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 .
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code on pg. 36 in reference (3). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Applicant is reminded of the proper language and format for an abstract of the disclosure.
The abstract should be in narrative form and generally limited to a single paragraph on a separate sheet within the range of 50 to 150 words in length. The abstract should describe the disclosure sufficiently to assist readers in deciding whether there is a need for consulting the full patent text for details.
The language should be clear and concise and should not repeat information given in the title. It should avoid using phrases which can be implied, such as, “The disclosure concerns,” “The disclosure defined by this invention,” “The disclosure describes,” etc. In addition, the form and legal phraseology often used in patent claims, such as “means” and “said,” should be avoided.
The abstract of the disclosure is objected to because it is too short (currently ~23 words) and contains the implicit phrase “This disclosure provides”. A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
Claim Objections
Claim 6 objected to because of the following informalities: in lines 3-4, “the second proximity protein” should read “the second proximity inducing protein” for consistency. 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.
Claim 8 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 8 recites the limitation “the second portion” in lines 1-2. There is insufficient antecedent basis for this limitation in the claim. In the interest of compact prosecution, Examiner interprets claim 8 to be dependent on claim 6 rather than claim 1.
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.
Claim(s) 1-3, 6, 17-19 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (Non-Patent Literature, J. Am. Chem. Soc., Vol. 143, pgs 16700-16708, 09/30/2021, as cited in the IDS submitted on 10/04/2024) in view of Ge et al. (Non-Patent Literature, Biochemical Society Transactions, Vol. 49, pgs. 2891-2901, 11/16/2021). For image clarity, figures captured from Wang et al. are sourced from the final published version of the article that is provided with this office action.
Regarding claim 1, Wang teaches a method of labeling a protein of interest (POI) with an acetyl group (abstract), comprising:
Contacting, in a sample (pg. 3 last paragraph) (we appended the previously reported FKBP12F36V-binding ligand to the… p300/CBP BRD inhibitor to generate a series of compounds, AceTAG 1-3, with different linker compositions):
a first fusion protein comprising an acetyl transferase (Figure 1a, p300/CBP) fused to a first proximity inducing protein (Figure 1a, Bromodomain),
a second proximity inducing protein comprising a protein of interest (Figure 1a, Protein of interest) fused to a second proximity inducing protein (Figure 1a, FKBP12F36V),
a proximity inducing ligand (Figure 1a, AceTAG),
incubating the sample for a duration sufficient for the first and second proximity inducing proteins to both to bind to the proximity inducing ligand and for the acetyl transferase to label the POI with an acetyl group (pg. 4 last paragraph and pg. 5 second paragraph) (confirmed increased H3.3-FKBP12F36V acetylation upon incubation of cells and we observed that induced acetylation occurs almost immediately).
See figure 1a:
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Wang is silent to a glycan being transferred to a POI via a glycan transferase protein.
In the analogous art of labelling proteins of interest with post translational modifications, Ge teaches a method of labelling a POI with a glycan using a glycan transferase, O-GlcNAc, (pg. 2893 first paragraph) (use various affinity molecules to write and erase other PTMs on target proteins in living cells [49–54], including ‘proteolysis targeting chimeras (PROTACs)’ for targeted protein degradation [55], have inspired the design of proximity-induced enzymatic reactions for engineering O-GlcNAc). Ge teaches that this is advantageous for studying the function of O-GlcNAc mediated post-translational modifications on proteins of interest (pg. 2893 first paragraph) (writing and erasing O-GlcNAc on a target protein in living cells is crucial for functional interrogation in the O-GlcNAc field). Accordingly, the prior art references teach that it is known that p300/CBP and O-GlcNAc are elements that are known in the art for providing posttranslational modifications by labelling proteins of interest.
It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to substitute the acetyl transferase protein (p300/CBP) to label a POI with an acetyl group as taught by Wang with a O-GlcNAc transferase to label a POI with a glycan as taught by Ge to lead to the predictable outcome of functional interrogation of the effects of O-GlcNAc modification on proteins of interest and because both elements were known equivalents for providing post translational modifications via labelling within the protein labelling art (see pgs. 3-5 and Figure 1a of Wang and pg. 2893 first paragraph of Ge).
Regarding claim 2, Modified Wang teaches the method of claim 1 as rejected above. Modified Wang teaches wherein the glycan transferase is O-GlcNAc (Ge, p. 2893, first paragraph).
Regarding claim 3, Modified Wang teaches the method of claim 1 as rejected above. Wang teaches wherein the first proximity inducing protein is bromodomain (BRD) (pg. 3 last paragraph) (we appended the previously reported FKBP12F36V-binding ligand to the… p300/CBP BRD inhibitor to generate a series of compounds, AceTAG 1-3, with different linker composition).
Wang does not teach wherein the first proximity inducing protein is a FKB3P12F36V. Wang teaches wherein the second proximity inducing protein is a FKB3P12F36V (pg. 3 last paragraph) (we appended the previously reported FKBP12F36V-binding ligand to the… p300/CBP BRD inhibitor to generate a series of compounds, AceTAG 1-3, with different linker compositions). As such, the proximity inducing ligands reported by Wang (AceTAG 1-3) are capable of simultaneously binding to both the first and second proximity inducing ligand. Furthermore, the proximity inducing proteins are installed on the desired proteins (proteins of interest or glycan transferases) by conjugation procedures known in the protein labelling art (see pg. 3 second and third paragraph).
It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to substitute the first proximity inducing protein of bromodomain with the second proximity inducing protein of FKB3P12F36V because they may both be appended to desired proteins and bind to the proximity inducing ligand with a reasonable expectation of success (see pg. 3 last paragraph and Figure 1 of Wang). See MPEP 2143(I)(B).
Regarding claim 6, Modified Wang teaches the method of claim 1 as rejected above. Wang teaches wherein the proximity inducing ligand comprises a first portion that is recognized and bound by the first proximity inducing protein and a second portion that is recognized an bound by the second proximity inducing protein (pg. 3 last paragraph) (we appended the previously reported FKBP12F36V-binding ligand to the… p300/CBP BRD inhibitor to generate a series of compounds, AceTAG 1-3, with different linker compositions). See also annotated Figure 1a:
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Regarding claim 17, Wang teaches a method of labeling a protein of interest (POI) with an acetyl group (abstract), comprising:
Contacting, in a sample (pg. 3 last paragraph) (we appended the previously reported FKBP12F36V-binding ligand to the… p300/CBP BRD inhibitor to generate a series of compounds, AceTAG 1-3, with different linker compositions):
a fusion protein comprising an acetyl transferase (Figure 1a, p300/CBP) fused to a proximity inducing protein (Figure 1a, Bromodomain),
a protein of interest (Figure 1a, Protein of interest),
with a proximity inducing ligand (Figure 1a, AceTAG),
incubating the sample for a duration sufficient for the first and second proximity inducing proteins to both to bind to the proximity inducing ligand and for the acetyl transferase to label the POI with an acetyl group (pg. 4 last paragraph and pg. 5 second paragraph) (confirmed increased H3.3-FKBP12F36V acetylation upon incubation of cells and we observed that induced acetylation occurs almost immediately).
See figure 1a:
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Wang is silent to a glycan being transferred to a POI via a glycan transferase protein.
In the analogous art of labelling proteins of interest with post translational modifications, Ge teaches a method of labelling a POI with a glycan using the glycan transferase, O-GlcNAc, (pg. 2893 first paragraph) (use various affinity molecules to write and erase other PTMs on target proteins in living cells [49–54], including ‘proteolysis targeting chimeras (PROTACs)’ for targeted protein degradation [55], have inspired the design of proximity-induced enzymatic reactions for engineering O-GlcNAc). Ge teaches that this is advantageous for studying the function of O-GlcNAc mediated post-translational modifications on proteins of interest (pg. 2893 first paragraph) (writing and erasing O-GlcNAc on a target protein in living cells is crucial for functional interrogation in the O-GlcNAc field). Accordingly, the prior art references teach that it is known that p300/CBP and O-GlcNAc are elements that are known in the art for providing posttranslational modifications by labelling proteins of interest.
It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to substitute the acetyl transferase protein (p300/CBP) to label a POI with an acetyl group as taught by Wang with a O-GlcNAc transferase to label a POI with a glycan as taught by Ge because both elements were known equivalents for providing post translational modifications via labelling within the protein labelling art (see pgs. 3-5 and Figure 1a of Wang and pg. 2893 first paragraph of Ge).
Regarding claim 18, Modified Wang teaches the method of claim 17 as rejected above. Modified Wang teaches wherein the glycan transferase is O-GlcNAc (Ge, pg. 2893, first paragraph).
Regarding claim 19, Modified Wang teaches the method of claim 17 as rejected above. Modified Wang teaches a proximity inducing protein comprising glycan transferase fused to a FKB3P12F36V (pg. 3 last paragraph) (we appended the previously reported FKBP12F36V-binding ligand to the… p300/CBP BRD inhibitor to generate a series of compounds, AceTAG 1-3, with different linker compositions). Wang teaches wherein another proximity inducing protein is bromodomain (BRD) (pg. 3 last paragraph) (we appended the previously reported FKBP12F36V-binding ligand to the… p300/CBP BRD inhibitor to generate a series of compounds, AceTAG 1-3, with different linker composition). As such, the proximity inducing ligands reported by Wang (AceTAG 1-3) are capable of simultaneously binding to both the first and second proximity inducing ligand. Furthermore, the proximity inducing proteins are installed on the desired proteins (proteins of interest or glycan transferases) by conjugation procedures known in the protein labelling art (see pg. 3 second and third paragraph).
Wang does not teach wherein the first proximity inducing protein, that is bound to glycan transferase, is a FKB3P12F36V.
It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to substitute the first proximity inducing protein of bromodomain with the second proximity inducing protein of FKB3P12F36V because they may both be appended to desired proteins and bind to the proximity inducing ligand with a reasonable expectation of success (see pg. 3 last paragraph and Figure 1 of Wang). See MPEP 2143(I)(B).
Claim 4 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (Non-Patent Literature, J. Am. Chem. Soc., Vol. 143, 2021, pgs 16700-16708, as cited in the IDS submitted on 10/04/2024) in view of Ge et al. (Non-Patent Literature, as cited in the IDS submitted on 10/04/2024) further in view of Los et al. (Non-Patent Literature, ACS chemical biology, Vol 3., No. 6, Pg. 373-382, 06/06/2008).
Regarding claim 4, Modified Wang teaches the method of claim 1 as rejected above. Wang teaches a method using a second proximity inducing protein comprising a protein of interest (Figure 1a, Protein of interest) fused to a second proximity inducing protein (Figure 1a, FKBP12F36V). Wang teaches that the second proximity inducing protein is designed to bind to a synthetic proximity inducing ligand (see Figure 1a and pg. 3 last paragraph) (we appended the previously reported FKBP12F36V-binding ligand to the… p300/CBP BRD inhibitor to generate a series of compounds, AceTAG 1-3, with different linker compositions).
Wang is silent to wherein the second proximity inducing protein is a Halotag.
In the analogous art of labelling proteins of interest, Los teaches a method of using a Halotag to covalently target and bind to synthetic ligands (abstract) (The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (Halo-Tag ligands)). Los teaches that this method is advantageous due to Halotag’s ability to bind to several types of synthetic molecules as well as its utility in imaging and studying protein-protein interactions (abstract) (The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles and the utility of this system for cellular imaging and protein immobilization by analyzing… capture of protein-protein).
It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to modify the second proximity inducing protein of Wang with the Halotag protein of Los because it would enable the usage of several types of synthetic proximity inducing ligands and the imaging of protein-protein interactions with a reasonable expectation of success (see pg. 3 last paragraph and figure 1a of Wang and abstract of Los). See MPEP 2143(I)(G). It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to substitute the second proximity inducing protein of Wang with the Halotag protein of Los because they may both be appended to desired proteins and bind to the proximity inducing ligand with a reasonable expectation of success. See MPEP 2143(I)(B).
Claim 5, 8, 10, 20, 22, 24, 26 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (Non-Patent Literature, J. Am. Chem. Soc., Vol. 143, 2021, pgs 16700-16708, as cited in the IDS submitted on 10/04/2024) in view of Ge et al. (Non-Patent Literature, as cited in the IDS submitted on 10/04/2024) as applied to claims 1, 6, or 17 above further in view of Raina et al. (Non-Patent Literature, bioRxiv, pgs. 1-26, 01/02/2023, as cited in the IDS submitted on 10/04/2024).
Regarding claim 5, modified Wang teaches the method of claim 1 as rejected above. Wang teaches a second proximity inducing protein comprising a protein of interest (Figure 1a, Protein of interest) fused to a second proximity inducing protein (Figure 1a, FKBP12F36V).
Wang does not specifically mention wherein the second proximity inducing protein is fused to the N-terminus or the C-terminus of the POI.
In the analogous art of inducing post-translational modifications on a protein of interest, Raina teaches a proximity inducing protein that is fused to the protein of interest (pg. 3 third paragraph) (To generate a model system, we engineered… Flag-tagged HaloTag7-FKBPF36V (hereafter HaloTag-FKBP) with a C-terminal P2A-EGFP sequence as target protein). Raina teaches that this approach allowed for both covalent and non-covalent interactions to induce the proximity of the target protein (TP) (pg. 3 third paragraph) (This allowed access to both covalent (via HaloTag) and non-covalent (via FKBP) means of recruiting the same TP).
It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to modify the second proximity inducing protein of Wang to be fused at the C-terminus as taught by Raina because it would allow for both covalent and non-covalent means of inducing the proximity of the protein of interest with a reasonable expectation of success (see Figure 1a and pg. 3 last paragraph of Wang and pg. 3 third paragraph of Raina).
Regarding claim 8, Modified Wang teaches the method of claim [6] as rejected above (recall that Examiner is interpreting claim 8 to be dependent on claim 6 and not claim 1). Wang teaches a proximity inducing ligand (AceTAG 1-3) that has a second portion that is a multicyclic heteroaromatic group, see annotated figure 1b:
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Wang is silent to wherein the second portion is a haloalkane.
In the analogous art of inducing post-translational modifications on a protein of interest, Raina teaches a method of using a series of proximity inducing ligands to bind to targeted proteins (abstract) (heterobifunctional small molecules called, Regulated Induced Proximity Targeting chimeras or RIPTACs, which elicit a stable ternary complex between a target protein… and a pan-expressed protein). Raina teaches wherein a portion of the ligand is a haloalkane, see annotated figure 2A below:
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Raina teaches that the Haloalkane portion of the molecule is advantageous for binding to certain proteins such as Halotag (pg. 4 third paragraph) (we confirmed the role of the TP in the observed viability effects by testing des-chloro RIPTAC negative controls (HLDA-120 and HLDA-110) that cannot bind HaloTag). See also supplementary Figure 4 for structures of the negative controls (HLDA-120 and HLDA-110) that lack a haloalkane and are ineffective at binding Halotag.
It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to modify the multicyclic heteroaromatic second portion of Wang with the haloalkane second portion of Raina because it would lead to binding to certain proteins such as Halotag with a reasonable expectation of success (See figure 1a and pg. 3 third paragraph of Wang and pg. 4 third paragraph, Figure 2A, and supplemental figure 4 of Raina).
Regarding claim 10, Modified Wang teaches the method of claim 6 as rejected above. Wang teaches wherein the proximity inducing ligand further comprises a chemical linker between the first portion and the second portion (pg. 3 last paragraph) (generate a series of compounds, AceTAG 1-3, with different linker compositions (Figure 1b and Figure S1a)). See also Figure 1b:
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Regarding claim 20, Modified Wang teaches the method of claim 17 as rejected above. Wang teaches a method of using a proximity inducing ligand comprising a first portion that recognizes and binds to a first proximity inducing protein and a second portion that binds to a second proximity inducing protein (see pg. 3 third paragraph and figure 1a).
Wang does not teach wherein second portion that is recognized and bound by the POI.
In the analogous art of inducing post-translational modifications on a protein of interest, Raina teaches a method of using a proximity inducing ligand (‘RIPTAC’) that comprises a second portion that binds to a protein of interest, for example, (abstract third paragraph) (we describe RIPTACs that incorporate a covalent or non-covalent target ligand connected via a linker to effector ligands such as JQ1 (BRD4), BI2536 (PLK1), or multi-CDK inhibitors such as TMX3013). Raina teaches that this approach is advantageous in the treatment of cancers that are known to selectively express a specific intracellular protein (abstract third paragraph) (therapeutic modality to treat cancers that are known to selectively express a specific intracellular protein).
It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to modify the second portion of the proximity inducing ligand that binds to a second proximity inducing protein as taught by Wang by instead using a second portion that binds to a protein of interest as taught by Raina because it would lead to treating cancers that are known to selectively express a specific intracellular protein with a reasonable expectation of success (see pg. 3 third paragraph of Wang and see abstract of Raina).
Regarding claim 22, Modified Wang teaches the method of claim 20 as rejected above. Wang teaches a method of using a proximity inducing ligand comprising a first portion that recognizes and binds to a first proximity inducing protein and a second portion that binds to a second proximity inducing protein (see pg. 3 third paragraph and figure 1a). As such, Modified Wang meets the limitations of claim 20 because they are optional.
In the interest of compact prosecution, this rejection is made in the alternative. Wang does not teach wherein the second portion is an optionally substituted BET bromodomain inhibitor (JQ 1), an optionally substituted EZH2 inhibitor, an optionally substituted BRD inhibitor, or an optionally substituted kinase inhibitor.
In the analogous art of inducing post-translational modifications on a protein of interest, Raina teaches a method of using a proximity inducing ligand (‘RIPTAC’) that comprises a second portion that is a substituted bromodomain inhibitor (JQ1) (abstract third paragraph) (we describe RIPTACs that incorporate… ligands such as JQ1 (BRD4), BI2536 (PLK1), or multi-CDK inhibitors such as TMX3013). Raina teaches that this approach is advantageous in the treatment of cancers that are known to selectively express a specific intracellular protein (abstract third paragraph) (therapeutic modality to treat cancers that are known to selectively express a specific intracellular protein).
It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to substitute the second portion of the proximity inducing ligand that binds to a second proximity inducing protein as taught by Wang by instead using a second portion that is a substituted bromodomain inhibitor (JQ1) as taught by Raina because both may be used to bind to a desired protein with a reasonable expectation of success.
Regarding claim 24, Modified Wang teaches the method of claim 20 as rejected above. Wang teaches wherein the proximity inducing ligand further comprises a chemical linker between the first portion and the second portion (pg. 3 last paragraph) (generate a series of compounds, AceTAG 1-3, with different linker compositions (Figure 1b and Figure S1a)). See also Figure 1b:
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Regarding claim 26, Modified Wang teaches the method of claim 17 as rejected above. Wang teaches a method of using a proximity inducing ligand (AceTAG-3) with an alkane linker that flanked by amide bonds (C=ONH) and FKBP12F36V and p300/CBP binding terminuses, see annotated figure 2A below:
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Wang is silent to wherein the structure of the proximity inducing ligand is:
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In the analogous art of inducing post-translational modifications on a protein of interest, Raina teaches a method of using a proximity inducing ligand (‘RIPTAC’) (abstract third paragraph) (we describe RIPTACs that incorporate… ligands such as JQ1 (BRD4), BI2536 (PLK1), or multi-CDK inhibitors such as TMX3013), comprising the claimed structure with a polyethylene glycol linker (HLDA-221,222,223), see modified figure 2A:
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Raina teaches that this method is advantageous because the resulting ternary complex between the proximity inducing ligand and targeted proteins is highly stable and may be observed several days after contacting the proximity inducing ligand and proteins (pg. 5 first paragraph) (cellular ternary complexes formed by RIPTACs were profoundly stable
by conducting a washout experiment… even with the non-covalent RIPTAC HLDA-222, were readily detectable even 72h following washout).
It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to substitute the p300/CBP binding terminus of Wang with the second binding terminus as taught by Raina because both binding terminuses may be used to bind to a proximity inducing protein with a reasonable expectation of success (see figure 2a and abstract of Wang, see abstract and figure 2A of Raina).
Claim 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (Non-Patent Literature, J. Am. Chem. Soc., Vol. 143, 2021, pgs 16700-16708, as cited in the IDS submitted on 10/04/2024) in view of Ge et al. (Non-Patent Literature, as cited in the IDS submitted on 10/04/2024) further in view of Hu et al. (Non-Patent Literature, Journal of the American Chemical Society, Vol. 145, pgs. 4045-4055, 02/08/2023).
Regarding claim 12, Wang teaches the method of claim 1 as rejected above. Wang teaches wherein the proximity inducing ligand is AceTAG 1-3 (pg. 3 last paragraph) (generate a series of compounds, AceTAG 1-3, with different linker compositions (Figure 1b and Figure S1a)). See also Figure 1b:
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Wang is silent to wherein the proximity inducing ligand is:
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Wherein n is an integer between 1 and 8.
In the analogous art of inducing post-translational modifications onto a target protein using proximity inducing ligands (abstract) (We sought to further develop our phosphorylation targeting chimera (PhosTAC) technology… PhosTACs induced the formation of a stable ternary complex), Hu teaches the proximity inducing ligand of claim 12 wherein n is 6 (PhosTAC7). See figure 1E left structure, where the differences between Wang’s reported proximity inducing ligand and Hu’s proximity inducing ligand are highlighted in red:
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Note that PhosTAC7 meets the limitations of the claimed structure; it is simply displayed in a different rotational conformation. Hu teaches that the PhosTAC7 ligand is advantageous in rapidly inducing the formation of the ternary protein-ligand-protein complex and the desired post-translational modification on a target protein (abstract) (PhosTACs induced the formation of a stable ternary complex, leading to rapid, efficient, and sustained tau dephosphorylation).
It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to substitute the p300/CBP binding terminus and linker of the proximity inducing ligand of Wang by instead using the polyethylene glycol linker and haloalkane binding terminus as taught by Hu because both binding terminuses and linkers may be used to form a ternary protein-ligand-protein complex and a desired post-translational modification on a target protein with a reasonable expectation of success (see pg. 3 last paragraph and Figure 1A of Wang and see abstract and figure 1E of Hu).
Claim 14 and 27 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (Non-Patent Literature, J. Am. Chem. Soc., Vol. 143, 2021, pgs 16700-16708, as cited in the IDS submitted on 10/04/2024) in view of Ge et al. (Non-Patent Literature, as cited in the IDS submitted on 10/04/2024) as applied to claims 1 or 17 above and further in view of Merrill et al. (Non-Patent Literature, Journal of Biological Chemistry, Vol. 294, No. 44, pgs. 16164-16171, 09/11/2019).
Regarding claim 14, Modified Wang teaches the method of claim 1 as rejected above. Wang measured the formation of the protein-protein-ligand-protein-protein complex (‘ternary complex’) using an AlphaScreen assay that evaluates protein turnover (pg. 3 fourth paragraph) (we assessed the ability of the AceTAG 1-3 to mediate complex formation between soluble recombinant FKBP12F36V and the… p300 (BRD-p300) in vitro using an AlphaScreen assay). See also figure 1c, which shows the formation of the ternary complex:
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Wang is silent to the step (c) of contacting the sample with a chase ligand that is recognized and bound by the second proximity protein.
In the analogous art of labelling targeted proteins, Merrill teaches a method of contacting the sample with a chase ligand that is recognized and bound by a second proximity protein (abstract) (we screened several inexpensive, low-molecular-weight haloalkanes as blocking agents in pulse-chase labeling experiments with the cell-permeable tetramethyl rhodamine HaloTag ligand). Merrill teaches that this method is advantageous because it provides an economical and non-toxic means of controlling when the formation of protein-ligand complexes with the proximity inducing ligand stops, leading to greater control of the reaction products formed during incubation (pg. 16169 left column first paragraph) (identified 7BRO as an economical HaloTag-blocking agent that can compete with commercially available HaloTag ligands attached to highly sensitive reporters… 7BRO chase is nontoxic and offers advantages over traditional methods of protein turnover determination). See also figure 1A:
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It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to modify the method of Wang by incorporating the additional step of contacting the sample with a chase ligand that is recognized and bound by a second proximity protein as taught by Merrill because it would provide an economical and non-toxic means of controlling when the formation of protein-ligand complexes with the proximity inducing ligand stops, leading to greater control of the reaction products formed during incubation with a reasonable expectation of success (see pg. 3 fourth paragraph and figure 1c of Wang; see abstract and pg. 16169 left column first paragraph and figure 1a of Merrill).
Regarding claim 27, modified Wang teaches the method of claim 17 as rejected above. Wang measured the formation of the protein-protein-ligand-protein-protein complex (‘ternary complex’) using an AlphaScreen assay that evaluates protein turnover (pg. 3 fourth paragraph) (we assessed the ability of the AceTAG 1-3 to mediate complex formation between soluble recombinant FKBP12F36V and the… p300 (BRD-p300) in vitro using an AlphaScreen assay). See also figure 1c, which shows the formation of the ternary complex:
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Wang is silent to the step (c) of contacting the sample with a chase ligand that is recognized and bound by the second proximity protein.
In the analogous art of labelling targeted proteins, Merrill teaches a method of contacting the sample with a chase ligand that is recognized and bound by a second proximity protein (abstract) (we screened several inexpensive, low-molecular-weight haloalkanes as blocking agents in pulse-chase labeling experiments with the cell-permeable tetramethyl rhodamine HaloTag ligand); see also figure 1A:
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Merrill teaches that this method is advantageous because it provides an economical and non-toxic means of controlling when the formation of protein-ligand complexes with the proximity inducing ligand stops, leading to greater control of the reaction products formed during incubation (pg. 16169 left column first paragraph) (identified 7BRO as an economical HaloTag-blocking agent that can compete with commercially available HaloTag ligands attached to highly sensitive reporters… 7BRO chase is nontoxic and offers advantages over traditional methods of protein turnover determination).
It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to modify the method of Wang by incorporating the additional step of contacting the sample with a chase ligand that is recognized and bound by a second proximity protein as taught by Merrill because it would provide an economical and non-toxic means of controlling when the formation of protein-ligand complexes with the proximity inducing ligand stops, leading to greater control of the reaction products formed during incubation with a reasonable expectation of success (see pg. 3 fourth paragraph and figure 1c of Wang; see abstract and pg. 16169 left column first paragraph and figure 1a of Merrill).
Claim 15-16 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (Non-Patent Literature, J. Am. Chem. Soc., Vol. 143, 2021, pgs 16700-16708, as cited in the IDS submitted on 10/04/2024) in view of Ge et al. (Non-Patent Literature, as cited in the IDS submitted on 10/04/2024) further in view of Merrill et al. (Non-Patent Literature, Journal of Biological Chemistry, Vol. 294, No. 44, pgs. 16164-16171, 09/11/2019) as applied to claim 14 above and further in view of Liu et al. (Non-Patent Literature, Biochemistry, Vol. 57, pgs. 4663-4674, 02/23/2018).
Regarding claim 15, Modified Wang teaches the method of claim 14 as rejected above. Modified Wang teaches of using chase ligands that do not have a detectable portion, but do have a portion that is recognized and bound by the second proximity protein (figure 1A of Merrill):
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Modified Wang clarifies that it is the haloalkane portion of the chase ligands that are recognized and bound by the second proximity inducing protein (Halotag) (pg. 16164 right column last paragraph of Merrill) (we identified a list of haloalkanes that may also interact with HaloTag).
Modified Wang is silent to wherein the chase ligand comprises a detectable portion and a portion that is recognized and bound by the second proximity protein.
In the analogous art of labelling proteins of interest (abstract) (we present a multicolor HaloTag-based sensor (named AgHalo) to visualize and quantify proteome stresses in live cells), Liu teaches a method of using a ligand (AgHalo) that recognizes and binds to a second proximity inducing protein (figure 2 legend) (AgHalo probes have three components: HaloTag warhead for bioorthogonal conjugation, extended linker to minimize background fluorescence upon conjugation, and environmentally sensitive fluorophores), see also figure 2A:
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Liu teaches that this method allows for two-color imaging of cells before/after labelling the protein as well as, for pulse-chase experiments, discerning changes to the cellular proteome in live cells (abstract) (enable two-color imaging, allowing for direct visualization of the AgHalo sensor both before and after cells are subjected to stress conditions… pulse−chase experiments can be performed to discern changes in the cellular proteome in live cells).
It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to modify the method of modified Wang by using chase ligands that contain both a detectable portion and a portion that recognizes and binds to a second proximity protein because it would lead to two-color imaging of cells before/after labelling the protein as well as, for pulse-chase experiments, discerning changes to the cellular proteome in live cells with a reasonable expectation of success (see figure 1a and pg. 16164 right column last paragraph of Merrill and see abstract and figure 2 of Liu).
Regarding claim 16, Modified Wang teaches the method of claim 14 as rejected above. Modified Wang teaches of using the following chase ligands (from figure 1A of Merrill):
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Modified Wang is silent to wherein the chase ligand is:
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In the analogous art of labelling proteins of interest (abstract), Liu teaches a method of using a chase ligand (‘AgHalo probe’) that is (from figure 2B):
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Liu teaches that this always-fluorescent chase ligand is advantageous to use as a chase ligand because it does not require radioactive metabolic labelling or translational attenuation to detect the chase ligand (pgs. 4670-4671, beginning on right column last paragraph of pg. 4670) (Classic pulse−chase experiments often involve inconvenient radioactive metabolic labeling or translational attenuation… AgHalo sensor exhibits technical advantages in pulse−chase experiments without the need for radioactive labeling or translational attenuation).
It would have been obvious for a person having ordinary skill in the art before the effective filing date of the instant application to modify the method of using the chase ligands of Modified Wang (Merrill Figure 1A structures) by instead using the always-fluorescent chase ligand of Liu because it does not require radioactive metabolic labelling or translational attenuation to detect the chase ligand (see figure 1A of Merrill and figure 2B and pgs. 4670-4671 of Liu).
Conclusion
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/H.D.C./Examiner, Art Unit 1758
/MARIS R KESSEL/Supervisory Patent Examiner, Art Unit 1758
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