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.
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 Claim 20 is withdrawn as directed to the nonelected invention.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, of record) in view of Alexander et al. (US2013/0260379A1, published 10/03/2013, of record) and Chukka et al. (US2015/0347702A1, published 12/03/2015, of record).
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]). Murillo teaches in Fig. 1 that an immobilized tissue sample; a primary antibody 120 binds to an epitope 130 within an immobilized tissue sample; a secondary antibody is introduced and binds to the primary antibody (also see para. [0242]). Murillo teaches that horseradish peroxidase-antibody conjugate includes a secondary antibody and the hapten-tyramide conjugate is added and in the presence of HRP and peroxide, the hapten-tyramide conjugate 100 becomes covalently bound proximal to the enzyme site, which would read on steps (a-c). Murillo teaches horseradish peroxidase catalyzes the dimerization of phenolic compounds and generating free radicals (see para. [0235]). Murillo teaches 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 by digital image analysis (see para. [0018]).
Further in Fig. 2, Murillo teaches a method for detecting hapten-tyramide/tyrosine dimers and an anti-hapten antibody is introduced and the anti-hapten antibody is conjugated to HRP and the antibody bindings to the hapten portion of the hapten-tyramide/tyrosine dimer (see para. [0244]). 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]), which would read on steps (d)-(e). Murillo teaches that the target is located in the sample when the hapten is detected via detectable label and the target is located by brightfield microscopy (see paras. [0018] and [0286]). As stated above, Murillo teaches anti-hapten antibody is conjugated to HRP and DAB is used for chromogenic detection of the HRP with brightfield microscopy and the multiplexed RNA-ISH assay produces punctate signals for each target in the sample, allowing simultaneous evaluation of the presence and relative amounts of each target within the tissue sample, which would read on visible as a punctate dot using brightfield microscopy wherein the punctate dot is indicative of the target protein biomarker (see para. [0253]). Murillo teaches in some embodiments the signals are quantified by counting the number of pixels above background in each image (see para. [0253] and Example 5). Murillo teaches using an Olympus fluorescent microscope fitted with a Spectral Imaging camera (Applied Spectral Imaging (ASI) Vista, Calif.) (Fig.48) (for example, see para. [0299]).
Murillo teaches that hapten conjugates include a peroxidase-activatable aryl moiety capable of forming a free radical when combined with a peroxidase enzyme and peroxide and subsequently forming a dimer with a phenol-containing compound (see para. [0010]). Murillo teaches the method that allows sub-cellular structures to be distinguished e.g., nuclear membrane versus the nuclear region, cellular membrane versus the cytoplasmic region (see bottom of para. [0235]). Murillo further teaches that the hapten conjugate is a hapten-tyramide conjugate that can be utilized in a tyramide signal amplification assay and tyramide signal amplification is a peroxidase-based signal amplification system (see para. [0236]). Fig. 1 illustrates a dimer formed when the phenol group of tyramine binds to the phenol group of a tyrosine residue in the protein (also see bottom of para. [0242]). Murillo teaches the results shown in Table 4 include a subjective score of the signal strength (e.g., the intensity of the staining) on a scale of 1-4 and with 4 being the most intensely stained and the background (BG) score and signal to noise ratio (see para. [0286] and Table 4 and Figs 6-33).
Even though Murillo teaches the steps of amplifying a signal for a target protein biomarker as claimed (see Figs. 1-2) and the signals are quantified by counting the number of pixels and subjectively score the signal strength (see Table 4), the reference does not explicitly teach the steps (a-c) and (steps d-e) as a single embodiment; (f) automatically identifying one or more regions of interest in a first obtained digital image of the sample; and the automated object identifier and automated scoring engine in steps (g-i).
Alexander teaches 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]). For example, Figs. 31-34 teaches recognizing punctuate dots using microscopy. Alexander teaches color can be measured in several different ways, for example, intensity values (see para. 0124]). Alexander teaches that there is a need for automated in situ hybridization assays which target mRNA that enables robust and reproducible evaluation of biomarker expression while preserving tissue context and specificity, as well as cell-cell relationships (see para. [0213]). Alexander teaches automated mRNA-ISH assays for FFPE samples have been developed which enable simultaneous analysis of biomarker expression and an internal control gene expression to monitor assay performance and sample integrity (see para. [0230]).
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 (see para. [0014]). Alexander teaches 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 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 a two-tiered amplification procedure was used to amplify the signal for each of the binding events and HRP was used to catalyze deposition of a chromophore and tyramide conjugate (see para. [0222]). Alexander teaches the tyramide-based detection reagents yielding a chromogenic signal detectable using bright-field light microscopy (see para. [0144]).
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 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 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]).
First, 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 combined dimerization of tyramine and tyrosine in the presence of HRP-antibody (steps a-c) with anti-hapten antibody and 3,3’diaminobenzidine DAB of steps (d-e) for signal amplification assays (Figs. 1 and 2) as taught by Murillo because Murillo teaches anti-hapten antibodies are added after the presence of dimerization at proximal to the HRP enzyme sites and the steps would allow sub-cellular structures to be distinguished such as nuclear membrane versus the nuclear region, cellular membrane versus the cytoplasmic region in a single process.
With respect to automatically identifying a digital image with an automated object identifier and automated scoring engine, it would have been obvious to the person to have used the signal amplification assays wherein the signals are quantified by counting the number of pixels as taught by Murillo with automated identification, counting and scoring of the samples as taught by Alexander and Chukka because Alexander teaches that the automated, facilitated by computer analysis and image analysis system, in situ hybridization assays would enable robust and reproducible evaluation of biomarker expression and Chukka teaches the use of automated image analysis for each region are computed through computer automation tools and automatically generates immunohistochemistry scores. Thus, it would have been obvious to have used the digital images from the assay of Murillo with the automated system of Chukka because it has been recognized in the art that automation is consistent and reproducible for analyzing the punctate signals and processing tools are require for automating immunohistochemistry.
The person would have reasonably expected success in using the assay through an automatic system for identification of the punctuate dots because it has been well understood in the art to produce digital images for the assays of Murillo, Alexander, and Chukka.
With regard to claim 2, Murillo teaches tissue sample contains proteins (see para. [0238]).
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 the antibody is a native and unmodified antibody.
With regard to claim 8, Murillo teaches primary antibodies are mouse monoclonal antibodies specific for their respective targets (see para. [0248]).
With regard to claim 9, Murillo teaches a secondary antibody is introduced and binds to 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 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 09/29/2025 have been fully considered but they are not persuasive under 35 U.S.C. 103 rejection.
Applicant argues on page 6 that Murillo does not disclose that punctate signals may be generated via an immunohistochemical technique; and certainly, does not disclose or suggest any assays that would produce punctate signals indicative of target protein biomarkers as required by the claimed invention. Applicant argues that there is absolutely no basis in the art that Murillo’s disclosure of punctate signals resulting from an RNA-ISH assay would “read on” punctate does indicative of the target protein biomarker. Applicant further argues that Murillo does not disclose or suggest that a detectable moiety may be visible as one or more punctate dots using brightfield microscopy. Applicant further argues that Alexander has no bearing on the generation and/or identification of punctate dots indicative of target protein biomarkers in a sample. Chukka does not teach counting as claimed and punctate dots visible using brightfield microscopy.
The arguments are not found persuasive for the following reasons. Murillo does teach that the molecule of interest or target includes protein and nucleic acid sequences (see para. [0119] and Figs. 4-5), which would read on target protein biomarker. In particular, the reaction of Murillo produces punctate signals for each target in the sample, allowing simultaneous evaluation of the presence and relative amount of each target within the tissue sample (see above or para. [0253]). Murillo also teaches that the detectable label and the target is detected by brightfield microscopy. Thus, Murillo’s tyramide signal amplification assay has the ability to count the punctate signals to correlate to amounts of target (i.e., target protein biomarker). Murillo also teaches that the system is compatible with immunohistochemical detection. With respect to Alexander, the reference teaches tyramide signal amplification and states that it is known to amplify the visibility of targets (similar to Murillo). Alexander also 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]). Because digital images can be analyzed through digital camera software (i.e., automated object identifier), it would have been obvious to the person to have used the digital images from the assay of Murillo with the digital camera software because it has been recognized in the art that automation is consistent and reproducible for analyzing the punctate signals and processing tools are require for automating immunohistochemistry.
Because Murillo teaches that the punctate signals are related to the amounts of the target (i.e., target protein biomarker) in the sample and digital images are produced, the automated system and scoring of that sample would be based on Murillo’s punctate signals for measuring the amounts of targets in Murillo’s samples.
Conclusion
No claim is allowed.
THIS ACTION IS MADE FINAL. 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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/N.P.N/Examiner, Art Unit 1678
/SHAFIQUL HAQ/Primary Examiner, Art Unit 1678