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 .
Status of Claims / Response to Amendment
This office action is in response to an amendment filed on May 15, 2026 (in response to a Notice to the applicant regarding a non-compliant amendment ; mail date 05/13/2026); as well as the remarks filed on April 27, 2026.
For clarity of the record: it is noted that the amendment filed on May 15, 2026 still contains clerical errors, which Applicant clarified in written communication by email on May 21, 2026 (records attached).
Specifically:
In claim 1, line 7 of the claims amendment submitted on 05/15/2026, it contains texts that are both underlined and strikethrough: see claim 1, line 7.
Applicant clarified that this text is intended to be stricken.
The remarks submitted on 05/15/2026 refers to incorrect dates for the prior OA and remarks. There are no remarks filed on May 2nd 2026, nor Office Action mailed on Oct 2, 2025 in this application.
Applicant acknowledges that the dates are inaccurate on page 6 of the response. And further clarified they should be corrected as: "Applicant’s April 27, 2026 Response to the Non-Final Office Action of November 3, 2025."
Claims 1-2, 4, 7-9, 12-22, 24-25, and 56-58 were previously pending in Amendment filed on 05/20/2025. Applicant amended claims 1, 9, 18, 25; added new claims 59-60.
Claims 1-2, 4, 7-9, 12-22, 24-25, and 56-60 are currently pending, with claims 2, 12, 15-16, 20, 22, 24 withdrawn from consideration.
Claims 1, 4, 7-9, 13-14, 17-19, 21, 25, and 56-60 are under consideration.
All of the previously presented rejections have been withdrawn as either being addressed or obviated by the amendment of the claims, which introduces new combinations of elements that were not previously considered in the prior rejection (e.g., the amended claim 1 now require a combination of analyzing circulating tumor cells with a labeling moiety comprising a double-stranded polynucleotide linker, wherein the polynucleotide linker comprises an oligomer sequence, designed to be cleavable by a CRISPR-Cas9 reagent system, and wherein the oligomer sequence is positioned in the polynucleotide linker so as to release the detectable lag from the labeling moiety when cleaved by the CRISPR-Cas9 reagent system, which were not presented in prior claims and not considered in the prior office action).
Thus, the scope of the claims has been changed in a manner that were not considered in the previous rejections.
Applicant' s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
This office action contains new grounds for rejection necessitated by amendment.
Response to Arguments
Applicant's arguments filed on April 27, 2026 have been fully considered.
Claim Rejections - 35 USC § 103
In the prior Office Action (Non-Final Office Action-11/03/2025):
Claims 1, 4, 7-9, 17, 19, 21, 25 and 56-58 are rejected under 35 U.S.C. 103 as being unpatentable over Gupta, in view of GAO, as evidenced by Stehr.
Claims 13-14 are rejected under 35 U.S.C. 103 as being unpatentable over Gupta in view of GAO, as applied to claim 1 above and further in view of Hennek.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Gupta in view of GAO, as applied to claims 1 and 17 above and further in view of Lu.
The prior rejections set forth above have been withdrawn as being obviated by the amendment of the claims, which added new limitations to the claims, that were not considered in the previous rejections.
However, as discussed in detail in the new grounds of rejections below, the claims are still considered obvious in view of Gupta, GAO, Hennek, Lu and additional prior art references.
To the extent applicable to the current rejections set forth in this Office Action, Applicant's arguments are addressed below.
Regarding GAO, Applicant asserts it teaches away from the claimed invention because "GAO enables multiplexed labeling via a protein G adaptor using DNA programmable tags. It does not reference replacement for a sequential staining paradigm." (Remarks, page 9, para. 2).
Regarding Hennek, Applicant asserts it teaches away from cleavage of double-stranded duplexes by CRISPR-Cas9 because Hennek indicates using Cas9 nickase that leads to single-strand cuts. (Remarks, page 9, para. 1)
These arguments are not persuasive. The references do not teach away because they do not explicitly criticize, discredit, or otherwise discourage the claimed features. Mere silence regarding a feature does not constitute teaching away. Nor do Applicants consider the prior art as a whole.
Applicant's arguments are also not commensurate in scope with the claimed invention. Regarding Applicant's assertion concerning GAO, claim 1 does not recite, either implicitly or explicitly, "replacement for a sequential staining paradigm."
Regarding Applicant's assertion concerning Hennek, claim 1 recites only that "the polynucleotide linker comprises an oligomer sequence, designed to be cleavable by a CRISPR-Cas9 reagent system" and "the cleavage moiety effects cleavage of the polynucleotide linker to release the detectable tag from the binding moiety."
The claim does not specify whether the cleavage is double-stranded or single-stranded. Therefore, the claimed cleavage encompasses both double-stranded and single-stranded cleavage.
Claim Objections
Claim 1 is objected to because of the following informalities:
In claim 1, line 14, "detectable lag" should be "detectable tag."
Priority -- Updated in view of Amendment
The priority date of the instant claims 1, 4, 7-8, 13-14, 17-18, 21, 25 and 57-60 is 04/01/2021, filling date of the provisional application PRO 63/169,566.
The priority date of the instant claims 9, 19, and 56 is 03/31/2022, because the priority document (PCT application PCT/US2022/022742) filed that date is the first to disclose the subject matter in these claims.
Claim Interpretation -- Updated in view of Amendment
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP § 2111.
For the purpose of applying prior art, claim 1 has been amended to recite a "CRISPR-Cas9 reagent system," a term that is not expressly defined in the application's disclosure.
Paragraph [0017] of the specification provides a relevant description as follows:
"the CRISPR-Cas system (e.g., CRISPR-Cas9) system comprises the Cas endonuclease (e.g., Cas9 protein) and a guide nucleic acid molecule (e.g., a small guide RNA or sgRNA)."
Accordingly, under BRI and in light of the specification, "CRISPR-Cas9 reagent system" is interpreted to comprise a Cas9 endonuclease and a guide RNA.
For the purpose of applying prior art, claim 1 has been amended to recite:
"wherein the high-affinity binding domain comprises a crosslinker which covalently, upon exposure to a select wavelength light source, binds the labeling moiety to a target ligand."
Paragraphs [0068] and [0071] of the specification provide the only relevant descriptions of this limitation:
"Antibody labeling reagents, that allow site-specific and covalently couple a DNA oligomer with the Fc region of various off-the-shelf antibodies, can be used. For example, oYo Link reagents contain low molecular weight, high-affinity antibody-binding domains embedding a photo-crosslinker within their Fc-binding site. Upon illumination with non-damaging 365 light, oYo-Link forms a covalent bond with the antibody (Light-Activated Site-Specific Conjugation (LASIC)). This site-specific antibody labeling ensures that the label does not interfere with antigen binding with the target antigen." ([0068])
"FIG. 2 schematically illustrates an example process of using the labeling moiety and the cleavage moiety, as described herein, for imaging a biological sample, e.g., a cell. The example process can comprise the following steps:
· 1. Generate DNA oligomer with custom sequence.
· 2. Generate CRISPR-Cas9 guide RNAs (gRNA) targeting the DNA oligomer sequence.
· 3. Create oYo-Link oligo coupled antibodies.
· Utilizing the oYo-Link Oligo custom kit conjugate DNA oligomer with custom antibody. This construct can be linked to (e.g., covalently conjugated to via using a polynucleotide linker as disclosed herein) a fluorophore, such as HyperBright 488, 647, Alexa Fluor 488, 647 etc.
· Attach the oYo-Link to Fc region of selected antibodies.
§ a. Potential fluorescence amplification can be implemented using DNA labels.
· 4. Mix a first cocktail of probes and contact immobilized cell suspension
· 5. Image immobilized cells.
· 6. Process with CRISPR-Cas9 reagents to cleave oYo-link oligomers and make cells available for staining with a follow-up staining with probes and imaging of previously identified target cells." ([0071])
Accordingly, under BRI and in light of the specification, this limitation is interpreted to mean that the crosslinker (e.g.,oYo-Link, which forms a covalent bond with the antibody via Light-Activated Site-Specific Conjugation (LASIC)), upon light activation with a specific wavelength, forms a covalent bond within the labeling moiety, thereby forming a labeling moiety comprising a detectable tag (e.g., antibody conjugated to a fluorophore). However, the actual binding of the labeling moiety to the target ligand ꟷ for example, antibody conjugate binding to an antigen ꟷ need not itself be covalent.
For the purpose of applying prior art, claim 19 recites the term "selective plane imaging microscopy," which is not defined nor clearly described in the applicant's disclosure.
In view of Applicant's Remarks filed on May 20, 2025 (page 6) and definition provided in Huisken (Huisken et al. Selective plane illumination microscopy techniques in developmental biology. Development. 2009 Jun;136(12):1963-75. doi: 10.1242/dev.022426. PMID: 19465594; PMCID: PMC2685720.), this term "selective plane imaging microscopy" is interpreted under BRI to mean "fluorescence microscopy technique that uses a focused light-sheet to illuminate the specimen from the side."
Claim Rejections - 35 USC § 112(b) -- New Grounds
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.
Claims 1, 4, 7-9, 13-14, 17-19, 21, 25, and 56-60 are rejected under 35 U.S.C. 112(b), 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.
A) Claim 1 has been amended to recite a "high affinity binding domain."
This limitation is indefinite because the metes and bounds of "high affinity binding domain" are unclear. The application's disclosure does not clearly define the term by any structural features, nor does it provide any guidance or criteria for determining what qualifies as "high affinity," which appear to be a relative and subjective description.
For the purpose of compact prosecution and applying prior art under 35 USC§ 102 and 103, because the application's disclosure does not define "high affinity binding domain" by any structural features that distinguish it from binding domains known in the art, the descriptive language "high affinity" is interpreted as not further distinguishing the claimed binding domain from binding domains in the prior art.
Claims 4, 7-9, 13-14, 17-19, 21, 25, and 56-60 are rejected for depending from claim 1 and not remedying the indefiniteness.
B) Claim 1 has been amended in the preamble to recite a method for analyzing "circulating tumor cells." However, parts (b) and (c) of the claim recite "the biological sample," which lack proper antecedent basis.
The claim is indefinite because the claim recites both a narrower scope directed to circulating tumor cells and a broader scope directed to "the biological sample," without clearly defining the relationship between the two.
It is thus unclear whether "the biological sample" refers to a sample containing circulating tumor cells, or whether it refers more broadly to any biological sample subjected to the claimed steps. Accordingly, the metes and bounds of the claim are unclear.
Dependent claims 4, 7-8 and 17 also recite "the biological sample" and require correction for the same reason.
Claim Rejections - 35 USC § 103 -- New Grounds
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.
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, 4, 7-9, 13-14, 17-19, 21 and 56-60 are rejected under 35 U.S.C. 103 as being unpatentable over Gupta (WO2019152391A1- Sequential staining for multiplex analyses of tissues and cells; Published August 08, 2019), in view of
Lu (Lu et al., Identifying cancer origin using circulating tumor cells. Cancer Biol Ther. 2016 Apr 2;17(4):430-8. doi: 10.1080/15384047.2016.1141839. PMID: 26828696; PMCID: PMC4910938.);
Hui (Hui et al., LASIC: Light Activated Site-Specific Conjugation of Native IgGs. Bioconjugate Chem. 2015, 26, 1456-1460);
GAO (GAO et al. Efficient Small-Scale Conjugation of DNA to Primary Antibodies for Multiplexed Cellular Targeting. Bioconjug Chem. 2019 Sep 18;30(9):2384-2392. doi: 10.1021/acs.bioconjchem.9b00490. Epub 2019 Sep 3. PMID: 31438665; PMCID: PMC6753658);
Hennek (US20180372736A1- Compositions for multiplex imaging using labeled nucleic acid imaging agents; Published on December 27, 2018);
Laboda (WO2021081466A1- Polynucleotide-linked bioconjugates and methods of making and using; effective filing date 2020-10-24) as evidenced by
Stehr (Stehr et al. Flat-top TIRF illumination boosts DNA-PAINT imaging and quantification. Nat Commun 10, 1268 (2019). doi.org/10.1038/s41467-019-09064-6).
A) The claimed method is an obvious application of a known multiplex imaging method using analyte detection agents comprising removable labels for analysis (i.e., SeqProbe disclosed in Gupta) to a known biological sample type (i.e. circulating tumor cells, disclosed in Lu). The claimed method further includes obvious modifications using known alternatives in the art for antibody conjugation (i.e., Light Activated Site-Specific Conjugation (LASIC), as disclosed in Hui and GAO, in place of general covalent linkage disclosed in Gupta); and for enzymatic cleavage of polynucleotides (i.e., CRISPR-Cas cleavage in place of restriction enzyme cleavage, as disclosed in Hennek and Laboda).
Gupta teaches a probe i.e., SeqProbe, and its use in sequential staining for multiplex analyses of tissues and cells ([0007]-[0008]; FIGs. 1-2, , 6, 18, 26; [0086]; [0091] ). Each SeqProbe comprises a label tag coupled to an analyte detection agent. Each label tag is removable without destroying a sample to which the plurality of analyte detection agents is applied, thereby allowing further analysis of the sample with additional analyte detection agents.
Regarding claim 1, Gupta teaches a method comprising:
(a) contacting one or more cells with a labeling moiety ([0086]lines1-6; [0078], line1, SeqProbe; [0091]; FIG. 1; FIG. 18; FIG. 26) ,
wherein the labeling moiety comprises a high affinity binding domain coupled to a double-stranded polynucleotide linker having attached thereto a detectable tag (FIG. 1, a cross-linker attached to a double-stranded DNA comprising restriction site, and fluorescent label; [0078] ; see also FIG. 18; FIG. 26),
wherein the high-affinity binding domain comprises a crosslinker which covalently ([0079] analyte recognizing agent is linked to the fluorescent tag via a non-covalent, metal coordination chemistry ; [0051] Fluorescent tags can be attached to analyte recognizing agents via covalent or non-covalent linkage ) binds the labeling moiety to a target ligand, and
wherein the polynucleotide linker comprises an oligomer sequence, designed to be cleavable (FIG. 1; FIG. 18; FIG. 26; [0078]lines 20-23 “FIG. 1A also depicts region that contains a restriction enzyme recognition sequence (R). Treatment with either a nuclease or a restriction enzyme would release the fluorescent tag) , and
wherein the oligomer sequence is positioned in the polynucleotide linker so as to release the detectable lag from the labeling moiety when cleaved (FIG. 1; FIG. 6; FIG. 18; FIG. 26; [0078] lines 20-23 );
(b) subsequent to (a), imaging the biological sample to obtain an image ([0086]; [0091]lines20-21 ; FIG13A, Anti-CDF11b-AF488),
wherein the image is indicative of presence or absence of the target ligand in the biological sample based on staining or lack of staining by the labeling moiety ([0091]lines20-21 ; FIG13A, Anti-CDF11b-AF488); and
( c) subsequent to (b ), contacting the biological sample with a cleavage moiety ([0091]lines21-22, EcoRV restriction enzyme),
wherein the cleavage moiety forms a complex with the polynucleotide linker ([0091]lines21-22; FIG.18, the restriction enzyme binds to the DNA and forms a complex),
wherein, after formation of the complex, the cleavage moiety effects cleavage of the polynucleotide linker to release the detectable tag from the binding moiety ([0091]lines21-22; claim 24 ; FIG 20, destain; FIG. 6).
Regarding the specific sample type of circulating tumor cells, Gupta does not explicitly teach analyzing circulating tumor cells using its method. However, a skilled artisan would have found it obvious to apply Gupta's method to circulating tumor cells in view of the known utility of circulating tumor cells as a sample type for cancer detection, as supported by Lu.
Lu teaches a method for isolating and analyzing circulating tumor cells using immunofluorescent imaging to identify the origin of cancer cells (Abstract).
Lu also teaches potential advantages of analyzing circulating tumor cells for cancer diagnosis. For example, the quantity of circulating tumor cells can indicate cancer severity, and further characterization of circulating tumor cells may provide the source of the primary tumor as a simple alternative to tumor biopsy:
"Cancer constitutes an enormous burden and is a leading cause of death worldwide due to the growth and aging of the population. The majority of cancer deaths are caused by metastasis, when circulating tumor cells (CTCs) leave the primary tumor site, travel in blood through the circulatory system, and lead to the formation of distant, secondary tumors. Quantity of CTCs has shown to correlate with the severity of the cancer disease, and the emerging evidence showed that the characteristic of CTCs may provide the source of primary tumors as a simple, fast alternative to tumor biopsy. Thus, the combination of both could be a powerful tool in the ongoing battle against cancer." (introduction, para 1)
Therefore, a person of ordinary skill in the art before the effective filing date of the claimed invention would have found it prima facie obvious to apply Gupta's multiplex sequential staining method to analyze circulating tumor cells in view of Lu, in order to analyze multiple characteristics of circulating tumor cells and leverage benefits of using circulating tumor cells for advancing cancer diagnostics, as highlighted by Lu.
The person of ordinary skill would have had a reasonable expectation of success because Lu provides a specific application (circulating tumor cell analysis) that is a natural extension of the general cell analysis taught in Gupta. The method described in Gupta is technically compatible to the circulating tumor cells detailed in Lu ꟷ Gupta's method is applicable to immuno-staining and immunofluorescent imaging of cells, and Lu's teaching confirms that circulating tumor cells can be analyzed by immuno-staining and immunofluorescent imaging. Doing so would have yielded the predictable result of an enhanced method for cancer diagnostics using circulating tumor cells, allowing for more precise and multiplexed data collection.
Regarding the limitation "wherein the high-affinity binding domain comprises a crosslinker which covalently, upon exposure to a select wavelength light source, binds the labeling moiety to a target ligand," Gupta teaches its SeqProbe as a labeling moiety comprising a binding moiety such as an antibody ([0078], lines2-3, analyte recognizing agent; [0091]lines12-20, antibodies) coupled to a detectable tag, such as a florescent tag (([0078], line3, florescent tag; [0091]lines12-20, Alexflour488) via a double-stranded polynucleotide linker, such as DNA ([0078],lines17-20, double-stranded DNA attached to analyte recognizing agent; [0091]lines12-20, dsDNA oligo).
Gupta further teaches SeqProbe comprises a cross-linker (FIG. 1A) attaching the polynucleotide linker to the analyte recognizing agent, thereby allowing for the labeling of the analyte with the detectable tag through binding between the analyte recognizing agent and the analyte.
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Gupta teaches that such linker attachment can be performed in “many different ways,” including covalent linkage and non-covalent linkage ([0051]).
“Fluorescent tags can be attached to analyte recognizing agents in many different ways. For example, for antibodies as analyte recognizing agents that are tagged with fluorescently labeled oligonucleotide, the fluorescent oligonucleotide can be attached to the antibody either using a covalent linkage, a non-covalent linkage (such as via streptavidin) or via a metal-coordinate bonds (such as via chloroplatinum-based cross-linkers). In some embodiments, the tagging agent may include DNA prelabeled with fluorophores such as cleavable fluorophores, cleavable DNA and/or ULS-labeled DNA.” ([0051]).
Thus, Gupta teaches covalent linkage of detectable tag to antibodies.
Although the combined teachings of Gupta and Lu do not explicitly teach that the covalent linkage is formed “upon exposure to a select wavelength light source,” this feature would have been obvious in view of the knowledge in the prior art.
Photo-activated UV crosslinking for antibodies labeling were well-known in the art, as supported by Hui and GAO.
Hui teaches methods for Light Activated Site-Specific Conjugation, using a Protein G adapter and a UV-active site. When bound to the Fc region of IgG and activated by long wavelength UV light (365 nm), a covalent bond is formed between Protein G and IgG.(Scheme 1). The adapter domain can be customized to contain a variety of moieties (Abstract).
GAO similarly teaches Light Activated Site-Specific Conjugation for efficient conjugation of DNA to Antibodies for Multiplexed Cellular Targeting (Figure 1; Abstract; p. 2385, left-hand col. Para 2). GAO teaches protein G adaptor as a UV cross-linker, and illuminating the cross-linker with long-wavelength UV light (365 nm) to form a covalent bond between the binding moiety (e.g., antibody) to a detectable tag (e.g., oligonucleotide conjugated to antibody, Fig.1 and legends; Fig. 4).
GAO further teaches benefits of its antibody conjugation method, such as no cross-activity, preservation of antibody function, compatibility with universal purification strategy, and directly applicable to commercially available antibodies. Gao additionally highlights the method's potential to support more quantitative imaging applications in multiplex cellular assays (page 1289, conclusion):
"In this work, we have developed a generally applicable ODN–antibody coupling method using a small protein G adaptor that site-selectively targets the heavy chain of an IgG antibody. We successfully demonstrated pG-ODN labeling of antibodies from different host species without cross-reactivity towards BSA. Importantly, we showed that the pG-ODN labeling did not affect the native function of the antibody. In combination with the universal, benchtop-compatible purification strategy using magnetic beads, this ODN labeling method is directly applicable to commercially available primary antibodies. Because multiple pG-ODN conjugates can be constructed in parallel and lyophilized without a loss of function, the potential of the pG-ODN labeling strategy lies in the synthesis of a library of pG-ODN constructs that can directly be used for antibody labeling. This could eventually facilitate the implementation of the multiplexing abilities of DNA-based read-out methods for the detection of a large variety of subcellular components and make these methods accessible for a broader scientific community. Additionally, we envision the use of pG-ODN–antibody constructs in more quantitative imaging applications, owing to the unique ability of pG-ODN conjugates to selectively label an antibody with a controlled number of ODNs."
Accordingly, a skilled artisan would have found it prima facie obvious to substitute the general crosslinker used in the combined teachings of Gupta and Lu with a Light Activated UV crosslinker for antibody labeling, as taught in Hui and GAO.
This modification represents a simple substitution of one known crosslinking approach for another to achieve the same predictable result ꟷ covalently linking a label to an antibody. see MPEP 2141.
There would have been a reasonable expectation of success because Gupta, Hui and GAO all teach approaches for labeling antibodies with detectable labels. A skilled artisan would have recognized the Light Activated UV crosslinking approaches of Hui and GAO as functionally equivalent to the linkage approach of Gupta, as they perform the same function of attaching a label to an antibody. Hui and GAO further show that Light Activated UV crosslinkers are suitable for labeling antibodies with a variety of conjugates, including DNA.
Regarding the limitation "wherein the polynucleotide linker comprises an oligomer sequence, designed to be cleavable by a CRISPR-Cas9 reagent system, and wherein the oligomer sequence is positioned in the polynucleotide linker so as to release the detectable lag from the labeling moiety when cleaved by the CRISPR-Cas9 reagent system, " Gupta teaches releasing the detectable tag from labeling moiety by endonuclease enzyme cleavage, such as cleavage by restriction enzymes (FIG. 6; [0078]lines17-20).
Although the combined teachings of Gupta, Lu, Hui and GAO do not explicitly teach using a CRISPR-Cas9 reagent system for this releasing step, this feature would have been obvious, as a skilled artisan would have readily understood that CRISPR-Cas9 can be used as an alternative endonuclease for sequence-specific polynucleotide cleavage.
The use of CRISPR-Cas endonuclease for cleaving DNA labels conjugated to antibodies was known in the art as being alternative to restriction cleavage, as supported by Hennek and Laboda.
Hennek teaches an immunofluorescent imaging method and compositions for multiplex imaging analysis of analytes in biological samples, using antibody-DNA conjugate with corresponding fluorescent labels (entire document, [0043] for instance).
Hennek teaches releasing a detectable tag via cleavage can be achieved by either using a restriction endonuclease or CRISPR-Cas9 endonuclease ([0154]; claims 19-20).
Laboda similarly teaches a binding molecule (e.g., an antibody) linked to a conjugate component (e.g., fluorescent label) via a nucleic acid linker (FIG. 2; Abstract; [0013]).
Laboda teaches its nucleic acid linkers are cleavable through enzymatic cleavage, and provides CRISPR and restriction enzymes as alternative examples. ([0072] lines 16-25)
Accordingly, a skilled artisan would have found it prima facie obvious to modify the combined method of Gupta, Lu, Hui and GAO by cleaving the polynucleotide linker using a CRISPR-Cas9 system instead of restriction enzyme. This modification represents a simple substitution of one known endonuclease cleavage approach for another to achieve the same predictable result of releasing the detectable tag.
There would have been a reasonable expectation of success because Hennek and Laboda teach CRISPR-Cas and restriction enzymes as alternative approaches for cleaving polynucleotide linkers attached to antibodies. Thus, a skilled artisan would have readily appreciated that these approaches to be functional equivalents and would have had sufficient knowledge and skill to use CRISPR-Cas9 to cleave the polynucleotide linker in Gupta and achieve the same result of releasing the detectable tag.
Regarding claim 4, Gupta teaches contacting the biological sample with an additional labeling moiety ([0091]lines12-20, anti-CD45 antibodies conjugated with oligo2 and a terminal Alexaflour594), wherein the additional labeling moiety comprises an additional binding moiety ([0091]lines12-20, anti-CD45 antibody) that is coupled to an additional detectable tag ([0091]lines12-20, terminal Alexaflour594) via an additional polynucleotide linker (([0091]lines12-20, oligo2), wherein (i) the additional binding moiety exhibits specific binding to an additional target ligand that is different from the target ligand ([0091]lines12-20, CD45 is a different target from CD11 ) and (ii) the additional detectable tag is different from the detectable tag ([0091]lines12-20, Alexaflour594 is different from Alexaflour488);
imaging the biological sample to obtain an additional image ([0091]lines20-21 ; FIG13A, Anti-CD45-AF594), wherein the additional image is indicative of presence or absence of the additional target ligand in the biological sample based on staining or lack of staining by the additional labeling moiety([0091]lines20-21 ; FIG13A, Anti-CD45-AF594); and
contacting the biological sample with an additional cleavage moiety ([0091]lines21-22, SmaI restriction enzyme), wherein the additional cleavage moiety forms an additional complex with the additional polynucleotide linker ([0091]lines21-22; FIG.12, the restriction enzyme binds to the DNA and forms a complex), wherein, after formation of the additional complex, the additional cleavage moiety effects cleavage of the additional polynucleotide linker to release the additional detectable tag from the additional binding moiety ([0091]lines21-22; claim 24 ; FIG 20, destain; FIG. 6).
Regarding claim 7, it recites “wherein the cleavage moiety is not expressed by a cell of the biological sample.” This is obvious in view of the combined teachings of Gupta, Hennek
Hennek teaches CRISPR-Cas9 endonuclease ([0154]; claims 19-20), which are derived from bacteria, not expressed by mammalian cells used in Gupta ([0091])
Regarding claim 8, Gupta teaches the labeling moiety is disposed at an extracellular space of a cell of the biological sample ([0091]lines18-20), and wherein the cleavage moiety is disposed at the extracellular space of the cell ([0091]lines21-22).
Regarding claim 9, LABODA teaches polynucleotide linker has a length of at least 10 nucleobases per strand (page 17, lines 23-25).
Regarding claim 13, Hennek teaches a cleavage moiety comprises a complex, wherein the complex comprises a Cas protein (claims 19-20; [0154]lines6-9) and a guide nucleic acid molecule ([0154]lines6-9, RNA-guided), wherein the guide nucleic acid molecule exhibits specific binding to the polynucleotide linker ([0154]lines6-9, the guide RNA binds specifically to recognition site).
Regarding claim 14, Hennek teaches guide nucleic acid molecule without a dye (claims 19-20; [0154]).
Regarding claim 17, Gupta teaches a cell (FIG.20; FIG.19; [0061]line4).
Regarding claim 18, GAO teaches live cells staining (p. 2391, left-hand col. “Cellular Labeling for Flow Cytometry,” cells are directly stained and analyzed). Lu also teaches cultured cells and blood cells from patients, a skilled artisan would have expected these cells to be alive (materials and methods, “Cell culture” and “Blood sample collection and processing”).
Regarding claim 19, GAO teaches imaging comprises selective plane imaging microscopy, as evidenced by Stehr.
GAO teaches imaging using total internal reflection fluorescence (TIRF) microscopy (page 2391, left-hand col, para 2). TIRF is a form of selective plane imaging microscopy, as evidenced by Stehr:
"DNA-PAINT experiments are typically performed using some sort of selective plane illumination and/or detection, such as total internal reflection fluorescence (TIRF) microscopy, oblique illumination, or spinning disk confocal microscopy." (Stehr, page 2, left-hand col, lines 19-23)
Regarding claim 21, Gupta teaches the binding moiety is covalently coupled to the detectable tag ([0051]line4) via the polynucleotide linker ([0051]lines6-8).
Regarding claim 56, Gupta teaches the target polynucleotide linker had a length of at most 50 nucleobases (FIG.17, Linker Oligo Sequence, 28 nucleobases).
Regarding claim 57, Gupta teaches wherein the polynucleotide linker comprises an oligonucleotide sequence configured to be recognized by a cleavage moiety (FIG 1A; [0078] lines 20-21).
Regarding claim 58, Gupta teaches wherein the polynucleotide linker comprises an oligonucleotide sequence configured to be cleaved by a cleavage moiety (FIG 1A; [0078] lines 20-24).
Regarding claim 59, Gupta teaches the detectable tag is linked to the polynucleotide linker by a covalent bond ([0051] “DNA prelabeled with fluorophores such as cleavable fluorophores, cleavable DNA and/or ULS-labeled DNA.” Nucleotides within a DNA are connected via covalent bonds, thus a fluorophore labeled to DNA via cleavable DNA is link via a covalent bond.)
Regarding claim 60, Gupta teaches a fluorescent label ([0051]).
Claim 25 is rejected under 35 U.S.C. 103 as being unpatentable over Gupta, in view of Lu, Hui, GAO, Hennek and Laboda, as applied to claims 1, 17-18 above and further in view of
Pradhan (Pradhan et al., Fundamentals of Laser-Based Hydrogel Degradation and Applications in Cell and Tissue Engineering. Adv Healthc Mater. 2017 Dec;6(24):10.1002/adhm.201700681. doi: 10.1002/adhm.201700681. Epub 2017 Oct 24. PMID: 29065249; PMCID: PMC5797692) and
Brandenberg (Brandenberg et al., (2016), In Situ Patterning of Microfluidic Networks in 3D Cell-Laden Hydrogels. Adv. Mater., 28: 7450-7456. doi.org/10.1002/adma.201601099).
Claim 25 has been amended to recite "wherein the cell is visualized in a 3D immobilized
preparation perfused with media." Claim 25, as currently written, describes the cell as being visualized. However, this visualization does not relate to any of the steps previously recited in the base claims. Therefore, the visualizing of cell in claim 25 is interpreted as being performed in conjunction with the steps in claim 1 as a separate approach.
Although the claimed feature in claim 25 is not expressly taught by the combined teachings of Gupta, in view of Lu, Hui, GAO, Hennek and Laboda, which involve analyzing circulating tumor cells using multiplex fluorescence analysis, a skilled artisan would have found the feature obvious because 3-D cell culture and imaging were well-known in the art and had specific benefits applicable to studying circulating tumor cells.
Pradhan is a review article that teaches visualizing cells embedded in 3-D hydrogels, with artificial fabrication of vasculature that allows for perfusion within the gel matrix (Figure 8). Pradhan teaches that 3-D cell culture for in vitro vascular studies "could provide a useful platform for studying vascular phenomena including interactions with immune cells, circulating tumor cells, and other blood components." (p.16, left-hand col., para 4, lines 8-9 to right-hand col., lines 1-2)
Pradhan teaches:
"The generation of hydrogel embedded vascular networks is essential for long-term culture of large-volume tissue constructs and maintenance of high cell viability via adequate transport of nutrients and cellular metabolites." (p. 15, right-hand col, "3.1.2. Fabrication of Vascular Networks" lines 3-7).
Therefore, a skilled artisan would have readily understood that the hydrogel embedded vascular network in Pradhan are designed to be perfused with cell culture media comprising necessary nutrients, to support long-term culture by providing adequate transport of nutrients and cellular metabolites.
This is supported by Brandenberg, which is cited by Pradhan and shown in Pradhan's Figure 8A. Brandenberg teaches perfusion of a 3-D cell culture with media (Figure 1; page 7450, right-hand col, para 2).
Accordingly, it would have been prima facie obvious to also perform 3-D cell culture and imaging with long-term culture while maintaining cell viability, in order to further study circulating tumor cells in the method taught by the combined teachings of Gupta, Lu, Hui, GAO, Hennek and Laboda. A skilled artisan would have been motivated to use an in vitro vascular 3-D culture platform to further study interaction within tissue environments involving circulating tumor cells and other blood components, as suggested by Pradhan.
There would have been a reasonable expectation of success because as supported by the literature review in Pradhan, 3-D cell culture and visualization by fluorescent microscopy were well-established in the art.
Prior Art
Below are relevant prior art not used in rejection but pertinent to the claims or disclosure.
Song (Song et al. DNA Hydrogel with Aptamer-Toehold-Based Recognition, Cloaking, and Decloaking of Circulating Tumor Cells for Live Cell Analysis. Nano Lett. 2017 Sep 13;17(9):5193-5198. doi: 10.1021/acs.nanolett.7b01006. Epub 2017 Aug 10. PMID: 28771008)) also teaches immobilizing circulating tumor cells in 3-D matrix for imaging and live-cell analysis.
Stiller (Stiller et al; Fast and Efficient Fc-Specific Photoaffinity Labeling To Produce Antibody-DNA Conjugates. Bioconjug Chem. 2019 Nov 20;30(11):2790-2798. doi: 10.1021/acs.bioconjchem.9b00548. Epub 2019 Oct 25. PMID: 31609586) also teaches light-activated production of Antibody-DNA conjugates.
Guo (US20160054308A1- System and method for iterative detection of biological molecules; published 2016-02-25) also teaches iterative imaging and detection of target analytes using multiplexed immunofluorescence with cleavable fluorescent antibodies.
Conclusion
Claim 1 is objected to; claims 1, 4, 7-9, 13-14, 17-19, 21, 25, and 56-60 are rejected. No claims are allowed.
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 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.
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/TIAN NMN YU/Examiner , Art Unit 1681 /AARON A PRIEST/Primary Examiner, Art Unit 1681
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