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 .
Drawings
The drawings were received on 3/18/2024. These drawings are accepted.
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.
Claim(s) 1-4 and 7-22 is/are rejected under 35 U.S.C. 103 as being unpatentable over Prunicki et al (Air pollution exposure is linked with methylation of immunoregulatory genes, altered immune cell profiles, and increased blood pressure in children," Scientific Reports, Volume 11, Number 1, Article 4067, February 18, 2021, 12 pages) in view of Hillson et al (US 2011/0117590 A1) (both provided by applicant in IDS dated 3/18/2024).
With respect to claim 1 Prunicki teaches a method for detecting an amount of one or more heavy metals in a biological sample (an effect of one or more pollutants on a biological sample), the method comprising:
(a) contacting the biological sample with one or more antibodies conjugated to a heavy metal (See Pg. 3, Para. 6 - 'Cell type measurement on unstimulated PBMCs was performed using a high-throughput, time-of-fight mass cytometry approach, CyTOF, to characterize immune cell subsets at a single-cell level. CyTOF is based on inductively coupled plasma mass spectrometry and time of fight mass spectrometry used to identify properties of cells using heavy metal ion tags rather than fluorochromes. See Supplemental Table S2 for the CyTOF panel. Premade MAXPJ\R antibodies against the immune cell markers of interest were purchased through Fluidigm Corporation (South San Francisco, CA, USA). PBMCs were stained for surface markers using validated, published methods'; Note, Supplemental Table S2 (See Pg. 3, Supplementary Information) shows the marker antibody and associated metal tag used for CyTOF); and
(b) detecting the amount of heavy metals conjugated to the one or more antibodies (See Pg.3, Para. 6 - 'Cell type measurement on unstimulated PBMCs was performed using a high-throughput. time-of-fight mass cytometry approach, CyTOF, to characterize immune cell subsets at a single-cell level. CyTOF is based on inductively coupled plasma mass spectrometry and time-of-flight mass spectrometry used to identify properties of cells using heavy metal ion tags rather than fluorochromes. See Supplemental Table S2 for the CyTOF panel. Pre made MAXPAR antibodies against the immune ce!I markers of interest were purchased through Fluidigm Corporation (South San Francisco, CA, USA). PBMCs were stained for surface markers using validated, published methods'; Note, Supplemental Table S2 (See Pg. 3, Supplementary Information) shows the marker antibody and associated metal tag used for CyTOF; See Pg. 4, Para. 2 - 'Using JMP Genomics v6.0, we applied partial least squares (PLS) to identify patterns of average air pollution exposures linked with methylation of immunoregulatory genes, immune cell typology and BP levels.'; See Pg. 6, final paragraph for discussion of - 'In multivanate PLS, recent exposure to PM2.5, CO, 03, and PAH456 were main predictors of tile immune cell profiles, particularly of Th1, Th2, and Th17 cell percentages (See Table 3; Supplemental Fig. S4). PLS explained only 3.5% of the variance in the immune cell types, likely due to the heterogeneous outcomes. Indeed, when relaunching PLS with only Th1, Th2, Th17 and T regulatory cells as outcomes, the same key predictors were identified (recent exposure to PM2.5, CO, 03. CO and PAH456) but with much higher variance of outcome explained (9%).').
Prunicki fails to teach detecting an amount of one or more heavy metals in a biological sample.
Hillson teaches detecting an amount of one or more heavy metals in a sample (See Para. 0020 - 'Compositions and methods are provided for the biological detection of heavy metals with a biosensor organism, comprising at least one heavy metal responsive element, which comprises a polynucleotide sequence encoding a fluorescent reporter protein operably linked to a promoter sequence that is active in presence of these heavy metals. and inactive in its absence.'; See Para. 0090 - 'In the work presented here, we have constructed a whole cell uranium biosensor that can report the presence of micro molar amounts of the uranyl cation in-situ with nothing other than a handheld UV lamp.').
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 Prunicki to detect heavy metals present in a sample, as taught by Hillson, as long as the metal ions were distinct ( i.e., using distinct metal ions to discriminate between antibody label and a pollutant or analyte heavy metal contaminating the biological sample) in order to detect impact of heavy metal ions on immune cells of the subject from whom the biological sample is derived (See Paras. 0020 and 0090 of Hillson).
With respect to claim 2 the combination of Prunicki and Hillson teaches that the heavy metal conjugated to the one or more antibodies is different (See Pg. 3, Para. 6 of Prunicki- 'Cell type measurement on unstimulated PBMCs was pe1formed using a higi1-throughput, time-of-fight mass cytometry approach, CyTOF, to characterize immune cell subsets at a single-cell level. CyTOF is based on inductively coupled plasma mass spectrometry and time-of-flight mass spectrometry used to identify properties of cells using heavy metal ion tags rather than fluorochromes. See Supplemental Table S2 for the CyTOF panel. Premade MAXPAR antibodies against the immune cell markers of interest were purchased through Fluidigm Corporation (South San Francisco, CA. USA). PBMCs were stained for surface markers using validated, published methods'; Note, Supplemental Table S2 (See Pg. 3, Supplementary Information) shows the marker antibody and associated metal tag used for CyTOF) than the one or more heavy metals detected as taught by Hillson (See Para. 0090 of Hillson - 'In the work presented here, we have constructed a whole cell uranium biosensor that can report the presence of micro molar amounts of the uranyl cation in situ with nothing other than a handheld UV lamp.') in the biological sample.
With respect to claim 3 the combination of Prunicki and Hillson teaches the one or more heavy metals are detected by mass cytometry, cytometry by time-of-flight (CyTOF), inductively coupled plasma-mass spectrometry (1 CPMS). inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS) or epigenetic landscape profiling using cytometry by Time -of-flight (EpiTOF) (See Pg. 3, Para. 6 of Prunicki - 'Cell type measurement on unstimulated PBMCs was performed using a high-throughput. time-of-fight mass cytometry approach, CyTOF, to characterize immune cell subsets at a single-cell level. CyTOF is based on inductively coupled plasma mass spectrometry and time of fight mass spectrometry used to identify properties of cells using heavy metal ion tags rather than fluorochromes. See Supplemental Table S2 for the CyTOF panel. Premade MAXPAR antibodies against the immune cell markers of interest were purchased through Fluidigm Corporation (South San Francisco, CA, USA). PBMCs were stained for surface markers using validated, published methods'; Note, Supplemental Table S2 (See Pg. 3, Supplementary Information) shows the marker antibody and associated metal tag used for CyTOF).
With respect to claim 4 the combination of Prunicki and Hillson teaches the one or more heavy metals comprise one or more isotopes in the range of 75-209 atomic mass units (AMU) (See Supplemental Table S2 of Prunicki (Pg. 3, Supplementary Information) shows the marker antibody and associated metal lag used for CyTOF in the range of 141-176 atomic mass units (AMU)).
With respect to claim 7 the combination of Prunicki and Hillson teaches the one or more antibodies are conjugated to a heavy metal isotope in the range of 75-209 atomic mass units (AMU) (See Supplemental Table S2 of Prunicki (See Pg. 3, Supplementary Information) shows the marker antibody and associated metal lag used for CyTOF in the range of 141-176 atomic mass units (AMU)).
With respect to claim 8 the combination of Prunicki and Hillson teaches the antibody is conjugated to a heavy metal isotope, specifically 146Nd (See Supplemental Table S2 of Prunicki (See Pg. 3, Supplementary Information) shows the marker antibody and associated metal lag used for CyTOF in the range of 141-176 atomic mass units (AMU) including 146Nd).
With respect to claim 9 the combination of Prunicki and Hillson teaches that the antibody specifically binds to CD3 and CD14 (See Supplemental Table S2 of Prunicki (Pg. 3, Supplementary Information) shows the marker antibody and associated metal tag used for CyTOF, including antibodies to CD3 and CD 14).
With respect to claim 10 the combination of Prunicki and Hillson teaches that the biological sample comprises a cell or tissue (See Pg. 3, Para. 6 of Prunicki - 'Cell type measurement on unstimulated PBMCs was performed using a high-throughput, time-of-flight mass cytometry approach, CyTOF, to characterize immune cell subsets at a single--ceil level.').
With respect to claim 11 the combination of Prunicki and Hillson teaches that the cell is selected from the group consisting of an immune cell, a peripheral blood mononuclear cell (PBMC), a leukocyte. a dendritic cell, and combinations thereof (See Pg. 3, Para. 6 of Prunicki- 'Cell type measurement on unstimulated PBMCs was performed using a high-throughput, time-of-fight mass cytometry approach, CyTOF, to characterize immune cell subsets at a single-cell level.').
With respect to claim 12 the combination of Prunicki and Hillson teaches that the leukocyte is selected from the group consisting of a 6 cell, a T cell, a myeloid cell, a granulocyte, a natural killer cell, and combinations thereof (See Pg. 3, Para. 6 of Prunicki - 'Cell type measurement on unstimulated PBMCs was performed using a high-throughput, time-of-flight mass cytometry approach, CyTOF, to characterize immune cell subsets at a single--cell level'; Supplemental Table S2 (Pg. 3, Supplementary Information) shows the marker antibody and associated metal tag used for CyTOF including antibodies to CD3 and CD14'; Note, CD3 is a T cell marker).
With respect to claim 13 the combination of Prunicki and Hillson teaches that the T cell is selected from the group consisting of a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell (Treg), a natural killer T cell, and combinations thereof (See Pg. 6, final Para. in Prunicki - 'In multivariate PLS, recent exposure to PM2.5, CO, 03, and PAH456 were main predictors of the immune ceil profiles, particularly of Th1, Th2, and Th17 cell percentages (See Table 3; Supplemental Fig. 84)').
With respect to claim 14 the combination of Prunicki and Hillson teaches that the helper T cell is a subtype selected from the group consisting of Th1, Th2, Th9, Th17, Tfh, and combinations thereof (See Pg. 6, final Para. in Prunicki - 'In multivariate PLS, recent exposure to PM2.5, CO, 03, and PAH456 were main predictors of the immune ceil profiles, particularly of Th1, Th2, and Th17 cell percentages (See Table 3; Supplemental Fig. 84)').
With respect to claim 15 the combination of Prunicki and Hillson teaches that the cell is a single cell (See Pg. 3, Para. 6 of Prunicki - 'Cell type measurement on unstimulated PBMCs was performed using a high-throughput time-of-flight mass cytometry approach, CyTOF, to characterize immune ceil subsets at a single-cell level.').
With respect to claim 16 the combination of Prunicki and Hillson teaches that the cell is a living cell (See Pg. 3, Para. 6 of Prunicki - 'Computational approaches were utilized on bead normalized data files gated for live singlets using the t-SNE algorithm to analyze and visualize the multidimensional data in two dimensions as unsupervised clustering (viSNE. Cytobank). Supplemental Fig. S2 illustrates the CyTOF workflow from PBMCs through data analysis.').
With respect to claim 17 the combination of Prunicki and Hillson teaches that the cell is a dead cell, a cryopreserved cell, a fixed cell. or obtained from cadaveric tissue (See Pg. 3, Para. 4 of Prunicki- 'Blood samples were shipped overnight to Stanford University and peripheral blood mononuclear cells (PBMCs) and plasma were extracted from blood samples via Ficoll procedure; PBMCs were stored in liquid nitrogen and plasma stored at -- 80 °C, per published techniques18').
With respect to claim 18 the combination of Prunicki and Hillson teaches that the cell or tissue is selected from an animal, a mammal, a human, a plant, a single cell eukaryotic organism, or a bacterium (See Abstract of Prunicki - 'We therefore determined the associations between exposure to multiple air pollutants and both immunological outcomes (methylation and protein expression of immune cell types associated with immune regulation) and cardiovascular outcomes (blood pressure) in a cohort of school aged children (6-8 years. N=221) living in a city with known elevated pollution levels').
With respect to claim 19, the combination of Prunicki and Hillson does not expressly teach that the heavy metal is detected on the cell surface. However, Prunicki teaches detecting markers of a cell surface (See Pg. 3, Para. 6 – ‘PBMCs were stained for surface markers using validated, published methods22-24. Briefly, cryopreserved PBMCs were thawed and incubated in RPMI 1640 media (ThermoFisher) supplemented with ·10% FBS at 37 "C. Unstimulated PBMCs were stained for surface markers using validated, published methods'). Thus, it would have been obvious to one of ordinary skill in the art that the method of combined Prunicki and Hillson could be used wherein heavy metal detected is on the cell surface.
With respect to claim 20, the combination of Prunicki and Hillson does not expressly teach that the heavy metal detected is intracellular. However, Prunicki teaches detecting markers of cell surfaces (See Pg. 3, Para. 6 – ‘PBMCs were stained for surface markers using validated, published methods22-24. Briefly, cryopreserved PBMCs were thawed and incubated in RPMI 1640 media (ThermoFisher) supplemented with ·10% FBS at 37 "C. Unstimulated PBMCs were stained for surface markers using validated, published methods'). Adaptation of cell surface staining methods for intracellular staining by adding fixing and permeabilization steps is well known in the art, and as such, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to adapt the method of combined Prunicki and Hillson such that the heavy metal detected is intracellular.
With respect to claim 21 the combination of Prunicki and Hillson teaches that the biological sample is obtained from a subject exposed to heavy metals (See Para. 0018 of Hillson for discussion of the study of galactosidase activity after two hours of heavy metal exposure).
With respect to claim 22 the combination of Prunicki and Hillson teaches that the biological sample is obtained from a subject exposed lo air pollution (See Pg. 2, Para. 5 - 'From 2015 to 2016, eligible children (6 to 8 years) attending school in Fresno, California, visited our University of California San Francisco-Fresno clinical site, where we obtained a detailed health and demographics questionnaire, BPs, and blood samples. Over the 1-year study period, we measured air pollutants using a combination of continuous daily pollutant concentrations measured at central air monitoring stations in Fresno, daily concentrations from periodic spatial sampling, and meteorological and geophysical data. Average air pollution exposures were estimated for 1 day, 1 week and 1, 3, 6, and ·12 months prior to each participant visit. Using high-dimensional mass cytometry (CyTOF), we determined immune cell markers from unstimulated participant peripheral blood mononuclear cells (PBMCs). Details of pollution and cell measures are described below.').
Claim(s) 5 and 6 is/are rejected under 35 U.S.C. 103 as being unpatentable over Prunicki et al (Air pollution exposure is linked with methylation of immunoregulatory genes, altered immune cell profiles, and increased blood pressure in children," Scientific Reports, Volume 11, Number 1, Article 4067, February 18, 2021, 12 pages) and Hilson et al (US 2011/0117590 A1) in view of Maecker et al (US 2017/0059574 A1) (provided by applicant in IDS dated 3/18/2024).
Refer above for the combined teachings of Prunicki and Hillson.
With respect to claim 5 the combination of Prunicki and Hillson fails to teach that the one or more heavy metals are selected from the group consisting of arsenic, selenium, bromide, rhodium, cadmium, antimony, barium, tantalum, tungsten, osmium, platinum, mercury, lead, and combinations thereof.
Maecker teaches platinum labeled antibodies for analyte detection (See Abstract - 'The present disclosure provides analyte-specific binding reagents conjugated with a platinum-containing moiety, e.g., cisplatin, and methods, compositions, and kits for their production and use in assays for analyte detection.'), specifically, the use of platinum labeled antibodies for staining T cells (See Para. 0023 - 'FIG. 1, Panels A-O. Production and validation of cisplatin-antibody conjugates for mass cytometry. Numbers indicated percentages (Panel C) Anti-CD8 Ab was labeled with natural abundance cisplatin (Pt*) using indicated amounts of cisplatin in the labeling reaction').
As such, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to incorporate the use of the platinum labeled antibody of Maecker into method of combined Prunicki and Hillson, in light of the advantages of using platinum as a label compared to other heavy metals as taught by Maecker (See Para. 0003 of Maecker - 'Some channels are not suited for reporting of specific probes due to the overlap with the detection of traces of xenon in argon (AM 128-I 32, 134, 136) or barium (AM 134-138), given that it is an unwanted yet frequent metal contaminant. The use of some elements such as Pb, Hg and Tl appears limited due to their toxicity for cells and eventually users.').
With respect to claim 6, the combination of Prunicki and Hillson fails to teach that the one or more isotopes are selected from the group consisting of 75As, 78Se, 80Se, 79Br, 81Br, 103Rh, 106Cd, 108Cd, 110Cd, 111Cd, 112Cd, 114Cd, 116Cd, 121Sb, 123Sb, 138Ba, 180Ta, 181Ta, 182W, 184W, 186W, 187Os, 188Os, 189Os, 194Pt, 195Pt, 196Hg, 198Hg, 199Hg, 200Hg, 201Hg, 202Hg, 204Pb, 206Pb, 207Pb, 208Pb, and combinations thereof.
Maecker teaches the use of 194Pt labeled antibody for analyte detection (See Abstract - 'The present disclosure provides analyte-specific binding reagents conjugated with a platinum-containing moiety, e.g., cisplatin, and methods, compositions, and kits for their production and use in assays for analyte detection’; See Fig. 2 and Para. 0024 for discussion and depiction of Panels A-C. Cisplatin-Ab conjugates compare to lanthanide loaded polymer Ab conjugates. (Panel A) PBMC were labeled with CD4-Pt194 and CD8-Pt198 as well as several lineage markers used to distinguish T cells.'), in addition to the use of platinum labeled antibodies for staining T cells (See Para. 0023 - 'FIG. 1, Panels A-D. Production and validation of cisplatin-antibody conjugates for mass cytometry…Numbers indicated percentages (Panel C) Anti-CD8 Ab was labeled with natural abundance cisplatin (Pt*) using indicated amounts of cisplatin in tile labeling reaction').
Thus, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to incorporate the platinum labeled antibody of Maecker into the method of combined Prunicki and Hillson, considering the advantages of using platinum as a label compared to other heavy metals (See Para. 0003 of Maecker - 'Some channels are not suited for reporting of specific probes due to the overlap with the detection of traces of xenon in argon (AM 128-I 32, 134, 136) or barium (AM 134-138), given that it is an unwanted yet frequent metal contaminant. The use of some elements such as Pb, Hg and Tl appears limited due to their toxicity for cells and eventually users.').
Conclusion
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/BRITTANY I FISHER/Examiner, Art Unit 1796 July 11, 2026
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