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 .
Election/Restrictions
Applicant’s election without traverse of Group 1 (claims 1-10) in the reply filed on 30 June 2026 is acknowledged.
Claim 11 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected group, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 30 June 2026.
Claim Objections
Claim 1 is objected to because of the following informalities: in step f, the comma between “…probe” and “to the single…” should be removed. Appropriate correction is required.
Claim Rejections - 35 USC § 112
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 4 and 6 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.
These claims recite sites which are “about [X] to about [X] bases long”. The specification defines “about” in regards to the lengths of polynucleotides as “X ± 10%”. This can result in non-whole numbers (i.e., 15 ± 1.5 bases). However, nucleotide length is necessarily expressed as a whole number because an oligonucleotide cannot contain a fractional nucleotide. It is therefore unclear how the definition of “about” is to be applied to the discrete nucleotide numbers recited in the claims, rendering the metes and bound of the limitations unclear.
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 1-3, 5, 7, 8, and 10 are rejected under 35 U.S.C. 103 as being unpatentable over Horta [Horta S et al. Analytical chemistry. 2021 Apr 7;93(15):6169-77] in view of Samusik [US 20170107563 A1].
Horta teaches an immuno-rolling circle amplification assay for the multiplex detection of three antigen-specific antibody isotypes (IgG, IgA, and IgM). As depicted in Figure 1 (below), the assay involves (1) capturing the targeted antibody via antigen-coated slides, (2) applying target-specific antibody oligonucleotide conjugates (AOCs) to bind to the captured antibody, (3) hybridizing AOC specific padlock probes (PLP) to the AOC and circularizing via ligation, and (4) amplifying the PLP via rolling circle amplification (RCA) to yield a concatemeric linear DNA product which is (5) detected via two detection oligonucleotides (DOs).
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Figure S1 (below) provides another depiction of this process with a focus on the AOC/PCP binding and the result of RCA. The PCP begins as a linear probe with two arms on each end, a generic barcode site (green) and a isotype-specific barcode (red). The linear probe hybridizes to the oligonucleotide of the AOC via its arms. The RCA product is labeled with two DOs, one for each of the barcode sites. Therefore, the arms, the barcode sites, and the DOs are equivalent to the target recognition sites, the FISH probe binding sties, and the FISH probes, respectively, of the instant application.
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Horta does not teach that the padlock probe is annealed to the AOC prior to the binding of the AOC to its target nor does it specifically mention the oligonucleotide of the AOC acting as a primer.
Samusik discloses multiple methods for single-cell antigen cytometry which utilizes various capture agents, including antibodies, labeled with DNA that can be subsequently detected via primer extension [0003-0004]. In one such method, an antibody is linked to an oligonucleotide which is hybridized to a padlock probe with its ends positioned adjacent to each other. After the ends are ligated together, the circularized padlock probe can be amplified via an RCA reaction which is primed by the capture agent-linked oligonucleotide [Samusik, 0176]. This configuration is identical to that performed by Horta. Therefore, the skilled artisan would recognize the ability of the oligonucleotide of Horta’s AOC to serve as a primer for RCA as this is evidenced by Samusik.
Additionally, Example 2 of Samusik demonstrates hybridizing the padlock probe to the AOC prior to adding the construct to a sample does not affect the AOC’s ability to bind to its target [Samusik, 0220]. Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention that modifying Horta’s assay to include hybridizing the padlock probe to the AOC prior to binding of the AOC’s target, as evidenced by Samusik. As both configurations allow for subsequent padlock probe ligation and performance of RCA and thus serve the same function with the same expectation of success, the skilled artisan would recognize that choosing a configuration represents nothing more than an assay design choice. The Supreme Court decided that a claim can be proved obvious merely by showing that the combination of known elements was obvious to try. In this regard, the Supreme Court explained that, “[w]hen there is a design need or market pressure to solve a problem and there are a finite number of identified, predictable solutions, a person of ordinary skill in the art has a good reason to pursue the known options within his or her technical grasp.” An obviousness determination is not the result of a rigid formula disassociated from the consideration of the facts of the case. Indeed, the common sense of those skilled in the art demonstrates why some combinations would have been obvious where others would not. Therefore, choosing from a finite number of identified, predictable solutions, with a reasonable expectation for success, is likely to be obvious to a person if ordinary skill in the art. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, E.).
Regarding claim 3, by hybridizing to the oligonucleotide of the AOC, the ends of the PLP’s arms are placed adjacent to each other and perfectly match the template provided by the AOC (see the example sequences below provided by Horta’s Table S1). As ligase requires a perfect match between a template DNA strand and the strand to be ligated, this positioning can be considered the first, or initiating step, towards circularization [Horta, p6170].
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Regarding claim 7, Horta’s figure 1 teaches that the different DOs are labeled with different fluorophores (Cy3, FITC, Cy5, and Cy7).
Regarding claims 8 and 10, Horta teaches that after amplification a labeling buffer was added along with each DO. Therefore, Horta demonstrates the simultaneous contacting of each probe to the RCA product. After labeling, images were obtained via fluorescent microscopy using four different fluorescence channels allowing for colocalization of the antibody isotopes by relating the generic channel with each of the isotype-specific channels [Horta, Figure 1, p6171].
Claims 4, 6, and 9 are rejected under 35 U.S.C. 103 as being unpatentable over Horta and Samusik as applied to claim 1 above, and further in view of Soares [Soares RR et al. Accounts of Chemical Research. 2021 Oct 12;54(21):3979].
Horta does not teach target recognition sites and probe binding sites which are within the claimed ranges. Horta also does not teach contacting the imaging probes to the RCA product individually and imaging between.
Soares introduces PLP-RCA providing a detailed account of each of the three main assay steps: PLP design and ligation, RCA, and detection [Soares, abstract].
Regarding PLP design, Soares describes PLPs as linear oligonucleotides with their end-sequences complementary to a target sequence and a backbone with a linker sequence which can host functionality-dependent regions. Soares further highlights the length of the end sequences as one of the major PLP design considerations. This characterization of PLP design as a result effective parameter is supported by Horta’s disclosure which details the process used to develop their PLPs. Briefly summarized, random and unique nucleotide sequences were generated using publicly available tools and checked for cross-hybridization and secondary structures [Soares, p3980-3984].
Regarding detection, Soares teaches that detection of the RCA product can be readily achieved through the use of labeled probes which are complementary to the product. Furthermore, the ability to modify the backbone of the PLP, allows for extreme versatility, a high degree of multiplexing, and in situ and spatial analyses. For example, performing a single round of labeling can identify eight different targets in a single imagine step; by performing sequential probing and stripping cycles, this can be expanded to identify more than 200 different targets. These processes are depicted in figure 1A and 1B (below) [Soares, p3980-3984].
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Therefore, a person of ordinary skill prior to the effective filing date of the claimed invention would have recognized the PLP design, and consequently the specific lengths of its component parts, as a result effective parameter and it would have been customary for said person to determine the optimal length of each component part for their specific assay. Additionally, a skilled artisan looking to increase the multiplexing capabilities of Horta and Samusik’s assay would have been motivated to employ the sequential imaging technique taught by Soares. The use of a known technique to improve similar devices (methods or products) in the same way is likely to be obvious. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, C.).
Conclusion
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/K.N.K./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
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