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 .
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 07/10/2025 has been entered.
Election/Restrictions
Applicant’s election of Group I (i.e., claims 1-22 and 27 drawn to a method of profiling the energetic metabolism profile of a population of cells) in the reply filed on June 12th 2024, is acknowledged. Additionally, Applicant’s election of Species A (i.e., Single and specific method of profiling the energetic metabolism profile of a population of cells; 1. Single and specific type of cell and whether the population of cells is homogenous or heterogeneous. Applicants’ Election: immune cells and heterogeneous; 2. Single and specific inhibitor A, and whether acetate or pyruvate are present when contacting the sample with inhibitor a, Applicants’ Election: 2-Deoxy-D-Glucose as inhibitor A, and acetate or pyruvate are not present when contacting the sample; 3. Single and specific inhibitor B, and whether inhibitor B is an inhibitor of wild type or mutant enzyme, Applicants’ Election: Oligomycin as inhibitor B. This functions as an inhibitor of wild or mutated enzyme of mitochondrial metabolism pathways; 4. Single and specific inhibitor C, and if inhibitor C is present, a single and specific inhibitor, Applicants’ Election: Trimetazidine/TMZ as inhibitor C, and inhibitor C is optional; 5. Single and specific inhibitor D, and if inhibitor D is present, as a single and specific inhibitor, Applicants’ Election: Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide/BPTES as inhibitor D, and inhibitor D is optional; 6. Single and specific detectable label, Applicants’ Election: Fluorescent label as detectable label; 7. Single and specific quantification method to assess the protein synthesis level, Applicants’ Election: cytometry as a specific quantification method) in the reply filed on June 12th 2024, is acknowledged. Because Applicants did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claim 6 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, and claims 25-26 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Note: Species A (i.e., single and specific inhibitor A) has been expanded.
Status of Claims
Claims 1-26 were originally filed and amended on October 14th 2021. The amendment cancelled claims 23-24, amended claims 1, 3-4, 6, 10-15, 17-22, 26, and added new claim 27.
The amendment filed on December 19th 2024, amended claims 1-5, 7-18, 21-22 and 27; and cancelled claims 6 and 25-26.
The amendment filed on July 10th 2025, amended claim 1; and cancelled claim 18.
Claims 1-5, 7-17, 19-22 and 27 are currently pending and under consideration.
Priority
The present application claims status as a 371 (National Stage) of PCT/EP2020/060486 filed April 14th 2020, and claims priority under 119(a)-(d) to European Application No. 19305485.5 filed on April 15th 2019.
Receipt is acknowledged of papers submitted under 35 U.S.C. 119(a)-(d) for European Application No. 19305485.5, which papers have been placed of record in the file. Please note that the European application is in English and therefore no further action is needed.
Claim Interpretation
For purposes of applying prior art, the claim scope has been interpreted as set forth below per the guidance set forth at MPEP § 2111. If Applicant disputes any interpretation set forth below, Applicant is invited to unambiguously identify any alleged misinterpretations or specialized definitions in the subsequent response to the instant action. Applicant is advised that a specialized definition should be properly supported and specifically identified (see, e.g., MPEP § 2111.01(IV), describing how Applicant may act as their own lexicographer).
For claim 1, regarding the scope of “LCo”, it is noted that the specification does not define what constitutes “LCo,” and “LCo” is not a term known in the art. Pursuant to MPEP 2111.01, under a broadest reasonable interpretation, words of the claim must be given their plain meaning, unless such meaning is inconsistent with the specification. The plain meaning of a term means the ordinary and customary meaning given to the term by those of ordinary skill in the art at the time of the invention. The specification makes several recitations of the phrase “measuring the protein synthesis level” when referring to the inhibitors A or B and recites “protein synthesis level LA” or “protein synthesis level LB” or “protein synthesis level L(A+B)” (see specification, pg. 3, lines 24, 27-28 and 30). As such, the Examiner is interpreting the scope of “LCo” as the protein synthesis level of the control sample (i.e., S1).
Regarding the scope of “calculating,” it is noted that the instant specification does not define what constitutes “calculating.” Pursuant to MPEP 2111.01, under a broadest reasonable interpretation, words of the claim must be given their plain meaning, unless such meaning is inconsistent with the specification. The plain meaning of a term means the ordinary and customary meaning given to the term by those of ordinary skill in the art at the time of the invention. The Meriam Webster Dictionary defines “calculating” as to determine by mathematical processes (see Merriam Webster Dictionary, “calculating” available online at https://www.merriam-webster.com/dictionary/calculate, accessed on 09/24/2025). As such, the Examiner is interpreting the scope of “calculating” as to performing a mathematical process to determine the amount of something.
Regarding the scope of “monoclonal antibodies specific for puromycin,” it is noted that the instant specification does not define what constitutes “monoclonal antibodies specific for puromycin.” Instead the instant specification recites that the sample is contacted with an amount of puromycin and then after with an amount of monoclonal antibodies specific for puromycin that are typically conjugated with a detectable label (see instant specification, pg. 11, lines 13-15), but it fails to teach the specific complementarity-determining regions of the antibodies specific for puromycin.
Although the instant specification fails to teach any examples of monoclonal antibodies specific to puromycin, the prior art identifies a representative number of monoclonal antibodies specific to puromycin that exhibit the function of monitoring protein synthesis. Schmidt et al. report the use of monoclonal antibodies to puromycin to directly monitor translation using standard immunochemical methods (See Schmidt et al., Nature Methods. Vol. 6 No. 4 (2009), pp. 275-277 at pg. 275, left column, 1st paragraph). Schmidt et al., detected puromycin incorporation by immunoblotting with the 12D10 monoclonal antibody to puromycin (see Schmidt et al., pg. 275, left column, 2nd paragraph). Similarly, Kelleher et al., teach Anti-Puromycin [3RH11] Antibody, a monoclonal antibody to puromycin which provides a non-radioactive method to measure rates of global protein synthesis (mRNA translation) in cells or tissue slices incubated with puromycin, or animals treated with puromycin in vivo (see Kelleher et al., Puromycin Incorporation as a Measure of Global Protein Synthesis, 2013, pp. 1-10, retrieved from https://news.kerafast.com/2013/09/27/puromycin-incorporation-as-a-measure-of-global-protein-synthesis/ on 09/28/2024). Furthermore, Fisher Scientific teaches Anti-Puromycin antibody, clone 4G11 which detects puromycin incorporated into protein (see Fisher Scientific, Catalog No. MABE342MI, pp. 1-5, retrieved from https://www.fishersci.com/shop/products/anti-puromycin-clone-4g11-emd-millipore/MABE342MI on 09/28/2024). As such, given the pre-existing knowledge in the art demonstrating a representative number of species that fall within the claimed genus of monoclonal antibodies specific to puromycin that would exhibit the function of determining protein synthesis levels, an ordinary skilled artisan would have put one in possession of the genus of a monoclonal antibody specific to puromycin. Thus, an ordinary skilled artisan would conclude that the Applicants were in possession of the claimed genus at the time the application was filed.
For claims 1, 9-12, 14-15, 17, 20 and 27, regarding the scope of “assessing” or “assessed” or “assessment,” it is noted that the instant specification does not define what constitutes “assessing” or “assessed” or “assessment.” Pursuant to MPEP 2111.01, under a broadest reasonable interpretation, words of the claim must be given their plain meaning, unless such meaning is inconsistent with the specification. The plain meaning of a term means the ordinary and customary meaning given to the term by those of ordinary skill in the art at the time of the invention. The Meriam Webster Dictionary defines “assess” as to determine the rate or amount of something (see Merriam Webster Dictionary, “Assess” available online at https://www.merriam-webster.com/dictionary/assess accessed on 09/26/2024). As such, the Examiner is interpreting the scope of “assessing” or “assessed” or “assessment,” as determining the amount of something (i.e., glucose dependency, mitochondrial dependency, glycolytic capacity, oxidation capacity of fatty acid, oxidation capacity of amino acids and/or energetic metabolism) by finding or calculating a specific value quantity or result by using mathematical operations or reasoning.
Response to Arguments
1. Applicants’ arguments, see Remarks, filed 07/10/2025, with respect to 35 U.S.C. 101 because the claimed invention is directed to a judicial exception (i.e., abstract idea/mental step) without significantly more, have been fully considered but are not persuasive. The 35 U.S.C. 101 rejection to claims 1-5, 7-17, 19-22 and 27 has been maintained.
2. Applicants’ arguments, see Remarks, filed 12/19/2024, with respect to 35 U.S.C. 103 as being unpatentable over Neilson et al., US 2007/0087401 A1 published on Apr. 19, 2007 (herein after “Neilson”), in view of Argüello et al., Journal of Cell Science (2018) 131, pp. 1-11 (herein after “Argüello”), as evidenced by Fermentek., Oligomycin Complex -A mitochondrial Specific Reagent, 2024, pp. 1-3 retrieved from https://www.fermentek.com/oligomycin-complex-mitochondrial-specific-reagent on 9/29/24 (herein after “Fermentek”) and Pontes et al., Journal of Molecular Biology, 2015, vol. 427, issue 16, pp. 2586-2594 (herein after “Pontes”), have been fully considered but are not persuasive. The 35 U.S.C. 103 rejection to claims 1-5, 7-11, 19-21 and 27 is maintained.
3. Applicants’ arguments, see Remarks, filed 12/19/2024, with respect to 35 U.S.C. 103 as being unpatentable over Neilson et al., US 2007/0087401 A1 published on Apr. 19, 2007 (herein after “Neilson”), Argüello et al., Journal of Cell Science (2018) 131, pp. 1-11 (herein after “Argüello”), as evidenced by Fermentek., Oligomycin Complex -A mitochondrial Specific Reagent, 2024, pp. 1-3 retrieved from https://www.fermentek.com/oligomycin-complex-mitochondrial-specific-reagent on 9/29/24 (herein after “Fermentek”), as applied to claim 1, and further in view of Kantor et al., Circulation Research, 2000, pp. 580-588 (herein after “Kantor”), and Gowda et al., Frontiers in Molecular Biosciences, 2018, volume 5, article 49, pp. 1-13 (herein after “Gowda”), as applied to claims 12-17, have been fully considered but are not persuasive. The 35 U.S.C. 103 rejection to claims 1 and 12-17 is maintained.
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.
1. Claims 19-20 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. It is noted that claims 19 and 20 depend from base claim 18. However, base claim 18 has been cancelled, therefore claims 19 and 20 fail to particularly point out and distinctly claim the subject matter of the claimed invention.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
2. Claim 4 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 4 recites “wherein the population of immune cells comprises immune cells, natural killer cells, myeloid cells, neutrophils, eosinophils, mast cells, basophils, and/or granulocytes. However claim 4 is dependent upon claim 1, which limits the method of profiling the energetic metabolism profile to a population of immune cells. Thus, the scope of claim 4, fails to further limit the subject matter of independent claim 1 when the population of immune cells comprises immune cells. Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
2. Claim 5 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 5 is directed to inhibitor A; and recites that inhibitor A is selected from a group consisting of six different inhibitor alternatives (i.e., 2-Deoxy-Glucose, 2-[N-(7-Nitrobenz-2-oxa-1,3-diaxol-4- yl)amino]-2-deoxyglucose/2-NBDG, Phloretin, 3-Bromophyruvic acid, Iodoacetate, Fluoride and 6-Aminonicotinamide). However claim 5 is dependent upon claim 1, which limits inhibitor A to 2-Deoxy-Glucose. Thus, the scope of claim 5, fails to further limit the subject matter of independent claim 1. Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
3. Claim 7 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 7 is directed to inhibitor B; and recites that inhibitor B is selected from a group consisting of eight different inhibitor alternatives (i.e., Oligomycin (A/B/C/D/E//F and derivates), Rotenone, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone/FCCP, Trimetazidine/TMZ, 2[6(4-chlorophenoxy)hexyl]oxirane-2-carboxylate/etomoxir, Bis-2-(5-phenylacetamido-1,3,4- thiadiazol-2-yl)ethyl sulfide/BPTES, and enasidenib). However claim 7 is dependent upon claim 1, which limits inhibitor B to Oligomycin. Thus, the scope of claim 7 fails to further limit the subject matter of independent claim 1. Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
4. Claim 20 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 20 recites: “The method of claim 18 wherein the protein synthesis levels are assessed by cytometry, cytof or Cite-seq.” However, claim 20 is of improper dependent form because it depends on a cancelled claim and also because it fails to further limit the subject matter; since claim 1 (presuming that claim 20 ultimately depends upon claim 1 and not claim 18) recites that the protein synthesis levels are assessed by cytometry; and cytometry is a broader analytical technique than flow cytometry for cell analysis; and because Cite-Seq is a different technique that does not involve the use of a flow cytometer, instead Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) is a single-cell phenotyping method that uses antibody-derived tags (ADTs) to quantitatively detect cell surface protein expression and generate transcriptomic data at the single-cell level. Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
Maintained/Modified Rejections
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.
2. Claims 1-5, 7-17, 19-22 and 27 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception (i.e., abstract idea/mental step) without significantly more. Claim 1 recites a method of profiling the energetic metabolism profile of a population of cells, which encompasses in steps (ii) “measuring an intracellular protein synthesis level in the sample S1;” (iii) measuring an intracellular protein synthesis level LA in said sample S2;” (iv) “measuring an intracellular protein synthesis level LB in said sample S3;” and (v) “measuring an intracellular protein synthesis level L(A+B) in said sample S4;” Thus, the claimed invention is directed to a process, which is one of the statutory categories of invention.
Regarding step 2A, Prong One, of the patent eligible subject analysis, the claims recite a judicial exception of an abstract idea. Claim 1 recites in steps (vi) “calculating the glucose dependency of the population of immune cells;” (vii) “calculating the mitochondrial dependency of the population of immune cells;” (viii) “calculating the glycolytic capacity of the population of immune cells;” and (ix) “calculating the capacity for the oxidation of fatty acids and the oxidation of amino acids of the population of immune cells,” and x) determining the energetic metabolism profile of the population of immune cells based on assessments made in steps vi), vii), viii) and ix), wherein A is 2-Deoxy-D-Glucose, and wherein B is oligomycin, wherein the protein synthesis levels LCo, LA, LB and L(A+B) are determined by contacting the samples SL, S2, S3 and S4, respectively, with puromycin and then with a monoclonal antibody specific for puromycin, wherein the monoclonal antibody specific for puromycin is conjugated with a detectable label, and wherein flow cytometry is used to quantify the monoclonal antibody specific for puromycin conjugated with the detectable label at a single cell resolution.
These limitations therefore recite an abstract idea/mental step. The series of steps which describe the calculation (see steps vi-ix)/assessment(see step x) of different parameters by performing an arithmetic calculation which can be practically performed in the human mind, e.g., looking at the protein synthesis levels obtained from step (i) and comparing them to the protein synthesis levels obtained from steps (ii)-(v) in order to calculate the parameters recited in steps (vi)-(ix) and (x), where the calculation steps consists of a mathematical formula that involves arithmetic calculations (i.e., addition, subtraction, multiplication and division). Even though most humans would use a physical aid (e.g., pen and paper or a calculator) to help them perform the calculation/assessment steps, the use of such physical aid does not negate the mental nature of this limitation. Therefore, steps (vi) to (ix) and (x) in claim 1 recite a judicial exception (i.e., abstract idea/mental step). Thus, the answer to step 2A, Prong One is YES.
Regarding step 2A, Prong Two, of the patent eligible subject analysis, the claims do not integrate the judicial exception into a practical application. The judicial exception is not integrated into a practical application because claim 1 fails to recite a practical application of the abstract idea/mental step. Although claim 1 encompasses steps (ii) to (v) and (x), such steps are recited at a high level of generality and would be considered a nominal or tangential addition to the claims. In other words, steps (ii) to (v) and (x) recite additional elements beyond the judicial exception (i.e., “contacting the sample with an inhibitor”, “contacting the samples S1, S2, S3 and S4 with puromycin and then with a monoclonal antibody specific for puromycin, wherein the monoclonal antibody specific for puromycin is conjugated with a detectable label” and “wherein flow cytometry is used to quantify the monoclonal antibody specific for puromycin conjugated with the detectable label at a single cell resolution”). Although these limitations indicate that the population of cells is treated with an inhibiting substance (i.e., 2-Deoxy-D-Glucose as inhibitor A and/or Oligomycin as inhibitor B), the limitation does not provide any additional information because the use of 2-Deoxy-D-Glucose and oligomycin would have been well-understood, routine and conventional to the relevant audience (i.e., a scientist). Similarly, although claims 2-5, 7-18, 19-22 and 27 are directed to additional elements such as a specific population of cells, and specific inhibitors (e.g., natural and synthetic), such recitation of additional elements tell the relevant audience about which biological activity is being compared, and at most adds a suggestion that the scientist take those biological activities into account when comparing the population of cells. Moreover, although the newly added limitations to claim 1 are directed to where the protein synthesis levels are determined by contacting the samples with puromycin and then with monoclonal antibodies specific to puromycin, there is no recitation of a specific structure of the monoclonal antibodies that bind to puromycin. As such, the limitations of the claim recite a high level of generality without any specificity, and are nothing more than an attempt to generally link the judicial exception to a particular technological environment. Therefore, the answer to step 2A, Prong Two, is NO.
Regarding Step 2B of the patent eligible subject analysis, the claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because steps (vi) to (ix) and (x) of the claimed invention are directed to non-statutory subject matter. Although claim 1 encompasses steps (ii) to (v), these steps are drawn to adding a substance (i.e., inhibitor) to a population of cells and measuring the impact of the substance on target biological activities. Such steps are routine and conventional in the art. For instance, the instant specification teaches that the measurement of protein synthesis level is determined by any well-known method in the art (see instant specification pg. 11, lines 1-2). In other words, steps (i)-(v) appear to relate to nothing more than data gathering, because the steps comprise determining the amount/quantity/level of a product/result/process in a population of cells in the presence and/or absence of a substance (i.e., an inhibitor). Thus, the claims do not include addition elements that are sufficient to amount to significantly more than the judicial exception. Therefore, the answer to Step 2B is NO.
Accordingly, the claimed invention is directed to ineligible patent subject matter.
Maintained/Modified Rejections in Light of Amendments
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
103 - KSR Examples of 'Rationales' Supporting a Conclusion of Obviousness
(Consistent with the "Functional Approach" of Graham)
Further regarding 35 USC 103(a) rejections, the Supreme Court in KSR International Co. v. Teleflex Inc., 550 U.S. 398, 127 S. Ct. 1727, 82 USPQ2d 1385, 1395-97 (2007) (KSR) identified a number of rationales to support a conclusion of obviousness which are consistent with the proper "functional approach" to the determination of obviousness as laid down in Graham. The key to supporting any rejection under 35 U.S.C. 103 is the clear articulation of the reason(s) why the claimed invention would have been obvious. The Supreme Court in KSR noted that the analysis supporting a rejection under 35 U.S.C. 103 should be made explicit.
Exemplary rationales that may support a conclusion of obviousness include:
(A) Combining prior art elements according to known methods to yield predictable results;
(B) Simple substitution of one known element for another to obtain predictable results;
(C) Use of known technique to improve similar devices (methods, or products) in the same way;
(D) Applying a known technique to a known device (method, or product) ready for improvement to yield predictable results;
(E) "Obvious to try" - choosing from a finite number of identified, predictable solutions, with a reasonable expectation of success;
(F) Known work in one field of endeavor may prompt variations of it for use in either the same field or a different one based on design incentives or other market forces if the variations are predictable to one of ordinary skill in the art;
(G) Some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention.
Note that the list of rationales provided is not intended to be an all-inclusive list. Other rationales to support a conclusion of obviousness may be relied upon by Office personnel.
Also, a reference is good not only for what it teaches by direct anticipation but also for what one of ordinary skill in the art might reasonably infer from the teachings. (In re Opprecht 12 USPQ 2d 1235, 1236 (Fed Cir. 1989); In re Bode 193 USPQ 12 (CCPA) 1976).
3. Claims 1-5, 7-11, 17, 19-22 and 27 are rejected under 35 U.S.C. 103 as being unpatentable over Neilson et al., US 2007/0087401 A1 published on Apr. 19, 2007 (herein after “Neilson”), as evidenced by Pontes et al., Journal of Molecular Biology, 2015, vol. 427, issue 16, pp. 2586-2594 (herein after “Pontes”), and Fermentek., Oligomycin Complex -A mitochondrial Specific Reagent, 2024, pp. 1-3 retrieved from https://www.fermentek.com/oligomycin-complex-mitochondrial-specific-reagent on 9/29/24 (herein after “Fermentek”) in view of Argüello et al., Journal of Cell Science (2018) 131, pp. 1-11 (herein after “Argüello”).
Regarding claim 1, a method of profiling the energetic metabolism profile of a population of immune cells comprising: i) providing at least four samples of said population of immune cells; wherein the four samples are identified as S1, S2, S3 and S4; ii) measuring an intracellular protein synthesis level in the sample S1 in the absence of any inhibitor (control level, LCo); iii) contacting the sample S2 with an inhibitor A of energy production resulting from glycolysis and oxidative phosphorylation of glucose-derived pyruvate and measuring an intracellular protein synthesis level LA in said sample S2; iv) contacting sample S3 with an inhibitor B of energy production resulting from TCA cycle and oxidative phosphorylation comprising pyruvate oxidation, oxidation of fatty acids and oxidation of amino acids and measuring an intracellular protein synthesis level LB in said sample S3; v) contacting sample S4 with both inhibitors A and B and measuring an intracellular protein synthesis level L(A+B) in said sample S4; vi) calculating the glucose dependency of the population of immune cells; vii) calculating the mitochondrial dependency of the population of immune cells; viii) calculating the glycolytic capacity of the population of immune cells; and ix) calculating the capacity for the oxidation of fatty acids and the oxidation of amino acids of the population of immune cells, and x) determining the energetic metabolism profile of the population of immune cells based on assessments made in steps vi), vii), viii) and ix) wherein A is 2-Deoxy-D-Glucose, wherein B is oligomycin, as recited in instant claim 1:
Neilson’s invention provides ways of assessing the metabolic profile of cells in culture by measuring multiple extracellular concentration changes of components involved in metabolism, preferable simultaneously (see Neilson, pg. 2, para[0013]). Neilson also teaches that any type of animal cell, tissue or organelle may be used, and that the cells under analysis may be primary animal cells, cells growing on a surface in a well, neoplastic cells, cells disposed in suspension (see Neilson, pg. 2, para[0123]). Neilson’s method comprises bringing into contact with the cells a substance potentially capable of altering cellular metabolism (see Neilson, pg. 2, para[0014]), thereby constituting contacting the samples with an inhibitor as recited in instant claim 1. Neilson also teaches that the method may comprise the steps of incubating in parallel plural cultures of animal cells in plural wells, adding to the media in different wells different substances or different concentrations of the same substance, and measuring the rate of change in plural wells (see Neilson, pg. 2, para[0017]). In a specific example, Neilson compared the respiratory capacity of three distinct cell lines based on the response to two drugs that induce mitochondrial uncoupling (disruption of the electron transport chain) (see Neilson, pg. 14, para[0163]), thereby constituting contacting the sample S2 with an inhibitor A as recited in step (iii), and contacting the sample S3 with an inhibitor B as recited in step (iv) of instant claim 1. Neilson also teaches that the baseline metabolic rates were measured twice in wells containing the three different cell types tested (see Neilson, pg. 14, para[0168]), thereby constituting sample S1 in the absence of any inhibitor as recited in step (ii) of instant claim 1. Neilson’s Example 11, teaches that C2C12, CHO-Kl, and HEK-293 cells were profiled using the Seahorse XF instrument to compare basal metabolic rates and respiratory pathway preference, and then to compare the effects of two uncouplers of mitochondrial respiration (see Neilson, pg. 14, para[0164]), thereby constituting calculating the glucose dependency of the population of immune cells as recited in step (vi), constituting calculating the mitochondrial dependency of the population of immune cells as recited in step (vii), constituting calculating the glycolytic capacity of the population of immune cells as recited in step (viii) and constituting calculating for the oxidation of fatty acids and the oxidation of amino acids of the population of immune cells as recited in step (ix) of instant claim 1.
Neilson also teaches that the measured component of cellular aerobic metabolism is preferably extracellular oxygen and the measurement is oxygen consumption rate (OCR) (see Neilson, pg. 2, para[0017]). The measured component of cellular anaerobic metabolism is preferable extracellular proton concentration (extracellular acidification rate – ECAR), or carbon dioxide production rate (CPR) (see Neilson, pg. 2, para[0017]). Lactic acid production rate, or lactate production rate can also be used (see Neilson, pg. 2, para[0017]). Other molecules absorbed or secreted by animal cells and related to metabolic activities also may be exploited (see Neilson, pg. 2, para[0017]). Measuring in a cell medium the rate of change in concentration of both an extracellular solute which is a component of cellular aerobic metabolism, and an extracellular solute which is a component of cellular anaerobic metabolism (see Neilson, pg. 2, para[0014]). Profiling the metabolic function of living cells include comparing measurements of the extracellular flux rate of at least two analytes selected from gasses, ions, nutrients and byproducts of or component necessary for metabolism (see Neilson, pg. 3, para[0022]).
Neilson’s method also comprises adding a fatty acid to a well to assess a characteristic of fatty acid metabolic activity of the cell culture (see Neilson, pg. 2, para[0016]), or incubating the cells in cell media containing a substance suspected to alter the rate of fatty acid metabolic activity of the cell culture prior to measurement (see Neilson, pg. 2, para[0016]), or adding a known inhibitor of fatty acid transport or oxidation in order to more specifically determine the effect of the substance (see Neilson, pg. 2, para [0016]), thereby constituting inhibitor A or inhibitor B as recited in instant claim 1, and constituting where the oxidation of fatty acid capacity is calculated in a population of immune cells as recited in step (ix) of instant claim 1. Neilson’s method also comprises measuring, in the medium of a cell culture in a well separate from the cells under analysis and treated differently than the cells under analysis (i.e., instant sample S1), either or both the rate of change in concentration of extracellular solutes which are respectively components of cellular aerobic and anaerobic metabolism in order to establish control or baseline values, and then comparing the measurements of the rates of change in the separate cell cultures (see Neilson, pg. 2, para[0015]), thereby constituting where an intracellular protein synthesis level in the sample S1 in the absence of any inhibitor (control level, LCo) is measured as recited in instant claim 1. Neilson teaches that the method comprises incubating in parallel plural cultures of animal cells in plural wells, adding to the media in different wells different substances or different concentrations of the same substance, and measuring the rate of change in plural wells (see Neilson, pg. 2, para[0017]), thereby constituting contacting sample S2 with inhibitor A as recited in step (iii) of instant claim 1, and constituting contacting S3 with inhibitor B as recited in step (iv) of instant claim 1.
Neilson’s method does much more than take a measurement indicative of whether the cells in a culture are alive, as it can measure metabolic rate; measure relative contribution of aerobic (oxidative phosphorylation) versus anaerobic (glycolysis) processes for generation of ATP (see Neilson, pg. 3, para[0019]). In a related aspect, the invention permits the scientist to obtain data indicative of respiratory (or metabolic) capacity of a cell culture without cell counting (see Neilson, pg. 3, para[0020]). This is done by measuring a basal metabolic rate or rates (i.e., rates of change of OCR, ECAR etc.), before the addition of any metabolism altering substance (see Neilson, pg. 3, para[0020]). A class of substances suitable for this purpose are drugs known to uncouple the TCA cycle within a cell, thereby producing waste heat in lieu of providing energy via ADP to ATP conversion (see Neilson, pg. 3, para[0020]). These methods may be used to assess aerobic versus anaerobic metabolism (i.e., energetic metabolism profile), including situations: where at least one analyte is sensitive to aerobic metabolism and at least one second analyte is sensitive to anaerobic metabolism; where one analyte is sensitive to either aerobic or anaerobic metabolism alone and a second analyte is insensitive to both; where an analyte sensitive to anaerobic metabolism is O2, CO2, and the like; where an analyte sensitive to anaerobic metabolism is proton flux (pH change), lactate, and the like; or where two analytes, one sensitive to aerobic and another sensitive to anaerobic metabolism can be used to calculate the amount of ATP generated per unit of time and the percent uncoupled metabolism per unit time (see Neilson, pg. 3, para[0022]). Therefore Neilson implies that two analytes with different functions are contacted with a cell sample, as recited in step (v) where the sample [S4] is contacted with both inhibitors [A] and [B], of instant claim 1. Additionally, Neilson also suggest determining the energetic metabolism profile of the population of cells based on assessments made in steps vi, vii, viii and ix of instant claim 1. Therefore the teachings of Neilson are suggestive of the claim limitations as recited in steps (i)-(x) of instant claim 1.
As evidenced by Pontes, adenosine triphosphate (ATP) is the energy currency of living cells (see Pontes, pg. 2587, Abstract). Even though ATP powers virtually all energy-dependent activities, most cellular ATP is utilized in protein synthesis via tRNA aminoacylation and guanosine triphosphate regeneration (see Pontes, pg. 2586, Abstract). Proteins are largely responsible for the structural, transport and catalytic properties of all cells (see Pontes, pg. 2587, left column, paragraph 1). Consuming over 70% of the ATP pools utilized by biosynthetic processes, translation is the most expensive anabolic activity (see Pontes, pg. 2587, left column, paragraph 1). Since, Neilson’s invention permits scientist to obtain data indicative of respiratory (or metabolic) capacity of a cell culture by measuring the basal metabolic rate or rates (i.e., rates of change of OCR, ECAR etc.), before and after the addition of any metabolism altering substance, i.e., instant inhibitors A, B, C and D; thus reduced levels of OCR and ECAR due to the cell’s response to an inhibitor would result in reduced production of ATP which consequently would result in reduced protein synthesis levels. As such, the teachings of Neilson as evidenced by the teachings of Pontes are suggestive of the claim limitations as recited in steps (ii)-(iv) of instant claim 1, where the protein synthesis level in the sample S1 is measured in sample S1, the protein synthesis level LA is measured in sample S2, the protein synthesis level LB is measured in sample S3 and the protein synthesis level L(A+B) is measured in sample S4.
With respect to wherein inhibitor A is 2-Deoxy-D-Glucose and wherein inhibitor B is oligomycin, as recited in instant claim 1:
Neilson teaches that metabolic modulators include, but a are not limited to glycolysis inhibitors, mitochondrial uncouplers and inhibitors, and pentose cycle inhibitors (see Neilson, pg. 9, para[0108]). As shown in Neilson’s FIG 11, the elevated ECAR was then inhibited by the hexokinase inhibitor, 2-Deoxy-glucose (see Neilson, pg. 12, para[0143]). In Example 2, Neilson also teaches that a maximal increase of 150% in OCR over baseline was observed in H460 cells in response to glycolysis inhibitors (A), oxamate, 3-bromopyruvic acid, iodoacetate and fluoride (see Neilson, pg. 12, para[0146]), thereby constituting where A is 2-Deoxy-D-Glucose as recited in step x) of instant claim 1.
Neilson also teaches that the coupled respiration and proton leak were determined using the ATP synthase inhibitor oligomycin as shown in FIG 33 (see Neilson, pg. 16, para[0191]); thereby constituting wherein B is oligomycin as recited in step x) of instant claim 1. As evidenced by Fermentek, oligomycins are macrolides created by the Streptomyces species that act as strong antibacterial agents, but are often poisonous to other organisms, including humans (see Fermentek, pg. 1, 1st full paragraph). They serve as a classical mitochondrial reagent that binds to the Fo subunit of the Fo/F1 ATPase (ATP synthase) (see Fermentek, pg. 1, 1st full paragraph). These binding blocks proton conductance across the synthase complex and inhibit the synthesis of mitochondrial ATP, thus inhibiting proton translocation and O2 uptake (see Fermentek, pg. 1, 1st full paragraph). Oligomycin complex is a mixture of Oligomycins A, B, and C (see Fermentek, pg. 1, 2nd full paragraph). Different Oligomycin isomers are highly specific for the disruption of mitochondrial metabolism (see Fermentek, pg. 1, 2nd full paragraph). Oligomycin B is a nonselective inhibitor of ATP synthases, which can reduce the rate of ATP depletion in myocardial ischemia (see Fermentek, pg. 1, 2nd full paragraph).
However, Neilson does not expressly teach profiling the energetic metabolism profile of a population of immune cells, nor wherein the protein synthesis levels LCo, LA, LB and L(A+B) are determined by contacting the samples S1, S2, S3, and S4, respectively, with puromycin and then with monoclonal antibodies specific for puromycin, wherein the monoclonal antibodies are conjugated with a detectable label, and wherein flow cytometry is used to quantify the monoclonal antibody specific for puromycin conjugated with the detectable label at a single cell resolution, as recited in instant claim 1.
Argüello teaches using a combination of puromycilation detection and flow cytometry (a method call ‘SunRiSE’) to show that translation elongation can be measured accurately in primary cells in pure or heterogeneous populations isolated from blood or tissues (see Argüello, pg. 1, abstract). Puromycilation detection with ad hoc antibodies and measurement by flow cytometry has proven to be an extremely versatile method to replace radiolabeled amino acid incorporation as a means to monitor protein synthesis levels (see Argüello, pg. 1, right column, paragraph 2). Puromycin (puro) is an aminonucleoside Tyr-tRNA mimetic antibiotic that enters the A site of ribosomes, and is incorporated into nascent chains (see Argüello, pg. 1, right column, paragraph 2). By using puro as a nascent protein tag and anti-puro monoclonal antibodies for its detection, puro-immunomonitoring and, in particular, flow cytometry to measure translation in living cells and organisms (see Argüello, pg. 2, left column, paragraph 2). Argüello and coworkers used 12D10 anti-puro antibody conjugated directly to Alexa® Flour (AF) dyes (e.g., AF488 and AF647) (see Argüello, pg. 3, right column, paragraph 2), thereby constituting where monoclonal antibodies specific for puromycin are conjugated with a detectable label, and wherein flow cytometry is used to quantify the monoclonal antibody specific for puromycin conjugated with the detectable label at a single cell resolution as recited in instant claim 1. With respect to profiling the energetic metabolism profile of a population of immune cells, as recited in instant claim 1. Argüello performed flow cytometry using a gating strategy in which B cells were identified as CD4− CD8− CD11c− CD19+ B220+ MHC-II+ cells, and CD4+ T cells by a CD4+ CD8− CD19− CD11c− MHC-II− B220− phenotype (see Argüello, pg. 6, left column, paragraph 1); thereby constituting profiling a population of immune cells, as recited in instant claim 1.
From the teachings of the references, the Examiner recognizes that it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow the teachings of Neilson and measure rates of change of OCR and ECAR after the addition of a metabolism altering substance such as inhibitors, given that OCR and ECAR rates are common indicatives of respiration (or metabolic) capacity of a cell culture, where the addition of inhibitors such as 2-deoxy-glucose and oligomycin reduce OCR and/or ECAR levels thereby resulting in reduced ATP production, and subsequently, protein synthesis levels. One of ordinary skill in the art before the effective filing date of the claimed invention would have been motivated to do so because measuring multiple extracellular concentration changes of components involved in metabolism (i.e., OCR and ECAR rates) was known to be used in determining the metabolic profile of cells in culture exposed to different metabolic disturbing agents as taught by Neilson. One of ordinary skill in the art before the effective filing date of the claimed invention would have had a reasonable expectation of success given that the method of assessing the metabolic profile of cells in culture by measuring multiple extracellular concentration changes of components involved in metabolism of Neilson was used for profiling the metabolic function of living cells by assessing the aerobic versus anaerobic metabolism and by measuring the effects of metabolism altering substances such as inhibitors. Therefore, utilizing cell sample 1 (i.e., S1) to measure basal metabolic rates (i.e., in the absence of any inhibitor) as a control sample, sample 2 (i.e., S2) exposed to inhibitor 1 such as 2-Deoxy-D-Glucose (i.e., inhibitor A), sample 3 (i.e., S3) exposed to inhibitor 2 such as oligomycin (i.e., inhibitor B), and sample 4 (i.e., S4) exposed to inhibitors 1 and 2, and measuring the OCR and ECAR rates of each sample where such measurements allow for the determination of a metabolic profile based on assessments of glucose dependency, mitochondrial dependency, glycolytic capacity and oxidation of fatty acids and amino acids, which all correlate to inhibition of ATP production, which necessarily reduces protein synthesis levels would support a method for profiling the energetic metabolism profile of a population of cells via assessment of glucose dependency, mitochondrial dependency, glycolytic capacity and oxidation of fatty acids and amino acids by constituting some teaching, suggestion or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention and/or the use of known technique to improve similar devices (methods, or products) in the same way and/or the application of a known technique to a known device (method, or product) ready for improvement to yield predictable results pursuant to KSR.
Furthermore, it would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to modify the teachings of Neilson with those of Argüello and incorporate puromycilation detection and flow cytometry in the method for assessing the metabolic profile of cells, given that primary cells in pure or heterogeneous populations isolated from human blood or tissue samples are commonly used in protein synthesis experiments; given that B cells and CD4+ T cells are commonly known to