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 .
Applicant’s arguments and amendments have been thoroughly reviewed and considered. Claims 52-56 have been added. Claims 11-13, 19, 21-23, 30-31, 34-35, and 42 remain withdrawn. Claims 1-2, 5-7, 47, and 49-56 are pending and are examined on the merits herein.
Response to Applicant’s Amendments and Arguments
Regarding the 35 USC 103 Rejections, Applicant argues that the size selection of the instant invention and that of Alcaide’s AMPure XP bead clean up are different in that Alcaide only removes short sequences below 100 bp, while the instant invention size selects for a range of 100-270 bp (Remarks, pages 8-9). Additionally, Alcaide allegedly does not teach all of the elements of the present claims, particularly the size selection and newly amended portion of claim 1 (Remarks, pages 9-10). Additionally on page 10, Applicant argues that the size selection of 100-270 bp recited in claim 1 is “operationally critical” for the claimed method, and thus cannot be evaluated with an overlapping range analysis. Finally, Applicant argues against the secondary references, and particularly Dinakaran, which is used in the rejection of claim 50, as using this reference would allegedly destroy the native size distribution of cfDNA that is central to the claimed invention’s enrichment mechanism (Remarks, page 11).
Regarding the alleged operational criticality of the claimed size range, MPEP 2144.05 III (A) states that “…In such a situation, the applicant must show that the particular range is critical, generally by showing that the claimed range achieves unexpected results relative to the prior art range." In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990),” and provides further examples of ways in which a range may produce new and unexpected results in order to be deemed critical. This follows the analysis of MPEP 716.02, which describes evaluations for allegations of unexpected results. A showing of such results requires an explanation of why the results achieved by the claimed invention would be unexpected, a comparison with the closest prior art, and a showing that unexpected results are commensurate in scope with the claimed invention. In Applicant’s Remarks, they point to Example 1 of the instant specification, which is described in para. 112 of the application as published. This paragraph states that when size selecting for 100-237 bp, this results in a 2-5 fold enrichment of fetal cfDNA. In para. 123, it is stated that biased PCR can enrich shorter DNA and improve test performance, but no specific results are presented. In Example 1, the 100-237 bp size selection is recited in para. 227, and the 2-5 fold enrichment is repeated in para. 226. This example is not stated to produce unexpected results, nor is it clear why the results produced would be surprising. The results are compared to results produced without size selection, but this is not considered a comparison to the closest prior art. Additionally, this example focuses on fetal cfDNA within maternal samples, which, while potentially being encompassed by instant claim 1, does not read on the entirety of the scope of instant claim 1. The size selection range in this example is also more narrow than that of instant claim 1. Thus, the example is not currently commensurate in scope with the claimed invention.
Therefore, Applicant’s arguments related to the alleged operational criticality of the claimed invention are not persuasive, and the claim interpretation described in the Non-Final Rejection mailed 2/25/2026 related to the size selection range is maintained herein.
Regarding the size selection taught by Alcaide as evidenced by Beckman Coulter, the Examiner used the teachings of Alcaide regarding the size of the cfDNA fragments, adapters, and primers, and general method of the reference to approximate the total length of the final cfDNA library products (see para. 21 of the Non-Final Rejection). Applicant states that Table 2 shows peak bp sizes higher than 335 bp, but Table 2 appears to show the number of unique molecules obtained, and not fragment length. The bead-clean up method of Alcaide (which is evidenced by Beckman Coulter) performs a clean-up of fragments to remove those that are under 100 bp. Thus, the remaining sequences would be above 100 bp to the true maximum length of the cfDNA products. This range, due to its minimum length being 100 bp, must overlap with the range of the instant invention. As instant claim 1 does not require a particular size selection method, and there are no specific definitions of the claimed terms in the instant specification that would preclude the bead clean-up method from reading on the instant claim, said clean-up method is considered to, in light of the size selection range interpretation described above, produce fragments that overlap in size range with the claimed invention. As said claimed range is not noted to be critical or produce unexpected results, it is thus rendered obvious by the bead clean-up of Alcaide.
Regarding the newly amended portion of claim 1, the claim now states that the selectively enriched DNA must include an “increased fraction” of cfDNA from a target tissue. It is unclear what this “increased fraction” encompasses. See the 35 USC 112(b) Rejections below. However, this phrase is interpreted to mean that the selectively enriched DNA contains a proportionally higher amount of cfDNA relative to the total nucleic acids present as compared to the cfDNA proportion in an initial target tissue. As Alcaide is noted to utilize tumor tissue in their methods when analyzing individual-somatic mutations (Figure 1, page 2, para. 3, and page 12, para. 6), the reference is already focused on mutations associated with a target tissue. The selectively enriched DNA of Alcaide contains a cfDNA library, with cfDNA appended with primer and adapter sequences, while the initial tumor tissue contains cells, and thus proportionally contains far less cfDNA than the prepared library. Thus, the reference is considered to meet this new limitation.
Therefore, Applicant’s arguments regarding Alcaide in reference to the claimed limitations are not persuasive.
Finally, regarding the Dinakaran reference, the combination of Alcaide, as evidenced by Beckman Coulter, and further in view of Dinakaran and Invitrogen does not suggest that the whole genome amplification and multiple displacement amplification methods of Dinakaram should be used. Para. 40 of the Non-Final Rejection specifically states, “…it would have been prima facie obvious for one of ordinary skill in the art to substitute the size selection and sequencing method described in Alcaide as evidenced by Beckman Coulter with that of Dinakaran and Invitrogen. Specifically, Alcaide as evidenced by Beckman Coulter teaches size selection with paramagnetic beads and Illumina sequencing, while Dinakaran and Invitrogen detail size selection with gel electrophoresis and Ion Torrent Sequencing (see page 3 of Dinakaran, column 2, para. 1 and Invitrogen page 5).” In this combination, a motivation (i.e. that of MPEP 2143 I (B)) and reasonable expectation of success (“Both methods of size selection and sequencing are known in the art, as shown in the references, and utilizing size selection with gel electrophoresis with Ion Torrent sequencing still allows for the processing of sequencing reads (see page 3, column 2, “Processing of Sequence reads” in Dinakaran). Thus, this substitution would provide predictable results (and this substitution would also have a reasonable expectation of success). Specifically, this substitution would result in the same general steps of Alcaide as evidenced by Beckman Coulter being used (cfDNA extraction, adapter ligation, PCR with sequences corresponding to adapters required for Ion Torrent sequencing, hybrid capture, size selection with gel electrophoresis to remove short sequences, and Ion Torrent sequences). As Invitrogen teaches that the E-gel can detect sequences below 100 bp (e.g. a user can see sequences at or below 100 bp via the DNA ladder), size selection can still be used to eliminate unused primers and primer dimers, and so size selection in a similar range to that described above in the rejection of claim 1 (i.e. a size range overlapping with the claimed size range) would be performed.”) were provided. Applicant does not substantively address the Examiner’s specific rationale.
Therefore, the arguments against Dinakaran are not considered persuasive.
As claims 1-2, 5-7, 47, and 49-51 are still rendered obvious over the references recited in the Non-Final Rejection without altering the rationale of the rejections, these rejections have been maintained. New grounds of rejection are provided below for the newly added claims.
Claim Interpretation
Regarding the specific size selection range discussed in instant claim 1, it is noted that the instant specification does not state that this range is critical or produces unexpected results. Para. 88 of the instant specification states that the 100-270 bp range is included in “one illustrative example,” and para. 200 describes size selection of 100-237 bp. Thus, in finding prior art that reads on this limitation, the guidance provided in MPEP 2144.05 will be used, where prior art that details ranges similar to or overlapping with the claimed size selection range will be considered to render the claimed range obvious.
Additionally regarding claim 1, step (c) states “obtaining a plurality of DNA fragments comprising a plurality of target loci each encompassing at least one of a plurality of individual-specific somatic mutations.” Due to the repeated use of the word “comprising,” this phrase will be interpreted as the plurality of DNA fragments also encompassing additional loci that do not contain individual-specific somatic mutations.
Claim Rejections - 35 USC § 112(b)
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- 2, 5-7, 47, and 49-56 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 rejected due to the phrase “wherein the selectively enriched DNA comprises an increased fraction of cfDNA from a target tissue.” Firstly, it is unclear if/how the target tissue is related to the biological sample described in (a), from which the cfDNA is isolated from. As written, they appear to be two distinct elements of the claim (i.e. “a target tissue” is only mentioned once in the claim), and if they are distinct, it is unclear how cfDNA from said target tissue would be included in the selectively enriched DNA that originated from the biological sample, if such a limitation is intended to be required. Secondly, it is unclear what the “increased fraction” is in reference to – in other words, what the increase is relative to. Because the cfDNA is isolated and enriched, the selectively enriched DNA would naturally comprise almost entirely cfDNA sequences (excluding any adapter/primer/artifact sequences, etc.), and so would naturally have a higher fraction of cfDNA than a fraction of cfDNA in a target tissue, which also contains cellular information. Taken together, these issues render the scope of the claim indefinite. The phrase will be interpreted as though the biological sample need not be a target tissue, and that the “increased fraction” is inherently achieved during the selective enrichment process.
Claims 2, 5-7, 47, and 49-56 are rejected based on their dependence on rejected claim 1.
It is noted in claim 2 that the biological sample is specifically a liquid sample, providing further evidence to support that the target tissue is not intended to be the biological sample.
Claim 52 is also rejected for the “increased fraction of circulating tumor DNA (ctDNA) from the tumor” language, as, similar to the case in claim 1, it is unclear what this increased fraction is relative to. As the selectively enriched DNA would naturally comprise almost entirely cfDNA sequences (excluding any adapter/primer/artifact sequences, etc.), it would naturally have a higher fraction of cfDNA than a fraction of cfDNA in a target tumor, which also contains cellular information.
It is noted that claim 53, which depends from claim 52, alleviates this indefiniteness issue, as it clearly explains that the increased fraction is relative to the isolated cfDNA.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 5, 7, 49, 52-53, and 55-56 are rejected under 35 U.S.C. 103 as being unpatentable over Alcaide et al. (Scientific Reports, 2017) as evidenced by Beckman Coulter (“AMPure XP for PCR Purification”, 20191).
Alcaide teaches methods for detection of rare alleles in circulating tumor DNA (Abstract). This was done through the analysis of cell-free DNA (cfDNA) libraries (page 2, para. 2). Their general method is shown in Figure 1. First, tumor and/or liquid biopsies are used to identify somatic mutations for a particular sample. Then, utilizing a liquid sample, cfDNA is extracted, end-repaired, and ligated to adapters on each end. cfDNA is then amplified and subjected to two rounds of hybrid capture with biotinylated baits that are developed based on the specific somatic mutations in the samples (instant claim 7). After capture, NGS sequencing is performed to analyze sequence reads to determine if the somatic mutants identified in the first tumor/liquid sample are present (see Figure 1 caption). Pages 14-15 note that the hybrid capture of multiple sequences occurred in single reaction mixtures (“Targeted enrichment and sequencing of cfDNA libraries.”).
In their specific methods, Alcaide states that after the ligation reaction, magnetic beads were used to clean the reaction mixture to remove adapter dimers (page 14, para. 1). These same magnetic beads were then used again after PCR was performed (see the same para., “Libraries were then purified using 0.8× volumes (48 µl) of Agencourt AMPure XP beads ensuring again that beads were fully resuspended in 75% ethanol during the washes.”). The PCR primers used by Alcaide are 42-69 nt long (page 14, para. 1). The adapters used by Alcaide are 48-50 nt long (page 13, para. 4). On page 11, para. 4 notes that the average size of cfDNA fragments is around 170 bp. Thus, adapter-ligated cfDNA is, on average, a little over 270 bp long (due to end-repair and A-tailing, see page 13, para. 6 and the Figure 1 caption). It is noted in Figure 1 that the PCR primers and the adapters do overlap in sequence near the end of the primer index sequence, meaning the portion of the primers that would be added to the adapter-ligated cfDNA during amplification is around 65 nt total (around 30 nt for the first primer and around 35 nt for the second based on the primer sequences on page 14, para. 1). Thus, adapter-ligated and amplified cfDNA is on average around 335 bp long. The magnetic beads used in Alcaide are AMPure XP magnetic beads (page 14, para. 1). Beckman Coulter notes that these beads are paramagnetic beads that can specifically remove primer dimers, and provide recovery of sequences that are over 100 bp long (see the information under “Cleanup and Size Selection”; instant claim 5). Alcaide’s use of the paramagnetic beads after PCR (in order to remove unused primer and potential primer dimers) would thus involve size selection of nucleotide sequences of greater than 100 bp to around 335 bp, which overlaps with the claimed range of 100-270 bp.
MPEP 2144.05 I states, “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990).” As noted above in the “Claim Interpretation” section, as Applicant has not pointed out that their claimed range is critical or unexpected, the range for size selection described by Alcaide as evidenced by Beckman Coulter is considered to render obvious the claimed size selection range for both claim 1 and claim 56.
Additionally, as noted above in the “Response to Applicant’s Arguments” and 35 USC 112(b) Rejections sections, Alcaide is noted to utilize tumor tissue in their methods when analyzing individual-somatic mutations (Figure 1, page 2, para. 3, and page 12, para. 6), and so the reference is already focused on mutations associated with a target tissue. The size selection of the reference (i.e. the bead clean-up) accomplishes the “increased fraction” limitation of newly amended claim 1. Specifically, the selectively enriched DNA of the reference contains a cfDNA library, with cfDNA appended with primer and adapter sequences, while the initial tumor tissue contains cells, and thus proportionally contains far less cfDNA than the prepared library.
Thus, claims 1, 5, 7, and 56 are prima facie obvious over Alcaide as evidenced by Beckman Coulter.
Regarding claim 2, Alcaide states that the samples initially gathered from patients were blood samples, where cfDNA was later isolated from plasma (page 13, para. 2).
Regarding claim 49, Alcaide specifically notes that their methods are designed to target mutations comprising SNVs (Abstract, Table 3 caption, page 9, para. 5, and page 14, para. 2).
Regarding claim 52, as noted above, the methods of Alcaide are specifically drawn to analyzing mutations associated with tumor tissue (Figure 1, page 2, para. 3, and page 12, para. 6), and the reference specifically uses tumor tissue to identify individual somatic mutations (Figure 1). The tracking of ctDNA is of particular focus in the reference, and the final sequencing data is specifically analyzed for ctDNA (Figure 1 and caption). Table 3 also shows the ctDNA loci examined in particular patients, and pages 9-11 of the reference discuss ctDNA detection and analysis. Thus, the cfDNA of Alcaide contains ctDNA. Similar to the reasoning provided above in the rejection of claim 1, the size selection of the reference (i.e. the bead clean-up) accomplishes the “increased fraction” limitation of newly amended claim 52. Specifically, the selectively enriched DNA of the reference contains a cfDNA library, with cfDNA appended with primer and adapter sequences, while the initial tumor tissue contains cells, and thus proportionally contains far less cfDNA (and ctDNA) than the prepared library.
Regarding claim 53, it is noted that the “increased fraction” of selectively enriched DNA can be present in step (c) of the method of claim 1, and is not necessarily associated with the actual size selection process of step (b). In Alcaide, the ctDNA mutations are specifically hybridized to a biotinylated probe and then captured on streptavidin beads (Figure 1 and caption). Pages 14-15 (“Targeted enrichment and sequencing of cfDNA libraries.”) further explain this process. These captured sequences then undergo PCR. Thus, the final library would contain a majority ctDNA sequences, and would represent a large increase in fraction from the ctDNA present in the initial cfDNA sample, which in some cases was as low as 0.1% (Abstract and page 4, para. 1).
Regarding claim 55, Alcaide teaches that after utilizing the AMPure beads, the products were eluted in Tris-HCL salt (page 14, para. 1).
Claims 6, 47, and 51 are rejected under 35 U.S.C. 103 as being unpatentable over Alcaide et al. (Scientific Reports, 2017), as evidenced by Beckman Coulter (“AMPure XP for PCR Purification”, 2019), and further in view of Babiarz et al. (US 2016/0244838 A1).
Alcaide as evidenced by Beckman Coulter teaches the methods of claims 1-2, 5, 7, 49, 52-53, and 55-56, as described above.
Regarding claims 6 and 47, Alcaide does not teach multiplex amplification after size selection, and instead utilizes hybrid capture, as described above. Alcaide also does not explicitly state measuring a large number of loci (e.g. Figure 1 shows 4 loci).
Babiarz teaches analyzing cfDNA from liquid samples (Abstract). These methods include analyzing circulating tumor nucleic acids via multiplex amplification (para. 56), and the reference includes multiple examples involving multiplex amplification of thousands of targets (e.g. paras. 109, 175, 188-190). Babiarz also alludes to matching mutations obtained from tumor samples with mutations in plasma samples utilizing multiplex amplification methods (paras. 119-120). In Example 7 of their methods, multiplex PCR is used to analyze chromosomal aneuploidy and copy number variation (para. 178). Blood samples were taken from cancer patients and cfDNA was isolated (paras. 181-182). Libraries were then generated by ligating adapters to DNA fragments and amplifying them (para. 187). Then, additional multiplex PCR was used for 3,168 SNPs to be examined in a single reaction (para. 188). This PCR is noted to be different than the initial amplification following adapter ligation (para. 187 notes PCR conditions involving 15 cycles, while paras. 189-190 note separate multiplex cycling conditions). Multiple amplification cycles occurred with primer pairs for each SNP (para. 189). The amplification products were sequenced via the Illumina HiSeq 2500 (paras. 189-191). Babiarz teaches that 32 CNVs were detected in their plasma samples from cancer patients (para. 210), where each CNV corresponds to multiple SNPs, where some regions contained over 100 SNPs (para. 193).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art that the multiplex amplification of Babiarz could be used in place of the hybrid capture of Alcaide as evidenced by Beckman Coulter. Babiarz teaches a similar method for manipulating cfDNA and targeting particular mutations as in Alcaide, including cfDNA isolation from a liquid sample, and the use of adapters, amplification, and sequencing. Alcaide details the process for the generation of biotinylated baits, but this also requires the use of PCR (and therefore, additional primers; page 14, para. 2). Therefore, rather than use the biotinylated baits that later require incubation with streptavidin and rely on PCR, it would be obvious that PCR could simply be used on the target cfDNA sequences, cutting down on the methodology while utilizing equipment already involved in the method. Such a method could be used with Illumina sequencing, as noted in para. 191 of Babiarz, which corresponds with the sequencing used in Alcaide, providing a reasonable expectation of success. This would essentially substitute the hybrid capture of Alcaide as evidenced by Beckman Coulter with multiplex amplification. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.” Both hybrid capture and multiplex amplification are known in the art, as evidenced by the references, and the substitution would have predictable results, as there would be particular SNPs that would be amplified and prepared for sequencing.
Thus, claim 6 is prima facie obvious over Alcaide, as evidenced by Beckman Coulter, and further in view of Babiarz.
Additionally regarding claim 47, Babiarz shows that many different SNPs can be targeted at once. Alcaide teaches the use of patient-specific mutations (Figure 1), including SNVs (Abstract, Table 3 caption, page 9, para. 5, and page 14, para. 2), and the number of potential patient-specific mutations possible to examine is only limited by the number of somatic mutations present in a patient. Babiarz notes that a large of number of SNPs can be present in a particular sample, and so it would be obvious to target additional SNPs in Alcaide, as evidenced by Beckman Coulter, and further in view of Babiarz, as creating a full profile of patient-specific mutations allows for enhanced mutation tracking overtime, which has implications for further diagnostics, prognoses, and treatment plans. For example, if the number of SNVs in a particular gene changes as cancer progresses in a patient, then practitioners can be aware of this trend, and monitor this gene closely in other patients with that same cancer. As Babiarz teaches that particular regions of interest have more than 100 SNPs, and teaches the analysis of over 3,000 SNPs in the working example described above, it would be prima facie obvious to specifically detect at least 100 patient-specific SNPs in the method of Alcaide, as evidenced by Beckman Coulter, and further in view of Babiarz. There would be a reasonable expectation of success as this would not involve changing the methodology steps or principles of Alcaide, as evidenced by Beckman Coulter, and further in view of Babiarz, but would simply involve focusing on additional targets.
Thus, claim 47 is prima facie obvious over Alcaide, as evidenced by Beckman Coulter, and further in view of Babiarz.
Regarding claim 51, as noted above, Alcaide teaches measurement of target loci, though the reference does not explicitly state measuring a large number of loci (e.g. Figure 1 shows 4 loci).
Alcaide teaches the use of patient-specific mutations (Figure 1), including SNVs (Abstract, Table 3 caption, page 9, para. 5, and page 14, para. 2), and the number of potential patient-specific mutations possible to examine is only limited by the number of somatic mutations present in a patient. As noted above, Babiarz teaches measuring large numbers of SNPs in cfDNA and notes the utility of examining matching mutations in tumors and liquid samples for patients, and so with this guidance it would be obvious to target additional SNPs in Alcaide as evidenced by Beckman Coulter, as creating a full profile of patient-specific mutations allows for enhanced mutation tracking overtime, which has implications for further diagnostics, prognoses, and treatment plans. For example, if the number of SNVs in a particular gene changes as cancer progresses in a patient, then practitioners can be aware of this trend, and monitor this gene closely in other patients with that same cancer. As Babiarz teaches that particular regions of interest have more than 100 SNPs, and teaches the analysis of over 3,000 SNPs in their working example described above, it would be prima facie obvious to specifically detect at least 100 patient-specific SNPs in the method of Alcaide as evidenced by Beckman Coulter. There would be a reasonable expectation of success as this would not involve changing the methodology steps or principles of Alcaide as evidenced by Beckman Coulter but would simply involve focusing on additional targets.
Thus, claim 51 is prima facie obvious over Alcaide, as evidenced by Beckman Coulter, and further in view of Babiarz.
Claim 50 is rejected under 35 U.S.C. 103 as being unpatentable over Alcaide et al. (Scientific Reports, 2017), as evidenced by Beckman Coulter (“AMPure XP for PCR Purification”, 2019), and further in view of Dinakaran et al. (PLoS ONE, 2014) and Invitrogen (E-Gel SizeSelect II Agarose Gel, 2017).
Regarding claim 50, Alcaide as evidenced by Beckman Coulter teaches the methods of claims 1-2, 5, 7, 49, 52-53, and 55-56, as described above. This combination of references teaches size selection with paramagnetic beads, and not gel electrophoresis.
Dinakaran teaches measuring metagenomic profiling in cardiovascular disease patients (Abstract). Specifically, they focus on circulating cell-free DNA in patients (page 1, column 2, para. 1). Plasma DNA was extracted, amplified, and underwent barcoded shotgun sequencing (pages 2-3, “Extraction of plasma DNA,” “Whole genome amplification of circDNA,” and “Bar-coded shotgun sequencing of circDNA”). On page 3, the reference states that amplified DNA was fragmented to a size range of 100-600 bp with a maximum concentration around the 200 bp region. DNA fragments were then barcoded and adapters were ligated, and then additional size selection was performed with a 2% E-gel (“Bar-coded shotgun sequencing of circDNA”). Invitrogen notes that their E-Gel is a “fast and convenient method for DNA fragment library size selection as part of NGS library preparation workflows” (page 1, “Product description”). Page 4 notes the DNA ladder used with the gel goes from 1500-50 bp, and page 5 notes that library sizes with 100-400 base reads and peaks of 200-480 bp can be created.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to substitute the size selection and sequencing method described in Alcaide as evidenced by Beckman Coulter with that of Dinakaran and Invitrogen. Specifically, Alcaide as evidenced by Beckman Coulter teaches size selection with paramagnetic beads and Illumina sequencing, while Dinakaran and Invitrogen detail size selection with gel electrophoresis and Ion Torrent Sequencing (see page 3 of Dinakaran, column 2, para. 1 and Invitrogen page 5). Both types of size selection and sequencing are compatible with amplification and the use of adapters/barcodes, as is shown in both references. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.” Both methods of size selection and sequencing are known in the art, as shown in the references, and utilizing size selection with gel electrophoresis with Ion Torrent sequencing still allows for the processing of sequencing reads (see page 3, column 2, “Processing of Sequence reads” in Dinakaran). Thus, this substitution would provide predictable results (and this substitution would also have a reasonable expectation of success). Specifically, this substitution would result in the same general steps of Alcaide as evidenced by Beckman Coulter being used (cfDNA extraction, adapter ligation, PCR with sequences corresponding to adapters required for Ion Torrent sequencing, hybrid capture, size selection with gel electrophoresis to remove short sequences, and Ion Torrent sequences). As Invitrogen teaches that the E-gel can detect sequences below 100 bp (e.g. a user can see sequences at or below 100 bp via the DNA ladder), size selection can still be used to eliminate unused primers and primer dimers, and so size selection in a similar range to that described above in the rejection of claim 1 (i.e. a size range overlapping with the claimed size range) would be performed. Invitrogen also notes that their methods are fast and convenient, further motivating the ordinary artisan.
Thus, claim 50 is prima facie obvious over Alcaide, as evidenced by Beckman Coulter, and further in view of Dinakaran and Invitrogen.
Claims 54 is rejected under 35 U.S.C. 103 as being unpatentable over Alcaide et al. (Scientific Reports, 2017), as evidenced by Beckman Coulter (“AMPure XP for PCR Purification”, 2019), and further in view of Srinivasan et al. (US 2016/0017412 A1).
It is noted that the term “biased PCR” does not have a specific definition in the instant specification, nor does this appear to be a commonly used term in the prior art. This term will be considered to encompass any PCR that can preferentially amplify particular sequences over other sequences in a sample that would result in or could be used in a size selection process.
Alcaide as evidenced by Beckman Coulter teaches the methods of claims 1-2, 5, 7, 49, 52-53, and 55-56, as described above.
Srinivasan teaches methods of analysis of mother-and-fetus cfDNA, where the fetus may have a genetic disease and/or a genetic sequence anomaly (Abstract). The reference teaches enriching particular sequences of interest (e.g. paras. 13, 19, 116, and 244), where further amplification and sequencing may occur after enrichment (e.g. paras. 19, 154, and 244). Example 1 (specifically para. 377) teaches a scenario in which purified cfDNA from plasma was adaptor ligated, underwent AMPure XP purification, followed by 18 cycles of PCR to selectively enrich adaptor-ligated cfDNA.
This protocol described by Srinivasan is generally similar to the protocol described by Alcaide, in that cfDNA is isolated, adaptor-ligated, bead purified, and amplified. However, Srinivasan specifically notes that their PCR step selectively enriches the cfDNA. As this reference already performs the same bead purification (the AMPure XP purification) followed by PCR during library preparation as Alcaide, the ordinary artisan would link the teachings of the two references, despite Srinivasan being directed to maternal/fetal cfDNA and not tumor cfDNA. Additionally, the ordinary artisan would recognize from the teachings of Srinivasan that PCR could be used to further enrich the size selected, adaptor-ligated cfDNA that has gone through AMPure purification. As Alcaide teaches that ctDNA may be present in very small amount in initial samples (as little as 0.1%, see Abstract and page 4, para. 1), the ordinary artisan would recognize the benefits of additional enrichment, to ensure that desired ctDNA is presented in readily detectable amounts for analysis. As Alcaide is tracking ctDNA mutations, accurate detection is of paramount importance, as this can inform patient prognosis and treatment.
Thus, prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Srinivasan to inform Alcaide as evidenced by Beckman Coulter, specifically to perform additional enrichment PCR of the bead-purified, adaptor-ligated cfDNA before performing the subsequent PCR described by Alcaide. As noted above, increasing the amount of potential ctDNA in a sample would increase the efficacy and real-world applicability of the method of Alcaide. There would be a reasonable expectation of success as PCR protocols are well-known, as evidenced by Alcaide and Srinivasan, and because Alcaide already teaches the use of PCR, no new equipment (aside from additional PCR reagents) would be required. Additionally, the PCR protocols of Srinivasan are compatible with the bead purification methods of Alcaide, furthering this reasonable expectation of success.
Thus, claim 54 is prima facie obvious over Alcaide, as evidenced by Beckman Coulter, and further in view of Srinivasan.
Conclusion
No claims are currently allowable.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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/F.F.G./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
1 The attached webpage for this reference is from August 17, 2019. However, the reference itself notes that these beads were used in 2017 (see para. 2 under “Don’t Lose Critical Data”), and their use in Alcaide also indicates these beads were available in 2017, before the effective filing date of the claimed invention.
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