-47, Fan shows that one of ordinary skill in the art is capable of determining peptides to target for the immuno-affinity column, preparing antibodies for the targeted peptides and producing the immune-affinity column so that it would have been obvious to one of ordinary skill in the art at the time the application was filed to select appropriate dystrophin antibodies to create an immunoaffinity column for dystrophin for at least human dystrophin because Aartsma-Rus teaches Fan as showing a similar process for a different protein extracted from tissue that can be used as an example to follow and the various advantages for the use of a peptide immuno-affinity column in combination with mass spectral analysis.
Claims 48-49 and 51 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The following is a statement of reasons for the indication of allowable subject matter: the art of record fails to teach or fairly suggest the general requirement of claim 48 or the specific peptide/protein sequences of claims 49 and 51.
The description relied upon in the Aartsma-Rus reference appears to be a disclosure of a method/process being developed by Hendrik Neubert during a workshop on dystrophin quantification methodology for representatives of academia, patient organizations, industry and the United States Food and Drug Administration (see the abstract of the paper) was held in London on the 14th March 2018. Since this meeting was in a timeframe that would constitute a bar to patent anything which was disclosed, examiner is requesting information regarding what was actually disclosed (slides/posters/handouts) and if there are circumstances that would prevent the disclosure from being considered a public disclosure usable against the instant claims.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. The additionally cited art is related to peptide immune-affinity methods and dystrophin analysis methods. Of note is the Brown paper which has a table (Supplemental Table S1) listing tryptic peptides from dystrophin extracted from tissue by a RIPA buffer and a 10% SDS modified Laemmli buffer. The table lists the specific peptides of claims 45 and 47.
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/ARLEN SODERQUIST/ Primary Examiner, Art Unit 1797
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