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 1-3, 6-7, 10-11, 13, 20-21, 24-25, 30, 33, 36, 45-48, and 64 are pending and are examined on the merits herein.
Response to Applicant’s Amendments
Nucleotide and/or Amino Acid Sequence Disclosure Objections
The drawings were objected to because Figures 3 and 4 contained sequences without appropriate sequence identifiers. In light of Applicant’s new drawings submitted 11/6/2025, these objections have been withdrawn.
The specification was also objected to due to an allegedly defective Sequence Incorporation by Reference paragraph. Applicant’s Remarks point out that for the purposes of determining the requirements for sequence disclosures, the international filing date of the instant application must be used, and not the filing date of the application to the USPTO (Remarks, page 8). This argument is persuasive, and so this objection has been withdrawn.
Claim Objections
Claims 1-3, 7, 10, 20-21, 36, and 64 were objected to for various informalities. In light of Applicant’s amendments to the claims submitted 11/6/2025, these objections have been withdrawn, but see new grounds of objection below.
35 USC 112(b) Rejections
Claims 20-21 and 24 were rejected for various indefiniteness issues. In light of Applicant’s amendments to the claims submitted 11/6/2025, these rejections have been withdrawn, but see new grounds of rejection below.
35 USC 103 Rejections
Claims 1-3, 6-7, 10-11, 13, 20-21, 24-25, 30, 33, 36, 45-48, and 64 were rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (WO 2020/118046 A1) in view of Babiarz et al. (US 2016/0369333 A1). In light of Applicant’s amendments to the claims, these rejections have been withdrawn for all claims except claim 64, for which the previously set forth rejections have been maintained. See “Response to Applicant’s Arguments” and new grounds of rejection below.
Response to Applicant’s Arguments
Regarding the 35 USC 103 Rejections, Applicant argues that neither of the cited references teach the use of isolated cell-free DNA. Additionally, Zhang allegedly does not teach quantifying the total amount of cfDNA in a sample, and allegedly only teaches quantifying donor DNA (Remarks, pages 11, paras. 2-3). Applicant also notes that claims 1-2 have been amended to recite specific components/structures of the Tracer DNA, and alleges that none of the cited references teach these additional limitations (Remarks, page 11, para. 4). Regarding the Christians reference, Applicant states that this reference also does not teach the barcode positioning of newly amended instant claims 1, 2, and 11 or the required amplification of instant claim 21 (Remarks, page 13, paras. 2-3). Finally, Applicant argues that Knight does not teach multiple organ donors as recited in instant claim 48.
Firstly, Zhang, the primary reference used, teaches isolation of cell-free DNA. Para. 13 states “the cell-free DNA is isolated from whole blood”, and para. 22 teaches isolation of cell-free DNA from plasma. Figures 8 and 9 of Zhang were used to show quantification of total numbers of reads in a sample in order to identify the proportion of reads from that are foreign/donor-related. Generally, the reference also teaches sequencing cfDNA methods and analyzing sequence reads (e.g. paras. 19-21). Given that the examples of Zhang cited in the Non-Final Rejection teach finding a particular fraction of cfDNA within a sample using sequencing reads (see paras. 24-25), the ordinary artisan would recognize that this would also require knowing the total amount of sequence reads, and so is considered to meet the limitation of “quantifying the amount of total cell-free DNA using sequencing reads”. It is noted that in the Non-Final Rejection, these teachings were combined with the teachings of Examples 8 and 11 of Zhang to arrive at the claimed invention. Specifically, the rejection states “Zhang shows that spike-in sequences can be used to quantify foreign DNA in a sample, with particular uses for quantifying donor-derived cfDNA and making transplant rejection determinations. The ordinary artisan would thus be motivated to include these spike-in methods in the methods of Zhang described above, to aid in quantifying cfDNA and to monitor organ transplant patients, as methods involving the use of a standard (such as a spike-in sequence) can allow for more accurate quantification and better comparison of cfDNA values in a patient over time and/or between different patients, allowing analysis results to be more useful and widely-applicable,” (para. 26). This means that the teachings of Zhang involving the foreign spike-in DNA are analogous to the “Tracer DNA” cited in the instant claims, and though Zhang may not teach all aspects of the claim in a single embodiment (with the exception of the limitations involving the 100 or more different primer pairs, for which Babiarz is used), when taken together, the ordinary artisan would arrive at the previously claimed invention, without taking into account the newly added limitations to the claims. Thus, Zhang teaches cfDNA isolation, the addition of Tracer DNA, amplification of the cfDNA, and sequencing and analysis of amplification products, where sequencing reads are quantified. The fact that Zhang goes on to find the donor/foreign/spike-in specific fractions of reads in the sample is simply additional analysis that is not prohibited by the instant claims (as the claims comprise the listed steps).
The Examiner acknowledges that Zhang is directed to methods of universal amplification (as shown in para. 27 of the Non-Final Rejection, which states “Zhang appears to rely on the use of universal primers that hybridize to ligated adaptors (e.g. paras. 6, 9, and 11).”)
As to Babiarz, this reference is only used to add to the amplification methods of Zhang. The statement of rejection notes “Babiarz teaches that universal amplification can be followed by specific amplification involving primer pairs that target particular loci. Babiarz teaches a multitude of advantages to using this method that would motivate the ordinary, including reduced bias and increased accuracy. Zhang and Babiarz also both teach that their methods can analyze cfDNA in the context of organ transplants and transplant rejections. There would be a reasonable expectation of success because Babiarz shows that such multiplexed single reaction amplification can successfully be performed (e.g. para. 835 and Figure 15),” (see para. 29 of the Non-Final Rejection). The specific amplification of Babiarz involves a first universal step following by the use of at least 1,000 primer pairs to target at least 1,000 loci in a single reaction mixture, where this method can be used on cfDNA. Thus, combined with Zhang in the manner recited by the Examiner, these references are considered to have taught the limitations of the previous version of claims 1-2.
As to the arguments against the additional secondary references that are relevant to the previously presented and current claims, claim 48 specifically requires that the sample include donor-derived cfDNA from two donors, as well as recipient cfDNA. Recipient cfDNA would naturally be present in a cfDNA sample taken from the transplant recipient, and the two donor-derived portions would originate from two organ transplants, where each organ was donated by a different individual. It is noted that this claim depends on claim 2, and does not require parsing out how much donor-derived DNA is from the first and/or second donor – the total amount of donor-derived cfDNA is simply used. Thus, the claim simply amounts to performing the method of claim 2 on a recipient who has had two organ transplants where the organs came from different donors. The Examiner does agree with Applicant’s summary of the teachings of Knight, but argues that this combination of references would still lead the ordinary artisan to perform the method of claim 48. MPEP 2141.03 I states, “”A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton." KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 421, 82 USPQ2d 1385, 1397 (2007). "[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle." Id. at 420, 82 USPQ2d 1397. Office personnel may also take into account "the inferences and creative steps that a person of ordinary skill in the art would employ." Id. at 418, 82 USPQ2d at 1396.” The ordinary artisan would be aware of the existence of patients with multi-organ transplants, and in the case where two organs are given to a single recipient, there is either one donor (who gives two organs) or two (where each organ is from a separate donor). As instant claim 48 simply involves performing the method of claim 2 on a particular subject, and the ordinary artisan would recognize that such subjects exist, it would be prima facie obvious to perform the method of instant claim 2 on the subject specific in instant claim 48. Para. 55 of the Non-Final Rejection provides rationale for the use of such a subject that does not rely on the explicit teachings of Knight, but on logical conclusions the ordinary artisan, with ordinary creativity and knowledge, would come to. Knight is simply used because it discusses organ transplantation in the context of multiple organs.
Regarding the newly added limitations to the claims, it is first noted that though the Tracer DNA must now comprise one or more “target sequences,” the term “target” is not defined by the instant specification, and so this can simply mean any sequence. The claims go on to recite a particular structure for the targets (a barcode between two primer binding sequences) as well as a particular function during amplification (the primer binding sites are used with the 100 or more different primer pairs). The use of such target sequences was recited in previously presented claim 20. In the Non-Final Rejection, Christians was used to teach these limitations. Specifically, the reference teaches spike-in sequences that contain tags and adapters with primer binding sites (see para. 46 of the Non-Final Rejection). In para. 47 of the rejection, it is stated, “as Christians does not require a specific location for the tag sequences, and notes they may be embedded within sequences, it would be obvious to include the tag in the middle of the spike-in sequences.” In other words, given the scope of the teachings of the reference, including the tag in the middle of the spike-in sequence, where the primer binding sites would be on either side of it, is encompassed by the reference. Furthermore, this paragraph states, “As noted in the explanation of the teachings of Christians above, primer binding sites can be attached to the ends of the spike-in sequences, and it would be obvious to ensure that these primer binding sites could attach to one of the primers already present in the method of Zhang in view of Babiarz, as this would prevent the need for designing primers specifically for the spike-in sequences, saving time and resources in the overall methodology. Additionally, as noted above, Zhang in view of Babiarz teaches quantification of cfDNA via the use of sequence reads and spike-in sequences (e.g. Zhang Figure 10).” Therefore, these limitations have already been addressed by the combination of Zhang, in view of Babiarz, and in view of Christians. In Applicant’s Remarks, no actual teaching of Christians, nor the specific combination of reference teachings or the rationale used, is argued against. Therefore, this rejection in the Non-Final Rejection is considered proper, and is used in the new grounds of rejection below. It is noted that in this rejection (as presented previously and below), the specific spike-in sequences of Christians are used in the method of Zhang in view of Babiarz, and so Applicant’s notes that the spike-ins of Zhang do not meet the limitations of the newly amended claims (Remarks, page 11, para. 4) are moot.
Thus, in specifically relating Zhang, in view of Babiarz, and in view of Christians to the newly amended independent claims, the spike-ins, which are considered analogous to the claimed Tracer DNA, contain a barcode positioned between primer binding sites, where those primer binding sites bind to at least one of the primers used to amplify the cfDNA in the biological sample.
Finally, it is noted that Applicant has amended throughout the instant claims to change the word “preparation” to “non-naturally occurring composition.” Such a composition is not specifically defined in the instant specification, and in fact does not appear in the instant specification. However, the claimed compositions would inherently be non-naturally occurring, as they involve amplification methods and primer sequences that would not be found naturally.
Therefore, Applicant’s arguments against the references are generally considered not persuasive. New grounds of rejection are required to fully address the claim amendments, but the relevant teachings and rationales presented in the Non-Final Rejection are reiterated below.
Claim Objections
Claim 7 is objected to because of the following informalities: for the first two options presented by the claim, it is recommended to specify that the biological sample used would be a whole blood sample or plasma sample, to clearly connect the samples recited in this claim to the sample of claim 1. Appropriate correction is required.
Claim 20 is objected to because of the following informalities: in lines 4-5 of the claim, the phrase “the amplified isolated cell-free DNA is used.” However, this should read “amplified DNA” to better correspond to the terminology used in claim 1. Similarly, the phrase “reads of the Tracer DNA” in line 4 should read “reads derived from the first Tracer DNA composition” to better correspond to the terminology used in claim 1. Appropriate correction is required.
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 1-3, 6-7, 10-11, 13, 20-21, 24-25, 30, 33, 36, and 45-48 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 recites “the biological sample” in line 4 without first reciting “a biological sample” in the preamble or earlier in step (a). Therefore, this phrase lacks antecedent basis. Additionally, the claim recites “one of the primer pairs” in lines 7-8 without first reciting “primer pairs” in the preamble or earlier in step (a). Therefore, this phrase also lacks antecedent basis.
Claims 7, 10-11, 13, 20-21, 24-25, 30, and 33 are rejected due to their dependence on rejected claim 1.
Claim 2 recites “the biological sample of the transplant recipient” in line 5 without first reciting “a biological sample” or “a transplant recipient” in the preamble or earlier in step (a). Therefore, these phrases lack antecedent basis. Additionally, the claim recites “one of the primer pairs” in line 10 without first reciting “primer pairs” in the preamble or earlier in step (a). Therefore, this phrase also lacks antecedent basis.
Claims 3, 6, 36, and 45-48 are rejected due to their dependence on rejected claim 2.
Claim Interpretation
As noted above in the “Response to Applicant’s Arguments” section, Applicant has amended throughout the instant claims to change the word “preparation” to “non-naturally occurring composition.” Such a composition is not specifically defined in the instant specification, and in fact does not appear in the instant specification. However, the claimed compositions would inherently be non-naturally occurring, as they involve amplification methods and primer sequences that would not be found naturally.
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, 6-7, 10-11, 13, 20-21, 24-25, 30, 33, 36, and 45-47 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (WO 2020/118046 A1), in view of Babiarz et al. (US 2016/0369333 A1), and in view of Christians et al. (US 2017/0275691 A1).
Zhang teaches methods of quantifying foreign cell-free DNA in blood samples (Abstract). Para. 21 describes a method of monitoring organ rejection via extraction of cell-free DNA and genomic DNA from an organ transplant recipient, amplifying short fragments of cell-free DNA, obtaining sequence reads for at least 500 SNPs, and quantifying donor versus recipient cell-free DNA. The DNA may be extracted from whole blood and isolated from plasma (paras. 13 and 22; instant claim 46). The nucleic acids used in the invention can be from a human sample (paras. 54, 61, 73; instant claim 36). Zhang teaches methods of sequence analysis when donor genotype is both known and unknown (paras. 23-24), and shows workflows where donor genotype is explicitly not required (para. 42 and Figure 9; instant claim 45). These methods rely on quantifying total numbers of SNPs (i.e. total cell-free DNA) before determining which portions of sequence reads are from donor-derived cfDNA (Figures 8 and 9). Zhang describes methods for selectively amplifying cfDNA in a sample with genomic DNA (para. 36 and Figures 3A-3C, which involve incorporating adapters into the ends of the cfDNA). 500-1,000,000 SNPs may be targeted (para. 45).
Zhang also teaches the use of spike-in DNA to detect donor-derived cfDNA in a sample, where a known amount of foreign DNA is added to an existing DNA sample (paras. 119-120 and 130, Examples 8 and 11). By providing a known value of introduced foreign DNA and then later detecting that DNA, this can be used to determine the amount of foreign (i.e. donor) DNA in a sample (Figure 10). Zhang teaches that this information can be used to monitor the occurrence of transplant rejection (para. 130). It is noted that Example 11 in particular utilizes the methods of Example 10, which are shown in Figure 9 and clearly lay out a method for determining the amount of foreign DNA present in a sample. Table 2 also explicitly shows sequencing results when using spike-in sequences.
Thus, Zhang shows that spike-in sequences can be used to quantify foreign DNA in a sample, with particular uses for quantifying donor-derived cfDNA and making transplant rejection determinations. The ordinary artisan would thus be motivated to include these spike-in methods in the methods of Zhang described above, to aid in quantifying cfDNA and to monitor organ transplant patients, as methods involving the use of a standard (such as a spike-in sequence) can allow for more accurate quantification and better comparison of cfDNA values in a patient over time and/or between different patients, allowing analysis results to be more useful and widely-applicable.
However, Zhang does not teach that the amplification used in their method relies on the use of 100 or more different primer pairs in a single reaction volume – Zhang appears to rely on the use of universal primers that hybridize to ligated adaptors (e.g. paras. 6, 9, and 11). Additionally, Zhang does not teach that their spike-in sequences contain a barcode positioned between primer binding sites.
Babiarz teaches methods for simultaneously amplifying multiple nucleic acids of interest in a single reaction volume (Abstract). These methods can involve a first non-specific amplification step, followed by specific and simultaneous amplification of at least 1,000 non-identical loci in a reaction mixture (where up to 100,000 target loci can be amplified), and at least 1,000 primer pairs (and up to 100,000 primer pairs) can be used (paras. 12-13). The target DNA can come from DNA of a mixed origin (such as DNA resulting from transplantation; para. 395), and Babiarz specifically teaches analyzing cell-free DNA from transplant recipients, where some of the cfDNA is from donors, and the method can be used to, “prognose, diagnose, detect, or monitor a transplant status or outcome, such as a transplant, tolerance, non-rejection based allograft injury, transplant function, transplant survival, chronic transplant injury, or tittering of pharmacological immunosuppression,” (para. 408). The reference teaches that performing a universal amplification step before targeted amplification can have advantages, such as removing the risk of bottlenecking and reducing allelic bias (para. 454). A universal amplification step can also be used to increase overall sample quantity, which can aid in minimizing bias (para. 368). Babiarz also states, “Greater multiplexing allows more alleles to be targeted, giving more accurate results. Better uniformity results in more of the targeted alleles being measured, giving more accurate results. Lower rates of allelic bias result in lower rates of miscalls, giving more accurate results. More accurate results result in an improvement in clinical outcomes, and better medical care,” (para. 475).”
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 combine the methods of Zhang and Babiarz. Zhang teaches the targeting of at least 500 SNPs via universal amplification and sequencing methods, and Babiarz teaches that universal amplification can be followed by specific amplification involving primer pairs that target particular loci. Babiarz teaches a multitude of advantages to using this method that would motivate the ordinary, including reduced bias and increased accuracy. Zhang and Babiarz also both teach that their methods can analyze cfDNA in the context of organ transplants and transplant rejections. There would be a reasonable expectation of success because Babiarz shows that such multiplexed single reaction amplification can successfully be performed (e.g. para. 835 and Figure 15; instant claim 30).
However, Babiarz does not teach spike-in sequences that contain a barcode positioned between primer binding sites.
Christians teaches methods of making and using synthetic nucleic acids (Abstract). The synthetic nucleic acids may be spike-ins, where they can be used with targets for eventual sequencing (paras. 3-4 and 7), and they can be added at the start of sample processing (para. 10). The spike-ins may have an identifying tag sequence (para. 8), and can be generated via the use of PCR (paras. 144 and 147). Thus, the spike-in sequences would have primer binding sites on either end. The spike-in sequences can also have adapters (para. 143) and these adapters can be primer binding sites (para. 112). The spike-ins may be double-stranded DNA (para. 15), and can be less than 500 (or less than 200) nucleotides in length (para. 28; instant claim 25). Specifically, the spike-ins can contain genomic DNA sequences (paras. 144 and 147). Sets of spike-ins may be used, where the sets may have different species with different lengths, concentrations, and/or sequences (paras. 132-133). These spike-ins can be used for sample tracking, monitoring cross-contamination, tracking reagents, and normalization, among other uses (para. 132). The tags of Christians can be useful for analyzing sequence reads (para. 216), and these tags may be “embedded within each spiked molecule,” (para. 412). The spike-in sequences generally may have specifically designed sequences (paras. 92-93).
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 specific spike-in teachings of Christians in Zhang in view of Babiarz. Specifically, by including more than one spike-in sequence, the second spike-in could provide increased accuracy by ensuring the results match those of the first spike-in sequence, and could be used for the tracking and bias applications taught by Christians. It would also be obvious to have all the spike-in sequences have the same basic structure, so as to limit the time and resources put into designing said spike-in sequences while still ensuring desired function. Regarding the structure of the spike-ins of Christians, the reference clearly teaches use of genomic/double-stranded DNA spike-ins with the length requirements of instant claim 25, and as Christians does not require a specific location for the tag sequences, and notes they may be embedded within sequences, it would be obvious to include the tag in the middle of the spike-in sequences. This is because these tags are useful for identifying sequence reads, and by including them away from the ends of the spike-in sequences, it can ensure that these tag sequences remain intact and are not lost or degraded through the amplification and sequencing processes. Thus, the tags would be flanked by genomic DNA. As noted in the explanation of the teachings of Christians above, primer binding sites can be attached to the ends of the spike-in sequences, and it would be obvious to ensure that these primer binding sites could attach to one of the primers already present in the method of Zhang in view of Babiarz, as this would prevent the need for designing primers specifically for the spike-in sequences, saving time and resources in the overall methodology (instant claim 11). Additionally, as noted above, Zhang in view of Babiarz teaches quantification of cfDNA via the use of sequence reads and spike-in sequences (e.g. Zhang Figure 10; instant claim 20). In combining Christians with Zhang in view of Babiarz, there would be a reasonable expectation of success as Christians processes spike-ins in a method similar to that of Zhang in view of Babiarz successfully, and Zhang is not specific about required or prohibited spike-in structure (instant claims 1-2).
Thus, claims 1-2, 11, 20, 25, 30, 36, and 45-46 are prima facie obvious over Zhang, in view of Babiarz, and in view of Christians.
Regarding claim 6, Zhang teaches that “if the fraction of the short fragments of cell-free DNA that correspond to the DNA of the organ transplant donor is above a normal range or increases over time, then the organ transplant recipient is considered to be rejecting the transplanted organ,” (para. 27). Para. 97 notes that, “When the organ from the donor is rejected and attacked by the immune system, the concentration of cfDNA derived from the dying rejected organ’s cells will significantly increase.” Figure 11 also details the foreign molecule percentage in healthy individuals and non-rejection organ recipients (para. 44), which would require a determination of total amount of cell-free DNA present (see Figures 8-9 for details on how donor fraction is determined). This information would render obvious the limitation of instant claim 6. Specifically, Zhang teaches determining the level of cell-free DNA in a sample, as noted above in the rejection of claim 2. Zhang also notes that in cases of transplant rejection, donor cell-free DNA goes up, and thus, the total cell-free DNA level in a sample would also go up. Zhang also notes the normal range of donor cell-free DNA present in transplant recipients, which would require a determination of the normal range of total cell-free DNA present in transplant recipients. This information could be combined to determine a normal total cell-free DNA range, and then as samples are evaluated, numbers greater than this range could be used to further examine the associated subject for potential transplant rejection.
Regarding claim 7, Zhang teaches that their spike-in sequences are added to pure DNA samples (paras. 119 and 130). In combining the spike-in methods of Zhang with the methods of Zhang previously described in the rejection of claim 1 above, it would be obvious to add the spike-ins to the composition comprising the isolated cfDNA to ensure the spike-in sequences were subjected to the same processing as the cfDNA.
Regarding claim 10, Zhang teaches the use of universal adaptors attached to cfDNA sequences (see Figures 3A-C, as noted above). As these are added before amplification occurs, and as noted in the rejection of claims 1 and 7 above, the spike-in sequences are added before sample analysis begins, the spike-ins would be added before the adaptors are ligated.
Regarding claim 13, the spike-ins used by Zhang, in view of Babiarz, and in view of Christians are fragmented genomic DNA, and would therefore have different specific sequences and lengths. Christians also specifically notes that their spike-in sequences are unique (e.g. paras. 4, 8, 19, and 27-28), further supporting this notion.
Regarding claims 21 and 24, in the method of Zhang, in view of Babiarz, and in view of Christians described above, regarding the specific genomic DNA used for the spike-in sequences, as genomic DNA from the patient is already utilized, and the spike-ins taught by Zhang initially were fragmented genomic DNA, it would be obvious to use fragmented genomic DNA sequences from the patient as the core of the spike-in sequences, where these sequences would be surrounded by primer binding sites and would contain a barcode. These genomic sequences are already provided by the sample, making them easy to obtain, and would significantly cut down on the time required to design the spike-in sequences, as they could simply be modified by the ordinary artisan as needed to produce the spike-in structure described above (instant claim 24). Furthermore, as noted above, the spike-in sequences may be double-stranded, and so each portion of the sequence would have a corresponding complement/reverse complement. Christians teaches that unique sample identifiers can have “regions of shared sequence,” (para. 188), and that tags can have a common sequence (para. 30). Thus, it would be prima facie obvious that the tags in Zhang, in view of Babiarz, and in view of Christians could have regions that are reverse complementary to a portion of genomic DNA, from which the spike-in is made. This tag portion could be reverse complementary to the genomic DNA on the second strand of the spike-in, and could be a common or shared sequence among all of the spike-ins. By utilizing a sequence that is the same for all spike-ins, it would help to better distinguish said spike-ins from other sequences in the sample, making analyses more accurate.
Regarding claim 33, Zhang teaches that the short DNA fragments of their invention (e.g. the cfDNA) can have lengths of 50-400 nucleotides, where the range may be as small as 50-250 nucleotides. The reference also teaches that cfDNA from blood samples is typically 50-200 bp. Thus, the specific primers of Zhang, in view of Babiarz, and in view of Christians would target sequences within the length range described by instant claim 33.
Regarding claim 47, Zhang teaches noting the fraction of donor cfDNA over time to determine if an organ transplant recipient is rejecting the transplanted organ (para. 27). To determine if the level of donor cfDNA is increasing over time, it would be obvious to the ordinary artisan that the level of donor cfDNA would have to be measured at multiple time points, and thus, the method described above in the rejection of claim 2 would be repeated at each time point.
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (WO 2020/118046 A1), in view of Babiarz et al. (US 2016/0369333 A1), in view of Christians et al. (US 2017/0275691 A1), and further in view of Favalli (WO 2019/149673 A1).
Regarding claim 3, Zhang, in view of Babiarz, and in view of Christians teaches the method of claim 2, as described above.
However, neither Zhang, nor Babiarz, nor Christians teach the specific use of thresholds in relation to transplant rejection.
Favalli teaches the detection of donor cfDNA in organ transplant recipients to provide an early indication of transplant rejection (Abstract). The reference teaches that during rejection episodes, circulating donor DNA may increase from 1%-5% in organ recipients, and an increase of over 5% can indicate the beginning of a rejection (page 12, para. 1). Favalli teaches calculations for determining the amount of donor cfDNA in a sample (page 15). They teach that an increase in the level of cfDNA between 5-15% is indicative of the probable presence of rejection, and an increase of 10-25% can be indicative of clinically significant rejection (page 18, para. 4).
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 Favalli with the methods of Zhang, in view of Babiarz, and in view of Christians to arrive at the invention of claim 3. Specifically, the thresholds established by Favalli could be used to determine if a patient is at risk of transplant rejection (1-5%) or is likely experiencing transplant rejection (>5%). Zhang teaches comparing donor cfDNA levels within a patient over time (para. 27), but does not note a specific threshold. By providing a specific threshold, risk of rejection can be measured in a standardized way across multiple patients. A specific threshold also allows for invasive interventions to only be performed if they are necessary, and not simply because the amount of donor cfDNA in a sample increased marginally between testing windows. Because Zhang teaches quantification of total cfDNA, donor cfDNA is a function of total cfDNA, and the threshold taught by Favalli is a function of the amount of cfDNA, these combined teachings are considered to meet the teachings of instant claim 3.
Thus, claim 3 is rejected as prima facie obvious over Zhang, in view of Babiarz, in view of Christians, and further in view of Favalli.
Claim 48 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (WO 2020/118046 A1), in view of Babiarz et al. (US 2016/0369333 A1), in view of Christians et al. (US 2017/0275691 A1), and further in view of Knight et al. (Transplantation, 2019).
For this claim, it is noted that while at least two donors must be provided, there is no requirement that in the quantification of donor-derived cfDNA, that the contributions of the first and second donors be separately quantified.
Zhang, in view of Babiarz, and in view of Christians teaches the method of claim 2, as described above. However, neither reference teaches the use of multiple organ donors.
Knight teaches the measurement of donor cfDNA as a biomarker in solid organ transplantation (Abstract). The reference teaches the evaluation of data from various sources, and several studies included data from multiorgan transplants (Abstract). Table 1 shows the successful detection of cfDNA in multiple multiorgan samples from the plasma of patients.
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 information provided by Knight to evaluate multiorgan transplant recipients with the method of Zhang, in view of Babiarz, and in view of Christians. The presence of a second transplanted organ provides additional risk of transplant rejection, and so these patients require intensive monitoring. By providing a non-invasive monitoring method, this would improve patient comfort while still closely monitoring donor cfDNA levels in the blood, and would allow for quick action upon a noted increase in these levels.
Thus, claim 48 is rejected over Zhang, in view of Babiarz, in view of Christians, and further in view of Knight.
Claim 64 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (WO 2020/118046 A1), in view of Babiarz et al. (US 2016/0369333 A1), and further in view of Favalli (WO 2019/149673 A1).
Regarding claim 64, it is noted that, with the exception of step (d), the limitations of the claim are met by the teachings of Zhang in view of Babiarz described above in the rejection of claim 2 (claim 64 does not require the Tracer DNA of claim 2, and so the teachings of Christians are not required, see paras. 31-36 of the rejection above for the specific teachings of Zhang in view of Babiarz), in combination with the following: these references also teach analyzing over 500 loci (500 SNPs are noted in Zhang, para. 21 for example, and Babiarz teaches at least 1,000 loci in paras. 12-13) and the use of a blood sample (Zhang para. 22). Regarding step (d), Zhang does teach quantifying donor-derived cfDNA (para. 21), and teaches this quantification by finding the percentage of donor cfDNA over the total amount of cfDNA (e.g. Figures 8-9).
However, neither Zhang nor Babiarz teach the specific use of thresholds in relation to transplant rejection.
Favalli teaches the detection of donor cfDNA in organ transplant recipients to provide an early indication of transplant rejection (Abstract). The reference teaches that during rejection episodes, circulating donor DNA may increase from 1%-5% in organ recipients, and an increase of over 5% can indicate the beginning of a rejection (page 12, para. 1). Favalli teaches calculations for determining the amount of donor cfDNA in a sample (page 15). They teach that an increase in the level of cfDNA between 5-15% is indicative of the probable presence of rejection, and an increase of 10-25% can be indicative of clinically significant rejection (page 18, para. 4).
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 Favalli with the methods of Zhang in view of Babiarz to arrive at the invention of claim 64. Specifically, the thresholds established by Favalli could be used to determine if a patient is at risk of transplant rejection (1-5%) or is likely experiencing transplant rejection (>5%). Zhang teaches comparing donor cfDNA levels within a patient over time (para. 27), but does not note a specific threshold. By providing a specific threshold, risk of rejection can be measured in a standardized way across multiple patients. A specific threshold also allows for invasive interventions to only be performed if they are necessary, and not simply because the amount of donor cfDNA in a sample increased marginally between testing windows. Because Zhang teaches quantification of total cfDNA, donor cfDNA is a function of total cfDNA, and the threshold taught by Favalli is a function of the amount of cfDNA, these combined teachings are considered to meet the teachings of instant claim 64.
Thus, claim 64 is rejected as prima facie obvious over Zhang, in view of Babiarz, and further in view of Favalli.
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