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 .
Office Action: Notice
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 3/8/2026 has been entered.
Claim Status
Claims 74, 77, 81, 90, and 94-95 have been amended (3/8/2026). Claims 77, 91 and 96 are cancelled (3/8/2026). Claims 97-109 are new (3/8/2026). No new matter was added. Thus, claims 74, 81, 89-90, 94-95, and 97-109 are under examination.
Priority
Claims 74, 81, 89-90, 94-95, and 97-109 are given a priority date of 3/4/2019, the filing date of US Provisional 62/813,235.
Objections Withdrawn
Claims:
The objections to correct minor clerical issues to claims 74 and 94 are withdrawn due to Applicant’s amendments of the instant claims.
New Objections
Claims 89 and 90 are objected due to the following informalities:
Claims 89 and 90 are objected to because they improperly depend from subsequent claim 95. Under 37 CFR 1.75 (c), a dependent claim should refer to a preceding claim. Appropriate correction is required.
Rejections Withdrawn
Claim Rejections - 35 USC § 101
The rejection of claims 74-88, 90 and 92-93 under 35 U.S.C. 101 is withdrawn in view of Applicant’s significant amendments of independent claim 74 and not extended to resultant new independent claims 103 and 108 as a result of the Applicant’s incorporation of these amendments similarly. Specifically, the instant claim now recites a concrete laboratory protocol; obtaining blood, extracting white blood cells, isolating CD4 T cells, activating them with anti-CD3/anti-CD28 (or PMA/ConA), staining for a defined plurality of cytotoxic-CD4 biomarkers (i.e., EOMES, GZMB, perforin) pre-and post-activation, and determining a subject’s CD4 T-cell profile from measured levels. These limitations apply any correlation between CD4-subset distributions and immune status to a particular technological environment (flow-cytometry immunophenotyping) using specific sample manipulations and reagents, rather than merely claiming the correlation or a mental evaluation. Under Step 2A, Prong Two of the 2019 PEG (see MPEP 2106.04 (d)), the claim integrates any recited judicial exception into a practical application; therefore, the claim is not “directed to” the exception because eligibility is established at Step 2A Prong Two, a Step 2A analysis is not required.
Claim Rejections - 35 USC § 103
The rejection of claims 74, 77, 81, and 89-91 and 94-96 as being unpatentable over unpatentable over Alonso-Arias et al. (“NKG2D expression in CD4+ T lymphocytes as a marker of senescence in the aged immune system”, Age (Dordr), published 1/2011, from IDS 1/4/2022) and in view of Regev et al. (WO 2017/075478 A2, published 4/5/2017), is withdrawn in view of Applicant’s cancellation of claims 77, 91 and 96 as well as Applicant’s amendments of independent claim 74 and resultant dependent claims that now recite a more specific CD4 T-cell population defined by co-expression of particular biomarkers, including EOMES, granzyme B, and perforin. Upon further consideration of the Applicant’s argument (3/8/2026), the prior rejection did not sufficiently establish that one of ordinary skill would have a reason to select the claimed combination of biomarkers with a reasonable expectation of success for detecting the claimed biomarker-defined CD4 T-cell population.
New Rejections
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 74, 80, 89-90, 94-95, 97-102 and 103-107 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.
Claim 74 is rejected. Claim 74 recites the limitation "the percentage” at step (e), line 1. There is insufficient antecedent basis for this limitation in the claim.
Claim 74 is further rejected. Claim 74 recites the limitation "the binding” at step (e), line 2. There is insufficient antecedent basis for this limitation in the claim.
Claims 80, 89-90, 94-95, 97-102 are included in this rejection due to their dependency on claim 74.
Claim 97 is further rejected. Claim 97 recites the limitation "the levels” at step (h), line 1. There is insufficient antecedent basis for this limitation in the claim.
Claim 103 is rejected. Claim 103 recites the limitation "the percentage” at step (e), line 1. There is insufficient antecedent basis for this limitation in the claim.
Claims 104-107 are included in this rejection due to their dependency on claim 103.
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 text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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 74, 81, 89-90, 94-95, and 97-109 are rejected under 35 U.S.C. 103 as being unpatentable over Alonso-Arias et al. (“NKG2D expression in CD4+ T lymphocytes as a marker of senescence in the aged immune system”, Age (Dordr), published 1/2011, from IDS 1/4/2022) and in view of Regev et al. (WO 2017/075478 A2, published 4/5/2017) and further in view of Dervovic (“Requirements for Notch Signaling in Positive Selection and Effector Function of CD8 T Cells”, A thesis for the degree of Doctor of Philosophy at the Department of Immunology for the University of Toronto”, published 2012).
Regarding claims 74, 81, 89-90, 94-95, and 97-109 Alonso-Arias teaches that human aging is characterized by changes in the immune system which have a profound impact on the T-cell compartment, including a CD4+ subset, characterized by the aberrant expression of NKG2D in CD4+ T cells and can be used to evaluate age-related expression of NKG2D in CD4+ T cells between elderly and young adults (Abstract). Alonso-Arias further teaches that elderly donors (i.e., minimum age of 55 for the young group, and the range of the elderly group from 68-105 years) display a marked increase in the proportion of highly differentiated effector and memory T cells due to a lifetime of exposure to a variety of pathogens versus thymic involution which implies a decreased output of naïve T cells, which is evident in peripheral blood and the lymph nodes (Introduction: Paragraph 1; Study Population: Paragraph 1). Specifically, Alonso-Arias teaches that in the elderly, a significant proportion of T cells loses the expression of costimulatory molecules and acquires inhibitory receptors (Introduction: Paragraph 1). Additionally, Alonso-Arias teaches that CD4+ NKG2D+ subset, a type of antibody as a biomarker subset, was clearly related to the status of the T cells via higher frequencies of the NKG2D+ subset were accompanied with a gradual decrease of naive and central memory cells, but also with a higher level of more differentiated subsets of CD4+ T cells showcasing that CD4+ NKG2D+ represent a subset of highly differentiated T cells which characterizes the senescence of the immune system (Abstract). Further, Alonso-Arias teaches that the human immune system progressively deteriorates with age, which leads to a greater incidence or reactivation of infectious diseases, as well as to the development of autoimmune disorders and cancer (Introduction: Paragraph 1).
Further, Alonso-Arias teaches that human aging is characterized by changes in the immune system which have a profound impact on the T-cell compartment, including a CD4+ subset, characterized by the aberrant expression of NKG2D in CD4+ T cells and can be used to evaluate age-related expression of NKG2D in CD4+ T cells between elderly and young adults (Abstract).
Alonso-Arias does not teach or suggest detecting a level of pluralities of biomarkers to identify the presence of cytotoxic, activated regulatory (aTreg), naïve_Ig15, exhausted or rTregs CD4 T cells. Further, Alonso-Arias does not teach the presence of cytotoxic or exhausted cells above 5% indicating a mature immunological age without expression of CD8 following isolation.
Alonso-Arias does not teach or suggest that the previously mentioned plurality of biomarkers is selected from; CD137, CD134, FOX3P+, GITR+, Helios+, CD74, HLA-DR for identification of an aTreg CD4 T cells; or Nkg, Runx3, Eomes, Gzmk, IFN-b, IL-27, IL-21, IL-17A, Ccl3, Ccl4, Ccl5 for identification of a cytotoxic CD4 T cells; or Cd200, Lag3, Hifla, Nfatc1, Pdcd1 for the identification of exhausted CD4 T cells, or that these T cells are further incubated with antibodies capable of detecting a plurality of biomarkers comprising EOMES, GzmB, perforin, FOXP3, CD44, CD81, PD1 and CD62L.
Alonso-Arias also does not teach or suggest incubating CD4 T cells with antibodies capable of detecting at least one biomarker selected from IL-10 and TGF-beta, followed by determination of higher biomarker levels prior to cell activation.
Alonso-Arias also does not teach the use of a kit for the determination of immunological age via detecting a level of a plurality of biomarkers to identify the presence of cytotoxic, activated regulatory (aTreg), naïve_Ig15, exhausted or rTregs CD4 T cells, and further administering a subject a specific therapeutic based on the identified immunological age of the immune system, comprising a siRNA, microRNA, or a CD7, CD134, CD137 or GITR antibody. Specifically, Alonso-Arias does not teach or suggest that the previously mentioned therapeutic is an age-related frailty comprising a statin or aspirin-based combination.
Additionally, Alonso-Arias does not or suggest that the isolation of the CD4 T cells is accomplished via fluorescence.
Regarding claims 74, 81, 89-90, 94-95, and 97-109, Regev teaches a method of evaluating dysfunction of immune cells, marker signatures and molecular targets (Abstract). Regev also teaches that even dysfunctional T cells can exist in various functional states; and a biological sample is analyzed via a plethora of different CD8 or CD4 T cell dysfunctional states, regulated by multiple molecular pathways, and present across different diseases and during different stages of disease progression, distinctively contributing to immune control (Paragraph 10, lines 1-5; Paragraph 18, lines 10-15). Further, Regev teaches that the plurality of biomarkers or plethora of dysfunctional T cells include cytotoxic and activated regulatory (aTreg) CD4 T cells (Paragraph 119, lines 1-10; Paragraph 122, lines 1-4). Regev also teaches that the plurality of biomarkers or plethora of dysfunctional states includes gene modules that are uniquely associated with the dysfunctional T cell state and activated T cell state, and key molecular nodes that control them, providing present markers, marker signatures and molecular targets that provide for new ways to evaluate and modulate immune responses, such as to specifically evaluate and target the dysfunctional T cell state while leaving T cell activation programs intact (Paragraph 17, lines 1-10). Also, Regev teaches the analysis of expression profiles of single-cells within a population of cells from isolated samples (i.e., blood samples), thus allowing the discovery of novel cell subtypes or cell states (Paragraph 82, lines 5-10). Additionally, Regev teaches that differentially expressed genes across the Tim-3 / PD-1 defined subpopulations define a dysfunctional signature in CD8, TILs, and presents a heatmap of specified genes determined as differentially expressed across the TILs subpopulations with cells from spleens of non-tumor-bearing Balb/c mice, Efflv1enr Effector memory CD8+ CD62L10wCD44hi cells extracted from non-tumor bearing Balb/c mice, DN: CD8+Tim3-PD-1", SP: CD8+Tim3-PD-l ..,_, DP: CD8-'-Tim3+PD-l ..,_ TILs) (Figures 1 and 2; Paragraph 54, lines 5-10), and further analyzed via the expression of MTl and MT2 as determined by qPCR in sorted CD8 TILs isolated from mice bearing CT26 colon carcinoma and B 16 melanoma tumors (Figure 2A; Paragraph 55, lines 1-5). Regev also teaches that in one preferred embodiment, T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3x28)-conjugated beads, or XCYTE DYNABEADS for a time period sufficient for positive selection of the desired T cells where the time period is about 30 minutes (Paragraph 96, lines 5-10). Regev teaches that furthermore depletion of exhausted T cells by targeting known regulators such as Tbet and Eomes resulted in high viral load suggesting that dysfunctional T cells provide partially effective immune control (Paragraph 8, lines 10-15).
Regev teaches that Alonso-Arias’ previously described method of evaluating immunological age includes various T-cell subsets; such as, exhausted/dysfunctional immune cells, such as T cells, that produce reduced amounts of IFN-gamma, TNF-alpha and/or one or more immunostimulatory cytokines, such as IL-2, compared to functional immune cells (Paragraph 152, lines 1-20). Further, Regev teaches that other types of T cell subsets include; naïve (Paragraph 502, lines 20-25), effector memory (TEM) (Figure 1A-1J; Paragraph 54, lines 1-10), and regulatory (rTregs) cells (Paragraph 119, lines 1-10). Further, Regev teaches that percentage of polyfunctional T cells, in specific populations, consistent with tumor growth, could be evaluated (Figure 3A; Paragraph 20, lines 10-15).
Regev also teaches that Alonso-Arias’ previously described method of evaluating immunological age includes the evaluation of dysfunctional or exhausted T-cells (i.e., CD4), where the cell does not perform its usual function or activity in response to normal input signals, and includes refractivity of immune cells to stimulation, such as stimulation via an activating receptor or a cytokine (i.e., cytotoxic) (Paragraph 152, lines 1-5). Further, Regev teaches that exhausted/dysfunctional or cytotoxic T cells do not respond adequately to stimulation, and display poor effector function (i.e., mature), sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells and prevents optimal control of infection and tumors, thereby contributing to local immunosuppression (Paragraph 152, lines 10-15). Specifically, Regev teaches that the presence of an exhausted or cytotoxic state (i.e., >1% T dysfunctional T cell population) unresponsive immune cells can reduce cytokine production by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% in cytotoxic activity, cytokine production, proliferation, trafficking, phagocytotic activity, or any combination thereof, relative to a corresponding control immune cell of the same type (Figure 4D; Paragraph 152, lines 10-15; Paragraph 749, lines 5-10).
Regev teaches that that the previously described method of evaluating immunological age includes an enzyme-linked immunosorbent assay, or ELISA, used to measure the differential expression of a plurality of signature genes based on the immobilization of an antigen or antibody on a solid surface, generally a microtiter plate and comprises or consists of three or more genes or biomarkers (Paragraph 83, lines 1-5; Paragraph 110, lines 1-10).
Regev teaches that the isolated immune cells used for Alonso-Arias’ previously described method of evaluating immunological age, such as a specialized T cell (i.e., regulatory), may be further modified to comprise an agent or biomarker capable of altering expression or activity; including, CD137, CD134, FOX3P+, GITR+, Helios+ and CD74 (Figure 5A-5H; Paragraph 58, lines 15-20; Paragraph 166, lines 10-15; Paragraph 179, lines 1-5; Paragraph 279, lines 1-10; Paragraph 838, lines 1-5).
Regev teaches that the isolated immune cells used for Alonso-Arias’ previously described method of evaluating immunological age, such as a specialized T cell (i.e., cytotoxic), may be further modified to comprise an agent or biomarker capable of altering expression or activity; including, Nkg7, IFN-b, IL-21, IL-17A, Ccl3, Ccl4 and Ccl5 subsets (Paragraph 152, lines 10-15; Paragraph 279, lines 1-10; Paragraph 707, lines 1-30).
Regev teaches that the isolated immune cells used for Alonso-Arias’ previously described method of evaluating immunological age, such as a specialized T cell (i.e., exhausted), may be further modified to comprise an agent or biomarker capable of altering expression or activity; including, Lag3, Pdcd1, and Cd200 (Paragraph 18, lines 15-20; Paragraph 21, lines 1-10).
Regev teaches a kit comprising means for detection of the signature or level of dysfunction, activation, activation and/or dysfunction, or memory (Paragraph 23, lines 1-5), via the plurality of biomarkers or plethora of dysfunctional T cells include cytotoxic and activated regulatory (aTreg) CD4 T cells (Paragraph 119, lines 1-10; Paragraph 122, lines 1-4). Regev also teaches that the plurality of biomarkers or plethora of dysfunctional states includes gene modules that are uniquely associated with the dysfunctional T cell state and activated T cell state, and key molecular nodes that control them, providing present markers, marker signatures and molecular targets that provide for new ways to evaluate and modulate immune responses, such as to specifically evaluate and target the dysfunctional T cell state while leaving T cell activation programs intact (Paragraph 17, lines 1-10).
Regev teaches a method of treatment for an age-related condition, disease or disorder where an enhanced immune response is required, such as but not limited to a cancer, or a condition, disease or disorder where a decreased immune response is required, such as but not limited to an autoimmune disease where the immune cell may be modified, such that expression of a gene signature is altered (Paragraph 47, lines 1-8). Regev further teaches that the condition is treated via the plurality of biomarkers or plethora of dysfunctional T cells include cytotoxic and activated regulatory (aTreg) CD4 T cells (Paragraph 119, lines 1-10; Paragraph 122, lines 1-4). Regev also teaches that the plurality of biomarkers or plethora of dysfunctional states includes gene modules that are uniquely associated with the dysfunctional T cell state and activated T cell state, and key molecular nodes that control them, providing present markers, marker signatures and molecular targets that provide for new ways to evaluate and modulate immune responses, such as to specifically evaluate and target the dysfunctional T cell state while leaving T cell activation programs intact (Paragraph 17, lines 1-10).
Regev teaches that the previously described method of evaluating age-based therapies includes various T-cell subsets; such as, exhausted/dysfunctional immune cells, such as T cells, that produce reduced amounts of IFN-gamma, TNF-alpha and/or one or more immunostimulatory cytokines, such as IL-2, compared to functional immune cells (Paragraph 152, lines 1-20). Further, Regev teaches that other types of T cell subsets include; naïve (Paragraph 502, lines 20-25), effector memory (TEM) (Figure 1A-1J; Paragraph 54, lines 1-10), and regulatory (rTregs) cells (Paragraph 119, lines 1-10).
Regev teaches that the previously described method of evaluating age-based therapies includes the evaluation of dysfunctional or exhausted T-cells (i.e., CD4), where the cell does not perform its usual function or activity in response to normal input signals, and includes refractivity of immune cells to stimulation, such as stimulation via an activating receptor or a cytokine (i.e., cytotoxic) (Paragraph 152, lines 1-5). Further, Regev teaches that
exhausted/dysfunctional or cytotoxic T cells do not respond adequately to stimulation, and display poor effector function (i.e., mature), sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells and prevents optimal control of infection and tumors, thereby contributing to local immunosuppression (Paragraph 152, lines 10-15). Specifically, Regev teaches that the presence of an exhausted or cytotoxic state (i.e., >1% T dysfunctional T cell population) unresponsive immune cells can reduce cytokine production by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% in cytotoxic activity, cytokine production, proliferation, trafficking, phagocytotic activity, or any combination thereof, relative to a corresponding control immune cell of the same type (Figure 4D; Paragraph 152, lines 10-15; Paragraph 749, lines 5-10).
Regev teaches that the previously described method of evaluating age-based therapies includes a combination of a chemical entity or biological product is preferably, but not necessarily a low molecular weight compound, but may also be a larger compound, or any organic or inorganic molecule effective in the given situation, including modified and unmodified nucleic acids such as antisense nucleic acids, RNAi, such as siRNA or shRNA, , microRNA (mRNAi), CRISPR-Cas systems, peptides, peptidomimetics, receptors, ligands, and antibodies, aptamers, polypeptides, nucleic acid analogues (Paragraph 134, lines 1-5). Regev also teaches that the detection of signature genes may involve a cell sorting step to enrich for cells of interest and thus facilitate or enhance their sensitive and specific detection based on tagging the cell with an antibody (i.e., CD7, CD134, GTIR) against the cell membrane antigen specific to the target subpopulation of cells and the antibody is conjugated to a magnetic bead and/or fluorophore or other label to enable cell sorting and detection (Paragraph 112, lines 1-5; Paragraph 166, lines 5-10; Paragraph 179, lines 1-5; Paragraph 279, lines 1-10).
Regev teaches that the previously described method of evaluating age-based therapies includes a specific application, neuro and muscular degenerative diseases develop due to abnormal gene expression, including those based on age and gender (i.e., anti-apoptotic, anti-inflammatory and anti-degenerative drugs including small drugs and macromolecules may also optionally be therapeutic (Paragraph 458, lines 1-5).
Regev teaches that the age-based therapies include RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector that expresses angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the web form of age-related macular degeneration is also contemplated (Paragraph 373, lines 1-5).
Additionally, Regev teaches that Alonso-Arias’ previously described method of evaluating immunological age includes various T-cell subsets; such as, exhausted/dysfunctional immune cells, such as T cells, that produce reduced amounts of IFN-gamma, TNF-alpha and/or one or more immunostimulatory cytokines, such as IL-2, compared to functional immune cells (Paragraph 152, lines 1-20). Further, Regev teaches that other types of T cell subsets include; naïve (Paragraph 502, lines 20-25), effector memory (TEM) (Figure 1A-1J; Paragraph 54, lines 1-10), and regulatory (rTregs) cells (Paragraph 119, lines 1-10). Further, Regev teaches that percentage of polyfunctional T cells, in specific populations, consistent with tumor growth, could be evaluated (Figure 3A; Paragraph 20, lines 10-15).
Regev also teaches that Alonso-Arias’ previously described method of evaluating immunological age includes the evaluation of dysfunctional or exhausted T-cells (i.e., CD4), where the cell does not perform its usual function or activity in response to normal input signals, and includes refractivity of immune cells to stimulation, such as stimulation via an activating receptor or a cytokine (i.e., cytotoxic) (Paragraph 152, lines 1-5). Further, Regev teaches that exhausted/dysfunctional or cytotoxic T cells do not respond adequately to stimulation, and display poor effector function (i.e., mature), sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells and prevents optimal control of infection and tumors, thereby contributing to local immunosuppression (Paragraph 152, lines 10-15).
Regev teaches that the previously described method of evaluating age-based therapies includes a combination of a chemical entity or biological product is preferably, but not necessarily a low molecular weight compound, but may also be a larger compound, or any organic or inorganic molecule effective in the given situation, including modified and unmodified nucleic acids such as antisense nucleic acids, RNAi, such as siRNA or shRNA, , microRNA (mRNAi), CRISPR-Cas systems, peptides, peptidomimetics, receptors, ligands, and antibodies, aptamers, polypeptides, nucleic acid analogues (Paragraph 134, lines 1-5). Regev also teaches that the detection of signature genes may involve a cell sorting step to enrich for cells of interest and thus facilitate or enhance their sensitive and specific detection based on tagging the cell with an antibody (i.e., CD7, CD134, GTIR) against the cell membrane antigen specific to the target subpopulation of cells and the antibody is conjugated to a magnetic bead and/or fluorophore or other label to enable cell sorting and detection (Paragraph 112, lines 1-5; Paragraph 166, lines 5-10; Paragraph 179, lines 1-5; Paragraph 279, lines 1-10). Regev also teaches that in one preferred embodiment, T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3x28)-conjugated beads, or XCYTE DYNABEADS for a time period sufficient for positive selection of the desired T cells where the time period is about 30 minutes (Paragraph 96, lines 5-10). Regev teaches that furthermore depletion of exhausted T cells by targeting known regulators such as Tbet and Eomes resulted in high viral load suggesting that dysfunctional T cells provide partially effective immune control (Paragraph 8, lines 10-15).
Further, Regev teaches that exhausted/dysfunctional or cytotoxic T cells do not respond adequately to stimulation, and display poor effector function (i.e., mature), sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells and prevents optimal control of infection and tumors, thereby contributing to local immunosuppression (Paragraph 152, lines 10-15). Specifically, Regev teaches that the presence of an exhausted or cytotoxic state (i.e., >1% T dysfunctional T cell population) unresponsive immune cells can reduce cytokine production by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% in cytotoxic activity, cytokine production, proliferation, trafficking, phagocytotic activity, or any combination thereof, relative to a corresponding control immune cell of the same type (Figure 4D; Paragraph 152, lines 10-15; Paragraph 749, lines 5-10).
Dervovic teaches that the evidence that the signals that direct CD4 or CD8 lineage differentiation operate at the CD4+CD8lo stage was highlighted above in the cell suspension section and as pointed out, Brugnera et al. demonstrated that CD4+ CD8lo T cells undergo coreceptor reversal as they give rise to CD832 lineage cells, which is facilitated by IL7R signaling and cessation of TCR signaling (91) and further support showing that IL7R signaling is critical for CD8-lineage commitment came from studies utilizing FTOCs and specifically, FTOCs isolated from B6 mice, which contained DP but not SP thymocytes, were cultured with or without blocking antibodies to the IL7R complex (IL7Rα and γc), and pronase treatment was employed to identify effects on coreceptor expression (p. 31). Dervovic further teaches that while DP thymocytes differentiated into both CD4 and CD8 SP T cells when cultured in media alone, they failed to differentiate into CD8 SP cells when cultured with IL7R blocking antibodies and since antibodies were used against both IL7Rα and γc (common chain for IL2, IL4, IL7, IL9 and IL21 cytokines), the authors concluded that the CD8 lineage commitment was uniquely dependent upon γc-dependent cytokine receptor signaling rather than IL7R
signaling alone and this conclusion was complemented with studies by Yu et al., in which FTOCs from WT and Bcl2-Tg mice were cultured with blocking antibodies against the IL7R complex (IL7Rα and γc) (92) (p. 32). Dervovic teaches that as described above, IL7R blocking antibodies inhibited CD8 differentiation in WT FTOCs, however this treatment had no effect on CD8 SP development in Bcl2-Tg FTOCs and these results indicated that the ectopic expression of Bcl2 was sufficient to overcome the loss of IL7R signaling in CD8 SP differentiation where together, these findings revealed that cytokine-induced signals play a critical role at the transitional CD4+CD8lo stage to support and enable CD8 lineage commitment (p. 31-32).
Dervovic also teaches that a subsequent study by the same group demonstrated that T cell commitment requires signals transduced by TCR/CD3 complex or TCRζ chains of sufficient intensity to upregulateCD5 expression (89) and as a result, an asymmetric model of selection was proposed in which TCR/CD3 or TCRζ signals direct the transition from CD5lo DP to CD5hi DP thymocytes to CD4+CD8- committed cells, while additional lineage specific signals are needed for final CD4- CD8+ Tcell commitment to occur. Indeed, Cibotti et al. illustrated that coupling of TCR signaling with different ‘coinducer’ molecules (CD2, CD5, CD1, CD28, CD49d, CD81 and TSA-1) resulted in selective transition from CD4+ CD8+ through CD4lo CD8lo to CD4+ CD8- T cells in vitro (90) and given the likely prominent role of thymic epithelium in CD4 T cell commitment, it was surprising that a default pathway for CD4 T cell development could be induced in the absence of additional lineage-specific signals (p. 17-18).
Additionally, Dervovic teaches that expression of activated/memory phenotype by CD8 T cells generated in vitro and further differentiation of effector to memory CD8 T cells, plays an important role in secondary responses against viral and tumor challenges and hence, in vitro-derived CD8 T cells were compared to ex vivo CD8 T cells for the expression of known activation/memory markers after anti-CD3/CD28 stimulation followed by IL15 treatment and found that majority of activation/memory CD8 T cell markers (upregulation of CD11a, CD27, CD44, CD69 and downregulation of CD62L) were maintained throughout the period of 10 days (3 days of stimulation followed by treatment with IL15 for 7 days) (Fig. 12A). Dervovic also teaches that however, one of the differences observed was upregulation of CD279 (PD1) marker by in vitro-derived CD8 T cells (Fig. 12A) and as of note, upregulation of this marker has been associated with exhaustion of Agspecific CD8 T cells in people with chronic infections (272), but, we wanted to test if Notch signaling may perhaps regulate expression of PD1 (p. 86).
Also, Dervovic teaches that although conventional CD4 and CD8 T cells arise from the same DP precursor and utilize the same TCR-induced signaling pathways, recent studies identified a requirement for TEC kinases, Itk (interleukin-2 (IL2)-inducible T-cell kinase) and Rlk (resting lymphocyte kinase), as independent signaling pathway implicated in the development of conventional CD8 T cells (135, 165, 239, 301), where mice deficient in Itk or Itk and Rlk fail to develop conventional CD8 T cells, and instead support the development of CD8 T cells that have an innate-like phenotype (CD44hi, CD122hi, IL15-dependance), and resemble T cells selected by non-classical MHC-Ibmolecules. Later findings revealed that these non-conventional CD8 T cells from Itk-/- or Itk-/-Rlk-/- deficient mice are selected by classical MHC-Ia molecules expressed on hematopoietic cells in the thymus (135, 241, 247, 267), where additional non-conventional lineages selected by classical pMHC-II expressing hematopoietic cells comprise of natural forkhead box P3 (FOXP3)+ CD4+CD25+ regulatory T (Treg), and innate-like CD4+ T - cells as seen in mice expressing exogenous MHC-II activator transcription factor (CIITA) in thymocytes and in humans expressing endogenous MHC-II on immature thymocytes (59, 60, 302) (p. 108). Dervovic conversely teaches that other non-conventional lineages that originate from DP precursors and acquire innate-like characteristics include cells with TCRs specific for ligands presented by non-classical MHC-Ib molecules expressed on hematopoietic cells in the thymus (p. 108).
Additionally, Dervovic teaches that CD8+ TCRβhi cells were sorted from day 35 HSC/OP9-DL1 cocultures initiated with BM-derived HSCs isolated from SAP-/-, Itk-/-Rlk-/- and B6 mice. A, F) Flow cytometry analysis of CD4, CD8, and TCRβ expression from day 35 cocultures. B, G) Flow cytometry analysis of the expression of CD44, CD122,and β7 integrin on gated CD8 T cell population is illustrated by black- and corresponding isotype Abs by
gray –lines. C, H, I, E, J) IFN-g, IL17, and Gzm-B production from anti-CD3/CD28 stimulated vs. unstimulated culture-derived CD8+ TCRβhi cells (SAP-/-, Itk-/-Rlk-/- or WT) were detected by ELISA(IGN-g, IL17) or FACS analysis (GzmB), respectively; where data are representative of at least 3 independent experiments D) Proliferation of SAP-/- or WT (B6) in vitro-derived CD8+TCRβhi cells with or without stimulation with plate-bound anti-CD3/CD28 antibodies was measured by 3H-thymidine incorporation assay and represent average values and error bars represent standard deviation (p. 114, Figure 18).
Also, Dervovic teaches that the differentiation from BM-HSCs to naïve T cells in the thymus and subsequently to effector/memory T cells in the periphery is regulated at multiple stages and it is well established that Notch signaling plays a key role in regulating T versus B cell lineage commitment, αβ vs. γδ T cell lineage commitment, specification, progression and survival throughout DN (DN1 through DN4) stages of T cell development, TCR β gene rearrangement, and potentially CD4/CD8 lineage commitment, where in the periphery, ligation of different Notch ligands (Dll1 or Jagged 1) with Notch receptors (Notch2 or Notch1) have been implicated to influence differentiation of CD4 T cells to type 1 helper (Th1) - versus type 2 helper (Th2) - cells upon activation (p. 157). Dervovic also teaches that correspondingly,
Notch signaling has been suggested to promote activation and proliferation of peripheral CD4
and CD8 T cells and in parallel, Notch-induced activation might be involved in suppression of
TGFβ-induced CD4+CD25+ Foxp3+ T-regulatory cells (T-regs), and thus maintenance of the
peripheral immune homeostasis where similarly, Notch signaling appears to influence polarization of CD4 T helper IL17-producing (Th17) cells, with a direct role in regulating the Th17-lineage specific transcription factor ROR-γt (p. 157). Dervovic teaches that in addition, there is increasing evidence that Notch signaling might govern effector function (IFN-γ, GranzymeB, perforin) of CD8 T cells through either eomes-dependent or eomes-independent regulation and thus, Notch signaling imparts different functions at each stage of CD4/CD8 T cell development in the thymus as well as in the periphery (p. 157).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Alonso-Arias’ method of evaluating age-related immune changes to include detecting a level of multiple biomarkers to identify various CD4 T cell subsets (i.e., cytotoxic, aTreg, naïve, exhausted), using specific percentage thresholds to determine immunological age, and further administering therapeutics based on the identified immunological age profile to treat age-related frailties as taught by Regev. One would have been motivated to make these modifications because Alonso-Arias already establishes the relationship between aging and changes in T cell populations, teaching that these changes lead to increased susceptibility to diseases including infectious diseases, autoimmune disorders, and cancer. Regev provides the specific methodology to quantify these age-related immune changes through biomarker detection and percentage thresholds, as well as the therapeutic approach to address the resulting age-related conditions. The combination would provide a more comprehensive approach to not only evaluate immunological age with greater precision, but also to treat the associated age-related conditions.
There would have been a reasonable expectation of success because both references deal with evaluating T cell populations and their relationship to immune system status and age-related conditions. Alonso-Arias establishes the correlation between T cell subset distributions and aging, while Regev provides detailed methodology for detection and quantification of these cell populations, as well as therapeutic applications based on the findings. Further, the technical approaches in both references are compatible, involving analysis of T cell subsets and their functional characteristics. Combining the age-related immune evaluation of Alonso-Arias with the specific detection methods, quantitative thresholds, and therapeutic applications of Regev would predictably result in a more comprehensive method to evaluate and tread age-related immune dysfunction.
Further, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the combined teachings of Alonos-Arias and Regev to include the teachings of Dervovic regarding differentiation, activation, exhaustion, and lineage commitment of CD4/CD8 T-cell populations, including EOMES-associated cytotoxic T-cell function, PD1 expression, granzyme B production, perforin-mediated effector function, and CD4/cD8 lineage characterization. One would have been motivated to combine Dervovic with Alonso-Arias and Regev because Alonso-Arias teaches age-associated alterations in CD4+ T-cell populations and immune senescence, while Regev teaches multiplex biomarker profiling and characterization of dysfunctional, cytotoxic, activated and exhausted immune-ell states using molecular signatures. Dervovic further teaches that T-cell lineage commitment, activation state, exhaustion markers, cytotoxic function, and EOMES-dependent signaling pathways are associated with expression of biomarkers including granzyme B, perforin, PD1, FOXP3, and relate immune regulatory markers in differentiated T-cell subsets. Specifically, it would have been obvious to one of ordinary skill in the art to utilize IL-10 as one of the detected immune biomarkers because Dervovic teaches characterization of differentiated, activated, regulatory and exhausted T-cell states using cytokine-associated immune profiling, and IL-10 was a known immunoregulatory cytokine conventionally associated with dysfunctional, suppressive and exhausted immune-cell phenotypes within multiplex T-cell analyses.
The combination would have predictably resulted in methods for identifying and characterizing specific CD4 T-cell subpopulations using known biomarker combinations because all three references are directed toward immune-cell phenotyping and functional characterization of T-cell subsets using known analytical and flow cytometric techniques. Further, Dervovic expressly teaches that cytotoxic T-cell effector functions may proceed through EOMES-dependent pathways and those markers such as PD1, granzyme B, perforin, FOXP3, and activation/memory markers are associated with differentiated and exhausted T-cell states, thereby providing additional rationale to employ such markers within the multiplex immune profiling framework taught by Regev and immune-aging taught by Alonso-Arias.
Thus, one of ordinary skill in the art would have had a reasonable expectation of success because the cited references employ compatible immune phenotyping methodologies, including isolation of CD4/CD8 populations, flow cytometric biomarker detection, cytokine and cytotoxin marker analysis, and characterization of differentiated or exhausted T-cell states using known immune markers and conventional laboratory techniques.
Applicant’s Response: The Applicant argues that Alonso-Arias does not teach detecting a plurality of biomarkers for identifying CD4 T-cell subsets or using percentage thresholds to determine immunological age. The Applicant further argues that Regev only broadly discloses dysfunctional T-cell profiling and does not teach the claimed specific biomarker combinations, isolation scheme or therapeutic limitations.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered and were found to be partially persuasive, as discussed below.
Specifically, the Applicant’s argument regarding the absence of the claimed biomarker combinations in Alonso-Arias and Regev were persuasive in part. However, the arguments are not persuasive as the rejection has been modified using Dervovic. Dervovic teaches characterization of differentiated and exhausted T-cell populations using biomarkers including EOMES, granzyme B, perforin, PD1, and cytokine-associated immune profiling. Further, the Applicant’s argument regarding the alleged absence of a quadrupole positive population is unpersuasive because the combined references teach multiplex biomarker profiling of T-cell subsets, rendering the claimed biomarker-defined profiling methods obvious under the broadest reasonable interpretation.
Conclusion
No claim is allowed.
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/ELIZABETH ROSE LAFAVE/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
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