DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. This action is in response to the papers filed May 14, 2026. Applicant’s remarks and amendments have been fully and carefully considered but are not found to be sufficient to put the application in condition for allowance. Any new grounds of rejection presented in this Office Action are necessitated by Applicant's amendments. Any rejections or objections not reiterated herein have been withdrawn. This action is made FINAL.
Claims 13, 15, 37, 41, 43-44, and 93-97 are currently pending and have been examined herein.
Claim Rejections - 35 USC § 103
3. 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 13, 15, 37, 41, 43-44, and 93-97 are rejected under 35 U.S.C. 103 as being unpatentable over Mitchell (US 2015/0086477 Pub 3/26/2015) in view of Zimmermann (US 2014/0141981 Pub 5/2014), and Schutz (US 2016/0115541 Pub 4/28/2016).
Regarding Claim 13 Mitchell teaches determining the amounts of cell free DNA, native, total, and/or non-native concentrations from biological samples. Mitchell teaches that non-native cell free DNA includes donor specific cf-DNA that is shed from a donated transplanted organ into the blood, plasma, serum, or urine of a transplant recipient (para 0064). Mitchell teaches measuring cell free DNA in samples drawn at three time points after aortic clamp removal in each of 11 new heart transplant recipients. Mitchell teaches the following time points: time point 1 (14-36 hours, day 1), time point 2 (84-126 hours, day 3-5), and time point 3 (160-206 hours, day 6-9) (para 0177). Mitchell teaches extracting cell free DNA (para 0165). Mitchell teaches that the amounts of total cf-DNA were obtained by TaqMan quantitative real time PCR (para 0166). Mitchell teaches that the amount of DS cf-DNA was determined by DANSR amplification followed by sequencing (para 0167). Mitchell teaches that for DANSR, 192 genomic loci were targeted for amplification (para 0167). The 192 genomic loci are SNVs since recipient genotypes were homozygous and donor genotypes were either heterozygous or homozygous for the other allele (para 0171). Figure 10 is a series of bar graphs showing the percent DS cf-DNA and total cf-DNA at three time points post-surgery. Thus Mitchell teaches a method comprising: extracting cf-DNA from each of at least two samples, wherein the at least two samples are each a blood, plasma or serum sample from a transplant subject and are each taken from the transplant subject at a different time within 8 days of the transplant and performing quantitative PCR to quantify the amount of total cf-DNA in each of the at least two samples, quantifying the amount of DS cf-DNA in each of the at least two samples based on biallelic SNPs that are distinct between the DS cf-DNA and transplant subject specific cf-DNA, and comparing the amount of DS cf-DNA and the amount of total cf-DNA of each sample to the amount of the other sample (comparison of timepoints 1-3 in Fig 10).
Mitchell does not teach preparing a preparation of amplified DNA by performing multiplexed targeted PCR amplification on the cfDNA to amplify at least 50 SNV targets together in the same reaction (clm 13).
However Zimmermann discloses multiplexed targeted PCR (para 0202). Zimmermann teaches a multiplex pool of PCR assays are designed to amplify potentially heterozygous SNP or other polymorphic or non-polymorphic loci on one or more chromosomes and these assays are used in a single reaction to amplify DNA. The number of PCR assays may be between 50 and 200 PCR assays (para 0223).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Mitchell by preparing a preparation of amplified DNA by performing multiplexed targeted PCR amplification on the cfDNA to amplify at least 50 SNVs. One of skill in the art would have been motivated to preamplify the DNA using multiplex targeted PCR since the amount of cfDNA and particularly DS-cfDNA is low in blood, plasma, and serum samples obtained from transplant patients. Further one of skill in the art would have been motivated to amplify at least 50 SNVs since the SNVs allow one to distinguish native cfDNA from DS-cfDNA.
Mitchell does not teach performing quantitative PCR on the amplified DNA to quantify the amount of DS cf-DNA in each of the at least two samples based on biallelic SNPs that are distinct between the DS cf-DNA and the transplant subject specific cf-DNA (clm 13).
However Schultz discloses a method wherein a cfDNA sample is obtained from a blood sample and is analyzed to determine the level of donor material by identifying the presence of donor SNP alleles in the cfDNA. Schultz teaches that any method can be used to evaluate the sample. In typical embodiments, digital PCR, such as a microfluidics-based digital PCR or droplet-based PCR is employed. Other methods can be based on direct hybridization of detection probes (without prior amplification) or sequencing, e.g., sequencing of an amplicon defined in Table 1. For example, the SNP region is amplified by PCR and then the percentage of the minor allele is determined by amplicon sequencing. The percentage of donor cfDNA (also referred to as graft cfDNA) in the cfDNA sample can be determined. In other embodiments, the copy number of donor cfDNA is determined (para 0057). It is noted for the record that digital PCR is a quantitative PCR method.
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Mitchell and Zimmerman by performing digital PCR (a type of quantitative PCR) on the amplified DNA to quantify the amount of DS cf-DNA based on biallelic SNPs that are distinct between the DS cf-DNA and the transplant subject specific cf-DNA as suggested Schultz. In the instant case Mitchell discloses sequencing to determine the amount of DS cf-DNA (para 0167). Schultz discloses digital PCR to determine the amount of DS cf-DNA (para 00578). The claim would have been obvious because the substitution of one method of quantifying the amount of DS cf-DNA (sequencing) for another method (digital PCR) would have yielded predictable results to one of ordinary skill in the art at the time of the invention.
Regarding Claim 15 Mitchell teaches correlating an increase in total cf-DNA and/or an increase in non-native cf-DNA (e.g., DS cf-DNA) with an increased risk of a condition such as transplant rejection, transplant injury, bacterial, fungal and/or viral infection and/or systemic disease associated with transplant (para 0074). Mitchell further teaches providing therapy to these patients, wherein the therapy is an anti-rejection therapy, therapy for a systemic condition, or a therapy for treating a bacterial, fungal, or viral infection (para 0088). Thus Mitchell teaches a method comprising: determining a treatment regimen for the subject based on the amounts of DS cf-DNA and total cf-DNA in each of the at least two samples.
Regarding Claim 37 Mitchell teaches analyzing nucleic acids from cell-free DNA extracted from a biological sample obtained from the subject to identify a plurality of loci, the nucleic acids comprising first nucleic acids of the subject and second nucleic acids not native to the subject; determining an allele of each of the plurality of loci; selecting at least one informative locus from the plurality of loci based on the determining of the allele; calculating an estimated allele frequency of a first allele at the at least one informative locus using a statistical distribution; determining an amount of cell-free DNA not native to the subject in the cell-free DNA based on the estimated allele frequency (para 0002).
Regarding Claim 41 Mitchell teaches correlating an increase in total cf-DNA and/or an increase in non-native cf-DNA (e.g., DS cf-DNA) with an increased risk of a condition such as transplant rejection, transplant injury, bacterial, fungal and/or viral infection and/or systemic disease associated with transplant (para 0074). Mitchell further teaches providing therapy to these patients, wherein the therapy is an anti-rejection therapy, therapy for a systemic condition, or a therapy for treating a bacterial, fungal, or viral infection (para 0088). Thus Mitchell teaches administering an anti- rejection treatment to a subject of whom the amount of DS cf-DNA and the amount of total cf-DNA have increased over time in the at least two samples.
Mitchell does not specifically teach a method further comprising administering an anti-rejection treatment to a subject of whom the amount of DS cf-DNA is not decreasing by at least 0.98% each day post-transplant, within the first 8 days (clm 43). Mitchell does not specifically teach a method further comprising administering an anti-rejection treatment to a subject of
whom the amount of total cf-DNA is not decreasing by at least 7% each day post-transplant, within the first 8 days (clm 44).
However Mitchell further teaches thirty three samples were drawn at three time points after aortic clamp removal in each of 11 new heart transplant recipients (median age was 1 year, range 0-18 years): time point 1 (14-36 hours, day 1), time point 2 (84-126 hours, day 3-5), and time point 3 (160-206 hours, day 6-9). Increased levels of DS cf-DNA were observed in all samples at time point 1 (FIG. 5A). In each case these levels rapidly declined with subsequent samples showing a significant decrease between time points 1 and 2 (FIG. 5A). The samples at time point 3 (day 6-9) were not different from baseline levels found in asymptomatic heart transplant recipients undergoing scheduled surveillance biopsy (para 0177). Mitchell also teaches correlating an increase in total cf-DNA and/or an increase in non-native cf-DNA (e.g., DS cf-DNA) with an increased risk of a condition such as transplant rejection, transplant injury, bacterial, fungal and/or viral infection and/or systemic disease associated with transplant (para 0074). Mitchell further teaches providing therapy to these patients, wherein the therapy is an anti-rejection therapy, therapy for a systemic condition, or a therapy for treating a bacterial, fungal, or viral infection (para 0088).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Mitchell by administering an anti-rejection treatment to a subject whom the amount of total cf-DNA or DS cf-DNA is not decreasing each day post-transplant within the first 8 days. In the instant case Mitchell demonstrates that levels of total cf-DNA or DS cf-DNA are supposed to be increased following a transplant and then rapidly decrease over the first week following surgery. Mitchell further teaches that increased levels of total cf-DNA or DS cf-DNA are a sign of increased risk of rejection or systemic disease. Thus one of skill in the art would have been motivated to administer an anti-rejection treatment to a subject of whom the amount of total cfDNA or DS cfDNA is not decreasing over the first week because these subjects would be considered as having a higher risk of rejection or systemic disease. Further it would have been obvious and well within ones skill in the art to determine the percentage that total cf-DNA and DS cf-DNA would need to decrease each day to return to the baseline level within the first week.
Regarding Claim 93 Mitchell teaches samples were drawn at three time points after aortic clamp removal in each of 11 new heart transplant recipients. Mitchell teaches the following time points: time point 1 (14-36 hours, day 1), time point 2 (84-126 hours, day 3-5), and time point 3 (160-206 hours, day 6-9) (para 0177). Thus Mitchell teaches a method wherein at least one sample is taken within 7, 6, 5 or 4 days of the transplant.
Mitchell does not teach a method wherein at least one sample is taken on day 0 after the transplant (clm 94). Mitchell does not teach a method wherein at least one sample is taken during cross clamp removal (clm 95). However in addition to the time points disclosed above, Mitchell teaches that the amount of non-native cf-DNA, such as DS cf-DNA, or total cf-DNA or native cf-DNA, may also be determined at any other time following the transplant, and may be utilized for short- or long-term surveillance (para 0086). Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Mitchell by taking at least one sample on day 0 after the transplant and at least on sample during cross clamp removal. In the instant case Mitchell teaches that the amount of non-native cf-DNA, such as DS cf-DNA, or total cf-DNA or native cf-DNA, may also be determined at any other time following the transplant, and may be utilized for short- or long-term surveillance (para 0086). Mitchell further teaches that levels of DS-cf DNA are elevated during rejection and cardiac allograft injury and decrease during recovery (para 0183). One of skill in the art would have been motivated to take at least one sample on day 0 after the transplant and at least on sample during cross clamp removal for the benefit of having baseline samples to monitor rejection and cardiac allograft injury.
Regarding Claim 96 teaches samples were drawn at three time points after aortic clamp removal in each of 11 new heart transplant recipients. Mitchell teaches time point 1 is (14-36 hours, day 1). Thus Mitchell teaches a method wherein at least one sample is taken 36 hours or less from the time of cross clamp removal (para 0177).
Regarding Claim 97 Mitchell teaches samples were drawn at three time points after aortic clamp removal in each of 11 new heart transplant recipients. Mitchell teaches the following time points: time point 1 (14-36 hours, day 1), time point 2 (84-126 hours, day 3-5), and time point 3 (160-206 hours, day 6-9) (para 0177). Thus Mitchell teaches a method wherein one or more further amounts of DS cf-DNA and total cf-DNA are determined each from a sample taken from the subject at a different point in time (in addition to the two samples required by claim 1).
Response To Arguments
4. In the response the Applicants traversed the rejections under 35 USC 103. In the response the Applicants argue that the claims do not solely recite determining the level of DS cf- DNA, but rather, require the quantification of total as well as DS cf-DNA within certain timeframes, performing quantitative PCR on DNA amplified for at least 50 single nucleotide variant (SNV) targets, and comparing amounts for both total as well as DS cf-DNA amounts. Why one of ordinary skill in the art would combine the features of the methods of the claims, which include the foregoing features, and expect a predictable result is not at all clear. It appears that in order to arrive at the instant claims, the Examiner has to use impermissible hindsight to pick and choose the features of the methods of the claims and combine them from the references cited. The Examiner's assertion of obviousness in view of the cited references stems from an improper application of hindsight reasoning starting from the teachings of the present application rather than starting from the teachings in the prior art. Instead, the present application has been used as a blueprint to search and identify the elements of the instant claims in the prior art.
These arguments have been fully considered but are not persuasive. In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). In the instant case the rejection only relies on knowledge which was withing the level of ordinary skill at the time of the invention. The rejection does not include any knowledge gleaned from applicants disclosure. The rejection sets forth the relevant teachings of the primary reference (Mitchell). The primary reference discloses a method of detecting total cfDNA and DS-cfDNA in samples obtained from transplant patients. The primary reference discloses Mitchell used quantitative PCR to quantify the amount of total cfDNA and sequencing to quantify the amount of DS-cfDNA (paras 0166-0167). The primary reference discloses comparing the amounts of total cfDNA and DS cf-DNA over three post-surgery timepoints (Fig 10). The rejection sets forth the differences in the claim over the primary reference. The rejection sets forth the relevant teachings of the additional references (Zimmermann and Schutz). The rejection proposes modification of the primary reference to arrive at the claimed subject matter and explains why the claimed invention would have been obvious to one of ordinary skill in the art at the time the invention was made. Thus motivation is present and is provided. Applicants assert that the claims do not solely recite determining the level of DS cf-DNA, but rather, require the quantification of total as well as DS cf-DNA. However it is noted that the primary reference (Mitchell) teaches quantification of total cfDNA AND DS cfDNA. The Applicants state that the claims require performing quantitative PCR on DNA amplified for at least 50 SNV. Zimmerman is being relied upon to teach performing multiplex targeted PCR to detect at least 50 SNV for the benefit of increasing the amount of cfDNA in the sample since cfDNA is usually present in low amounts in blood, plasma, and serum samples obtained from transplant patients. Further one of skill in the art would have been motivated to amplify at least 50 SNVs since the SNVs allow one to distinguish native cfDNA from DS-cfDNA. Finally it is noted that the primary reference (Mitchell) also teaches comparing the amounts of total cfDNA and DS cf-DNA over three post surgery timepoints (Fig 10). Based on the teachings in the prior art there was more than a reasonable expectation of success. The rejections are maintained.
5. 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).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMANDA HANEY whose telephone number is (571)272-8668. The examiner can normally be reached Monday-Friday, 8:15am-4:45pm EST.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Wu-Cheng Shen can be reached on 571-272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/AMANDA HANEY/Primary Examiner, Art Unit 1682
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