DETAILED ACTION
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 .
Election/Restrictions
Applicant’s election with traverse of target proteins Apolipoproteins and Interleukins, specifically APOA1, IL-8, and IL-6, in the reply filed 02/11/2026 is acknowledged. Applicant timely traversed the restriction requirement in the reply filed on 12/13/2022. The traversal is on the ground that the applicant alleges that the claimed invention is indeed linked to form a single general inventive concept under the Unity of Invention Requirement because the prior art (Patel et al US20210033622) does not teach binding both target and standard protein to a binding agent on a solid support and then digesting the target protein and the standard protein. This argument is found persuasive. The requirement for election of species is withdrawn.
Priority
The present application was filed 12/15/2022. Acknowledgement is made of the present application as a proper National Stage (371) entry of PCT Application No. PCT/EP2021/067375, filed 06/24/2021, which in turn claims foreign priority to EP20182679.9, filed 06/26/2020 with the European Patent Office.
Information Disclosure Statement
The information disclosure statements (IDS) filed 02/02/2023 and 12/15/2022 have been considered, initialed and are attached hereto.
Claim Status
Claims 1-15 are pending and under examination. Claims 3-15 are amended.
Objection
The title of the invention is not descriptive. A new title is required that is clearly indicative of the invention to which the claims are directed.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 11 and 14 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 11 is indefinite because it recites one “target protein suspected to be present” followed “is present […] in a concentration of between”. It is unclear if the target is present or is suspected to be present.
Further, regarding claim 11, the phrase "such as" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Regarding claim 14, the phrase "such as" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). Claim 14 is further indefinite because it is unclear how a standard curve, the establishment of which precedes the method can be used to approximate the amount of target protein in the sample, considering a standard curve is established using known amounts of standard.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
Claim 15 is rejected under 35 U.S.C. 102(a)(2) as being anticipated by Ray et al., US20200378986A1.
Regarding claim 15, Ray teaches kits for detecting and/or quantitating target proteins in biological samples comprising capture and immobilization of the target protein (Ray, ‘Abstract’, lines 1-3). Ray teaches that the kit comprises a capture entity directed to the target protein of interest (binding agent) and at least one stable -isotope labeled internal standard protein (Ray, page 4, paragraph [0054], lines 7-9 and 17-18). Ray further teaches that the kit generally includes instructions (Ray, page 4, paragraph [0055], lines 1-2). Regarding the limitation “instructions for carrying out the method”, MPEP §2112.01(III) states that “[w]here the only difference between a prior art product and a claimed product is printed matter that is not functionally related to the product, the content of the printed matter will not distinguish the claimed product from the prior art. In re Ngai, 367 F.3d 1336, 1339, 70 USPQ2d 1862, 1864 (Fed. Cir. 2004) (Claim at issue was a kit requiring instructions and a buffer agent. The Federal Circuit held that the claim was anticipated by a prior art reference that taught a kit that included instructions and a buffer agent, even though the content of the instructions differed, explaining "[i]f we were to adopt [applicant’s] position, anyone could continue patenting a product indefinitely provided that they add a new instruction sheet to the product.").” In the instant case, the limitation is not considered to contribute to the overall patentability of the claimed invention.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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 1-12 and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Kuhn et al. Developing multiplexed assays for troponin I and interleukin-33 in plasma by peptide immunoaffinity enrichment and targeted mass spectrometry. Clinical chemistry. 2009 Jun 1;55(6):1108-17 and Supplemental Material, Boström et al. Antibodies as means for selective mass spectrometry. Journal of Chromatography B. 2016 May 15;1021:3-13 (of record, see IDS 12/15/2022) and Edfors et al. Immunoproteomics using polyclonal antibodies and stable isotope–labeled affinity-purified recombinant proteins. Molecular & cellular proteomics. 2014 Jun 1;13(6):1611-24, as evidenced by Lee et al. Fabrication of troponin I biosensor composed of multi-functional DNA structure/Au nanocrystal using electrochemical and localized surface plasmon resonance dual-detection method. Nanomaterials. 2019 Jul 11;9(7):1000.
Regarding claims 1 and 12, Kuhn teaches a method of quantifying proteins by peptide immunoaffinity enrichment coupled with stable isotope dilution mass spectrometry (Kuhn, page 1108, see ‘Background’ and ‘Methods’). Kuhn teaches a ‘stable isotope standards and capture by anti-peptide antibodies’ (SISCAPA) assay comprising the steps of combining digested plasma samples, magnetic beads bound with antibody (solid support with binding agent) and internal standard peptide in phosphate buffered saline, isolating the magnetic beads using a magnet and removing unbound material, washing twice, before analyzing the peptides by mass spectrometry (Kuhn, pages 1109-1110, ‘Peptide immunoaffinity enrichment and SID-MRM Quantification’, entire paragraph). Kuhn further teaches quantifying peptides using labeled peptides as internal standards (Kuhn, page 1108, ‘Methods’, lines 5-7). Kuhn further teaches the use of stable isotope peptides as standards (Kuhn, page 1109, ‘Preparation of standards and LC-MRM-MS Method’, 2nd column, lines 6-8). Kuhn further teaches preparing plasma samples from patient blood (body fluid; Kuhn, page 1109, ‘Patient sample collection protocol’, lines 4-6). Kuhn further teaches using the internal standard to calculate the concentration of the protein in the original sample (Kuhn, page 1110, ‘Data Analysis and Protein Quantification’, lines 4-8). Kuhn further teaches that adding a suitably labeled protein standard instead of adding the labeled peptide could correct for interassay variability and normalize for incomplete protein digestion (Kuhn, page 1115, 2nd column, 2nd paragraph, lines 10-17). Kuhn further teaches that the method employs the ideal internal standard, a labeled version of the same peptide that has identical physiochemical properties, including retention time, and differs from the analyte only in its parent and fragment masses (Kuhn, page 1115, 1st column, 4th paragraph, line 8 -2nd column, line 4).
Kuhn does not teach that the SISCAPA method comprises a standard that is a fragment of the target protein and does not teach that the target is added to the sample before immunoenrichment, followed by digestion of the target protein and the sample.
Boström teaches that SISCAPA can detect targets in plasma and saliva and can be multiplexed to enable simultaneous detection of up to 50 targets (Boström, page 5, ‘2.1. SISCAPA’, lines 4-14). Boström teaches that immuno-SILAC is similar to the SISCAPA workflow (Boström, page 7, ‘immuno-SILAC’, lines 4-6). Boström further teaches that the production of full length proteins is quite troublesome compared to the expression of protein fragments or generation of synthetic peptides (Boström, page 5, ‘2. Peptide enrichment coupled to mass spectrometry’, lines 10-13). Boström further teaches that immunoenrichment on protein level as compared to enrichment on peptide level has several advantages. If enzymatic digestion is applied prior to mass spectrometry analysis, multiple peptides originating from the target protein will hopefully be detected and the increased protein sequence coverage acquired using enrichment on the protein level can result in a more reliable identification compared to peptide level enrichment where only one target peptide is detected. Boström further teaches that performing immunoenrichment prior to trypsin cleavage is advantageous from an economical perspective especially in samples of high protein content such as plasma or serum because the sample complexity is significantly decreased, lowering the amount of costly enzyme needed (Boström, page 7, ‘3. Protein enrichment coupled to mass spectrometry’, see entire paragraph).
Edfors teaches the details of immuno-SILAC (Edfors, page 1611, ‘Abstract’, lines 3-4). Edfors teaches antibodies raised against human recombinant proteins called protein epitope signature tags (Edfors, page 1612, 3rd paragraph, lines 13-16). Edfors further teaches quantitative proteomics using immunoenrichment using stable isotope approaches which is built on detection of peptides generated by protease cleavage of proteins in a sample and quantification by reading out the ratio between endogenous peptide and heavy-labeled spiked-in peptide. Edfors further teaches that the endogenous protein and the labeled internal standard behave identically throughout the sample preparation including the immuno-enrichment, the relative ratio provides quantitative information, as the peptides can be distinguished by the mass spectrometer because of the shift in mass (Edfors, page 1612, 4th paragraph, lines 1-14). Put another way, by teaching that the endogenous peptides behave identically to the standard in immuno-enrichment, one of ordinary skill in the art would expect that the binding agent is capable of binding both the target and standard protein (claim 1) and further that the target protein and the standard protein would bind to the binding agent with comparable affinity, i.e. at a ratio of KD values close to 1:1 (claim 12).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the SISCAPA method taught by Kuhn, comprising immunoenrichment of a peptide sample and isotope-labeled peptide standard before analysis with mass spectrometry, by using a protein fragment standard because of the teaching of Boström that the production of full length proteins is quite troublesome compared to the expression of protein fragments and that the variation introduced by digestion of the sample and the protein standard will be lower when digested together and because a standard containing a longer stretch of the protein will give rise to both fully cleaved peptides and peptides containing missed cleavage sites that can also be used for quantification. One of ordinary skill in the art would be motivated to do so because this method would give a more accurate picture of the true protein concentration within the sample.
It would have further been prima facie obvious to have modified the SISCAPA method as taught by Kuhn by performing the immunoaffinity step before protein digestion because of the teaching of Boström that this is advantageous from an economical perspective because the sample complexity is significantly decreased, lowering the amount of costly enzyme needed.
It would have further been prima facie obvious to have applied the teaching of Edfors to the assay of Boström of using an assay with an internal standard that behaves identical to the target protein in immunoenrichment and as such also comprising a binding agent that binds both the standard and the target protein because of the teaching of Edfors that this allows for quantification of the target protein.
One of ordinary skill in the art would have a reasonable expectation of success because the methods of Kuhn, Boström and Edfors are all applied in similar assays (such as SISCAPA and immuno-SILAC) as taught by Boström and are all used in methods combining immunoenrichment with mass spectrometry analysis.
Regarding claims 2 and 3, Kuhn teaches quantitatively detecting Interleukin-33 (secreted protein; interleukin; Kuhn, page 1108, ‘Background’, lines 10-11).
Regarding claim 4, Kuhn teaches antibodies linked to magnetic beads (Kuhn, page 1109, ‘Polyclonal Antibody Production and Magnetic Bead Preparation’, lines 1-2; see also Boström, page 5, Figure 1, ‘Peptide immunoenrichment’).
Regarding claim 5, Kuhn teaches that plasma samples and standards are digested using the enzyme trypsin, which is a type of proteolytic enzyme (Kuhn, page 1108, ‘Results:’, lines 1-2 and page 1109, ‘Protein Standard Curve Preparation’, lines 6-7). Put another way, Boström as evidenced by Kuhn teaches digesting proteins with an enzyme and as such teach a proteolytic enzyme.
Regarding claim 6, Kuhn teaches two IL-33 signature peptides comprising 10 and 22 amino acids respectively (Kuhn, page 1110, ‘Selection of Peptides for SISCAPA Assay Development’, lines 12-14).
Regarding claim 7, Kuhn teaches selecting two of the IL-33 signature peptides (TDPGVFIGVK and VLLSYYESQHPSNESGDGVDGK; Kuhn, page 1110, ‘Selection of Peptides for SISCAPA Assay Development’, lines 12-1) and supplemental Data Figure 1 teaches that the at least two tryptic peptides are selected after digestion of protein standards. Put another way, Kuhn teaches more than one non-overlapping tryptic peptide and as such teaches that the standard protein comprises more than two cleavage site for a proteolytic enzyme used to digest the standard (Kuhn, page 16 of 18, supplemental data figure 1 and figure legend).
Regarding claim 8¸ Kuhn teaches employing an internal standard which is a 13C and/or 15N-labeled version of the same peptide (Kuhn, page 1115, 4th paragraph line 8 -2nd column, line 2).
Regarding claim 9¸ Kuhn teaches enriching peptides from digested human plasma using antipeptide antibodies (Kuhn, page 1108, ‘Methods’, lines 1-3).
Regarding claim 10, Kuhn teaches a multiplexed assay for Troponin I and Interleukin-33 (at least two target proteins; Kuhn, page 1108, see Title).
Regarding claim 11, Kuhn teaches measuring the plasma concentration of cardiac Troponin I over 24 h and further teaches the levels of said protein range from 3 µg/l to 8 µg/l in patients 24 hours after undergoing therapeutic planned heart attack for hypertrophic cardiomyopathy (Kuhn, page 1114, ‘Temporal Measurements of cTn1 in Cardiovascular Patient Plasma’, see entire paragraph). Considering troponin 1 has a molecular weight of about 23.9 kDa (as evidenced by Lee, page 2 of 13, 2nd paragraph, line 7), this range teaches that troponin is present in the samples at 1.25 x 10-10-3.35 x 10-10 mol of troponin I which falls within the claimed range of between 10-4 and 10-10 and therefore teaches the limitation of the claim.
Regarding claim 14, Kuhn teaches preparing peptide and protein response curves for IL-33 using analyte peptide standards spiked directly or peptides derived from the spiked proteins and analyzing the eluates after enrichment to calculate protein concentrations from the analyte to internal standards (Kuhn, page 1110, ‘SISCAPA Assay Development and Characterization’, see entire paragraph).
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Kuhn et al. in view of Boström et al. and Edfors et al. as applied to claim 1 above and further in view of Buus et al. High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays. Molecular & Cellular Proteomics. 2012 Dec 1;11(12):1790-800.
Regarding claim 13, Kuhn and the prior art above teach a method of measuring an amount of target protein in body fluid substantially as claimed.
Kuhn teaches raising an antibody against two of the IL-33 signature peptides (TDPGVFIGVK and VLLSYYESQHPSNESGDGVDGK; Kuhn, page 1110, ‘Selection of Peptides for SISCAPA Assay Development’, lines 12-1).
Kuhn does not teach that the epitope is at least 4 to at least 15 amino acids long.
Buus teaches mapping linear antibody epitopes using an ultrahigh density peptide microarray technology (Buus, page 1792, ‘The Location and Length of Linear Epitopes’, lines 1-3). Buus teaches generating antibodies to a 145 amino acid long protein epitope signature tag (PrEST) and that this revealed that the shortest recognizable sequences of the most dominant reactivities are 6-mer and 7-mer peptides and that there were no signals for peptides shorter than six amino acid residues (Buus, page 1793, line 1- second column, line 4). Buus further teaches comparing the above method with bacterial expression epitope mapping and teaches that the smallest epitope length observed is at least four amino acids (Buus, page 1796, ‘Comparisons with a Bacterial Expression Epitope Mapping Strategy’, lines 23-24 and Fig. 5).
It would have been prima facie obvious for one having ordinary skill in the before the effective filing date of the claimed invention to have applied the method of Kuhn and the prior art to a sample that after digestion with a proteolytic enzyme comprises an epitope with at least 4 amino acids or more because of the teaching of Buus that the smallest linear antibody epitope is at least 4 amino acids and the most dominant reactivities comprised 6- and 7-mers and Kuhn teaches using an antibody to bind the peptide.
One of ordinary skill in the art would have a reasonable expectation of success using a digested sample that comprises epitopes of at least 4 amino acids in length because of the teaching of Buus that epitopes of that length (and longer) are the minimum length to be recognized by antibodies.
Communication
Any inquiry concerning this communication or earlier communications from the examiner should be directed to STEFANIE J KIRWIN whose telephone number is (571)272-6574. The examiner can normally be reached Monday - Friday 7.30 - 4 pm.
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/STEFANIE J. KIRWIN/Examiner, Art Unit 1677
/Soren Harward/Primary Examiner, TC 1600
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