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 .
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.
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 04/16/2026 has been entered.
Status of Claims
Claims 1-20 are pending. Claim 20 is withdrawn as directed to the nonelected invention. Claims 1-19 are currently under examination.
Maintained Rejection
Claim Rejections - 35 USC § 103
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.
Claims 1-19 are rejected under 35 U.S.C. 103 as being unpatentable over Murillo et al. (US2013/0109019A1, published 05/02/2013, 892 dated 09/28/2022) in view of Alexander et al. (US2013/0260379A1, published 10/03/2013, 892 dated 09/28/2022) and Chukka et al. (US2015/0347702A1, published 12/03/2015, 892 dated 05/13/2024).
Murillo teaches a method that includes the steps of (a) providing a sample comprising targets; (b) immobilizing a peroxidase on a first target in the sample, (c) contacting the sample with a solution comprising a first hapten conjugate and a solution comprising peroxide, wherein the first hapten conjugate includes the hapten bound to a peroxidase-activatable aryl moiety (see para. [0020]). Murillo teaches that the peroxidase-activatable aryl moiety is tyramine (see abstract). Murillo further teaches in step (d) that immobilizing a subsequent peroxidase on a subsequent target in the sample; (e) contacting the sample with a solution comprising a subsequent hapten conjugate and a solution comprising peroxide, wherein the subsequent hapten is bound to a peroxidase-activatable aryl moiety and in certain embodiments, the first hapten conjugate and the subsequent hapten conjugate are hapten-tyramide conjugates (see para. [0020]).
In particular, Murillo teaches in Fig. 1 that an immobilized tissue sample; a primary antibody 120 binds to an epitope 130 within an immobilized tissue sample 110; a secondary antibody 140 is introduced and binds to the primary antibody 120 wherein the secondary antibody 140 is a horseradish peroxidase-antibody conjugate (see para. [0242]). Murillo further teaches that the hapten-tyramide conjugate 100 is added and in the presence of HRP and hydrogen peroxide of the secondary antibody 140, the hapten-tyramide conjugate 100 becomes covalently bound proximal to the enzyme site (see para. [0242]), which would read on the claimed steps of (a-c). Murillo teaches after the hapten conjugate is bound to the sample, in the presence is detected by an anti-hapten antibody that has antibody and a detectable label (see para. [0243]). In Fig. 2, Murillo further teaches a method that is for detecting the covalently bound hapten-tyramide/tyrosine dimers 160 and an anti-hapten antibody is introduced and the anti-hapten antibody 170 is an HRP-antibody and the antibody binds to the hapten portion of the hapten-tyramide/tyrosine dimer and 3,3’-diaminobenzidine (DAB) assay is used for chromogenic detection of the HRP (see para. [0244] and Fig. 2), which would read on step (d) and applying to the sample a first detectable moiety, wherein the tertiary enzyme catalyzes a first reaction with the first detectable moiety to make the first detectable moiety visible of step (e). Additionally, Murillo teaches in other embodiments, the anti-hapten antibody may be a fluorophore-antibody conjugate and the fluorophore (e.g., quantum dot) may be detected by its fluorescence (see para. [0244]). Murillo teaches horseradish peroxidase catalyzes the dimerization of phenolic compounds and generating free radicals and that only peroxidase-activatable aryl moieties in close proximity to the immobilized enzyme will react and form dimers with tyrosine residues in the vicinity of, or proximal to, the immobilized enzyme (see para. [0235]).
Murillo teaches the target may be located in the sample when the hapten is detected directly or indirectly (via a detectable label) and the target is located by brightfield microscope, fluorescence microscopy or spectroscopy, and/or digital image analysis (see para. [0018]). In particular, Example 2 teaches the evaluating bc124 antibody on tonsil tissue for the comparison of tyramide-hapten conjugates on the slides with the added HRP-anti-hapten monoclonal antibody and DAB and H2O2 (hydrogen peroxide) (also see paras. [0282]-[0285]) and the slides were viewed through a brightfield microscope for Figs. 6-33 and the results shown in Table 4 include a subjective score of the signal strength (e.g., the intensity of the staining) (see para. [0286]). Murillo teaches automated protocol is used (see para. [0283]). In addition, Murillo teaches hapten labeled probes produce punctate signals for each target in the sample and the signals are quantified (see para. [0253]). Murillo teaches data was plotted for each hapten/native anti-hapten pair (Fig. 34) and the variability in the signal suggests a range of native- anti-hapten antibody detection efficiencies (see para. [0293]).
In particular, the instant specification discloses a detectable moiety via brightfield microscope is DAB (see para. [0062], as filed 06/14/2019). Because the visible “one or more punctate dots” is based from enzyme catalyzes with a detectable moiety, Murillo’s assay of using HRP-antibody to react with DAB detectable moiety in brightfield microscope would produce the claimed visible punctate dots using brightfield microscope (for example, see para. [0286]).
Even though Murillo teaches automation with digital image analysis with the steps of amplifying a signal for a target protein biomarker in Figs. 1-2 by reacting a tertiary enzyme catalyzes with DAB detectable moiety to produce visible signals with brightfield microscopy (see Figs. 6-33 and para. [0286]) (i.e., steps a-e) and scoring signal strength (Table 4), the reference does not teach (f) automatically identifying one or more regions of interest in a first obtained digital image of the sample; using the automated object identifier to identify and count the visible punctate dots (in steps g-h) and using an automated scoring engine for scoring the sample (in step i).
Alexander teaches detecting the signal conjugates remains simple from a brightfield microscope or an equivalent digital scanner (see para. [0013]). Alexander further teaches that all aspects of the disclosed embodiments can be automated and facilitated by computer analysis and/or image analysis system (see para. [0124]). Alexander further teaches acquiring digital images which can be done by coupling a digital camera to a microscope and the digital images obtained of stained samples are analyzed using image analysis software (see para. [0124]). Alexander further teaches involving using brightfield imaging with the signaling conjugates (see para. [0125]). Alexander also teaches detecting targets within the sample includes contacting the biological sample with a first amplifying conjugate that is covalently deposited proximally to or directly on the first labeling conjugate and the enhanced signaling conjugate deposition enables easier visual identification of the chromogenic signal, the amplification makes the color darker and easier to see (see para. 0014]). Alexander teaches a two-tiered amplification procedure was used to amplify the signal for each of the binding events and the reagents included HRP-conjugated antibody to catalyze deposition of a tyramide-hapten conjugate wherein the chromophore moiety is DAB (see para. [0222]). Alexander teaches enzyme-labeled targets and signaling conjugates wherein the signaling conjugates are configured to bind proximally to or directly on the one or more target in the biological sample and are configured to provide a brightfield signal (see para. [0015]).
Chukka teaches an imaging tool can support a digital pathologist workflow that includes designating fields of view in an image of the tissue sample and based on the fields of view, a heterogeneity metric can be calculated and combined with an immunohistochemistry combination score (see abstract, para. [0005], and Fig. 1). Chukka teaches digital image depicting a subject detectably labeled with antibodies for a particular biomarker (see para. [0127]). Chukka teaches detectable label is enzyme that permits detection of the binding to the protein (see para. [0300]). Chukka teaches in Fig. 1 an image acquisition device with input device, image processing application, and display through computing system. Further, Fig. 19 shows an image analyzer and a difference engine for calculating a heterogeneity score for a biomarker (also see para. [0034] and para. [0081]), which would read on automated object identifier and automated scoring engine. Chukka teaches prognosis, prediction and/or treatment recommendation based on the output value is communicated to interested parties as soon as possible after the assay is completed and the prognosis is generated and the results of the tests can be generated and delivered automatically to interested parties using a combination of computer hardware and software (see para. [0330]). Chukka teaches the slides can be digitized and automated image analysis algorithms can be computed the percent positivity, binned score (see para. [0337]). Chukka further teaches the digitized slide and the pathologist annotates specific regions to quantify the inter-region intensity and regional heterogeneity and using the automated image analysis for each region, positive and negative stained cell counts, percent positivity, and binned score (0,1+,2+, 3+) are computed (see para. [0338]). Chukka teaches H-score can be calculated from a field of view or fields of view collectively via automated techniques (see para. [0217]). Chukka teaches, for example, given the counts of cells in a plurality of bins associated with respective staining intensities and the H-score can be calculated by summing the product of the percentage (e.g., percentage of cell count, such as the number of cells in a bin divided by the total number of cells) of cells in a bind by the respective intensity level associated with the bin (see para. [0219]). Chukka further teaches computing systems as described herein can be used to implement automated functionality such as processes and actions (see para. 0340]).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have used the signal amplification assay through brightfield microscopy as taught by Murillo with automated identification, counting and scoring of the samples as taught by Alexander and Chukka because Murillo teaches brightfield microscope captures the reaction of the enzyme catalyze with DAB detectable moiety for scoring signal strengths in the samples (see para. [0286] and Figs. 6-33) and Alexander teaches brightfield microscopy can acquire digital images by coupling a digital camera and scanner for automation and facilitated by computer analysis and image analysis system and Chukka teaches the use of automated image analysis for each region are computed through computer automation tools and automatically generates scores from enzyme labeling. Meanwhile, Murillo does recognize the combination of brightfield microscopy and digital image analysis for analyzing the assays (see para. [0018]). Therefore, it would have been obvious to have used the signal amplification assays of Murillo with the automation system for imaging enzyme labeling as taught by Chukka because Chukka teaches the effectiveness of automatically generating scores from imaging tissue labeling and delivering the results to interested parties as soon as the generated results are available.
The person would have reasonably expected success in using the Murillo’s assays through an automatic system for identifying signal strengths (i.e., punctate dots) from the brightfield microscope because it has been well understood by Chukka to automate imaging of tissue labels and Murillo and Alexander teach digitizing brightfield microscope for assay detection.
With regard to claim 2, Murillo teaches tonsil tissue sample contains proteins (see paras. [0282]-[0286]).
With regard to claim 3, Murillo teaches formalin-fixed, paraffin-embedded calu-3 xenograft tissue mounted on slides (see para. [0305]).
With regard to claim 4, Murillo teaches the molecules of interest include proteins (see para. [0119]).
With regard to claim 5, Murillo teaches that hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides) or phospholipids (see para. [0097]). Murillo teaches HER2, a protein linked with higher aggressiveness in breast cancers (see paras. [0114]-[0115]), which would read on the claimed modifications. The instant specification has indicated in the Experiment 1 that HER2 is detected.
With regard to claim 6, Murillo teaches a primary antibody binds to an epitope within an immobilized tissue sample (see para. [0242]).
With regard to claim 7, Murillo teaches the primary antibody is a mouse IgG antibody (i.e., Y shape antibody, no fragments), which would read on native and unmodified antibody.
With regard to claim 8, Murillo teaches primary antibodies are monoclonal antibodies (see para. [0239]).
With regard to claim 9, Murillo teaches a secondary antibody for the primary antibody (see para. [0242] and Fig. 1).
With regard to claims 10-11, Murillo teaches peroxidase is horseradish peroxidase (see para. [0016] and Figs. 1-2).
With regard to claim 12, Murillo teaches digoxigenin (DIG) is a hapten (see para. [0107]). Murillo teaches dinitrophenyl (DNP) haptens (see para. [0299]).
With regard to claims 13-14, Murillo teaches primary antibodies are monoclonal antibodies (see para. [0239]). Murillo further teaches dinitrophenyl (DNP) haptens were detected by anti-DNP monoclonal antibody (see para. [0299]).
With regard to claims 15-16, Murillo does not explicitly teach the detectable moiety comprises silver or tyramide-rhodamine dye. Alexander teaches techniques that include silver in situ hybridization (see para. [0005]). Alexander teaches 5-TAMRA-tyramide conjugate (see para. [0033]). Alexander teaches that TAMRA refers to carboxytetramethyl-rhodamine, a pink rhodamine chromophore (see para. [0092]). Alexander teaches the HRP enzyme catalyzed the deposition of tyramide-TAMRA, which stains the slide with a pink chromogen (see para. [0142]). Alexander teaches the detectable moiety of the detection probe is fluorescein derivatives such as TAMRA and Texas Red (see para. [0154]). Thus, it would have been obvious to the person to have used tyramide-rhodamine dye of Alexander for the signal amplification assays of Murillo because Alexander teaches that the HRP was used to catalyze deposition of chromophore and tyramide, as tyramide-based detection reagents yield a chromogenic signal detectable in amplification assays. Additionally, it would have been obvious to have used tyramide-TAMRA because tyramide-TAMRA produces a distinct pink chromogen.
The person would have reasonably expected success in using tyramide-rhodamine dye because it has been understood by Murillo and Alexander to conjugate tyramide in signal amplification assays.
With regard to claim 17, Murillo teaches that when the anti-hapten antibody is an HRP-antibody conjugate, 3,3’-diaminobenzidine (DAB) assay may be used for chromogenic detection of the HRP (see bottom of para. [0244]).
With regard to claim 18, Murillo teaches multiplexing can be performed with immunohistochemistry (IHC) (see para. [0247]) and the method is suitable for detecting two or more targets in a sample (see paras. [0020] and [0247]).
With regard to claim 19, Murillo teaches HER2 expression and only some cells in the tissue sample are expressing HER2 at any given time (see bottom of para. [0325]).
Response to Arguments
Applicant's arguments filed 04/16/2026 have been fully considered but they are not persuasive over the obviousness rejection of Murillo in view of Alexander and Chukka.
Applicant argues on page 5, para. 4, that Murillo does not teach the claimed punctate-dot detection of a target. Applicant argues on page 6, para. 2, that paras. [0242] and [0244] of Murillo likewise do not cure this deficiency. Applicant argues that while these disclosures may relate to signal amplification chemistry, they do not disclose that the resulting detectable moiety is visible as one or more punctate dots using brightfield microscopy. None of these passages teaches or suggests that a target protein biomarker is visualized as punctate dots, that such dots are indicative of individual molecules of the target protein biomarker. Applicant argues on page 8, paras 2-3, that even assuming a general motivation to automate, the rationale does not cure the absence of the claimed elements. The missing elements here are not generic automation, but rather the claimed punctate-dot protein biomarker quantitation. Applicant argues on page 9 that the Office has still not identified any disclosure in Murillo that a target protein biomarker is visualized as one or more punctate dots using brightfield microscopy.
The arguments are not found persuasive for the following reasons. Murillo does teach brightfield microscope was performed in the assay of enzyme catalyzes with DAB detectable moiety and the slides are shown in Figs. 6-33 (also see para. [0286]). Meanwhile, Figs. 1-2 of Murillo teach all the elements of steps (a)-(d) and wherein the tertiary enzyme catalyzes a detectable moiety to produce a visible reaction (also see para. [0286]), which would read on all the claimed limitations to produce the “one or more punctate dots”. In particular, the instant specification discloses a detectable moiety via brightfield microscope is DAB (see para. [0062], as filed 06/14/2019). Because the visible “one or more punctate dots” is based from enzyme catalyzes with a detectable moiety, Murillo’s assay of using HRP-antibody to react with DAB detectable moiety for a reaction in brightfield microscope would produce the claimed visible punctate dots using brightfield microscope. Therefore, Murillo’s assay would read on the punctate dots using brightfield microscopy wherein the one or more punctate dots are indicative of individual molecules of the target protein biomarker.
Additionally, Applicant argues on page 7, paras. 1-3, that Alexander does not cure Murillo’s deficiencies as the punctate-type disclosures concern ISH gene targets, not target protein biomarkers in a quantitative immunohistochemistry assay. Further, Applicant also argues on pages 7-8 that Chukka likewise does not cure the deficiencies of Murillo or Alexander. Chukka is directed to cell-based and intensity-based scoring metrics, not punctate-dot counting. None of the passages teaches identifying punctate dots and counting those punctate dots to quantitate an amount of a first target protein biomarker. Thus, the combination of Murillo, Alexander, and Chukka combined in the manner would not result in the claimed invention.
The arguments are not found persuasive for the following reasons. As stated above, Murillo’s assay would produce the one or more punctate dots because the “one or more punctate dots” is based from enzyme catalyzes with a detectable moiety and Murillo does teach using HRP-antibody to react with DAB detectable moiety for a reaction in brightfield microscope (see Figs 1-2 and para. [0286] of Example 2). Meanwhile, as stated above in the rejection, it would have been obvious to the person have used the signal amplification assay through brightfield microscopy as taught by Murillo with automated identification, counting and scoring of the samples of Alexander and Chukka because Murillo teaches brightfield microscope captures the reaction of the enzyme catalyze with DAB detectable moiety for scoring signal strengths in the samples and Alexander and Chukka recognize the effectiveness of automatically generating scores from image labeling (see above). Additionally, the person would have reasonably expected success i because it has been well understood by Chukka to automate imaging of tissue labels and Murillo and Alexander teach digitizing brightfield microscope for assay detection.
Conclusion
No claim is allowed.
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/N.P.N/Examiner, Art Unit 1678
/GREGORY S EMCH/Supervisory Patent Examiner, Art Unit 1678
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