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 OFFICE ACTION
This Office Action is in response to the papers filed on 30 June 2026.
CLAIMS UNDER EXAMINATION
Claims 1-2, 19, 22-23, 134-135, 140 and 361-371 are pending and have been examined on their merits.
PRIORITY
Provisional Application 63/053939, filed on 20 July 2020, is acknowledged.
WITHDRAWN REJECTIONS:
Claim 1 has been amended to further limit the aqueous wash solutions. The claim recites measuring a concentration of an active form of the NSP in the supernatant. The previous rejections are withdrawn.
NEW REJECTIONS
New grounds of rejection have been necessitated by claim amendment.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1-2, 19, 22-23, 134-135, 140, 361 and 365-371 are rejected under 35 U.S.C. 103 as being unpatentable over Kalupov et al. (previously cited; Structural Characterization of Mouse Neutrophil Serine Proteases and Identification of Their Substrate Specificities. JBC. Vol. 284, No. 49, pp. 34084–34091, 2009) in view of Wong et al. (The Role of Physical Stabilization in Whole Blood Preservation. Sci Rep 6, 21023 (2016) and Hamon et al. (Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases. J Biol Chem. 2016 Apr 15;291(16):8486-99) as evidenced by Dictionary.com.
Kalupov teaches a method of purifying neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (Pr3) (see page 34086, right column, second paragraph).
Peritoneal cavities are lavaged with phosphate buffered saline (i.e., “consisting of” PBS) (page 34084, left column, fifth paragraph). The art teaches lavage fluid contains neutrophils. Therefore PBS is added to a sample (lavage) comprising white blood cells of a subject. The art teaches cytospin (centrifugation). Kalupov teaches a cell pellet (see section cited above). Therefore a supernatant and pellet comprising the WBCs are obtained.
The cell pellet is resuspended in PBS containing Brij 35 (page 34084, left column, fifth paragraph). It is well known that Brij 35 is a surfactant. To purify neutrophil serine proteases, cell pellets were subjected to three cycles of freezing and thawing. The cell suspension was centrifuged after each cycle, and supernatants were collected and diluted in two volumes of 50 mM phosphate-buffered saline, pH 7.4. Protease fractions are recovered from pooled supernatants (page 34085, left column, last paragraph).
The art does not explicitly teach analyzing the supernatant obtained after the cytospin for active serine proteases.
The art does not teach lysing the pellet with octylphenoxypolyethoxyethanol.
Wong teaches a method of quantifying neutrophil elastase (a neutrophil serine protease). Blood samples were diluted with PBS before centrifugation. The supernatant was then transferred to a new centrifuge tube. The level of neutrophil elastase was quantified using the Neutrophil Elastase Activity Assay Kit (see page 8, third paragraph). Concentrations are determined (e.g., text of Figure 4d). Therefore the art measures the concentration of an active neutrophil elastase (a neutrophil serine protease).
Hamons studies a neutrophil protease. Hamon teaches purified neutrophils can be lysed in 50 mm HEPES buffer, 750 mm NaCl, and 0.05% Nonidet P-40, pH 7.4 (see page 8488, right column, fourth paragraph). As evidenced by the specification, Nonidet P-40 is (see [0047] of PG Pub).
It would have been obvious to measure the concentration of active NSP in the supernatant formed in Kalupov. One would have been motivated to do so to quantify the level of NSP in a white blood cell sample. One would have had a reasonable expectation of success since Wong teaches active NSP can be measured in a supernatant. One would have expected similar results since both references are directed to methods of analyzing NSPs.
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It would have been obvious to one of ordinary skill in the art at the time of the invention to use the surfactant taught by Hamon in Kalupov’s method. Kalupov lyses cells using a surfactant. Harmon teaches the surfactant Nonidet P-40 can be used to lyse cells. KSR B teaches it is rational to substitute one known, equivalent element for another to obtain predictable results. One would have had a reasonable expectation of success since Hamon teaches a neutrophil protease can be isolated from a neutrophil using Nonidet P-40. One would have expected similar results since Kalupov and Hamon are both directed to methods of isolating neutrophil proteases. Therefore claim 1 is rendered obvious.
Kalupov sonicates cells on ice to lyse cells (page 34085, left column, last paragraph). As evidenced by Dictionary.com, ice is a solid consisting of frozen water which forms at or below a temperature of 0°C (32°F). It would have been obvious to contact cells with the solution taught by Harmon at a temperature of about 0°C. KSR A teaches it is obvious to combine familiar elements according to known methods to yield predictable results. One would have had a reasonable expectation of success since Kalupov teaches cells can be lysed on ice a (temperature of 0°C). Therefore claim 2 is rendered obvious. Therefore claim 2 is rendered obvious.
Kalupov teaches repeating the cell lysis steps. Harmon teaches an aqueous medium comprising octylphenoxypolyethoxyethanol. It would have been obvious to use the same medium. One would have been motivated to do so to repeat the extraction steps as taught in Kalupov using the same conditions. One would have had a reasonable expectation of success since Harmon teaches Nonidet P-40 can lyse cells for NSP extraction. Therefore claim 19 is included in this rejection.
Kalupov teaches pooling (supra). Therefore claim 22 is rendered obvious.
Kalupov teaches centrifuging after each cycle, collecting the supernatant (hence, the lysate) and pooling to analyze protease activities (page 34086, left column, first paragraph). Wong teaches NSP can be quantified in a supernatant.
It would have been obvious to measure the concentration of active NSP in the pooled extract formed in Kalupov. One would have been motivated to do so to quantify the level of NSP. One would have had a reasonable expectation of success since Wong teaches active NSP can be measured in a supernatant. One would have expected similar results since both references are directed to methods of analyzed NSPs. Therefore claim 23 is included in this rejection.
Hamon teaches purified neutrophils can be lysed in 50 mm HEPES buffer, 750 mm NaCl (0.75 M), and 0.05% Nonidet P-40, pH 7.4. This reads on claim 134.
Kalupov teaches NE can be isolated (supra). Therefore claim 135 is included in this rejection.
Wong teaches “EasySep Human Neutrophil Enrichment Kit) (page 7, section titled “Leukocyte enrichment and yield quantification”). Therefore the cells can be obtained from a human subject. Claim 140 is rendered obvious.
Kalupov teaches PBS. This reads on the “consisting of” claim language. Claim 361 is included in this rejection.
Harmon teaches 0.05% Nonidet P-40 (supra). Claims 365-369 are included in this rejection.
Kalupov teaches a method of purifying neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (Pr3) (supra). Claims 370-371 are rejected.
Therefore Applicant’s Invention is rendered obvious as claimed.
Claim 362 is rejected under 35 U.S.C. 103 as being unpatentable over Kalupov in view of Wong and Harmon as applied to claim 1 above, and further in view of
Berkow et al. (Purification and Functional Evaluation of Mature Neutrophils From Human Bone Marrow, Blood, Volume 68, Issue 4,1986,Pages 853-860).
Claim 1 is rejected on the grounds set forth above. The teachings of the prior art are reiterated. Kalupov washes in PBS. The art does not teach washing in 0.9% NaCl.
Berkow teaches a method of isolating neutrophils from peripheral blood. Berkow centrifuges a leukocyte-rich supernatant. The pellet was suspended in 0.9% NaCI (see page 854, left column, third paragraph).
It would have been obvious to substitute the PBS wash taught by Kalupov with 0.9% NaCl. KSR B teaches that it is rational to substitute one known, equivalent element for another to obtain predictable results. Kalupov teaches an aqueous wash for a sample containing white blood cells and Berkow teaches 0.9% NaCl can be used to wash white blood cells. One would have had a reasonable expectation of success since Kalupov teaches 0.9% NaCl can wash a sample containing neutrophils. One would have expected similar results since both references are directed to neutrophil isolation. Claim 362 is rejected.
Therefore Applicant’s Invention is rendered obvious as claimed.
Claims 363-364 are rejected under 35 U.S.C. 103 as being unpatentable over Kalupov in view of Wong and Hamon as applied to claim 1 above, and further in view of Genaxxon Bioscience (previously cited; Tris buffered saline (TBS) 2014, pages 1-2).
Claim 1 is rejected on the grounds set forth above. The teachings of the art are reiterated. Kalupov does not teach the use of Tris buffered saline (claims 363-364).
Genaxxon teaches Tris buffered saline (TBS) is isotonic and non-toxic to cells and is suitable for molecular biology. The buffer is commonly used as a wash buffer (see page 1, first paragraph of Product Description). Genaxxon teaches a TBS buffer comprising 0.05M Tris. 0.138M NaCl at a pH 8.0 (see Product specifications on page 1; 1X TBS pH 8.0). Because the claims do not define the term “about”, the amount of saline in TBS is interpreted to read on claim 362
It would have been obvious to use TBS comprising NaCl to wash cells. Seren washes cells and Genaxxon teaches washing cells with a TBS buffer. One would have been motivated to do so since Genaxxon teaches TBS is non-toxic to cells. One would have had a reasonable expectation of success since Genaxxon teaches TBS comprising NaCl can be used to wash cells. One would have expected similar results since both references are directed to solutions for washing cells. Therefore claim 363 is rendered obvious. Because the claims nor the specification define the term “about”, the concentrations and pH taught by Genaxxon are interpreted to read on claim 364.
Therefore Applicant’s Invention is rendered obvious as claimed.
APPLICANT’S ARGUMENTS
The arguments made in the response filed on 10 June 2026 are acknowledged.
The Applicant argues Kalupov resuspends a neutrophil pellet in PBS supplemented with Brij 35, a surfactant. The Applicant argues this is distinct from a supernatant lacking a surfactant in the claimed method.
Response: Kalupov teaches peritoneal cavities are lavaged with phosphate buffered saline (i.e., “consisting of” PBS) (page 34084, left column, fifth paragraph). The art teaches lavage fluid contains neutrophils. Therefore PBS is added to a sample (lavage) comprising white blood cells of a subject. The art teaches cytospin (centrifugation). Kalupov teaches a cell pellet (see section cited above). The preceding steps do not require a surfactant. Kalupov used Brij 35 in a subsequent step. New grounds of rejection have been made to address the amended claims. Therefore the argument is not persuasive.
CONCLUSION
No Claims Are Allowed
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to NATALIE MOSS whose telephone number is (571) 270-7439. The examiner can normally be reached on Monday-Friday, 8am-5pm EST.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Sharmila Landau can be reached on (571) 272-0614. The fax phone number for the organization where this application or proceeding is assigned is (571) 273-8300.
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/NATALIE M MOSS/ Examiner, Art Unit 1653
/SHARMILA G LANDAU/ Supervisory Patent Examiner, Art Unit 1653
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