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 .
DETAILED ACTION
Status of the Claims
Claims 1, 3, 5-6, 8-13 and 16-24 are pending and the subject of this FINAL Office Action.
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 02/05/2026 has been entered.
Claim Rejections - 35 USC § 102 - Maintained
The following is a quotation of the appropriate paragraphs of 35 U.S.C. § 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 3, 5-6, 8-13 and 16-20 are rejected under 35 U.S.C. § 102(a)(2) as being anticipated by ALLIS (US20050069931, national stage entry of WO 03/070894 A2), as evidenced by AMIT (US20170037453), BURGESS (WO 02/14550 A2), Ren et al, Use of chromatin immunoprecipitation assays in genome-wide location analysis of mammalian transcription factors, Methods Enzymol. 2004;376:304-15. doi: 10.1016/S0076-6879(03)76020-0, ThermoFisher, Overview of the Immunoprecipitation (IP) Technique, avail. at https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/immunoprecipitation-ip.html, 07/25/2017; Ryan Hamnett, Chromatin Immunoprecipitation Guide, 4th June 2024, avail. at https://www.antibodies.com/applications/chromatin-immunoprecipitation.
As to claims 1 and 5-6, ALLIS teaches incubating a bead with immobilized H3K antibody (paras. 0039, 0053 & 0071-74, claims 1 & 10) in blood serum or plasma (Abstract, claims 1 & 9-10), isolating the bound proteins attached to cfDNA (Abstract, claims 1 & 10) and eluting the cfDNA (Claims 1 & 10). Paragraph 0053 specifically states “Alternatively, modified histone specific antibodies of the present invention can be linked to an insoluble support to provide a means of isolating cell-free nucleosomes from a sample. The support may be in particulate or solid form and could include, but is not limited to: a plate, a test tube, beads, a ball, a filter or a membrane. Methods for fixing antibodies to insoluble supports are known to those skilled in the art.”
As to claim 3, “linking” or “fixing” an antibody to a bead for ChIP means to a skilled artisan protein A/G beads with immobilized antibody, either covalent or non-covalent (See Hamnett and ThermoFisher; see also BURGESS, pgs. 36-37; Ren, pg. 309; AMIT, para. 0103).
As to claims 8-13 and 16-20, the cfDNA is NGS sequenced, and can be from any source and any “disease state” (paras. 0055, 0062, 0067, claims 3-4, 12-13).
Claims 1, 3, 5-6, 8-13 and 16-20 are rejected under 35 U.S.C. § 102(a)(2) as being anticipated by BAWDEN (US20070160989, national stage entry of WO 2005/019826), as evidenced by AMIT (US20170037453), BURGESS (WO 02/14550 A2), Ren et al, Use of chromatin immunoprecipitation assays in genome-wide location analysis of mammalian transcription factors, Methods Enzymol. 2004;376:304-15. doi: 10.1016/S0076-6879(03)76020-0, ThermoFisher, Overview of the Immunoprecipitation (IP) Technique, avail. at https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/immunoprecipitation-ip.html, 07/25/2017; Ryan Hamnett, Chromatin Immunoprecipitation Guide, 4th June 2024, avail. at https://www.antibodies.com/applications/chromatin-immunoprecipitation.
As to claims 1 and 5-6, BAWDEN teaches incubating a bead with immobilized H3K antibody (paras. 0069, 0026-29, claims 34, 36) in blood serum or plasma (Abstract, claims 15, para. 0011), isolating the bound proteins attached to cfDNA (Abstract, paras. 0012, 0074-78, claim 26) and eluting the cfDNA (para. 0012, 0076). Paragraph 0069 specifically states “One of said first and second antibodies may be immobilised and the binding of the other antibody may be detected. Preferably, the first antibody is immobilised. An antibody may be immobilised, for example, by attachment to an insoluble support. The support may be in particulate or solid form and may include a plate, a test tube, beads, a ball, a filter or a membrane. An antibody may, for example, be fixed to an insoluble support that is suitable for use in affinity chromatography. Methods for fixing antibodies to insoluble supports are known to those skilled in the art. An antibody may be immobilised, for example, to isolate cell-free nucleosomes from the biological fluid sample.”
As to claim 3, “linking” or “fixing” an antibody to a bead for ChIP means to a skilled artisan protein A/G beads with immobilized antibody, either covalent or non-covalent (See Hamnett and ThermoFisher; see also BURGESS, pgs. 36-37; Ren, pg. 309; AMIT, para. 0103).
As to claims 8-13 and 16-20, the cfDNA is NGS sequenced, and can be from any source and any “disease state” (claims 28, 37, 39, paras. 0084, 0092, 0102).
Response to Arguments
The Examiner does not agree that the prior art does not teach pre-binding. ALLIS specifically teaches
In accordance with one embodiment of the present invention the nucleosomes from an individual's body fluid are isolated by immunoprecipitation using one or more of the modified histone specific antibodies of the present invention. Alternatively, modified histone specific antibodies of the present invention can be linked to an insoluble support to provide a means of isolating cell-free nucleosomes from a sample. The support may be in particulate or solid form and could include, but is not limited to: a plate, a test tube, beads, a ball, a filter or a membrane. Methods for fixing antibodies to insoluble supports are known to those skilled in the art. In one embodiment an antibody of the current invention is fixed to an insoluble support that is suitable for use in affinity chromatography. After the sample has been contacted with the modified histone specific antibodies under conditions suitable to allow specific binding of the antibody to its target antigen, nucleosomes comprising the modified histones can be isolated using standard techniques known to hose skilled in the art
(para. 0053). It is clear from this paragraph that “modified histone specific antibodies of the present invention can be linked to an insoluble support to provide a means of isolating cell-free nucleosomes from a sample” means the antibodies are linked to supports (e.g. beads) before isolation. Not only is this clear from paragraph 0053, even more it is clear from the principle of the invention itself which requires “contacting the sample with an antibody that binds to a modified histone associated with active gene sequences; [then] isolating nucleosomes bound to said antibody” (claim 1). Thus, ALLIS clearly discloses embodiments in which “contacting the sample with an antibody that binds to a modified histone associated with active gene sequences” includes solid supports such as beads linked to the antibody before “isolating nucleosomes bound to said antibody.”
BAWDEN teaches similarly in paragraph 0069: “An antibody may be immobilised, for example, to isolate cell-free nucleosomes from the biological fluid sample.” Again, this is even more clear in the context of the invention in BAWDEN: “contacting biological fluid sample obtained from an individual suffering from a cancer condition with an antibody which binds specifically to a histone having a modification associated with silencing, [then] isolating nucleosomes bound to said antibody” (claim 37). In other words, BAWDEN clearly teaches that “contacting biological fluid sample obtained from an individual suffering from a cancer condition with an antibody which binds specifically to a histone having a modification associated with silencing” includes using solid-support-immobilized antibody before “isolating nucleosomes bound to said antibody.”
The only outstanding issue is whether Applicants can demonstrate that both ALLIS and BAWDEN fail to provide a reasonable expectation of success in directly isolating nucleosomes from plasma.
Claim Rejection - 35 USC § 103 - Maintained
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 1-3, 5-6, 8-13 and 16-24 are rejected under 35 U.S.C. § 103 as being unpatentable over AMIT (US20170037453), in view of ALLIS (WO 03/070894 A2) and BAWDEN (WO 2005/019826), as evidenced by MICALLEF (US 2018/0024141), ECCLESTON (US20190064184), BURGESS (WO 02/14550 A2), Ren et al, Use of chromatin immunoprecipitation assays in genome-wide location analysis of mammalian transcription factors, Methods Enzymol. 2004;376:304-15. doi: 10.1016/S0076-6879(03)76020-0, ThermoFisher, Overview of the Immunoprecipitation (IP) Technique, avail. at https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/immunoprecipitation-ip.html, 07/25/2017; Ryan Hamnett, Chromatin Immunoprecipitation Guide, 4th June 2024, avail. at https://www.antibodies.com/applications/chromatin-immunoprecipitation.
ALLIS and BAWDEN teach the bead-immobilized H3K antibody technique for isolating cfDNA. They do not explicitly teach ligating adaptors to the bead-attached cfDNA.
Further as to claims 1 and 3-20, AMIT teaches preparing cellular DNA for sequencing analysis (e.g. of disease state, etc.; Abstract, para. 0123) by providing a bodily fluid sample such as blood (para. 0087, for example), contacting the blood sample with magnetic beads containing covalently-immobilized histone antibodies (Fig. 1; Abstract; para. 0103), then isolating them (Fig. 1; Abstract), then ligating adaptors to the immobilized complex (Fig. 1; Abstract). As to covalent immobilization of beads, AMIT states that covalently cross-linking antibodies to beads is routine in the art, and an easy option:
An alternative method of attaching antibodies to magnetic beads or other solid phase support material contemplated by the present invention is the procedure of chemical cross-linking. Cross-linking of antibodies to beads may be performed by a variety of methods but may involve the utilization of a chemical reagent which facilitates the attachment of the antibody to the bead followed by several neutralization and washing steps to further prepare the antibody coated beads for immunoprecipitation. Yet another method of attaching antibodies to magnetic beads contemplated by the present invention is the procedure of UV cross-linking. A third method of attaching antibodies to magnetic beads contemplated by the present invention is the procedure of enzymatic cross-linking
(para. 0103). Thus, AMIT anticipates all but the use of cfDNA.
However, cfDNA was a very well-known option in the art of ChIP-seq analysis. For example, ALLIS teaches ChIP-seq using cell-free histones from blood or plasma (pg. 16, claims 1, 3, 24; see also Third-Party Submission Filed Under 37 CFR 1.290, US 16/980,497, 08/27/2021). BAWDEN also teaches ChIP-seq using cell-free histones from blood or plasma (pgs. 3, 9, 14, 19 and claims 1, 26-28; see also Third-Party Submission Filed Under 37 CFR 1.290, US 16/980,497, 08/27/2021). Furthermore, the motivation to use the method of AMIT on cfDNA is found explicitly in AMIT: “Comprehensive study of the chromatin events during hematopoiesis has been hampered by the low sensitivity and reproducibility for small cell numbers with current chromatin immunoprecipitation (ChIP) protocols” (para. 0003); yet “[t]he present inventors have now devised a novel method for analyzing chromatin derived from a small numbers of cells” using “barcoding [] directly on the total cellular chromatin, thereby avoiding the low input enzymatic reactions occurring in conventional ChIP” (para. 0068; see also paras. 0069-70). In other words, as clearly implied to any skilled artisan, the method of AMIT is well-suited to low-input samples. cfDNA was a very well-known low input (see e.g. ALLIS; BAWDEN). For example, MICALLEF teaches cfDNA, which is known to be present in “as little as 0.1%” in samples (US 2018/0024141, para.018).
Not only was cfDNA well-known to be available in low quantities, even more covalently-attached antibodies for ChIP were well-known options to allow capture of low/limited quantities of protein-DNA complexes. For example, ECCLESTON teaches that “Under normal conditions the level of circulating nucleosomes found in healthy subjects is reported to be low” (para. 0004; see also paras. 0016-18 (amounts of cfDNA and ctDNA known to be low)). Such teaching are found throughout the cfDNA art and have been known for decades.
To address this low amount of circulating nucleosomes in ChIP, a skilled artisan would have been well-aware of antibodies covalently-attached to magnetic beads to allow concentration of low amounts of nucleosomes. BURGESS teaches
The presently described invention utilizes magnetic beads linked covalently to either monoclonal or polyclonal antibodies specific for discrete and particular transcription factors (Dynal Corporation). It is clear that by implementing solid phase separation techniques for immunoprecipitation both the amount of material recovered as well as the specificity for real in vivo interactions is considerably enhanced. This is due primarily to the increased ability to recover the protein/DNA complexes of limited quantity and implementation of additional washing procedures as compared to immunoprecipitation in the absence of using a solid phase base. A diagrammatic illustration of the use of solid phase technology to increase yield and sensitivity is represented in Figure 3. Cross-linked DNA/protein material is combined with magnetically charged Dynabeads upon which antibodies to the protein of interest have been conjugated. Use of a magnet results in purification of protein/DNA complexes of interest. Subsequent washing steps allow for the removal of the unbound cellular debris, proteins and DNA fragments. Magnetic bead/protein/DNA complexes are subsequently subjected to further analysis as discussed below. In the presently described invention linkage of antibodies to the solid phase support magnetic beads is accomplished via standard protocol (Dynal Corporation product information and specifications) and those known and skilled in the art are capable of establishing this linkage successfully
(pgs. 36-37). Ren further demonstrates that antibodies covalently linked to magnetic beads in ChIP were routinely used to allow greater sensitivity and specificity (pg. 309). In other words, a skilled artisan would have been aware that by using well-known, commercially-available covalent antibody-conjugated magnetic beads, “amount of material recovered as well as the specificity for real in vivo interactions is considerably enhanced . . . . due primarily to the increased ability to recover the protein/DNA complexes of limited quantity and implementation of additional washing procedures as compared to immunoprecipitation in the absence of using a solid phase base increase amount of material recovered and increase.”
As to NGS, ECCLESTON teaches as much (para. 008). This is a routine technique in the art.
As to claims 5-6, ALLIS teaches histone-based ChIP using antibodies to H3K4me1, H3K4me2 or H3K4me3 (pg. 16). As does BAWDEN (pgs. 3, 5-6, 9, 12, 14-16, 18-19, 21, Table 1 and claims 1, 26-28, 46; see also Third-Party Submission Filed Under 37 CFR 1.290, 08/27/2021).
As to claim 10, ALLIS teaches dead cell (pgs. 2, 11, 12).
As to claim 11, ALLIS teaches cancer (pg. 14, for example). As does BAWDEN (pg. 7, for example).
As to claims 12 and 16, ALLIS teaches association of said DNA-associated protein with said genomic location is indicative of active transcription, and comparing to known “transcriptional programs” (pgs. 1-2, 6-7, 16). As does BAWDEN (pgs. 21).
As to claims 17-19, ALLIS teaches promoters, genes, mutations, etc. such as oncogenes (pg. 15). As does BAWDEN (pg. 1, 6, 17-20, 38-39).
As to claims 17-19, ALLIS teaches detecting residual disease, monitoring disease and/or early detection of disease (pgs. 12-13). As does BAWDEN (pgs. 14, 21-23).
As to claim 20, ALLIS teaches treating subjects (pg. 12). As does BAWDEN (pgs. 14, 21-23, claim 42).
In sum, it would have been prima facie obvious to a person of ordinary skill in the art before effective filing to apply familiar methods of conjugating antibodies to beads in ChIP to increase sensitivity and specificity in cfDNA samples with a reasonable expectation of success.
Response to Arguments
The Examiner finds Applicants Declaration deficient to overcome the obviousness rejections. The Examiner understands Applicants to argue that the data shows that pre-binding the antibody (any histone antibody) to a bead (whether covalent or native) before isolating nucleosomes directly (no buffer) in plasma yields “successful” sequencing. Although “successful” sequencing is not defined, yet the Declaration 09/08/2025 shows only 23% reads mapped to TSS (indicating nucleosomes pulldown) versus 80% for pre-bound antibody. In other words, pre-binding the antibody to a bead before isolating nucleosomes directly in plasma is essential to achieving high, relative pure amounts of nucleosome cfDNA for sequencing. The Examiner agrees that the data shows all of this. However, the issue remains that the Declaration evidence fails to overcome the anticipation rejections above because anticipation (based on prior art teaching “linking,” “immobilizing” or “fixing” an antibody to a bead for ChIP) cannot be overcome by unexpected results. In light of this, the obviousness rejection stands because BAWDEN and ALLIS already anticipate the technique of bead-immobilized antibodies used to isolate plasma nucleosomes containing cfDNA.
Prior Art
The following prior art is pertinent to ChIP-seq of cell-free nucleosomes: US 20140322719; US 20160145685; US 20170211143; US 20130310260; US 2013/0017958; US 2018/0024141; US 2019/0064184.
Conclusion
No claims are allowed.
All claims are identical to or patentably indistinct from, or have unity of invention with claims in the application prior to the entry of the submission under 37 CFR 1.114 (that is, restriction (including a lack of unity of invention) would not be proper) and all claims could have been finally rejected on the grounds and art of record in the next Office action if they had been entered in the application prior to entry under 37 CFR 1.114. Accordingly, THIS ACTION IS MADE FINAL even though it is a first action after the filing of a request for continued examination and the submission under 37 CFR 1.114. See MPEP § 706.07(b). 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 nonprovisional extension fee (37 CFR 1.17(a)) 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 mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Aaron Priest whose telephone number is (571)270-1095. The examiner can normally be reached 8am-6pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Gary Benzion can be reached at (571) 272-0782. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/AARON A PRIEST/Primary Examiner, Art Unit 1681