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 .
DETAILED ACTION
Claims 1 – 9 and 11 – 20 are pending in this application. Applicant’s preliminary amendment, submitted May 30, 2024, is entered, wherein claims 7, 9, 11 – 12, 14 – 16, and 18 – 20 are amended and claim 10 is canceled.
Claims 1 – 9 and 11 – 20 are examined on the merits herein.
Priority
This application is a national stage application of PCT/IB2022/061738, filed December 3, 2022, which claims benefit of provisional application 63/285,996, filed December 3, 2021.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 05/30/2024 was filed in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner.
Claim Objections
Claim 4 is objected to because of the following informalities:
Claim 4, line 12, “I” should read “i”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
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.
Claims 4 – 6 are 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 depends from claim 1. Claim 1 requires that R3 and R4 are each independently H or C1-4 alkyl. Claim 4, however, defines R3 and R4 as each independently H, C1-4 alkyl, or a peptide of 1 to 10 amino acids in length that is covalently linked using a bioconjugation reaction or click reaction. Thus, claim 4 encompasses embodiments in which R3 and/or R4 is a peptide, whereas such embodiments are outside the scope of claim 1, which limits R3 and R4 to H or C1-4 alkyl. Accordingly, claim 4 does not merely further limit the subject matter of claim 1, but introduces subject matter not encompassed by claim 1 and is therefore not in proper dependent form. Claims 5 – 6 depend directly from claim 4 and include embodiments based on the expanded definition of R3 and R4. Accordingly, claims 4 – 5 are also not in proper dependent form. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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 9, 11 – 13, and 15 – 17 are rejected under 35 U.S.C. 101 because the claimed invention is directed to judicial exception (e.g. law of nature, a natural phenomenon, or an abstract idea) without significantly more.
Claim 9 recites a method for determining or localizing GCase activity in a cel comprising providing a test cell and a control cell, contracting the test cell with the recited fluorescence-quenched substrate, and determining or visualizing fluorescence intensity, wherein an increase in fluorescence intensity of the test cell relative to the control cell, or an increase in fluorescence intensity at a location in the test cell relative to a corresponding location in the control cell, is indicative of GCase activity. The claim therefore recites the biological relationship between GCase-mediated processing of the substrate and the resulting increase in fluorescence intensity, whereby an observed increase in fluorescence relative to a control is used as an indication of the presence or location of GCase activity.
Step 1: Claims 9, 11 – 13, and 15 – 17 are drawn to methods, namely a process, and therefore fall within a statutory category of invention under 35 U.S.C. 101.
Step 2A1: Claim 9 recites judicial exceptions in the form of a law of nature or natural biological relationship and an abstract ideal in the mental process grouping. In particular, claim 9 relies upon the relationship between GCase enzymatic activity and cleavage or processing of a GCase substrate, which results in separation of the fluorophore and quencher and a corresponding increase in fluorescence. The claim then uses the observed fluorescence increase relative to a control as an indication that GCase activity is present or localized in the cell. Thus, the claim recites the biological correlation that the GCase-mediated cleavage/processing of the fluorescence-quenched GCase substrate produces increases fluorescence, such that an increase in fluorescence intensity relative to a control is indicative of GCase activity. This relationship reflects a naturally occurring enzymatic process and therefore constitutes a law of nature or natural biological relationship. Claim 9 further recites a mental process because step (c) requires determining, based on the observed fluorescence intensity in the test cell and control cell, that an increase in fluorescence is indicative of GCase activity, or determining the localization of GCase activity based on an increase in fluorescence at a location in the test cell relative to a corresponding location in the control cell. This determination involves evaluating the observed fluorescence information and drawing a conclusion regarding the presence or location of GCase activity, which is an evaluation that may be practically performed in the human mind. Accordingly, the determining portion of step (c) recites an abstract idea in the mental process grouping. The visualization or observation of the fluorescence signal does not create the underlying enzymatic relationship between GCase activity and GCase-mediated substrate processing; instead, it provides the fluorescence information used in the claimed analysis. The determination that the observed fluorescence increase is indicative of GCase activity, or that GCase activity is localized based on the observed fluorescence information, constitutes the mental process judicial exception discussed above.
Claims 11 – 13 and 15 – 17 depend directly or indirectly from claim 9 and therefore include the same judicial exceptions, which are the natural biological relationship between GCase activity and substrate-derived fluorescence and the mental process of determining GCase activity from the observed fluorescence information. Claim 11 further limits the location at which GCase activity is determined or localized. Claims 12 – 13 further concern determination of GCase modulation and specify the type of GCase modulator, including an inhibitor, activator, or chaperone. Claim 15 limits the cells to fibroblasts or PBMCs; claim 16 limits the cells to cells derived from tissue; claim 17 further limits the tissue to a skin punch. These additional limitations do not remove the recited relationship between fluorescence intensity and GCase activity.
Step 2A2: Claim 9, considered as a whole, does not integrate the recited judicial exception into a practical application. The steps of providing the cells, contacting the test cell with the fluorescence-quenched substrate, and observing or measuring fluorescence provide the information used in the claimed analysis. The further determination that the observed fluorescence increase is indicative of GCase activity constitutes the recited mental process judicial exception. Thus, the claim combines physical data gather steps with the mental evaluation of the resulting fluorescence information and the recited natural biological relationship. After the fluorescence is generated and observed, the claim concludes that an increase in fluorescence intensity is “indicative of GCase activity”. The claim does not require any further action based upon the determination or localization of GCase activity. Thus, the additional limitations merely provide the data used to observe and apply the recited biological relationship rather than meaningfully applying the result to effect a treatment, prophylaxis, modification of therapy, or other further result. Mere data gathering that is necessary to obtain information for application of a judicial exception is an example of insignificant extra solution activity that does not, by itself, integrate the exception into a practical application. The eligibility analysis considers the additional limitations individually and in combination and asks whether they impose a meaningful limit on the exception. The providing, contacting, and fluorescence observation visualization steps provide the information from which the recited GCase/fluorescence relationship is applied, but they do not require a further practical use of the GCase determination.
Claims 11 – 13 and 15- 17 also do not integrate the judicial exception into a practical application. Claim 11 merely further specifies the intracellular location at which the same fluorescence/GCase relationship is observed. Claims 12 – 13 merely further define the biological context in which fluorescence differences are used to identify GCase modulation and specify particular categories of GCase modulators. Claims 15 – 17 merely limit the cells or source of the cells to fibroblasts or PBMCs, tissue-derived cells, or skin punch-derived cells. These limitations narrow the circumstances under which the same biological relationship is observed but do not meaningfully later the manner in which the judicial exception is used. Accordingly, claims 9, 11 – 13, and 15 – 17 are directed to the recited judicial exceptions under Step 2A.
Step 2B: This part of the eligibility analysis evaluates whether the claim as a whole amounts to significantly more than the recited judicial exception, i.e., whether any additional element or combination of additional elements adds an inventive concept to the claim.
The additional elements of claims 9, 11 – 13, and 15 – 17, considered individually and as an ordered combination, do not amount to significantly more than the recited judicial exception. Claim 9 additionally requires providing a test cell and a control cell, contacting the test cell with a fluorescence-quenched GCase substrate, and physically observing or visualizing the resulting fluorescence signal. These assay steps generate and provide the fluorescence information to which the recited natural biological correlation and mental process determination are applied. As discussed above, the claim ultimately uses the measured or visualized fluorescence difference to determine or localize GCase activity and does not require a further technological or therapeutic action based on that determination. Yadav et al. (Journal of the American Chemical Society, 2015, Vol. 137, Issue 3, page 1181 – 1189, PTO-892) teach fluorescence-quenched GCase substrates for monitoring endogenous GCase activity in living cells, including contacting cells with such probes, observing fluorescence resulting from GCase-mediated processing, localizing GCase activity within lysosomal compartments, and assessing changes in GCase activity using fluorescence imaging (Abstract). Thus, the use of fluorescence-quenched GCase substrates together with cellular fluorescence measurements is known in the art as an approach for monitoring GCase activity. Accordingly, the additional elements of claim 9, considered individually and as an ordered combination, do not amount to significantly more than the recited biological relationship. The steps of providing a test cell and a control cell, contacting the test cell with the fluorescence-quenched substrate, and observing or visualizing fluorescence provide the data used in applying the recited judicial exceptions. These steps do not change the basic function of the claimed method, which remains determining or localizing GCase activity based on the observed fluorescence response.
Claims 11 – 13 and 15 – 17 also do not add significantly more. Claim 11 further limits the intracellular location at which GCase activity is determined or localized. Claims 12 – 13 further limit the method to determining modulation of GCase activity and specify particular classes of GCase modulars. Claims 15 – 17 further limit the type or source of the cells to fibroblasts or PBMCs, tissue-derived cells, and skin punch-derived cells, respectively. These additional limitations merely narrow the biological context in which the same relationship between GCase activity and fluorescence intensity is applied and do not add a further technological or therapeutic action beyond determining or localizing GCase activity.
When considered as an ordered combination, the additional elements do not transform the nature of the claim into a patent-eligible application. Instead, the claimed steps operate together to generate and observe fluorescence signal, which is then evaluated according to the recited mental process and biological relationship. Therefore, the additional elements of claims 9, 11 – 13, and 15 – 17, individually and in combination, do not amount to significantly more than the judicial exception. Accordingly, claims 9, 11 – 13, and 15 – 17 are not patent eligible under 35 U.S.C. 101.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or
nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 4 – 5, 7 – 9, 11 – 13, 15 – 16, and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Yadav et al. (Journal of the American Chemical Society, 2015, Vol. 137, Issue 3, page 1181 – 1189, PTO-892) in view of Kanie et al. (US6358919B1).
Yadav et al. teach fluorescence-quenched substrates for live cell imaging of human glucocerebrosidase activity (Title). Yadav et al. indicate that determining the levels of GCase activity present within the lysosomes of live cells, such as visualizing the activity of endogenous levels of any glycoside hydrolases, including GCase, within live mammalian cells, has been difficult. Yadav et al. teach the successful modular design and synthesis of fluorescence-quenched substrates for GCase, wherein the selection of a suitable fluorophore and quencher pair permits the generation of substrates that allow a convenient time-dependent monitoring of endogenous GCase activity within cells as well as localization of activity within lysosomes. These efficiently quenched (~99.9%) fluorescent substrates also permit assessment of GCase inhibition in live cells by either confocal microscopy of high content imaging (Abstract). Yadav et al. explicitly teach fluorescent quenched substrates 9b by the following synthesis (page 1183, Scheme 2):
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wherein substrate 9b comprises a glucopyranoside scaffold bearing a BHQ2 quencher and a BODIPY 576/589 fluorophore (page 1183, Right Col., para. 1). Thus, substrate 9b meets the claimed glucopyranoside core and fluorophore/quencher arrangement, wherein R1 is a fluorophore; R2 is
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, wherein k is 2 and R8 is a quencher; h is 1; i is 1; and Y is O. However, as discussed below, Yadav et al. do not teach the entire claimed substituent extending from the triazole toward the fluorophore-bearing amino acid-derived moiety. Substrate 9b is synthesized by first attaching a BocNHCH2CH2N3 linker. A copper (I) catalyzed azide-alkyne cycloaddition is performed between the linker and the alkyne moiety to yield the terminal Boc-protected amine. After removal of the Boc protecting group, the resulting amine intermediate is reacted with the N-hydroxysuccinimide ester of BODIPY 576/589 (BODIPY-NHS) to furnish the substrate 9b (page 1183, Right Col., para. 2). Thus, Yadav et al. explicitly teach substrate 9b as a fluorescence-quenched substrate for GCase, wherein the GCase is a human GCase, which read on the limitations of claims 7 – 8. Yadav et al. further teach that the fluorescence associated with cleavage of substrate 9b occurs in the lysosomal compartments of the cells, and that analysis of the Z-direction stack indicates substrate processing is occurring within lysosomes (page 1184, Right Col., para. 1). The disclosure reads on the limitation of claim 11, wherein the location is the lysosomal compartment. Yadav et al. further use substrate 9b for live cell imaging of GCase activity in human fibroblasts (page 1183, Right Col., para. 3), wherein the human fibroblasts are human primary skin fibroblasts (page 1184, Left Col., para. 1). Thus, Yadav et al. teach the cells are fibroblasts from skin, which reads on the limitations “fibroblasts” of claim 15 and “tissue” of claim 16. Yadav et al. incubate cells with conduritol B epoxide (CBE), a GCase inactivator, or vehicle alone for 16 h, then treated the cells with both substrate 9b and DAPI for 2 h. Thus, Yadav et al. indicate that CBE is a GCase inactivator, which reads on the limitation “GCase inhibitor” of claim 13. The CBE-treated cells show little fluorescence as compared to vehicle-treated control cells. These data indicate that substrate 9b is predominantly processed by GCase within cells, supporting its use as a tool to image GCase activity within live cells across a range of cell densities (page 1184, Right Col., para. 2). Thus, Yadav et al. teach the use of substrate 9b for determining GCase activity in a cell with the steps of providing a test cell and a control cell, contacting the test cell with substrate 9b, and determining fluorescence intensity in the test cell and control cell, which corresponds to the limitation of claims 9 and 12. The disclosure of Yadav et al. read on the limitation “wherein a difference in fluorescence intensity of the test cell when compared to the control cell is indicative of GCase modulation” of claim 12 because Yadav et al. compare cells treated with GCase inhibitor to vehicle-treated control cells and show that CBE-treated cells exhibit decreased fluorescence from substrate 9b relative to the control cells, demonstrating that a change in fluorescence intensity reflects modulation of GCase activity. Moreover, Yadav et la. disclose that the compatibility of this approach with high content imaging in live cells opens the door to screening for endogenous protein modifiers of GCase activity as well as screening for activators that function within cells to influence GCase activity (page 1185, Right Col., para. 1). Thus, Yadav et al. suggest that the same method disclosed is capable of screening for GCase activity activators, which corresponds to the “method of screening for a GCase activity enhancer” of claim 18.
However, Yadav et al. do not teach the entire claimed amino acid-derived amide-containing substituent extending from the triazole, including the –(CH2)iNH-C(O) linkage to the substituted carbon bearing the aminoalkyl side chain (CH2)jNR3R4, wherein j is an integer from 1 to 5; R3 and R4 are hydrogen.
Kanie et al. teach that a polymer compound comprising a saccharide-conjugated sphingosine (Col. 1 lines 6 – 7). Such polymer compound may contain a fluorescent functional group so that intracellular transmigration can be visually traced. The fluorescent functional group may preferably be bound to a spacer moiety. Kanie et al. identify BODIPY as a suitable fluorescent functional group and explicitly teach that a fluorescent functional group may be attached through a spacer, preferably a trifunctional amino acid, such as lysine, used as one amino acid spacer (Col. 4, lines 41 – 51). Thus, when BODIPY is attached through such a spacer, the lysine residue serves as the intervening spacer between the fluorescent functional group and the molecule to which the spacer is coupled. Kanie et al. further exemplify this by reacting BODIPY FL C3 succinimidyl ester with NƐ-Fmoc-lysine to obtain Nα-BODIPY- NƐ-Fmoc-lysine. Kanie et al. then convert the carboxyl group of the BODIPY-bearing lysine derivative to a succinimidyl ester and react that activated ester with lyso GM3 to obtain the corresponding BODIPY-lysine-Lyso GM3 conjugate in quantitative yield. Kanie et al. subsequently remove the Fmoc protecting group to provide the free lysine side chain amino group:
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wherein R3 represents hydrogen and R4 represents a fluorescent functional group (Col. 7, lines 57 – 67; Col. 8, lines 1 – 49; claim 12). Thus, Kanie et al. teach a BODIPY-bearing lysine-derived spacer having the BODIPY fluorophore attached through the α-amino functionality, a carboxyl functionality capable of coupling to an external amino group, and following deprotection, a free aminoalkyl side chain corresponding to (CH2)4NH2, wherein the claimed j is 4 and R3 and R4 are hydrogen recited in claims 1 and 4 – 5.
It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the fluorescence-quenched substrate 9b of Yadav et al. by substituting the BODIPY-bearing lysine spacer in view of Kanie et al. for the BODIPY-containing fluorophore reagent employed by Yadav et al. at the terminal amine coupling step because Yadav et al. teach a modular synthesis in which reaction of the alkyne-containing glucopyranoside with BocNHCH2CH2N3 forms the triazole-containing linker, followed by removal of the Boc protecting group to provide a terminal amine and thereafter couple the terminal amine with an NHS-activated BODIPY reagent to provide the BODIPY-containing fluorescence-quenched substrate 9b and Kanie et al., in turn, teach that a fluorescent functional group may preferably be bound through a spacer moiety, identify BODIPY as a suitable fluorescent functional group and explicitly teach that a trifunctional amino acid, such as lysine, may be preferably be employed as such a spacer. Thus, Kanie et al. explicitly identify a lysine-derived spacer as a preferred spacer for attachment of a BODIPY fluorescent functional group. Kanie et al. further demonstrate the manner in which the BODIPY-bearing lysine spacer may be incorporated into an amino-containing molecule. Kanie et al. teach a BODIPY-bearing lysine spacer having a carboxyl group that may be coupled to an external amino group. Coupling that carboxyl group to the terminal amine of the triazole-containing linker of Yadav et al. would provide the missing amide-linked amino acid-derived portion, including the aminoalkyl side chain. Specifically, Kanie et al. prepare Nα-BODIPY- NƐ-Fmoc-lysine, convert the lysine carboxyl group to a succinimidyl ester, and react the activated ester with the amino group of Lyso GM3 to form the corresponding amide-liked conjugate. Accordingly, one of ordinary skill in the art, following the explicitly teachings of Kanie et al., would have recognized that the terminal amine provided by Yadav et al. may similarly be reacted with the NHS-activated carboxyl group of the BODIPY-bearing lysine spacer of Kanie et al., rather than with the BODIPY-containing NHS ester of Yadav et al., thereby incorporating the lysine-derived spacer and its aminoalkyl side chain into the substrate of Yadav et al. One of ordinary skill in the art would have had a reasonable expectation of success in making this modification because both Yadav et al. and Kanie et al. demonstrate the same conventional amine/NHS-ester amide-forming coupling chemistry. Yadav et al. demonstrate that the terminal amine of the triazole-containing linker is suitable for coupling to an NHS-activated BODIPY reagent, while Kanie et al. demonstrate that the carboxyl functionality of the BODIPY-bearing lysine spacer may be activated as a succinimidyl ester and successfully coupled to an external amino group. The proposed modification therefore would not have required development of a new coupling reaction, but rather the predictable application of an explicitly taught BODIPY-bearing lysine spacer using a coupling reaction already demonstrated by both references. The resulting structure would retain the BODIPY fluorescent functionality while additionally providing the lysine-derived aminoalkyl side chain taught by Kanie et al.
Regarding “an increase in fluorescence intensity of the test cell when compared to the control cell is indicative of GCase activity” of claim 9, Yadav et al. teach determining GCase activity by measuring fluorescence intensity in cells contacted with the fluorescence-quenched GCase substrate. Because enzymatic cleavage of the substrate by GCase separates the fluorophore and quencher and generates fluorescence, an increase in fluorescence intensity relative to a control cell is indicative of increased GCase activity. Yadav et al. demonstrate this relationship by showing reduced fluorescence in cells treated with the GCase inhibitor CBE relative to vehicle-treated control cells, thereby establishing that differences in fluorescence intensity reflect differences in GCase activity.
Claims 2 and 19 – 20 are rejected under 35 U.S.C. 103 as being unpatentable over Yadav et al. (Journal of the American Chemical Society, 2015, Vol. 137, Issue 3, page 1181 – 1189, PTO-892) in view of Kanie et al. (US6358919B1) as applied to claims 1, 4 – 5, 7 – 9, 11 – 13, 15 – 16, and 18 above, and further in view of Vocadlo (WO2018/229663A1), Rudd et al. (Journal of the American Chemical Society, 2020, Vol. 142, Issue 42, page 17887 – 17891, PTO-892), and Rajopadhye et al. (WO2009/114776A1), with evidence provided by PubChem (PubChem 2026, PTO-892).
The combined teachings of Yadav et al. and Kanie et al. teach the limitations discussed above. As discussed above, Yadav et al. teach the specific BODIPY 576/589 used as a fluorophore. Accordingly to PubChem, BODIPY 576/589 corresponds to the 3-(5,5-difluoro-7-(1H-pyrrol-2-yl)-5H-dipyrrolo[1,2-c:2,1'-f][1,3,2]diazaborinin-4-ium-5-uid-3-yl)propanoyl (page 9, Synonyms 1 and 12).
However, Yadav et al. and Kanie et al. do not teach all of the particular selections from Formula (I) of claim 1 required by claims 2 and 19. In particular, Yadav et al. teach substrate 9b in which the BODIPY fluorophore is positioned on the triazole-containing side of the substrate and the BHQ2 quencher is positioned on the opposing anomeric side linker. Claims 2 and 19 require the converse relative fluorophore/quencher arrangement of Formula (I), wherein R1 is the specifically recited quencher and R8 is the specifically recited fluorophore. Yadav et al. and Kanie et al. also do not teach the recited value j = 3.
Vocadlo also teaches fluorescence-quenched substrates of exo-glycosidase enzymes, such as human exo-glycosidase, wherein the human exo-glycosidase is GCase (para. [0001]; para. [0056]). Vocadlo provides methods for imaging exo-glycosidase enzymes activity within cells (Abstract). The fluorescence-quenched substrate is a compound of Formula (I):
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wherein R6 is a fluorophore and R7 is a quencher, as well as the converse arrangement wherein R6 is a quencher and R7 is a fluorophore (para. 0008]). Vocadlo further teaches that the aldehyde of Formula (V) will be fluorescent, as the fluorophore R6 is no longer internally quenched by the quencher R7, and that when R7 is a suitable fluorophore, the alcohol of Formula (VI) will be fluorescent, as the fluorophore R7 is no longer internally quenched by the quencher R6:
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Vocadlo states that measuring fluorescence from either Formula (V) or Formula (VI) provides a method for monitoring exo-glycosidase activity (page 20, lines 12 – 17). Thus, Vocadlo teaches that the relative placement of the fluorophore and quencher either relative fluorophore/quencher arrangement is operable in a fluorescence-quenched glycosidase substrate because enzymatic cleavage relieves internal quenching and produces a fluorescent product in either orientation. .
Rudd et al. teach a live-cell fluorogenic probe (Title). The probe comprises BODIPY FL as the fluorophore and the Black Hole Quencher-1 (BHQ-1) as the quencher. BHQ-1 is selected as the quencher because it exhibits high quenching efficiency at the 509 nm emission maximum of BODIPY FL (page 17887, Right Col., para. 3). Thus, Rudd et al. teach that BHQ1 is a suitable complementary quencher for a BODIPY fluorophore based on spectral overlap and quenching efficiency.
Rajopadhye et al. teach imaging agents comprising an imaging reporter, including a fluorophore, chemically linked to a targeting moiety through an optional linker or spacer. Rajopadhye et al. teach that linker/spacer moieties may be used to chemically link imaging reporters to other components of the imaging agent and that useful linkers include natural and non-natural amino acids (para. [0012]; [0089]). More particularly, Rajopadhye et al. teach that a linker may be a derivative of a diamine, wherein the diamine provides a linker arm of varying lengths and chemistries for chemically linking molecules, and explicitly identify diamine-amino acids including homolysine, lysine, ornithine, diaminobutyric acid, and diaminopropionic acid as suitable examples (para. [0092]). Thus, Rajopadhye et al. teach lysine and ornithine as alternative amino acid-derived linkers in fluorescent imaging agents, with ornithine providing the shorter aminoalkyl side chain –(CH2)3NH2 corresponding to j = 3. Rajopadhye et al. further teach conventional attachment of imaging reporters through NHS-ester/amine coupling (para. [0091]; [0094]).
It would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the fluorescence-quenched GCase substrate of Yadav et al., as modified by Kanie et al., to employ the converse relative placement of the fluorophore and quencher required by claims 2 and 19 in view of Vocadlo because Yadav et al. teach substrate 9b having the BODIPY fluorophore on the triazole-containing side and the BHQ2 quencher on the anomeric side linker, whereas Vocadlo explicitly teaches fluorescence-quenched glycosidase substrates in which the fluorophore and quencher may occupy either relative position and further teaches that, when R6 is the fluorophore, the Formula (V) cleavage product is fluorescent because R6 is no longer internally quenched by R7, whereas when R7 is the fluorophore, the Formula (VI) cleavage product is fluorescent because R7 is no longer internally quenched by R6. One of ordinary skill in the art would have been motivated to make this modification because Vocadlo teaches fluorescence-quenched glycosidase substrates in which the fluorophore and quencher may occupy either relative position. Vocadlo further teaches that when R6 is the fluorophore, the Formula (V) product is fluorescent because R6 is no longer internally quenched by R7, whereas when R7 is the fluorophore, the Formula (VI) product is fluorescent because R7 is no longer internally quenched by R6. Thus, Vocadlo teaches the converse arrangement as a known alternative configuration for obtaining a fluorescent product following glycosidase cleavage. A person of ordinary skill in the art would have had a reasonable expectation of success because Vocadlo teaches that both relative fluorophore/quencher arrangements are functional in fluorescence-quenched glycosidase substrates, with enzymatic cleavage relieving internal quenching and producing a fluorescent product in either arrangement.
It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the fluorescence-quenched GCase substrate of Yadav et al., as modified by Kanie et al., by substituting the BHQ-1 for the quencher in view of Rudd et al. because Yadav et al. teaches that suitable fluorophore/quencher pairs may be selected and substituted in the modular probe design, while Rudd et al. specifically teach BHQ-1 as a quencher for a BODIPY fluorophore. Thus, the modification would have amounted to selection of a known alternative quencher within the fluorophore/quencher design framework already taught by Yadav et al. One of ordinary skill in the art would have been motivated to employ BHQ-1 because Yadav et al. teaches optimization and substitution of fluorophore/quencher pairs in its modular fluorescence-quenched probe architecture, and Rudd et al. teach that BHQ-1 is selected for use with a BODIPY fluorophore based on its high quenching efficiency at the emission wavelength of the fluorophore. One of ordinary skill in the art would have had a reasonable expectation of success because Yadav et al. demonstrate that its GCase substrate architecture accommodates alternative fluorophore/quencher selections, and Rudd et al. demonstrate operative quenching of a BODIPY fluorophore by BHQ-1 in a functional fluorogenic probe.
Regarding “j is 3”, Kanie et al. teach a lysine-derived spacer having an aminoalkyl side chain corresponding to –(CH2)4NH2, i.e., j = 4, but does not teach the –(CH2)3NH2 with 1 methylene shorter as required by the present claims. Rajopadhye et al. teach fluorescent imaging agents employing linker moieties and explain that a diamine moiety or derivative may provide a linker arm of varying lengths and chemistries for chemically linking molecules. Rajopadhye et al. explicitly identify diamino-amino acids including homolysine, lysine, ornithine, diaminobutyric acid and diaminopropionic acid as suitable linker materials. Ornithine has the amino acid structure HOOC-CH(NH2)-(CH2)3NH2, and therefore provides the aminoalkyl side chain –(CH2)3NH2 corresponding to j = 3. Rajopadhye et al. further teach conventional NHS-ester/amine coupling for chemically linking fluorophores or other imaging reporters to amino-functionalized moieties. It would have been obvious to substitute the ornithine-derived linker taught by Rajopadhye et al. for the lysine-derived linker of Kanie et al., thereby shortening the aminoalkyl side chain by one methylene and providing j = 3. Such modification would have amounted to selection of a known alternative diamino-amino acid linker of different length for the same linker function. One of ordinary skill would have been motivated to make the substitution because Rajopadhye et al. teach lysine and ornithine among a series of diamino-amino acid linkers capable of providing linker arms of varying lengths and chemistries. One of ordinary skill in the art would have had a reasonable expectation of success because both lysine and ornithine are taught as suitable linker materials and with only 1 methylene difference, and Rajopadhye et al. further teach the same type of conventional amine/NHS-ester conjugation chemistry used to attach imaging reporters.
Claims 3, 14, and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Yadav et al. (Journal of the American Chemical Society, 2015, Vol. 137, Issue 3, page 1181 – 1189, PTO-892) in view of Kanie et al. (US6358919B1) as applied to claims 1, 4 – 5, 7 – 9, 11 – 13, 15 – 16, and 18 above, and further in view of Vocadlo (WO2018/229663A1).
The combined teachings of Yadav et al. and Kanie et al. teach the limitations discussed above.
However, Yadav et al. and Kanie et al. do not teach the second specifically recited compound of claim 3. In particular, Yadav et al. and Kanie et al. do not teach the particular TAMRA fluorophore/BHQ2 quencher selection recited in the second compound of claim 3.
Vocadlo also teaches fluorescence-quenched substrates of exo-glycosidase enzymes, such as human exo-glycosidase, wherein the human exo-glycosidase is GCase (para. [0001]; para. [0056]). Vocadlo provides methods for imaging exo-glycosidase enzymes activity within cells (Abstract). The fluorescence-quenched substrate is a compound of Formula (I):
PNG
media_image4.png
164
387
media_image4.png
Greyscale
,
wherein R6 is a fluorophore and R7 is a quencher, as well as the converse arrangement wherein R6 is a quencher and R7 is a fluorophore (para. 0008]). Vocadlo further teaches that a suitable fluorophore may be carboxytetramethylrhodamine (TAMRA) (para. [0063]) and identifies TAMRA among suitable fluorophores (para. [0064]). Vocadlo also identifies NHQ2 as a suitable quencher at the R7 position (para. [0066]). In addition, Vocadlo exemplifies a glucose-based fluorescence-quenched substrate containing the TAMRA/BHQ2 fluorophore-quencher pair (page 31, Table 1, Compound 8), and evaluates the resulting probe in live cells (para. [00158 – 00160]). Thus, Vocadlo teaches TAMRA and BHQ2 not merely as separate possible components, but as an operative fluorophore-quencher pair in a fluorescence-quenched glycosidase substrate. Moreover, Vocadlo teaches a method for determining the efficacy of an exo-glycosidase-directed therapy by providing a test cell obtained from a subject treated with the exo-glycosidase-directed therapy and a control cell obtained from a subject not treated with the exo-glycosidase-directed therapy; contacting the test and control cells with a fluorescence-quenched exo-glycosidase substrate; measuring fluorescence intensity in the test cell and the control cell; and determining efficacy of the therapy based on a difference in fluorescence intensity between the test cell and the control cell (para. [0030]). Vocadlo further teaches that an exo-glycosidase-directed therapy may include enzyme replacement therapy, chaperone therapy, activator therapy, or activity-enhancer therapy, and that cells may be derived from a subject treated with such therapy (para. [00106 – 00107]). Vocadlo identifies GCase as a human exo-glycosidase encompassed by the disclosure. Thus, Vocadlo teaches the treated-subject/untreated-subject comparison and fluorescence-based assessment of therapy efficacy recited in claim 14. Finally, Vocadlo teaches that cells used in the disclosed exo-glycosidase assays may be derived from tissue and that the tissue may be a skin punch (para. [0030]). Vocadlo further teaches that suitable tissue includes skin punch tissue and specifically states that the tissue may be a skin punch derived from a human subject treated with an exo-glycosidase-directed therapy (para. [00107]; [00110]). Thus, Vocadlo teaches the additional limitation of claim 17 that the tissue is a skin punch.
It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the fluorescence-quenched GCase substrate of Yadav et al., as modified by Kanie et al., to employ the TAMRA/BHQ2 fluorophore-quencher pair in view of Vocadlo because Yadav et al. explicitly teach a modular fluorescence-quenched GCase probe design in which suitable fluorophore-quencher pairs may be selected to provide an operative quenched substrate, thereby establishing fluorophore/quencher identity as a design variable within the probe architecture. Vocadlo, in turn, specifically teaches and exemplified TAMRA/BHQ2 as an operative fluorophore-quencher pair in fluorescence-quenched glycosidase substrates. Thus, substituting the TAMRA/BHQ2 pair for the fluorophore-quencher pair used by Yadav et al. would have amounted to selection of a known alternative pair within the modular design framework already taught by Yadav et al., with the predictable result of maintaining fluorescence quenching prior to cleavage and fluorescence generation following enzymatic cleavage. One of ordinary skill would have been motivated to make this substitution because Yadav et al. teach selection and optimization of fluorophore-quencher pairs in its modular GCase probe design, while Vocadlo identifies and uses TAMRA/BHQ2 pair in the same general fluorescence-quenched glycosidase context. Vocadlo therefore provides a specific known alternative pair that a skilled artisan would have considered when selecting a suitable fluorophore and quencher for the modular probe of Yadav et al. One of ordinary skill in the art would have had a reasonable expectation of success because Yadav et al. demonstrate that its probe architecture accommodates selected fluorophore-quencher pairs and Vocadlo prepares and evaluates a TAMRA/BHQ2-containing fluorescence-quenched glycosidase substrate, demonstrating that this pair provides the intended quenching and fluorescence response upon enzymatic cleavage.
Conclusion
No claim is found to be allowable.
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/H.Y.L./Examiner, Art Unit 1693
/SCARLETT Y GOON/Supervisory Patent Examiner, Art Unit 1693
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