rtDETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 02/04/2026 has been entered.
Election/Restrictions
Applicant’s election without traverse of Group III (i.e., a method comprising administering to a subject a composition comprising CAII and copper) in the reply filed on 03/28/2025 is acknowledged.
Amendments Received
Amendments to the claims were received and entered on 12/31/2025.
Status of Claims
Claim 33 has been cancelled.
Claims 11-14, 17, 19-21, 26-27, and 31-32 and 34-39 are currently pending.
Claims 11-14 and 34-39 are under consideration, as claims 17, 19-21, 26-27, and 31-32 are withdrawn.
Priority
The present application claims status as a 371 (National Stage) of PCT/US21/13416 filed on January 14, 2021. Acknowledgment is made of applicant’s claim for benefit under 35 U.S.C. 119(e) of Provisional application No. 63/008,607, filed on April 10, 2020 and Provisional application No. 62/961,147, filed on January 14, 2020. The present application and all claims are being examined with an effective filing date of January 14, 2020. In future actions, the effective filing date may change due to amendments or further review of priority documents.
Withdrawn Objections
In view of Applicant’s amendments, the objection to claim 12 is hereby withdrawn.
Withdrawn Rejections
In view of Applicant’s amendments, rejection of claim 13 under 35 USC § 112(b) is hereby withdrawn.
In view of Applicant’s remarks filed on 12/31/2025, regarding limitations reciting “Ser2”, rejections of claims 35, 38, and 39 under 35 USC § 103 are hereby withdrawn.
Maintained/Modified Rejections Necessitated by Amendment
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 11-14, 34, 36 and 37 are rejected under 35 U.S.C. 103 as being unpatentable over Watkins et al. (Nitrate- nitrite- nitric oxide pathway in pulmonary arterial hypertension therapeutics. Circulation. 2012 Jun 12;125(23):2824-6, cited in the IDS), Nettles et al. (Characterization of the Copper(II) Binding Sites in Human Carbonic Anhydrase II. Inorg Chem. 2015 Jun 15;54(12):5671-80, cited in the IDS), Hanff et al. (GC-MS determination of nitrous anhydrase activity of bovine and human carbonic anhydrase II and IV. Anal Biochem. 2018 Jun 1;550:132-136, cited in the IDS), and Aamand et al. (Generation of nitric oxide from nitrite by carbonic anhydrase: a possible link between metabolic activity and vasodilation. Am J Physiol Heart Circ Physiol. 2009 Dec;297(6):H2068-74, cited in the IDS).
Regarding claims 11-13, Watkins et al. teaches that elevated vascular resistance in pulmonary arterioles is characteristic of pulmonary hypertension and “impairments in vasodilator (nitric oxide [NO] and prostaglandin signaling) and vasoconstrictor (endothelin-1, reactive oxygen species, angiotensin II) pathways underlie the evolution of early disease”. As such, drugs that enhance NO signaling have been developed for the treatment of pulmonary arterial hypertension (pg. 1, 1st para). It is noted here that “drugs that enhance NO signaling” necessarily result in vasodilation. Watkins et al. also teaches pathways to NO production, including the nitrate to nitrite to NO pathway, and that “the signaling properties of nitrite are largely mediated by nitrite reduction to NO” (pg. 2, last para). Watkins et al. further teaches that mammalian nitrite reductase candidates have been proposed, “divided into groups based on active site metal content”, including the copper containing carbonic anhydrase (pg. 3, 2nd para). Watkins et al. does not expressly teach administering to a subject, the copper containing carbonic anhydrase, nor does it expressly teach wherein two binding sites within the CAII are each bound to a copper atom; however it does establish the therapeutic motivation to enhance NO signaling for the treatment of pulmonary hypertension and specifically identifies copper-containing carbonic anhydrase as a mammalian enzyme potentially capable of mediating nitrite reduction to NO.
Nettles et al. also teaches that human carbonic anhydrase II is a zinc-containing metalloprotein, wherein the apo form binds to copper with high affinity (Abstract). Nettles et al. further teaches that “in the traditional (His) 3 metal binding site (Cu B) binds Cu 2+ with a higher affinity than Zn2+” (pg. 5676, right column, last para). Nettles et al. characterizes two thermodynamically distinct Cu2+ binding sites within carbonic anhydrase II (CAII) – Cu A (N-terminal) and Cu B (canonical) – and describes preparations wherein both sites are occupied. Specifically, Nettles et al. discloses three forms of carbonic anhydrase preparations; reconstituted CA (ZnCA, adding 1 equiv Zn2+ to apoCA), copper/zinc CA (Cu/ZnCA, addition of 1 equiv Cu2+ into ZnCA), and dicopper CA (Cu/CuCA, addition of 2 equiv of Cu2+ into apoCA) (pg. 5672, left column, “Sample Preparation”). Regarding the dicopper CA, Nettles et al. expressly teaches that “Cu2+ occupies both the CuA and CuB sites” (pg. 5672, NMR measurements). Thus, Nettles et al. confirms the structural and coordination feasibility for Cu 2+ substitution into CAII (including configurations “wherein two binding sites within the CAII are each bound to a copper atom”) and provides a reasonable expectation that Zn2+ could be replaced by Cu2+ without loss of enzyme integrity, favored by the higher binding affinity of Cu2+ (compared to Zn2+).
Hanff et al. teaches, through their own studies, that human erythrocytic carbonic anhydrase II bound to zinc does not exhibit nitrite reductase activity, likely because the zinc cation of CA has only a single oxidation state (Zn2+). These results were compared to those disclosed in Aamand et al., incorporated by reference, wherein bovine carbonic anhydrase II generated nitric oxide from nitrite, and induced vasodilation in aortic rings. Hanff et al. suggests that “a possible explanation for the discrepancy in the literature could be the use by Aamand et al. of a pharmaceutical formulation of dorzolamide that contained additional unspecified redox-active ingredients. This explanation is realistic because we found by means of an NO-sensitive electrode that CAII and CAIV can mediate conversion of nitrite to NO in the presence of L-cysteine which contains a readily oxidizable sulfhydryl (SH) group” (pg. 132, last para -133, first line). Furthermore, Hanff et al. explicitly states that “the Zn²⁺ in CA II is unlikely to exhibit nitrite reductase activity, most likely because its zinc cation lacks oxidation states other than +II,” and contrasts this with copper-containing enzymes such as ceruloplasmin and hemoglobin that possess redox-active ions capable of reducing nitrite to NO. This teaching would have further motivated a person of ordinary skill in the art to replace the redox-inert Zn²⁺ in CA II with Cu²⁺ to create a redox-active center and thereby impart nitrite-reductase functionality. Aamand et al. supports this reasoning by demonstrating that carbonic anhydrase can generate NO from nitrite and induce vasodilation in aortic rings, thereby providing a functional link between enzyme activity and the vasodilatory effect. Thus, the teachings of Hanff et al. and Aamand et al. are complementary, not contradictory, and together would have provided a skilled artisan with a clear mechanistic basis for substituting Cu²⁺ for Zn²⁺ in CA II to enable nitrite → NO conversion.
With respect to claim 14, Watkins et al. further teaches that “pulmonary arterial hypertension (PAH) is a disorder characterized by elevated vascular resistance in pulmonary arterioles,” and that progressive increases in pulmonary vascular resistance and arterial pressure lead to right heart failure and reduced cardiac output (pg. 1, 1st para.). Accordingly, the combined teachings of Watkins, Aamand, Hanff, and Nettles would have led a person of ordinary skill in the art to administer a composition comprising Cu-substituted CA II to a subject suffering from hypertension or pulmonary hypertension to achieve vasodilation through enhanced NO signaling. Such use would represent a predictable application of known principles, as taught in the prior art.
Regarding claims 34 and 37, Nettles et al. teaches that human carbonic anhydrase II possesses two distinct copper binding sites, including an active site CuB coordinated by His94, His96, and His119 (pg. 5675–5676). Nettles et al. further demonstrates that this CuB site binds Cu²⁺ with high affinity and that copper substitution for Zn²⁺ at this position is structurally and thermodynamically feasible. Accordingly, the limitation of claim 34 identifying copper coordination through His94, His96, and His119 is expressly taught by Nettles et al., and the recited percentages of copper‑bound enzyme in claim 37 (10-99%) merely define the degree of metal substitution, which would have been a matter of routine optimization to a person of ordinary skill in the art seeking to prepare copper‑substituted CAII compositions as taught by Nettles et al.
Regarding claim 36, while Nettles et al. teaches compositions comprising carbonic anhydrase II and copper, it does not explicitly describe a pharmaceutical formulation. However, Aamand et al. describes the use of commercial dorzolamide (Trusopt) – an FDA approved pharmaceutical preparation- in combination with purified carbonic anhydrase II in vasodilation studies, and further discloses the use of physiological saline (PSS) and phosphate-buffered saline (PBS) as the media for the enzyme administration and activity measurement (pg. 2, Sample Preparation). These represent pharmaceutically acceptable carrier systems widely used for protein based therapeutics. It would have therefore been obvious to a person of ordinary skill in the art to incorporate the compositions disclosed by Nettles et al., comprising CAII and copper, into a pharmaceutical formulation further comprising a pharmaceutically acceptable carrier, such as those described in Aamand et al., or other routine carrier known in the art, in or der to enable administration of the enzyme composition to a subject in need thereof, such as those with pulmonary hypertension, as described above.
An invention would have been obvious to a person of ordinary skill in the art if some teaching in the prior art would have led that person to combine prior art reference teachings to arrive at the claimed invention. Before the effective filing date of the claimed invention, the combined teachings of Hanff et al. and Watkins et al., that a CAII enzyme having a redox-active center, such as a copper-containing CAII, can reduce nitrite to NO, and that pulmonary hypertension is treated with drugs that enhance NO signaling, would have led that practitioner to administer the composition taught by Nettles et al., comprising CAII and copper, to a subject suffering from pulmonary hypertension. Moreover, because Hanff et al. explicitly contrasts Zn²⁺-bound CAII (inactive) with copper-containing enzymes (active for nitrite → NO conversion), and Nettles et al. demonstrates that Cu²⁺ binds CAII at two thermodynamically distinct, high-affinity binding sites (CuA and CuB), including the CuB site coordinated by His94, His96, and His119, and further demonstrates that both sites can be occupied, a person of ordinary skill in the art would have had both motivation and a reasonable expectation of success in substituting Cu²⁺ for Zn²⁺ to obtain a CAII enzyme wherein two binding sites are each bound to a copper atom. The resulting composition would have been predictably useful for enhancing NO signaling and inducing vasodilation in a subject as taught by Watkins et al. Given that redox-active metal centers are known to mediate nitrite reduction, and in view of the combined teachings of Hanff et al. and Aamand et al., which demonstrate that carbonic anhydrase can participate in NO generation from nitrite, there would have been a reasonable expectation that substitution of Cu²⁺ into CAII would impart nitrite reductase activity. Therefore, there would have been a reasonable expectation that the combination of these teachings would successfully result in a method comprising administering to a subject a composition comprising CAII and copper, wherein two binding sites within the CAII are each bound to a copper atom and wherein the composition has nitrite reductase activity. Accordingly, the claimed invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Response to Arguments for Prior Art Rejections
In the Response filed December 31, 2025, Applicants argue that:
1. The cited references teach away from copper-substituted CAII having nitrite reductase activity, as Hanff et al. teaches that CAII/CAIV induces NO from nitrite only with L-cysteine, Nettles et al. teaches that CuA has no reactivity and sequesters Cu(II), and Aamand et al. teaches that zinc-bound CAII produces NO and is ubiquitous.
2. The combination lacks motivation, as a skilled artisan would either follow Hanff (no reason to use Cu-CAII) or Aamand (no need to modify zinc-CAII), and Watkins et al.’s mention of copper-containing CA as a nitrite reductase candidate is a “mistake” unsupported by its sole cited reference (Aamand).
3. Results of the combination would not have been predictable, as prior artisans believed CAII lacked nitrite reductase activity absent additives like L-cysteine.
4. Nettles et al. does not teach the specific copper binding site of claim 35 (requiring His3, His4, and Ser2).
Applicants’ arguments have been considered in full. Applicants’ arguments are persuasive as to claim 35, but are not persuasive as to claim 11, for the reasons set forth below.
Applicants’ arguments improperly conflate claim 35’s narrow residue-specific coordination requirements with claim 11’s broader limitations of a composition comprising CAII and copper, wherein two binding sites within CAII are each bound to a copper atom and the composition has nitrite reductase activity. For claim 11, Nettles et al. expressly teaches dicopper CAII with two distinct Cu(II) binding sites, CuA in the N-terminus and CuB at the canonical active site, and thus teaches CAII compositions in which two binding sites are occupied by copper. No hindsight is required to rely on this express structural teaching.
The references are complementary not contradictory - Applicants argue that Hanff et al., Nettles et al., and Aamand et al. teach away because they allegedly provide contradictory results. That position is not persuasive. The cited references are properly read as complementary teachings describing different aspects of the same overall problem: functional nitrite-to-NO conversion, the mechanistic limitations of zinc-bound CAII, and the structural feasibility of copper substitution in CAII. Aamand et al. teaches that carbonic anhydrase can generate nitric oxide from nitrite and induce vasodilation in aortic rings, thereby linking CA-associated nitrite processing to a physiological NO response. Hanff et al. then refines the mechanism by showing that zinc-bound CAII does not exhibit nitrite reductase activity, explaining that this is consistent with zinc’s redox-inactive nature, while contrasting that with copper-containing enzymes that possess redox-active metal centers capable of reducing nitrite to NO. Nettles et al. provides the structural solution by showing that apo-CAII binds two equivalents of Cu(II) at distinct sites, with CuA and CuB both capable of occupancy. Applicants emphasize Hanff’s statement that CAII and CAIV can induce formation of NO from nitrite only under specific conditions, namely in the presence of L-cysteine. That statement does not teach away from Cu-substituted CAII; rather, it underscores that nitrite reduction requires a redox-active context. Hanff explicitly contrasts zinc with copper-containing enzymes, thereby motivating substitution of Cu for Zn in CAII to create a redox-active center capable of nitrite reduction.
Motivation to combine was present - Watkins et al. teaches that pulmonary arterial hypertension is characterized by impaired NO signaling and that therapeutic strategies aimed at enhancing NO signaling are beneficial. Watkins et al. further teaches the nitrate-to-nitrite-to-NO pathway and identifies mammalian nitrite reductase candidates, including copper-containing carbonic anhydrase, based on active site metal content. That express teaching provided the motivation to look to copper-containing CAII as a potential nitrite reductase candidate in a therapeutic context. Applicants’ assertion that Watkins’ statement was merely a “mistake” is not persuasive. Watkins expressly includes copper-containing carbonic anhydrase among proposed mammalian nitrite reductase candidates, and the Office relies on the express teachings of the reference as written, not on speculation about whether Applicants would characterize that statement as erroneous. Watkins therefore supplies a proper prior-art teaching and motivation, particularly when read together with Hanff’s explicit redox rationale and Nettles’ demonstration that CAII can bind copper at two distinct sites.
Reasonable expectation of success was present - Applicants further argue that the results of the combination would not have been predictable because prior artisans believed CAII lacked nitrite reductase activity absent additives such as L-cysteine. That argument is not persuasive. Hanff’s discussion of zinc-bound CAII shows that the absence of nitrite reductase activity is tied to zinc’s redox-inactive nature, not to a general inability of carbonic anhydrase to participate in nitrite-to-NO chemistry. Hanff further contrasts zinc with copper-containing enzymes that are known to reduce nitrite to NO, which would have suggested to a skilled artisan that substitution of copper for zinc in CAII could impart the desired redox activity. That expectation is reinforced by Nettles, which demonstrates that CAII can accommodate copper at two distinct sites with high affinity, including dicopper preparations in which both CuA and CuB are occupied. This is additionally supported by Song et al. (Building reactive copper center(s) in human carbonic anhydrase II, (2013) Biochemistry 52, 4517–4526, cited in PTO-892) which further confirms that dicopper CAII is redox-competent by showing catalytic reactivity in oxidation chemistry, and Tabbi et al. (The copper(II) binding centres of carbonic anhydrase are differently affected by reductants that ensure the redox intracellular environment, Journal of Inorganic Biochemistry 199 (2019) 110759, cited in PTO-892) which demonstrates that the catalytic and peripheral (N-terminal) copper binding sites in carbonic anhydrase are distinct and are differently affected by naturally occurring intracellular reductants – confirming that the dicopper centers are chemically active and sensitive to the cellular redox milieu/environment. These teachings, taken together, provide a robust structural and mechanistic basis for a person of ordinary skill in the art to have a reasonable expectation that copper-substituted CAII could exhibit nitrite reductase activity.
No improper hindsight or personal opinion - Applicants’ hindsight argument is not persuasive. The rejection does not rely on the Examiner’s personal opinion or on Applicants’ later-disclosed invention as a template. Rather, it relies on the express teachings of Watkins, Hanff, Aamand, and Nettles, which together would have led a person of ordinary skill in the art to consider copper-substituted CAII as a candidate nitrite reductase in the context of enhancing NO signaling. Obviousness can be established where the prior art supplies a reason to combine or modify known teachings and there is a reasonable expectation of success; it does not require absolute predictability of success (see MPEP 2143.01 and 2143.02). Here, Watkins provides the therapeutic motivation, Hanff provides the redox rationale for copper over Zn, and Nettles provides the structural feasibility of copper substitution at two CAII binding sites, so the combination was reasonably predictable even though absolute certainty was not required.
Accordingly, claim 11 remains rejected under 35 U.S.C. § 103 as obvious over Watkins, Nettles, Hanff, and Aamand. Applicants’ argument regarding Nettles et al. not teaching His3/His4/Ser2 coordination, or the specific N-terminal motif required by claim 35, is persuasive. Nettles localizes CuA to the N-terminus but does not expressly teach the Ser2-containing coordination environment recited in claim 35. As such, rejections of claims reciting Ser2 coordination have been withdrawn.
Allowable Subject Matter
Claims 35, 38 and 39 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Conclusion
No claim is in condition for allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to NAGHMEH NINA MOAZZAMI whose telephone number is (703)756-4770. The examiner can normally be reached Monday-Friday, 9:00-5:00.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert Mondesi can be reached at 408-918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/NAGHMEH NINA MOAZZAMI/ Examiner, Art Unit 1652
/ROBERT B MONDESI/ Supervisory Patent Examiner, Art Unit 1652
Prosecution Insights