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 .
Priority
The present application was filed on 01/15/2021. This application claims benefit of U.S. Provisional Patent Application 62/699,618 filed on 07/17/2018.
Claim Status
Claims 5-7 are withdrawn. Claims 1 and 18 are amended. Claim 19 is canceled. Claims 20-21 are new. Claims 1-18 and 20-21 are pending. Claims 1-4, 8-18 and 20-21 are examined.
Objections/Rejections Status
The rejection of claim 1 under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter is updated in view of the amendment of the claims.
The rejections of claims 1-4 and 8-18 under 35 U.S.C. 103 are updated in view of the amendment of the claims.
Claim Objections
Claim 20 is objected to because of the following informalities: the phrase “wherein the” in line 1 is repeated twice. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-4, 8-18 and 20-21 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
The term “most closely match” in claims 1 and 18 is a relative term which renders the claim indefinite. The term “most closely match” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Since one having skilled in the art can’t define how is “most closely match”, he would not know how to select titers of the first and second biomarker-specific reagents to provide intensity matched staining.
Claim 21 recites “wherein the first and second predetermined concentrations are selected to provide intensity matched staining”. The expression "intensity-matched staining" is vague and does not provide the skilled person with the technical features to which it refers. The skilled person does not know, based upon his common general knowledge, which technical features need to be present in order to ensure that "intensity-matched staining" takes place. The lack of clarity is further aggravated due to the fact that the skilled person does not know what exactly is meant with "intensity-matched staining" and when a sample is to be considered as "intensity-matched stained". It is an undue burden for someone in the art using commonly known tests to identify those staining procedures resulting in "intensity-matched staining".
The definition provided in the description (page 6, lines 4-7) uses vague terms and does not help the skilled person in determining which technical features are referred to by the above expression.
Claims 2-4, 8-17 and 20 are rejected because they depend on the rejected claim.
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.
Claim(s) 1-4, 8 and 12-15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Holt (US20100035281) in view of Dako (Immunohistochemical Staining Methods, 2006).
Regarding claim 1, Holt teaches a method of preparing a histological or cytological sample of a tumor for detection of a fusion protein (see Abstract: teaching a method for identifying the state of a target protein known to associate with a genetic disorder; the target protein may have a DNA sequence change such as a mutation, deletion, insertion or substitution leading to generation of a termination codon and truncation of the protein (i.e., fusion protein); see par.31, disclosing that the genetic disorder is cancer).
The claimed method comprises:
affinity enzymatically staining a first portion of the sample with a first biomarker-specific reagent, wherein the first biomarker-specific reagent is specific for a retained portion of a wild-type protein, and
affinity enzymatically staining a second portion of the sample with a second biomarker-specific reagent, wherein the second biomarker-specific reagent is specific for a lost portion of the wild-type protein.
See pars.122-123: Holt teaches that the immunohistochemistry (IHC) is performed on a tissue sample. An antibody to N-terminal and an antibody to C-terminal are used to detect the BRCA protein in the one or more tissue samples, which helps in distinguishing BRCA2 hereditary cancers and sporadic breast cancers. See par.146: Holt discloses that each experiment uses horseradish peroxidase (HRP) which stains brown and a counterstain which stains metanil yellow. The teaching of IHC, the use of antibodies and the use of HRP for developing detectable color anticipate the affinity enzymatically staining process of the instant claim.
Furthermore, from the teaching of Hold in par.122-123, a retained or lost portion of a wild-type protein can be interpreted as a C-terminal or N-terminal portion of a target protein. Particularly, if BRCA gene is truncated at C-terminal portion, there is no immune staining for the C-terminus (due to the protein truncation) while normal tissues from these patient and control cancers and normal tissues without BRCA2 mutations all show positive immunostaining (see par.122). In this case, C-terminal antibody is specific for the lost portion of the wild type of protein, while N-terminal antibody is specific for the retain portion of the wild type of protein. In addition, this interpretation mentioned above corresponds with the explanation in the instant Specification paragraphs 14-15 and 67, and Fig.1.
Holt does not teach that the first and second biomarker-specific reagents are used at a first or second predetermined concentration. Holt does not teach “wherein the affinity enzymatically staining of the first portion of the sample and the affinity enzymatically staining of the second portion of the sample results in intensity- matched staining of the first portion of the sample and the second portion of the sample; and wherein affinity enzymatically staining of the first portion of the sample and affinity enzymatically staining of the second portion the sample results in intensity-matched staining of the first portion of the sample and the second portion of the sample, wherein the first and second predetermined concentrations are determined by testing the first and second biomarker-specific reagents at various titers on one or more serial sections of a tissue sample known to express a wild-type counterpart of the fusion protein, and selecting titers of the first and second biomarker-specific reagents that most closely match each other in intensity based on a comparison of the staining obtained with the various titers of the first and second biomarker- specific reagents.”
Dako teaches that the advantage of multiple staining which is defined as the detection of two or more targets on one slide is increasing the information obtained from each slide (Dako page 61 pars.1-2).
Dako teaches that the first and second biomarker-specific reagents are used at a first or second predetermined concentration. (Dako teaches that antibody titers and dilutions have an effect on staining quality in immunohistochemistry (IHC) (page 15 par.1). “Correct dilutions will contribute to the quality of staining if they are prepared accurately and consistently. Often a manufacturer offers ready-to-use (RTU) reagents, or recommends dilution ranges compatible with other variables such as method, incubation time and temperature. If this information is not provided, optimal working dilutions of immunochemical reagents must be determined by titration” (page 16 par.1). “Optimum antibody titer may be defined as the highest dilution of an antiserum (or monoclonal antibody) that results in maximum specific staining with the least amount of background under specific test conditions” (Dako page 15 par.2). Therefore, the concentration of the primary antibodies (i.e., first and second biomarker-specific reagents) are pre-determined before being used for IHC.)
Dako teaches wherein the first and second predetermined concentrations are determined by testing the first and second biomarker-specific reagents at various titers (Dako page 16 par.1: “Correct dilutions are determined best by first selecting a fixed incubation time and then by making small volumes of a series of experimental dilutions.”) on one or more serial sections of a tissue sample known to express a wild-type counterpart of the fusion protein (Dako page 16 par. 4: “Nine tissue sections are required for testing three dilutions.”), and selecting titers of the first and second biomarker-specific reagents that most closely match each other in intensity based on a comparison of the staining obtained with the various titers of the first and second biomarker- specific reagents (Dako page 62 Technical Challenges for Multiple staining Immunohistochemistry: “it is difficult to balance signals enabling rare targets to be visible in the same slide as highly abundant targets…An adjustment in concentration of the primary antibodies may solve this problem.”).
The teaching of Dako supports the purpose of pre-defining the concentration of the first and second biomarker-specific reagents in obtaining the balance signals between the staining regions in the sample, which encompasses the limitation “the affinity enzymatically staining of the first portion of the sample and the affinity enzymatically staining of the second portion of the sample results in intensity- matched staining of the first portion of the sample and the second portion of the sample”.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to pre-determine the concentration of the first and second biomarker-specific reagents in the method taught by Holt, by testing the first and second biomarker-specific reagents at various titers on one or more serial sections of a tissue sample known to express a wild-type counterpart of the fusion protein and selecting titers of the first and second biomarker-specific reagents that most closely match each other in intensity based on a comparison of the staining obtained with the various titers of the first and second biomarker- specific reagents as taught by Dako to ensure a success of multiple staining (i.e., the signals from rare targets and highly expressed targets can be observed clearly) (Dako page 61 par.1).
One having an ordinary skill in the art would have had a reasonable expectation of success in combining Holt and Dako because Holt is directed to perform multi-staining IHC on the tissue and Dako provides the principle of multi-staining IHC comprising the adjustment and pre-determination in concentration of the primary antibodies to obtain the balanced signals from targets of interest.
Regarding claim 2, Holt and Dako teach the method of claim 1, wherein affinity enzymatically staining of the sample comprises an affinity histochemical assay (Holt par.123: antibodies used for the IHC were rabbit polyclonal antibodies to N-terminal amino acids (R and D systems) and C-terminal amino acids 3245-3418 of BRCA2 (Oncogene Research); par.36: these antibodies specifically bind to their target protein, e.g., C-terminal or N-terminal end of the target protein). The teaching of Holt encompasses the limitation of the claim because according to the instant specification paragraph 71, affinity histochemical staining technique typically involves contacting a sample deposited on a slide or other solid support with a biomarker-specific reagent under conditions sufficient to permit specific binding between the biomarker-specific reagent and the biomarker of interest.
Regarding claim 3, Holt and Dako teach the invention as discussed above. Holt teaches step a and c of claim 3:
(a) contacting the first section with a first antibody, wherein the first antibody is immune-specific for an epitope located in the retained portion of the wild-type protein;
(c) contacting the second section with a second antibody, wherein the second antibody is immune-specific for an epitope located in the lost portion of the wild-type protein.
See the discussion in claim 1 above.
Holt further teaches:
(b) contacting the first section having the first antibody bound thereto with a first set of detection reagents under conditions sufficient to deposit a dye on the first section in proximity to the first antibody bound to the first section; (Holt pars.145-147: an IHC is performed on a tissue section with C-terminal mAb (i.e., a first antibody) and a horseradish peroxidase (i.e., a first set of detection reagent). Horseradish peroxidase stains brown at the site where the antibody binds to the section so that the cancer state of tissue is illustrated.)
(d) contacting the sample having the second antibody bound thereto with a second set of detection reagents under conditions sufficient to deposit the second dye on the sample in proximity to the second antibody bound to the sample. (Although Holt does not clearly teach a step of contacting a second set of detection reagent on the second antibody bound to the second section, Holt teaches that there is a detecting step for one or more N-terminal antibodies bound to the section (see page 32, right col step h), using a detection agent (see par.87). Also, Holt discloses a black color stain at the site where the antibody binds to the section so that the cancer state of tissue is illustrated (see par.129). Therefore, the teaching from Holt anticipates the claimed step.
Regarding claim 4, Holt and Dako teach the invention as discussed above. Holt (in par.93) further teaches that the detection agent (i.e., can be applied for both first and second claimed sets) is a molecule which is administered linked to or conjugated to an antibody moiety, i.e., antibody or antibody fragment, or sub-fragment, and is useful in diagnosing or detecting a disease by locating the cells containing the antigen. The detection agent of Holt encompasses the claimed first/second secondary detection reagent because the molecule can bind to the primary first/second antibody.
Holt also discloses that there is an enzyme, e.g., horseradish peroxidase, and a chromogenic or fluorescent reagent that causes an antibody peroxidase reaction (see par.49: teaching that the level of binding of a phosphor-specific antibody to a tissue sample target protein can be assessed using immunohistochemistry or immunoprecipitation and staining with fluorescent dyes; par.91: teaching that enzyme is horseradish peroxidase or alkaline phosphatase; par.125: showing that a color represents specific staining of the antibody peroxidase reaction).
Regarding claim 8, Holt and Dako teach the invention as discussed above. Holt teaches that a detection agent is linked to an antibody moiety, e.g., biotin-streptavidin complex (Holt par.87 or 93) and peroxidase is used to develop the color of the stained tissue (Holt par.146). Holt does not teach that the enzyme is bound to the tertiary detection reagent which is capable of specifically binding to the secondary detection reagent.
Dako teaches a method for immunohistochemistry staining comprising the secondary antibodies which are conjugated to biotin and function as links between tissue-bound primary antibodies and an avidin-biotin-peroxidase complex (Dako pages 48-49 par.2, Figures 7.2, 7.3). This teaching encompasses the enzyme peroxidase binding to the tertiary detection reagent, e.g., biotin-streptavidin complex, which is capable of specifically binding to the secondary detection reagent, e.g., the secondary antibody.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method taught by Holt, by the method taught by Dako because the detection methods taught by Holt and Dako, which are based on a product of a chemical reaction catalyzed by the enzyme (Holt pars.87, 93 and 146; Dako pages 48-49 par.2, Figures 7.2 and 7.3), are functionally equivalent. Moreover, a single primary antibody subsequently is associated with multiple peroxidase molecules and because of the large enzyme-to antibody ratio, a considerable increase in sensitivity is achieved compared to direct peroxidase-conjugate methods (Dako page 49). One having an ordinary skill in the art would have been motivated to improve sensitivity for detection of target antigens by using the method taught by Dako.
One having an ordinary skill in the art would have had a reasonable expectation of success in combining Holt and Dako because Holt is directed to perform IHC on the tissue and Dako provides the methods of improving sensitivity for target detection in IHC.
Regarding claims 12-15, Holt and Dako teach the invention as discussed above. Holt also discloses that the intensity from N-terminal and C-terminal antibody staining was rated from 0-3 and percent positive cells was rated from 0-3 and these two scores were added and then results were decoded and unblinded to compare with known BRCA2 mutation status (see par.144 and Table 2). Therefore, Holt determined an intensity of C-terminal and N-terminal antibody binding into the mutant protein and translated into scores and a ratio (between the binding of two specific antibodies on the sample). Holt also considered the percentage of positively-staining cells of two staining sections. The ratio of two scores was used to determine the presence of mutant protein. See paragraph 144 and Table 2.
Claim(s) 9-11 is/are rejected under 35 U.S.C. 103 as being unpatentable over Holt in view of Dako, as applied in claim 8, and further in view of Polaske et al. (Quinone Methide Signal Amplification: Covalent Reporter Labeling of Cancer Epitopes using Alkaline Phosphatase Substrates, Bioconjugate Chem. 2016, 27, 3, 660–666, PTO-892 07/03/2024).
Regarding claims 9-10, Holt and Dako teach the method of claim 8.
For claim 9, Holt fails to teach that a set of detection reagent further comprises a signaling conjugate. The signaling conjugate comprises: a latent reactive moiety reactive with the enzyme to generate a reactive species capable of binding to the sample; and an element conjugated to the latent reactive moiety wherein the element is selected from the group consisting of the dye, the enzyme, and a member of a specific binding pair.
For claim 10, Holt does not teach that the element conjugated to the latent reactive moiety of the signaling conjugate is the member of the specific binding pair, and wherein the set of detection reagents further comprises a second member of the specific binding pair, wherein the second member of the specific binding pair is selected from the group consisting of the first dye and the first enzyme.
Polaske teaches that a signaling conjugate comprises a latent reactive moiety (e.g., quinone methide precursor) and an element (e.g., reporter) (see Introduction par.3 page 661, and Fig. 1A). Polaske teaches the signaling conjugate reacting with the enzyme, e.g., alkaline phosphatase, to generate a reporter label for immunohistochemical assay (see Abstract, Introduction par.3 page 661, and Fig. 1A). Briefly, after the enzyme conjugated detection reagent is immobilized at the position of the target analyte, a reporter-labeled quinone methide precursor is applied (see Introduction par.3 page 661). The precursor reacts with the enzyme (e.g., alkaline phosphatase) to form a quinone methide which is already conjugated with an element, e.g., reporter. The quinone methide either reacts with immobilized nucleophiles in close proximity to the site of generation or are quenched by nucleophiles in the reaction media in order to identify the presence or absence of the target analyte (see Introduction par.3 page 661, and Fig. 1A).
The element in the signaling conjugate can comprise biotin reporter, which is considered as a first binding pair, thus integrating with a second binding pair avidin to form avidin–biotin–enzyme complex visualization techniques (see Abstract, Introduction par.1 page 660, Fig.1B).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the signaling system in the method taught by Holt for the signaling system taught by Polaske for the benefit of signal amplification, thereby improving the diagnostic immunohistochemical assay (Polaske Abstract).
One having an ordinary skill in the art would have had a reasonable expectation of success in combining Holt and Polaske because Holt and Polaske are directed to IHC method using the signaling system which comprises the enzymatic labeling moiety.
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Polaske Fig.1
Regarding claim 11, Holt, Dako and Polaske teach the method of claim 9. Holt fails to teach that: the secondary detection reagent comprises a hapten, the tertiary detection reagent is capable of specifically binding to the hapten, the first member of the specific binding pair is the hapten and the second member of the first specific binding pair is the tertiary detection reagent.
As discussed in claim 8, Dako teaches that the secondary detection reagent comprises a biotin, the tertiary detection reagent is capable of specifically binding to the biotin to form an enzyme complex on the secondary detection reagent (Dako pages 48-49 par.2, Figures 7.2, 7.3). Dako also teaches that the signaling system can be amplified by adding more layer on the primary detection reagent by using biotin or haptens, subsequently employing the corresponding antibody or streptavidin reagent (Dako page 64 par.1). For example, hapten is attached to the probe and recognized by a secondary molecule – normally an antibody; the secondary molecule can be conjugated to an enzyme for chromogenic detection; or form the basis for further amplification steps, depending on the required level of sensitivity (Dako page 91 par.2).
See discussion of Polaske in claim 9 above. Polaske further teaches that the reporter in the reporter-labeled QM precursor can be fluorophore reporters, biotin reporters or hapten reporters (Polaske Fig.1, pages 661-662).
From the teachings of Dako and Polaske, biotin or hapten can be alternatively used in the signal amplification system as a linker to connect the detection reagents.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the hapten for the biotin in the signaling system of Holt, Dako, and Polaske because hapten or biotin is functionally equivalent in forming the set of detection reagents in the signaling system (Dako page 64 par.1, page 91 par.2; Polaske pages 661-662, Fig.1). One having an ordinary skill in the art would have had a
reasonable expectation of success in combining Holt, Dako, and Polaske because they are directed to the IHC method which produces a highly amplified and specific signal to detect a target in a tissue sample (Polaske Conclusion).
Claim(s) 16-17 is/are rejected under 35 U.S.C. 103 as being unpatentable over Holt in view of Dako, as applied in claim 1, and further in view of Stransky et al. (The landscape of kinase fusions in cancer, Nature communications Sep 2014, IDS filed date 05/06/2022) and Crosby et al. (US20150056193).
Regarding claims 16-17, Holt and Dako teach the invention as discussed above. Holt does not teach the protein analyte is from the claimed group (ROS1, RET, ALK, NTRKA, NTRKB, NTRKC, RAF1, BRAF, PRKCA, PRKCB, and PKN1). Holt does not teach detecting ROS1 fusion protein by using the first biomarker-specific reagent binds to an epitope disposed in amino acids residues 1926-2347 of SEQ ID NO: 1, and the second biomarker-specific reagent binds to an epitope disposed in amino acids residues 1-1749 of SEQ ID NO: 1.
Stransky discloses a variety of fusion genes associated with cancer diagnosis and therapy (see Abstract, and page 2 Introduction section). Stransky surveys gene fusion across a variety of tumor types (see page 2 right col par.3). Stransky discloses that most known translocation mutations occur in genes ROS1, RET, ALK, NTRK, BRAF, PRKC (see at least page 3 left col par.2; page 4 left col par.2). Stransky also finds recurrent fusions in PKN1 and RAF1 in various tumor types (see page 3 right col par.3).
Crosby provides methods of detecting, in a biological sample from a patient having or suspected of having cancer, the presence of a polypeptide having ROS1 kinase activity (see Abstract). Crosby teaches that it would be useful to discover new ways to identify cancer at an early stage (see par.6). Accordingly, the detection of ROS oncogene 1 fusion allows for identification of patients whose tumors can benefit from therapy with one or both of an EGFR-inhibiting therapeutic or a ROS1-inhibiting therapeutic (see par.7).
In addition, Crosby discloses an IHC method to detect the expression and/or activation status of a polypeptide with ROS1 kinase activity (e.g., full length ROS1 polypeptide or a ROS1 fusion polypeptide) in tumor tissue (see pars.151-152). Crosby discloses that the antibodies from Abcam (ab108492) and from Cell Signaling Technology #3287 were used (see par.101). These two antibodies can bind to epitopes disposed in amino acid residues at the N and C terminal of ROS1 protein as disclosed in Table 5 of the instant specification.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use the method of detecting fusion protein taught by Holt, for detecting the lost and retained portion of wild-type proteins comprising ROS1, RET, ALK, NTRK, BRAF, PRKC, PKN1 and RAF1 as suggested by Stransky because these gene are associated with various tumor types. The motivation to do so also comes from Stransky, who teaches that the findings of the association of these fusion genes with tumor types would justify a rapid reassessment of current protocols for targeted genomic profiling of patients to cover therapeutically actionable fusion events across cancers. In addition, the combination of Holt and Stransky may motivate cancer drug discovery against targets. See Stransky page 7 left col par.2.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of detecting fusion protein taught by Holt, by using the antibodies to N-terminal and C-terminal of ROS1 protein to detect if there is a c-Ros oncogene 1 (ROS1) fusion as taught by Crosby because it may be co-expressed in tumors and it can identify a patient whose tumors can benefit from therapy with a ROS1-inhibiting therapeutic (see Crosby par.7). In addition, the combination of Holt and Crosby would result in a predictable outcome because Crosby also uses immunohistochemistry assay with anti-ROS1 antibody to detect fusion ROS1 in a sample.
Claim(s) 18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Holt in view of Dako and Day et al. (Covalently deposited dyes: a new chromogen paradigm that facilitates analysis of multiple biomarkers in situ, Laboratory investigation, vol.97, issue 1, 2017, 104-113, PTO-892 07/29/2025).
Regarding claim 18, Holt and Dako teach the method of claim 1, which encompasses the limitations (a), (b), (c) and partially step (d) of claim 18. Holt and Dako also teach “wherein the first and second predetermined concentrations are selected to provide intensity matched staining, and wherein the first and second predetermined concentrations are determined by testing the first and second biomarker-specific reagents at various titers on one or more serial sections of a tissue sample known to express a wild-type counterpart of the oncogenic fusion protein, and selecting titers of the first and second biomarker-specific reagents that most closely match each other in intensity based on a comparison of the staining obtained with the various titers of the first and second biomarker-specific reagents”. See discussion of Holt and Dako in claims 1 above (Holt pars.122-123 and 146; Dako pages 15-16 and 61-62).
Moreover, Holt teaches step (d1) that the first and the second dye are distinguishable from one another when co-localized on the sample (see Holt par.127, disclosing the immunohistochemistry of tissue section using C-terminal antibody (left panel), N-terminal antibody (middle panel); see par.129, disclosing that C-terminal staining shows dark grey color and N-terminal staining shows black color on the panels, thus the dyes are distinguishable).
However, Holt fails to teach step (d2) which the first dye generates a first detectable signal when not co-localized with the second dye, the second dye generates a second detectable signal when not co-localized with the first dye, and the first dye and the second dye generate a third detectable signal when co-localized.
Dako teaches that the first dye generates a first detectable signal when not co-localized with the second dye, the second dye generates a second detectable signal when not co-localized with the first dye, and the first dye and the second dye generate a third detectable signal when co-localized (Dako page 65 par.3: when selecting color combinations for multiple staining with chromogenic dyes, it is advisable to choose opposing colors in the color spectrum, to facilitate spectral differentiation. When working with co-localized targets, dyes must be chosen so that it is possible to distinguish the mixed color from the individual colors, which the mixed color is a third detectable signal when co-localized.)
Day discloses that multiplexed analysis of multiple biomarkers in a tissue sample requires use of reporter dyes with specific spectral properties that enable discrimination of signals (see Abstract). The analysis method comprises: using the dyes, individually or in blends to generate new color, providing signal sensitivity and dynamic range similar to conventional DAB chromogen, while enabling analysis of co-localized biomarkers. This new paradigm will enable generation of a wide variety of new chromogens, which is useful for both research and clinical biomarker analysis. See Abstract.
Day demonstrates that co-localization of two biomarkers, each one stained via a different cognate antibody with a different associated chromogenic stain, can produce a third distinctly colored stain. This method can be used in immunohistochemistry. See Abstract, page 108 column 2 paragraph 3-4, page 112 column 2 paragraph 1 and Figure 5d.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the immunohistochemistry assay taught by Holt, using an immunostaining with different chromogenic dye associated antibodies for the detection of biomarker of interest as taught by Day because Day’s method can be used for multiplexed analysis by enabling discrimination of signals and analysis of co-localized biomarkers (see Abstract). Moreover, the chromogen in Day’s method also generates a sensitive and specific signal compared with the gold standard DAB chromogen (see page 106 col.2).
One having ordinary skill in the art would have had a reasonable expectation of success in combining Holt, Dako and Day because they are directed to use immunohistochemistry in detecting biomarkers of interest. While Dako is generic to the method of using dyes to generate different signals for detecting antigens when they are co-localized, Day provides a specific example for using dyes to generate different signals for detecting two co-localized antigens in the sample.
Claim(s) 20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Holt in view of Dako, as applied in claim 1, and further in view of Crosby et al.
Regarding claim 20, Holt and Dako teach the method of claim 1. Holt does not specially teach wherein the first biomarker-specific reagent is capable of binding to an N-terminal portion of a wild-type human Ros1 protein; and wherein the second biomarker-specific reagent is capable of binding to a C-terminal portion of a wild-type human Ros1 protein.
Crosby provides methods of detecting, in a biological sample from a patient having or suspected of having cancer, the presence of a polypeptide having ROS1 kinase activity (see Abstract). Crosby teaches that it would be useful to discover new ways to identify cancer at an early stage (see par.6). Accordingly, the detection of ROS oncogene 1 fusion allows for identification of patients whose tumors can benefit from therapy with one or both of an EGFR-inhibiting therapeutic or a ROS1-inhibiting therapeutic (see par.7).
In addition, Crosby discloses an IHC method to detect the expression and/or activation status of a polypeptide with ROS1 kinase activity (e.g., full length ROS1 polypeptide or a ROS1 fusion polypeptide) in tumor tissue (see pars.151-152). Crosby discloses that the antibodies from Abcam (ab108492) and from Cell Signaling Technology #3287 can be used (see par.101). These two antibodies can bind to epitopes disposed in amino acid residues at the N and C terminal of ROS1 protein as disclosed in Table 5 of the instant specification.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of detecting fusion protein taught by Holt, by using the antibodies to N-terminal and C-terminal of ROS1 protein to detect if there is a c-Ros oncogene 1 (ROS1) fusion as taught by Crosby because it may be co-expressed in tumors and it can identify a patient whose tumors can benefit from therapy with a ROS1-inhibiting therapeutic (see Crosby par.7). In addition, the combination of Holt and Crosby would result in a predictable outcome because Crosby also uses immunohistochemistry assay with anti-ROS1 antibody to detect fusion ROS1 in a sample.
Claim(s) 21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Holt in view of Dako.
Regarding claim 21, Holt, in paragraphs 122-123 and 146, teaches a method of preparing a histological or cytological sample of a tumor for detection of a fusion protein, the method comprising:
- affinity enzymatically staining a first portion of the sample with a first biomarker-specific reagent, wherein the first biomarker-specific reagent is specific for a retained portion of a wild-type protein,
- affinity enzymatically staining a second portion of the sample with a second biomarker-specific reagent, wherein the second biomarker-specific reagent is specific for a lost portion of the wild-type protein.
See discussion of Holt in claim 1 above.
Holt further teaches:
- morphologically staining a third portion of the sample with a morphological stain; (Holt par.146: tissue was counterstained with metanil yellow; par.157: cell membrane with nuclei was counterstained with hematoxylin; Fig.11)
- identifying one or more regions of interest in the morphologically stained third portion of the sample;- scoring staining in the first portion of the sample and the second portion of the sample, wherein the scoring in the first portion of the sample and the second portion of the sample are in regions which correspond to the identified one or more regions of interest in the morphologically stained third portion of the sample; (Holt par.144: slides were then quantitated by a combined scoring scale which assesses both immunostaining intensity and percent positive cells)
- determining the presence of the fusion protein based on the scores. (Holt par.144 and Table 2)
Holt does not teach the concentrations of the first and second biomarker-specific reagents are predetermined, wherein the first and second predetermined concentrations are selected to provide intensity matched staining; and wherein the affinity enzymatically staining of the first portion of the sample and the affinity enzymatically staining of the second portion of the sample results in intensity- matched staining of the first portion of the sample and the second portion of the sample.
Holt does not teach the scoring step is done with an automated image scoring algorithm.
Dako teaches the concentrations of the first and second biomarker-specific reagents are predetermined, wherein the first and second predetermined concentrations are selected to provide intensity matched staining; and wherein the affinity enzymatically staining of the first portion of the sample and the affinity enzymatically staining of the second portion of the sample results in intensity- matched staining of the first portion of the sample and the second portion of the sample. See discussion of Dako in claim 1 above (Dako pages 15-16 and 61-62).
Dako teaches the scoring step can be done with human eye or automated image detector (Dako page 67). The advantages of digital image analysis are: allowing the combination fluorescent and immunoenzyme dyes, allowing compensation for overlapping emission spectra, setting a threshold on signal intensity to exclude unspecific staining or background from final images, allowing signal quantitation (Dako page 67 Automated Image Acquisition and Analysis).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to pre-determine the concentration of the first and second biomarker-specific reagents in the method taught by Holt, by testing the first and second biomarker-specific reagents at various titers on one or more serial sections of a tissue sample known to express a wild-type counterpart of the fusion protein and selecting titers of the first and second biomarker-specific reagents that most closely match each other in intensity based on a comparison of the staining obtained with the various titers of the first and second biomarker- specific reagents as taught by Dako to ensure a success of multiple staining (i.e., the signals from rare targets and highly expressed targets can be observed clearly) (Dako page 61 par.1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use an automated image scoring algorithm to analyze the IHC result taught by Dako in the method of Holt for the benefit of partially replacing a skilled pathologist (Dako page 68). Moreover, the automated image system has many advantages such as using a greater number of dyes (which can’t be detected with human eye), allowing the combination fluorescent and immunoenzyme dyes and allowing signal quantitation (Dako page 67).
One having an ordinary skill in the art would have had a reasonable expectation of success in combining Holt and Dako because Holt is directed to perform multi-staining IHC on the tissue and Dako provides the principle of multi-staining IHC comprising the adjustment and pre-determination in concentration of the primary antibodies to obtain the balanced signals from targets of interest, and the method of IHC image analysis.
Response to Arguments
Regarding the rejections under 35 U.S.C. 103:
Applicant's arguments filed 10/08/2025 have been fully considered. Claims 1 and 18 have been amended by adding the method of identifying the concentration of the biomarker-specific reagents which provide intensity matched staining of the first portion of the sample and the second portion of the sample.
Accordingly, a new ground(s) of rejection is made in view of the amendment of the claims.
Conclusion
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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/CHAU N.B. TRAN/Examiner, Art Unit 1677
/BAO-THUY L NGUYEN/Supervisory Patent Examiner, Art Unit 1677 February 18, 2026