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 2-4, 6-9, 11-12, 15-41, 49 are canceled. Claims 50-57 are new. Claims 1, 5, 10, 13-14, 42, 45, 46-48, and 50-57 are pending and under consideration.
Priority
The instant claims are entitled to an effective filing date of 06/26/2020.
Claim Objections
(New) Claim 45 is objected to because of the following informalities:
Claim 45 recites “cholera” in line 3, which should be italicized.
Claim 45 recites “; and propeptide, and” in lines 25-26, which should be replaced with “; and”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
(Maintained and extended to new claims 54-55) Claims 42 and 54-55 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method of identifying Vibrio cholera in a sample; does not reasonably provide enablement for identifying C. difficile or P. aeruginosa. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
The factors to be considered in determining whether a disclosure would require undue experimentation include:
A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
I The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
In re Wands, 8 USPQ2d, 1400 (CAFC 1988) and MPEP 2164.01.
The breadth of the claims and the nature of the invention:
Under the broadest reasonable interpretation, claim 42 and its dependent claims entail identifying V. cholera, C. difficile or P. aeruginosa in any sample by monitoring a detectable signal before and after a contact step in which the sample is contacted with a construct comprising a propeptide with at least one mutated cysteine residue that is conjugated to a detectable label, AMP or a therapeutically active small molecule. The propeptide within the construct is required to be selected from a group that consists of: SEQ ID NO: 1, SEQ ID NO : 2. SEQ ID NO: 3, residues 52-128 of SEQ ID NO: 1, residues 1-66 of SEQ ID NO: 2 and residues 25-211 of SEQ ID NO: 3.
According to the instant specification, SEQ ID NO: 1 is IvaP (in vivo-activated protease) from Vibrio cholerae. The I9 inhibitor domain (i.e. the propeptide) comprises residues 52-128 of SEQ ID NO: 1. See line 12 on page 16 and the paragraph spanning pages 16-17. SEQ ID NO: 2 is the C. difficile protease CspB. The I9 inhibitor domain comprises amino acid residues 1-66 of SEQ ID NO: 2. See lines 3 and 37-38 on page 17. SEQ ID NO: 3 is P. aeruginosa protease IV and the propeptide inhibitor domain comprises residues 25-211 of SEQ ID NO: 3. See line 40 on page 17 to line 22 on page 18. The specification discloses that proteases are produced in an inactive propeptide-bound form. Proteases are typically activated by cleavage of the propeptide during enzyme maturation. See page 8 lines 9-11 and figure 2A. Since, SEQ ID NOs: 1-3 are proteases that include propeptides, the specification suggests that the proteases of SEQ ID NOs: 1-3 are inactive.
The state of the prior art and the level of predictability in the art:
With respect to the state of the art on using protease propeptide constructs to identify pathogenic bacteria, Buss (EBioMedicine, 2018, 38, 248-256) teaches designing a nanoparticle sensor comprised of a peptide substrate for the Pseudomonas aeruginosa protease LasA, a virulence factor secreted by strain PA01. Buss indicates that the substrates within the nanoparticle sensor include a fluorophore-quencher FRET pair flanking the protease cleavable sequence. See the abstract and the first paragraph of section 3.1. Buss indicates that the activity-based nanoparticle sensor is deployable to the context of infection, both for the specific identification of P. aeruginosa lung infections and for the monitoring of their treatment. The strategy measures both host- and pathogen-derived protease activity, which facilitates the rapid identification of infection. Buss indicates that the nanosensors are able to detect PA01 infections, but it may not cover all P. aeruginosa strains and may not perfectly differentiate P. aeruginosa infections from those caused by other bacterial species. See section 4. Thus, Buss indicates that pathogenic bacterial infections can be identified by monitoring the protease activity. However, Buss indicates detection may be limited to specific strains; furthermore, Buss is silent regarding propeptide constructs. DeColli (Cell Chemical Biology, 2022, 29(10), 1505-1516), a post-filing date reference, teaches leveraging proteolytic activity to design molecular diagnostics. DeColli hypothesizes that I9 propeptides can be harnessed to selectively detect specific proteases in complex samples. See the first passage and paragraph on page 1506. DeColli teaches generating constructs via site-directed mutagenesis and subsequent bioconjugation of I9 with environmentally sensitive dyes including MDCC. The I9E123C-MDCC construct exhibits a V. cholerae dependent decreases in fluorescence when added to mixed cultures of bacteria, and the construct is resistant to nonspecific cleavage by host proteases produced by cultures of human intestinal cells. See the last 8 lines of the left column on page 1506. However, DeColli suggests that additional studies are needed to establish the applicability of this technology to microbes beyond V. cholerae. See the first full paragraph on page 1513. Moreover, DeColli acknowledges that the applicability of the I9E123C-MDCC probe may be limited because the fluorescent properties of MDCC are heavily influenced by its microenvironment. See the last section on page 1513. In summation, Buss illustrates the unpredictability in the art prior to the effective filing date because Buss suggests that protease activity-based nanoparticle sensors may be strain specific; similarly, DeColli illustrates the unpredictability in the art to date because DeColli suggests that additional studies are required before I9 propeptide probes can be used for detecting microbes beyond V. cholerae.
The amount of direction provided by the inventor and the existence of working examples:
In example 2, the specification teaches incubating an I9 E123C-MDCC probe with biofilm cultures of V. cholerae, S. enterica typhimurium, E. coli and Vibrio parahaemolyticus. See the last paragraph on page 38. The I9 E123C-MDCC probe comprises an I9 propeptide domain with an E123C mutation and a MDCC (i.e. [7-(diethylamino)-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-2-oxo-2H-chromene-3-carboxamide]) conformation-sensitive dye. See the second paragraph on page 38. The specification indicates that the cysteine mutations provide a selective handle for bioconjugation. See lines 14-15 on page 37. After the I9 E123C-MDCC probe is incubated with the biofilm cultures, there is a significant and specific decrease in the fluorescence intensity of the probe that is detected in the presence of V. cholerae, but not other tested bacteria (figure 10B). Moreover, the probe can selectively detect V. cholerae grown in mixed cultures with V. parahaemolyticus, as an ~8-fold decrease in probe intensity is observed only in the presence of V. cholerae (figure 10C). See the last paragraph on page 38. Thus the specification provides one working example in which an I9 E123C-MDCC propeptide construct is contacted with a sample comprising a V. cholerae pathogenic bacterium, and a fluorescent detectable signal is monitored before and after the contact step.
The quantity of experimentation needed to make or use the invention:
In view of the nature of the invention, the breadth of the claims, the guidance and
working examples in the specification, and the level of predictability within the art, as
evidenced above, one skilled in the art could not use a construct commensurate in scope with claim 42 to identify C. difficile or P. aeruginosa in any sample, without undue experimentation. Prior to the effective filing date of the instantly claimed invention, it was well known that protease activity probes may be used to detect infections caused by specific strains, as evidenced by Buss. However, instant claim 42 encompasses constructs comprising SEQ ID NOs: 1-3, which are inactivate proteases.
Accordingly claims 42 and 54-55 are enabled for a method comprising identifying Vibrio cholera in a sample, the method comprising: contacting a I9E123C-MDCC construct with a sample, and monitoring a detectable fluorescent signal before and after the contact step, whereby a qualitative or quantitative change in the detectable fluorescent signal indicates that V. cholerae is present in the sample.
(Maintained and extended to new claims 56-57) Claims 45-48 and 56-57 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The factors to be considered in determining whether a disclosure would require undue experimentation include:
A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
In re Wands, 8 USPQ2d, 1400 (CAFC 1988) and MPEP 2164.01.
The breadth of the claims and the nature of the invention:
Under the broadest reasonable interpretation, claims 45-48 entail killing, or reducing or preventing the growth of a Vibrio cholera, C. difficile or P. aeruginosa pathogenic bacterium by contacting the pathogenic bacterium with a construct comprising a propeptide and any antimicrobial peptide (AMP). The propeptide is selected from: SEQ ID NOs: 1, 2 or 3, residues 52-128 of SEQ ID NO: 1, residues 1-66 of SEQ ID NO: 2 and residues 25-211 of SEQ ID NO: 3. Claims 46-47 require the method to be performed in vivo in any subject infected by the pathogenic bacterium, wherein the pathogenic bacterium is in the digestive tract, respiratory tract, urinary tract or skin of the subject. Claim 48 requires any non-pathogenic bacteria in the gut of the subject to not be killed, reduced or prevented.
According to the instant specification, SEQ ID NO: 1 is IvaP (in vivo-activated protease) from Vibrio cholerae, and the I9 inhibitor domain (i.e. the propeptide) comprises residues 52-128 of SEQ ID NO: 1. See line 12 on page 16 and the paragraph spanning pages 16-17. SEQ ID NO: 2 is the C. difficile protease CspB and the I9 inhibitor domain comprises amino acid residues 1-66 of SEQ ID NO: 2. See lines 3 and 37-38 on page 17. SEQ ID NO: 3 is P. aeruginosa protease IV and the propeptide inhibitor domain comprises residues 25-211 of SEQ ID NO: 3. See line 40 on page 17 to line 22 on page 18. The specification discloses that proteases are produced in an inactive propeptide-bound form. Proteases are typically activated by cleavage of the propeptide during enzyme maturation. See page 8 lines 9-11. Thus, the specification indicates that SEQ ID Nos: 1-3 are the inactive propeptide-bound form of proteases.
The state of the prior art and the level of predictability in the art:
With respect to the state of the art on targeting proteases to kill pathogenic bacteria, Culp (The Journal of antibiotics, 2017, 70(4), 366-377) discloses that proteases have a number of key roles in bacterial physiology and biochemistry, as well as pathogenicity. See the second paragraph of the introduction section. Proteases are also essential to the ability of many bacteria to infect the host and cause disease. Blocking such virulence factors to prevent infection has been on the radar of leaders in infectious disease for decades; however, there is no approved drugs with this mode of action yet. See the third paragraph of the introduction section.
With respect to the state of the art on using protease cleavage to deliver AMPs in vivo, Drayton (Molecules, 2020, 25(13), 3048) discloses that AMPs have not found wide spread use in the clinic for a number of reasons, despite all their promise. First, most AMPs are susceptible to protease degradation and rapid kidney clearance. Furthermore, some AMPs are not specific to bacteria and hence display systemic toxicity. Oral administration of AMPs can lead to proteolytic digestion by enzymes in the digestive tract such as trypsin and pepsin. Moreover, systemic administration results in short half-lives in vivo and cytotoxic profiles in blood. To circumvent these issues, the use of delivery vehicles is being used. See the first paragraph on page 2. A number of characteristics unique to the microenvironment of an infection site can be utilized for targeted delivery of antibiotics. These include enzyme concentration. See the first paragraph on page 4. Enzymatically cleavable linkers enable targeting of both enzymes secreted by the bacteria themselves and/or enzymes released by the host in response to infection. The altered expression of these enzymes, e.g. proteases, can allow for drug accumulation at the site of infection. Protease targeting is particularly appealing for AMP development as the sequences can be directly adjoined to the N- or C-terminus of the peptide during expression or chemical synthesis. See the third paragraph on page 4. Yet, Drayton concludes that many hurdles block the successful translation of AMPs into clinical practice. See the paragraph spanning pages 15-16.
The amount of direction provided by the inventor and the existence of working examples:
The specification does not teach a working example of a construct capable of killing a pathogenic bacterium, or reducing or preventing growth of a pathogenic bacterium. Rather, the specification provides a prophetic example. Specifically, in example 3, the specification provides a prophetic example of a protease-activated antimicrobial for selectively killing pathogens in the gut. The specification describes propeptides from pathogen proteases that are fused to the C-terminal of antimicrobial peptides (AMP), potent antibiotics that punch holes in bacterial membranes. Propeptide cleavage releases the AMP. The specification discloses that the AMP release should not significantly impact the microbiota because most commensal microbes are naturally resistant to AMPs. The specification suggests that a non-limiting example is a Vibrio-toxic AMP fused to a propeptide. See the last full paragraph on page 39. However, the specification does not demonstrate using any specific AMP-propeptide construct to kill any bacterium.
The quantity of experimentation needed to make or use the invention:
In view of the nature of the invention, the breadth of the claims, the guidance and
working examples in the specification, and the level of predictability within the art, as
evidenced above, one skilled in the art could not use the disclosed construct to kill Vibrio cholera, C. difficile or P. aeruginosa, or reduce or prevent their growth without undue experimentation. The specification does not provide any working examples of a propeptide-AMP construct capable of killing pathogenic bacteria or reducing or preventing their growth. Culp illustrates the unpredictability in the art because Culp indicates that there are no clinically available drugs that target protease activity. Although Dayton suggests that using protease cleavage to deliver AMPs to an infection site is promising, Dayton also suggests that the use of AMPs in vivo is unpredictable. Consequently, there is no indication that the instantly claimed construct can kill any pathogenic bacteria or reduce or prevent their growth.
Accordingly claims 45-48 are not enabled.
Response to Arguments
Applicant's arguments filed 12/11/2025 have been fully considered but they are not persuasive.
§ 112(a) rejection of claim 42 and new claims 54-55
Applicant argues that the scope of amended claim 42 focuses on three particular pathogenic bacteria, V. cholerae, C. difficile and P. aeruginosa, such that the claim is no longer drawn to any pathogenic bacterium. Applicant asserts that examples 1-3 of the specification describe selective detection and killing of V. cholera. Example 4 notes that the selective detection and killing of C. difficile by adapting the present propeptide-based technology to target proteases from C. difficile. See the second and third paragraph under the ‘Claims 42-44’ subheading on page 12 of the remarks.
This argument is not persuasive because it is not commensurate in scope with the instant claims. The claims do not require contacting the V. cholerae, C. difficile or P. aeruginosa with targeted propeptides that are specific to proteases from that species. The specification teaches detecting V. cholera with an I9 E123C-MDCC propeptide construct. However, the specification does not indicate that the same construct can be used to identify C. difficile or P. aeruginosa. Furthermore, the I9 E123C-MDCC propeptide construct disclosed in the specification includes MDCC, which is a detectable label. Whereas, claim 42 does not require the construct to include a detectable label. Furthermore, the claimed construct encompasses embodiments comprising SEQ ID NO: 1-3, which are inactive forms of proteases.
§ 112(a) rejection of claims 45-48 and new claims 56-57
Applicant argues that the rejection largely mirrors the basis for the rejection of claim 42, in that the claims are alleged to entail killing any pathogenic bacterium or reducing or preventing the growth of any pathogenic bacterium. See the last full paragraph on page 12 of the remarks. Applicant asserts that claim 45 now recites propeptide sequences of SEQ ID NO: 1, 2 and 3 as well as specific subsets of these sequences. Applicant asserts that SEQ ID NOs: 1-3 correspond to V. cholerae, C. difficile and P. aeruginosa respectively. Applicant argues that, based on example 3 of the specification, a skilled artisan would be able to adapt an appropriate AMP for use in killing or reducing or preventing the growth of V. cholerae, C. difficile and P. aeruginosa. See the paragraph spanning pages 12-13 of the remarks.
This argument is not persuasive because claims 45-48 are not enabled by the specification. Example 3 is a prophetic example, and the specification does not provide any working examples of killing or reducing or preventing the growth of V. cholerae, C. difficile or P. aeruginosa. Furthermore, the claims encompass an in-vivo contact step, which would have been unpredictable because there are no clinically available drugs that target protease activity, as evidenced by Culp.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 5, 10, 13-14, 42, 45, 46-48, and 50-57 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.
Claims 1, 42 and 45 are indefinite for five reasons. First, claims 1, 42 and 45 recite “a pathogenic bacterium protease propeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3”, which render the claims indefinite because SEQ ID NOs: 1-3 are not propeptides. Rather, SEQ ID NOs: 1-3 are, respectively, the protease IvaP from V. cholerae, protease CspB from C. difficile, and the protease IV from P. aeruginosa. Therefore, it is unclear whether the claim intends for SEQ ID NOs: 1-3 to serve as a propeptide, or whether the claim intends to encompass any propeptide subsequence within SEQ ID Nos: 1-3. Second, claims 1, 42, and 45 recite “(a) the propeptide binds to its corresponding protease, at least one mutated cysteine is present in the interface of the propeptide-protease complex”, which is indefinite because the proximity encompassed by the recited interface is unclear, and because SEQ ID NOs: 1-3 are already propeptide-protease complexes, as discussed above. Third, it is unclear whether the one to three residues mutated to cysteine are in addition to any preexisting cysteine residues in the propeptide. Claims 1, 42 and 45 and recite “the pathogenic bacterium protease propeptide has one to three residues independently mutated to cysteine”. See lines 5-6 of claim 1, lines 8-9 of claim 42 and lines 9-10 of claim 45. However, SEQ ID NO: 1, 2 and 3 have 7, 3 and 7 cysteine residues respectively. Residues 52-128 of SEQ ID NO: 1, residues 1-66 of SEQ ID NO: 2, and residues 25-211 of SEQ ID NO: 3 do not contain any cysteine residues. As such, it is unclear whether the structure of the propeptide is required to have 1-3 cysteine residues overall, or 1-3 additional residues mutated to cysteine. Fourth, claims 1, 42 and 45 recite a narrow range of “one to three” residues mutated to cysteine, but later the claims refer to “at least one mutated cysteine” (lines 7, 8 and 10 of claim 1, lines 10, 12, and 14 of claim 42, lines 11,13 and 15 of claim 45), which is a broader limitation with no upper limit. Therefore, it is unclear whether the claims intend to limit the number of residues mutated to cysteine to 1-3. As such, the structure of the required propeptide is unclear. Fifth, claims 1, 42 and 45 recite the broad recitation “the propeptide” (see lines 7 and 18 of claim 1; lines 10 and 24 of claim 42; lines 11 and 24 of claim 45), and the claims also recite “pathogenic bacterium protease propeptide” (see lines 2, 5, 10 of claim 1; lines 5, 8 and 13 of claim 42; and lines 6, 9 and 14 of claim 45) which is the narrower statement of the limitation. Thus 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.
Claims 5, 10, 13-14, 46-48, and 50-57 depend from claims 1, 42 or 45 and are rejected for the reason set forth above.
Claim 45 recites “the detectable label” in lines 17 and 21. There is insufficient antecedent basis for this limitation in the claim. Claim 45 requires contacting the pathogenic bacterium with a construct that comprises a pathogenic bacterium protease propeptide that is conjugated to an antimicrobial peptide. As such, it is unclear which detectable label is being referenced in claim 45 because there is no earlier recitation of a detectable label.
Claims 46-48 depend from claim 45 and are rejected for the reason set forth above.
Claims 50 and 51 are indefinite because the claims recite residues that are not present in SEQ ID NO: 1. Specifically, the A35, A112, E123, N125, Q126, I128, N131 and N144 residues are not present in SEQ ID NO: 1. Rather, SEQ ID NO: 1 has glutamine (Q) at position 35, glycine (G) at position 112, proline (P) at position 123, valine (V) at position 125, alanine (A) at position 126, leucine (L) at position 128, and glycine (G) at position 131. Therefore, it is unclear which residues are being referenced. Moreover, claim 50 references positions 35, 44, 42, 154, 131, 135 and 144 of SEQ ID NO:1, which are not relevant to the propeptide that that has residues 52-128 of SEQ ID NO: 1. Therefore, it is unclear whether claim 50 intends to encompass embodiments in which residues 52-128 of SEQ ID NO: 1 do not include one to three residues replaced by cysteine.
Claims 50-53 are indefinite because it is unclear whether the claims intend to encompass additional mutations besides the one to three residues replaced by cysteine. To clarify, claims 50-53 specifically recite “the pathogenic bacterium protease propeptide is” [SEQ ID NO: 1-3, or residues thereof]; however then the claims require specific residue(s) within those sequences to be replaced by cysteine. Therefore, the propeptide sequence can no longer be 100% identical to SEQ ID NO: 1-3, or the recited residues thereof. Consequently, it is unclear whether the propeptide is limited to only embodiments with 1-3 mutations to cysteine.
Claims 51 and 53 recite “the one residue replaced by cysteine”, which is indefinite because it is unclear which one residue is being referenced. In other words, it is unclear whether claims 51 and 53 intend to limit one of the 1-3 residues mutated to cysteine, or whether the claims are requiring only one residue to be replaced by cysteine.
Claim Interpretation
Claim 1 is a product comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, residues 52-128 of SEQ ID NO: 1, residues 1-66 of SEQ ID NO: 2 or residues 25-211 of SEQ ID NO: 3; and at least one mutated cysteine residue (in addition to any existing cysteine residues) conjugated to a detectable label, antimicrobial peptide (AMP) or a therapeutically active small molecule. The mutated cysteine residues are not required to be located within the claimed propeptide sequences, e.g. within residues 52-128 of SEQ ID NO: 1. The detectable label is selected from a group that includes a conformation-sensitive label (e.g. MDCC). Claim 1 recites “wherein (a) once the propeptide binds to its corresponding protease, at least one mutated cysteine is present in the interface of the propeptide-protease complex or (b) at least one mutated cysteine is present at a protease cleavage site on the pathogenic bacterium protease propeptide”. However, claim 1 is not a method claim, and the way in which the cysteine is required to be present [at the propeptide-protease interface or at the protease cleavage site] is not specified. As such, this recitation does not limit the structure of the propeptide because any cysteine within a propeptide could reasonably be considered as present at an interface.
In claim 5, the structure of the claimed conformation-sensitive label is a coumarin derivative. The specification teaches 7-diethylamino-3-((((2 maleimidyl) ethyl) amino) carbonyl) coumarin, referred to as MDCC, which includes the required structure of claim 5. See figure 5B.
In claim 10, the pathogenic bacterium protease propeptide can be conjugated to the detectable label through a linker. The linker is not limited in anyway, and encompasses maleimide.
In claim 13, the linker structure claimed is inherent to the structure of MDCC.
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.
Claims 1, 5, 10, 13-14 and 50-51 are rejected under 35 U.S.C. 103 as being unpatentable over Howell, (Journal of Biological Chemistry, 2019 294(25), 9888-9900; provided herein with the supplemental information) in view of Goodey (Biochem Biophys Res Commun. 2011).
Regarding claim 1, Howell teaches a Y9C V. cholerae strain containing a Δivap::ivaPY9C plasmid, which encodes a cysteine at position 9 of the IvaP protein. See tables 1 and S2. Howell teaches that both wild-type IvaP from V. cholerae Haiti and IvaP79C remain incompletely processed in culture supernatant. See the first full paragraph on page 9891. As shown in figure S1, the IvaP sequence of Howell comprises residues 52-128 of instant SEQ ID NO: 1. See the alignment provided below. Thus, Howell teaches an IvaP sequence comprising residues 52-128 of instant SEQ ID NO: 1 and one residue mutated to cysteine at position 9.
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[AltContent: textbox (Alignment between IvaP of Howell (top sequence) and residues 52-128 of instant SEQ ID NO: 1 (bottom sequence). Note that in the bottom sequence residues 1-77 correspond to residues 52 to 128 of instant SEQ ID NO: 1.)]
Howell does not teach at least one mutated cysteine that is conjugated to a detectable label, an antimicrobial peptide or a therapeutically active small molecule; wherein the at least one mutated cysteine is present in the interface of the propeptide-protease complex (relevant to instant (a)) or at least one cysteine that is present at a protease cleavage site (relevant to instant (b)).
Goodey teaches introducing an S131C mutation in Bacillus stearothermophilus dihydrofolate reductase (DHFR). See sections 2.1 and 2.2. The distal residue S131 is replaced by a Cys residue and labeled with the environmentally sensitive fluorescent N-[2-(1-maleimidyl)ethyl]ethyl]-7-(diethyl-amino)coumarin-3-carboxamide (MDCC) to create a labeled construct. See the last paragraph of section 1. Goodey suggests that the MDCC fluorescence senses conformation changes that influence the environment. See the left column on page 445.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to attach the MDCC label of Goodey to any cysteine residue within the IvaPY9C of Howell including the mutated cysteine residue at position 9. One would be motivated to do so because Goodey suggests that the MDCC label detects conformational changes that influence the environment; and Howell suggests that the IvaP maturation process is influenced by its structure (first passage page 9895). There would be a reasonable expectation of success because Goodey demonstrates labeling an enzyme with MDCC at a mutated cysteine residue and Howell demonstrates mutating the IvaP enzyme to include a cysteine residue at position 9.
Regarding claim 5, Goodey teaches N-[2-(1-maleimidyl)ethyl]ethyl]-7-(diethyl-amino)coumarin-3-carboxamide (MDCC). See the last paragraph of section 1.
Regarding claim 10, Goodey teaches a N-[2-(1-maleimidyl)ethyl]ethyl]-7-(diethyl-amino)coumarin-3-carboxamide (MDCC) label. See the last paragraph of section 1 and figure 1. As shown in the structure above, MDCC includes one unit of a maleimide linker (relevant to instant (iii) and (v)).
Regarding claim 13, Goodey teaches a N-[2-(1-maleimidyl)ethyl]ethyl]-7-(diethyl-amino)coumarin-3-carboxamide (MDCC) label. See the last paragraph of section 1 and
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[AltContent: textbox (MDCC shown in figure 1 of Goodey)]figure 1. As shown in the structure above, the O=CNHCH2CH-2-[maleimide] portion of the MDCC is identical to the instantly claimed linker structure.
Regarding claim 14, Howell discloses that IvaP is homologous to subtilisin-like enzymes. See the first full paragraph on page 9889.
Regarding claims 50, Howell teaches an IvaP sequence that comprises instant residues 52-128 of SEQ ID NO: 1. Howell teaches mutating the IvaP sequence to include a Y9C mutation. See tables 1 and S2. Howell, discloses that IvaP contains up to seven cysteine residues that may minimize the enzyme’s dependence on calcium for structural stability. See the last full paragraph of the left column on page 9895.
Howell and Goodey do not teach a pathogenic bacterium protease propeptide that is instant SEQ ID NO: 1 or residues 52-128 of SEQ ID NO: 1, wherein the one to three residues replaced by cysteine are selected from the group consisting of A35, A44, K42, 154, V79, F94, A97, S102, A112, Y121, E123, N125, Q126, 1128, N131, S135, and N144.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to apply the cysteine mutation technique of Howell to any residue within the IvaP. One would be motivated to do so because Howell suggests that cysteine residues may minimize IvaP’s dependence on calcium for structural stability. There would be a reasonable expectation of success because Howell demonstrates mutating IvaP.
Regarding claim 51, Howell teaches an IvaP sequence that comprises instant residues 52-128 of SEQ ID NO: 1. Howell teaches mutating the IvaP sequence to include a Y9C mutation. See tables 1 and S2. As shown in figure S7, the IvaP of Howell includes an E123 residue.
Howell and Goodey do not teach a pathogenic bacterium protease propeptide that is instant SEQ ID NO: 1 or residues 52-128 of SEQ ID NO: 1, wherein the one residue replaced by cysteine E123.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to apply the cysteine mutation technique of Howell to any residue within the IvaP including the E123 residue of the IvaP. One would be motivated to do so because Howell suggests that cysteine residues may minimize IvaP’s dependence on calcium for structural stability. There would be a reasonable expectation of success because Howell demonstrates mutating IvaP.
Response to Arguments
Applicant’s arguments filed 12/11/2025 have been fully considered but they do not apply to the new grounds of rejection set forth above.
Conclusion
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 nonprovisional extension fee (37 CFR 1.17(a)) 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 mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KIMBERLY C BREEN whose telephone number is (571)272-0980. The examiner can normally be reached M-Th 7:30-4:30, F 8:30-1:30 (EDT/EST).
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/K.C.B./Examiner, Art Unit 1657