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 .
Applicant’s amendment, filed on 7/05/2024, is acknowledged.
Claims 8-16, 19-23, 25-30, 34-37, 40-45, 47, 50, 53-57, and 59-65 are cancelled.
Claims 1-5, 7, 17, 18, 24, 31-33, 38, 39, 46, 48, 49, 51, 52, and 58 are currently pending.
Claims 1, 2 and 46 are independent claims.
Election/Restrictions
Applicants’ election without traverse of Group II, claims 46, 48, 49, 51, 52, and 58, directed to a method of improving the predictive value of a biomarker, and the species of: i) PD-1, CD8, and FoxP3 biomarkers; and ii) CD8+FoxP3+ cells, filed on 5/29/2026, is acknowledged.
Claims 1-5, 7, 17, 18, 24, 31-33, 38, 39, 46, and 49 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to nonelected inventions.
Claims 46, 48, 51, 52, and 58 are under examination as reading on the elected invention of to a method of improving the predictive value of a biomarker, the biomarker combination of PD-1, CD8, and FoxP3, and the cell species of CD8+FoxP3+ cells.
Priority
Applicant’s claim for the benefit of a prior-filed U.S. Provisional Patent Application No. 63/208,829 filed on June 9, 2021, is acknowledged.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 6/10/2026 and 7/29/2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner in their entireties.
Drawings
The drawings are objected to under 37 CFR 1.83(a) because they fail to show in detail the following (Specification Example 10): “…density of all CD8+FoxP3+ cells had the highest predictive value for a positive response for an individual feature identified on the mIF assay (FIG. 25A)…second analysis was performed to determine pre-treatment features that predicted the degree of pathologic response to therapy (FIG. 25B). The heat map showed some potential cell populations identified by this method and their association with pathologic response values…” as described in the specification. The heat maps disclosed in the drawings require further detail to determine which cell population(s) express or do not express PD-1 in Fig 25A, and which cell population(s) were predicted to have different responses to therapy in Fig 25B. Any structural detail that is essential for a proper understanding of the disclosed invention should be shown in the drawing. MPEP § 608.02(d).
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Specifically, browser-executable code is disclosed in the specification on pg. 37, line 13 and pg. 38, line 31. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
The use of the terms:
VectaMount® (pg. 34, line 28); and
ProLong™ (pg. 35, line 15);
which are trade names or marks used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claims 46 is objected to because the claim recites two instances of “…improving predictive value…” and should most likely recite “…improving the predictive value…” to correct minor typographical errors.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claim 46, 48, 51, 52, and 58 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a law of nature/natural phenomenon without significantly more. The judicial exception is not integrated into a practical application and the claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception for the reasons that follow:
Claims 46, 48, and 51 are directed to methods of improving the predictive value of a biomarker comprising detecting a biomarker in HPF images from a sample, detecting a biomarker in the images/sample, selecting a subset of images, and analyzing the images to improve the predictive value of the biomarker. Claims 52 and 58 are directed to the same methods, wherein the analysis comprises steps to identify cell types in the biological sample based on the presence of a biomarker.
The claims actually are directed to an abstract idea (correlating cell expression of a biomarker with its predictive value), or a law of nature/natural phenomenon (cell expression of a biomarker indicates or “predicts” response to therapy), or both.
Note that the unpatentability of laws of nature was confirmed by the U.S. Supreme Court in Mayo Collaborative Services v. Prometheus Laboratories, Inc., No. 10-1150 (March 20, 2012).
The unpatentability of abstract ideas was confirmed by the U.S. Supreme court in Bilski v. Kappos, No. 08-964, 2010 WL 2555192 (June 28, 2010) and in Alice Corp. v. CLS Bank Int’l, 134 S. Ct. 2347, 2354 (2014).
Applicant’s attention is directed to the USPTO January 7, 2019 Revised Patent Subject Matter Eligibility Guidance (i.e., “PEG”) available at URL: <https://www.govinfo.gov/content/pkg/FR-2019-01-07/pdf/2018-28282.pdf>.
Regarding Step 1 of the PEG, the claims are directed to the statutory category of a process.
Regarding Step 2A, prong one, the claims recite the patent-ineligible concept of an abstract idea/process and a law of nature/natural phenomenon.
The claims encompass performing image acquisition, biomarker detection, and image analysis to improve the predictive value of a biomarker, for example the biomarkers of PD-1, CD8, and FoxP3. Neither the specification nor the claims set for a limiting definition for “improving predictive value”, and the claims do not set forth how this step is accomplished. As broadly recited, one may perform the recited steps of “improving predictive value of the biomarker”, claim 46(d), the step of “identifying a cell type in the biological sample” in claim 52, and/or the step of “detecting expression of the biomarker” in claim 52 mentally. For example, one may identify a cell or cells expressing all of PD-1, CD8, and FoxP3 in a sample by eye and determine that the biomarkers PD-1, CD8, and FoxP3 have an improved value to predict the present of such tri-expressing cells in a sample, which can be performed in the human mind.
Additionally, see MPEP § 2106.04(a)(1)(III)(A): “…claims do recite a mental process when they contain limitations that can practically be performed in the human mind, including for example, observations, evaluations, judgments, and opinions. Examples of claims that recite mental processes include:
a claim to "collecting information, analyzing it, and displaying certain results of the collection and analysis," where the data analysis steps are recited at a high level of generality such that they could practically be performed in the human mind, Electric Power Group v. Alstom, S.A., 830 F.3d 1350, 1353-54, 119 USPQ2d 1739, 1741-42 (Fed. Cir. 2016);…”
Here, the claims can be considered collecting data about cells expression biomarkers, and analyzing it in a way that they could be performed in the human mind, for example, by locating and identifying cells that express the biomarker or biomarkers by eye, thus improving the “predictive value” of that biomarker to identify cells in a sample via biomarker expression.
Additionally, the claims are directed to a natural law/phenomenon. Here the natural phenomenon refers to a correlation between detecting a biomarker or multiple biomarkers in a sample and the biomarker’s “predictive value” for detecting the biomarker(s) in a sample. These limitations set for a judicial exception, because this type of correlation is a consequence of a natural process, similar to a correlation between the presence of a biomarker in a sample and the biomarker’s “predictive value” in determining disease rise. See MPEP § 2106.04(b), part of which reads as follows (emphasis added):
“The courts have identified the following concepts and products as examples of laws of nature or natural phenomena:
i. isolated DNA, Ass’n for Molecular Pathology v. Myriad Genetics, Inc., 569 U.S. 576, 589-91, 106 USPQ2d 1972, 1978-79 (2013);
ii. a cloned farm animal such as a sheep, In re Roslin Institute (Edinburgh), 750 F.3d 1333, 1337, 110 USPQ2d 1668, 1671 (Fed. Cir. 2014);
iii. a correlation between variations in non-coding regions of DNA and allele presence in coding regions of DNA, Genetic Techs. Ltd. v. Merial LLC, 818 F.3d 1369, 1375, 118 USPQ2d 1541, 1545 (Fed. Cir. 2016);
iv. a correlation that is the consequence of how a certain compound is metabolized by the body, Mayo Collaborative Servs. v. Prometheus Labs., 566 U.S. 66, 75-77, 101 USPQ2d 1961, 1967-68 (2012);
v. a correlation between the presence of myeloperoxidase in a bodily sample (such as blood or plasma) and cardiovascular disease risk, Cleveland Clinic Foundation v. True Health Diagnostics, LLC, 859 F.3d 1352, 1361, 123 USPQ2d 1081, 1087 (Fed. Cir. 2017);
vi. electromagnetism to transmit signals, O’Reilly v. Morse, 56 U.S. 62, 113 (1853);
vii. qualities of bacteria such as their ability to create a state of inhibition or non-inhibition in other bacteria, Funk Bros., 333 U.S. at 130, 76 USPQ at 281;
viii. single-stranded DNA fragments known as "primers", University of Utah Research Foundation v. Ambry Genetics Corp., 774 F.3d 755, 761, 113 USPQ2d 1241, 1244 (Fed. Cir. 2014);
ix. the chemical principle underlying the union between fatty elements and water, Tilghman v. Proctor, 102 U.S. 707, 729 (1880);
x. the existence of cell-free fetal DNA (cffDNA) in maternal blood, Ariosa Diagnostics, Inc. v. Sequenom, 788 F.3d 1371, 1373, 115 USPQ2d 1152, 1153 (Fed. Cir. 2015); and
xi. the natural relationship between a patient’s CYP2D6 metabolizer genotype and the risk that the patient will suffer QTc prolongation after administration of a medication called iloperidone, Vanda Pharmaceuticals Inc. v. West-Ward Pharmaceuticals, 887 F.3d 1117, 1135-36, 126 USPQ2d 1266, 1281 (Fed. Cir. 2018).”
Therefore, the claims as currently recited are considered to involve the patent-ineligible concepts of an abstract idea/process and a law of nature/natural phenomenon.
Regarding Step 2A, prong two, having determined that the claims recite a judicial exception, it is then determined whether the claims recite additional elements that integrate the judicial exception into a practical application.
Herein, the claims do not recite additional steps or elements that integrate the recited judicial exceptions into a practical application of the exception(s). The claims only recite the judicial exceptions and broad method steps that can be completed in the human mind, as discussed supra.
Regarding Step 2B, the next question is whether the remaining elements/steps – i.e., the non-patent-ineligible elements/steps – either in isolation or in combination, amount to significantly more than the judicial exception.
Here, the claims as a whole are not considered to recite any additional steps or elements
that amount to significantly more than adding insignificant extra-solution activity to the judicial
exception. Steps (a)-(c) in claim 46 and steps (i)-(iii) in claim 52 are recited at a high level of generality and encompass many methods of image acquisition, biomarker detection, image selection, cell phenotyping, image normalization , and biomarker detection. Furthermore, they are data-gathering steps that feed into the law of nature recited in step (d). The recited steps and elements do not provide the inventive concept necessary to render the claims patent eligible. Also see Mayo, 566 U.S. at 79, 101 USPQ2d at 1968 and MPEP § 2016.05(g).
For the reasons set forth above, when the claims are considered as a whole, the claims are not considered to recite something significantly more than a judicial exception and thereby are not directed to patent eligible subject matter.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 46, 48, 51, 52, and 58 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claims contain subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventors, at the time the application was filed, had possession of the claimed invention.
Claims 46, 48, 51, 52, and 58 comprise methods of improving the predictive value of a generic biomarker (claims 46, 51, 52, and 58), or the biomarkers recited in claim 48 which includes a generic “tumor cell marker”. As currently recited, the claims encompass improving the predictive value of any and all biomarkers, or any and all tumor cell markers, comprising the recited method steps.
However, the specification fails to provide adequate written description support for the recited methods having the desired functional properties required to practice the claimed functions of improving the predictive value of any and all biomarkers (claims 46, 51, 52, and 58), or any and all tumor cell markers (claim 48), comprising the recited method steps.
The claims are not supported by a description that satisfies 35 U.S.C. § 112(a) or 35 U.S.C. § 112, first paragraph. "[T]he test for sufficiency [of the written description] is whether the disclosure of the application relied upon reasonably conveys to those skilled in the art that the inventor had possession of the claimed subject matter as of the filing date." Ariad Phanns., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1351 (Fed. Cir. 2010) (en bane).
A "sufficient description of a genus ... requires the disclosure of either a representative number of species falling within the scope of the genus or structural features common to the members of the genus so that one of skill in the art can 'visualize or recognize' the members of the genus." Id. at 1350. "[A]n adequate written description requires a precise definition, such as by structure, formula, chemical name, physical properties, or other properties, of species falling within the genus sufficient to distinguish the genus from other materials." Id.
"[F]unctional claim language can meet the written description requirement when the art has established a correlation between structure and function." Id. "But merely drawing a fence around the outer limits of a purported genus is not an adequate substitute for describing a variety of materials constituting the genus and showing that one has invented a genus and not just a species." Id.
"A sufficient description of a genus ... requires the disclosure of either a representative number of species falling within the scope of the genus or structural features common to the members of the genus so that one of skill in the art can "visualize or recognize" the members of the genus" (AbbVie, 759 F.3d at 1297, reiterating Eli Lilly, 119 F.3d at 1568-69) (emphasis added).
The specification discloses the AstroPath multiplex IF assay to detect the presence of the PD-1, PD-L1, CD8, FoxP3, CD163, S100/Soxl0 biomarkers to predict patient responses to tumor therapy (Example 1): “[o]nce the index mIF assay (PD-1, PD-L1, CD8, FoxP3, CD163, S100/Soxl0) was optimized, a retrospective analysis was performed on a Discovery cohort of pre-treatment FFPE tumor specimens from 53 patients with metastatic melanoma who went on to receive anti-PD-I-based therapy… Patients were classified as responders (complete response or partial response) or non-responders on the basis of RECIST 1.1 criteria. 5-year overall and progression free survival information was also determined”.
The instant specification further discloses after analysis of cell types based on the multiplex IF assay, there is a correlation between the presence of CD8+, FoxP3+, and PD-1+ tri-expressing cells (Example 9): “…density of PD-1 and PD-L1 expressing cell populations within the HPF tiles were assessed for their predictive value for objective response (determined by the area under the curve (AUC) of a receiver operator characteristic curve). Of those populations studied, the population showing the closest association with a positive response to therapy was the CD8+FoxP3+ cells expressing PD-1…” This association therefore improves the “predictive value” of the combination of these three biomarkers using the disclosed methods.
With respect to representative number of species, see AbbVie Deutschland GmbH & Co. v. Janssen Biotech, Inc. (Fed. Cir. 2014). Also, see MPEP 2163 Il(A)(3)(a))(ii):
A representative number of species means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. A "representative number of species" means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. See Abb Vie Deutschland GmbH & Co., KG v. Janssen Biotech, Inc., 759 F.3d 1285, 1300, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014) (Claims directed to a functionally defined genus of antibodies were not supported by a disclosure that "only describe[d] one type of structurally similar antibodies" that "are not representative of the full variety or scope of the genus.").
Satisfactory disclosure of a "representative number" depends on whether one of skill in the art would recognize that the applicant was in possession of the necessary common attributes or features possessed by the members of the genus in view of the species disclosed. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus. See, e.g., Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406. Instead, the disclosure must adequately reflect the structural diversity of the claimed genus, either through the disclosure of sufficient species that are "representative of the full variety or scope of the genus," or by the establishment of "a reasonable structure-function correlation." Such correlations may be established "by the inventor as described in the specification," or they may be "known in the art at the time of the filing date." See AbbVie, 759 F.3d at 1300-01, 111 USPQ2d 1780, 1790-91 (Fed. Cir. 2014) (Holding that claims to all human antibodies that bind IL-12 with a particular binding affinity rate constant (i.e., koff) were not adequately supported by a specification describing only a single type of human antibody having the claimed features because the disclosed antibody was not representative of other types of antibodies in the claimed genus, as demonstrated by the fact that other disclosed antibodies had different types of heavy and light chains, and shared only a 50% sequence similarity in their variable regions with the disclosed antibodies.).
In the instant case, the application is claiming methods of increasing the predictive value of any and all biomarkers (claims 46, 51, 52, and 58), or any and all tumor cell markers (claim 48), while the instant specification only discloses one example of increasing the predictive value of the combination of the PD-1, FoxP3, and CD8 biomarkers. This one combination of three biomarkers, which includes biomarkers that are not tumor cell markers, does not sufficiently represent the broadly claimed methods of improving the predictive value of the claimed genera of biomarkers or combination of biomarkers.
Moreover, there is insufficient written description of the required kind of structure-identifying information about the corresponding makeup of the claimed methods of improving the predictive value of biomarker(s) to demonstrate possession. Also, see Amgen Inc. v. Sanofi, Aventisub LLC, No. 2017-1480 (Fed. Cir. 2017). The Court reiterated that adequate written description must “contain enough information about the actual makeup of the claimed products . . . .” The Court simultaneously suggested that the “newly characterized antigen” test “flouts” section 112 because it “allows patentees to claim antibodies by describing something that is not the invention, i.e., the antigen.” The Court concluded that for written description of an antibody to be adequate when presented with “functional” terminology, there must be an established correlation in the art between structure and function.
Given the broadly claimed class of antigen binding constructs, and in the absence of sufficient disclosure of relevant identifying characteristics for the broadly claimed class of antigen binding molecules that bind to a ligand, the patentee must establish “a reasonable structure-function correlation” either within the specification or by reference to the knowledge of one skilled in the art with functional claims. AbbVie Deutschland GmbH & Co. v. Janssen Biotech, Inc. (Fed. Cir. 2014), MPEP 2163.
The specification provides no guidance on associating biomarkers with a disease type or outcome, thus improving its “predictive value”, other than the disclosed examples of biomarkers. Neither the instant specification nor the prior art provides sufficient guidance to allow one with ordinary skill in the art to determine which other biomarkers will have their “predictive value” improved by the instantly claimed methods other than the biomarkers and combinations of biomarkers disclosed in the instant specification. Additionally, neither the instant specification nor the prior art provide sufficient guidance to allow one with ordinary skill in the art to determine which biomarker(s) would have their predictive value improved by the claimed methods and which would not. The instant specification does not provide adequate written description support to demonstrate possession of the claimed methods of improving the broadly claimed genera of biomarkers.
Possession is not be shown by merely describing how to obtain possession of members of the claimed genus or how to identify their common structural features. See University of Rochester, 358 F.3d at 927, 69 USPQ2d at 1895. Sufficient description to show possession of such a genus may be achieved by means of a recitation of a representative number of anti-TMPRSS6 antibodies or antigen binding fragments thereof falling within the scope of the genus or of a recitation of structural features common to members of the genus, which features constitute a substantial portion of the genus. See Eli Lilly, 119F.3d at 1568, 43 USPQ2d at 1406.
Claims 46, 48, 51, 52, and 58 do not meet the requirements of 35 U.S.C. 112(a) for written description.
Vas-Cath Inc. v. Mahurkar, 19 USPQ2d 1111, makes clear that "applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the written description inquiry, whatever is now claimed." (See page 1117.) The specification does not "clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed." (See Vas-Cath at page 1116.). Consequently, Applicant was not in possession of the instant claimed invention. See University of California v. Eli Lilly and Co. 43 USPQ2d 1398.
Applicant is invited to point to clear support or specific examples of the claimed invention in the specification as-filed.
To overcome this rejection, it is recommended to amend the claimed to recite the species of biomarkers or combinations of biomarkers disclosed in the instant specification that had their predictive value improved via the claimed methods.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 46, 51, and 52 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Lopes et al. (Lab Invest 100, 120–134 (2020)).
Claim 46 recites a method of improving the predictive value of a biomarker comprising:
obtaining a plurality of HPF images
detecting a biomarker in each image
selecting a sub-plurality of images, and
analyzing the sub-plurality of images to improve the predictive value of the biomarker
Lopes et al. teaches multispectral IHC staining and imaging methods for cancer biomarkers (Abstract): “…(mIHC) combined with multispectral imaging and digital image analysis (DIA) is a quantitative high-resolution method for determination of protein expression in tissue. We applied this method for five biomarkers (CDX2, SOX2, SOX9, E-cadherin, and β-catenin)…”
Lopes et al. teaches this method comprising first staining for biomarkers and imaging colon cancer tissue microarray to generate a plurality of images and detect the biomarkers in the images (pg. 122-125): “…three 3-plex stains (for analysis of SOX9, β catenin, and E-cadherin), one 4-plex stain (for analysis of CDX2 and SOX2), and one 5-plex stain (for analysis of CDX2 and two unpublished markers) were performed in the study (see also Table 2 for a list of all stains and Table S1 for an overview of the staining procedure for each multiplex stain and included biomarkers). Tissue samples were incubated for 30 min with the following primary antibodies: CDX2 (1:50, clone 88, Abcam, UK; detected by Opal 520 at 1:100), SOX2 (1:25, clone SP76, Cell Marque/Sigma Aldrich, Germany; detected by Opal 570 at 1:100), SOX9 (1:500, Sigma-Aldrich; detected by Opal 570 at 1:100), E cadherin (1:16000, clone 36, Becton Dickinson, USA; detected by Opal 570 at 1:100), and β catenin (1:3000, clone 14, Becton Dickinson; detected by Opal 570 at 1:100)… Multispectral images were acquired at ×20 magnification using the Vectra 3.0 Automated Quantitative Pathology Imaging System, 200 slides (Vectra software version 3, PerkinElmer/Akoya). Standard settings were used for multispectral image acquisition…”
Lopes et al. teaches this is followed by image analysis to remove images of technical artifacts that could skew image analysis and measurements, i.e., “selecting a sub-plurality of images” (pg. 125): “…images were reviewed after batch processing; normal glands, necrotic tissue, tissue folds, and other technical artefacts were excluded from further image analyses…”
Lopes et al. teaches analysis of the sub-plurality of the images, including tissue microarrays stained for CDX2, and improves the predictive value of CDX2 in determining colorectal cancer prognosis (pg. 130): “…mIHC combined with DIA is an efficient approach to assess the prognostic value of CDX2 protein expression, highlighting the potential clinical utility of this technology to assess nuclear and cytoplasmic markers in a more standardized fashion…”
Therefore, Lopes et al. teaches a method of biomarker image analysis to improve its predictive value, comprising obtaining a plurality of HPF images, detecting a biomarker CDX2 in each image, selecting a sub-plurality of images, and analyzing the sub-plurality of images to improve the predictive value of the biomarker, anticipating the limitations of instant claim 46.
Regarding instant claim 51, Lopes et al. teaches per immunofluorescence microscopy (Fig. 4), anticipating the claim limitations:
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Regarding instant claim 52, Lopes et al. teaches that tumor microarray slides were imaged with an automated imaging system (pg. 124): “…images were acquired at ×20 magnification using the Vectra 3.0 Automated Quantitative Pathology Imaging System, 200 slides (Vectra software version 3, PerkinElmer/Akoya). Standard settings were used for multispectral image acquisition…” The images for the microarrays were compiled into a single image of the entire slide/sample, which is displayed in Fig. S2A:
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Lopez et al. further teaches image processing further comprises segmenting the cells in the tissue into tumor and non-tumor tissue (pg. 124-125): “…a machine learning algorithm was trained by user-specified tissue annotations aided by the signal from the epithelial markers to accurately segment tumor tissue versus stromal tissue and background, as well as individual cells using the nuclear DAPI signal…”, followed by cell phenotyping by detecting the presence of biomarkers in the cells (pg. 125): “…protein expression levels and patterns of CDX2, SOX2, SOX9, E-cadherin, and β-catenin were evaluated visually by single plex chromogenic-based (DAB) IHC and by fluorescence-based mIHC. Both staining methods showed that all markers were expressed predominantly in epithelial cells (all but SOX2 were also expressed both in the normal mucosa and the cancer cells); CDX2, SOX2, and SOX9 were expressed predominantly in the cell nuclei…” Therefore, Lopez et al. teaches the limitations recited in instant claim 52.
The reference teachings anticipate the instantly claimed invention.
Claim Rejections - 35 USC § 103
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.
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.
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 46, 48, and 51 are rejected under 35 U.S.C. 103 as being unpatentable over Heeren et al. (J Immunother Cancer. 2019 Feb 12;7(1):43. doi: 10.1186/s40425-019-0526-z).
Claim 46 recites a method of improving the predictive value of a biomarker comprising:
obtaining a plurality of HPF images
detecting a biomarker in each image
selecting a sub-plurality of images, and
analyzing the sub-plurality of images to improve the predictive value of the biomarker
Heeren et al. teaches methods of identifying different T-cell subtypes in a sample via flow cytometry and multiplex immunofluorescence (Abstract): “[t]o identify therapeutic targets for local immune modulation, multi-parameter flow cytometric T-cell profiling of primary cervical tumors…as well as multiplexed immunofluorescence histochemistry staining…”
Heeren et al. teaches the multiplex immunofluorescence comprises staining sections for different biomarkers including CD8, FoxP3, and PD-1 (Multiplexed immunohistochemistry): “[m]ultiplexed immunofluorescence staining was performed on LN+ (n = 4) and PT (n = 4) samples…slides were incubated with primary antibody diluted in Normal Antibody Diluent o/n at RT (anti-PD-1), for 30 min at RT (anti-CD8, anti-CD3, and anti-GrB), and for 60 min at RT (anti-FoxP3) and 30 rounds per minute (rpm) on a shaker. Primary antibodies were separately added and incubated in the following order: anti-PD-1, anti-CD8, anti-CD3, anti-FoxP3, and anti-GrB…slides were washed 3x in 1x TBST and incubated with Opal fluorochromes diluted in amplification buffer (all provided by the OPAL 7-color fIHC kit…”
Heeren et al. teaches that the mIF comprises collection of a plurality of images of a HFP from the samples via a slidescanner with a 3x3 tile scan at 40x (Imaging and quantification): “…multiplex immunofluorescence stainings were visualized with Leica TCS SP8 microscope (Leica), tile-scan (3 × 3, 40x oil objective with 1.3 NA) images were generated and viewed using LAS AF Lite software…” Heeren et al. teaches that the images were divided into pub-pluralities based on whether the images were intratumoral or peritumoral areas (Statistical analysis): “…paired t-test (for normally distributed data) or the Wilcoxon test was used to compare frequencies of CD8+FoxP3+ vs. CD8+FoxP3− populations and to compare T cell subset frequencies in intratumoral vs. peritumoral areas. Correlations were determined by the Pearson r test. Data were analyzed using Prism 7 Software. P-values below 0.05 were considered statistically significant.”
Heeren et al. teaches that this multiplex immunofluorescence allows for identification of PD-1 expression CD8+/FoxP3+ T-cells (Fig. 5, pg. 8): “…sub-phenotyping showed low percentages of GrB-expressing CD8+FoxP3+PD-1+ T cells, indicative of a functionally suppressed state of this subset in vivo…”
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Heeren et al. does not teach the analysis improves the predictive values of the CD8, FoxP3, and PD-1 biomarkers, however Heeren et al. suggests that T-cells expressing the biomarkers can be used as a biomarker for adaptive immunity in a tumor site (Discussion): “…CD8+FoxP3+ T cells were PD-1-positive and had an activated non-exhausted phenotype, with a high expression of other immune checkpoint molecules. Of note, the CD8+FoxP3+ T-cell subset was the only subset able to produce IFNγ…these cells may represent a valuable biomarker for monitoring ongoing adaptive immunity in the tumor. Our finding of this subset being present in LN+ with detectable HPV16 E6 reactive T cells, but not in LN-, in which we failed to detect any such responses, is consistent with this suggestion…these proliferating “early effector cells” could be tumor-reactive effector cells in melanoma in the earliest stage after priming…high expression of the immune checkpoints on CD8+FoxP3+ T cells might also indicate that this is a more differentiated antigen-experienced population”
It would have been obvious to one with ordinary skill in the art, before the effective filing date of the instant application, to have modified the imaging/analysis method taught by Heeren et al. to improve the predictive value of the CD8, FoxP3, and PD-1 biomarkers in predicting the immune response to a tumor (i.e., the limitations of instant claim 46 and 48), for example by identifying the presence of T-cells expressing all three biomarkers to help predict how a patient’s immune system is reacting to a tumor. One would have a reasonable expectation of success, as Heeren et al. teaches this imaging and analysis method and suggests that these biomarkers can be used to predict immune responses to a tumor, for example by aiding in identifying if the tumor microenvironment is immune suppressive or if it contains tumor-reactive effector cells. One would have been motivated to make this change for the purposes of identifying new biomarkers or combinations of biomarkers that can be used to predict a patient’s response to a tumor.
Regarding instant claim 51, Heeren et al. teaches immunofluorescent microscopy to image the sample (Multiplexed immunohistochemistry section), meeting the claim limitations.
Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the time the invention was made, as evidenced by the references, especially in the absence of evidence to the contrary.
Claims 52 and 58 are rejected under 35 U.S.C. 103 as being unpatentable over Heeren et al. (supra), as applied to claims 46, 48, and 51 above, and further in view of Blom et al. (Sci Rep. 2017 Nov 14;7(1):15580. doi: 10.1038/s41598-017-15798-4).
The teachings of Heeren et al. are discussed supra. Heeren et al. does not teach whole-sample image acquisition followed by cell segmentation and phenotyping by identifying cells that express the PD-1, FoxP3, and CD8 biomarkers.
Blom et al., in the same field of endeavor, teaches a multiplex IF/IHC protocol to allow for staining and detection of dix protein markers and nuclei (Abstract).
Blom et al. further teaches (Introduction): “…we applied the mIHC method for the whole-slide analysis of immune cells (Fig. 1) and prostate epithelial cells (Fig. 2) in human prostate cancer (PCa) samples. The method utilizes a combination of fluorescent and chromogenic tissue staining for the simultaneous detection of six proteins and nuclei in formalin-fixed tissue section…”
Blom et al. further teaches the entire sample is imaged using a slide scanner, which imaged the entire sample at 20x magnification and stitched the images into a composition multichannel image of the whole sample (Imaging, Fig, 2):
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Blom et al further teaches the image analysis comprises cell segmentation of phenotyping based on biomarker expression (Fig. 2, Immune cell classification and analysis): “…classification of immune cells in prostate cancer epithelium and stroma, we detected cells by nuclear staining, and identified leukocytes and epithelial cells using markers for pan-leukocyte (CD45) and pan-epithelium (Pan-Epi = pan-cytokeratin + E-cadherin). We classified the CD45+ leukocytes as stromal or epithelial depending on their location within the tissue segments, and further classified each leukocyte as T regulatory (CD4+FoxP3+CD8−), T helper (CD4+FoxP3−CD8−), or T effector (CD8+CD4−FoxP3−) cell…” Blom et al. teaches that the fluorescent image normalization for each biomarker (Image processing, cell segmentation and classification): “…mean intensity values per object area (pixels), and were normalized…”
Blom et al. teaches the benefits of such a whole-sample mIHC imaging and analysis method (Discussion): “…there is a need for open-source systems and methods that would allow high-resolution whole-slide multiplexed readouts. For instance, it has been shown that analysis of small regions of interest causes significant variation and error in the assessment of molecular markers in breast cancer…we present an mIHC method for the whole-slide analysis of immune cells and epithelial cells in human prostate cancer combining fluorescent and chromogenic detection of six proteins and nuclei in the same tissue section. As the system is designed for a fixed set of secondary reagents and unlabelled primary antibodies, introducing new primary antibodies is rapid allowing flexible design of custom mIHC target panels. The presented mIHC system enables not only a multiplexed analysis but also an open-source high-resolution digital image analysis across entire tissue sections…”
It would have been obvious to one with ordinary skill in the art, before the effective filing date of the instant application, to have modified the image acquisition method of Heeren et al. in view of Blom et al. to add whole sample imaging and cell segmentation/phenotyping of the biomarkers of Heeren et al. (i.e., the limitations of instant claim 52) with a reasonable expectation of success, as Blom et al. teaches such a method that can be applied to the method taught by Heeren et al. One would have been motivated to make this change to image the whole tumor samples of Heeren et al. to remove potential variation/error that can arise from imaging and analyzing only a small number of regions, as taught by Blom et al.
Regarding the limitations of instant claim 58, Heeren et al. teaches identification of PD-1 expressing CD8+FoxP3+ cells (Fig. 5, pg. 8): “…sub-phenotyping showed low percentages of GrB-expressing CD8+FoxP3+PD-1+ T cells, indicative of a functionally suppressed state of this subset in vivo…”, meeting the claim limitations.
Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the time the invention was made, as evidenced by the references, especially in the absence of evidence to the contrary.
Conclusion
No claim is allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure:
Berry (John Hopkins Dissertation: MULTISPECTRAL IMMUNOFLUORESCENCE FOR IMMUNO-ONCOLOGY BIOMARKER DISCOVERY: MAPPING CO-EXPRESSION AND INTENSITY ON WHOLE-SLIDES USING THE ‘ASTROPATH’ PLATFORM, jhir.library.jhu.edu/handle/1774.2/62536) teaches the instantly claimed invention and was uploaded on 3/30/2020, however this is not considered prior art because the dissertation was under a publication embargo until 5/01/2024.
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/ALEC JON PETERS/Examiner, Art Unit 1641
/MISOOK YU/Supervisory Patent Examiner, Art Unit 1641
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