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 .
Priority
This present application was filed 02/07/2025. Acknowledgement is made of the present application as a proper National Stage (371) entry of PCT Application No. PCT/IB2023/059313, filed 09/20/2023, which claims benefit under 35 U.S.C. 119(e) to provisional applications 63/414,883, filed 10/10/2022, and 63/410,438, filed 09/27/2022.
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed applications, Application Nos. 63/414,883 and 63/410,438, fail to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. Claim 25 recites “wherein the one or more molecular reactions is done in the presence of additional secondary antibodies”. Although the prior-filed application does recite that unlabeled secondary antibodies for stabilizing the binding of primary antibodies, the prior filed application does not disclose that step (c), the performing of one or more molecular reactions to produce a nucleic acid product, is performed in the presence of additional secondary antibody. Therefore claim 25 and the dependent claims 26-40 do not comply with one or more conditions for receiving the benefit of the earlier filing date.
The effective filing date of claims 25-40 is 09/20/2023.
Status of the Claims
Claims 25-40 and 42-49 are pending in the application. Claims 25 – 28 are amended, claims 42-49 are new, and claims 1-24 and 41 are cancelled. Claims 25-40 and 42-49 are examined below.
Withdrawn Objections
The objection to the specification has been withdrawn due to the amendment of the specification.
The objections to claims 27-28 are withdrawn due to the amendment of the claims.
The objection to claim 41 is withdrawn due to the cancellation of the claim.
Claim Objections
Claim 25 is objected to because of the following informalities:
In line 2, cross-linked is spelled “crosslinked”, whereas in lines 6 and 7 it is spelled “cross-linked”. In the interest of consistency it is suggested to amend “crosslinked” to ---cross-linked---.
Appropriate correction is required.
Withdrawn rejections
The rejection of claim 26 under 35 U.S.C. 112(b) has been withdrawn due to the amendment of the claim.
After consideration of the arguments put forth by applicant, the rejection under 35 USC § 103 has been withdrawn, please see new grounds of rejection below.
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 25, 31, 34, and 37-40 are rejected under 35 U.S.C. 103 as being unpatentable over Weibrecht et al. Proximity ligation assays: a recent addition to the proteomics toolbox. Expert review of proteomics. 2010 Jun 1;7(3):401-9, in view of Lamvik et al. Nonlabeled secondary antibodies augment/maintain the binding of primary, specific antibodies to cell membrane antigens. Cytometry: The Journal of the International Society for Analytical Cytology. 2001 Nov 1;45(3):187-93 (of record, IDS 02/07/2025) and Vernay et al. Immunofluorescence labeling of cell surface antigens in Dictyostelium. BMC research notes. 2013 Aug 12;6(1):317.
Regarding claim 25¸ Weibrecht teaches proximity ligation assays as a method for detection of proteins or protein-protein interactions (Weibrecht, page 401, Abstract, lines 8-9). Weibrecht teaches that in studies aimed to detect a particular protein or protein-protein interaction the target molecule is typically labeled by using a specific affinity reagent or a direct label (labeling a sample; Weibrecht, page 402, ‘Probe-based targeting’, lines 2-8). Weibrecht further teaches an example of a probe based method, such as immunostaining which allows detection of endogenous proteins in situ, providing information at a single cell level and that the protein of interest is targeted by antibodies and then visualized by a reporter molecule bound to the antibodies. Weibrecht further teaches that to make intracellular proteins accessible to the antibodies, samples need to be fixed and permeabilized (sample has been crosslinked; Weibrecht, page 402, 2nd column, see entire 3rd paragraph). Weibrecht further teaches a proximity ligation assay, which combines a probe based assay with a split-reporter approach, where a pair of proximity probes, i.e. antibodies to which an oligonucleotide has been conjugated, is used to target the protein of interest (Weibrecht, page 404, ‘Proximity ligation assays, lines 10-16). Specifically, Weibrecht teaches cells or tissues fixed to a slide prior to addition of the proximity probes and further teaches that for localized detection of the protein complexes, oligonucleotides conjugated to proximity probes provide the template for hybridization and ligation of two subsequently added connector oligonucleotides. Successful ligation results in a circular DNA molecule (Weibrecht, page 404, 2nd column, 2nd paragraph, lines 3-8). The circular molecule can serve as a template for rolling circle amplification (Weibrecht, page 405, lines 1-3) the product of which can be visualized by hybridization of fluorescently labeled detection oligonucleotides to a sequence encoded in the DNA-circle and are easily distinguishable from potential background fluorescence in an epifluorescence microscope (detecting the product of c; Weibrecht, page 405, 2nd paragraph, lines 1-8).
Weibrecht fails to teach incubating the labeled sample with a secondary antibody that recognizes the primary antibody-oligonucleotide conjugates. Weibrecht further fails to teach that the one or more molecular reactions is done in the presence of additional secondary antibodies.
Lamvik teaches staining peripheral blood mononuclear cells with antibodies specific for cell surface antigen with a second-step application of nonconjugated goat anti-mouse IgG antibodies (anti-species antibody; Lamvik, page 187, ‘Material and Methods’, lines 1-4). Lamvik further teaches that the labeled primary antibody are monoclonal mouse IgG antibodies (Lamvik, page 188, ‘Antibodies and Reagents’, lines 1-4). Lamvik further teaches that when cells are being stained with labeled antibodies followed by weak or no fixation, combined with permeabilization it appears that direct staining of the membrane antigens gives no or only weak fluorescence (Lamvik, page 187, lines 1-12). Lamvik further teaches that nonlabelled secondary antibody is able to influence the ability of primary, specific antibody to be or remain bound to cell membrane antigens when cell membrane permeabilization techniques are applied in order to stain cytoplasmatic or nuclear components in addition to surface membrane staining (Lamvik, page 192, ‘Discussion’, lines 1-11). Lamvik further teaches that the observed effect of the secondary antibody may be explained by the formation of a network of primary and secondary antibodies (crosslinked; Lamvik, page 192, ‘Discussion’, 2nd paragraph, lines 1-3). Lamvik further teaches that with the nonlabelled antibody added, the staining of membrane antigens is clearly augmented (Lamvik, page 189, 2nd column, 2nd paragraph, lines 4-6). Lamvik further teaches that the application of nonlabelled secondary antibody in addition to specific primary antibodies may be useful in combined studies on surface membrane proteins and intracellular components when no or only weak fixatives are permitted (Lamvik, page 192, 2nd column, 5th column, line 10- page 193, line 2).
Vernay teaches a method of detecting the presence of a protein and determine its intracellular localization (Vernay, page 1 of 4 lines 1-2). Vernay further teaches fixing cells expressing csA-SA in paraformaldehyde (Vernay, page 2 of 4, see entire 1st paragraph) and incubating the fixed cells with mouse anti-antibody (primary antibody) followed by Alexa-488-coupled anti-mouse antibody (secondary antibody; Vernay, page 2 of 4, 3rd paragraph, lines 1-5). Vernay further teaches that after surface labeling, cells were fixed again in paraformaldehyde, permeabilized and intracellular csA was then labeled with mouse anti-csA antibody followed by Alexa-488-coupled anti-mouse antibody and mounted in Möwiol (Vernay, page 2 of 4, see entire 4th paragraph). Cells were visualized using a confocal microscope (Vernay, page 2 of 4, 2nd paragraph, lines 12-13). As such, Vernay teaches detecting the target antigen in the presence of additional secondary antibody. Vernay further teaches that in contrast to the two-step method, if cells were first fixed and permeabilized, and then incubated with anti-csA antibody followed by anti-mouse Alexa-488 antibody this resulted in detection of only a very small amount of csA on the cell surface suggesting that the procedure results in the loss of a significant amount of csA protein, particularly on the cell surface. Fixing cells and incubating said cells with antibodies prior to permeabilization, then fixing the cells again, permeabilizing said cells and labeling the intracellular antigens resulted in prominent staining of the cell surface. Vernay teaches that these results indicate that the csA antigen was lost from the cell surface during cell permeabilization, unless it was stabilized by the binding of two layers of antibodies (Vernay, page 2 of 4, 2nd column, see entire 2nd paragraph).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Weibrecht comprising the binding of proximity probes to label proteins in fixed cells or tissue with the method of Lamvik of crosslinking primary labeled antibodies bound to cells by applying nonlabelled secondary anti-species antibodies that crosslink the primary antibodies because of the teaching of Weibrecht and Lamvik that cells have to be permeabilized in order to detect intracellular or nuclear antigens and the teaching of Lamvik that the technique may be useful in combined studies on surface membrane proteins and intracellular components when no or only weak fixatives are permitted.
It would have further been prima facie obvious to have modified the method of Weibrecht in view of Lamvik of cross-linking primary proximity-probe labeled antibodies to cells by applying secondary anti-species antibodies to the sample with the method of Vernay of adding additional secondary antibodies to the sample after permeabilizing the cells and labeling the intracellular targets with primary antibody because of the teaching of Vernay that this method allows for detecting an antigen on the surface of the cells and its intracellular localization without a loss of cell surface antigen during permeabilization of the cells.
One of ordinary skill in the art would have a reasonable expectation of success crosslinking the primary labeled antibody of Weibrecht with a secondary anti-species antibody as taught by Lamvik, because Weibrecht teaches adding oligonucleotide-labeled primary antibody to a sample and Lamvik teaches crosslinking primary labeled antibody with an anti-species secondary antibody.
One of ordinary skill in the art would further have a reasonable expectation of success adding additional labeled secondary antibody (as taught by Vernay) to the method of Weibrecht in view of Lamvik because Vernay teaches adding secondary anti-species antibody to a fixed sample, the same type of antibody as taught by Söderberg and Lamvik and as such the additional secondary antibody would not be expected to interfere with the method of Weibrecht in view of Lamvik.
As such, one of ordinary skill in the art would have a reasonable expectation of success that modifying the method of Weibrecht by adding anti-species antibody, that is capable of binding all primary antibodies, would crosslink the primary labeled antibodies as taught by Lamvik and Vernay and therefore augment detection of cell surface antigen by primary antibody in permeabilized cells.
Regarding claim 31, Weibrecht teaches that for localized detection of the protein complexes, oligonucleotides conjugated to proximity probes provide the template for hybridization and ligation of two subsequently added connector oligonucleotides. Successful ligation results in a circular DNA molecule (Weibrecht, page 404, 2nd column, 2nd paragraph, lines 3-8).
Regarding claim 34, Weibrecht teaches a proximity ligation assays (Weibrecht, page 401, Abstract, lines 8-9).
Regarding claim 37, Weibrecht in view of Lamvik and Vernay as applied to claim 25 also applies to claim 37.
Lamvik teaches a fluorescein isothiocyanate-conjugated mouse monoclonal anti-CD45, anti-CD3, and anti-CD19 primary antibody and a polyclonal goat anti-mouse IgG secondary antibody (Lamvik, page 188, ‘Antibodies and Reagents’, lines 1-6). As such, Lamvik teaches a secondary antibody that is made in another species, namely goat.
Regarding claims 38-39, Weibrecht in view of Lamvik and Vernay as applied to claim 25 also applies to claims 38 and 39.
Lamvik teaches a polyclonal goat anti-mouse IgG secondary antibody (Lamvik, page 188, ‘Antibodies and Reagents’, lines 5-6).
Regarding claim 40¸ Weibrecht teaches that the method can be used for detection of protein-protein interactions in tissue sections (Weibrecht, page 401, abstract, line 15).
Claim 26 is rejected under 35 U.S.C. 103 as being unpatentable over Weibrecht in view of Lamvik and Vernay as applied to claim 25 above, and further in view of Söderberg et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nature methods. 2006 Dec;3(12):995-1000 (see PTO-892; 09/22/2025).
Regarding claim 26, Weibrecht teaches a method for labeling a biological sample with oligonucleotide labeled antibodies and crosslinking the primary labeled antibodies with secondary antibodies essentially as claimed.
Weibrecht does not teach exposing the primary antibody-oligonucleotide conjugates to disassociating conditions while they are bound to the sample, the conditions comprising one or more reactions in at least 150 mM salt at 37°C for at least 1 min.
Söderberg teaches a P-LISA reaction comprising blocking the zinc-fixed cells before overnight incubation with proximity probes (primary antibody-oligonucleotide conjugates), followed by adding two connector oligonucleotide probes in a buffer comprising 250 mM NaCl (at least 150 mM salt; disassociating conditions while bound to the sample) and ligating the probes (one or more reactions) to form circles using as templates the two oligonucleotides attached to the antibodies (Söderberg, page 999, ‘P-LISA reactions’, lines 1-16). Söderberg further teaches that the ligation were performed at 37°C for 30 min (at least 1 minute). As such, Söderberg teaches dissociation conditions of 37°C for at least 1 min comprising a reaction. Söderberg further teaches that the P-LISA method enables the localized detection of individual, endogenous, interacting protein pairs in fixed cultured cells, cytospin preparation, and tissue sections, thus providing an important new tool for use in research (Söderberg, page 995, 2nd column, 2nd paragraph, lines 23-26).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Weibrecht with the P-LISA method as taught by Söderberg, comprising the dissociating condition as taught by Söderberg because of the teaching of success of Söderberg of detecting individual, endogenous, interacting protein pairs in fixed cultured cells and tissue sections with the P-LISA method. One of ordinary skill in the art would have been motivated to do so because of the teaching of Söderberg that this technique provides an important new tool for research.
Claims 27 and 28 are rejected under 35 U.S.C. 103 as being unpatentable over Weibrecht in view of Lamvik and Vernay as applied to claim 25 above, and further in view of Morozova et al. Force differentiation in recognition of cross-reactive antigens by magnetic beads. Analytical biochemistry. 2008 Mar 15;374(2):263-71.
Regarding claims 27 and 28, Weibrecht and the cited art above teach a method of labeling a biological sample comprising primary antibody-oligonucleotide conjugates and a non-labeled secondary antibody substantially as claimed.
The combination of Weibrecht in view of Lamvik and Vernay further teaches that the labeled primary antibody is a monoclonal mouse IgG antibody (Lamvik, page 188, ‘Antibodies and Reagents, lines 1-4).
Weibrecht and the cited art above fails to teach that the primary antibody-oligonucleotide conjugates are exposed to a physical shearing stress by tethering to a rolling circle amplification product or bead.
Morozova teaches binding antibodies to the surface of magnetic beads through protein G, which binds IgG molecules via their Fc domain (Morozova, page 265, ‘Binding of IgG to magnetic beads’, lines 1-3). Morozova further teaches testing microarrays containing spots of nine albumins from sera of different mammals for their interaction with the magnetic beads functionalized with monoclonal antibodies (Morozova, page 263, ‘Abstract’, lines 3-5). Morozova further teaches subjecting the tethered beads to increasing shear flow (physical shearing stress (claim 27); by tethering to bead (claim 28)) in order to remove beads first from the weakest cross-reactive antigens and then from more strong ones in order to decrease the effect of cross-reactivity in the assay (Morozova, page 263, ‘Abstract’, lines 7-9). Morozova further teaches a critical shear rate and that the critical shear stress is determined as the stress at which 50% of the beads are removed in 15 s (Morozova, page 270, Figure 7, see figure legend). As such, Morozova teaches shear stress. Morozova further teaches that subjecting magnetic beads to increasing shear flow can effectively distinguish between closely related antigens by the strength of their binding to antibodies and that this detection technique may be used to rapidly evaluate cross-reactivity of antibodies, evaluate recombinant antibodies, and may further be used for ultra-rapid microarray-based immunoassays (Morozova, page 270, see ‘Conclusion’).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Weibrecht and the prior art by attaching the antibodies to magnetic beads and subjecting the tethered beads to shear stress as taught by Morozova, because of the teaching of Morozova that subjecting the bead bound antibody-antigen complexes to shear stress removes the beads first from the weakest cross-reactive antigens and that this method can effectively distinguish between closely related antigens. Put another way, subjecting the bound antibodies to shear stress avoids binding of the same antibody to two different, though related, antigens avoiding misidentification of closely related antigens.
The ordinary artisan would have a reasonable expectation of success in doing so because Weibrecht and the cited art above teaches a method of labeling a sample comprising IgG as primary antibody conjugated to oligonucleotides and Morozova teaches success applying shear stress to antigen bound IgG antibody with a method comprising the binding of antibody to beads via protein G which binds the Fc domain of the IgG primary antibody.
Claims 29-30, 32-33, and 35 are rejected under 35 U.S.C. 103 as being unpatentable over Weibrecht in view of Lamvik and Vernay as applied to claim 25 above, and further in view of Fredriksson et al. WO2022137047A1 (published 06/30/2022).
Regarding claim 29, Weibrecht and the cited art above teach a method of labeling a biological sample comprising primary antibody-oligonucleotide conjugates and a non-labeled secondary antibody substantially as claimed.
Even though Weibrecht teaches oligonucleotide tagged antibodies, Weibrecht does not specifically teach that each primary antibody is identified by a unique oligonucleotide identifier sequence.
Fredriksson teaches a method of a proximity assay comprising antibody-oligonucleotide conjugates (antibodies labeled with oligonucleotides comprising a unique identifier sequence) bound to cell surface markers. Fredriksson further teaches a capture agent that can be an antibody-oligonucleotide conjugate (see above) and that the sequence of an oligonucleotide that is conjugated to a binding agent uniquely identifies the epitope or sequence to which the binding agent binds, for example, if the method is performed using 10 different antibodies, then each antibody is tethered to a different sequence that identifies the epitope to which the antibody binds. This feature allows the method to be multiplexed and can result in at least 5 to at least 50 different antibodies binding to different markers in or on the surface of a cell. The unique identifier sequences allow the binding events for a particular antibody to be mapped (Fredriksson, pages 23, line 17- page 24, line 15). Fredriksson further teaches that the method can be used to analyze cells in suspension, e.g. immune cells isolated from a body fluid, blood or a tissue, or fixed tissues or tissue sections (Fredriksson, page 29, lines 4-6). Fredriksson further teaches that the method may comprise making a physical map of the immobilized particles using pairs of unique particle identifier sequences and mapping the binding agents to the physical map of the immobilized particles. The binding agents can be placed on the map of particles described above thereby providing a two or three dimensional map of the binding events, where the map may correspond to the surfaces of one or more cells (Fredriksson, page 25, line 22- page 26, line 8). Fredriksson further teaches that the method can be used to analyze cells in suspension or fixed tissues or tissue sections that have been immobilized on a surface (Fredriksson, page 29, lines 5-7).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Weibrecht and the prior art above comprising labeling antibodies with nucleotide sequences with the technique of Fredriksson using unique antibody identifier sequences for each antibody because of the teaching of Fredriksson that it allows the method to be multiplexed. The ordinary artisan would be motivated to do so by the teaching of Fredriksson that this allows mapping of individual antibodies to the surfaces of one or more cells.
The ordinary artisan would have a reasonable expectation of success, because Weibrecht teaches a method for labeling proteins in fixed tissues comprising antibodies labeled with oligonucleotide sequences and Fredriksson similarly teaches success labeling proteins in fixed tissues using antibodies comprising oligonucleotide labels for each antibody. Put another way, the ordinary artisan would have a reasonable expectation of success applying antibodies labeled with unique oligonucleotide sequences in the method of Weibrecht and Lamvik, because the methods differ not in type of label but only in the sequence of the oligonucleotide label.
Regarding claim 30, Weibrecht and the cited art above teach a method of labeling a biological sample comprising primary antibody-oligonucleotide conjugates and a non-labeled secondary antibody substantially as claimed.
Weibrecht teaches a method that comprises en masse consistent with the present claims, see the originally filed specification of the instant application on page 7, lines 8-10, the specification recites en masse is intended to mean labeling the sample with a plurality of primary antibody oligonucleotide conjugates that recognize different targets at the same time.
Weibrecht teaches a pair of oligonucleotide-labeled antibodies to target a protein of interest and binding of two such oligonucleotide-labeled antibodies to adjacent epitopes on one protein or two molecules in a complex (plurality of primary antibody-oligonucleotide conjugates that recognize different targets; Weibrecht, page 404, ‘Proximity ligation assay’, lines 13-21). As such Weibrecht teaches labeling a sample with a plurality of primary antibody oligonucleotide conjugates at the same time, i.e. Weibrecht teaches en masse labeling.
Weibrecht fails to teach sequencing at least the antibody identifier sequences in the nucleic acid reaction product.
As discussed previously in detail above, Fredriksson teaches a method of performing a proximity assay comprising antibody-oligonucleotide conjugates (Fredriksson, pages 23, line 17-22). Fredriksson further teaches performing a ligation, polymerization and/or gap-fill/ligation reaction and that the reaction products are sequenced and the sequences are analyzed to identify which pairs of unique particle identifier sequences or complements thereof have been copied and/or ligated together (Fredriksson, page 2, lines 6-11). Fredriksson further teaches that the bridging moieties have been extended to add the unique identifiers from adjacent barcoded particles, the extended bridging moieties are sequenced. As such, Fredriksson teaches sequencing at least the antibody identifiers (Fredriksson, page 22, lines 6-8). Fredriksson further teaches that in this method the spatial relationships between the barcoded particles are determined and the sites to which the capture agent binds are mapped (Fredriksson, page 10, paragraph [0099], lines 10-14). Fredriksson further teaches that one or more physical maps of the barcoded particles can be made using the identified pairs of sequences (Fredriksson, page 2, lines 11-12). Fredriksson further teaches that the spatial resolution of target proteins on the surface of each single cell can provide valuable diagnostic information (Fredriksson, page 30, lines 8-9).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Weibrecht and Lamvik with the method of Fredriksson of sequencing the reaction products to identify the unique particle identifiers in order to analyze which pairs of targets are ligated together. The ordinary artisan would have been motivated to do so because this allows for the creation of a physical map of the various barcoded particles which can provide valuable diagnostic information.
The ordinary artisan would have a reasonable expectation of success, because Weibrecht teaches a method for labeling proteins in fixed tissues comprising antibodies labeled with oligonucleotide sequences and Fredriksson similarly teaches success labeling proteins in fixed tissues using antibodies comprising oligonucleotide labels for each antibody. Therefore one would expect success when performing a ligation, polymerization and/or gap-fill/ligation reaction on the nucleotide labeled barcode and further would expect success in sequencing the reaction products.
Regarding claims 32 and 35, Weibrecht and the cited art above teach a method of labeling a biological sample comprising primary antibody-oligonucleotide conjugates and a non-labeled secondary antibody substantially as claimed.
Weibrecht does not teach a molecular reaction that is a hybridization, ligation, primer extension nor that detection of the reaction product is done by sequencing.
As discussed previously in detail above, Fredriksson teaches a method for making a physical map of a population of barcoded particles wherein the barcoded particles are uniquely barcoded by oligonucleotides (Fredriksson, ‘Abstract’, lines 1-3). Fredriksson further teaches that in this method a ligation, polymerization, and/or gap-fill/ligation reaction is performed (claim 32) thereby producing reaction products that comprise pairs of unique particle identifier sequences from adjacent barcoded particles, or complements thereof and that these reaction products are sequenced (claim 35) and the sequences are analyzed to identify which pairs of unique particle identifier sequences or complements thereof have been copied and /or ligated together. Then one or more physical maps of the barcoded particles can be made using the identified pairs of sequences (detecting (step (d)) comprises sequencing the product) and the sequences analyzed to identify which pairs of unique particle identifier sequences have been ligated together which can be used to make a physical map using the identified pairs of sequences (Fredriksson, page 2, lines 6-12). Fredriksson further teaches that the method may comprise making a physical map of the immobilized particles using pairs of unique particle identifier sequences and mapping the binding agents to the physical map of the immobilized particles. The binding agents can be placed on the map of particles described above thereby providing a two or three dimensional map of the binding events, where the map may correspond to the surfaces of one or more cells (Fredriksson, page 25, line 22- page 26, line 8). Fredriksson further teaches that the method can be used to analyze cells in suspension or fixed tissues or tissue sections that have been immobilized on a surface (Fredriksson, page 29, lines 3-7). Fredriksson further teaches that the spatial resolution of target proteins on the surface of each single cell can provide valuable diagnostic information and that the present method is capable of quantifying the abundance and relative positions of hundreds to thousands of cell surface markers on millions of immune cells (Fredriksson, page 30, line 8-19).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Weibrecht and Lamvik with the method of Fredriksson by performing ligation, polymerization, and/or gap-fill/ligation and then use sequencing in order to detect which products of this reaction are ligated together because the reaction products of each of these reactions comprise pairs of unique particle identifier sequences from adjacent barcoded particles (claim 32). The information gained, namely which barcoded particles are adjacent on the sample surface, for example a cell, can be used to make a physical map after the sequences are identified (see below).
It would have further been obvious to have modified the method of Weibrecht and Lamvik with the method of Fredriksson by sequencing the identifier sequences from adjacent barcoded particles because this allows for the identification of pairs of unique particle identifier sequences that have been ligated together which can be used to make a physical map using the pairs of sequences (claim 35), because of the teaching of Fredriksson that the spatial resolution of target proteins on the surface of each single cell can provide valuable diagnostic information.
The ordinary artisan would have a reasonable expectation of success, because Weibrecht teaches a method of labeling proteins in fixed tissue comprising oligonucleotide labeled antibodies and Fredriksson similarly teaches success labeling proteins in fixed tissues using an oligonucleotide-labeled antibody, both methods comprising oligonucleotide labeled antibodies and differing only in the sequence of the attached label. Therefore, the ordinary artisan would expect success labeling the antibodies of Söderberg with nucleotide comprising sequences as taught by Fredriksson which allow for multiplexing of the assay and further ligation, polymerization, and/or gap-fill/ligation and to be detected by sequencing as taught by Fredriksson.
Regarding claim 33, Weibrecht and the cited art above teach a method of labeling a biological sample comprising primary antibody-oligonucleotide conjugates and a non-labeled secondary antibody substantially as claimed.
Weibrecht does not teach a splinted ligation to a nucleic acid.
Fredriksson teaches a method for making a physical map of a population of barcoded particles wherein the barcoded particles are uniquely barcoded by oligonucleotides (Fredriksson, ‘Abstract’, lines 1-3). Fredriksson further teaches that the method may comprise making a physical map of the immobilized particles using pairs of unique particle identifier sequences and mapping the binding agents to the physical map of the immobilized particles. The binding agents can be placed on the map of particles described above thereby providing a two or three dimensional map of the binding events, where the map may correspond to the surfaces of one or more cells (Fredriksson, page 12, lines 16-24). Fredriksson further teaches a method that uses a bridging moiety as a splint. Fredriksson further teaches that the ligation product can be readily amplified and sequenced using forward and reverse primers. Fredriksson further teaches that after binding of the target-specific probes (i.e., the antibodies) to the sample, the barcoded particles are added to the sample, whereon type of particle may be pre-hybridized with the bridging moiety, which acts as a ligation splint and that a single ligation then ligates the particles together and ligates each target-specific oligonucleotide to a particle. The ligation products can be amplified by PCR, sequenced and analyzed to provide a map of barcoded particles as well as the targets that bind to those particles (Fredriksson, pages 35-36, see entire ‘Example 12’). Fredriksson further teaches that “splint” refers to an oligonucleotide that hybridizes to the ends of two other oligonucleotides and brings those ends together to produce a ligatable junction (Fredriksson, page 10, lines 21-22). Fredriksson further teaches that a splint mediated proximity ligation assay step ensures that the grid oligonucleotide will be a single molecule if two binding events to the same target molecule occur and that this increases the specificity of the assay (Fredriksson, page 34, lines 5-7).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Weibrecht with the method of Fredriksson of using a bridging moiety for a splinted ligation because a splint mediated proximity ligation assay step increases the specificity of the assay.
The ordinary artisan would have a reasonable expectation of success, because Weibrecht teaches a method of labeling proteins in fixed tissue comprising oligonucleotide labeled antibodies and Fredriksson similarly teaches success labeling proteins in fixed tissues using an oligonucleotide-labeled antibody, both methods comprising oligonucleotide labeled antibodies and differing only in the sequence of the attached label. The ordinary artisan would therefore have a reasonable expectation of success modifying the oligonucleotide labels with oligonucleotide labels comprising sequences as taught by Fredriksson that allow for splinted ligation because this would not change the principal of the assay as taught by Weibrecht, as both the assay of Weibrecht as well as Fredriksson teach an oligonucleotide labeled antibody and by modifying the nucleotide label one would expect to merely improve and not disrupt the detection of the nucleotide labeled antibody.
Claims 42, 46-47, and 49 are rejected under 35 U.S.C. 103 as being unpatentable over Weibrecht et al., in view of Lamvik et al., and Shahi et al. Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding. Scientific reports. 2017 Mar 14;7(1):44447.
Regarding claim 42¸ Weibrecht teaches proximity ligation assays as a method for detection of proteins or protein-protein interactions (Weibrecht, page 401, Abstract, lines 8-9). Weibrecht teaches that in studies aimed to detect a particular protein or protein-protein interaction the target molecule is typically labeled by using a specific affinity reagent or a direct label (labeling a sample; Weibrecht, page 402, ‘Probe-based targeting’, lines 2-8). Weibrecht further teaches an example of a probe based method, such as immunostaining which allows detection of endogenous proteins in situ, providing information at a single cell level and that the protein of interest is targeted by antibodies and then visualized by a reporter molecule bound to the antibodies. Weibrecht further teaches that to make intracellular proteins accessible to the antibodies, samples need to be fixed and permeabilized (sample has been crosslinked; Weibrecht, page 402, 2nd column, see entire 3rd paragraph). Weibrecht further teaches a proximity ligation assay, which combines a probe based assay with a split-reporter approach, where a pair of proximity probes, i.e. antibodies to which an oligonucleotide has been conjugated, is used to target the protein of interest (Weibrecht, page 404, ‘Proximity ligation assays, lines 10-16). Specifically, Weibrecht teaches cells or tissues fixed to a slide prior to addition of the proximity probes and further teaches that for localized detection of the protein complexes, oligonucleotides conjugated to proximity probes provide the template for hybridization and ligation of two subsequently added connector oligonucleotides. Successful ligation results in a circular DNA molecule (Weibrecht, page 404, 2nd column, 2nd paragraph, lines 3-8). The circular molecule can serve as a template for rolling circle amplification (Weibrecht, page 405, lines 1-3) the product of which can be visualized by hybridization of fluorescently labeled detection oligonucleotides to a sequence encoded in the DNA-circle and are easily distinguishable from potential background fluorescence in an epifluorescence microscope (detecting the product of c; Weibrecht, page 405, 2nd paragraph, lines 1-8).
Weibrecht fails to teach incubating the labeled sample with a secondary antibody that recognizes the primary antibody-oligonucleotide conjugates. Weibrecht further fails to teach that the one or more primary antibody-oligonucleotide conjugates each comprises an antibody identifier sequence that uniquely identifies the primary antibody to which it is conjugated.
Lamvik teaches staining peripheral blood mononuclear cells with antibodies specific for cell surface antigen with a second-step application of nonconjugated goat anti-mouse IgG antibodies (anti-species antibody; Lamvik, page 187, ‘Material and Methods’, lines 1-4). Lamvik further teaches that the labeled primary antibody were monoclonal mouse IgG antibodies (Lamvik, page 188, ‘Antibodies and Reagents’, lines 1-4). Lamvik further teaches that when cells are being stained with labeled antibodies followed by weak or no fixation, combined with permeabilization it appears that direct staining of the membrane antigens gives no or only weak fluorescence (Lamvik, page 187, lines 1-12). Lamvik further teaches that nonlabelled secondary antibody is able to influence the ability of primary, specific antibody to be or remain bound to cell membrane antigens, especially when cell membrane permeabilization techniques are applied in order to stain cytoplasmatic or nuclear components in addition to surface membrane staining (Lamvik, page 192, ‘Discussion’, lines 1-11). Lamvik further teaches that the observed effect of the secondary antibody may be explained by the formation of a network of primary and secondary antibodies (crosslinked; Lamvik, page 192, ‘Discussion’, 2nd paragraph, lines 1-3). Lamvik further teaches that with the nonlabelled antibody added, the staining of membrane antigens is clearly augmented (Lamvik, page 189, 2nd column, 2nd paragraph, lines 4-6). Lamvik further teaches that the application of nonlabelled secondary antibody in addition to specific primary antibodies may be useful in combined studies on surface membrane proteins and intracellular components when no or only weak fixatives are permitted (Lamvik, page 192, 2nd column, 5th column, line 10- page 193, line 2).
Shahi teaches a method to profile proteins in single cells that combines the speed of flow and mass cytometry with markedly increased sensitivity accuracy and multiplexing potential. Shahi teaches replacing the usual fluorophore or heavy metal tagged antibodies with DNA sequence tags that can be read out at the single-cell level using droplet microfluidic barcoding and DNA sequencing (Shahi, page 1, 3rd paragraph- page 2, line 7). Shahi further teaches that cells are bound with antibodies against different target epitopes as in conventional immunostaining, except that the antibodies are labeled with unique sequence tags (uniquely identifies the primary antibody; Shahi, page 2, 2nd paragraph, lines 2-3). Shahi further teaches that a modest tag length of ten bases provides over a million unique sequences, sufficient to label an antibody against every epitope in the human proteome (oligonucleotide label; Shahi, page 2, 1st paragraph, lines 2-4). Shahi further teaches that DNA tags afford a number of valuable advantages for labeling antibodies such as amplifying them from low levels to make them detectable. A further advantage, according to Shahi, is that the tags provide a combinatorial tag space sufficient to label an antibody against every epitope in the human proteome. Shahi further teaches that the accuracy, sensitivity, and essentially limitless multiplexing make it valuable for characterizing heterogeneous populations of single cells across biology (Shahi, page 1, 3rd paragraph- page 2, line 7).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Weibrecht comprising the binding of oligonucleotide labeled primary antibodies to label proteins in fixed cells or tissue with the method of Lamvik of crosslinking primary labeled antibodies bound to cells by applying nonlabelled secondary anti-species antibodies that crosslink the primary antibodies. The ordinary artisan would be motivated to do so because of the teaching of Lamvik that the nonlabelled secondary antibody augments binding of the primary antibody to cell membrane proteins in cells that are permeabilized in order to stain cytoplasmic or nuclear components and because of the teaching of Weibrecht and Lamvik that cells have to be permeabilized in order to detect intracellular or nuclear antigens and further because of the teaching of Lamvik that the technique may be useful in combined studies on surface membrane proteins and intracellular components when no or only weak fixatives are permitted.
It would have been further prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have applied the method of Shahi of added DNA-labeled antibodies in the method of Weibrecht comprising oligonucleotide probes to two different antigen because of the teaching of Shahi that the tags provide a combinatorial tag space sufficient to label an antibody against every epitope in the human proteome. One of ordinary skill in the art would be motivated to do so because of the teaching of Shahi that the accuracy, sensitivity, and essentially limitless multiplexing make it valuable for characterizing heterogeneous populations of single cells across biology.
One of ordinary skill in the art would have a reasonable expectation of success crosslinking the primary labeled antibody of Weibrecht with a secondary anti-species antibody as taught by Lamvik, because Lamvik teaches crosslinking primary labeled antibody with anti-species antibodies.
One of ordinary skill in the art would further have a reasonable expectation of success applying the method of Shahi to the method of Weibrecht because both Shahi and Weibrecht teach a method of labeling cells with oligonucleotide-tagged antibodies.
As such, one of ordinary skill in the art would have a reasonable expectation of success that modifying the method of Weibrecht by adding anti-species antibody, that is capable of binding all primary antibodies, because of the teaching of Lamvik that anti-species secondary antibodies crosslink the primary labeled antibodies and augment detection of cell surface antigen by primary antibody in permeabilized cells.
Regarding claim 46, Weibrecht and the cited art above teach a method of labeling a biological sample comprising primary antibody-oligonucleotide conjugates and a non-labeled secondary antibody substantially as claimed.
Weibrecht teaches a method that comprises en masse consistent with the present claims, see the originally filed specification of the instant application on page 7, lines 8-10, the specification recites en masse is intended to mean labeling the sample with a plurality of primary antibody oligonucleotide conjugates that recognize different targets at the same time.
Weibrecht teaches a pair of oligonucleotide-labeled antibodies to target a protein of interest and binding of two such oligonucleotide-labeled antibodies to adjacent epitopes on one protein or two molecules in a complex (plurality of primary antibody-oligonucleotide conjugates that recognize different targets; Weibrecht, page 404, ‘Proximity ligation assay’, lines 13-21). As such Weibrecht teaches labeling a sample with a plurality of primary antibody oligonucleotide conjugates at the same time, i.e. Weibrecht teaches en masse labeling.
Weibrecht fails to teach sequencing at least the antibody identifier sequences in the nucleic acid reaction product.
As discussed previously in detail above, Fredriksson teaches a method of performing a proximity assay comprising antibody-oligonucleotide conjugates (Fredriksson, pages 23, line 17-22). Fredriksson further teaches performing a ligation, polymerization and/or gap-fill/ligation reaction and that the reaction products are sequenced and the sequences are analyzed to identify which pairs of unique particle identifier sequences or complements thereof have been copied and/or ligated together (Fredriksson, page 2, lines 6-11). Fredriksson further teaches that the bridging moieties have been extended to add the unique identifiers from adjacent barcoded particles, the extended bridging moieties are sequenced. As such, Fredriksson teaches sequencing at least the antibody identifiers (Fredriksson, page 22, lines 6-8). Fredriksson further teaches that in this method the spatial relationships between the barcoded particles are determined and the sites to which the capture agent binds are mapped (Fredriksson, page 10, paragraph [0099], lines 10-14). Fredriksson further teaches that one or more physical maps of the barcoded particles can be made using the identified pairs of sequences (Fredriksson, page 2, lines 11-12). Fredriksson further teaches that the spatial resolution of target proteins on the surface of each single cell can provide valuable diagnostic information (Fredriksson, page 30, lines 8-9).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Weibrecht and Lamvik with the method of Fredriksson of sequencing the reaction products to identify the unique particle identifiers in order to analyze which pairs of targets are ligated together. The ordinary artisan would have been motivated to do so because this allows for the creation of a physical map of the various barcoded particles which can provide valuable diagnostic information.
Regarding claim 47¸ Weibrecht and the cited art above as applied to claim 42 also applies to claim 47.
Shahi teaches that to read out single cell protein expression the antibody tag sequences bound to cells are conjugated with unique cell barcode sequences via splicing by PCR and the chimeric products are pooled and sequenced (Shahi, page 2, figure legend, Figure 1, lines 2-5).
Claim 43 is rejected under 35 U.S.C. 103 as being unpatentable over Weibrecht in view of Lamvik and Vernay as applied to claim 42 above, and further in view of Söderberg et al.
Regarding claim 43, Weibrecht teaches a method for labeling a biological sample with oligonucleotide labeled antibodies and crosslinking the primary labeled antibodies with secondary antibodies essentially as claimed.
Weibrecht does not teach exposing the primary antibody-oligonucleotide conjugates to disassociating conditions while they are bound to the sample, the conditions comprising one or more reactions in at least 150 mM salt at 37°C for at least 1 min.
Söderberg teaches a P-LISA reaction comprising blocking the zinc-fixed cells before overnight incubation with proximity probes (primary antibody-oligonucleotide conjugates), followed by adding two connector oligonucleotide probes in a buffer comprising 250 mM NaCl (at least 150 mM salt; disassociating conditions while bound to the sample) and ligating the probes (one or more reactions) to form circles using as templates the two oligonucleotides attached to the antibodies (Söderberg, page 999, ‘P-LISA reactions’, lines 1-16). Söderberg further teaches that the ligation were performed at 37°C for 30 min (at least 1 minute). As such, Söderberg teaches dissociation conditions of 37°C for at least 1 min comprising a reaction. Söderberg further teaches that the P-LISA method enables the localized detection of individual, endogenous, interacting protein pairs in fixed cultured cells, cytospin preparation, and tissue sections, thus providing an important new tool for use in research (Söderberg, page 995, 2nd column, 2nd paragraph, lines 23-26).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Weibrecht with the P-LISA method as taught by Söderberg, comprising the dissociating condition as taught by Söderberg because of the teaching of success of Söderberg of detecting individual, endogenous, interacting protein pairs in fixed cultured cells and tissue sections with the P-LISA method. One of ordinary skill in the art would have been motivated to do so because of the teaching of Söderberg that this technique provides an important new tool for research.
Claims 44 and 45 are rejected under 35 U.S.C. 103 as being unpatentable over Weibrecht in view of Lamvik and Shahi as applied to claim 42 above, and further in view of Morozova et al. Force differentiation in recognition of cross-reactive antigens by magnetic beads. Analytical biochemistry. 2008 Mar 15;374(2):263-71.
Regarding claims 44 and 45, Weibrecht and the cited art above teach a method of labeling a biological sample comprising primary antibody-oligonucleotide conjugates and a non-labeled secondary antibody substantially as claimed.
Weibrecht in view of Lamvik further teaches that the labeled primary antibody is a monoclonal mouse IgG antibody (Lamvik, page 188, ‘Antibodies and Reagents, lines 1-4).
Weibrecht and the cited art above fails to teach that the primary antibody-oligonucleotide conjugates are exposed to a physical shearing stress by tethering to a rolling circle amplification product or bead.
Morozova teaches binding antibodies to the surface of magnetic beads through protein G, which binds IgG molecules via their Fc domain (Morozova, page 265, ‘Binding of IgG to magnetic beads’, lines 1-3). Morozova further teaches testing microarrays containing spots of nine albumins from sera of different mammals for their interaction with the magnetic beads functionalized with monoclonal antibodies (Morozova, page 263, ‘Abstract’, lines 3-5). Morozova further teaches subjecting the tethered beads to increasing shear flow (physical shearing stress (claim 27); by tethering to bead (claim 28)) in order to remove beads first from the weakest cross-reactive antigens and then from more strong ones in order to decrease the effect of cross-reactivity in the assay (Morozova, page 263, ‘Abstract’, lines 7-9). Morozova further teaches a critical shear rate and that the critical shear stress is determined as the stress at which 50% of the beads are removed in 15 s (Morozova, page 270, Figure 7, see figure legend). As such, Morozova teaches shear stress. Morozova further teaches that subjecting magnetic beads to increasing shear flow can effectively distinguish between closely related antigens by the strength of their binding to antibodies and that this detection technique may be used to rapidly evaluate cross-reactivity of antibodies, evaluate recombinant antibodies, and may further be used for ultra-rapid microarray-based immunoassays (Morozova, page 270, see ‘Conclusion’).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Weibrecht and Lamvik by attaching the antibodies to magnetic beads and subjecting the tethered beads to shear stress as taught by Morozova, because of the teaching of Morozova that subjecting the bead bound antibody-antigen complexes to shear stress removes the beads first from the weakest cross-reactive antigens and that this method can effectively distinguish between closely related antigens. Put another way, subjecting the bound antibodies to shear stress avoids binding of the same antibody to two different, though related, antigens avoiding misidentification of closely related antigens.
The ordinary artisan would have a reasonable expectation of success in doing so because Weibrecht and the cited art above teaches a method of labeling a sample comprising IgG as primary antibody conjugated to oligonucleotides and Morozova teaches success applying shear stress to antigen bound IgG antibody with a method comprising the binding of antibody to beads via protein G which binds the Fc domain of the IgG primary antibody.
Response to Arguments
Applicant’s arguments, see page 10, ‘Claim Rejections – 35 USC § 103’, lines 10-11 and ‘New claims 42-49’, lines 7-10, filed 12/22/2025, with respect to the rejection(s) of claim(s) 25-40 under 35 USC § 103 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of the teaching of Vernay et al., teaching the addition of additional secondary antibody to the sample.
Regarding the argument that AIA 35 U.S.C. 102(b)(2)(C) can be applied to the prior art of Fredriksson (US20240336959)) as applied to claims 29-30, 32-33, and 35, this argument is persuasive.
See new grounds of rejection above.
The amended claims include subject matter previously recited in claim 41. The limitation of claim 41, “wherein step (c) is done in the presence of additional secondary antibodies” was not disclosed in the prior filed application and does not receive the benefit of the earlier filing date. Therefore the effective filing date of amended claim 25 is 09/20/2023. The new grounds of rejection rely on Fredriksson et al. WO2022137047A1, which was published 06/30/2022. Therefore AIA 35 U.S.C. 102(b)(2)(C) does not apply to the new rejection of amended claim 25 and the dependent claims.
Claims 42-47 and 49 do not comprise the limitation of former claim 41 and are rejected over Weibrecht in view of Lamvik and Shahi.
Allowable Subject Matter
Claims 36 and 48 appear to be free of the prior art.
Communication
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/STEFANIE J. KIRWIN/Examiner, Art Unit 1677
/ELLEN J MARCSISIN/Primary Examiner, Art Unit 1677