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
Status of the Claims
Claims 1-15 are pending and examined herein.
Priority
The present application, filed 01/11/2024, is a 371 of PCT/JP2022/008360, filed 02/28/2022, which claims foreign priority of JP2021-123441, filed 07/28/2021. Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Information Disclosure Statement
The Information Disclosure Statement(s) filed 01/11/2024 are acknowledged and have been considered.
Claim Rejections - 35 USC § 103
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 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.
Claims 1-15 are rejected under 35 U.S.C. 103 as being unpatentable over Pezzi et al. (Integration of Magnetic Bead-Based Cell Selection into Complex Isolations. ACS Omega. Vol. 3, No. 4, April 2018) in view of Lee et al. (US 20140134645 A1) as evidenced by the CELLection™ Biotin Binder Kit (Thermo Fisher. Invitrogen. Manual. Catalog No. 11533D. Rev. Date: June 2012).
Regarding claim 1, Pezzi discloses a reagent for preparing a cell sample using magnetic bead-based analyte capture for highly specific isolation of target cell populations from cell-containing samples (Abstract, p. 3908; Introduction, p. 3909).
With regard to “the reagent comprising a solid support,” Pezzi teaches magnetic bead solid supports, including Dynabeads, CELLection Biotin Binder beads, FlowComp Dynabeads, and Sera-Mag SpeedBeads, for antibody-mediated cell isolation (Introduction, p. 3909; Materials and Methods, p. 3914).
With regard to “a trapping substance,” Pezzi teaches that antibodies as trapping substances, including goat polyclonal anti-EpCAM antibody conjugated to magnetic beads for capture of EpCAM-expressing target cells (Materials and Methods, p. 3914).
With regard to “linked to the solid support by a cleavable linker,” Pezzi teaches CELLection beads in which a DNA linker connects the antibody to the bead, and DNase I cleaves the DNA linker attaching the bead to the antibody to release captured cells from the bead (Results and Discussion, p. 3911).
With regard to “and traps a target cell,” Pezzi teaches antibody-coated magnetic bead capture of EpCAM-expressing cell lines and direct isolation by incubating antibody-coated magnetic beads with cells (Results and Discussion, p. 3910; Materials and Methods, p. 3915).
Pezzi also teaches a labeling system, although not in the same manner as claimed. Specifically, Pezzi teaches antibody-based magnetic bead labeling/conjugation systems including biotin-streptavidin antibody conjugation and CELLection DNA-linker bead systems. Pezzi further uses fluorescently labeled antibody systems to characterize bead-antibody density and release, and evaluates compatibility of magnetic bead-based cell isolation with downstream fluorescence-based assays, including fluorescence imaging, fluorescent staining, protein localization analysis, and fluorescent immunohistochemistry. (Introduction and Results and Discussion pp.3909-3910; Materials and Methods, p. 3914-3916).
Hence, although Pezzi teaches magnetic bead cell-capture reagents having antibody-based labeling/conjugation chemistry and downstream fluorescence compatibility, Pezzi does not expressly teach or specify “wherein a fluorescent dye is bound to a site on the linker closer to the trapping substance than a cleavage position in the linker or a site on the trapping substance except for a binding site to the target cell in the trapping substance.”
However, Lee teaches the missing fluorescent labeling arrangement. Specifically, Lee teaches isolating or counting target cells using a dye complex that includes a target-binding material, a cleavable linker linked to the target-binding material, and a fluorescent dye linked to the cleavable linker (paragraph [0021], p. 12). Lee further teaches that the target-binding material may be an antibody, the cleavable linker may be a photocleavable linker, and the fluorescent dye may include FITC, Rhodamine, and other FACS-compatible fluorescent dyes (paragraphs [0024]-[0026], p. 12).
Lee also teaches an antibody-PC linker-FITC complex in which PC linker-FITC is conjugated to an amino group of the antibody (paragraph [0019], p. 11; FIG. 7), and Figure 1 below illustrates a fluorescent dye coupled to a cleavable linker that is coupled to an antibody target-binding material (paragraph [0047], p. 14; FIG. 1.) Accordingly, Lee teaches or at least suggests placing a fluorescent dye within an antibody-side cleavable-linker construct.
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Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Pezzi’s antibody-coated CELLection magnetic bead reagent, in which a DNA linker connects the antibody trapping substance to the magnetic bead solid support and DNase cleaves the DNA linker to release captured target cells, to further incorporate Lee’s fluorescent dye/cleavable-linker antibody labeling arrangement. Pezzi itself provides the reason to make the modification because Pezzi expressly evaluates magnetic bead-based cell isolation systems for downstream fluorescence-based compatibility, including fluorescence imaging, fluorescent staining, protein localization analysis, and downstream imaging endpoints. Although Pezzi teaches antibody-conjugated bead labeling systems and downstream fluorescence compatibility, Pezzi does not provide the claimed fluorescent dye placement integrated into the antibody/linker capture construct. Lee provides that specific improvement by teaching a fluorescent dye coupled to a cleavable linker attached to an antibody target-binding material for fluorescent detection, isolation, and FACS-based analysis. A person of ordinary skill in the art would therefore have recognized Lee’s labeling strategy as a suitable modification to Pezzi’s releasable antibody-DNA-linker magnetic bead platform to improve the downstream fluorescence-based analysis and sorting applications expressly contemplated by Pezzi while preserving Pezzi’s magnetic bead capture and DNase-release functionality. Incorporating Lee’s fluorescent dye/cleavable-linker arrangement into Pezzi’s bead-DNA-linker-antibody capture reagent would have positioned the fluorescent dye on the trapping-substance side of the cleavable linker, i.e., closer to the antibody trapping substance than the linker cleavage position, thereby providing fluorescently identifiable captured cells for downstream imaging, analysis, and cell sorting without altering Pezzi’s target-cell capture or linker-cleavage mechanism.
A person of ordinary skill in the art would have had a reasonable expectation of success because Pezzi and Lee both employ antibody-mediated target-cell capture systems using cleavable linker technology for isolating and analyzing target cells from biological samples. Pezzi demonstrates that its antibody-DNA-linker-bead construct captures target cells, releases them by cleavage of the DNA linker, and remains compatible with downstream fluorescence-based analyses. Lee demonstrates that fluorescent dyes can be incorporated into antibody/cleavable-linker target-cell binding complexes while preserving target-cell binding and enabling fluorescent detection, isolation, and FACS analysis. Because Lee’s fluorescent labeling arrangement is incorporated into the antibody/linker portion of the capture construct without changing the antibody target-binding function or the cleavable-linker release function relied upon by Pezzi, a person of ordinary skill in the art would have reasonably expected the modified Pezzi reagent to retain target-cell capture and release performance while additionally providing fluorescent identification, downstream fluorescence analysis, and fluorescence-assisted cell sorting.
Regarding claims 2 and 3, as discussed above, Pezzi teaches that the solid support is a bead and that the bead is a magnetic bead (Introduction, p. 3909; Materials and Methods, p. 3914).
Regarding claim 4, as discussed above, Pezzi teaches that the linker is a DNA linker (Results and Discussion, p. 3911).
Regarding claims 5 and 6, as discussed above, Pezzi teaches that the trapping substance is a receptor-binding site, antibody, or antibody fragment (Materials and Methods, p. 3914).
Regarding 7, Lee teaches fluorescent dyes suitable for separating target cells by a cell sorter. Specifically, Lee teaches that the fluorescent dye may include FITC, Rhodamine, and other fluorescent dyes compatible with flow cytometry/FACS (paragraph [0026], p. 12). Lee further teaches isolating dye-complex-bound target cells by flow cytometry, including fluorescence-activated cell sorting (FACS), and demonstrates use of fluorescent antibody-PC linker complexes with a BD FACSAria II Cell Sorter for target-cell isolation (paragraphs [0031], [0046], and [0048], pp. 12, 14). Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to select Lee’s FACS-compatible fluorescent dyes in the modified Pezzi reagent because Pezzi expressly evaluates magnetic bead-based cell isolation for downstream fluorescence-based analysis, and Lee teaches fluorescent dye/cleavable-linker antibody complexes specifically adapted for fluorescence-based target-cell sorting. A person of ordinary skill in the art would have reasonably expected success because Lee demonstrates that fluorescent dye-containing antibody-PC linker complexes label target cells and can be used with a FACS cell sorter for target-cell isolation.
Regarding claim 8, as discussed above, Pezzi discloses a reagent kit (CeLLection Biotin Binder) for preparing a cell sample that includes a magnetic bead solid support, an antibody trapping substance, and a releasable DNA-linker attachment. The CELLection Biotin Binder Kit literature further describes this reagent kit as comprising streptavidin-coated magnetic beads configured with a cleavable DNA linker for coupling biotinylated antibodies to the bead while permitting enzymatic release of viable captured cells for downstream applications (pp. 1-2).
However, although Pezzi teaches a releasable magnetic bead reagent kit that incorporates a biotin/streptavidin-based labeling system together with a cleavable DNA-linker attachment, Pezzi does not disclose incorporating a fluorescent dye into the antibody/linker capture construct as recited in claim 8. As discussed above with respect to claim 1, Lee teaches fluorescent labeling of antibody/cleavable-linker complexes in which the fluorescent dye is incorporated into the antibody-side linker arrangement for fluorescent detection, isolation, and cell sorting.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the CELLection-based reagent kit taught by Pezzi by incorporating Lee’s fluorescent dye/cleavable-linker antibody labeling arrangement. As discussed with respect to claim 1, Pezzi expressly evaluates magnetic bead-based cell isolation systems, including CELLection beads, for compatibility with downstream fluorescence-based analytical techniques, including fluorescent staining, fluorescence imaging, protein localization analysis, and downstream imaging endpoints. Lee provides a fluorescent antibody/cleavable-linker labeling arrangement suitable for fluorescent target-cell detection and FACS-based isolation. Thus, a person of ordinary skill in the art would have been motivated to incorporate Lee’s fluorescent labeling strategy into Pezzi’s CELLection reagent kit to provide fluorescently identifiable captured or released target cells for downstream fluorescence-based analysis and cell sorting while retaining Pezzi’s established bead-based capture and DNase-release functionality. A person of ordinary skill in the art would have had a reasonable expectation of success because Pezzi demonstrates that the CELLection antibody-DNA-linker bead system captures and releases target cells, and Lee demonstrates that fluorescent dye-containing antibody/cleavable-linker complexes preserve target-cell binding and enable fluorescence-based cell isolation.
Regarding claims 9 and 10, as discussed above, Pezzi teaches that the solid support is a bead and that the bead is a magnetic bead (Introduction, p. 3909; Materials and Methods, p. 3914).
Regarding claim 11, as discussed above, Pezzi teaches that the linker is a DNA linker (Results and Discussion, p. 3911).
Regarding claims 12 and 13, as discussed above, Pezzi teaches that the trapping substance is a receptor-binding site, antibody, or antibody fragment (Materials and Methods, p. 3914).
Regarding claim 14, as discussed above, Lee teaches that the fluorescent dye is a fluorescent dye available for separating a target cell by a cell sorter (paragraphs [0026], [0031], [0046], and [0048]). Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use Lee’s fluorescent dye in the modified Pezzi/CELLection reagent kit because Pezzi expressly teaches a CELLection-based releasable magnetic bead reagent kit compatible with downstream fluorescence-based analysis, while Lee teaches fluorescent antibody/cleavable-linker complexes specifically adapted for fluorescence-based target-cell detection and FACS separation. A person of ordinary skill in the art would have reasonably expected success because Lee demonstrates that fluorescent dye-containing antibody-PC linker complexes label target cells and can be used with a FACS cell sorter for target-cell isolation.
Regarding claim 15, refer to the discussion above. Pezzi teaches a method of preparing a cell sample using antibody-coated magnetic beads. Pezzi teaches forming a composite of the bead reagent and cells by incubating antibody-coated magnetic beads with target-cell-containing samples (Materials and Methods, p. 3915). Pezzi further teaches separating bead-bound cells using the magnetic bead solid support and collecting the bead-bound cell population by magnetic manipulation (Materials and Methods, p. 3915). Pezzi also teaches cleaving the DNA linker in CELLection beads with DNase to release captured cells from the bead (Results and Discussion, p. 3911; Materials and Methods, p. 3915). Thus, Pezzi teaches steps (a)-(c) of claim 15.
However, Pezzi does not expressly teach using the cell stained with a fluorescent dye and obtained after linker cleavage as a cell sample for separating a target cell by a cell sorter. As discussed above, Lee teaches fluorescent dye-containing antibody/cleavable-linker complexes for labeling target cells and isolating dye-complex-bound target cells by flow cytometry, including FACS. Lee also demonstrates use of fluorescent antibody-PC linker complexes with a BD FACSAria III Cell Sorter for target-cell isolation (paragraphs [0031], [0046], and [0048], pp. 12, 14).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Pezzi’s CELLection-based magnetic bead cell-isolation method, in which antibody-coated magnetic beads bind target cells, the bead-bound cells are magnetically separated, and a DNA linker is cleaved with DNase to release captured cells, to further include Lee’s fluorescent dye/cleavable-linker antibody labeling and FACS-based separation approach because Pezzi expressly evaluates magnetic bead-based cell isolation for downstream fluorescence-based analysis and imaging, while Lee teaches fluorescent antibody/cleavable-linker complexes specifically adapted for fluorescent target-cell detection and FACS-based separation. The modification would have allowed cells released after linker cleavage in Pezzi’s workflow to remain fluorescently identifiable and suitable for subsequent cell-sorter separation, while preserving Pezzi’s antibody-mediated magnetic capture, magnetic separation, and DNase-linker release steps. A person of ordinary skill in the art would have reasonably expected success because Pezzi demonstrates successful capture, magnetic separation, and release of target cells using antibody-coated CELLection beads, and Lee demonstrates successful fluorescent labeling and FACS-compatible separation of target cells using antibody/cleavable-linker dye complexes.
Ultimately, claims 1-15 are rejected under 35 U.S.C. § 103 as being unpatentable over Pezzi et al. in view of Lee et al., as evidenced by the CELLection™ Biotin Binder Kit where applicable. Pezzi teaches antibody-conjugated magnetic bead capture/release reagents, kits, and methods, including magnetic bead solid supports, antibody-mediated target-cell capture, biotin/streptavidin-based conjugation, DNA-linker cleavage, magnetic separation, and compatibility with downstream fluorescence-based analytical workflows. Lee teaches a fluorescent antibody/cleavable-linker labeling arrangement and use of fluorescently labeled target cells for FACS/cell-sorter separation. Thus, the proposed combination is supported by the references themselves: Pezzi provides the releasable antibody-conjugated magnetic bead platform and identifies downstream fluorescence compatibility as a relevant objective, while Lee provides the specific fluorescent antibody/linker labeling strategy for fluorescence-based detection and sorting. Therefore, the claimed subject matter as a whole would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ELIZABETH OGUNTADE whose telephone number is (571)272-6802. The examiner can normally be reached Monday-Friday 6:00 AM - 3 PM.
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/E.O./Examiner, Art Unit 1677
/BAO-THUY L NGUYEN/Supervisory Patent Examiner, Art Unit 1677 June 25, 2026
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