DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 3-11, 13-18, 28, 31-34, 38-50, and 54-79 are canceled.
Claims 1, 2, 12, 19-27, 29, 30, 35-37, and 51-53 are pending and examined on the merits.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 2, 12, 19-27, 29, 30, 35-37, and 51-53 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites the limitation "said β-hexosaminidase" in line 3. There is insufficient antecedent basis for this limitation in the claim. There is no prior recitation that the sample comprises β-hexosaminidase, or that the sample and/or β-hexosaminidase substrate are combined with β-hexosaminidase. Since claim 1 is indefinite, then its dependent claims, claims 2, 12, 19-27, 29, 30, and 35-37, are rendered indefinite. Thus, claims 1, 2, 12, 19-27, 29, 30, and 35-37 are rejected under 35 U.S.C. 112(b). For the purpose of applying prior art, the sample is being interpreted as comprising β-hexosaminidase.
Regarding claim 19, a broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 19 recites the broad recitation “said β-hexosaminidase substrate comprises a β-hexosaminidase A substrate,” and the claim also recites “said β-hexosaminidase substrate is a substrate for β-hexosaminidase A” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. Regarding the recitation “said β-hexosaminidase substrate comprises a β-hexosaminidase A substrate,” since the transitional term “comprises” is inclusive or open-ended and does not exclude additional, unrecited elements (see MPEP 2111.03(I)), then the β-hexosaminidase substrate can include at least one additional element other than a β-hexosaminidase A substrate. On the other hand, the recitation “said β-hexosaminidase substrate is a substrate for β-hexosaminidase A” limits the β-hexosaminidase substrate to being a β-hexosaminidase A substrate, which not does include any additional, unrecited elements, thus having a narrower scope as compared to the other recitation. Since claim 19 is indefinite, then its dependent claims, claims 20-24, are rendered indefinite. Thus, claims 19-24 are rejected under 35 U.S.C. 112(b). For the purpose of applying prior art, claim 19 is being interpreted as requiring that the β-hexosaminidase substrate comprises a β-hexosaminidase A substrate.
Claim 20 recites the limitation "said β-hexosaminidase A" in line 1. There is insufficient antecedent basis for this limitation in the claim. Parent claims 1 and 19 do not require that the sample comprises β-hexosaminidase A, nor do they require that the sample and/or β-hexosaminidase substrate are combined with β-hexosaminidase A. Since claim 20 is indefinite, then its dependent claims, claims 21-24, are rendered indefinite. Thus, claims 20-24 are rejected under 35 U.S.C. 112(b). For the purpose of applying prior art, claim 20 is being interpreted as requiring that the sample comprises β-hexosaminidase A.
Claims 21 and 22 are each rendered indefinite by the recitation “wherein detection of said β-hexosaminidase A enzymatic reaction product.” This recitation is confusing because parent claim 20 does not require detecting the β-hexosaminidase A enzymatic reaction product. Though parent claim 1 recites detecting a β-hexosaminidase enzymatic reaction product via mass spectrometry, parent claim 20 does not require that the β-hexosaminidase A enzymatic reaction product is the β-hexosaminidase enzymatic reaction product that is detected via mass spectrometry according to claim 1 (it is noted that “detection of said β-hexosaminidase A enzymatic reaction product” of claims 21 and 22 does not require detection via mass spectrometry). Since claim 22 is indefinite, then its dependent claims, claims 23 and 24, are rendered indefinite. Thus, claims 21-24 are rejected under 35 U.S.C. 112(b).
Regarding claim 25, a broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 25 recites the broad recitation “said β-hexosaminidase substrate comprises a β-hexosaminidase A/B substrate,” and the claim also recites “said β-hexosaminidase substrate is a substrate for β-hexosaminidase A and β-hexosaminidase B” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. A β-hexosaminidase A/B substrate is a substrate for both β-hexosaminidase A and β-hexosaminidase B, according to page 9, line 30 through page 10, line 1 of the instant specification. Regarding the recitation “said β-hexosaminidase substrate comprises a β-hexosaminidase A/B substrate” in claim 25, since the transitional term “comprises” is inclusive or open-ended and does not exclude additional, unrecited elements (see MPEP 2111.03(I)), then the β-hexosaminidase substrate can include at least one additional element other than a β-hexosaminidase A/B substrate. On the other hand, the recitation “said β-hexosaminidase substrate is a substrate for β-hexosaminidase A and β-hexosaminidase B” limits the β-hexosaminidase substrate to being a β-hexosaminidase A/B substrate (i.e. a substrate for β-hexosaminidase A and β-hexosaminidase B), which not does include any additional, unrecited elements, thus having a narrower scope as compared to the other recitation. Since claim 25 is indefinite, then its dependent claims, claims 26 and 27, are rendered indefinite. Thus, claims 25-27 are rejected under 35 U.S.C. 112(b). For the purpose of applying prior art, claim 25 is being interpreted as requiring that the β-hexosaminidase substrate comprises a β-hexosaminidase A/B substrate.
Claim 26 recites the limitation "said β-hexosaminidase A and said β-hexosaminidase B" in lines 1-2. There is insufficient antecedent basis for this limitation in the claim. Parent claims 1 and 25 do not require that the sample comprises β-hexosaminidase A and β-hexosaminidase B, nor do they require that the sample and/or β-hexosaminidase substrate are combined with β-hexosaminidase A and β-hexosaminidase B. Since claim 26 is indefinite, then its dependent claim, claim 27, is rendered indefinite. Thus, claims 26-27 are rejected under 35 U.S.C. 112(b). For the purpose of applying prior art, claim 26 is being interpreted as requiring that the sample comprises β-hexosaminidase A and β-hexosaminidase B (i.e. The method of claim 25, wherein the sample comprises β-hexosaminidase A and β-hexosaminidase B, and said β-hexosaminidase A and said β-hexosaminidase B enzymatically react with said β-hexosaminidase A/B substrate to product a β-hexosaminidase A/B enzymatic reaction product”).
Claim 27 is rendered indefinite by the recitation “wherein detection of said β-hexosaminidase A/B enzymatic reaction product.” This recitation is confusing because parent claim 26 does not require detecting the β-hexosaminidase A/B enzymatic reaction product. Though parent claim 1 recites detecting a β-hexosaminidase enzymatic reaction product via mass spectrometry, parent claim 26 does not require that the β-hexosaminidase A/B enzymatic reaction product is the β-hexosaminidase enzymatic reaction product that is detected via mass spectrometry according to claim 1 (it is noted that “detection of said β-hexosaminidase A/B enzymatic reaction product” of claim 27 does not require detection via mass spectrometry).
Claim 51 recites the limitation "said β-hexosaminidase A" in line 4. There is insufficient antecedent basis for this limitation in the claim. There is no prior recitation that the sample comprises β-hexosaminidase A, or that the sample and/or β-hexosaminidase A substrate are combined with β-hexosaminidase A. Since claim 51 is indefinite, then its dependent claims, claims 52 and 53, are rendered indefinite. Thus, claims 51-53 are rejected under 35 U.S.C. 112(b). For the purpose of applying prior art, claim 51 is being interpreted as follows:
A method for screening an individual for a deficiency in β-hexosaminidase A enzymatic activity, comprising:
combining a sample from said individual with a β-hexosaminidase A substrate under conditions sufficient for β-hexosaminidase A, if present in the sample, and said β-hexosaminidase A substrate to enzymatically react to produce a β-hexosaminidase A enzymatic reaction product; and
detecting the presence of said β-hexosaminidase A enzymatic reaction product via mass spectrometry;
wherein the absence of said β-hexosaminidase A enzymatic reaction product indicates said deficiency in said β-hexosaminidase A enzymatic activity.
Notice Re: Prior Art Available Under Both Pre-AIA and AIA
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.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 2, 12, 19-22, 25-27, 29, 35, and 36 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Fitterer (Molecular Genetics and Metabolism. 2014. 11: 382-389. Listed on IDS filed 6/6/24).
Fitterer discloses a β-hexosaminidase assay using two β-hexosaminidase substrates and internal standards (page 383, paragraph bridging left and right columns). Blood was eluted from a newborn screening card with an extraction buffer to obtain an extraction plate (page 383, right column, third paragraph). The extracted blood is directed to a sample comprising a blood sample, meeting the limitation of instant claim 2. A Total Hex reaction was started by adding each extract from the extraction plate to wells of a Total Hex plate, and the plate was incubated (page 383, right column, fourth paragraph). Each well of the Total Hex plate comprises Total Hex reagent cocktail (page 383, right column, fourth paragraph). The Total Hex reagent cocktail comprises a β-hexosaminidase substrate and an internal standard as shown in Figure 1 (page 383, paragraph bridging left and right columns). Therefore, performing the Total Hex reaction as taught in Fitterer is directed to the first step of instant claim 1 of combining a sample (extracted blood sample which comprises β-hexosaminidase) with a β-hexosaminidase substrate (the substrate for the Total Hex reaction) under conditions sufficient for β-hexosaminidase (as present in the sample) and the β-hexosaminidase substrate to enzymatically react to produce a β-hexosaminidase enzymatic reaction product (since the Total Hex reaction is performed).
Another reaction is performed in the assay of Fitterer which also is directed to the first step of instant claim 1. Fitterer also teaches re-sealing and incubating the extract plate at a higher temperature in order to heat inactivate β-hexosaminidase A (page 383, right column, fourth paragraph). After the incubation, heat-inactivated extract was added to the wells of a HexB plate comprising HexB reagent cocktail in each well, and then incubated (page 383, right column, fourth paragraphs). The HexB reagent cocktail is for measuring HexB (i.e. β-hexosaminidase B) and comprises a β-hexosaminidase substrate and an internal standard as shown in Figure 1 (page 383, paragraph bridging left and right columns). The incubation with the HexB reagent cocktail is directed to the first step of instant claim 1 of combining a sample (heat-inactivated extracted blood sample comprising β-hexosaminidase) with a β-hexosaminidase substrate (the substrate for HexB, i.e. β-hexosaminidase B) under conditions sufficient for β-hexosaminidase (β-hexosaminidase B as present in the sample) and the β-hexosaminidase substrate to enzymatically react to produce a β-hexosaminidase enzymatic reaction product.
After incubation the reaction in each plate (Total Hex plate, HexB plate) was stopped and the total volumes from the wells of the Total Hex and HexB plates were combined in a plate for product purification (page 383, right column, fourth paragraph). Product detection was carried out using a mass spectrometer (page 384, left column, first paragraph). Therefore, Fitterer teaches the second step of instant claim 1 of detecting the β-hexosaminidase enzymatic reaction product (product from the Total Hex reaction or product from the HexB reaction). As such, Fitterer anticipates instant claims 1 and 2.
Regarding instant claim 12, for each of the Total Hex reaction and the HexB reaction, the plate was incubated at 37ºC for about 22 hours (page 383, right column, fourth paragraph). This is directed to incubating the sample (extracted blood sample or heat-inactivated extracted blood sample) with the β-hexosaminidase substrate (substrate for the Total Hex reaction or substrate for the HexB reaction) at a pre-determined temperature (37ºC) for a pre-determined amount of time (about 22 hours) sufficient for the β-hexosaminidase (as present in the sample) and the β-hexosaminidase substrate to enzymatically react to produce the β-hexosaminidase enzymatic reaction product. Thus, instant claim 12 is anticipated.
Regarding instant claims 19-22, the substrate for the Total Hex reaction of Fitterer is directed to a β-hexosaminidase substrate comprising a β-hexosaminidase A substrate since it is used for assaying Total Hex, i.e. total β-hexosaminidase, which includes β-hexosaminidase A, meeting the limitation of instant claim 19. In the Total Hex reaction, all β-hexosaminidase, including β-hexosaminidase A, in the extracted blood sample reacts with the substrate for the Total Hex reaction (directed to a β-hexosaminidase A substrate) to produce a β-hexosaminidase enzymatic reaction product that includes a β-hexosaminidase A enzymatic reaction product, meeting the limitation of instant claim 20. See page 384, paragraph bridging left and right paragraphs in which HexA (β-hexosaminidase A) enzyme activity was calculated by subtracting the HexB (β-hexosaminidase B) activity calculated from the HexB reaction product, from the Total Hex activity calculated from the Total Hex reaction product. The detection of the Total Hex reaction product includes detection of the β-hexosaminidase A enzymatic reaction product (since it is included in the Total Hex reaction product). Through the subtraction of the HexB activity from the Total Hex activity (which is obtained from the Total Hex reaction product that includes HexA reaction product), then a measure of only β-hexosaminidase A activity is obtained. In other words, in that calculation, detection of β-hexosaminidase A enzymatic reaction product (as part of the Total Hex reaction product that is used to calculate the Total Hex activity) provides a measure of only β-hexosaminidase A activity, meeting limitations of instant claims 21 and 22. Thus, instant claims 19-22 are anticipated.
Regarding instant claims 25-27, the substrate for the Total Hex reaction of Fitterer is directed to a β-hexosaminidase substrate comprising a β-hexosaminidase A/B substrate (i.e. a substrate for both β-hexosaminidase A and β-hexosaminidase B, according to page 9, line 30 through page 10, line 1 of the instant specification) since it is used for assaying Total Hex, i.e. total β-hexosaminidase which includes both β-hexosaminidase A and β-hexosaminidase B, meeting the limitation of instant claim 25. In the Total Hex reaction, all β-hexosaminidase, including β-hexosaminidase A and β-hexosaminidase B, enzymatically reacts with the substrate for the Total Hex reaction (directed to a β-hexosaminidase A/B substrate) to produce a total β-hexosaminidase enzymatic reaction product (directed to a β-hexosaminidase A/B enzymatic reaction product), meeting the limitation of instant claim 26. Fitterer teaches calculating the β-hexosaminidase enzyme activities from the peak areas for the products and internal standards (page 384, paragraph bridging left and right columns). Therefore, detection of the Total Hex reaction product, directed to a β-hexosaminidase A/B enzymatic reaction product, provides a measure of the Total Hex activity, directed to β-hexosaminidase A activity and β-hexosaminidase activity (as a total), meeting the limitation of instant claim 27. Thus, instant claims 25-27 are anticipated.
Regarding instant claim 29, Fitterer discloses β-hexosaminidase product detection by MS/MS analysis (page 384, left column, first paragraph). Peaks areas for products and internal standards were used to calculate β-hexosaminidase enzyme activities (page 384, left column, last paragraph). The ratio of peak area for the products to the peak area of the internal standard for each reaction was used to determine the enzyme activity relative to the amount of internal standard added to each reaction (page 384, left column, last paragraph). The MS/MS analysis is directed to a quantitative method and the peak areas of the total β-hexosaminidase reaction product (Total Hex reaction product) and the HexB reaction product, in comparison to the peak area of the internal standard, are each directed to the amount of β-hexosaminidase enzymatic reaction product. Thus, instant claim 29 is anticipated.
Regarding instant claim 35, as pointed out above, product detection was carried out using a mass spectrometer, and MS/MS analysis was performed (page 384, left column, first paragraph). MS/MS is known in the art as tandem mass spectrometry (see also the title of the reference). Thus, instant claim 35 is anticipated.
Regarding instant claim 36, as discussed above, instant claim 1 is anticipated by Fitterer’s teaching of performing each of the Total Hex reaction and the HexB reaction in separate plates and performing product detection using a mass spectrometer on each of their reaction products. The two reactions use different β-hexosaminidase substrates (substrate for Total Hex reaction, substrate for HexB reaction) and produced different β-hexosaminidase enzymatic reaction products which are detected by mass spectrometry (see page 384, left column, first paragraph). Thus, instant claim 36 is anticipated.
Claims 1, 12, 19-22, 25-27, 29, and 35 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Ng (Master’s thesis, University of Ottawa. 2010. [retrieved on 2026-01-23]. Retrieved from the Internet <URL: http://hdl.handle.net/10393/28924>).
Ng developed a MS/MS (i.e., tandem mass spectrometry; see page 47, first paragraph) based quantitative assay for determining exoglycosidase activities by measuring derivatized oligosaccharide products (page 26, last paragraph). Ng discloses one assay using a PNP (paranitrophenol; see page 89, first paragraph) substrate for β-hexosaminidase such that β-hexosaminidase activity is determined by MS/MS through detection of released PNP product (Figure 27 on page 90). In particular, 4-nitrophenyl N-acetyl-β-glucosaminide was used as the target for the determination of β-hexosaminidase activity through the detection of PNP product (page 91). The assay involves reacting β-hexosaminidase with the PNP substrate (4-nitrophenyl N-acetyl-β-glucosaminide) reconstituted in buffer, boiling for 5 minutes to stop the enzymatic reaction, and processing to obtain a product which was injected for analysis by MS/MS (page 91; page 29 as incorporated by page 91). PNP was detected by MS/MS (pages 92 and 93). Also, to demonstrate enzyme activity, the β-hexosaminidase assay compared 5 different reaction samples containing increasing amounts of active enzyme (AE) from fibroblasts to a control with inactive enzyme (IE), which was a fibroblast lysate that had been heat inactivated (page 94). The activity in the β-hexosaminidase assay was monitored by MS/MS detection of the PNP product, with PNP expressed as a percentage of the highest signal found in the 200 µl active enzyme (AE) sample (page 94).
Therefore, Ng anticipates the claimed invention since Ng discloses a method for assaying β-hexosaminidase enzymatic activity, comprising combining a sample (β-hexosaminidase-containing sample, e.g. fibroblast sample) with a β-hexosaminidase substrate (4-nitrophenyl N-acetyl-β-glucosaminide) under conditions sufficient for the β-hexosaminidase in the sample and the substrate to enzymatically react to produce a β-hexosaminidase enzymatic reaction product (PNP); and detecting said β-hexosaminidase enzymatic reaction product (PNP) via mass spectrometry (MS/MS, i.e., tandem mass spectrometry). As such, instant claims 1 and 35 are anticipated.
Regarding instant claim 12, Ng discloses that the reaction was incubated for 2 hours (page 91). The reaction inherently is performed at a temperature below boiling temperature since the reaction was stopped by boiling for 5 minutes after the incubation (page 91). That temperature is directed to a ‘pre-determined temperature,’ and the incubation period of 2 hours is directed to a ‘pre-determined amount of time’ that are sufficient for the β-hexosaminidase and the β-hexosaminidase substrate (4-nitrophenyl N-acetyl-β-glucosaminide) to enzymatically react to produce the β-hexosaminidase enzymatic reaction product (PNP). As such, instant claim 12 is anticipated.
Regarding instant claims 19-22, the PNP substrate, 4-nitrophenyl N-acetyl-β-glucosaminide, is directed to a β-hexosaminidase substrate comprising a β-hexosaminidase A substrate since it is used for assaying β-hexosaminidase in general, which includes β-hexosaminidase A, meeting the limitation of instant claim 19. In the reaction, all β-hexosaminidase, including β-hexosaminidase A, in the sample reacts with 4-nitrophenyl N-acetyl-β-glucosaminide to produce a β-hexosaminidase enzymatic reaction product that includes a β-hexosaminidase A enzymatic reaction product (PNP), meeting the limitation of instant claim 20. The detection of the total PNP includes detection of the PNP that results from the β-hexosaminidase A reacting with 4-nitrophenyl N-acetyl-β-glucosaminide. In measuring the β-hexosaminidase from the PNP detected by MS/MS, then β-hexosaminidase A activity is measured as it is part of the total β-hexosaminidase activity. Thus, instant claims 21 and 22 are anticipated (β-hexosaminidase A activity, i.e. ‘only β-hexosaminidase A activity,’ is part of the total β-hexosaminidase activity).
Regarding instant claims 25-27, 4-nitrophenyl N-acetyl-β-glucosaminide is directed to a β-hexosaminidase substrate comprising a β-hexosaminidase A/B substrate (i.e. a substrate for both β-hexosaminidase A and β-hexosaminidase B, according to page 9, line 30 through page 10, line 1 of the instant specification) since it is used for assaying β-hexosaminidase in general which includes both β-hexosaminidase A and β-hexosaminidase B, meeting the limitation of instant claim 25. In the reaction, all β-hexosaminidase, including β-hexosaminidase A and β-hexosaminidase B, enzymatically reacts with 4-nitrophenyl N-acetyl-β-glucosaminide (directed to a β-hexosaminidase A/B substrate) to produce PNP (directed to a β-hexosaminidase A/B enzymatic reaction product), meeting the limitation of instant claim 26. Detection of PNP (paranitrophenol), directed to a β-hexosaminidase A/B enzymatic reaction product, provides a measure of β-hexosaminidase activity, directed to β-hexosaminidase A activity and β-hexosaminidase activity (as a total), meeting the limitation of instant claim 27. Thus, instant claims 25-27 are anticipated.
Regarding instant claim 29, the method of Ng is a quantitative method which measures the amount of the β-hexosaminidase enzymatic reaction product (PNP) since PNP is expressed as a percentage of the highest signal found in the 200 µl active enzyme (AE) sample (page 94). As such, instant claim 29 is anticipated.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim 30 is rejected under 35 U.S.C. 103 as being unpatentable over Fitterer in view of Bateman (Journal of Medicinal Chemistry. 2011. 54: 1421-1429).
As discussed above, Fitterer anticipates claims 1, 2, 12, 19-22, 25-27, 29, 35, and 36. With respect to claim 29, Fitterer teaches that peaks areas for products and internal standards were used to calculate β-hexosaminidase enzyme activities (page 384, left column, last paragraph). The ratio of peak area for the products to the peak area of the internal standard for each reaction was used to determine the enzyme activity relative to the amount of internal standard added to each reaction (page 384, left column, last paragraph). Claim 29 is anticipated by Fitterer because the peak areas of the total β-hexosaminidase reaction product (Total Hex reaction product) and the HexB reaction product, in comparison to the peak area of the internal standard, are each directed to the amount of β-hexosaminidase enzymatic reaction product. This meets the limitation of claim 30 requiring that the amount of the β-hexosaminidase enzymatic reaction product (the Total Hex reaction product or the HexB reaction product) is determined by use of an internal standard. Additionally, Fitterer states that the HexB cocktail contained deuterated substrate with internal standard (page 383, right column, first paragraph). See Figure 1(B) showing the structure of the substrate for the HexB reaction. Thus, the β-hexosaminidase B substrate comprises isotopes.
Fitterer differs from the claimed invention in that Fitterer does not expressly disclose that the β-hexosaminidase enzymatic reaction product (the Total Hex reaction product or the HexB reaction product) and the internal standard have identical structures but are isotopically different.
However, Bateman teaches that β-hexosaminidases are a group of glycosyl hydrolase isozymes (abstract). Based on that teaching, the skilled artisan would have expected that the HexB reaction of Fitterer would have cleaved the glycosidic bond of the substrate of the HexB reaction of Figure 1(B) such that the HexB reaction product has the same structure as the internal standard of Figure 1(C), except being deuterated. Therefore, with respect to the process involving the HexB reaction of Fitterer, the internal standard and the β-hexosaminidase enzymatic reaction product (the product of the HexB reaction) have identical chemical structures but are isotopically different. As such, instant claim 30 is rendered obvious.
Claims 23, 24, 37, and 51-53 are rejected under 35 U.S.C. 103 as being unpatentable over Fitterer in view of Chamoles (US 2004/0166554) and Gelb (US 2016/0298166).
As discussed above, Fitterer anticipates claims 1, 2, 12, 19-22, 25-27, 29, 35, and 36. Fitterer differs from claim 23 in that Fitterer does not expressly disclose that β-hexosaminidase B does not enzymatically react with their β-hexosaminidase A substrate (the Total Hex reaction substrate is directed to this). Fitterer further differs from claim 24 in that Fitterer does not expressly disclose that the enzymatic reaction of β-hexosaminidase A with their β-hexosaminidase A substrate (the Total Hex reaction substrate is directed to this) does not provide a measure of β-hexosaminidase B activity.
Regarding claim 37, Fitterer discloses combining a sample with multiple β-hexosaminidase substrates, including the substrate for the Total Hex reaction substrate which is directed to a β-hexosaminidase substrate comprising a β-hexosaminidase A/B substrate with which β-hexosaminidase A and β-hexosaminidase B enzymatically react to produce a β-hexosaminidase A/B enzymatic reaction product which is detected via mass spectrometry. However, Fitterer differs from claim 37 in that Fitterer does not expressly disclose further combining the sample with a β-hexosaminidase substrate comprising a β-hexosaminidase A substrate with which β-hexosaminidase A enzymatically reacts to produce a β-hexosaminidase A enzymatic reaction product.
Additionally, Fitterer differs from claim 51 in that Fitterer does not expressly disclose a method for screening an individual for a deficiency in β-hexosaminidase A enzymatic activity, comprising combining a sample from an individual with a β-hexosaminidase A substrate under conditions sufficient for the β-hexosaminidase A and the substrate to enzymatically react to produce a β-hexosaminidase A enzymatic reaction product, and detecting the presence of the enzymatic reaction product via mass spectrometry, wherein the absence of the enzymatic reaction product indicates deficiency in β-hexosaminidase A enzymatic activity. Fitterer further differs from claim 52 in that Fitterer does not expressly disclose that the deficiency in the β-hexosaminidase A enzymatic activity indicates that the individual may be diagnosed with Tay-Sachs disease. Fitterer further differs from claim 53 in that Fitterer does not expressly disclose that the deficiency in β-hexosaminidase A enzymatic activity indicates that the individual may be a candidate for treatment for Tay-Sachs disease.
In the discussion of the paper, Fitterer discloses that identification of Tay-Sachs affected individuals was not possible using the assay conditions described in the paper, but a modified protocol of these substrates and internal standard allowed for the identification of Tay-Sachs patients (page 388, left column, fourth paragraph).
Chamoles discloses a method for assaying the activity of lysosomal enzymes present in dried bodily fluids, including hexosaminidase A and B (abstract). Tays-Sachs disease is produced by the deficiency of the lysosomal enzyme β-hexosaminidase α-subunit, which is a component of hexosaminidase A (paragraph [0012]). Individuals afflicted by this disorder show a low or non-existent activity of hexosaminidase A only (paragraph [0012]). Therefore, Chamoles teaches detection of Tay-Sachs disease comprising reacting a punch of human blood with 4-methylumbelliferyl-N-acetyl-β-glucopyranoside sulfate as a substrate of β-hexosaminidase A to detect for the enzyme product 4-methylumbelliferone (Example 10, particularly paragraphs [0180]-[0186] and [0190]; paragraph [0043] for defining MU in substrate name).
Gelb discloses methods for assaying one or more enzymes associated with a lysosomal storage disease, comprising contacting one or more lysosomal enzymes in a solution with an enzyme substrate for each lysosomal enzyme to be analyzed and incubating the substrates with the enzymes for a time sufficient to provide a solution comprising an enzyme product for each lysosomal enzyme present in the sample (paragraphs [0010]-[0013]). The enzyme substrate for each lysosomal enzyme is a compound having a carbohydrate moiety and an aglycone moiety and having the formula as shown in paragraph [0014], wherein S is the carbohydrate moiety covalently coupled to the aglycone moiety (paragraph [0015]). In certain embodiments, one or more substrates (S) and their corresponding internal standards (IS) are incubated in a suitable buffer with a suitable source of enzymes such as a dried blood spot from a newborn screening card for a sufficient time to form one or more products (P) that are subsequently detected by tandem mass spectrometry (paragraph [0132]).
Gelb points out that human hexosaminidase A, which is endogenous in a dried blood spot sample, can act on the sulfated-sugar-aglycone substrate to cleave the glycosidic linkage (paragraph [0481]). Gelb discusses a representative assay for mucopolysaccharidosis IVA (MPS-IVA) using a substrate in which N-acetyl-galactose-6-sulfate is attached to the aglycone where R1 is n-butyl, L2 is -CH2CH2-, L3 is -(CH2)5-, and R3 is phenyl (paragraph [0526]). That substrate is shown in paragraph [0530]. For that assay, the biological sample may contain human hexosaminidase A and other human hexosaminidases (paragraph 0526]). It was determined that human hexosaminidase A can cleave the glycoside even when the sugar is sulfated at the 6-position (paragraph [0526]). Table 1 in paragraph [0527]) shows assay results using that substrate, which includes peaks areas of the aglycone (product without sugar), and the internal standard. Given that peak areas were detected, then the product of the reaction of human hexosaminidase A with the sulfated-sugar-aglycone substrate was detected via mass spectrometry.
Before the effective filing date of the claimed invention, it would have been obvious to the person of ordinary skill in the art to additionally perform an assay of β-hexosaminidase A with a substrate specific for β-hexosaminidase A but not for β-hexosaminidase B, in particular a sulfated-sugar-aglycone substrate according to Gelb, in combination with the assays performed in Fitterer, wherein the product of the reaction of β-hexosaminidase A with the substrate is detected via mass spectrometry. One of ordinary skill in the art would have been motivated to do this in order to identify Tay-Sachs disease in the subject by detecting low or non-existent β-hexosaminidase A activity (as indicated in Chamoles as characteristic of the disease) in the blood sample, which was sought in Fitterer (page 388, left column, fourth paragraph). There would have been a reasonable expectation of assaying β-hexosaminidase A by treating the blood sample from a newborn screening card of Fitterer with the sulfated-sugar-aglycone substrate of Gelb because Gelb found that hexosaminidase A can cleave the glycoside when the sugar is sulfated at the 6-position, and its aglycone product can successfully be detected by mass spectrometry. Therefore, instant claims 23, 24, and 37 are rendered obvious.
Since the motivation to further detect β-hexosaminidase A is to identify Tay-Sachs disease in the subject by detecting low or non-existent β-hexosaminidase A activity in the blood sample when performing the method rendered obvious by Fitterer in view of Chamoles and Gelb, then it would have been obvious to the person of ordinary skill in the art that the method is for screening an individual for a deficiency in a β-hexosaminidase A enzymatic activity, specifically identifying the subject as having Tay-Sachs disease, wherein the absence of β-hexosaminidase A enzymatic reaction product indicates said deficiency indicative of Tay-Sachs disease. Therefore, instant claims 51 and 52 are rendered obvious. A subject that is identified as having Tay-Sachs disease is an individual that is a candidate for treatment for Tay-Sachs disease. Thus, instant claim 53 is rendered obvious.
Claims 23, 24, 36, 37, and 51-53 are rejected under 35 U.S.C. 103 as being unpatentable over Ng in view of Ben-Yoseph (Am. J. Hum. Genet. 1985. 37: 733-748. Listed on IDS filed 6/7/24).
As discussed above, Ng anticipates claims 1, 12, 19-22, 25-27, 29, and 35. Ng differs from claim 23 in that Ng does not expressly disclose that β-hexosaminidase B does not enzymatically react with their β-hexosaminidase A substrate (4-nitrophenyl N-acetyl-β-glucosaminide is directed to this). Ng further differs from claim 24 in that Ng does not expressly disclose that the enzymatic reaction of β-hexosaminidase A with their β-hexosaminidase A substrate (the 4-nitrophenyl N-acetyl-β-glucosaminide is directed to this) does not provide a measure of β-hexosaminidase B activity.
Ng differs from claim 36 in that Ng does not expressly disclose combining the sample with more than one β-hexosaminidase substrate to correspondingly produce more than one β-hexosaminidase enzymatic reaction product, and detecting the β-hexosaminidase enzymatic reaction products via mass spectrometry. Regarding claim 37, Ng meets the claimed limitation regarding the second β-hexosaminidase substrate. In particular, 4-nitrophenyl N-acetyl-β-glucosaminide is directed to the claimed second β-hexosaminidase substrate comprising a β-hexosaminidase A/B substrate since 4-nitrophenyl N-acetyl-β-glucosaminide is a substrate for β-hexosaminidase in general, thus being a substrate for both β-hexosaminidase A and β-hexosaminidase B, and being a substrate with which β-hexosaminidase A and β-hexosaminidase B enzymatically react to produce a β-hexosaminidase A/B enzymatic reaction product (PNP). However, Ng differs from claim 37 in that Ng does not expressly disclose further combining the sample with a first β-hexosaminidase substrate comprising a β-hexosaminidase A substrate with which β-hexosaminidase A enzymatically reacts to produce a β-hexosaminidase A enzymatic reaction product (in addition to the combining with the first β-hexosaminidase A substrate, i.e. 4-nitrophenyl N-acetyl-β-glucosaminide).
Additionally, Ng differs from claim 51 in that Ng does not expressly disclose a method for screening an individual for a deficiency in β-hexosaminidase A enzymatic activity, comprising combining a sample from an individual with a β-hexosaminidase A substrate under conditions sufficient for the β-hexosaminidase A and the substrate to enzymatically react to produce a β-hexosaminidase A enzymatic reaction product, and detecting the presence of the enzymatic reaction product via mass spectrometry, wherein the absence of the enzymatic reaction product indicates deficiency in β-hexosaminidase A enzymatic activity. Ng further differs from claim 52 in that Ng does not expressly disclose that the deficiency in the β-hexosaminidase A enzymatic activity indicates that the individual may be diagnosed with Tay-Sachs disease. Ng further differs from claim 53 in that Ng does not expressly disclose that the deficiency in β-hexosaminidase A enzymatic activity indicates that the individual may be a candidate for treatment for Tay-Sachs disease.
Ben-Yoseph discloses Tay-Sachs disease (TSD) as being a disease caused by deficient activity of hexosaminidase A (HEX A) (paragraph bridging pages 733 and 734). Ben-Yoseph teaches that two studies reported that p-nitrophenyl-6-sulfo-2-acetamido-2-deoxy-β-D-glucopyranoside (pNP-β-GlcNac-6-S) can be specifically hydrolyzed by HEX A to yield free p-nitrophenol and N-acetylglucosamine-6-sulfate, and this assay is suitable for diagnosis and carrier detection of TSD (page 734, second paragraph).
Before the effective filing date of the claimed invention, it would have been obvious to the person of ordinary skill in the art to substitute the 4-nitrophenyl N-acetyl-β-glucosaminide substrate with p-nitrophenyl-6-sulfo-2-acetamido-2-deoxy-β-D-glucopyranoside when performing the method of Ng, wherein p-nitrophenyl-6-sulfo-2-acetamido-2-deoxy-β-D-glucopyranoside is the sulfated (at the 6th position of the sugar N-acetyl-β-D-glucosaminide) form of 4-nitrophenyl N-acetyl-β-glucosaminide, using a sample obtained from a subject. One of ordinary skill in the art would have been motivated to do this in order to diagnose Tay-Sachs disease from a sample of a subject, specifically by determining the absence of PNP (p-nitrophenol) through MS/MS because of deficiency in β-hexosaminidase A activity. There would have been a reasonable expectation of performing the method of Ng, which involves MS/MS, with the sulfated substrate because both the sulfated substrate and the non-sulfated substrate yield PNP as a product when enzymatically reacted with the functional enzyme (β-hexosaminidase or β-hexosaminidase A), wherein PNP can be detected by MS/MS. Since 4-nitrophenyl N-acetyl-β-glucosaminide is specifically hydrolyzed by β-hexosaminidase A, thus not by β-hexosaminidase B, then Ng in view of Ben-Yoseph renders obvious instant claims 23, 24, 51, 52, and 53 (an individual diagnosed with Tay-Sachs disease is directed to an individual who may be a candidate for treatment for Tay-Sachs disease).
Alternatively, before the effective filing date of the claimed invention, it would have been obvious to the person of ordinary skill in the art to additionally perform the method of Ng in which the 4-nitrophenyl N-acetyl-β-glucosaminide substrate is substituted with p-nitrophenyl-6-sulfo-2-acetamido-2-deoxy-β-D-glucopyranoside, using a sample obtained from a subject that is also subjected to the method of Ng using the 4-nitrophenyl N-acetyl-β-glucosaminide substrate. One of ordinary skill in the art would have been motivated to do this in order to additionally diagnose Tay-Sachs disease from a sample of a subject, specifically by determining the absence of PNP (p-nitrophenol) through MS/MS because of deficiency of β-hexosaminidase A activity. There would have been a reasonable expectation of performing the method of Ng, which involves MS/MS, with the sulfated substrate because both the sulfated substrate and the non-sulfated substrate yield PNP as a product when enzymatically reacted with the functional enzyme (β-hexosaminidase or β-hexosaminidase A), wherein PNP can be detected by MS/MS. In additionally performing the method of Ng to diagnose Tay-Sachs disease, then the sample from a subject is combined with more than one β-hexosaminidase substrate (4-nitrophenyl N-acetyl-β-glucosaminide and p-nitrophenyl-6-sulfo-2-acetamido-2-deoxy-β-D-glucopyranoside), wherein p-nitrophenyl-6-sulfo-2-acetamido-2-deoxy-β-D-glucopyranoside is directed to the ‘first said β-hexosaminidase substrate’ of instant claim 37 and 4-nitrophenyl N-acetyl-β-glucosaminide is directed to the ‘second said β-hexosaminidase substrate’ of instant claim 37. As such, instant claims 36 and 37 are also rendered obvious.
Conclusion
No claims are allowed.
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