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 .
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.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 9/4/2025 has been entered.
Withdrawn Rejections
The rejection of the claims under 112b, 112d, 101 and 102 are withdrawn in response to the amendments.
Priority
The present application was filed as a proper National Stage (371) entry of PCT Application No. PCT/IB2020/053360, filed 10/11/2021. Acknowledgment is also made of applicant's claim for foreign priority under 35 U.S.C. 119(a)-(d) to Application No. 102019000005700, filed on 04/12/2019 in Italy.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Status of the Claims
Claims 46-48, 52, 54 and 56-57 are pending; claims 46-48, 54 and 56-57 are amended, claims 1-45, 49-51, 53, 55 and 58-72 are canceled; Claims 46-48, 52, 54 and 56-57 are examined below.
Information Disclosure Statement
The information disclosure statement filed on 9/4/2025 is being considered by the examiner.
Claim Objections
Claim 57 is objected to because of the following informalities: In line 1, "the amound" appears to be a typographical error, namely it is suggested that "the amound" read as "the amount". Appropriate correction is required.
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)(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 46, 52 and 54 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Angelini et al. COMMUNICATIONS BIOLOGY | (2018) 1:69 | DOI: 10.1038/s42003-018-0069-8 (Cite No. NPL1 of IDS 10/11/2021) (“Angelini”).
Although Angelini was published within the grace period of the foreign application (IT102019000005700 effectively filed on 4/12/2019) and includes the instant inventor, Geltrude Mingrone, this reference does not fall into the 102(b)(1)(A) exception because the journal article includes other authors not listed as inventors in the present application, specifically: Giulia Angelini, Serenella Salinari, Alessandro Bertuzzi and Amerigo Iaconelli.
Note that the Angelini reference includes an author contribution statement. Although it is stated that an inventor of the present application, Geltrude Mingrone, “designed the study… performed the clinical studies…drafted the manuscript… participated in the paper writing… [and] is a main guarantor of the study” (page 10 col. 1 para. 4); the author contribution statement also declares that “S.S. and A.B. designed the study… A.I. performed the clinical studies. G.A. did the in vitro studies with HepG2 and PBMC and made the HSP serum level analyses. S.S. and A.B. computed insulin sensitivity. G.A.…drafted the manuscript. All the authors actively participated in the paper writing. [and] The main guarantors of the study are Professor Mingrone and Dr. Angelini” (emphasis added).
Therefore, it is clear that a significant contribution of the Angelini reference was made by others not included as inventors of the present application.
Angelini teaches a method of measuring a level of perilipin-2 (Plin2) protein in a blood sample of an individual (“Plin2 expression is increased in PBMC and monocytes of IR subjects” page 3 col. 2 para. 2), comprising: obtaining the blood sample from the individual (“We enrolled 14 obese IR, NAFLD subjects…Blood samples and PBMCs were taken from all subjects after 12 h of fasting” page 8 col. 1 paras. 4-5); extracting leukocytes from the blood sample (page 8 col. 1 para. 5); and measuring an amount of Plin2 protein in the leukocytes extracted from the blood sample to determine a level of Plin2 protein in the blood sample (“To determine if Plin2 protein expression was increased in obese IR, NAFLD subjects, flow cytometry analysis was performed in monocyte subpopulations (CD14+CD16−, CD14++CD16+ and CD14+CD16+) and in total PBMCs of obese IR, NAFLD subjects and of healthy controls. Plin2 protein expression was markedly increased in monocyte subpopulations and PBMCs of obese IR subjects compared to healthy controls (Fig. 2d)” page 3 col. 2 para. 2 and page 4 col. 1 para. 1). Note that although Angelini fails to use the language “extracting leukocytes from the blood sample “ the teaching of taking blood and PBMCs from the subjects inherently provides a step of extracting leukocytes from the blood sample because PBMCs, which are leukocytes, are obtained from blood samples.
Regarding claim 52, Angelini teaches wherein the blood sample is a peripheral blood sample (page 8 col. 1 para. 5). Note that although Angelini fails to use the language “peripheral blood sample”, the teaching of “PBMCs” inherently provides a peripheral blood sample because PBMCs are peripheral blood mononuclear cells (“peripheral blood mononuclear cells (PBMCs)” page 2 col. 1 para. 5).
Regarding claim 54, Angelini teaches wherein the leukocytes are polymorphonuclear and/or monocytes (page 3 col. 2 para. 2 and page 4 col. 1 para. 1).
Claim(s) 46, 52 and 54 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by G. Niccoli et al. / International Journal of Cardiology 258 (2018) 55–58 (“Niccoli”) as evidenced by Appendix A. Supplementary data of G. Niccoli et al. / International Journal of Cardiology 258 (2018) 55–58 (retrieved online https://www.sciencedirect.com/science/article/pii/S0167527317379512?via%3Dihub on 11/14/2025).
Regarding claim 46, Niccoli teaches a method of measuring a level of perilipin-2 (Plin2) protein in a blood sample of an individual, comprising: obtaining the blood sample from the individual, extracting leukocytes from the blood sample; and measuring an amount of Plin2 protein in the leukocytes extracted from the blood sample to determine a level of Plin2 protein in the blood sample (“[t]he aim of the study was to determine PLIN2 protein levels in peripheral monocytes of enrolled subjects… Patients with ISR due to neoatherosclerosis had significantly higher PLIN2 protein levels in peripheral blood monocytes compared to patients with native CAD (342.47±75.63[SE] versus 119.51±20.95, p b 0.001)….PLIN2 protein levels are significantly increased in patients with neoatherosclerosis,” Abstract, “The information on methods used in the making of this study and relevant ethical disclosures are provided in the Appendix A submitted as supplemental file along with this manuscript” page 56 col. 1 para. 2). As evidenced by Appendix A, the method of Niccoli comprises obtaining the blood sample from the individual (“Peripheral blood was collected in EDTA vacutainer at the time of patients’ enrollment” page 3 para. 3), extracting leukocytes from the blood sample (“Peripheral blood monocytes (PBMCs) were obtained from whole blood samples by standard gradient centrifugation over Ficoll-Hypaque” page 3 para. 3); and measuring an amount of Plin2 protein in the leukocytes extracted from the blood sample (“The obtained PBMCs were lysed in RIPA buffer. Protein content was determined using Bradford Protein Assay” ” page 3 para. 4).
Regarding claim 52, Niccoli teaches wherein the blood sample is a peripheral blood sample (“Peripheral blood was collected in EDTA vacutainer at the time of patients’ enrollment” page 3 para. 3 of Appendix A).
Regarding claim 54, Niccoli teaches wherein the leukocytes are polymorphonuclear and/or monocyte cells (“Peripheral blood monocytes (PBMCs)” page 3 para. 3 of Appendix A).
Claim Rejections - 35 USC § 103
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.
Claims 47 and 56 are rejected under 35 U.S.C. 103 as being unpatentable over Angelini as applied to claim 46 above, and further in view of Palucka et al. (AU 2012261593 A1) (Cite No. O of PTO-892 1/3/2025) (hereinafter Palucka) .
Regarding claim 47, Angelini teaches the method of claim 46 as discussed above.
Angelini fails to teach further measuring Rab14.
Palucka teaches “monitoring indicators of immunosuppression through blood leukocyte microarray analysis” (Title). Palucka further teaches that “[b]lood microarray analyses were carried out in 25 healthy volunteers, 35 patients with metastatic melanoma, and 39 liver transplant recipients” (page 4, lines 1-2). Among the patients who had received a liver transplant, one had “Nonalcoholic Steatohepatitis” and many had “Cirrhosis” and one had “Hepatocellular Carcinoma with Cirrhosis” (Table 4, pages 38-39). Palucka teaches that “[t]he sample may be screened by quantitating the mRNA, protein or both mRNA and protein level of the expression vector” (page 4 lines 34-35). Palucka teaches measuring Rab14 (“a module array that includes at least one pair of first and second probe groups, each group having
one or more probes as defined by Table 1” page 7 lines 5-6). Table 1 shows the “M 2.11 module” which “[i]ncludes kinases…RAB14” (Table 1, page 14). Palucka further teaches that “[i]t should be pointed out that there is advantage to combining data from several domains” (page 11 lines 2-3). Palucka further teaches that “[t]he skilled artisan will appreciate that using the modules of the present invention it is possible to rapidly develop one or more disease specific arrays that may be used to rapidly diagnose or distinguish between different disease and/or conditions”(page 7 lines 10-12).
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 teachings of Angelini to include measuring Rab14 taught by Palucka because Palucka teaches that measuring Rab14 may be used to diagnose or distinguish between different diseases and or conditions and that there is an advantage in combining data from several domains. A person having ordinary skill in the art would have had a reasonable expectation of success because both Angelini and Palucka teach measuring proteins in blood samples from subjects with liver disease.
Regarding claim 56, Angelini teaches the method of claims 46 as discussed above.
Angelini fails to teach wherein the one or more proteins is measured by ELISA.
Palucka teaches wherein the one or more proteins is measured by ELISA (“the term "expression profile" refers to the relative abundance of RNA, DNA or protein abundance or activity levels. The expression profile can be a measurement…using…enzyme linked immunosorbent assays (ELISA)” page 16 lines 29-34).
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 teachings of Angelini to rely on the measuring of one or more proteins by ELISA taught by Palucka because it would be a matter of simply substituting the flow cytometry technique for measuring proteins used by Angelini for the art recognized technique, i.e., ELISA technique taught by Palucka for measuring proteins (base method). Both references teach measuring proteins from blood samples, therefore it would have been obvious to a person having ordinary skill in the art to simple use ELISA instead of flow cytometry. A person having ordinary skill in the art would have had a reasonable expectation of success because both Palucka and Angelini teach methods comprising measuring one or more proteins in a blood sample of an individual, wherein the individual is suspected of having NAFLD or NASH.
Claim 48 is rejected under 35 U.S.C. 103 as being unpatentable over Angelini in view of Palucka as applied to claim 47 above, and further in view of Rulifson et al. (WO 2019118638 A2) (Cite No. N of PTO-892 1/3/2025) (hereinafter Rulifson).
Regarding claim 48, Angelini in view of Palucka teach the method of claim 47 as discussed above.
Angelini in view of Palucka fail to teach further measuring Pnpla3.
Rulifson teaches “RNAi constructs for reducing expression of the PNPLA3 gene. Methods of using such RNAi constructs to treat or prevent liver disease, nonalcoholic fatty liver disease (NAFLD) are also described” (Abstract). Rulifson further teaches measuring Pnpla3 (“PNPLA3 expression can be assessed by measuring the amount or level of PNPLA3 mRNA, PNPLA3 protein” paragraph 55, “Reduction in gene expression can be assessed in peripheral blood sample… a blood sample serves as the tissue material for monitoring the reduction in PNPLA3 gene and/or protein expression” paragraph 170). Rulifson further teaches that “[t]he consensus among numerous GWAS indicate the association of PNPLA3 rs738409 with NAFLD is independent of age, gender, ethnicity, metabolic syndrome, body mass index, insulin resistance, and serum lipids” (paragraph 18). Rulifson further teaches that “in vivo mouse model data points to expression of the mutant Pnpla3.sup.I148M protein, and not over expression of the wild type protein, as the driver of the disease phenotype. These findings, in addition to the high frequency of the minor allele in NAFLD-affected individuals and prevailing association with the disease, underline PNPLA3 rs738409 as a prime therapeutic target for NAFLD” (paragraph 19).
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 teachings of Angelini in view of Palucka to include measuring Pnpla3 taught by Rulifson because Rulifson teaches that Pnpla3 is associated with NAFLD and is a therapeutic target of NAFLD. One would have been motivated to make such a modification because Angelini is concerned with NAFLD and Pnpla3 is an alternate marker for NAFLD. A person having ordinary skill in the art would have had a reasonable expectation of success because both Angelini in view of Palucka and Rulifson teach measuring proteins in blood samples from subjects with NAFLD.
Claim 57 is rejected under 35 U.S.C. 103 as being unpatentable over Angelini as applied to claim 46 above, and further in view of Carrillo et al. Medicine 97(39):p e12392, September 2018. | DOI: 10.1097/MD.0000000000012392 (Cite No. V of PTO-892 1/3/2025) (hereinafter Carrillo) as evidenced by ABCAM (https://www.abcam.com/en-us/search?facets.categoryType=Primary+Antibodies&sorting=relevance&keywords=plin2+antibody&utm_source=google&utm_medium=cpc&gad_source=1&gclid=Cj0KCQiA1Km7BhC9ARIsAFZfEIvaB_wmoLMURunKVZYom7JkRmD_q2DjaENx5BXV8smTBHlLpnpgGXMaAhveEALw_wcB&gclsrc=aw.ds), retrieved online on 12/24/2024 (Cite No. U of PTO-892 1/3/2025).
Regarding claim 57, Angelini teaches the method of claim 46 as discussed above.
Angelini further teaches wherein the amount of Plin2 protein is measured by cytofluorimetry using an antibody for the Plin2 protein (“Plin2 antibody was obtained from LS-BIO (Seattle, WA), AlexaFluor 488 from Life Technology (Carlsbad, CA)” page 8 col. 2 para. 3, “To assess Plin2 basal expression, PBMCs from obese IR, NAFLD subjects and healthy controls were fixed and permeabilized in FIX/PERM buffer (eBioscience San Diego, CA), and stained for Plin2 using AlexaFluor 488 as secondary Antibody. Results were expressed as MFI” page 9 col. 1 para. 2). Note that although Angelini fails to use the language “using an antibody for the Plin2 protein” in the cytofluorimetry experiments, the teaching of an antibody for the Plin2 protein as the antibody reagents together with the teaching of staining Plin2 “using AlexaFluor 488 as secondary Antibody” inherently provides the use of the antibody for Plin2 protein as the primary antibody.
Angelini fails to teach wherein the antibody for the Plin2 protein is a monoclonal antibody.
Carrillo teaches immunofluorescence imaging (“Optical tissue clearing in combination with perfusion and immunofluorescence for placental vascular imaging” Title). Carrillo further teaches using “[t]he primary antibody Perilipin 2 (Cat No. ab181452, Abcam; New York, NY)” together with “secondary antibody, Alexa 488” for fluorescence detection of Plin2 (page 2 column 1 paragraph 3). Note that as evidenced by ABCAM, the antibody (ab181452) is a monoclonal antibody to Plin2 for flow cytometry applications (page 1).
It would have been further prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Angelini to rely on the antibody being a monoclonal antibody targeting Plin2 taught by Carrillo because it would be a simple matter of applying a known reagent to a known method. In this case, both Angelini and Carrillo teach methods comprising measuring Plin2. Carrillo teaches the art-recognized monoclonal antibody targeting Plin2 (as evidenced by ABCAM), therefore, using said antibody for the method of measuring Plin2 would have been obvious to a person having ordinary skill in the art (using an art recognized, and available reagent, for its intended purpose). A person having ordinary skill in the art would have had a reasonable expectation of success because this antibody is commercially available and used for flow cytometry applications as evidenced by ABCAM.
Claim 46-47, 52, 54 and 56 are rejected under 35 U.S.C. 103 as being unpatentable over Cherrington (WO 2019164749 A1)-Cite No. FP4 of IDS filed on 10/11/2021 in view of Palucka.
Regarding claims 46, 52 and 54, Cherrington suggests a method comprising measuring Plin2 (“measuring…PLIN-2 lipid droplet proteins” claim 33) in a blood sample of an individual (“in a plasma sample” claim 25 “wherein the biological fluid sample is selected from the group consisting of blood” claim 17), comprising: obtaining a blood sample from the individual (“diagnosing a hepatic disorder in a human subject at risk of a hepatic disorder, b) obtaining a plasma sample…from the human subject…c) determining an amount of…” claim 1, “method according to any one of claims 1-32, further comprising measuring…PLIN-2 lipid droplet proteins” claim 33, “wherein the biological fluid sample is selected from the group consisting of blood” claim 17) and measuring an amount of Plin2 protein in the blood sample to determine a level of Plin2 protein in the blood sample (claims 17, 25 and 33). Note that although Cherrington fails to use the exact language “measuring an amount of Plin2 protein in the blood sample to determine a level of Plin2 protein in the blood sample”, claim 33 of Cherrington inherently suggests “measuring an amount of Plin2 protein in the blood sample to determine a level of Plin2 protein in the blood sample” because claim 33 teaches measuring Plin2 protein in a “a method according to any one of claims 1-32”. Combining any of claims 1, 17 or 25, for example, with claim 33 inherently provides “measuring an amount of Plin2 protein in the blood sample to determine a level of Plin2 protein in the blood sample” because claim 1, 17 or 25 are drawn to methods of measuring and determining a level of ezetimibe (EZE) and/or ezetimibe-glucuronide (EZE-Gluc) in a blood sample and claim 33 recites “further comprising measuring an amount of…PLIN-2”. The language “further comprising” suggests that the measurement of PLIN-2 is included in the method of measuring and determining a level of EZE and/or EZE-Gluc in the blood sample, i.e. Plin2 is also measured in the blood sample to determine a level of Plin2 in the blood sample.
Cherrington fails to teach extracting leukocytes from the blood sample; and measuring an amount of Plin2 protein in the leukocytes extracted from the blood sample, wherein the blood sample is a peripheral blood sample (claim 52) wherein the leukocytes are polymorphonuclear and/or monocyte cells (claim 54).
Palucka teaches “monitoring indicators of immunosuppression through blood leukocyte microarray analysis” (Title). Palucka further teaches that “[b]lood microarray analyses were carried out in 25 healthy volunteers, 35 patients with metastatic melanoma, and 39 liver transplant recipients” (page 4, lines 1-2). Among the patients who had received a liver transplant, one had “Nonalcoholic Steatohepatitis” and many had “Cirrhosis” and one had “Hepatocellular Carcinoma with Cirrhosis” (Table 4, pages 38-39). Palucka teaches that “[t]he sample may be screened by quantitating the mRNA, protein or both mRNA and protein level of the expression vector” (page 4 lines 34-35). Palucka further teaches wherein the blood sample is a peripheral blood sample and extracting leukocytes from the blood sample prior to measuring one or more proteins wherein the measurement is based on the amount of the one or more proteins in the extracted leukocytes, and wherein the leukocytes are polymorphonuclear and/or monocyte cells (“[c]ells that may be analyzed using the present invention, include, e.g., peripheral blood mononuclear cells (PBMCs )” page 3 lines 23-24, “[p]rocessing of blood samples. All blood samples were collected in acid citrate dextrose tubes (BD Vacutainer) and immediately delivered at room temperature to the Baylor Institute for Immunology Research, Dallas, TX, for processing. Peripheral blood mononuclear cells (PBMCs) from 3-4 ml of blood were isolated via Ficoll gradient and immediately lysed” page 28 lines 20-23). Palucka further teaches that “[o]ther data processed using this approach can be employed for instance in mechanistic studies, or screening of drug compounds” (page 2 lines 29-30). Palucka further teaches that “[l]eukocytes isolated from the peripheral blood of patients constitute an accessible source of clinically-relevant information” (page 49 lines 22-23).
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 teachings of Cherrington to rely on the blood sample being a peripheral blood sample, and to extract leukocytes from the blood sample and to measure the Plin2 protein in the extracted leukocytes, and wherein the leukocytes are polymorphonuclear and/or monocyte cells taught by Palucka because Palucka teaches that data processed using this approach can be employed for instance in mechanistic studies, or screening of drug compounds. Furthermore Palucka teaches that leukocytes from the peripheral blood of patients constitute an accessible source of clinically-relevant information. Also, making such a modification would be a simple matter of applying the known technique, i.e., an art-recognized technique, of extracting PBMCs from the blood prior to measuring one or more proteins in the PBMCs to the base method of measuring proteins from blood taught by Cherrington and Palucka. A person having ordinary skill in the art would have had a reasonable expectation of success because both Palucka and Cherrington teach methods comprising measuring one or more proteins in a blood sample of an individual, wherein the individual is suspected of having a hepatic disorder.
Regarding claim 47, Cherrington in view of Palucka teach the method of claim 46 as discussed above.
Cherrington fails to teach further measuring Rab14.
Palucka further teaches measuring Rab14 (page 7 lines 5-6, Table 1, page 14). Palucka further teaches that “[i]t should be pointed out that there is advantage to combining data from several domains” (page 11 lines 2-3). Palucka further teaches that “[t]he skilled artisan will appreciate that using the modules of the present invention it is possible to rapidly develop one or more disease specific arrays that may be used to rapidly diagnose or distinguish between different disease and/or conditions”(page 7 lines 10-12).
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 teachings of Cherrington in view of Palucka to include measuring Rab14 taught by Palucka because Palucka teaches that measuring Rab14 may be used to diagnose or distinguish between different diseases and or conditions and that there is an advantage in combining data from several domains. A person having ordinary skill in the art would have had a reasonable expectation of success because both Cherrington and Palucka teach measuring proteins in blood samples from subjects with a hepatic disorder.
Regarding claim 56, Cherrington in view of Palucka teaches the method of claim 46 as discussed above.
Cherrington teaches “[c]ytokeratin-18 levels will be determined using a commercially available ELISA kit” (paragraph 58). Cherrington further teaches “[u]sing the collected blood and urine samples, ezetimibe and metabolite quantification can be performed according to standard procedures…For example, the LC- MS/MS system” (paragraph 59).
Cherrington fails to explicitly teach wherein the amount of Plin2 protein in the leukocytes is measured by ELISA.
Palucka suggests measuring the protein by ELISA (page 16 lines 29-34).
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 teachings of Cherrington to rely on the ELISA taught by Palucka because it would be a matter of simply substituting the mass spectrometry technique for measuring proteins used by Cherrington for the art recognized technique, i.e., ELISA technique taught by Palucka for measuring proteins (base method). Both references teach measuring proteins from blood samples, therefore it would have been obvious to a person having ordinary skill in the art to simple use ELISA instead of mass spectrometry. A person having ordinary skill in the art would have had a reasonable expectation of success because both Palucka and Cherrington teach methods comprising measuring one or more proteins in a blood sample of an individual, wherein the individual is suspected of having a hepatic disorder.
Claim 48 is rejected under 35 U.S.C. 103 as being unpatentable over Cherrington in view of Palucka as applied to claim 47 above, and further in view of Rulifson.
Regarding claim 48, Cherrington in view of Palucka teach the method of claim 47 as discussed above.
Cherrington in view of Palucka fail to teach further measuring Pnpla3.
Rulifson teaches “RNAi constructs for reducing expression of the PNPLA3 gene. Methods of using such RNAi constructs to treat or prevent liver disease, nonalcoholic fatty liver disease (NAFLD) are also described” (Abstract). Rulifson further teaches measuring Pnpla3 (paragraph 55, paragraph 170). Rulifson further teaches that “[t]he consensus among numerous GWAS indicate the association of PNPLA3 rs738409 with NAFLD is independent of age, gender, ethnicity, metabolic syndrome, body mass index, insulin resistance, and serum lipids” (paragraph 18). Rulifson further teaches that “in vivo mouse model data points to expression of the mutant Pnpla3.sup.I148M protein, and not over expression of the wild type protein, as the driver of the disease phenotype. These findings, in addition to the high frequency of the minor allele in NAFLD-affected individuals and prevailing association with the disease, underline PNPLA3 rs738409 as a prime therapeutic target for NAFLD” (paragraph 19).
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 teachings of Cherrington in view of Palucka to include measuring Pnpla3 taught by Rulifson because Rulifson teaches that Pnpla3 is associated with NAFLD and is a therapeutic target of NAFLD. One would have been motivated to make such a modification because Cherrington is concerned with hepatic disorders and Pnpla3 is an alternate marker for hepatic disorders. A person having ordinary skill in the art would have had a reasonable expectation of success because both Cherrington in view of Palucka and Rulifson teach measuring proteins in blood samples from subjects with hepatic disorders.
Claim 57 is rejected under 35 U.S.C. 103 as being unpatentable over Cherrington in view of Palucka as applied to claim 46 above, and further in view of (JP 2004535410 A) (Cite No. P of PTO-892 1/3/2025) and Carrillo as evidenced by ABCAM.
Regarding claim 57, Cherrington in view of Palucka teach the method of claim 46 as discussed above.
Cherrington teaches “[c]ytokeratin-18 levels will be determined using a commercially available ELISA kit” (paragraph 58). Cherrington further teaches “[u]sing the collected blood and urine samples, ezetimibe and metabolite quantification can be performed according to standard procedures…For example, the LC- MS/MS system” (paragraph 59).
Cherrington fails to teach wherein the amount of Plin2 protein in the extracted leukocytes is measured by cytofluorimetry using a monoclonal antibody for the Plin2 protein.
JP 2004535410 A teaches that “the modification of impaired thiol metabolism is fundamentally important as a basic treatment in the treatment of many diseases of different origin” (paragraph 14) such as “liver diseases” (paragraph 17). JP 2004535410 A teaches measuring a protein by cytofluorimetry using a monoclonal antibody for that protein (“specific markers of cell activation were quantitatively verified by cytofluorimetry by testing with monoclonal antibodies. The effect of the combination used according to the invention on the activation markers CD69 (early activation antigen), CD25 (intermediate activation antigen) and CD71 (late activation antigen) of T-lymphocytes was investigated” paragraph 58).
Carrillo teaches immunofluorescence imaging (Title). Carrillo further teaches using “[t]he primary antibody Perilipin 2 (Cat No. ab181452, Abcam; New York, NY)” together with “secondary antibody, Alexa 488” for fluorescence detection of Plin2 (page 2 column 1 paragraph 3). Note that as evidenced by ABCAM, the antibody (ab181452) is a monoclonal antibody to Plin2 for flow cytometry applications (page 1).
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 teachings of Cherrington in view of Palucka to rely on the measuring of the Plin2 protein in the leukocytes by cytofluorimetry using a monoclonal antibody, this technique as taught by JP 2004535410 A, because it would be a simple matter of applying the known technique, i.e., an art-recognized technique, of using cytofluorimetry using monoclonal antibodies taught by JP 2004535410 A, to the base method of measuring proteins taught by both Cherrington in view of Palucka and JP 2004535410 A. A person having ordinary skill in the art would have had a reasonable expectation of success because both JP 2004535410 A and Cherrington in view of Palucka teach methods comprising measuring one or more proteins in the field of liver diseases.
It would have been further prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Cherrington in view of Palucka and JP 2004535410 A to rely on the monoclonal antibody being a monoclonal antibody targeting Plin2 taught by Carrillo because it would be a simple matter of applying a known reagent to a known method. In this case, both Cherrington, Palucka, JP 2004535410 A and Carrillo teach methods comprising measuring one or more proteins. Carrillo teaches the art-recognized monoclonal antibody targeting Plin2 (as evidenced by ABCAM), therefore, using said antibody for the method of measuring proteins would have been obvious to a person having ordinary skill in the art (using an art recognized, and available reagent, for its intended purpose). A person having ordinary skill in the art would have had a reasonable expectation of success because this antibody is commercially available and used for flow cytometry applications as evidenced by ABCAM; and JP 2004535410 A teaches using monoclonal antibodies for cytofluorimetry.
Response to Arguments
Applicant's arguments filed 9/4/2025 have been fully considered but they are not persuasive.
Regarding the 103 rejections, Applicant argues that “Cherrington does not teach "a method comprising measuring Plin2 ... in a blood sample of an individual,"… Claim 33 of
Cherrington does not state that the Plin2 amount is measured from a blood sample or from
leukocytes extracted from the blood sample (page 9 para. 5). However, Cherrington does suggest a method that includes measuring the level of Plin2 protein in a blood sample (see rejection above). Applicant further argues that “the presently claimed method recites "extracting leukocytes from the blood sample," which is not possible to do using the plasma sample recited in claim 1 of Cherrington” (page 10 para. 2). However, Palucka is relied upon for the teaching of extracting leukocytes from the blood sample (see rejection above). Applicant further argues that “the Examiner removed important context from part c) of claim 1 of Cherrington, which
actually recites, "determining an amount of EZE and/or ezetimibe-glucuronide (EZE-Gluc) in
the plasma sample…sample obtained from a human subject … EZE and EZE-Gluc are not Plin2, so it is clear that claim 1 of Cherrington is not teaching "measuring an amount of Plin2 protein" in a blood sample” (page 10 para. 2). However, even though claim 1 of Cherrington recites measuring EZE and/or EZE-Gluc in the blood sample, claim 33 of Cherrington provides the further measuring of Plin2 in the blood sample (see rejection above for a complete analysis). Similarly, Applicant argues that “Claim 17 of Cherrington depends from claims 2, 3, or 4 of Charrington, each of which recites, "measuring an amount of EZE and/or ezetimibe-glucuronide (EZE-Gluc) in the fluid sample" (emphasis added). Thus, claims 2, 3, and 4 of Charrington, combined with claim 17 of Cherrington, disclose measuring an amount of EZE and/or EZE-Gluc in a blood sample. But, again, EZE and EZE-Gluc are not Plin2” (page 10 para. 3). However, as stated above, even though claims 2-4 of Cherrington recite measuring EZE and/or EZE-Gluc in the blood sample, claim 33 of Cherrington provides the further measuring of Plin2 in the blood sample (see rejection above for a complete analysis). Applicant further remarks a similar argument as above, “Claim 25 of Cherrington recites, "determining an amount of EZE and/or ezetimibe-glucuronide (EZE-Gluc) in a plasma sample." However, EZE and
EZE-Gluc are not Plin2” (page 10 last paragraph and page 11 para. 1). However, as stated above, even though claim 25 of Cherrington recites measuring EZE and/or EZE-Gluc in the blood sample, claim 33 of Cherrington provides the further measuring of Plin2 in the blood sample (see rejection above for a complete analysis). Applicant further reiterates that “Claims 17, 25, and 33 refer to measuring EZE and/or EZE-Gluc in a fluid sample, such as a plasma sample, but make no reference to measuring Plin2 in a blood sample. While claim 33 discloses measuring an amount of Plin2, it does not state that such a measurement is made in a blood sample. Instead, Cherrington only teaches that Plin2 can be measured from a liver biopsy” (page 11 para. 2). However, as stated above, claim 33 of Cherrington suggests measuring Plin2 in the blood sample because claim 33 depends on any of claims 1-32 and by combining either claim 1, 17 or 25 with claim 33 the teaching of measuring Plin2 in the blood sample is effectively provided (see rejection above for a complete analysis). In short, claim 33 uses the language “further comprising” therefore it follows that Plin2 is also measured in the blood sample as was done in claims 1, 17 and 25 of Cherrington. Applicant further argues that “Palucka, Rulifson, Tager, Carrillo, and Caldwell is completely silent about Plin2, and accordingly does not cure the deficiencies of Cherrington. Thus, claim 46 is not obvious over Cherrington in view of Palucka, Rulifson, Tager, Carrillo, and Caldwell” (page 11 para. 3). However, Cherrington in view of Palucka teach claim 46 (see rejection above).
Conclusion
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/Fernando Ivich/ Examiner, Art Unit 1678
/CHRISTOPHER L CHIN/Primary Examiner, Art Unit 1677