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 .
Election/Restrictions
Applicant’s election without traverse of Group IV in the reply filed on 7 July 2026 is acknowledged.
Specification
The use of multiple trade names or a marks used in commerce, such as TaqMan on page 18, has been noted in this application. The terms should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever they appear or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the terms.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claims 2, 6, 15, 17, 19, 21- 23, and 27 are objected to because of the following informalities:
When abbreviations are used in the claims, the first instance should include the full terminology. Claim 2 contains the abbreviation “MGB” and should be accompanied by the full name, “minor groove binder”.
Claim 6, L2 should read as “…composition of the reference gene…”.
Claim 15 should read as “…anticancer drug is selected from a compound with one of the following structures:…”.
Claim 15 is further objected to because the molecular structures of the compounds are illegible.
Claim 17, (2) should read as “…PCR amplification with the cDNA …”
Claim 17, (3) should read as “obtaining the AKR1C3 RNA content of the ex vivo sample to be detected according to the qPCR or…”
Claim 19 should read as “…the primer-probe composition of claim 1 is mixed with the primer-probe composition of a reference gene;”
Claim 21 should read as “…AKR1C3 and a reference gene are amplified by a AKR1C3 digital PCR detection system and a reference gene….”
Claim 22, L2 should read as “…AKR1C3 RNA content of the ex vivo sample…”
Claim 22, (A) should read as “…AKR1C3 and a reference gene of the ex vivo…”
Claim 22, (B) should read as “…AKR1C3 RNA content of the ex vivo sample…”
Claim 23, (A1), L2 should read as “…value of a reference gene…”
Claim 23, (A1), L1-2: “…copy number lg value…” should be corrected to “…copy number log value…”
Claim 27 should read as “…administering an AKR1C3 activated anticancer drug[[s]] to the patient with…”
Appropriate correction is required.
Claim Interpretation
For the purposes of prior art and in the spirit of compact prosecution, the claims are being interpreted as follows:
Any limitation described as “preferable” will be interpreted as optional and non-limiting.
The polymerase mixture of claim 8 will be interpreted as if it was comprised of the subsequent components, rather than “mainly” comprised of said components.
In claim 17, it will be assumed that the steps of amplification include an additional step of creating a reaction system.
Claim 17, (2) requires the use of the primer-probe composition of claim 1 in order to perform amplification. However, it does not specify which parts of the composition are required. Therefore, this step can and will be broadly interpreted to include any amplification in which (1) one of the primers is used, (2) both primers are used, (3) only the probe is used, (4) one of the primers and the probe are used, or (5) both primers and the probe are used.
Claim 17(3) recites “obtaining the AKR1C3 RNA content…according to qPCR or digital PCR amplification results…”. This can and will be broadly interpreted to include amplified RNA itself or any data pertaining to RNA content as long as the primer-probe composition of claim 1 was used in a qPCR or digital PCR amplification.
Claim 18 can and will be broadly interpreted to require only one of either (1) detecting the concentration of extracted RNA or (2) the reverse transcription system comprises reverse transcriptase.
Claim 23 will be interpreted as depending from claim 22 rather than claim 17.
Claim 27 recites “…obtaining AKR1C3 content…”. This can and will be broadly interpreted to include template RNA, amplified RNA, or data pertaining to RNA content. Furthermore, the data could have been obtained through any means including, but not limited to, PCR, qPCR, and sequencing as long as the primer-probe composition of claim 1 is used.
Claim Rejections
35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 2, 4-6, 8-10, 13, 15-23, 25, and 27 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.
Regarding claims 2, 5, 6, 8- 10, 13, 16-21, 25, and 27, the phrase "preferably", or “more preferably” and the like, renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
The term “mainly comprising” in claim 8 is a relative term which renders the claim indefinite. The term “mainly” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. As a result, the composition of the polymerase mixture is called into question. The transitional term "comprising", which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. “Mainly” on the other hand implies a certain degree of completeness; however, the exact degree is unclear and would depend on the circumstances surround its use. Therefore, “mainly comprising” is of unclear scope.
Claims 1, 2, 6, 13, 17, 19, 20, 23, 25, and 27 recite the following limitations for which there is insufficient antecedent basis:
Claim 1: for each primer-probe composition, in the second line “…the nucleotide sequences…”
Claim 2, L4-5: “…the 5`-end reporters…” and “…the 3`-end quenchers…”
Claim 6: “…the nucleotide sequences…” in L3 and “…the 5`-end reporters…” and “…the 3`-end quenchers…” in L6
Claim 13, L2: “…the ratio of AKR1C3…”
Claim 17, L3-4: “…the reverse transcription system…”
Claims 19 and 20 make multiple references to a “qPCR reaction system” which lacks antecedent basis because the parent claim only mentions performing qPCR amplification and does not introduce a reaction system.
Claim 19, L3: “…the primer-probe composition of reference gene…”
Claim 19, L10-18 and claim 21, L11-16 describe various molar ratios for ACTB-F1, ACTB-R1, and ACTB-P1. However, these primers have not yet been introduced. While a primer-probe composition for a reference gene is mentioned within this claim, ACTB is not indicated to be said reference gene nor are these primers described as being the same as those present in the composition.
Claim 23 aims to further limit step (A); however, this step is not present in the parent claim 17.
Claim 23 (A1): “… the standard curve…” in lines 1 and 2.
Claim 23 (A2): “…the detected Ct values…” in line 2.
Claim 25, L1: “…the expression level…”
Claim 27, L5: “…the predetermined content…”
Claim 20 is additionally indefinite for the use of “also added” in line 3. It is unclear to what the recited components are being added as well as what was added before them.
Claims 8, 9, and 18 contain the trademarks/trade names “KAPA PROBE FAST RT-PCR Master Mix (2x)”, “Superscript VILO MASTER MIX”, and “Qubit RNA HS Assay Kits”, respectively. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe various proprietary reagents and a proprietary kit and, accordingly, the identification/description is indefinite.
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.
Claims 1, 2, 4-6 and 8-10 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a natural phenomenon (including a product of natural) without significantly more.
The claims recite a primer-probe composition containing SEQ ID NOs: 3, 5, and 7. These sequences are taught by Waters (US 20030186871 A1) as being segments of a polynucleotide which encodes an AKR1C polypeptide [Waters, 0021]. This judicial exception is not integrated into a practical application because primers and probes are nothing more than strands of nucleic acids whose characteristics are innate to naturally occurring DNA and thus lack markedly different characteristics from nature (see MPEP 2106.04(C).II.C.2; Ambry Genetics, 774 F.3d at 760-61, 113 USPQ2d at 1244). The claims do not include/require additional elements that are sufficient to amount to significantly more than the judicial exception because there are no additional elements.
Claim 4 introduces including this composition in a kit. However, merely placing a product(s) of nature in a kit does nothing more than attempt to generally link it to a technological environment without altering its structure or function. Therefore, this is considered to be insignificant extra-solution activity.
Claims 5, 6, and 8-10 introduce limitations in which additional components are added to the kit, such as an additional primer-probe composition, a polymerase, a reverse transcriptase, and reaction controls. However, these are also judicial exceptions (i.e., products of nature) and thus cannot amount to significantly more than the judicial exception.
Therefore, these claims do not contain eligible subject matter.
35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim 1 is rejected under 35 U.S.C. 103 as being unpatentable over Waters [US20030186871 A1].
Waters discloses SEQ ID NOs: 2-6, 8-16, 18-22, and 24-29. SEQ ID NOs: 3, 5, and 7 of the instant application are 100% concordant with SEQ ID NOs: 26, 22, and 29 of Waters, respectively (see Table 1 at the end of this office action for the alignments). According to Waters, appropriate primers and probes for identifying the genes encoding an AKR1C polypeptide from mammalian tissues can be derived from these sequences which serve as references for PCR primers and defines suitable regions for isolating probes [Waters, 0088, 0092]. Waters further discloses designing primers and probes specifically to measure human AKR1C1, AKR1C2, AKR1C3, and AKR1C4 mRNA levels in complex mixtures [Waters, 0252].
Therefore, a person of ordinary skill in the art prior to the effective filing date of the claimed invention, would have been capable of arriving at the claimed sequences as Water’s sequences provide for a finite number of possibilities from which primer and probe sequences can be selected from and which would be obvious to try. The Supreme Court decided that a claim can be proved obvious merely by showing that the combination of known elements was obvious to try. In this regard, the Supreme Court explained that, “[w]hen there is a design need or market pressure to solve a problem and there are a finite number of identified, predictable solutions, a person of ordinary skill in the art has a good reason to pursue the known options within his or her technical grasp.” An obviousness determination is not the result of a rigid formula disassociated from the consideration of the facts of the case. Indeed, the common sense of those skilled in the art demonstrates why some combinations would have been obvious where others would not. Therefore, choosing from a finite number of identified, predictable solutions, with a reasonable expectation for success, is likely to be obvious to a person if ordinary skill in the art. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, E.).
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Waters, as applied to claim 1 above, and as evidenced by ThermoFisher 1 [ThermoFisher Scientific. How TaqMan Assays Work. 2018 May:1-7. Accessed from the WayBack Machine on 21 July 2026].
Waters is applied to the relevant teachings of claim 1 as discussed above and is incorporated herein by reference. The probes designed by Waters were TaqMan MGB (minor groove binding) probes labeled on the 5` end with 6-FAM [0252-0253]. While Waters does not explicitly disclose which end the MGB is attached, ThermoFisher 1 teaches that TaqMan probes are labeled on the 5` end with a FAM or VIC dye and on the 3` end with a MGB and a nonfluorescent quencher [ThermoFisher 1, p1].
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Waters, as applied to claim 1 above, in view of Polansky [US 20040023207 A1].
Waters is applied to the relevant teachings of claim 1 as discussed above and is incorporated herein by reference.
Waters further describes a number of assays which would use the taught primers and probes, including hybridization assays, in situ hybridization assays, real-time PCR assays, and gene expression assays [Waters, 0136, 0137, 0139, 0188]. Example 2 demonstrates one such assay in which primers and probes were developed to specifically measure human AKR1C1, AKR1C2, AKR1C3, and AKR1C4 mRNA levels in complex mixtures [Waters, 0252].
While Waters does disclose the provision of kits for carrying out these assays, they do not explicitly state that said kits contain primers and/or probes. However, Polanksy teaches that the inclusion of individual components in a commercial kit imparts well known advantages such as convenience and reproducibility due to manufacturing standardization, quality control, and validation procedures [Polanksy, 0919]. Accordingly, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to package Waters’ primers and probes into a kit in order to provide the reagents required for performing the disclosed assays (e.g., gene expression assay, etc.) while also realizing the well-known advantages taught by Polansky.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Waters and Polanksy, as applied to claims 1 and 4 above, and further in view of Bio-Rad 1 [Bio-Rad. Normalization Methods for Quantitative PCR. BioRadiations 121. 2007:1-4.].
Waters and Polansky are applied to the relevant teachings of claims 1 and 4 as discussed above and are incorporated herein by reference. However, neither disclose the inclusion of a reference gene in the described kit.
Bio-Rad 1 teaches that most real-time PCR gene expression assays are based on the comparison of two or more samples which requires uniform sampling conditions in order for the comparison to be valid. However, many factors contribute to sample variability, including quantity and quality of extracted RNA, extraction and reverse transcription efficiency, and sample degradation and contamination. As such, in order to ensure the accuracy of the results, samples must be normalized. One method of sample normalization involves normalization to a reference gene that has little variability, such as β-actin. As the mRNA of the reference gene is extracted, reverse transcribed, and assessed via qPCR alongside the gene of interest, this method accounts for both differences in starting mRNA quantity, as well as differences in the efficiency of laboratory steps [Bio-Rad, p2].
Therefore, one of ordinary skill in the art prior to the effective filing date of the claimed invention, would have been motivated to include primers and probes specific for a reference gene, such as β-actin, in the kit of Waters and Polansky in order to normalize the data obtained by the gene expression assay described in Example 2 of Waters so that accurate conclusions can be drawn when comparing between samples. Applying a known technique to a known device (method or product) ready for improvement to yield predictable results is likely to be obvious. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, D.).
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Waters, Polanksy, and Bio-Rad 1, as applied to claims 1, 4, and 5 above, and further in view of Wang [US 6504010 B1], ThermoFisher 1, and Biosearch Technologies [Biosearch Technologies. Know Your Oligo Mod: BHQ® (Black Hole Quencher ®) Dyes. 2015 July 23: 1-4. Accessed from the WayBack Machine on 21 July 2026] as evidenced by Yates [Yates TM et al. Clinical genetics. 2017 Jul;92(1):3-9].
Waters, Polansky, and Bio-Rad are applied to the relevant teachings of claims 1, 4, and 5 as discussed above and are incorporated herein by reference; however, none disclose a primer-probe set for the reference gene comprising SEQ ID NOs: 17-19 of the instant application.
Wang discloses SEQ ID NO: 439 which is 100% concordant with SEQ ID NOs: 17-19 of the instant application. When this sequence was searched, it was found to be 98.74% similar to Homo sapiens ACTB mRNA (NM_001001.5), the gene for β-actin, as described by Yates [Yates, abstract, Fig. 3]. Wang teaches that any of their disclosed polynucleotide sequences can be used as probes or primers for nucleic acid hybridization, stating that nucleic acid segments which have 15 contiguous nucleotides that have identical or complementary sequences to segments of the same contiguous length within the disclosed sequences will be of particular utility. However, longer contiguous sequences can be of use, including those of about 20 and 30 nucleotides (including all intermediate lengths) [Wang, C58L19-30].
Therefore, a person of ordinary skill in the art prior to the effective filing date of the claimed invention, seeking to include primers and probes specific for β-actin so that it could serve as a reference gene as discussed in the claim 5 rejection, would have found it obvious to develop them using Wang’s SEQ ID NO: 439, identified by Yates as corresponding to ACTB the transcript, as Wang expressly teaches that contiguous segments of the disclosed sequence are useful as primers and probes. Instant SEQ ID NOs: 17-19 are contiguous subsequences of Wang’s SEQ ID NO: 439 and are in the proper orientation to serve their intended functions, thus providing a reasonable expectation of success (see figure below).
In Example 7, Wang performs real-time PCR on lung tumor sequences in order to evaluate their expression in tumor and normal tissues using TaqMan probes labeled on the 5` end with FAM and on the 3` end with TAMRA, the results of which were normalized against internal actin [Wang, C109-110]. While Wang does not teach the reporter and quencher labels used for the reference gene probe, Wang does indicate that a wide variety of labels and conjugation techniques for producing labeled PCR probes are known in the art [Wang, C70L58-60]. ThermoFisher 1 teaches that TaqMan probes are routinely labeled on the 5` end with a FAM or VIC dye and on the 3` end with a MGB and a nonfluorescent quencher [ThermoFisher 1, p1]. Biosearch Technologies further describes the quenching mechanisms of TAMRA and BHQ dyes (BHQ-0, BHQ-1, BHQ-2, and BHQ-3) and teaches that the absorption range of the fluorescent report will play a large role in which quencher is picked [Biosearch Technologies, p1-3].
Therefore, it would have been obvious to modify the probe of Wang to include a 5` VIC reporter and a 3` BHQ1 quencher as suggested by ThermoFisher and Biosearch Technologies, as the specific probe labels used represent nothing more than a design choice for which there is a finite number options. The Supreme Court decided that a claim can be proved obvious merely by showing that the combination of known elements was obvious to try. In this regard, the Supreme Court explained that, “[w]hen there is a design need or market pressure to solve a problem and there are a finite number of identified, predictable solutions, a person of ordinary skill in the art has a good reason to pursue the known options within his or her technical grasp.” An obviousness determination is not the result of a rigid formula disassociated from the consideration of the facts of the case. Indeed, the common sense of those skilled in the art demonstrates why some combinations would have been obvious where others would not. Therefore, choosing from a finite number of identified, predictable solutions, with a reasonable expectation for success, is likely to be obvious to a person if ordinary skill in the art. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, E.).
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Claims 8-10 are rejected under 35 U.S.C. 103 as being unpatentable over Waters and Polanksy, as applied to claims 1 and 4 above, and further in view of Qiagen [Qiagen. QuantiTect® Multiplex RT-PCR Handbook. 2011 May: 1-68].
Waters and Polansky are applied to the relevant teachings of claims 1 and 4 as discussed above and are incorporated herein by reference. However, neither disclose additionally including a positive or negative control, a reverse transcriptase mixture, or a polymerase mixture comprised of DNA polymerase, MgCl2, buffer, and dNTPs.
Qiagen sells the QuantiTect® Multiplex RT-PCR Kit which is designed to provide real-time RT-PCR quantification of RNA targets for gene expression analysis in a multiplex format [Qiagen, p8]. Included in this kit are a polymerase, two reverse transcriptases, a dNTP mix, a buffer containing MgCl2, RNAase-free water, and a passive reference dye [Qiagen, p4,7]. Qiagen also advises the use of a no template control, a no RT control, and a positive control when conducting these assays. The no template control, which includes all reagents except for the template, and the no RT control, which includes all components including template but does not include reverse transcriptase, allow for detection of contamination. The positive control, which contains a known concentration or copy number of template, tests for the presence or absence of the target ensuring that the reaction proceeded as intended [Qiagen, p32].
This demonstrates that the claimed components are either commonly included in commercially available RT-qPCR kits used for gene expression assays or recommended to be used when performing said assays.
Having already been motivated by Polansky to incorporate the primer-probe set of claim 1 into a kit, one of ordinary skill in the art prior to the effective filing date of the claimed invention would have further found it obvious to include these conventional RT-qPCR reagents so that the resultant kit would contain all the reagents necessary to perform the intended assay. A rationale to support a conclusion that a claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination would have yielded nothing more than predictable results to one of ordinary skill in the art. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395 (2007) (see MPEP §§ 2143, A. and 2143.02).
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Claims 15, 16, and 27 are rejected under 35 U.S.C. 103 as being unpatentable over Evans [Evans K et al. Clinical Cancer Research. 2019 Jul 15;25(14):4493-503], Waters, and Irwin [Irwin RJ, Irwin TC. European Journal of Internal Medicine. 2011 Jun 1;22(3):230-4].
Evans investigated the efficacy of a novel AKR1C3-activated prodrug, OBI-3424, in preclinical models of pediatric acute lymphoblastic leukemia (ALL) [Evans, abstract]. They first carried out cytotoxicity assays on a broad range of leukemia cell lines and found that OBI-3424 exerted potent cytotoxicity particularly against cell lines derived from T-lineage ALL (T-ALL) with high AKR1C3 expression. Additionally, the difference in efficacy between cell lines with high/medium AKR1C3 expression and those with low expression was statistically significant.
Next, Evans assessed AKR1C3 expression in the bone marrow aspirates of diverse pediatric ALL subtypes with respect to OBI-3424 sensitivity. From this they determined that AKR1C3 expression was significantly higher in T-ALL than in B-lineage ALL (B-ALL) [Evans, p4495].
This expression pattern was further studied in 90 ALL patient-derived xenographs (PDXs) using RNA-seq data which was confirmed by RT-qPCR and immunoblotting and showed significantly higher expression of AKR1C3 in T-ALL samples versus B-ALL as well as T-ALL versus most normal tissue samples. These expression levels were determined to be significantly correlated with AKR1C3 protein expression, which was itself found to be inversely correlated with cell survival in PDXs treated with OBI-3424. An important take away from these studies was that the level of AKR1C3 expression appeared to be a more important determinant of in vitro OBI-3424 sensitivity than cell lineage [Evans, p4496].
Therefore, Evans clearly shows that AKR1C3 mRNA expression in an ex vivo patient-derived sample can be evaluated via RT-qPCR and is an important consideration in determining whether treatment of ALL with OBI-3424 will be effective.
However, in performing RT-qPCR, Evans does not use the primer-probe set of instant claim 1, and does not disclose administering OBI-3424 only if AKR1C3 mRNA expression is above a predetermined threshold.
As discussed above, Waters discloses the primer-probe set of claim 1 which is capable of identifying the genes encoding an AKR1C polypeptide, including AKR1C3, from mammalian tissues. As discussed in the rejection of claim 4, Waters teaches that their primers-probe set can be used in a variety of assays including real-time PCR and gene expression assays indicating the primer-probe set of Waters is capable of performing the same RT-qPCR reaction performed by Evans, as well as targeting the same gene. Therefore, a person of ordinary skill in the art prior to the effective filing date of the claimed invention, would have recognized the potential for these primer-probe sets to be substituted for one another as they both perform the same function within the same technological environment.
Irwin reviewed two methods commonly used in medical science for locating diagnostic threshold on a continuum, ROC and indifference curves, and presents a third option which combines them resulting in a method that identifies thresholds which maximize the diagnostic utility of a given biomarker [Irwin, abstract, p230]. He specifically states that this combined method identifies diagnostic decision thresholds which achieve any sensible goal [Irwin, p233]. As a result, Irwin clearly shows that processes for choosing biomarker thresholds at which diagnostic decisions can be made are well established within the art.
As mentioned, Evans recognizes that AKR1C3 mRNA expression is predictive of sensitivity to OBI-3424. Therefore, a person of ordinary skill in the art prior to the effective filing date of the claimed invention would have been motivated to apply the threshold selection methods taught by Irwin to the AKR1C3 expression values obtained using the RT-qPCR assay of Evans, in which the primer-probe set of Waters was used, in order to identify an objective expression threshold at which administration of OBI-34234 would be expected to provide clinical benefit. Doing so amounts to applying a known technique to a known device (method or product) ready for improvement to yield predictable results which is likely to be obvious. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, D.).
Regarding claim 15, OBI-3424 appears to have the same chemical structure as one of the claimed compounds. A portion of Evans, Fig. 1A is replicated below to demonstrate this. Due to the image quality of the chemical structures in both the claims and the specification of the instant application, it is unclear which of the three claimed compounds exactly matches. However, page 1 of the instant specification names these compounds OBI-3424, OBI-3423, and OBI-2870 respectively. Therefore, based upon the apparent similarity in structure and the sharing of name, it can be assumed that the first claimed compound and Evan’s OBI-3424 are identical.
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Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Evans, Waters, and Irwin, as applied to claim 27 above, and further in view of ThermoFisher 2 [ThermoFisher Scientific. Absolute vs. Relative Quantification for qPCR. 2018 Jun 27:1-5. Accessed from the WayBack Machine on 23 July 2026].
Evans, Waters, and Irwin are applied to the relevant teachings of claim 27 as discussed above and are incorporated herein by reference. When Evans performed RT-qPCR, AKR1C3 mRNA expression was quantified with TaqMan Gene Expression Assays and elongation factor-1α was used as an internal standard. mRNA levels were quantified using the 2-ΔΔCt method and normalized to control HeLa cells [Evans, Supplemental p3].
According to ThermoFisher 2, the original gene amount in a PCR reaction can be deduced from cycle threshold (Ct) values due to a mathematical relationship that exists between Ct and quantity. Two methods exist to transform Ct values into quantities: the standard curve method and the ΔΔCt method. If desired, these transformed values can be normalized using an endogenous control. To create a standard curve, a dilution series is run and a best fit line is calculated where the y axis represents the Ct value and the x axis represents the log(quantity). This line of best fit can then be used to calculate the log(quantity) of an unknown sample by inputting the obtained Ct value. Then, to normalize to an endogenous control, you would simply divide the quantities [ThermoFisher 2, p2, 5].
On the other hand, the ΔΔCt method does not require the use of standard curves. While variations in this method exist, the traditional version involves performing normalization first before transforming Ct values into quantities. This normalization simply involves subtracting the Ct of the target gene by the Ct of the reference gene and/or a calibrator sample, producing either ΔCt or ΔΔCt values. These values are then converted to quantity using the equation (Quantity = 2-ΔΔCt) [ThermoFisher 2, p5].
Therefore, because ThermoFisher 2 teaches that gene expression may be quantified using any of these methods, a person of ordinary skill in the art prior to the effective filing date of the claimed invention would have recognized that each performs the same fundamental function of quantifying nucleic acids and are well known in the art. Accordingly, modifying the method of Nakara 1 and Waters to calculate gene expression use the standard curve method would amount to nothing more than the combination of known methods to yield predictable results. A rationale to support a conclusion that a claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination would have yielded nothing more than predictable results to one of ordinary skill in the art. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395 (2007) (see MPEP §§ 2143, A. and 2143.02).
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Nakarai 1 [Nakarai C et al. Clin Exp Med. 2014 June 17;15:333-341] in view of Waters.
Nakarai 1 performed a study in an effort to identify a set of biomarkers which could be used to predict lymph node metastasis in human colorectal cancer. By comparing the whole genomes of two colorectal cancer cell lines, they initially identified eight contenders. These genes were then interrogated by RT-qPCR and significant results were obtained for both AKR1C3 and CNN3 [Nakarai 1, p333].
To perform RT-qPCR, RNA was extracted and cDNA synthesized from tissue samples. The cDNA was then amplified using gene specific primers, including primers for AKR1C3 and ACTB, in a qPCR reaction. ACTB was used to calculate the relative level of expression for each of the target genes using the 2-ΔΔCt method [Nakarai 1, p335].
While Nakarai 1 targeted the AKR1C3 gene, they did not use the same primers as those described by claim 1. As discussed above, Waters discloses the primer-probe set of claim 1 which are capable of identifying the genes encoding an AKR1C polypeptide, including AKR1C3, from mammalian tissues. The primers of Nakarai 1 and Waters are both directed toward the same target. As discussed in the rejection of claim 4, Waters teaches that their primers can be used in a variety of assays including real-time PCR and gene expression assays indicating that Waters’ primers are capable of performing the same RT-qPCR reaction performed by Nakarai 1, as well as targeting the same gene. Therefore, a person of ordinary skill in the art prior to the effective filing date of the claimed invention, would have recognized the potential for these primers to be substituted for one another as they both perform the same function within the same technological environment.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Nakarai 1 and Waters, as applied to claim 17 above, as evidenced by Nakarai 2 [Nakarai C, et al. Anticancer research. 2012 Sep 1;32(9):3753-8].
Nakarai 1 and Waters are applied to the relevant teachings of claim 17 as discussed above and are incorporated herein by reference.
The steps of tissue preparation, RNA extraction, and cDNA synthesis performed by Nakarai 1 were conducted as described in Nakarai 2. Nakarai 2 extracted and purified total cellular RNA from tissue samples. The purified RNA was quantified and assessed for purity by UV spectrophotometry. Finally, cDNA was synthesized using the ReverTra Ace qPCR RT Kit according to the manufacturer’s protocol [Nakarai 2, p3754].
Claims 19 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Nakarai 1 and Waters, as applied to claim 17 above, and further in view of Hawkins [Hawkins SF, Guest PC. Multiplex Biomarker Techniques: Methods and Applications. 2016 Nov 29:125-33], Troutman [Troutman M. What’s the Right Master Mix for Your qPCR Experiment? ThermoFisher. 2015 July 22: 2pg], and Qiagen, as evidenced by Bio-Rad 2 [Bio-Rad. SsoFast™ EvaGreen® Supermix product page. 2018 Mar 31: 1pg. Accessed from the WayBack Machine on 23 July 2026].
Nakarai 1 and Waters are applied to the relevant teachings of claim 17 as discussed above and are incorporated herein by reference. However, neither teach mixing the primer-probe composition for the target gene with the primer-probe composition of the reference gene nor discuss the specific reaction components added to the qPCR reaction system.
Regarding mixing of the primer-probe sets, Hawkins teaches that multiplexing qPCR reactions allows for measurement of the expression levels of several targets or genes of interest quickly. However, in order to do this, probe-based assays must be used in which each probe is labeled with a unique fluorescent dye [Hawkins, abstract]. Nakarai 1 used the SsoFast™ EvaGreen Supermix® system which detects PCR products using EvaGreen, a fluorescent nucleic acid dye similar to SYBR® Green I per the product page, rather than fluorescent probes [Nakarai 1, p335; Bio-Rad 2, p1].
However, Troutman describes the similarities and differences between qPCR detection methods using DNA binding dyes and fluorescent probes. According to Troutman, the deciding factor in choosing a detection method will likely lie in your intended application as probe-based assays impart great levels of specificity, while DNA binding assays are more flexible. For gene expression experiments, for example, the high specificity of the probe-based system may be preferred [Troutman, p2].
The skilled artisan would therefore recognize that use of either a probe-based or DNA binding dye system represents an assay design choice and that modifying the RT-qPCR reaction of Nakarai 1 to use fluorescent probes as opposed to a DNA binding dye in order to achieve the increased specificity as described by Troutman, as well the ability to multiplex as described by Hawkins, would amount to nothing more than the application of a known technique to a known device ready for improvement to yield predictable results.
Regarding the reaction components, Qiagen sells the QuantiTect® Multiplex RT-PCR Kit which was specifically designed to provide real-time RT-PCR quantification of RNA targets for gene expression analysis in a multiplex format [Qiagen, p8]. Included in this kit are a polymerase, two reverse transcriptases, a dNTP mix (which includes dUTP), a buffer containing MgCl2, RNAase-free water, and a passive reference dye [Qiagen, p4,7]. While uracil-N-glycosylase (UNG) is not included in this kit, Qiagen provides instructions for the inclusion of this enzyme in the reaction mix if desired by the user [Qiagen, p10]. This demonstrates that the claimed components are commonly included in multiplexed RT-qPCR assays and would be obvious to include in a reaction mix designed to perform the same type of assay. The combination of familiar elements is likely to be obvious when it does no more than yield predictable results. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, A.).
Claims 21-23 are rejected under 35 U.S.C. 103 as being unpatentable over Nakarai 1 and Waters, as applied to claim 17 above, and further in view of ThermoFisher 2 and ThermoFisher 3 [ThermoFisher Scientific. Efficiency of Real-Time PCR. 2018 Aug 13:1-5. Accessed from the WayBack Machine on 23 July 2026].
Nakarai 1 and Waters are applied to the relevant teachings of claim 17 as discussed above and are incorporated herein by reference. However, while Nakarai 1 states that they used the ΔΔCt method to calculate mRNA expression, neither discuss the use of a digital PCR (dPCR) system, generating a standard curve, or calculating a ratio of AKR1C3/ACTB copy number.
According to ThermoFisher 2, the original gene amount in a PCR reaction can be deduced from cycle threshold (Ct) values due to a mathematical relationship that exists between Ct and quantity. Two methods exist to transform Ct values into quantities: the standard curve method and the ΔΔCt method. If desired, these transformed values can be normalized using an endogenous control. To create a standard curve, a dilution series is run and a best fit line is calculated where the y axis represents the Ct value and the x axis represents the log(quantity). This line of best fit can then be used to calculate the log(quantity) of an unknown sample by inputting the obtained Ct value. Then, to normalize to an endogenous control, you would simply divide the quantities [ThermoFisher 2, p2, 5].
On the other hand, the ΔΔCt method does not require the use of standard curves. While variations in this method exist, the traditional version involves performing normalization first before transforming Ct values into quantities. This normalization simply involves subtracting the Ct of the target gene by the Ct of the reference gene and/or a calibrator sample, producing either ΔCt or ΔΔCt values. These values are then converted to quantity using the equation (Quantity = 2-ΔΔCt) [ThermoFisher 2, p5].
As an alternative to both methods, digital PCR can be used to determine the absolute copy number of a target gene within a sample and does not rely upon a standard curve or endogenous controls. Each sample is partitioned into many individual real-time PCR reactions so that some partitions contain the target gene and are positive while others do not and are negative. The ratio of positive and negative reactions is used to count the exact number of target molecules in the sample [ThermoFisher 3, p2].
Each method has their own benefits [ThermoFisher 3, p4]:
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Therefore, because ThermoFisher 2 and 3 teach that gene expression may be quantified using any of these methods, a person of ordinary skill in the art prior to the effective filing date of the claimed invention would have recognized that each method possesses its own advantages and disadvantages, performs the same fundamental function of quantifying nucleic acids, and are well known in the art. Accordingly, modifying the method of Nakara 1 and Waters to calculate gene expression using either dPCR or the standard curve method would amount to nothing more than the combination of known methods to yield predictable results.
A rationale to support a conclusion that a claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination would have yielded nothing more than predictable results to one of ordinary skill in the art. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395 (2007) (see MPEP §§ 2143, A. and 2143.02).
Claim 25 is rejected under 35 U.S.C. 103 as being unpatentable over Nakarai 1 and Waters, as applied to claim 17 above, and further in view of Evans.
Nakarai 1 and Waters are applied to the relevant teachings of claim 17 as discussed above and are incorporated herein by reference. Neither discuss using the expression levels of AKR1C3 mRNA in the samples to determine the expression of the AKR1C3 enzyme in the sample.
Evans investigated the efficacy of a novel AKR1C3-activated prodrug, OBI-3424, in preclinical models of pediatric acute lymphoblastic leukemia (ALL) [Evans, abstract]. As part of this investigation, Evans assessed AKR1C3 expression in the bone marrow aspirates of diverse pediatric ALL subtypes with respect to OBI-3424 sensitivity. From this they determined that AKR1C3 expression was significantly higher in T-ALL than in B-lineage ALL (B-ALL).
This expression pattern was further studied in 90 ALL patient-derived xenographs using RNA-seq data which was confirmed by RT-qPCR and immunoblotting and showed significantly higher expression of AKR1C3 in T-ALL samples versus B-ALL and most normal tissue samples. These expression levels were determined to be significantly correlated with AKR1C3 protein expression (see Evans, Supp. Fig. 4 below), which was itself found to be inversely correlated with cell survival in PDXs treated with OBI-3424 [Evans, p4495-96].
This proven relationship demonstrates that measuring the amount of AKR1C3 mRNA in a sample can be used to determine the amount of AKR1C3 enzyme in said sample. Therefore, a person of ordinary skill in the art prior to the effective filing date of the claimed invention, would have been motivated to use the method taught by Nakarai 1 and Waters to determine AKR1C3 enzyme expression because Evans teaches that mRNA presence is predictive of enzyme presence.
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Conclusion
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/K.N.K./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
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