DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 08/20/2026 has been entered.
Examiner’s Note
Please note that the examiner of record has changed. Please address all subsequent correspondences to Georgiana Reglas, Examiner, AU1651.
Status of claim rejections
The rejections of record under 35 USC 102 have been maintained in view of Applicant’s amendments/arguments in the response filed 08/20/2026.
Claim Interpretation
Newly added claim 24 recites “The responsive microbubble according to claim 16, wherein stiffness of the microbubble shell is changed by the cleavage of the peptide substrate and the de-crosslinking of the components of the microbubble shell.” The examiner notes that this limitation is interpreted to be, absent evidence to the contrary, an inherent property of the microbubble with the claimed structural features. Put simply, a microbubble containing the claimed structural elements/features and cleavage functionality resulting in a change in ultrasound signal would have a change in stiffness by the cleavage of the peptide substrate and de-crosslinking of the components of the microbubble shell.
New 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 16, 18-21 and 24 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 16 is replete with narrative language. Please note that the dependent claims are also indefinite due to dependency on indefinite claim 16.
Maintained Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 16, 18-21 and 24 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Lux et al. (ACS Appl. Mater. Interfaces, 2017, Vol. 9, pp.37587-37596; of record).
Lux teaches perfluorobutane-filled microbubbles conjugated with thrombin-sensitive activatable cell-penetrating peptides (ACPPs) that detect acute thrombosis with ultrasound imaging (see Abstract, see paragraph bridging pp.37588-89, p.37592, paragraph bridging left and right columns, right column, 1st paragraph, and Figs. 2-4 and 6). Lux teaches the microbubble shell is made of distearoylglycerophosphocholine (DSPC), disteroylglycerophosphoethanolamine-maleimide(polyethelene glycol)-5000 (DSPE-PEG(5000)-mal), reading on a microbubble made of phospholipids with surfactants (see p.37592, paragraph bridging left and right columns, and Fig. 3A). Lux further teaches the microbubble is conjugated to activatable cell-penetrating peptide that is cleavable by the serine protease thrombin, reading on a peptide substrate for an enzyme of interest (see p.37592, left column, 1st-5th paragraphs, right column, 1st paragraph, and Figs. 1, 4, and S4A). Paragraph [0047] of the originally-filed specification provides evidence that substrates (e.g. peptides) may be covalently bond to the material of the shell using maleimide as crosslinking agents. ACPP comprises two terminal amino ends that crosslinks to the shell of the microbubble (see p.37592, left column, 1st paragraph, and Fig. S4A). Thus, the ACPPs conjugated to maleimide microbubbles are covalently crosslinked, reading on wherein the peptide substrate for an enzyme of interest is a crosslinking element bound by covalent bonds to the microbubble shell. Lux further teaches ACPP is cleaved by thrombin between the arginine and serine amino acids (see p.37588, left column, last paragraph, p.37592, left column, 1st and 5th paragraph, and Figs. 2, 3A, 4A, and S3A-B). Cleavage of ACPP conjugated to the maleimide microbubbles would eliminate cross linkages, thus reading on wherein cleavage of the peptide substrate by the enzyme of interest eliminates cross linkages. Lux incubated the ACPP conjugated maleimide microbubbles in vitro with thrombin and ultrasound imaged the interaction, demonstrating a shift in ultrasound intensity post incubation and cleavage of ACPP, reading on wherein the microbubble alters its interaction with ultrasound waves in response to elimination of cross linkages and wherein the altered interaction is an observable alteration in resonance frequency of the microbubble or attenuation due to a persistent change of mechanical properties of a shell of the microbubble (see p.37593, paragraph bridging left and right columns, and Fig. S8).
Regarding claim 18, Lux teaches the ACPP-MBs can be used to diagnose acute deep vein thrombosis, which is known to have a thrombin-rich environment, reading on for use in a method of diagnosis in vivo of a disease associated with or caused by an altered activity of the enzyme of interest (see p.37587, passage bridging left and right columns).
Regarding claim 19, Lux demonstrates the ACPP-MBs are suitable as an ultrasound contrast agent showing thrombin activity (see p.37593, paragraph bridging left and right columns, and Fig. S8).
Regarding claim 20, Lux teaches the ACPP-MBs have a mean diameter of 2.1 μM, which is within the claimed range of 0.5 – 4 μM (see p.37592, right column, 1st paragraph, and Fig. S4C).
Regarding claim 21, Lux teaches the ACPP-MBs have a shell that is made of distearoylglycerophosphocholine (DSPC), disteroylglycerophosphoethanolamine-maleimide(polyethelene glycol)-5000 (DSPE-PEG(5000)-mal), a perfluorobutane gas core, and the shell is covered by the ACPP thrombin substrate, reading on claim 21 (see Abstract, p.37592, left column, 1st paragraph,-right column, 1st paragraph, and Figs. 1, 3A, 4, and S4A).
Regarding claim 24, as discussed above, the claimed limitation was interpreted to be, absent evidence to the contrary, an inherent property of the microbubble with the claimed structural features. Put simply, a microbubble containing the claimed structural elements/features and cleavage functionality resulting in a change in ultrasound signal would have a change in stiffness by the cleavage of the peptide substrate and de-crosslinking of the components of the microbubble shell. Lux, as discussed above, teaches the claimed structural features and would inherently have a change in stiffness by the cleavage of the peptide substrate and de-crosslinking of the components of the microbubble shell, absent evidence to the contrary.
Accordingly, the claimed invention anticipated by the teachings of Lux.
Response to Arguments
Applicant's arguments filed 08/20/2026 have been fully considered but they are not persuasive.
On pg. 7-10, Applicant argues Lux discloses none of the limitations claimed because the peptide in Lux is a single-point tether rather than a crosslinking element whose cleavage decrosslinks the components of the microbubble shell. Specifically, Applicant argues “crosslinking” in the field of peptides means forming covalent bonds that link amino acid sidechains within the same protein/peptide or between peptides/molecules, which is different from normal linkage between a carbon in the non-side chain carboxylic group of one amino acid and a nitrogen in the non-side chain amino group of another amino acid which forms the backbone of the peptide. Applicant argues, inter alia, any number of amino acids can chain together by successive peptide bonds, and that the ACPP is attached to the microbubble shell at a single point, arguing that Lux states the C-terminus cysteine allows easy conjugation of ACPP to maleimide-functionalized microbubbles and depicts a single thiol-maleimide coupling reaction and that ACPP extends freely from that single anchor and released upon cleavage. Applicant argues the bond in FIG. 1A is cleaved and is not a crosslink and the peptide protrudes from the lipid shell. Applicant then argues, inter alia, “cutting parts of the linear arms as disclosed by Lux does not alter the stiffness of the bubble or its acoustic properties. The inventors assume that the size of the bubble also changed as a result of the cutting which meant that the effect was negated” and points to the specification regarding the crosslinking element, the interconnecting shell components into a network, and the responsive constructs in the examples (see pg. 39 and Fig. 12-15 and 20).
In response, the examiner disagrees. First, the examiner notes that a “crosslink” is generally known in the relevant art of crosslinking proteins as “the process of chemically joining two or more molecules by a covalent bond” (emphasis added; see, e.g., https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-crosslinking-protein-modification.html; available online as early as 10 Sept 2015), such that a crosslink only requires at least one bond (i.e., one or more bonds). BOC Sciences (“Maleimide Conjugation” retrieved online 29 Sept 26 from https://bioconjugation.bocsci.com/resources/maleimide-conjugation.html) evidences “maleimide conjugation is a term employed in biochemistry and molecular biology that describes the covalent entanglement of maleimide functional groups onto a protein, peptide or other biomolecule” and the bond is shown below in Fig. 2 of BOC Sciences (reproduced below):
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Lux discloses this same bond within the maleimide-APCC conjugation (see FIG. S4A of Lux), and further note that Lux discloses “The C-terminus cysteine allows an easy conjugation of ACPP to maleimide-functionalized microbubbles (mal-MBs)” (i.e., the C-terminus cysteine required in the maleimide reaction shown above to create a stable, covalent thioether bond), such that the maleimide-APCC conjugated microbubble of Lux does constitute a crosslink. Lux then specifically teaches that there is a 4.7-fold increase in US signal intensity upon introducing thrombin to the APCC-maleimide microbubbles (see pg. 6).
Second, regarding Applicant’s arguments about “cutting parts of the linear arms as disclosed by Lux does not alter the stiffness of the bubble or its acoustic properties. The inventors assume that the size of the bubble also changed as a result of the cutting which meant that the effect was negated”, [a]n argument by the applicant is not evidence unless it is an admission, in which case, an examiner may use the admission in making a rejection. See MPEP § 2129 and § 2144.03 for a discussion of admissions as prior art. Arguments presented by applicant cannot take the place of evidence in the record. See In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984); In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965); In re Geisler, 116 F.3d 1465, 43 USPQ2d 1362 (Fed. Cir. 1997) ("An assertion of what seems to follow from common experience is just attorney argument and not the kind of factual evidence that is required to rebut a prima facie case of obviousness."). If Applicant is asserting that the size of the bubble of Lux would change because of the cleavage, Applicant must provide empirical evidence on the record to substantiate such an argument.
On pg. 10-13, Applicant argues Lux does not support the characterization of the Office Action and that ACPP is bound to the shell only through the C-terminal cysteine and because the ACPP is bound at a single point, it is not a crosslinking element and there are no crosslinkages to eliminate. Applicant further argues the limitation of claim 16 requiring “the altered interaction is an observable alteration in resonance frequency of the microbubble, attenuation due to a persistent change of mechanical properties of the microbubble shell or is detectable as an onset of harmonic responses” and that the Fig. s8 reports a stability experiment measuring ultrasound signal of the microbubbles upon exposure to thrombin other than “no significant change in US signal was observed after 30 min of incubation” and that thrombin-triggered ACPP does not alter the acoustic or mechanical properties of the microbubbles. Applicant argues the signal enhancement of Lux arises from a different mechanism, reversal of the polyanionic arm that reverses the microbubble surface charge and the stronger signal results from greater number of bubbles bound after washing and not change in the property in any individual bubble. Applicant further urges the citation to the specification regarding maleimide bioconjugation chemistry is misplaced and that the claim requires the peptide to be a “crosslinking element” as a “linker that forms crosslinkages whose elimination changes shell mechanics” its cleavage de-crosslinks the components of the microbubble, and the de-crosslinking changes the mechanical properties of the bubble, Lux does not teach the altered interaction and argues much of the same for the dependent claims.
In response, the examiner disagrees for much of the same reasons as set forth above. First, the examiner notes there is no explicit definition of a “crosslinking element” used within the specification (see specification as filed). Applicant’s specification explicitly recites “possible mechanisms for introducing a substrate to the microbubble in a way that cleavage alters the mechanical properties including the ultrasound signal to include, but are not limited to . . . The substrate of the enzyme is a crosslinking element incorporating components that alters mechanical properties. Cleavage of the substrate eliminates cross linkage, resulting in changed ultrasound signals.” (see pg. 18 of the specification). This is a preferred embodiment of a crosslinker. Furthermore, under a broadest reasonable interpretation (BRI), words of the claim must be given their plain meaning, unless such meaning is inconsistent with the specification. The plain meaning of a term means the ordinary and customary meaning given to the term by those of ordinary skill in the art at the relevant time” (see MPEP 2111.01). The plain meaning of the term crosslink (see Thermofisher above) is not inconsistent with the specification.
As discussed above, Lux teaches the claimed structural elements of the responsive microbubble of claim 16. Specifically, Lux teaches perfluorobutane-filled microbubbles conjugated with thrombin-sensitive activatable cell-penetrating peptides (ACPPs) that detect acute thrombosis with ultrasound imaging (see Abstract, see paragraph bridging pp.37588-89, p.37592, paragraph bridging left and right columns, right column, 1st paragraph, and Figs. 2-4 and 6). Lux teaches the microbubble shell is made of distearoylglycerophosphocholine (DSPC), disteroylglycerophosphoethanolamine-maleimide(polyethelene glycol)-5000 (DSPE-PEG(5000)-mal), reading on a microbubble made of phospholipids with surfactants (see p.37592, paragraph bridging left and right columns, and Fig. 3A). Lux further teaches the microbubble is conjugated to activatable cell-penetrating peptide that is cleavable by the serine protease thrombin, reading on a peptide substrate for an enzyme of interest (see p.37592, left column, 1st-5th paragraphs, right column, 1st paragraph, and Figs. 1, 4, and S4A). Paragraph [0047] of the originally-filed specification provides evidence that substrates (e.g. peptides) may be covalently bond to the material of the shell using maleimide as crosslinking agents. ACPP comprises two terminal amino ends that crosslinks to the shell of the microbubble (see p.37592, left column, 1st paragraph, and Fig. S4A). Thus, the ACPPs conjugated to maleimide microbubbles are covalently crosslinked, reading on wherein the peptide substrate for an enzyme of interest is a crosslinking element bound by covalent bonds to the microbubble shell. Lux further teaches ACPP is cleaved by thrombin between the arginine and serine amino acids (see p.37588, left column, last paragraph, p.37592, left column, 1st and 5th paragraph, and Figs. 2, 3A, 4A, and S3A-B). Lux incubated the ACPP conjugated maleimide microbubbles in vitro with thrombin and ultrasound imaged the interaction, demonstrating a shift in ultrasound intensity post incubation and cleavage of ACPP, reading on wherein the microbubble alters its interaction with ultrasound waves in response to elimination of cross linkages and wherein the altered interaction is an observable alteration in resonance frequency of the microbubble or attenuation due to a persistent change of mechanical properties of a shell of the microbubble (see p.37593, paragraph bridging left and right columns, and Fig. S8). Furthermore, Fig. 6 explicitly shows that ACPP-MBs show increased ultrasound signal (i.e., a change in mechanical property) compared to control microbubbles). Thus, Lux teaches an equivalent responsive microbubble comprising the claimed structural features and cleavage functionality resulting in a change in ultrasound signal as recited in claim 16.
Conclusion
NO CLAIMS ALLOWED.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure:
Willmann JK, Kimura RH, Deshpande N, Lutz AM, Cochran JR, Gambhir SS. Targeted contrast-enhanced ultrasound imaging of tumor angiogenesis with contrast microbubbles conjugated to integrin-binding knottin peptides. J Nucl Med. 2010 Mar;51(3):433-40.
Fan Z, Kumon RE, Deng CX. Mechanisms of microbubble-facilitated sonoporation for drug and gene delivery. Ther Deliv. 2014 Apr;5(4):467-86.
Church CC. The Effects of an Elastic Solid-Surface Layer on the Radial Pulsations of Gas-Bubbles. J Acoust Soc Am. 1995;97(3):1510–1521
Any inquiry concerning this communication or earlier communications from the examiner should be directed to GEORGIANA C REGLAS whose telephone number is (571)270-0995. The examiner can normally be reached M-Th: 8:00am-2:00pm.
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/G.C.R./Examiner, Art Unit 1651
/THOMAS J. VISONE/Supervisory Patent Examiner, Art Unit 1672
