DETAILED ACTION
Status of Application, Amendments and/or Claims
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
The amendment of 3/9/26 has been entered in full. Claims 9, 11, 13, 15 and 17 are amended. Claims 1-24 are pending.
Election/Restrictions
Applicants' election without traverse of Group I, claims 1-21, in the reply filed on 3/9/26 is acknowledged. Claims 22-24 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
The elections of (1) SEQ ID NO: 1 as the species of OmpG protein, and (2) “6) L123 to D228” as the species of insertion location, are also acknowledged. Applicants indicate that the elected species read on claims 1-6 and 17-21. Claims 7-16 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim.
Claims 1-6 and 17-21 are under consideration, as they read upon the elected species.
Drawings
Corrected drawings in compliance with 37 CFR 1.121(d) are required because:
Figure 20 includes an extraneous character at the end of the identifier for the sequence labeled “A0A7H0FHK8”. Specifically, the specification at ¶ 68 (published application), indicates that the name for this sequence is “A0A7H0FHK8”, but Figure 20 shows it as “A0A7H0FHK8 |” (bolding adding for emphasis). The extraneous character should be removed in each place that it appears (four instances).
Applicants are advised to employ the services of a competent patent draftsperson outside the Office, as the USPTO no longer prepares new drawings. The corrected drawings are required in reply to the Office action to avoid abandonment of the application. The requirement for corrected drawings will not be held in abeyance.
Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d).
Specification
The disclosure is objected to because of the following informalities:
(1) The title of the invention, “Nanopore Biosensors and Uses Thereof” is not descriptive because it is directed to any type of nanopore biosensor, and the claims are limited to a OmpG-based nanopore biosensor. A new title is required that is clearly indicative of the invention to which the claims are directed. The following title is suggested: “OmpG Nanopore Biosensors and Uses Thereof”.
(2) Figures 8A, 11 and 12A each show one or more amino acid sequences that are not identified by the appropriate sequence identifier in the brief description of the figure in the specification, or in the figure itself, as required by the rules for sequences (37 CFR 1.821(d)). If each sequence is already present in the sequence listing as an entire sequence, or a subset of an entire sequence, the objection can be overcome by adding the appropriate sequence identifier, or residues within the appropriate sequence identifier, to the brief description of Figure 11 or the figure itself. Otherwise, the sequence listing must first be updated to include the sequence, and then the appropriate sequence identifier must be added.
(3) Figure 20 shows 8 amino acid sequences that are identified in the brief description of the figure as “SEQ ID NOs: 1 and 3-9”. However, the first sequence that is shown clearly does not match the sequence of SEQ ID NO: 1, which begins with the amino acids “MEERND”, and is 281 amino acids in length. The first sequence shown in Figure 20 instead begins with the amino acids “MKKLLP” and is approximately 300 amino acids in length. In order to overcome this objection, corrections must be made such that the first amino acid sequence shown in Figure 20 is accompanied by a sequence identifier identifying a sequence in the sequence listing that matches what is shown in Figure 20. Applicants are also requested to check that each of other sequences in Figure 20 also match the corresponding sequences (SEQ ID NO: 3-9) in the sequence listing.
Appropriate correction is required.
Claim Objections
Claims 5, 6 and 17-18 are objected to because of the following informalities:
In claims 5 and 6, lines 1-2 of each, “the amino acids sequence” should be “the amino acid sequence”.
The remaining claim(s) are objected to for depending from an objected claim.
Appropriate correction is required.
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 5, 17 and 18 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 pre-AIA the applicant regards as the invention.
Claim 5 limits the composition of claim 1 to one wherein the OmpG comprises an amino acid sequence of one of SEQ ID NO: 1-9. However, the OmpG of parent claim 1 is an OmpG “of E. coli origin”, and the amino acid sequences of SEQ ID NO: 3-9 all appear to be OmpG proteins from bacterial species other than E. coli. For example, SEQ ID NO: 3 is identified as A0A0F1B6I1, which is identified in the UniProt database as a protein from Enterobacter sichauensis instead of E. coli (see the Record for UniProt A0A0F1B6I1; dated 6/24/15; no author listed, 1 page as printed). SEQ ID NO: 4-9 also appear to be sequences from others species. It is unclear how an OmpG sequence can be sourced from another species and still be “of E. coli origin” as required in parent claim 1.
Claim 17 recites “amino acids L213 to D228 of SEQ ID NO: 1”; however, SEQ ID NO: 1 does not includes an “L” (leucine) at position 213 or a “D” (aspartate) at position 228. Therefore the claim is indefinite as to which amino acids are being modified.
Claim 18 depends from claim 18 and further limits the reference amino acids to a series of amino acids from L213 to R231. First, none of the listed amino acids correspond to the amino acids at positions 213-231 of SEQ ID NO: 1. Furthermore, positions 229-231 are outside the range recited in parent claim 17. Therefore the claim is indefinite as to which amino acids are being modified.
The remaining claim(s) included in the rejection are dependent claims that depend from one of the claims rejected above, and encompass the same indefinite subject matter.
Claim Rejections - 35 USC § 112, enablement
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.
Claims 1-5 and 19-21 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while enabling for
A composition comprising a nanopore disposed in a membrane preparation, wherein the nanopore comprises an OmpG of E. coli origin comprising the amino acid sequence SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9, wherein a heterologous peptide is inserted within one or more of the flexible loops of the OmpG,
does not reasonably provide enablement for comprising a nanopore disposed in a membrane preparation, wherein the nanopore comprises an OmpG of E. coli origin having fourteen β-strands connected by seven flexible loops on a first side of the membrane preparation and seven short turns on a second side of the membrane preparation, wherein a heterologous peptide is inserted within one or more of the flexible loops of the OmpG.
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 considered when determining if the disclosure satisfies the enablement requirement and whether any necessary experimentation is “undue” include, but are not limited to: 1) nature of the invention, 2) state of the prior art, 3) relative skill of those in the art, 4) level of predictability in the art, 5) existence of working examples, 6) breadth of claims, 7) amount of direction or guidance by the inventor, and 8) quantity of experimentation needed to make or use the invention. In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988).
The claims are product claims; specifically, a composition comprising a nanopore dispersed in a membrane preparation, wherein the nanopore comprises an outer membrane protein G (OmpG) of E. coli origin having fourteen β-strands connected by seven flexible loops on a first side of the membrane preparation and seven short turns on a second side of the membrane preparation, wherein a heterologous peptide is inserted within one or more of the flexible loops of the OmpG. The structure recited in claim 1 represents the native structure of the OmpG as expressed in E. coli; see Figure 11, which depicts the structure of OmpG, including the seven extracellular loops. The specification teaches that the OmpG nanopores are “useful as sensors for detecting intramolecular actions” (¶ 50). The elected species of OmpG protein under consideration is SEQ ID NO: 1.
The specification does not provide a limiting definition of the term “outer membrane protein G” or “OmpG” as used in independent claim 1. However, the term clearly encompasses not just defined OmpG sequences (e.g., SEQ ID NO: 1), but also variants of such, such as those of dependent claim 4, which expressly includes variants; e.g., a variant with at least 80% identity to SEQ ID NO: 1. Such includes up to 20% of any of the reference sequence changed; for example, the genus of variants that are at least 80% identical to SEQ ID NO: 1 includes variants with changes in up to 56 of the amino acids of the 281 amino acid sequence, in any combination. As such, the claims are directed to genus claims encompass a composition comprising a genus of OmpG variants.
In support of the scope of the claims, specification discloses several OmpG sequences. Figure 20 shows a sequence labeled “OmpG” that corresponds to the full-length E.coli OmpG sequence of 301 amino acids known in the prior art. SEQ ID NO: 1, not shown in this figure, represents a sequence of 281 amino acids wherein the OmpG signal peptide sequence has been removed except for the initiating methionine. SEQ ID NO: 2 is 99.6% identical to SEQ ID NO: 1, with just one less amino acid at the C-terminus. SEQ ID NO: 3-9 are longer sequences (304 amino acids), each including a signal peptide sequence, that appear to originate from other bacterial species (see the rejection of Claim 5 under 35 U.S.C. 112(b) set forth above).
The prior art appreciates that "Mutations … are generally destabilizing, and can reduce protein … fitness" and "In general, more comprehensive understanding of how mutations affect protein fitness within living cells is needed, including their combined effects on function, thermodynamic and kinetic stability, and clearance through aggregation and degradation" (pg 602 of Tokuriki et al, 2009, Current Opinion in Structural Biology. 19: 596-604). The prior art published also appreciates that "the range of possible SNV [single nucleotide variation] effects at the protein level are significantly greater than currently assumed by existing software prediction methods, and that correct prediction of consequences remains a significant challenge" (pg 18 of Bhattacharya et al, 2017. Plos One. 12(3): e0171355, pages 1-22 as printed). Thus, knowledge of the amino acid sequence of OmpG from E. coli alone is not sufficient for the skilled artisan at the time of the effective filing date to predict which mutations (substitutions, additions and deletions) will result in a variant of OmpG that retains its structure and functions as a nanopore.
While the specification provides the sequence of the native E. coli OmpG sequence, with or without a full-length signal sequence (SEQ ID NO: 1), the specification does not provide further guidance as to the range of mutations that can be made in this sequence and still retains its structure and functionality as a nanopore in a membrane. There is no guidance provided as to how single or multiple mutations in the E. coli OmpG will affect the structure and function of the protein, or as to compensatory mutations to be made to retain activity when a destabilizing mutation is introduced into the structure. The specification does not teach a minimal length for variants of OmpG that will retain structure and functionality; i.e., the ability to bind to form a nanopore.
Thus, the limited examples provided by the specification fail to correspond to the scope of the genus of variants of OmpG encompassed by the claims. The specification fails to provide guidance as to the scope of amino acid changes that can be made to SEQ ID NO: 1 and still retain function and the ability to form a nanopore.
Due to the large quantity of experimentation necessary to generate the large number E. coli OmpG variants encompassed by the claims and to screen the same for activity, the lack of direction/guidance presented in the specification regarding which mutations can be made to the sequence that will retain structure and activity, the absence of working examples directed to same, the complex nature of the invention, the state of the prior art which establishes the unpredictability of the effects of mutation on protein structure and function, and the breadth of the claims, undue experimentation would be required of the skilled artisan to make and use the claimed invention in its full scope.
Note on Prior Art Rejection(s)
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.
Claim Rejections - 35 USC § 102
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 1-5 and 19-21 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Chen et al, U.S. Patent Application Publication 2015/0080242, published 3/19/15. The earliest date to which the instant application claims priority is 6/22/22.
Claim 1 encompasses a composition comprising a nanopore disposed in a membrane preparation, wherein the nanopore comprises an OmpG protein of E. coli origin having fourteen β-strands connected by seven flexible loops on a first side of the membrane preparation and seven short turns on a second side of the membrane preparation, wherein a heterologous peptide is inserted within one or more of the flexible loops. The term “membrane preparation” is not provided with a limiting definition in the specification, but examples are given that include whole cells, e.g., a bacterium (¶ 17 of the instant specification).
Chen teaches a “nanopore” that is “disposed in a membrane preparation” (¶ 4), and that “[i]n certain embodiments, the nanopore comprises 14 β-strands connected by seven flexible loops on a first side of the membrane preparation and seven short turns on a second side of the membrane preparation” (¶ 7) and further that the nanopore can be “outer membrane protein G (OmpG)” that is “of E. coli origin” (¶ 7). Chen further teaches that the membrane preparation can be in a composition (¶ 12). Chen further teaches that a ligand may be “incorporated directly into a nanopore (e.g., an epitope in a nanopore loop)” (¶ 40). Chen also teaches “[i]n some embodiments, a library of nanopores (e.g., OmpG) is provided with the extracellular loops (e.g., 7 in total) of the nanopores substituted by randomized sequences. In some embodiments, these loops with randomized sequence may constitute high-affinity sites for target molecules to bind” (¶ 41). A ligand that is an epitope incorporated directly into a loop, i.e., as part of the polypeptide sequence, and a randomized sequence substituted into loop, each represents a heterologous peptide inserted within the loop. As such, the teachings of Chen anticipate instant claim 1.
Claim 2 encompass a composition of claim 1 wherein the heterologous peptide is inserted between two amino acids within the flexible loop. In the teachings of Chen described above, the incorporated epitope or the substituted randomized sequence is inherently placed between two amino acids of the loop because it is part of the sequence of the loop. As such, the teachings of Chen also anticipate claim 2.
Claims 3 and 4 encompass a composition of claim 2 wherein the heterologous peptide replaces one or more amino acids in the loops (claim 3) and further wherein the number of amino acids replaced is the same as the number of amino acids in the heterologous peptide (claim 4). The teachings of Chen set forth above for parent claim 1 include loop sequences “substituted by randomized sequences”, and substitution, by definition, represents a one-for-one replacement of an amino acid with another amino acid. As such, substitution of a loop sequence with a random sequence represents an insertion of a heterologous peptide between two amino acids within the loop that replaces the loop sequence with the same number of amino acids as the heterologous peptide. As such, the teachings of Chen also anticipate claims 3 and 4.
Claim 5 encompasses a composition of claim 1 wherein the OmpG comprises the amino acid sequence SEQ ID NO: 1 or a variant thereof having at least 80% sequence identity to SEQ ID NO: 1. Chen further teaches an OmpG sequence of SEQ ID NO: 5, which is 281 amino acids in length and 99.7% identical to instant SEQ ID NO: 1 (¶ 45). As such, the teachings of Chen also anticipate claim 5.
Claim 19 encompasses a method of claim 1 wherein the OmpG comprises two distinct heterologous peptides independently inserted within one or more flexible loops. As taught above for claim 1, Chen teaches a library of OmpG proteins with extracellular loops substituted by randomized sequences. These teachings refer to multiple OmpG proteins, and each randomized sequence will have a different sequence and thus represent a distinct heterologous peptide. Thus, such a library encompasses at least two OmpG proteins with different heterologous sequences substituted in loops. As such, the teachings of Chen also anticipate claim 19.
Claims 20 and 21 encompass a composition of claim 1 wherein the membrane preparation comprises a planar lipid bilayer (claim 20) that is a bacterium (claim 21). Chen further teaches that the membrane preparation is a planar lipid bilayer (¶ 11) or a bacterium (¶ 11). As such, the teachings of Chen also anticipate claims 20 and 21.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
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 at the time any inventions covered therein were effectively filed 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 at the time a later invention was effectively filed 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.
Claim 6 is rejected under 35 U.S.C. 103(a) as being unpatentable over Chen et al, U.S. Patent Application Publication 2015/0080242, published 3/19/15, as applied to claim 1 above, and further in view of Fahie et al, 2021, Chapter 7 of Nanopore Technology: Methods and Protocols, Methods of Molecular Biology, vol 2186, pages 77-91, published September 2020 as an eBook.
Claim 6 encompasses a composition of claim 1, wherein the OmpG comprises the amino acid sequence SEQ ID NO: 1, and wherein loops 1-7 comprise amino acids E16-A32, Q52-D68, G96-N109, F138-T150, E175-I194, R212-R236, and E258-V275, respectively.
The teachings of Chen that anticipate claim 1 are set forth above. Chen further teaches that the membrane preparation can be a lipid bilayer (¶ 11). Chen further teaches production of OmpG in an inclusion body and purification (¶ 58), refolding (¶ 59), formation of a lipid bilayer comprising the OmpG (¶ 61) and use as a “Biosensor for Detecting Ligand-Target Interactions” (¶ 61). Chen does not teach use of an OmpG protein comprising the amino acid sequence of SEQ ID NO: 1 as recited in dependent claim 6.
Fahie teaches that “Nanopore sensing is a powerful lab-on-a-chip technique that allows for the analysis of biomarkers present in small sample sizes” and specifically describe an “OmpG-based nanopore system” (see Abstract). Fahie further teaches techniques for creating this nanopore by expressing OmpG in inclusion bodies (page 82) and placement in “an artificial bilayer in a two chamber chip” (page 85). Fahie further teaches the amino acid sequence of OmpG in Figure 1 (page 79), which shows a sequence identical to SEQ ID NO: 1 except that it does not include the initiating methionine present in SEQ ID NO: 1. Fahie further teaches that OmpG can be modified so that the “signal peptide is removed from the open reading frame of the gene so that it expresses in the inclusion body and not secreted to the outer membrane. The signal peptide of OmpG consists of amino acids 1-21 MKKLLPCTALVMCAGMACAQA; therefore maintain the start methionine and delete amino acids 2-21”. An OmpG as with all of the signal sequence removed except the initiating methionine as taught by Fahie results in an OmpG amino acid sequence that is 100% identical to SEQ ID NO: 1. Such a sequence would inherently comprise loops 1-7 that comprise amino acids E16-A32, Q52-D68, G96-N109, F138-T150, E175-I194, R212-R236, and E258-V275, respectively; see Figure 1 of Fahie.
It would have been obvious to the person of ordinary skill in the art before the effective filing date of the claimed invention to take the composition comprising a nanopore in a membrane wherein the nanopore comprises OmpG of E. coli origin with a heterologous peptide inserted in one or more of the flexible loops as taught by Chen, and to modify such to an OmpG sequence as taught by Fahie where the signal peptide sequence has been removed except for the initiating methionine (i.e., an OmpG of SEQ ID NO: 1). The person of ordinary skill in the art would have been motivated to use the sequence as taught by Fahie that omits the signal peptide sequence of OmpG, other than the initiating methionine, in order to employ the superior properties of such a sequence with respect to expression the protein in an inclusion body. The person of ordinary skill in the art would have had a reasonable expectation of success in applying the sequence of Fahie to the teachings of Chen because each teaches expression of OmpG for the same purposes; i.e., production of OmpG using inclusion bodies to generate an OmpG nanopore composed in an artificial lipid bilayer. This rationale supports a prima facie conclusion of obviousness in accord with KSR International Co. v. Teleflex Inc., 82 USPQ2d 1385 (2007).
Conclusion
No claims are allowable.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ZACHARY C HOWARD whose telephone number is (571)272-2877. The examiner can normally be reached on Monday to Friday from 9 AM to 5 PM. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Vanessa Ford, can be reached at telephone number (571) 272-0857. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of an application may be obtained from Patent Center. Status information for published applications may be obtained from Patent Center. Status information for unpublished applications is available through Patent Center for authorized users only. Should you have questions about access to Patent Center, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free).
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) Form at https://www.uspto.gov/patents/uspto-automated-interview-request-air-form.
/ZACHARY C HOWARD/Primary Examiner, Art Unit 1674
Prosecution Insights