DETAILED 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 . 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 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.
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 submissions filed on 29 Sept, 2025 and 11 Jan, 2026 have been entered.
Election/Restrictions
Applicants elected group I (method of detecting a protease) using microbubbles constituting SEQ ID 133 and SEQ ID 101, by detecting collapse pressure without traverse in the reply filed on 7 March, 2023.
Claims Status
Claims 1-22, 24, 26, and 37-42 are pending.
Claims 1, 10, and 37 have been amended.
Claims 4, 10, 12, 16-20, and 37-41 have been withdrawn from consideration due to an election/restriction requirement.
Withdrawn Rejections
The rejection of claims 1-3, 5, 7-9, 11, 14, 15, 21, 22, and 24 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite due to uncertainty as to what constitutes a protease is hereby withdrawn due to argument.
Maintained/Modified Rejections
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.
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.
first rejection
Claim(s) 1, 3, 5-9, 11, 21, 22, and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Lakshmanan et al (US 20180028693, cited by applicants) in view of Lux et al (ACS Appl. Mater. Interfaces (2017) 9(43) p37587-37596).
The claims are drawn to a method of detecting a protease, comprising contacting an engineered protein gas vesicle (constructed with a GvpA/B protein and an engineered GvpC protein) with a protease and measuring its ultrasound response. The engineered GvpC protein has been designed with a protease recognition site for the protease to be detected.
Lakshmanan et al discuss gas vesicles that provide contrast for imaging which is modifiable by selectable acoustic collapse pressure value of the gas vesicle (abstract). This is done by engineering gas vesicles made by a modified GvpC protein attached to other gas vesicle proteins, where the engineering of the GvpC protein gives a lower acoustic collapse pressure (paragraph 11). Note that these GvpC proteins comprise an N-terminal region and a C-terminal region, with repetitions of a repeat unit between them (paragraph 166). Among the endogenously expressed gas vesicles to be modified are those of Anabaena floc-aquae (paragraph 149), from which applicants derived their elected species. Among the modifications of GvpC proteins discussed are deletions of the N-terminal sequence, the C-terminal sequence, one or more repeats, addition of amino acids (such as a functional tag), and substitutions of a subsequence in the native sequence (paragraphs 179-184). Vesicles modified from the native Anabaena floc-aquae vesicles by truncation of the GvpC protein decrease the acoustic collapse pressure by about 30% (truncation location not specified), while completely removing it decreases this parameter by about 65% (paragraph 191), indicating that truncated forms of the protein are a reasonable embodiment of the invention and that different amounts removed will vary the acoustic collapse pressure. An example is given where native Anabaena floc-aquae gas vesicles are isolated (example 4, paragraph 266), then stripped of their GvpC proteins, which are replaced with engineered versions (example 16, paragraph 302). Note that this will generate gas vesicles with the native Anabaena floc-aquae GvpA/B sequence, but variant GvpC proteins.
The difference between this reference and the instant claims is that this reference does not discuss a protease cleavage site.
Lux et al discuss thrombin activated microbubbles as ultrasound contrast agents (title). This is to distinguish between acute deep vein thrombosis (DVT), where anticoagulants are useful, and chronic DVT, where anticoagulants provide no benefit, but pose a risk of side effects (2nd pate, 1st paragraph). The construct is very different than those of Lakshmanan et al; here, an inhibitory sequence is linked to a cell binding sequence by a thrombin sensitive linker (3d page, 3d paragraph). A sample of these microbubbles were incubated with thrombin in vitro in an ultrasound scanner to visualize the reaction and show a proof of principle (10th page, 3d paragraph). This reference discusses microbubbles sensitive to a protease.
Therefore, it would be obvious to add a thrombin protease sequence to the GvpC proteins of Lakshmanan et al, to generate particles that can detect the presence of this protease. As Lakshmanan et al describe the addition of various sequences and show that deletions and truncations of the GvpC protein affect the acoustic collapse pressure (a measure by which the reference detects a difference), an artisan in this field would attempt this modification with a reasonable expectation of success.
Lakshmanan et al discuss microbubbles with GvpA/B and an engineered GvpC sequence to vary the acoustic collapse pressure, including truncated and missing GvpC sequences, rendering obvious modification of this protein. Lux et al discuss making microbubbles with a thrombin cleavage site to make the signal dependent on the presence of that protease. Both references discuss ultrasound examination. Thus, the combination of references renders obvious claims 1, 5, 6, and 21.
Lakshmanan et al teach that an engineered vesicle with a truncated GvpC sequence has a reduced acoustic collapse pressure, which is reduced even further when the protein is absent. This means that where it is truncated will affect this parameter, making it an optimizable variable. The MPEP states that “Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or working ranges by routine experimentation" In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 (“The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.”) (MPEP2144.05.II). In other words, it is obvious to vary the placement of the cleavage sequence to find a location that optimizes the change in properties when cleaved. Thus, the combination of references renders obvious claims 3 and 22.
Lakshmanan et al discuss taking an Anabaena floc-aquae gas vesicle, striping off the native GvpC, and adding an engineered GvpC. Thus, the combination of references renders obvious claims 7-9, 11, and 24.
response to applicant’s arguments
Applicants argue that Lakshmanan et al describes a fundamentally different structure than Lux et al, that the rigidity of the system would lead a person of skill in the art to assume a protease would not cleave GvpC, and that the references do not describe increased non-linear acoustical behavior. Applicants also point out sections of the MPEP that discuss motivation to combine, rendering the art unsuitable for its intended purpose, changing the principle of operation of a reference, and improper obvious to try, but do not tie those to arguments or the rejection.
Applicant's arguments filed 11 Jan, 2026 have been fully considered but they are not persuasive.
Applicants argue that the bubbles of Lux et al are fundamentally different than those of Lackshmanan et al. It is not clear what this argument is. It could be an argument of non-analogous art, inability to physically combine, or some other argument. Without some explanation of how this argument overcomes the rejection, it is not persuasive.
Applicants argue that a person of skill in the art would assume that the system is too rigid and the GvpC is too firmly bound for a protease to cleave a cleavage site engineered in that protein. Applicants state that the vesicle is very rigid, with no evidence, and that GvpC requires harsh conditions to strip it and has multiple sites of binding. It is clear that the vesicle is not totally rigid from the prior art. Lakchmanan et al (Nat. Protoc. (2017) 12(10) p2050-2080) discusses the same bubbles as the reference by the same author cited in the rejection with no mention of modified GvpC (2nd page, 2nd paragraph) and can produce harmonic ultrasound signals (3d page, 1st paragraph). Gaudron et al (J. Fluid Mech. (2015) 766 p54-75) shows that harmonics is from the vesicles oscillating (p68, 4th paragraph to end of page), which is movement of the vesicle’s walls. In other words, if the vesicles were completely rigid, as applicants are arguing, Lakchmanan et al would see no harmonics because the vesicles would lack the flexibility to oscillate. Applicants argue that GvpC is so firmly bound, the protease would not cleave. The evidence is that Lakchmanan et al uses urea to remove the protein from the vesicle, and that it is bound at multiple points. Note that there is no evidence about the strength of this binding, or evidence that other, less forceful methods to remove the protein would be ineffective. However, multiple sites of binding are very common in proteins – Kuster et al (PloS One (2015) 10(4) e0123146) states that helices have long been known to be optimized for hydrogen bonding (2nd page, 1st paragraph) – in other words, any helix (a very common motif in proteins) has multiple sites of binding to itself, yet such structures are routinely cleaved by proteases. And, while applicants have disparaged them by pointing out differences compared to their invention, there is an extensive set of references discussing numerous constrained, rigid, blocked, and otherwise restricted polypeptides cleaved by proteases.
Applicants argue that the rejection has not met the limitation regarding non-linear signal. This limitation has been interpreted as producing a non-linear signal compared to non-cleaved vesicles, which is mentioned by Lachmannan et al.
Applicants have cited MPEP locations discussing various ways a rejection can be improper, but have not stated that they believe these citations are relevant to the rejection, or explain how the rejection fits any of these scenarios.
second rejection
Claim(s) 1-3, 5-9, 11, 14, 15, 21, 22, and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Lakshmanan et al (US 20180028693, cited by applicants) in view of Lux et al (ACS Appl. Mater. Interfaces (2017) 9(43) p37587-37596) and Aguino et al (ACS Omega (2018) 3 p15829-15836).
Lakshmanan et al discuss gas vesicles that provide contrast for imaging which is modifiable by selectable acoustic collapse pressure value of the gas vesicle (abstract). This is done by engineering gas vesicles made by a modified GvpC protein attached to other gas vesicle proteins, where the engineering of the GvpC protein gives a lower acoustic collapse pressure (paragraph 11). Note that these GvpC proteins comprise an N-terminal region and a C-terminal region, with repetitions of a repeat unit between them (paragraph 166). Among the endogenously expressed gas vesicles to be modified are those of Anabaena floc-aquae (paragraph 149), from which applicants derived their elected species. Among the modifications of GvpC proteins discussed are deletions of the N-terminal sequence, the C-terminal sequence, one or more repeats, addition of amino acids (such as a functional tag), and substitutions of a subsequence in the native sequence (paragraphs 179-184). Vesicles modified from the native Anabaena floc-aquae vesicles by truncation of the GvpC protein decrease the acoustic collapse pressure by about 30% (truncation location not specified), while completely removing it decreases this parameter by about 65% (paragraph 191), indicating that truncated forms of the protein are a reasonable embodiment of the invention and that different amounts removed will vary the acoustic collapse pressure. An example is given where native Anabaena floc-aquae gas vesicles are isolated (example 4, paragraph 266), then stripped of their GvpC proteins, which are replaced with engineered versions (example 16, paragraph 302). Note that this will generate gas vesicles with the native Anabaena floc-aquae GvpA/B sequence, but variant GvpC proteins.
Lux et al discuss thrombin activated microbubbles as ultrasound contrast agents (title). This is to distinguish between acute deep vein thrombosis (DVT), where anticoagulants are useful, and chronic DVT, where they provide no benefit, but have a risk of side effects (2nd pate, 1st paragraph). The construct is very different than those of Lakshmanan et al; here, an inhibitory sequence is linked to a cell binding sequence by a thrombin sensitive linker (3d page, 3d paragraph). A sample of these microbubbles were incubated with thrombin in vitro in an ultrasound scanner to visualize the reaction and show a proof of principle (10th page, 3d paragraph). This reference discusses microbubbles sensitive to a protease.
Please note that, as noted above, these two references render obvious claims 1, 3, 5-9, 11, 21, 22, and 24.
The difference between the teachings of these references and the remaining claims is that these references do not specify a linker between the protease recognition site and the remainder of the GvpC sequence.
Aguino et al discuss fused proteins (title). Various fluorescent proteins were generated, separated by a flexible glycine serine linker, GSGSGSG, to increase the probability of proper protein folding (p15834, 1st column, 2nd paragraph). This reference teaches the advantage of using a linker sequence.
Therefore, it would be obvious to add the flexible glycine serine linker to either or both ends of the protease recognition site, to increase the probability of proper protein folding, as discussed by Aguino et al. As that reference discussed fusion proteins, which is similar to the construct made by the combination of Laksmanan et al and Lux et al, an artisan in this field would attempt this addition with a reasonable expectation of success.
Aguino et al render obvious adding the sequence GSGSGSG, applicant’s elected linker sequence, to one or both ends of the protease recognition site, rendering obvious claims 2, 14, and 15.
response to applicant’s arguments
Applicants use the same arguments for all rejections under 35 USC 103 and non-statutory double patenting, which were answered above.
third rejection
Claim(s) 1-3, 5-9, 11, 13-15, 21, 22, 24, 26, and 42 are rejected under 35 U.S.C. 103 as being unpatentable over Lakshmanan et al (US 20180028693, cited by applicants) in view of Lux et al (ACS Appl. Mater. Interfaces (2017) 9(43) p37587-37596), Aguino et al (ACS Omega (2018) 3 p15829-15836), To et al (PNAS (2015) 112(11) p3338-3343) and Cesaratto et al (J. Biotech. (2015) 212 p159-166). Please note that this rejection reads on applicant’s elected species.
Lakshmanan et al discuss gas vesicles that provide contrast for imaging which is modifiable by selectable acoustic collapse pressure value of the gas vesicle (abstract). This is done by engineering gas vesicles made by a modified GvpC protein attached to other gas vesicle proteins, where the engineering of the GvpC protein gives a lower acoustic collapse pressure (paragraph 11). Note that these GvpC proteins comprise an N-terminal region and a C-terminal region, with repetitions of a repeat unit between them (paragraph 166). Among the endogenously expressed gas vesicles to be modified are those of Anabaena floc-aquae (paragraph 149), from which applicants derived their elected species. Among the modifications of GvpC proteins discussed are deletions of the N-terminal sequence, the C-terminal sequence, one or more repeats, addition of amino acids (such as a functional tag), and substitutions of a subsequence in the native sequence (paragraphs 179-184). Vesicles modified from the native Anabaena floc-aquae vesicles by truncation of the GvpC protein decrease the acoustic collapse pressure by about 30% (truncation location not specified), while completely removing it decreases this parameter by about 65% (paragraph 191), indicating that truncated forms of the protein are a reasonable embodiment of the invention and that different amounts removed will vary the acoustic collapse pressure. An example is given where native Anabaena floc-aquae gas vesicles are isolated (example 4, paragraph 266), then stripped of their GvpC proteins, which are replaced with engineered versions (example 16, paragraph 302). Note that this will generate gas vesicles with the native Anabaena floc-aquae GvpA/B sequence, but variant GvpC proteins.
Lux et al discuss thrombin activated microbubbles as ultrasound contrast agents (title). This is to distinguish between acute deep vein thrombosis (DVT), where anticoagulants are useful, and chronic DVT, where they provide no benefit, but have a risk of side effects (2nd pate, 1st paragraph). The construct is very different than those of Lakshmanan et al; here, an inhibitory sequence is linked to a cell binding sequence by a thrombin sensitive linker (3d page, 3d paragraph). A sample of these microbubbles were incubated with thrombin in vitro in an ultrasound scanner to visualize the reaction and show a proof of principle (10th page, 3d paragraph). This reference discusses microbubbles sensitive to a protease.
Aguino et al discuss fused proteins (title). Various fluorescent proteins were generated, separated by a flexible glycine serine linker, GSGSGSG, to increase the probability of proper protein folding (p15834, 1st column, 2nd paragraph). This reference teaches the advantage of using a linker sequence.
Please note that, as noted above, these two references render obvious claims 1-3, 5-9, 11, 14, 15, 21, 22, and 24.
The difference between these references and the remaining claims is that these references do not discuss a TEVp recognition site.
To et al discuss a fluorogenic protease reporter (title). As a proof of concept, the sensor was designed with a TEV protease cleavage sequence (p3339, 1st column, 2nd paragraph), a 7 amino acid sequence (table S1). This was used to test the specificity of the construct (p3339, 2nd column, 4th paragraph), and for optimizing the various parameters, such as linker lengths (p3339, 2nd column, 3d paragraph) binding behavior (p3339, 1st column, 4th paragraph), and how it was expressed (p3339, 2nd column, 2nd paragraph). After optimization, a construct with a different protease cleavage sequence was generated and used in vitro and in vivo (p3339, 2nd column, 5th and 6th paragraphs). This reference discusses using a TEV protease cleavage sequence in a protease detection construct.
Cesaratto et al discuss the TEV protease (title). This protease is very specific, stable, and is active in a number of different buffers (p160, 1st column, 1st paragraph), and found safe to express in cells and living organisms (p160, 1st column, 2nd paragraph). The natural substrate for the protease is ENLYFQG/S, although variation of this sequence at the 2nd, 3d, and 5th positions is tolerated (p159, 1st column, 2nd paragraph). This reference gives the sequence of the TEV protease and give reasons why it may be used.
Therefore, it would be obvious to generate a microbubble system with the TEV protease recognition site, as a proof of concept and to optimize various parameters (such as cleavage location and number of linker sequences) as exampled by To et al. As Cesaratto et al teach that the protease is stable, selective, and not very picky (i.e. unlikely to fail due to an issue with the protease), an artisan in this field would attempt this optimization with a reasonable expectation of success.
To et al and Cesaratto et al render obvious using a TEV protease recognition sequence, rendering obvious claims 13 and 26.
The TEV protease sequence is 7 AAs, and the linker of Aguino et al is 7 amino acids, totaling less than 20 amino acids and rendering claim 42 obvious.
response to applicant’s arguments
Applicants use the same arguments for all rejections under 35 USC 103 and non-statutory double patenting, which were answered above.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/process/file/efs/guidance/eTD-info-I.jsp.
first rejection
Claims 1-3, 5-9, 11, 13-15, 21, 22, 24, and 26 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 10, 14-16 of U.S. Patent No. 10,493,172 in view of Lux et al (ACS Appl. Mater. Interfaces (2017) 9(43) p37587-37596), Aguino et al (ACS Omega (2018) 3 p15829-15836), To et al (PNAS (2015) 112(11) p3338-3343) and Cesaratto et al (J. Biotech. (2015) 212 p159-166).
Competing claim 1 describes an ultrasound imaging method using a gas vesicle protein structure with a selectable acoustic collapse pressure, and collapsing the structure with ultrasound to provide both a contrasted an uncontracted image of the relevant area. Competing claim 10 specifies additional details of the experiment, with competing claims 14 and 15 requiring that the gas vesicle be from Anabaena floc-aquae and competing claim 16 specifying that the selectable acoustical collapse pressure is modified by varying GvpC.
The difference between the competing claims and the instant claims is that the competing claims do not specify a protease recognition site in the GvpC.
Lux et al discuss thrombin activated microbubbles as ultrasound contrast agents (title). This is to distinguish between acute deep vein thrombosis (DVT), where anticoagulants are useful, and chronic DVT, where they provide no benefit, but have a risk of side effects (2nd pate, 1st paragraph). The construct is very different than those of Lakshmanan et al; here, an inhibitory sequence is linked to a cell binding sequence by a thrombin sensitive linker (3d page, 3d paragraph). A sample of these microbubbles were incubated with thrombin in vitro in an ultrasound scanner to visualize the reaction and show a proof of principle (10th page, 3d paragraph). This reference discusses microbubbles sensitive to a protease.
Aguino et al discuss fused proteins (title). Various fluorescent proteins were generated, separated by a flexible glycine serine linker, GSGSGSG, to increase the probability of proper protein folding (p15834, 1st column, 2nd paragraph). This reference teaches the advantage of using a linker sequence.
To et al discuss a fluorogenic protease reporter (title). As a proof of concept, the sensor was designed with a TEV protease cleavage sequence (p3339, 1st column, 2nd paragraph). This was used to test the specificity of the construct (p3339, 2nd column, 4th paragraph), and for optimizing the various parameters, such as linker lengths (p3339, 2nd column, 3d paragraph) binding behavior (p3339, 1st column, 4th paragraph), and how it was expressed (p3339, 2nd column, 2nd paragraph). After optimization, a construct with a different protease cleavage sequence was generated and used in vitro and in vivo (p3339, 2nd column, 5th and 6th paragraphs). This reference discusses using a TEV protease cleavage sequence in a protease detection construct.
Cesaratto et al discuss the TEV protease (title). This protease is very specific, stable, and is active in a number of different buffers (p160, 1st column, 1st paragraph), and found safe to express in cells and living organisms (p160, 1st column, 2nd paragraph). The natural substrate for the protease is ENLYFQG/S, although variation of this sequence at the 2nd, 3d, and 5th positions is tolerated (p159, 1st column, 2nd paragraph). This reference gives the sequence of the TEV protease and give reasons why it may be used.
Therefore, it would be obvious to add a protease recognition site to the GvpC sequence to generate a protease sensitive microbubble. As the variation in acoustical pressure collapse values is due to modifying this protein, an artisan in this field would make this modification with a reasonable expectation of success.
Furthermore, it would be obvious to add a flexible linker sequence to the protease cleavage sequence, to increase the likelihood of proper folding, as described by Aguino et al. As that reference uses fusion proteins, an artisan in this field would attempt this modification with a reasonable expectation of success.
Finally, it would be obvious to use a TEV protease sequence as proof of concept and to optimize the various parameters of the microbubble. As Cesaratto et al teach that this is a forgiving protease, an artisan in this field would attempt this process with a reasonable expectation of success.
response to applicant’s arguments
Applicants use the same arguments for all rejections under 35 USC 103 and non-statutory double patenting, which were answered above.
second rejection
Claims 1-3, 5-9, 11, 13-15, 21, 22, 24, and 26 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 3 and 5 of U.S. Patent No. 11,504,438 in view of Lux et al (ACS Appl. Mater. Interfaces (2017) 9(43) p37587-37596), Aguino et al (ACS Omega (2018) 3 p15829-15836), To et al (PNAS (2015) 112(11) p3338-3343) and Cesaratto et al (J. Biotech. (2015) 212 p159-166).
Competing claim 3 describes a method to tune the acoustical collapse properties of a gas vesicle protein structure (which requires using an ultrasound method), comprising replacing the GvpC protein with a genetically engineered GvpC protein. Competing claim 5 specifies that the gas vesicles come from a Markush group of critters, including Anabaena floc-aquae.
The difference between the competing claims and the instant claims is that the competing claims do not specify a protease recognition site in the GvpC.
Lux et al discuss thrombin activated microbubbles as ultrasound contrast agents (title). This is to distinguish between acute deep vein thrombosis (DVT), where anticoagulants are useful, and chronic DVT, where they provide no benefit, but have a risk of side effects (2nd pate, 1st paragraph). The construct is very different than those of Lakshmanan et al; here, an inhibitory sequence is linked to a cell binding sequence by a thrombin sensitive linker (3d page, 3d paragraph). A sample of these microbubbles were incubated with thrombin in vitro in an ultrasound scanner to visualize the reaction and show a proof of principle (10th page, 3d paragraph). This reference discusses microbubbles sensitive to a protease.
Aguino et al discuss fused proteins (title). Various fluorescent proteins were generated, separated by a flexible glycine serine linker, GSGSGSG, to increase the probability of proper protein folding (p15834, 1st column, 2nd paragraph). This reference teaches the advantage of using a linker sequence.
To et al discuss a fluorogenic protease reporter (title). As a proof of concept, the sensor was designed with a TEV protease cleavage sequence (p3339, 1st column, 2nd paragraph). This was used to test the specificity of the construct (p3339, 2nd column, 4th paragraph), and for optimizing the various parameters, such as linker lengths (p3339, 2nd column, 3d paragraph) binding behavior (p3339, 1st column, 4th paragraph), and how it was expressed (p3339, 2nd column, 2nd paragraph). After optimization, a construct with a different protease cleavage sequence was generated and used in vitro and in vivo (p3339, 2nd column, 5th and 6th paragraphs). This reference discusses using a TEV protease cleavage sequence in a protease detection construct.
Cesaratto et al discuss the TEV protease (title). This protease is very specific, stable, and is active in a number of different buffers (p160, 1st column, 1st paragraph), and found safe to express in cells and living organisms (p160, 1st column, 2nd paragraph). The natural substrate for the protease is ENLYFQG/S, although variation of this sequence at the 2nd, 3d, and 5th positions is tolerated (p159, 1st column, 2nd paragraph). This reference gives the sequence of the TEV protease and give reasons why it may be used.
Therefore, it would be obvious to add a protease recognition site to the GvpC sequence to generate a protease sensitive microbubble. As the variation in acoustical pressure collapse values is due to modifying this protein, an artisan in this field would make this modification with a reasonable expectation of success.
Furthermore, it would be obvious to add a flexible linker sequence to the protease cleavage sequence, to increase the likelihood of proper folding, as described by Aguino et al. As that reference uses fusion proteins, an artisan in this field would attempt this modification with a reasonable expectation of success.
Finally, it would be obvious to use a TEV protease sequence as proof of concept and to optimize the various parameters of the microbubble. As Cesaratto et al teach that this is a forgiving protease, an artisan in this field would attempt this process with a reasonable expectation of success.
response to applicant’s arguments
Applicants use the same arguments for all rejections under 35 USC 103 and non-statutory double patenting, which were answered above.
New Rejections
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 1-3, 5, 7-9, 11, 14, 15, 21, 22, and 24 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, and claims dependent on it, has the limitation that the vesicles exhibit a protease induced non-linear ultrasound response having a magnitude of non-linearity that is higher than a magnitude of the baseline non-linearity. This makes no sense. It is not clear if what is required is a change in the shape of the curve or the magnitude of the signal, or if it requires an order of magnitude change in whatever is changing, or how a difference in non-linearity is measured, or what the other variable whatever signal is varied by (power, frequency, time, etc).
Applicants have attempted to explain how a person of skill in the art would interpret this phrase in their arguments of 11 Jan, 2026, but that explanation does not make sense. Applicants have pulled from different parts of the disclosure discussing non-linear signals from vesicle collapse and non-linear signals from harmonics, and equated them, even though the antecedent support for this phrase clearly is discussing vesicle collapse. They point to a graph showing a signal amplification (two lines, one approx. 2x higher than the other, but showing the same shape) as an example of increased non-linearity, but as the curve shape is the same, the non-linearity (however it is measured) must be the same. In other words, applicant’s explanation of this phrase is also incomprehensible.
In addition, it is clear that the signal is dependent on the ultrasound parameters. Lakshmanan et al (cited in rejection) discusses a collapse pressure profile (paragraph 11); if insufficient energy is imputed, there will be no collapse. Gaudron et al discusses harmonic oscillations in terms of the driving frequency (p63, 2nd paragraph); if the driving frequency is not close to a harmonic frequency, there will be no harmonics. In other words, the parameters mentioned in the claims may or may not be within the claimed ranges dependent on a feature not defined by the claims.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to FRED REYNOLDS whose telephone number is (571)270-7214. The examiner can normally be reached M-Th 9-3:30.
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, Melissa Fisher can be reached at 571-270-7430. 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.
/FRED H REYNOLDS/Primary Examiner, Art Unit 1658