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 .
Priority
The present application filed on 04/08/2024, claims benefit of U.S. Provisional Application No. 63/601,041, filed on 11/20/2023.
The limitations of instant claims 1-7 are supported in the original disclosure provided in U.S. Provisional Application No. 63/601,041, filed on 11/20/2023, thus instant claims 1-7 have an effective filing date of 11/20/2023.
Instant claims 8-15 recite “Gro-alpha, INF-gamma, IL-15, RANTES, SCGF-beta, and VEGF,” which is/are not supported in the original disclosure. Further, limitations reciting measuring expression levels of these proteins and comparing their expression levels are not supported in the original disclosure. Thus instant claims 8-15 have an effective filing date 04/28/2024.
Information Disclosure Statement
One Information Disclosure Statement(s) (IDS), filed on 09/19/2024, is acknowledged and considered.
Claim Status
Claims 1-15 are pending and examined herein below.
Claim Objections
Claims 8-15 objected to because of the following informalities: the term “INF-gamma” has a typographical error and should be corrected to reads as “IFN-gamma”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 3 and 14 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 3 is dependent on claim 1 or 2 and recites “comprising measuring expression levels of at least one of WWP2, CLEC1B, HSP-27, CD40, and/or FR-alpha,”. In the instant that claim 3 is read together with claim 2, the limitations in claim 3 is fully recited in independent claim 2, from which claim 3 depends. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Further, claim 14 depends on claim 1 or 8 and recites “comprising measuring expression levels of Gro-alpha, INF-gamma, IL-15, RANTES, SCGF-beta, and/or VEGF”. In the instant that claim 14 is read together with claim 8, the limitations in claim 14 is fully recited in independent claim 8, from which claim 14 depends. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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 1-15 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 1 is indefinite because it fails to incorporate specific criteria/parameters and/or distinct steps to carry out the “method for diagnosing autism spectrum [ASD].” For instance, though the active steps for the recited method comprise isolating extracellular vesicles (EVs) and measuring expression levels, how to integrate these steps into a diagnostic for ASD is not clearly defined or described. Thus, a skilled artisan would not be able to determine, with a reasonable degree of certainty, how to perform the method and/or implement the instant limitation(s) to make and use the claimed invention. Therefore, the metes and bounds of the claim cannot be ascertained.
Claim 8 is indefinite because it fails to define “abnormal expression of proteins.” Specifically, it is unclear whether upregulation of protein expression, downregulation of protein expression, or a combination thereof would be defined as abnormal for the proteins recited in the instant claims. Thus, a skilled artisan would not be able to determine, with a reasonable degree of certainty, how to perform the method and/or implement the instant limitation(s) to make and use the claimed invention. Therefore, the metes and bounds of the claim cannot be ascertained.
Claims 3-7 and 9-15 are rejected as being dependent on independent claims 1 and 8.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-15 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter. The claim(s) does/do not fall within at least one of the four categories of patent eligible subject matter because the claimed invention is directed to natural phenomenon/correlation and an abstract idea without significantly more.
The claimed invention is directed to and law of nature/natural phenomenon and abstract mental process without significantly more. The claim(s) recite a correlation between protein expression levels and a diagnosis and comparisons between measured expression levels and a control or reference value. These judicial exceptions are not integrated into a practical application because the steps for measuring expression levels of different numbers and types of proteins are insignificant extra-solution activities. The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception because methods used for measuring expression levels are well-known, routine, and/or conventional in the art.
The U.S. Patent and Trademark Office recently revised the MPEP with regard to § 101
(see the MPEP at 2106). Regarding the MPEP at 2106, in determining what concept the
claim is “directed to,” we first look to whether the claim recites:
(1) any judicial exceptions, including certain groupings of abstract ideas (i.e.,
mathematical concepts, certain methods of organizing human activity such as a fundamental
economic practice, or mental processes); and
(2) additional elements that integrate the judicial exception into a practical application
(see MPEP § 2106.05(a)-(c), (e)-(h)). Only if a claim
(1) recites a judicial exception and
(2) does not integrate that exception into a practical application, do we then look
to whether the claim contains an “‘inventive concept’ sufficient to ‘transform’” the
claimed judicial exception into a patent-eligible application of the judicial exception.
Alice, 573 U.S. at 221 (quoting Mayo, 566 U.S. at 82). In so doing, we thus consider
whether the claim:
(3) adds a specific limitation beyond the judicial exception that is not “well-
understood, routine, conventional” in the field (see MPEP § 2106.05(d)); or
(4) simply appends well-understood, routine, conventional activities previously
known to the industry, specified at a high level of generality, to the judicial exception.
See MPEP 2106.
ELIGIBILITY STEP 2A: WHETHER A CLAIM IS DIRECTED TO A
JUDICIALEXCEPTION
Step 2A, Prong 1
Claim 1 recites “[a] method for diagnosing autism spectrum disorder comprising…. measuring protein expression levels in the extracellular vesicles.” Further, claims 2, 8 recite “diagnosing autism spectrum disorder comprising… measuring expression levels of proteins… wherein increased expression of proteins… is diagnostic of autism spectrum disorder,” which is drawn to making a correlation between the presence/amount of one or more naturally occurring proteins with a diagnosis, which is directed to a law of nature or natural phenomenon/correlation.
The court has recognized that the correlation between detecting naturally produced
biomarkers with presence of a disease state is directed to a law of nature. For example, in
Athena Diagnostics, Inc. v. Mayo Collaborative Services, LLC, a 2019 U.S. Federal Circuit case,
later denied review by the Supreme Court, the court held that claims for diagnosing a
neurological disorder by detecting naturally-produced antibodies to a specific naturally occurring
protein (MuSK) to be ineligible for patent protection. The court ruled that the correlation
between anti-MuSK autoantibodies and a disease state, although novel and non-obvious, was
directed to a law of nature.
Claims 2, 8, and 15 recite “increased expression of protein… compared to control is diagnostic of autism spectrum disorder,” “abnormal expression of proteins… compared to control, is diagnostic of autism spectrum disorder,” and “abnormal expression of proteins… compared to control comprises decreased [and/or increased] expression of” certain proteins, respectively, which are drawn to making a comparison against a control or reference value and is directed to an abstract idea, namely an abstract mental process.
‘CyberSource Corp. v. Retail Decisions, Inc., 654 F.3d 1366, 1372, 99 USPQ2d 1690, 1695 (Fed. Cir. 2011). As the Federal Circuit explained, "methods which can be performed mentally, or which are the equivalent of human mental work, are unpatentable abstract ideas the ‘basic tools of scientific and technological work’ that are open to all.’" 654 F.3d at 1371, 99 USPQ2d at 1694 (citing Gottschalk v. Benson, 409 U.S. 63, 175 USPQ 673 (1972)). See also Mayo Collaborative Servs. v. Prometheus Labs. Inc., 566 U.S. 66, 71, 101 USPQ2d 1961, 1965 (2012) ("‘[M]ental processes[] and abstract intellectual concepts are not patentable, as they are the basic tools of scientific and technological work’" (quoting Benson, 409 U.S. at 67, 175 USPQ at 675)); Parker v. Flook, 437 U.S. 584, 589, 198 USPQ 193, 197 (1978) (same). Accordingly, the "mental processes" abstract idea grouping is defined as concepts performed in the human mind, and examples of mental processes include observations, evaluations, judgments, and opinions.' Other examples of claims that recite a mental process when they contain limitations that can practically be performed in the human mind include ‘claims to “comparing BRCA sequences and determining the existence of alterations,” where the claims cover any way of comparing BRCA sequences such that the comparison steps can practically be performed in the human mind, University of Utah Research Foundation v. Ambry Genetics, 774 F.3d 755, 763, 113 USPQ2d 1241, 1246 (Fed. Cir. 2014) and a claim to collecting and comparing known information (claim 1), which are steps that can be practically performed in the human mind, Classen Immunotherapies, Inc. v. Biogen IDEC, 659 F.3d 1057, 1067, 100 USPQ2d 1492, 1500 (Fed. Cir. 2011).’ See MPEP 2106.04(a)(2).
Step 2A, Prong 2
The judicial exceptions are not integrated into a practical application because the additional elements of isolating extracellular vesicles (EVs) [claims 1-2 and 8] and measuring expression levels of additional protein or of a different number of proteins (claims 3-7 and 9-14) are insignificant extra-solution activities, namely data-gathering steps, that do not add meaningful limitations to the judicial exception.
ELIGIBILITY STEP 2B: WHETHER ADDITIONAL ELEMENTS AMOUNT TO
SIGNIFICANTLY MORE THAN THE JUDICIAL EXCEPTION (INVENTIVE CONCEPT)
Step 2B
The instant claims do not include additional elements that are sufficient to amount to significantly more than the judicial exceptions because using Enzyme-linked Immunosorbent Assay (ELISA) as the method for detecting and measuring expression levels of ASD markers (claims 2, 8 and instant Specification paras 0009, 0029, 0031, 0042), is well-known, routine, and conventional in the art. For instance, Ellina et al. used ELISA to measure the levels of several markers, including IFN-gamma, CD40, and VEGF, which are recited as ASD biomarkers in the instant claims, to determine if levels of these biomarker differed for samples of patients diagnosed with type 1 diabetes compared to healthy controls (see Abstract in Ellina et al., Extracellular matrix-associated (GAGs, CTGF), angiogenic (VEGF) and inflammatory factors (MCP-1, CD40, IFN- γ) in type 1 diabetes mellitus nephropathy, 2012, Clin Chem Lab Med, 50, 1, 167-174). Further, Beyer et al. used ELISA to measure the levels of cytokines and heat shock proteins, including RANTES and HSP-27, to investigate their role in muscle performance in geriatric patients with acute infection-induced inflammation (see pg. 3 in Beyer et al., Effects on muscle performance of NSAID treatment with Piroxicam versus placebo in geriatric patients with acute infection-induced inflammation. a double blind randomized controlled trial, 2011, BMC Musculoskeletal Disorders, 12, 292, 1-12). Additionally, Stefanantoni et al. used ELISA to measure levels of SCGF-beta, an ASD marker recited in the instant claims, to determine if SCGF-beta levels were elevated in serum samples of patients diagnosed with pulmonary arterial hypertension (PAH) [see Abstract in Stefanantoni et al., Elevated serum levels of macrophage migration inhibitory factor and stem cell growth factor b in patients with idiopathic and systemic sclerosis associated pulmonary arterial hypertension, 2014, Reumatismo, 66, 3, 270-276). Further, Bhat et al. used ELISA to measure Gro-alpha expression levels to investigate the role of Gro-alpha in triple negative breast cancer (TNBC) pathogenesis (see pgs. 22-24 in Bhat et al., GROα overexpression drives cell migration and invasion in triple negative breast cancer cells, 2017, Oncology Reports, 38, 21-30).
As discussed herein above, the prior art teaches that, at the time of filing, using ELISA to measure the levels of biomarkers, including most of the ASD markers recited in the instant claims, was routine and conventional in the art. A skilled artisan would have recognized that the well-known and conventional ELISA method would be an appropriate method for measuring expression levels of the recited ASD markers in the claimed invention.
For the reasons stated above, claims 1-15 stand rejected as being directed to a natural phenomenon/correlation and an abstract idea without additional elements that amount to significantly more.
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.
Claim(s) 1 is rejected under 35 U.S.C. 103 as being unpatentable over Hollenbeck et al., (US 20220357346 A1, Date:11/10/2022), in view of Tsilioni et al., (Tsilioni et al., Extracellular vesicles are increased in the serum of children with autism spectrum disorder, contain mitochondrial DNA, and stimulate human microglia to secrete IL-1β, 2018, Journal of Neuroinflammation, 15, 239, 1-8, provided in IDS filed on 09/19/2024, NPL Cite No. 4).
Throughout the disclosure, Hollenbeck teaches methods and kits for detecting and diagnosing Autism Spectrum Disorder (ASD) in a subject by measuring protein expression levels of a panel of proteins disclosed as ASD markers. Hollenbeck teaches, in some embodiments, methods comprise obtaining a biological sample from the subject; determining the levels of one or more ASD marker; and diagnosing the subject as having ASD if marker(s) is/are differentially expressed as compared to a control, wherein the control can be a biological sample obtained from a subject diagnosed as not having ASD. Hollenbeck teaches, in some embodiments, the level of at least one marker of ASD can be determined by measuring the protein expression level of the at least one marker of ASD. Hollenbeck further teaches, in some embodiments, methods can have a biological sample obtained from a subject wherein the biological sample can be a whole blood sample, a blood serum sample, a blood plasma sample, or any combination thereof.
Regarding claim 1, Hollenbeck teaches a method for diagnosing autism spectrum (ASD) disorder comprising measuring expression levels of protein marker of ASD from plasma samples of subjects suffering from autism (paras 0004-0005, 0008). Hollenbeck does not teach isolating extracellular vesicles (EVs) and does not teach measuring protein expression levels in the extracellular vesicles.
Throughout the article, Tsilioni teaches isolating serum from children diagnosed with ASD to characterize membrane-associated markers. Tsilioni teaches levels of extracellular vesicle-associated protein is significantly elevated in serum samples from children diagnosed with autism spectrum disorder (ASD) compared to healthy controls. Tsilioni teaches results may help explain brain inflammation in children diagnosed with ASD.
Tsilioni teaches isolating extracellular vesicles (EVs) from plasma samples of subjects suffering from autism spectrum disorder (ASD) [pg. 1, Abstract ; pg. 2, full paras 3; pg. 3, full para 1].
It would have been prima facie obvious, at the time of filing, to combine the method for diagnosing autism spectrum (ASD) taught by Hollenbeck with the method of measuring expression levels of proteins in extracellular vesicles (EVs), as taught by Tsilioni. At the time of filing, the prior art taught that a significantly increased levels of EV-associated protein are found in children with ASD when compared to healthy normotypic cohorts/controls and can stimulate release of a pro-inflammatory cytokine (see Tsilioni et al., 2018, Journal of Neuroinflammation, 15, 239, 1-8). Thus, a skilled artisan would have been motivated to combine these teachings and modify the method for diagnosing ASD by measuring protein expression levels in EVs, as taught by Tsilioni, because results would enable a skilled artisan to distinguish between a healthy normotypic subject and a subject likely to have ASD. A person having ordinary skill in the art would have a reasonable expectation of success because combining these teachings amounts to combining known element/methods, known to function and/or are performed the same separately as they would when combined, to yield expected and predictable results.
Claim(s) 2 and 3 are rejected under 35 U.S.C. 103 as being unpatentable over Hollenbeck et al., (US 20220357346 A1 , Date:11/10/2022), in view of Tsilioni et al., (Extracellular vesicles are increased in the serum of children with autism spectrum disorder, contain mitochondrial DNA, and stimulate human microglia to secrete IL-1β, 2018, Journal of Neuroinflammation, 15, 239, 1-8, provided in IDS filed on 09/19/2024, NPL Cite No. 4), Romano et al., (Romano et al., Extracellular Vesicles lysis: a guide for the release of biomarkers to be used in cancer diagnostics, 2023, ChemRxiv, 1-28), Bao et al., (Bao et al., Olink proteomics profiling platform reveals non-invasive inflammatory related protein biomarkers in autism spectrum disorder, 2023, Front. Mol. Neurosci. 16, 1185021, 1-11), and Yao et al., (Yao et al., Protein Biomarkers of Autism Spectrum Disorder Identified by Computational and Experimental Methods, 2021, Front. Psychiatry, 12, 1-13, provided in IDS provided in IDS filed on 09/19/2024, NPL Cite No. 1).
The teachings of Hollenbeck and Tsilioni are discussed herein above.
Regarding claim 2, Hollenbeck teaches a method for diagnosing autism spectrum (ASD) disorder comprising measuring expression levels of at least one and up to five markers of ASD from plasma samples of subjects suffering from autism, wherein levels are differentially expressed as compared to a control is diagnostic of ASD (paras 0004-0005, 0008, 0010). Hollenbeck does not teach isolating extracellular vesicles (EVs); lysing or permeabilizing the EVs; and measuring expression levels of proteins selected from WWP2, CLEC1B, HSP-27, CD40, and/or FR-alpha using an enzyme-linked immunosorbent assay (ELISA). Though Hollenbeck teaches wherein increased expression of ASD protein markers have different expression levels when compared to a control is diagnostic of ASD (para 0010), Hollenbeck does not teach wherein increased expression levels of specific proteins WWP2, CLEC1B, HSP-27, CD40, and/or FR-alpha compared to a control is diagnostic of ASD.
Tsilioni teaches isolating extracellular vesicles (EVs) from plasma samples of subjects suffering from autism spectrum disorder (ASD) [pg. 1; pg. 2, full paras 3, 5, 6; pg. 3, full para 1]. Tsilioni does not teach lysing or permeabilizing the EVs; measuring expression levels of proteins selected from WWP2, CLEC1B, HSP-27, CD40, and/or FR-alpha using an enzyme-linked immunosorbent assay (ELISA); wherein increased expression of proteins WWP2, CLEC1B, HSP-27, CD40, and/or FR-alpha compared to control is diagnostic of ASD.
Throughout the review article, Romano teaches extracellular vesicles (EVs) contain important biochemical information about their mother cells in the form of proteins, lipids, and nucleic acids. Romano teaches that lysing EVs is an important step for analysis of proteins, lipids, and nucleic acids inside the EVs. Romano summarizes well-known, routine, and conventional lysis reagents and protocols for characterizing the content of EVs. Romano provides practical guidance for those new to this area. Romano further teaches all major chemical lysis, highlighting the strengths and limitations of each, while accounting for the possibility of integrating these reagents and protocols into portable sensing devices for cancer EVs in the future.
Romano teaches lysing extracellular vesicles (EVs) [Abstract and Table 1]. Romano does not teach measuring expression levels of proteins selected from WWP2, CLEC1B, HSP-27, CD40, and/or FR-alpha using an enzyme-linked immunosorbent assay (ELISA); wherein increased expression of proteins selected from WWP2, CLEC1B, HSP-27, CD40, and/or FR-alpha compared to control is diagnostic of ASD.
Throughout the article Bao teaches applying proteomics to identify differentially expressed proteins (DEPs) in children with ASD compared to healthy children. Bao teaches identifying 13 DEPs that are differentially expressed in children with ASD compared to a control group (i.e. healthy children), including CD40, which is found to be up-regulated in the ASD group compared to the healthy group.
Bao teaches measuring expression levels of proteins selected from WWP2, CLEC1B, HSP-27, CD40, and/or FR-alpha, wherein increased expression of proteins selected from WWP2, CLEC1B, HSP-27, CD40, and/or FR-alpha compared to control is diagnostic of autism spectrum disorder (pg. 4, full para 1; pg. 6, Table 1 and full paras 1-2). Bao does not teach measuring expression levels of proteins using an enzyme-linked immunosorbent assay (ELISA).
Throughout the article Yao teaches combining computational predictions with experimental work to identify blood protein biomarkers for autism spectrum disorder (ASD). Yao teaches using bioinformatics to analyze brain tissue-based data of subjects with ASD from the Gene Expression Omnibus database to find ASD-related genes and a blood-secretory program to predict ASD-related protein in blood. Yao teaches using ELISA to verify levels of these ASD-related proteins in plasma samples of subjects diagnosed with ASD.
Yao teaches measuring expression levels of proteins using an enzyme-linked immunosorbent assay (ELISA) [pgs. 4 and 8].
It would have been prima facie obvious, at the time of filing, to combine the method for diagnosing ASD, as taught by Hollenbeck, with the method of measuring expression levels of proteins in extracellular vesicles (EVs), as taught by Tsilioni, with the method of lysing EVs, as taught by Romano, with the method of measuring expression levels of CD40, as taught by Bao, with the ELISA method for measuring protein levels, as taught by Yao. A skilled artisan would have been motivated to combine these prior art teachings because it would enable a skilled artisan to characterize, analyze, and detect ASD markers contained in EVs. A skilled artisan would have been further motivated to combine these teachings and substitute the ASD markers taught by Hollenbeck with CD40, as taught by Bao, because it would enable characterization and diagnosis of ASD in a subject. The prior art teaches that at the time of filing, lysing EVs to characterize, analyze, and detect markers contained inside EVs was conventional and routine in the art (see Romano et al., 2023, ChemRxiv, 1-28). Additionally, at the time of filing, using ELISA to measure expression levels of proteins was conventional and routine in the art (see Yao et al., 2021, Front. Psychiatry, 12, 1-13). Thus, a person having ordinary skill in the art would have a reasonable expectation of success because combining these teachings amounts to combining known element/methods, known to function and/or are performed the same separately as they would when combined, to yield expected and predictable results.
Regarding claim 3, Hollenbeck, Tsilioni, Romano, Bao, and Yao teach all the limitations of claims 1 and 2. Bao further teaches comprising measuring expression levels of at least one of WWP2, CLEC1B, HSP-27, CD40, and/or FR-alpha (pg. 4, full para 1; pg. 6, Table 1 and full paras 1-2).
It would have been prima facie obvious, at the time of filing, to combine the method for diagnosing ASD as taught by Hollenbeck, in view of Tsilioni, with the well-known and routine method of lysing EVs, as taught by Romano, with the method of measuring expression levels of CD40, as taught by Bao, with the well-known and routine ELISA method for measuring protein levels, as taught by Yao. Bao teaches CD40 is upregulated in subjects with ASD compared to healthy samples/control, and though Bao teaches other potential diagnostic biomarkers for ASD, a skilled artisan would have been motivated to try the finite number of markers with differential expression in ASD subjects compared to healthy subjects, taught by Bao, to determine if CD40 is an optimal ASD diagnostic marker and arrive at the claimed invention. Further, skilled artisan would have been motivated to combine these teachings and substitute the ASD markers taught by Hollenbeck with CD40 because it would enable early diagnosis of ASD if a certain level of CD40 is detected in a subject’s sample. A person having ordinary skill in the art would have a reasonable expectation of success firstly because the claimed method for diagnosing ASD comprising measuring expression levels of ASD markers and comparing to a control; lysing EVs; and measuring expression levels of markers contained in EVs using ELISA were already taught at the time of filing, thus combining these teachings amounts to combining known elements/methods, known to function and/or are performed the same separately as they would when combined, to yield expected and predictable results. Further, a person having ordinary skill in the art would have a reasonable expectation of success when combining these teachings to arrive at CD40 as an ASD marker to measure expression levels for in the method for diagnosing ASD because Bao teaches a finite number of known solutions (i.e. markers with differential expression in ASD samples compared to a control) that would yield expected and predictable results.
Claim(s) 4 and 5 are rejected under 35 U.S.C. 103 as being unpatentable over Hollenbeck et al., (US 20220357346 A1 , Date:11/10/2022), in view of Tsilioni et al., (Extracellular vesicles are increased in the serum of children with autism spectrum disorder, contain mitochondrial DNA, and stimulate human microglia to secrete IL-1β, 2018, Journal of Neuroinflammation, 15, 239, 1-8, provided in IDS filed on 09/19/2024, NPL Cite No. 4), Romano et al., (Romano et al., Extracellular Vesicles lysis: a guide for the release of biomarkers to be used in cancer diagnostics, 2023, ChemRxiv, 1-28), Bao et al., (Bao et al., Olink proteomics profiling platform reveals non-invasive inflammatory related protein biomarkers in autism spectrum disorder, 2023, Front. Mol. Neurosci. 16, 1185021, 1-11), and Yao et al., (Yao et al., Protein Biomarkers of Autism Spectrum Disorder Identified by Computational and Experimental Methods, 2021, Front. Psychiatry, 12, 1-13, provided in IDS provided in IDS filed on 09/19/2024, NPL Cite No. 1), as applied to claims 1 and 2, and further in view of Courchesne et al., (US 10002230 B2, Date: 06/19/2018), and as evidenced by Ambrozkiewicz et al., (Ambrozkiewicz et al., Polarity Acquisition in Cortical Neurons Is Driven by Synergistic Action of Sox9-Regulated Wwp1 and Wwp2 E3 Ubiquitin Ligases and Intronic miR-140, 2018, Neuron, 100, 1097-1115), and Liu et al., (Liu et al., Biomarker Exploration in Human Peripheral Blood Mononuclear Cells for Monitoring Sulforaphane Treatment Responses in Autism Spectrum Disorder, 2020, Scientific Reports, 10, 5822, 1-11).
Regarding claim 4, the teachings of Hollenbeck, Tsilioni, Romano, Bao, and Yao are discussed herein above. Hollenbeck, Tsilioni, Romano, Bao, and Yao teach all the limitations of claims 1 and 2. Hollenbeck, Tsilioni, Romano, Bao, and Yao do not teach measuring expression levels of at least two of WWP2, CLEC1B, HSP-27, CD40, and/or FR-alpha.
Throughout the disclosure, Courchesne teaches combining functional genomic signatures and multimodality signatures for screening, diagnosing, and prognosticating autism spectrum disorder (ASD). Courchesne teaches, in some embodiments, expression patterns can be used alone or in combination with clinical measures for diagnosing and prognosticating ASD. Courchesne teaches, in some embodiments, selecting two or more genes from a finite number of ASD-associated genes listed in Tables 16-25, which are differentially expressed in toddlers with ASD when compared to a control group of toddlers.
Courchesne teaches measuring expression levels of at least two of WWP2 and HSP-27 (col. 5, lines 7-8; col. 6, lines 15-27; Fig. 4E; col. 25, Table 1;cols. 69-70, Table 16; col. 73-74, Table 18).
It would have been prima facie obvious, at the time of filing, to combine the method for diagnosing ASD as taught by Hollenbeck, in view of Tsilioni, Romano, Bao, and Yao, with the method of measuring the expression levels of two or more ASD markers including WWP2 and HSP-27 for the diagnosis and prognosis of ASD in pediatrics, as taught by Courchesne. In the art, it is known that WWP2 plays a critical role in proper polarization of developing neurons, which then undergo migration, differentiation, and morphological changes. Changes in WWP2 levels may cause disruptions to these regulatory processes and the prior art teaches that such disruptions lead to severe neurodevelopmental disorders and likely autism spectrum disorder (ASD) [see Ambrozkiewicz et al., 2018, Neuron, 100, 1097-1115]. Thus, a skilled artisan would have been motivated to try WWP2 to determine if it would be an optimal ASD marker from the finite number of ASD markers taught by Ambrozkiewicz. A skilled artisan would have been further motivated to combine these teachings and substitute the ASD markers taught by Hollenbeck with CD40 and WWP2 and arrive at the claimed invention because it would enable early diagnostic and prognosis of ASD. A person having ordinary skill in the art would have a reasonable expectation of success when combining the teachings of Hollenbeck, in view of Tsilioni, Romano, Bao, and Yao with the teachings of measuring expression levels of two or more ASD markers including WWP2 because Courchesne teaches a finite number of known solutions (i.e. ASD markers known to have differential expression in ASD samples compared to a control) which would yield expected and predictable results. A person having ordinary skill in the art would have a further reasonable expectation of success because combining these teachings amounts to combining known elements/methods, known to function and/or are performed the same separately as they would when combined, to yield expected and predictable results.
Regarding claim 5, Hollenbeck, Tsilioni, Romano, Bao, and Yao teach all the limitations of claims 1 and 2. Bao further teaches measuring the expression levels of CD40. Hollenbeck, Tsilioni, Romano, Bao, and Yao do not teach measuring expression levels of at least three of WWP2, CLEC1B, HSP-27, CD40, and/or FR-alpha.
Courchesne teaches measuring expression levels of two or more markers of ASD, including WWP2 and HSP-27 (col. 5, lines 7-8; col. 6, lines 15-27; Fig. 4E; col. 25, Table 1;cols. 69-70, Table 16; col. 73-74, Table 18).
It would have been prima facie obvious, at the time of filing, to combine the method for diagnosing ASD as taught by Hollenbeck, in view of Tsilioni, Romano, Bao, and Yao, with the method of measuring the expression levels of two or more ASD markers including WWP2 and HSP-27 for diagnosis and prognosis of ASD in pediatrics, as taught by Courchesne. The motivations for trying and determining if CD40 and WWP2 are optimal ASD markers for the method of diagnosing ASD are discussed herein above. Further, at the time of filing, it was known in the art that HSP-27 expression levels are increased in ASD samples compared to healthy samples after sulforaphane treatment, indicating HSP-27 expression levels is a promising marker for ASD and for monitoring treatment response in ASD subjects (see Liu et al., 2020, Scientific Reports, 10, 5822, 1-11). Thus, a skilled a skilled artisan would have been motivated to try HSP-27 from the finite number of ASD markers taught by Courchesne to determine if HSP-27 would be an optimal ASD marker to measure expression levels for in the method for diagnosing ASD and arrive at the claimed invention. A skilled artisan would have been further motivated to combine these teachings and substitute the ASD markers taught by Hollenbeck with CD40, WWP2, and HSP-27 because it would enable accurate prediction of risk of and/or accurate diagnosis of ASD in a subject at a young age when intervention and treatment are the most effective (see Courchesne et al., col. 6, lines 38-50; col. 1, lines 60-67; and col. 2, lines 1-14). Further, a skilled artisan would have been motivated to measure the expression levels of at least three markers because it would enable a more specific and sensitive method especially for monitoring treatment response for ASD patients relative to measuring expression levels of only a single marker (see Liu et al., 2020, Scientific Reports, 10, 5822, pg. 8). A person having ordinary skill in the art would have a reasonable expectation of success when combining these teachings to arrive at CD40, WWP2, and HSP-27 as an ASD markers to measure expression levels for in the method for diagnosing ASD because the prior art teaches a finite number of known solutions (i.e. markers with differential expression in ASD samples compared to a control) that would yield expected and predictable results. A person having ordinary skill in the art would have a further reasonable expectation of success because combining these teachings amounts to combining known elements/methods, known to function and/or are performed the same separately as they would when combined, to yield expected and predictable results.
Claim(s) 6 is rejected under 35 U.S.C. 103 as being unpatentable over Hollenbeck et al., (US 20220357346 A1 , Date:11/10/2022), in view of Tsilioni et al., (Extracellular vesicles are increased in the serum of children with autism spectrum disorder, contain mitochondrial DNA, and stimulate human microglia to secrete IL-1β, 2018, Journal of Neuroinflammation, 15, 239, 1-8, provided in IDS filed on 09/19/2024, NPL Cite No. 4), Romano et al., (Romano et al., Extracellular Vesicles lysis: a guide for the release of biomarkers to be used in cancer diagnostics, 2023, ChemRxiv, 1-28), Bao et al., (Bao et al., Olink proteomics profiling platform reveals non-invasive inflammatory related protein biomarkers in autism spectrum disorder, 2023, Front. Mol. Neurosci. 16, 1185021, 1-11), and Yao et al., (Yao et al., Protein Biomarkers of Autism Spectrum Disorder Identified by Computational and Experimental Methods, 2021, Front. Psychiatry, 12, 1-13, provided in IDS provided in IDS filed on 09/19/2024, NPL Cite No. 1), as applied to claims 1 and 2, further in view of Courchesne et al., (US 10002230 B2, Date: 06/19/2018), and Kunkel et al., (WO 2011112961 A1, Date: 09/15/2011), and as evidenced by Leyser et al., (Leyser et al., 12p deletion spectrum syndrome: a new case report reinforces the evidence regarding the potential relationship to autism spectrum disorder and related developmental impairments, 2016, Molecular Cytogenetics, 9, 75, 1-12).
Regarding claim 6, the teachings of Hollenbeck, Tsilioni, Romano, Bao, and Yao are discussed herein above. Hollenbeck, Tsilioni, Romano, Bao, and Yao teach all the limitations of claims 1 and 2. Bao further teaches measuring expression levels of CD40. Hollenbeck, Tsilioni, Romano, Bao, and Yao do not teach measuring expression levels of at least four WWP2, CLEC1B, HSP-27, CD40, and FR-alpha.
The teachings of Courchesne are taught herein above. Courchesne teaches measuring expression levels of two or more ASD markers, including WWP2 and HSP-27 (col. 5, lines 7-8; col. 6, lines 15-27; Fig. 4E; col. 25, Table 1;cols. 69-70, Table 16; col. 73-74, Table 18). Courchesne does not teach measuring the expression levels of CLEC1B and/or FR-alpha.
Throughout the article Kunkel teaches methods and kits for characterizing and diagnosing autism spectrum disorder (ASD) in a subject. Kunkel teaches, in some embodiments, the method may comprise: (a) obtaining a clinical sample from the subject; (b) determining expression levels of a plurality of autism spectrum disorder-associated genes in the sample, wherein the autism spectrum disorder-associated genes comprise genes selected from Tables 4, 5, 6, 7 or 10; and (c) comparing each expression level determined in (b) with an appropriate reference level/control, wherein the results of comparing in (c) characterize the ASD status of the individual. Kunkel further teaches, in some embodiments, the methods comprise diagnosing ASD in the individual based on the ASD status or expression levels of proteins compared to the reference or control.
Kunkel teaches measuring expression levels of CLEC1B (pg. 65, Table 4).
It would have been prima facie obvious, at the time of filing, to combine the method for diagnosing ASD as taught by Hollenbeck, in view of Tsilioni, Romano, Bao, and Yao, with the method of measuring the expression levels of two or more ASD markers including WWP2 and HSP-27 for diagnosis and prognosis of ASD in pediatrics as taught by Courchesne, and with the teachings of measuring expression levels of CLEC1B for characterizing and diagnosing ASD, as taught by Kunkel. Kunkel teaches several ASD markers, however, at the time of filing, the prior art taught nine genes, including CLEC1B, that may play a role in the pathogenesis of autism spectrum disorder (ASD) because of their role in inflammatory, immunologic and neuro trafficking routes (see Leyser et al., 2016, Molecular Cytogenetics, 9, 75, 1-12). Kunkel teaches five of these nine genes, including CLEC1B, as genes differentially expressed in ASD patients and as potential markers for characterizing and diagnosing ASD. Thus, a skilled artisan would have been motivated to try CLEC1B from the finite number of overlapping genes taught in the prior art and by Kunkel as potential ASD markers to determine if CLEC1B is an optimal ASD marker to measure and arrive at the claimed invention. Motivations for trying and determining if CD40, WWP2, and HSP-27 are optimal ASD markers to measure are discussed herein above. A skilled artisan would have been further motivated to combine these teachings to substitute the ASD markers taught by Hollenbeck in the method for diagnosing ASD with WWP2 CLEC1B, HSP-27, and CD40 because it would enable accurate prediction of risk for and/or accurate diagnosis of ASD in a subject at a young age when intervention and treatment are the most effective. A person having ordinary skill in the art would have a reasonable expectation of success for combining prior art teachings with Kunkel to arrive at CLEC1B as an ASD marker to measure because a skilled artisan would be trying a finite number of known solutions, especially if a skilled artisan focused on potential ASD markers overlapping with the prior art, that would yield expected and predictable results. A person having ordinary skill in the art would have a further reasonable expectation of success because combining these teachings amounts to combining known elements/methods, known to function and/or are performed the same separately as they would when combined, to yield expected and predictable results.
Claim(s) 7 is rejected under 35 U.S.C. 103 as being unpatentable over Hollenbeck et al., (US 20220357346 A1 , Date:11/10/2022), in view of Tsilioni et al., (Extracellular vesicles are increased in the serum of children with autism spectrum disorder, contain mitochondrial DNA, and stimulate human microglia to secrete IL-1β, 2018, Journal of Neuroinflammation, 15, 239, 1-8, provided in IDS filed on 09/19/2024, NPL Cite No. 4), Romano et al., (Romano et al., Extracellular Vesicles lysis: a guide for the release of biomarkers to be used in cancer diagnostics, 2023, ChemRxiv, 1-28), Bao et al., (Bao et al., Olink proteomics profiling platform reveals non-invasive inflammatory related protein biomarkers in autism spectrum disorder, 2023, Front. Mol. Neurosci. 16, 1185021, 1-11), Yao et al., (Yao et al., Protein Biomarkers of Autism Spectrum Disorder Identified by Computational and Experimental Methods, 2021, Front. Psychiatry, 12, 1-13, provided in IDS provided in IDS filed on 09/19/2024, NPL Cite No. 1), as applied to claims 1 and 2, further in view of Courchesne et al., (US 10002230 B2, Date: 06/19/2018), and Frye et al., (Fyre et al., The Soluble Folate Receptor in Autism Spectrum Disorder: Relation to Autism Severity and Leucovorin Treatment, 2022, J. Pers. Med., 12, 2033, 1-8), and as evidenced by Frye et al, (Frye et al., Blocking and Binding Folate Receptor Alpha Autoantibodies Identify Novel Autism Spectrum Disorder Subgroups, 2016, Frontiers in Neuroscience, 10, 80, 1-10).
Regarding claim 7, the teachings of Hollenbeck, Tsilioni, Romano, Bao, and Yao are discussed herein above. Hollenbeck, Tsilioni, Romano, Bao, and Yao teach all the limitations of claims 1 and 2. Bao further teaches measuring the expression levels of CD40. Hollenbeck, Tsilioni, Romano, Bao, and Yao do not teach measuring expression levels of WWP2, CLEC1B, HSP-27, CD40, and FR-alpha.
The teachings of Courchesne are taught herein above. Courchesne teaches measuring expression levels of two or more markers of ASD, including WWP2 and HSP-27 (col. 5, lines 7-8; col. 6, lines 15-27; Fig. 4E; col. 25, Table 1;cols. 69-70, Table 16; col. 73-74, Table 18). Courchesne and Bao do not teach measuring the expression levels of CLEC1B and/or FR-alpha.
Throughout the article Frye teaches soluble folate binding proteins (sFBPs) have been detected in serum samples of some patients diagnosed with autism spectrum disorder (ASD). Frye teaches a study comprising patients with severe ASD having levels of sFBPs, including binding and blocking FR-alpha autoantibodies (FRAA), detected in their serum, and not in healthy subjects, which leads to cerebral folate deficiency (CFD). CFD is associated with dysfunction in FR-alpha. Frye teaches ASD symptoms improving in ASD patients with detected levels of FRAAs that were treated with leucovorin, a prescription version of folic acid.
Frye teaches measuring levels of FR-alpha autoantibodies (pg. 2, full para 1 and Table 1; and pg. 3, section “2.1. sFBP Assay”).
It would have been prima facie obvious to combine the method for diagnosing ASD as taught by Hollenbeck, in view of Tsilioni, Romano, Bao, and Yao, with the method of measuring the expression levels of two or more ASD markers including WWP2 and HSP-27 for the diagnosis and prognosis of ASD in pediatrics, as taught by Courchesne, with the teachings of measuring FRAAs, as taught by Frye, and arrive at measuring expression levels of FR-alpha for the method of diagnosing ASD. In the art, at the time of filing, it was known that both types of FR-alpha autoantibodies (FRAAs), binding FRAA and blocking FRAA, are prevalent in subjects with ASD and studies indicate both types are involved in ASD pathogenesis. In the art, at the time of filing, it was further known that FRAAs binding to FR-alpha receptors inhibits FR-alpha function (Frye et al., 2016, Frontiers in Neuroscience, 10, 80, pg. 1, Abstract; and pg. 2). A skilled artisan would have recognized that in the presence of binding and/or blocking FRAAs, levels of FR-alpha, detected through the structural-sensitive ELISA method, could appear altered compared to levels in healthy subjects, or a control, because ELISA capture and/or detection antibodies may be blocked from recognizing and/or binding FR-alpha due to steric hinderance, receptor degradation, and/or conformational/structural changes from FRAA binding to FR-alpha. Thus, a skilled artisan would have been motivated to try and determine if FR-alpha would be an optimal marker to measure expression levels for in the method for diagnosing ASD. A skilled artisan would have been further motivated to make this modification and measure expression levels of FR-alpha instead of measuring levels of both binding FRAA and blocking FRAA because detecting altered levels of FR-alpha can be performed with a single ELISA, which is a simpler/less complex method compared to the additional steps and/or assays required for detecting the two types of FRAAs (see Fyre et al., 2022, J. Pers. Med., 12, 2033, pg. 3, section “2.1. sFBP Assay”). Motivations for trying and determining if WWP2, CLEC1B, HSP-27, and CD40 would be optimal markers for diagnosing ASD are discussed herein above. Thus, a skilled artisan would have been further motivated to substitute the five ASD markers for diagnosing and prognosing ASD in the method taught by Hollenbeck with WWP2, CLEC1B, HSP-27, CD40, and FR-alpha because would enable accurate prediction for risk of and/or accurate diagnosis of ASD in a subject at a young age when intervention and treatment are the most effective. A person having ordinary skill in the art would have a further reasonable expectation of success because combining these teachings amounts to combining known elements/methods, known to function and/or are performed the same separately as they would when combined, to yield expected and predictable results.
Claim(s) 8-12 and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Hollenbeck et al., (US 20220357346 A1 , Date:11/10/2022), in view of Tsilioni et al., (Extracellular vesicles are increased in the serum of children with autism spectrum disorder, contain mitochondrial DNA, and stimulate human microglia to secrete IL-1β, 2018, Journal of Neuroinflammation, 15, 239, 1-8, provided in IDS filed on 09/19/2024, NPL Cite No. 4), Romano et al., (Romano et al., Extracellular Vesicles lysis: a guide for the release of biomarkers to be used in cancer diagnostics, 2023, ChemRxiv, 1-28), Masi et al., (Masi et al., Cytokine levels and associations with symptom severity in male and female children with autism spectrum disorder, 2017, Molecular Autism, 8, 63, 1-11), and Yao et al., (Yao et al., Protein Biomarkers of Autism Spectrum Disorder Identified by Computational and Experimental Methods, 2021, Front. Psychiatry, 12, 1-13, provided in IDS provided in IDS filed on 09/19/2024, NPL Cite No. 1).
Regarding claims 8-12 and 14, the teachings of Hollenbeck are discussed herein above. Hollenbeck teaches a method for diagnosing autism spectrum (ASD) disorder comprising measuring expression levels of protein marker(s) of ASD from plasma samples of subjects suffering from autism and wherein abnormal expression of proteins compared to control, is diagnostic of ASD (paras 0004-0005, 0008, 0010). Hollenbeck does not teach isolating extracellular vesicles (EVs); lysing or permeabilizing the EVs; measuring expression levels of at least one, of at least two, of at least three, of at least four, and of at least five proteins selected from Gro-alpha, INF-gamma, IL-15, RANTES, SCGF-beta, and/or VEGF, using an enzyme-linked immunosorbent assay (ELISA); and wherein abnormal expression of proteins selected from Gro-alpha, INF-gamma, IL-15, RANTES, SCGF-beta, and/or VEGF compared to control, is diagnostic of autism spectrum disorder.
The teachings of Tsilioni are discussed herein above. Tsilioni teaches isolating extracellular vesicles (EVs) from plasma samples of subjects suffering from autism spectrum disorder (ASD) [pg. 1; pg. 2, full paras 3, 5, 6; pg. 3, full para 1]. Tsilioni does not teach lysing or permeabilizing the EVs; measuring expression levels of at least one, of at least two, of at least three, of at least four, and of at least five proteins selected from Gro-alpha, INF-gamma, IL-15, RANTES, SCGF-beta, and/or VEGF, using an enzyme-linked immunosorbent assay (ELISA); and wherein abnormal expression of proteins selected from Gro-alpha, INF-gamma, IL-15, RANTES, SCGF-beta, and/or VEGF compared to control, is diagnostic of autism spectrum disorder.
The teachings of Romano are discussed herein above. Romano teaches lysing extracellular vesicles (EVs) [Abstract and Table 1]. Romano does not teach measuring expression levels of at least one, of at least two, of at least three, of at least four, and of at least five proteins selected from Gro-alpha, INF-gamma, IL-15, RANTES, SCGF-beta, and/or VEGF, using an enzyme-linked immunosorbent assay (ELISA); and wherein abnormal expression of proteins selected from Gro-alpha, INF-gamma, IL-15, RANTES, SCGF-beta, and/or VEGF compared to control, is diagnostic of autism spectrum disorder.
Throughout the article Masi teaches there is a negative association between platelet-derived growth factor (PDGF)-BB and severity of autism spectrum disorder (ASD). Masi teaches using well-characterized repository of biological samples and multiplexed assays to probe the correlation between cytokine levels and ASD symptom severity. Masi further teaches collecting peripheral blood samples from subjects to detect and quantify levels of cytokines in plasma samples including, but not limited to, INF-gamma, IL-15, RANTES, and VEGF. Masi further teaches ASD symptom severity is negatively associated with levels of cytokines including IL-1β, IL-8, MIP-1β, and VEGF in females but not in males.
Masi teaches the limitation(s) of claims 8-12 and 14 reciting measuring expression levels of at least one, of at least two, of at least three, of at least four, and of at least five proteins selected from Gro-alpha, INF-gamma, IL-15, RANTES, SCGF-beta, and/or VEGF [pg. 1, Abstract; pg. 3; pg. 5, Table 1; pg. 7, Table 4]. Masi does not teach measuring expression levels of proteins using an enzyme-linked immunosorbent assay (ELISA).
The teachings of Yao are discussed herein above. Yao teaches measuring expression levels of proteins using an enzyme-linked immunosorbent assay (ELISA) [pg. 4].
It would have been prima facie obvious, at the time of filing, to combine the method for diagnosing ASD, as taught by Hollenbeck, with the method of measuring expression levels of proteins in extracellular vesicles (EVs), as taught by Tsilioni, with the method of lysing EVs, as taught by Romano, with the ELISA method for measuring protein levels, as taught by Yao, with the teachings of measuring expression levels of at least one and up to four of INF-gamma, IL-15, RANTES, and VEGF, as taught by Masi. A skilled artisan would have been motivated to combine these prior art teachings because it would enable a skilled artisan to characterize, analyze, and detect ASD markers contained in EVs. A skilled artisan would have been further motivated to combine these teachings and substitute the ASD markers taught by Hollenbeck with INF-gamma, IL-15, RANTES, and VEGF, as taught by Masi because it would enable diagnosis of ASD in a subject as well as understanding and/or classifying severity of ASD-related symptoms based on these levels. Masi teaches measuring levels of more than four ASD markers. A skilled artisan would have been motivated to try the finite number of markers taught by Masi to determine the optimal set of ASD markers for diagnosing ASD and arrive at the claimed invention. As discussed herein above, the prior art teaches that that the time of filing, lysing EVs to characterize, analyze, and detect markers contained inside EVs and using ELISA to measure expression levels of proteins were conventional and routine methods/techniques in the art at the time of filing (see Romano et al., 2023, ChemRxiv, 1-28 and Yao et al., 2021, Front. Psychiatry, 12, 1-13). A person having ordinary skill in the art would have a reasonable expectation of success, with respect to arriving at measuring the levels of INF-gamma, IL-15, RANTES, and VEGF, because a skilled artisan would have been trying a finite number of known solutions already taught in the prior art at the time of filing and would thus yield expected and predictable results. A person having ordinary skill in the art would have a further reasonable expectation of success because combining these teachings amounts to combining known elements/methods, known to function and/or are performed the same separately as they would when combined, to yield expected and predictable results.
Claim(s) 13 is rejected under 35 U.S.C. 103 as being unpatentable over Hollenbeck et al., (US 20220357346 A1 , Date:11/10/2022), in view of Tsilioni et al., (Extracellular vesicles are increased in the serum of children with autism spectrum disorder, contain mitochondrial DNA, and stimulate human microglia to secrete IL-1β, 2018, Journal of Neuroinflammation, 15, 239, 1-8, provided in IDS filed on 09/19/2024, NPL Cite No. 4), Romano et al., (Romano et al., Extracellular Vesicles lysis: a guide for the release of biomarkers to be used in cancer diagnostics, 2023, ChemRxiv, 1-28), Masi et al., (Masi et al., Cytokine levels and associations with symptom severity in male and female children with autism spectrum disorder, 2017, Molecular Autism, 8, 63, 1-11 ), and Yao et al., (Yao et al., Protein Biomarkers of Autism Spectrum Disorder Identified by Computational and Experimental Methods, 2021, Front. Psychiatry, 12, 1-13, provided in IDS provided in IDS filed on 09/19/2024, NPL Cite No. 1), as applied to claims 1 and 8, and further in view of Sreenivas et al., (Sreenivas et al., Comprehensive immunoprofiling of neurodevelopmental disorders suggests three distinct classes based on increased neurogenesis, Th-1 polarization or IL-1 signaling, 2024 (available online 11 Nov 2023), Brain, Behavior, and Immunity, 115, 505-516).
Regarding claim 13, the teachings of Hollenbeck, Tsilioni, Romano, Masi, and Yao are discussed herein above. Hollenbeck, Tsilioni, Romano, Masi, and Yao teach all the limitations of claims 1 and 8. Hollenbeck, Tsilioni, Romano, Masi, and Yao do not teach measuring expression levels of at least five of Gro-alpha, INF-gamma, IL-15, RANTES, SCGF-beta, and/or VEGF.
Throughout the article, Sreenivas teaches a study comprising measuring levels of 48 immune pathway-related markers, including SCGF-beta, detected in plasma of children diagnosed with autism spectrum disorder (ASD, attention deficit disorder (ADHD), and intellectual disability disorder (IDD). Sreenivas teaches measuring levels of these 48 biomarkers and comparing to a control group comprising typically developing children to determine if neurodevelopmental disorders share a common immunopathogenic mechanism.
Sreenivas teaches measuring levels of SCFG-beta (pg. 507, section 2.3).
It would have been prima facie obvious, at the time of filing, to combine the method for diagnosing ASD, as taught by Hollenbeck, in view of Tsilioni, Romano, Masi, and Yao, with the method for measuring levels of SCGF-beta, as taught by Sreenivas. Motivations for trying and determining whether INF-gamma, IL-15, RANTES, and VEGF are optimal ASD markers for the method of diagnosing ASD are discussed herein above. Further, Sreenivas teaches 48 markers, including SCGF-beta, found in patients diagnosed with neurodevelopmental disorders, including ASD. Sreenivas focuses on determining common markers between three different neurodevelopmental disorders. A skilled artisan would have been motivated to try the finite number of potential ASD markers taught by Sreenivas to determine if SCGF-beta would be an optimal marker to measure levels for in a subject’s sample in the method for diagnosing ASD. A skilled artisan would have been further motivated to combine the teachings of Hollenbeck, in view of Tsilioni, Romano, Masi, and Yao, with the teachings of Sreenivas to substitute the five ASD markers taught by Hollenbeck with INF-gamma, IL-15, RANTES, SCGF-beta, and VEGF in the method for diagnosing ASD taught by Hollenbeck because it would enable diagnosis of ASD in a subject as well as understanding and/or classifying severity of ASD-related symptoms based on these markers’ expression levels relative to a control. Masi and Sreenivas teach measuring levels of more than four ASD markers. A person having ordinary skill in the art would have a reasonable expectation of success, with respect to arriving at measuring the levels of INF-gamma, IL-15, RANTES, VEGF, and SCGF-beta because a skilled artisan would have been trying a finite number of known solutions already taught in the prior art at the time of filing and the prior art teaches and suggests these as ASD markers, therefore, combining these teachings would yield expected and predictable results. A person having ordinary skill in the art would have a further reasonable expectation of success because combining these teachings amounts to combining known elements/methods, known to function and/or are performed the same separately as they would when combined, to yield expected and predictable results.
Claim(s) 15 is rejected under 35 U.S.C. 103 as being unpatentable over Hollenbeck et al., (US 20220357346 A1 , Date:11/10/2022), in view of Tsilioni et al., (Extracellular vesicles are increased in the serum of children with autism spectrum disorder, contain mitochondrial DNA, and stimulate human microglia to secrete IL-1β, 2018, Journal of Neuroinflammation, 15, 239, 1-8, provided in IDS filed on 09/19/2024, NPL Cite No. 4), Romano et al., (Romano et al., Extracellular Vesicles lysis: a guide for the release of biomarkers to be used in cancer diagnostics, 2023, ChemRxiv, 1-28), Masi et al., (Masi et al., Cytokine levels and associations with symptom severity in male and female children with autism spectrum disorder, 2017, Molecular Autism, 8, 63, 1-11 ), and Yao et al., (Yao et al., Protein Biomarkers of Autism Spectrum Disorder Identified by Computational and Experimental Methods, 2021, Front. Psychiatry, 12, 1-13, provided in IDS provided in IDS filed on 09/19/2024, NPL Cite No. 1), as applied to claim 8, further in view of Zerbo et al., (Zerbo et al., Neonatal cytokines and chemokines and risk of Autism Spectrum Disorder: the Early Markers for Autism (EMA) study: a case-control study, 2014, Journal of Neuroinflammation, 11, 113, 1-9).
Regarding claim 15, the teachings of Hollenbeck, Tsilioni, Romano, Masi, and Yao are discussed herein above. Hollenbeck, Tsilioni, Romano, Masi, and Yao teach the method of claim 8. Hollenbeck, Tsilioni, Romano, Masi, and Yao do not teach wherein abnormal expression of proteins selected from Gro-alpha, INF-gamma, IL-15, RANTES, SCGF-beta, and/or VEGF compared to control comprises decreased expression of Gro-alpha and/or RANTES, and/or increased expression of INF-gamma, IL-15, SCGG-beta and/or VEGF.
Throughout the article, Zerbo teaches results from a population-based case-control study comprising children diagnosed with autism spectrum disorder (ASD), developmental delay not ASD, and general population controls. Zerbo teaches that while cytokines were not detected in most newborn samples in all cohorts, chemokine monocyte chemotactic protein-1 (MCP-1) was elevated and levels of Regulated upon Activation Normal T-Cell Expressed and Secreted (RANTES) were decreased in children with ASD.
Zerbo teaches wherein abnormal expression of proteins selected from Gro-alpha, INF-gamma, IL-15, RANTES, SCGF-beta, and/or VEGF compared to control comprises decreased expression of RANTES (pg. 1, Abstract; pg. 4, Table 2; pg. 7).
It would have been prima facie obvious, at the time of filing, to combine the method for diagnosing ASD, as taught by Hollenbeck, in view of Tsilioni, Romano, Masi and Yao, with the method for measuring decreased expression levels of RANTES, as taught by Zerbo. A skilled artisan would have been motivated to combine these prior art teachings with the teachings of considering decreased levels of RANTES when compared to a control as taught by Zerbo because it would enable early detection of ASD in an infant subject. A person having ordinary skill in the art would have a further reasonable expectation of success because combining these teachings amounts to combining known elements/methods, known to function and/or are performed the same separately as they would when combined, to yield expected and predictable results.
Conclusion
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/MELISSA LIZETTE LIRIANO-NG/Examiner, Art Unit 1677
/BAO-THUY L NGUYEN/Supervisory Patent Examiner, Art Unit 1677 August 10, 2026
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