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 .
Claims 38, 57, 59, 64-65, and 67-87 are pending. Claim 38 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claims 1-37, 39-56, 58, 60-63, and 66 are canceled. Claims 57, 59, 64-65, 67-68, 73, 77, 84, and 86-87 are amended. Claims 57, 59, 64-65, and 67-87 are currently under examination.
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 2/6/26 has been entered.
Priority
The instant application 17/736,570 filed on 5/4/22 is a CON of PCT/CA2020/051507 filed on 11/6/20, and claims domestic priority to provisional application 62/931,411 filed
on 11/6/19. The priority date is determined to be 11/6/19.
Response to Arguments
Applicant’s arguments, see pages 9-10, filed 2/6/26, with respect to the rejections of claims 57, 59, 64-65, 67-83, and 85-87 under 35 USC 103 have been fully considered are found unpersuasive, and the 103 rejections documented in the Final mailed 8/8/25 have been revised to address claim amendments filed 2/6/26 in this Non-Final Office Action. More detailed responses to Applicant’s arguments are provided at the end of each maintained rejection.
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 57, 59, 64-65, 67-83, and 85-87 remain/are rejected under 35 U.S.C. 103 as being unpatentable over De Carvalho et al. (2017; WO 2017/190215 A1; FOR citation 002 on IDS filed 9/14/2023) in view of Diagenode MagMeDIP qPCR Kit Manual (version 2 - March 2019; NPL citation V in PTO-892 filed 8/8/25) and Diagenode DNA Methylation Control Package Manual (March 28, 2019; NPL citation U in PTO-892 filed 8/8/25).
This rejection is revised/updated in response to claim amendments filed 2/6/26.
(i) De Carvalho et al. specifically teaches limitations relevant to claims 57, 64-65, 67, 70-83, and 85-87.
Relevant to claim 57, De Carvalho et al. Figure 2 teaches A method of processing a cell-free sample, comprising:(a) generating a mixture comprising (1) a plurality of deoxyribonucleic acid (DNA) molecules derived from the cell-free sample and (2) a plurality of control synthetic nucleic acid molecules, wherein the plurality of control synthetic nucleic acid molecules comprises… (b) enriching for methylated DNA molecules of the plurality of DNA molecules, thereby yielding a plurality of enriched methylated DNA molecules; (c) sequencing (i) the plurality of enriched methylated DNA molecules and (ii) the plurality of control synthetic nucleic acid molecules to generate a plurality of sequencing reads comprising a first plurality of sequencing reads of the plurality of enriched methylated DNA molecules and a second plurality of sequencing reads of the plurality of control synthetic nucleic acid molecules.
The De Carvalho et al. Figure 2 includes a plurality of “Original Cell-free DNA” mixed with a plurality of control synthetic nucleic acid molecules of “Spiked-in A. thaliana DNA QC1 control” and “Spiked-in λ DNA (Filler DNA)”, wherein the control sequences contain both methylated and unmethylated populations. De Carvalho et al. Figure 2 further includes “Immunoprecipitation”, reading on (b) enriching for methylated DNA and “Sequencing” of methylated DNA.
Further relevant to claim 57, De Carvalho et al. teaches “Cancer DNA is frequently hypermethylated at CpG-rich regions [citation]. Since cfMeDIP-seq specifically targets methylated CpG~rich sequences, we hypothesized that ctDNA would be preferentially enriched during the immunoprecipitation procedure. To test this, we generated patient-derived xenografts (PDXs) from two colorectal cancer patients and collected the mouse plasma. Tumor-derived human cfDNA was present at less than 1% frequency within the total cfDNA pool in the input samples and at 2-fold greater abundance following immunoprecipitation (Figure 1 G). These results suggest that through biased sequencing of ctDNA, the cfMeDIP procedure could further increase ctDNA detection sensitivity” (page 22, line 29 – page 23, line 4).
This teaching reads on claim 57 (c)… wherein the first plurality of sequencing reads comprises a bias associated with CpG dinucleotide content values.
Further relevant to claim 57, De Carvalho et al. teaches “Figure 6 shows quality controls for cfMeDIP-seq from circulating cfDNA from pancreatic adenocarcinoma patients (cases) and healthy donors (controls). A-B) Specificity of reaction for each case (A) and each control (B) sample was calculated using methylated and unmethylated spiked-in A. thaliana DNA. Fold enrichment ratio was not calculated due to the very limited amount of DNA available. C-D) CpG Enrichment Scores of the sequenced samples show a strong enrichment of CpGs within the genomic regions from the immunoprecipitated samples” (page 5, lines 29-33 continued to page 6, lines 1-2).
This teaching reads on claim 57 (d) computer processing the first plurality of sequencing reads and the second plurality of sequencing reads to determine the bias based at least in part on the second plurality of sequencing reads.
Relevant to claim 64, De Carvalho et al. teaches “Cancer DNA is frequently hypermethylated at CpG-rich regions [citation]. Since cfMeDIP-seq specifically targets methylated CpG-rich sequences, we hypothesized that ctDNA would be preferentially enriched during the immunoprecipitation procedure. To test this, we generated patient-derived xenografts (PDXs) from two colorectal cancer patients and collected the mouse plasma. Tumor-derived human cfDNA was present at less than 1% frequency within the total cfDNA pool in the input samples and at 2-fold greater abundance following immunoprecipitation (Figure 1G). These results suggest that through biased sequencing of ctDNA, the cfMeDIP procedure could further increase ctDNA detection sensitivity” (page 22, line 29 – page 23, line 4).
This De Carvalho et al. teaching includes hypermethylated cancer DNA, or the first plurality of sequencing reads of the plurality of enriched methylated DNA. De Carvalho et al. computer-processed results of their cfMeDIP procedure (Figure 1G) reads on the instant bias correction, as De Carvalho et al. teaches that the method could “increase ctDNA detection sensitivity”, thus reading on claim 64 limitations.
Relevant to claim 65, De Carvalho et al. teaches that including methylated filler DNA “minimizes the amount of non-specific binding by the antibody and also minimizes the amount of DNA lost due to binding to plasticware,” (page 21, lines 4-5).
Relevant to claim 67, De Carvalho et al. Figure 2 “Spiked-in A. thaliana DNA QC1 control” are of plant – not human – origin, and thus reads on claim 67.
Relevant to claim 70, De Carvalho et al. claim 4 recites that the “first amount of filler DNA comprises about… 15%... methylated filler DNA”.
Relevant to claim 71, De Carvalho et al. claim 5 recites that the “first amount of filler DNA is from 20 ng to 100 ng”.
Relevant to claim 72, De Carvalho et al. claim 6 recites that “the cell-free DNA from the sample and the first amount of filler DNA together comprises at least 50 ng of total DNA”.
Relevant to claim 73, De Carvalho et al. claim 7 recites that the filler DNA is “50 bp to 800 bp long”, reading on claim 73.
Relevant to claims 74-75, De Carvalho et al. teaches that cell-free DNA libraries can be pooled with filler DNA in the form of exogenous Enterobacteria phage λ DNA (page 8, lines 26-27).
Relevant to claim 76, De Carvalho et al. Table 1 (page 26) contains multiple Enterobacteria phage λ DNA primers intended to generate claim 76 plurality of control synthetic nucleic acids comprises at least 3 fragment lengths.
Relevant to claim 77, De Carvalho et al. Figure 2 teaches that the spiked-in λ DNA and spiked-in A. thaliana DNA comprises a methylated sequence or an unmethylated sequence.
Relevant to claim 78, De Carvalho et al. Figure 2 teaches that each nucleic acid molecule in the plurality of methylated control synthetic nucleic acid molecules is at least partially methylated.
Relevant to claim 79, De Carvalho et al. claim 13 recites that the “binder is a protein comprising a Methyl-CpG-binding domain”.
Relevant to claim 80, De Carvalho et al. claim 14 specifies that “the protein is MBD2 protein”.
Relevant to claim 81, De Carvalho et al. claim 15 recites “immunoprecipitating the cell-free methylated DNA using an antibody”.
Relevant to claim 82, De Carvalho et al. claim 16 recites “adding at least 0.05 µg of the antibody to the sample”.
Relevant to claim 83, De Carvalho et al. claim 17 recites that the antibody is a “5-MeC antibody [5-methylcytosine] or 5-hydroxymethyl cytosine antibody”.
Relevant to claims 85-86, De Carvalho et al. teaches “In some embodiments, this method further comprises the step of amplifying and subsequently sequencing the captured cell-free methylated DNA” (page 9, lines 13-14).
This teaching reads on claim 85 amplifying the plurality of enriched methylated DNA molecules and the plurality of control synthetic nucleic acid molecules,thereby generating a plurality of amplified enriched methylated DNA molecules and a plurality of amplified control synthetic nucleic acid molecules, respectively; and claim 86 sequencing the plurality of amplified enriched methylated DNA molecules and the plurality of amplified control synthetic nucleic acid molecules to generate another plurality of sequencing reads comprising a first plurality of sequences of the plurality of amplified enriched methylated DNA molecules and a second plurality of sequences of the plurality of amplified control synthetic nucleic acid molecules.
Relevant to claim 87, De Carvalho et al. teaches “Figure 6 shows quality controls for cfMeDIP-seq from circulating cfDNA from pancreatic adenocarcinoma patients (cases) and healthy donors (controls). A-B) Specificity of reaction for each case (A) and each control (B) sample was calculated using methylated and unmethylated spiked-in A. thaliana DNA. Fold enrichment ratio was not calculated due to the very limited amount of DNA available. C-D) CpG Enrichment Scores of the sequenced samples show a strong enrichment of CpGs within the genomic regions from the immunoprecipitated samples” (page 5, lines 29-33 continued to page 6, lines 1-2).
Further relevant to claim 87, the skilled artisan would find it obvious to combine the teachings relevant to claim 57 and claims 85-86 to arrive at the limitations in claim 87.
(ii) De Carvalho et al. does not teach specifics regarding control sequence limitations relevant to claims 57, 59, and 68-69. However, these limitations were known in the prior art and referenced by De Carvalho et al.
De Carvalho et al. teaches “For MeDIP [methylated DNA immoprecipitation], the Diagenode MagMeDIP kit (Cat# C02010021) was used following the manufacturer’s protocol with some modifications. After the addition of 0.3 ng of the control methylated and 0.3ng of the control unmethylated A. thaliana DNA, the filler DNA (to complete the total amount of DNA [cfDNA + Filler + Controls] to 100ng) and the buffers…” (page 15, lines 1-5).
The Diagenode MagMeDIP qPCR Kit Manual teaches that the A. thaliana methylated and unmethylated spike-in controls are available within their DNA Methylation Control Package, which “are exactly the same as in the MagMeDIP qPCR kit” (page 46, paragraph 2).
The Diagenode DNA Methylation Control Package Manual teaches that the unmethylated control contains 92 bp (A. thaliana chr3:20074482-20074573) and the methylated control contains 81 bp (A. thaliana chr1:30084240-30084160). The regions’ GC% and CpG contents are in below excerpts.
Unmethylated control: 92bp, 41.3% GC, 3 CpG
PNG
media_image1.png
604
1467
media_image1.png
Greyscale
Methylated control: 81bp, 48.15% GC, 4 CpG
PNG
media_image2.png
602
1467
media_image2.png
Greyscale
Relevant to claim 57, the De Carvalho et al. control DNA have two different CpG dinucleotide content values.
Relevant to claim 59, the De Carvalho et al. control DNA contain 41.3% and 48.15% GC content (see above excerpt).
Relevant to claim 68, the De Carvalho et al. control DNA contain fragment lengths of 92bp and 81bp.
Relevant to claim 69, the De Carvalho et al. control DNA contain 3 and 4 CpGs.
Although De Carvalho et al. did not specify the Diagenode control sequence limitations, it would have been prima facie obvious to the skilled artisan. De Carvalho et al. and Diagenode Manuals are analogous disclosures to the instant MeDIP-seq invention.
The skilled artisan would have been motivated to combine the analogous disclosures. A skilled artisan would recognize that the De Carvalho et al. use of the “Diagenode MagMeDIP kit (Cat# C02010021)” (page 15, lines 1-2) would involve the contents of the Diagenode MagMeDIP kit and associated Diagenode DNA Methylation control package, and therefore, would involve the disclosures within the manufacturer’s manuals. The skilled artisan would have been motivated to include the Diagenode Manuals’ teachings within the methodology rendered obvious by De Carvalho et al. because De Carvalho et al. discloses that their method included the Diagenode products. Therefore, the skilled artisan would have been motivated to include the manufacturer’s teachings in order to provide a more complete disclosure to the original De Carvalho et al. methodologies.
The skilled artisan would have a reasonable expectation of success based on the disclosures of De Carvalho et al. in view of Diagenode MagMeDIP qPCR Kit Manual and Diagenode DNA Methylation Control Package Manual.
Applicant’s Arguments and Response to Applicant’s Arguments
Applicant argues that De Carvalho et al. and Diagenode materials do not teach all the elements of amended claim 57, including the computer processing… to determine the bias, wherein the bias is associated with CpG dinucleotide content values (Remarks 2/6/26, page 10).
The Examiner respectfully disagrees, as De Carvalho et al. teaches “Figure 6 shows quality controls for cfMeDIP-seq from circulating cfDNA from pancreatic adenocarcinoma patients (cases) and healthy donors (controls). A-B) Specificity of reaction for each case (A) and each control (B) sample was calculated using methylated and unmethylated spiked-in A. thaliana DNA. Fold enrichment ratio was not calculated due to the very limited amount of DNA available. C-D) CpG Enrichment Scores of the sequenced samples show a strong enrichment of CpGs within the genomic regions from the immunoprecipitated samples” (page 5, lines 29-33 continued to page 6, lines 1-2).
Additionally, De Carvalho et al. teaches “Cancer DNA is frequently hypermethylated at CpG-rich regions [citation]. Since cfMeDIP-seq specifically targets methylated CpG~rich sequences, we hypothesized that ctDNA would be preferentially enriched during the immunoprecipitation procedure. To test this, we generated patient-derived xenografts (PDXs) from two colorectal cancer patients and collected the mouse plasma. Tumor-derived human cfDNA was present at less than 1% frequency within the total cfDNA pool in the input samples and at 2-fold greater abundance following immunoprecipitation (Figure 1 G). These results suggest that through biased sequencing of ctDNA, the cfMeDIP procedure could further increase ctDNA detection sensitivity” (page 22, line 29 – page 23, line 4).
The argued limitations are embraced by the De Carvalho et al. teaching of the CpG Enrichment Score (computer processing to determine the CpG dinucleotide content bias) generated from sequencing reads of ctDNA (which contain CpG dinucleotide biases).
Claim 84 is rejected under 35 U.S.C. 103 as being unpatentable over De Carvalho et al. (2017; WO 2017/190215 A1; FOR citation 002 on IDS filed 9/14/2023) in view of Diagenode MagMeDIP qPCR Kit Manual (version 2 - March 2019; NPL citation V in PTO-892 filed 8/8/25) and Diagenode DNA Methylation Control Package Manual (March 28, 2019; NPL citation U in PTO-892 filed 8/8/25), as applied to claims 57, 59, 64-65, 67-83, and 85-87 above, and further in view of Lienhard et al. (2013; NPL citation 087 in IDS filed 6/24/25; “MEDIPS: genome-wide differential coverage analysis of sequencing data derived from DNA enrichment experiments”. Bioinformatics. 2014 Jan 15;30(2):284-6. doi: 10.1093/bioinformatics/btt650) and Chavez et al. (2014; R package MEDIPS Reference Manual; https://bioconductor.org/packages/2.13/bioc/html/MEDIPS.html).
This rejection is necessitated by claim amendments filed 2/6/26.
The teachings of De Carvalho et al. in view of MagMeDIP qPCR Kit Manual and Diagenode DNA Methylation Control Package Manual are applied to instantly rejected claim 84 as they were previously applied to claims 57, 59, 64-65, 67-83, and 85-87 as rendering obvious a method.
De Carvalho et al. in view of MagMeDIP qPCR Kit Manual and Diagenode DNA Methylation Control Package Manual is silent to claim 84 specifics of determination of bias in G+C content. However, these limitations were known in the prior art and taught by Lienhard et al. and Chavez et al.
Relevant to claim 84, De Carvalho et al. teaches “Figure 6 shows quality controls for cfMeDIP-seq from circulating cfDNA from pancreatic adenocarcinoma patients (cases) and healthy donors (controls). A-B) Specificity of reaction for each case (A) and each control (B) sample was calculated using methylated and unmethylated spiked-in A. thaliana DNA. Fold enrichment ratio was not calculated due to the very limited amount of DNA available. C-D) CpG Enrichment Scores of the sequenced samples show a strong enrichment of CpGs within the genomic regions from the immunoprecipitated samples” (page 5, lines 29-33 continued to page 6, lines 1-2).
Further relevant to claim 84, De Carvalho et al. teaches “we calculate the CpG
Enrichment Score as a quality control measure for the immunoprecipitation step [Lienhard et al. citation]” (page 21, lines 26-27).
Lienhard et al. Abstract section “Availability and implementation” teaches that MEDIPS version 1.12.0 is part of Bioconductor 2.13 and available with the manual on the Bioconductor website. Chavez et al. MEDIPS v.1.12.0 function MEDIPS.CpGenrich “counts the number of Cs, the number of Gs, the number CpGs, and the total number of bases within the stated reference genome” (page 9).
Thus, it is obvious to the skilled artisan that the De Carvalho et al. teaching of a CpG Enrichment Score via Lienhard et al.’s and Chavez et al.’s MEDIP algorithm reads on claim 84 determination of bias in G+C content.
Although De Carvalho et al. in view of Diagenode does not explicitly disclose the Lienhard et al./Chavez et al. MEDIP algorithm determination of bias in G+C content, it would have been prima facie obvious to the skilled artisan. De Carvalho et al., Diagenode Manuals, Lienhard et al., and Chavez et al. are analogous disclosures to the instant MeDIP-seq invention.
The skilled artisan would have been motivated to combine the analogous disclosures. A skilled artisan would recognize that the De Carvalho et al. use of the Lienhard et al. calculation of a CpG Enrichment Score using the MEDIP package would involve the Chavez et al. manual teachings of the MEDIP package. The skilled artisan would have been motivated to include the Lienhard et al. and Chavez et al. teachings within the methodology rendered obvious by modified-De Carvalho et al. because De Carvalho et al. discloses that their method included the Lienhard et al. and Chavez et al. package and algorithm. Therefore, the skilled artisan would have been motivated to include the package and algorithm manual’s teachings in order to provide a more complete disclosure to the modified-De Carvalho et al. methodologies.
The skilled artisan would have a reasonable expectation of success based on the disclosures of De Carvalho et al. in view of Diagenode MagMeDIP qPCR Kit Manual and Diagenode DNA Methylation Control Package Manual, and further in view of Lienhard et al. and Chavez et al.
Conclusion
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/SARAH JANE KENNEDY/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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