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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 31 July 2025 has been entered.
Response to Amendment
Amendments filed on 31 July 2025 are acknowledged. Claims 81, 84-88, 90, 91, 99, and 100 are amended; claims 82, 98, and 99 are newly canceled; and claims 101 and 102 are newly added. The status marker of claim 84, which is withdrawn for being drawn to non-elected species of lysine trimethylation, fails to include "withdrawn" (refer to page 2 of non-final office action mailed on 09/27/2024). Claims 81, 83-93, 96, 97, and 100-102 are pending; claim 84, 96, and 97 are withdrawn; and claims 81, 83, 85-93, and 100-102 are examined herein on the merits.
In response to the amendments filed on 31 July 2025, the priority statement is changed; an objection to the specification is added; the rejections under 35 U.S.C. 112(a) are changed; the rejections under 35 U.S.C. 112(b) are withdrawn; and the rejections over the prior art are modified and supplemented.
Priority
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 application, Application No. 62/702318, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) for one or more claims of this application.
Independent claim 81 recites the following limitations:
(a) treating the peptide with a labeling reagent, thereby covalently coupling a label to the one or more amino acid residues to yield a labeled peptide;
… wherein the post-translational modification is selected from the group consisting of phosphorylation, glycosylation, and trimethylation and (1) the labeling reagent comprises a thiol group or (2) treating the peptide comprises using a cycloaddition reaction.
Dependent claim 90 recites that "said treating comprises chemically converting said post-translational modification into a reactive group, and coupling said labeling reagent with said reactive group."
Claim 91 depends from claim 90 and recites that "said chemically converting comprises eliminating said post-translational modification from said one or more amino acid residues, thereby generating said reactive group."
Claim 92 depends from claim 91 and recites that "said reactive group comprises an olefin."
Claim 93 depends from claim 91 and recites that "said eliminating comprising base- catalyzed phosphoryl elimination or silver(I) oxide catalyzed amine elimination."
The disclosure of 62/702318 fails to provide adequate support for the combination of the limitation that "the post-translational modification is selected from the group consisting of phosphorylation, glycosylation, and trimethylation" with the limitation "(2) treating the peptide comprises using a cycloaddition reaction." Moreover, the disclosure of 62/702318 fails to provide adequate support for the combination of the limitation "(2) treating the peptide comprises using a cycloaddition reaction" with the limitations of dependent claims 91-93.
The disclosure of 62/702318 contains two mentions of the word "cycloaddition." The first mention is in the specification ([0009], page 4):
In some embodiments, the reactive peptide or protein of interest is reacted with the labeling reagent through a cycloaddition reaction to form a labeled peptide or protein of interest.
The second mention of the word "cycloaddition" is in claim 36, which is as follows:
36. The method according to any one of claims 21-29 and 33, wherein the reactive peptide or protein of interest is reacted with the labeling reagent through a cycloaddition reaction to form a labeled peptide or protein of interest.
The limitation "the reactive peptide or protein of interest" of claim 36 of 62/702318 has antecedent basis in claim 21 of 62/702318:
(i) reacting the peptide or protein of interest under conditions such that the post translational modification on the peptide or protein of interest is converted to a reactive group to form a reactive peptide or protein of interest;
The "reactive peptide or protein" recited in claim 21 of 62/702318 is formed by elimination reaction conditions (claims 21-29 of 62/702318) or oxidation reaction conditions (claims 30-32 of 62/702318).
The disclosure of 62/702318 teaches that "the post translational modification on the amino acid is phosphorylation, glycosylation, or trimethylation" ([0006] of specification; claim 2).
The disclosure of 62/702318 describes an elimination reaction to convert a residue having a phosphorylation PTM ([0059]) or a to a residue having a trimethylation PTM ([0073]) to reactive group that is an olefin for further reaction with a labeling reagent comprising a thiol group. Accordingly, for the choice of the PTM being phosphorylation or trimethylation, the disclosure of 62/702318 supports limitation (1) that "the labeling reagent comprises a thiol group" and also supports dependent claims 90-92.
The disclosure of 62/702318 supports converting a residue having a glycosylation PTM to reactive group that is an aldehyde ([0064]) for further reaction with a dithiol labeling reagent ([0067]). Accordingly, for the choice of the PTM being glycosylation, the disclosure of 62/702318 supports limitation (1) that "the labeling reagent comprises a thiol group."
The disclosure of 62/702318 (and the disclosure of PCT/US2019/042998) does not disclose a cycloaddition reaction involving an olefin or an aldehyde as a cycloaddition reactant. Instead, an olefin reactive group undergoes a thiolene (i.e., thiol-ene) reaction with a thiol (claim 34) or olefin metathesis (claim 35), and an aldehyde reactive group reacts with a dithiol reagent to form a 1,3-dithiane ([0067]). The reaction between an aldehyde and a dithiol to form a 1,3-dithiane is not a cycloaddition reaction according to the customary meaning of cycloaddition.
The disclosure of 62/702318 does not provide any examples of a cycloaddition reaction between a reactive group and a labeling reagent.
Unlike the disclosure of PCT/US2019/042998, the disclosure of 62/702318 does not mention the terms "alkyne," "azide," or their variants. Accordingly, for the choice of the PTM being phosphorylation, glycosylation, or trimethylation (or any other PTM), the disclosure of 62/702318 does not support limitation (2) that "treating the peptide comprises using a cycloaddition reaction." Moreover, the disclosure of 62/702318 does not support combining limitation (2) with the limitations of claims 91-93.
Regarding claim 93, disclosure of 62/702318 does not contain the term "catalyzed" or a variant thereof. Moreover, it does not describe a sub-stoichiometric amount of base or silver oxide or otherwise support catalysis by base or silver oxide.
Therefore, the Applicants did not show possession of the claimed invention in the disclosure of 62/702318 by describing the claimed invention with all of its limitations using such descriptive means as words, structures, figures, diagrams, and formulas that fully set forth the claimed invention, by description of an actual reduction to practice, or by the disclosure of drawings or structural chemical formulas that show that the invention was complete, or by describing distinguishing identifying characteristics sufficient to show that the applicant was in possession of the claimed invention in the disclosure of 62/702318.
Accordingly, the effective filing date of claims 81, 83, 85-93, and 100-102 is 23 July 2019, the filing date of PCT/US2019/042998.
Specification
The specification is objected to as failing to provide proper antecedent basis for the claimed subject matter of claim 102. See 37 CFR 1.75(d)(1) and MPEP § 608.01(o). Correction of the following is required:
Claim 102 recites that "the acid comprises trifluoroacidic acid (TFA)." The acid of claim 102, as set forth in step (d) of claim 81, is an acid used to treat an N-terminal amino acid residue to remove the N-terminal amino acid residue from said immobilized peptide.
The specification lacks sufficient antecedent basis for the claimed limitation that TFA is an acid used in this step of removing the N-terminal amino acid. The only other mentions of TFA in the specification are for deprotection of protecting groups and for non-specific peptide cleavage (e.g., [0097], which is not described as an Edman degradation or other cleavage of the N-terminal amino acid residue).
Sufficient written description support for new claim 102 is found in US 9,625,469 (IDS 11/29/2023), which the instant specification incorporates by reference in a paragraph describing an Edman degradation cycle (see [00104] of specification and page 6 of Applicant's arguments filed on 31 July 2025, which cites support for claim 102).
The specification must be amended to provide antecedent basis for claim 102.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 81, 83, 85-93, and 100-102 are rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, at the time the application was filed, had possession of the claimed invention.
The examiner respectfully reminds the Applicant that according to MPEP §2163:
"2163.02. Standard for Determining Compliance with Written Description Requirement:
The courts have described the essential question to be addressed in a description requirement issue in a variety of ways. An objective standard for determining compliance with the written description requirement is, “does the description clearly allow persons of ordinary skill in the art to recognize that he or she invented what is claimed.” In re Gosteli, 872 F.2d 1008, 1012, 10 USPQ2d 1614, 1618 (Fed. Cir. 1989). Under Vas-Cath, Inc. v. Mahurkar, 935 F.2d 1555, 1563-64, 19 USPQ2d 1111, 1117 (Fed. Cir. 1991), to satisfy the written description requirement, an applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention, and that the invention, in that context, is whatever is now claimed. The test for sufficiency of support in a parent application is whether the disclosure of the application relied upon “reasonably conveys to the artisan that the inventor had possession at that time of the later claimed subject matter.” Ralston Purina Co. v. Far-Mar-Co., Inc., 772 F.2d 1570, 1575, 227 USPQ 177, 179 (Fed. Cir. 1985) (quoting In re Kaslow, 707 F.2d 1366, 1375, 217 USPQ 1089, 1096 (Fed. Cir. 1983)). Whenever the issue arises, the fundamental factual inquiry is whether the specification conveys with reasonable clarity to those skilled in the art that, as of the filing date sought, applicant was in possession of the invention as now claimed. See, e.g., Vas-Cath, Inc. v. Mahurkar, 935 F.2d 1555, 1563-64, 19 USPQ2d 1111, 1117 (Fed. Cir. 1991). An applicant shows possession of the claimed invention by describing the claimed invention with all of its limitations using such descriptive means as words, structures, figures, diagrams, and formulas that fully set forth the claimed invention. Lockwood v. American Airlines, Inc., 107 F.3d 1565, 1572, 41 USPQ2d 1961, 1966 (Fed. Cir. 1997). Possession may be shown in a variety of ways including description of an actual reduction to practice, or by showing that the invention was “ready for patenting” such as by the disclosure of drawings or structural chemical formulas that show that the invention was complete, or by describing distinguishing identifying characteristics sufficient to show that the applicant was in possession of the claimed invention. See, e.g., Pfaff v. Wells Elecs., Inc., 525 U.S. 55, 68, 119 S.Ct. 304, 312, 48 USPQ2d 1641, 1647 (1998); Regents of the University of California v. Eli Lilly, 119 F.3d 1559, 1568, 43 USPQ2d 1398, 1406 (Fed. Cir. 1997); Amgen, Inc. v. Chugai Pharmaceutical, 927 F.2d 1200, 1206, 18 USPQ2d 1016, 1021 (Fed. Cir. 1991) (one must define a compound by “whatever characteristics sufficiently distinguish it”).
(I)
Non-original independent claim 81 recites the following limitations:
(a) treating the peptide with a labeling reagent, thereby covalently coupling a label to the one or more amino acid residues to yield a labeled peptide;
… wherein the post-translational modification is selected from the group consisting of phosphorylation, glycosylation, and trimethylation and (1) the labeling reagent comprises a thiol group or (2) treating the peptide comprises using a cycloaddition reaction.
Non-original dependent claim 90 recites that "said treating comprises chemically converting said post-translational modification into a reactive group, and coupling said labeling reagent with said reactive group."
Non-original claim 91 depends from claim 90 and recites that "said chemically converting comprises eliminating said post-translational modification from said one or more amino acid residues, thereby generating said reactive group."
Non-original claim 92 depends from claim 91 and recites that "said reactive group comprises an olefin."
Non-original claim 93 depends from claim 91 and recites that "said eliminating comprising base […] phosphoryl elimination or silver(I) oxide […] amine elimination."
The combination of the limitation that "the post-translational modification is selected from the group consisting of phosphorylation, glycosylation, and trimethylation" with the limitation "(2) treating the peptide comprises using a cycloaddition reaction" does not comply with the written description requirement. Moreover, the combination of the limitation "(2) treating the peptide comprises using a cycloaddition reaction" with the limitations of dependent claims 91-93 does not comply with the written description requirement [regarding claim 93, refer also to section (II) below].
The original disclosure contains two mentions of the word "cycloaddition" within two identical, grammatically incorrect sentences: "In some embodiments, the reactive peptide or protein is treated with the labeling reagent comprising a cycloaddition reaction to form a labeled peptide or protein" ([0016]; [0051]; specification paragraph numbering is the same as those found in WO 2020/023488 A1). This sentence is unclear because a composition (the labeling reagent) cannot comprise a reaction. It is noted that the disclosure of provisional application 62/702318 contains a related, and grammatically correct, teaching in its specification ([0009], page 4):
In some embodiments, the reactive peptide or protein of interest is reacted with the labeling reagent through a cycloaddition reaction to form a labeled peptide or protein of interest.
However, the original disclosure of PCT/US2019/042998 does not clearly provide any examples or explanation of a cycloaddition reaction between a "reactive peptide or protein of interest" (or a reactive group) and a labeling reagent. At best, the original specification mentions three labeling steps that use alkyne or azide or a click reaction that are generic to not only a Huisgen cycloaddition reaction but also to non-cycloaddition reactions. Moreover, each of these labeling steps that mention alkyne or azide are in the context of specific PTMs that are outside the scope of the choice of "phosphorylation, glycosylation, and trimethylation" recited in the independent claim. These three mentions of alkyne or azide or a click reaction are discussed below.
Regarding cysteine S-nitrosylation, the specification teaches a two-step reaction, beginning with a non-fluorescing reagent comprising an "organophosphine group with terminal handles (alkyne, azide)" followed by a "fluorophore reaction to the terminal handle" ([00121]). While alkynes and azides would be understood by one of ordinary skill to undergo a Huisgen cycloaddition reaction with one another, the specification does not explicitly teach that both an alkyne handle and an azide handle is used. Instead, the same paragraph teaches that this is a "fluorophore conjugate addition," and conjugate addition is a non-cycloaddition mechanism.
Regarding citrullination, the specification teaches reaction with a phenylglyoxal reagent under acidic conditions, and that the phenylglyoxal reagent can "contain a handle (click handle) for subsequent reaction with a fluorophore" ([00126]). The specification does not describe whether the click handle is alkyne or azide and that the subsequent reaction is a Huisgen cycloaddition reaction. In fact, the only example of a click reaction provide by the specification is a "thiolene-click reaction" (i.e., a thiol-ene click reaction) ([0016]), which is a non-cycloaddition reaction.
Regarding sulfenylation, the specification teaches labeling with a 1,3-cyclohexanedione reagent derivative ([00127]) and that the "reagent can be two halves—one with an azide handle and the second with a fluorophore that specifically reacts with the linker" ([00128]). The specification does not describe whether the second halve is an alkyne and therefore the specific reaction is a Huisgen cycloaddition reaction.
Even if the above teachings regarding citrullination and sulfenylation, in combination with grammatically incorrect sentences of [0016] and [0051], support using a cycloaddition reaction to label residues that are citrullinated or sulfenylated, the original disclosure does not support using a cycloaddition reaction to label residues that are phosphorylated, glycosylated, or trimethylated. As set forth below, the specific teachings and working examples of the original disclosure regarding PTMs that are phosphorylation or trimethylation involve the labeling reagent comprising a thiol group and which do not involve a cycloaddition reaction.
Regarding phosphorylation, the specification teaches an initial elimination reaction using barium hydroxide and 4M sodium hydroxide, followed by a Michael addition reaction with a dye-thiol reagent ([0099], [00103]), which does not use a cycloaddition reaction. The original disclosure does not disclose a cycloaddition reaction involving an olefin as a cycloaddition reactant. Accordingly, for the choice of the PTM being phosphorylation, the original disclosure supports that "(1) the labeling reagent comprises a thiol group" and claims 91-92 but not "(2) treating the peptide comprises using a cycloaddition reaction." Furthermore, the original disclosure supports that said eliminating comprising base-promoted phosphoryl elimination [refer to section (II) below].
Regarding glycosylation, the specification teaches an initial oxidation step, followed by reaction with a dithiol reagent to form a 1,3-dithiane ([00111]). The reaction between an aldehyde and a dithiol to form a 1,3-dithiane is not a cycloaddition reaction according to the customary meaning of cycloaddition. The original disclosure does not disclose a cycloaddition reaction involving an aldehyde as a cycloaddition reactant. Accordingly, for the choice of the PTM being glycosylation, the original disclosure supports that "(1) the labeling reagent comprises a thiol group" but not "(2) treating the peptide comprises using a cycloaddition reaction."
Regarding lysine trimethylation, the specification teaches an initial Hofmann elimination reaction treated with heat and silver oxide or DIPEA, followed by reaction with a thiol-linked fluorophore ([00116]; Scheme 6), which does not use a cycloaddition reaction. The original disclosure does not disclose a cycloaddition reaction involving an olefin as a cycloaddition reactant. Accordingly, for the choice of the PTM being trimethylation, the original disclosure supports that "(1) the labeling reagent comprises a thiol group" and claims 91-92 but not "(2) treating the peptide comprises using a cycloaddition reaction." Furthermore, the original disclosure supports that said eliminating comprising silver(I) oxide-promoted amine elimination [refer to section (II) below].
Claims 83, 85-93, and 100-102 are also rejected for inheriting the deficiencies of the independent claim.
(II)
Non-original dependent claim 93 recites that "said eliminating comprising base-catalyzed phosphoryl elimination or silver(I) oxide catalyzed amine elimination."
The specification does not contain the term "catalyzed" or a variant thereof. The original disclosure does not describe a sub-stoichiometric amount of base or silver oxide. The two recitations of "catalyzed" in claim 93 are new matter.
The disclosed phosphoryl elimination promoted by barium hydroxide and sodium hydroxide converts hydroxide to water, and therefore is not is catalytic with respect to the base.
The disclosed Hofmann elimination promoted by silver oxide (Ag2O) includes no teaching that Ag2O acts as a catalyst, and conventional Hofmann elimination reactions involve the conversion of Ag2O to insoluble AgI.
Therefore, the Applicants did not show possession of the claimed invention by describing the claimed invention with all of its limitations using such descriptive means as words, structures, figures, diagrams, and formulas that fully set forth the claimed invention, by description of an actual reduction to practice, or by the disclosure of drawings or structural chemical formulas that show that the invention was complete, or by describing distinguishing identifying characteristics sufficient to show that the applicant was in possession of the claimed invention.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 81, 83, 85-92, and 100-102 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Swaminathan ("Highly parallel single-molecule identification of proteins in zeptomole-scale mixtures," Nature Biotechnology, 22 October 2018; newly cited).
As noted above in the "Priority" section, the effective filing date of the claims under examination is 23 July 2019, the filing date of PCT/US2019/042998. The prior art of Swaminathan was published before, but within a year of, the effective filing date of the claims. This reference appears to be co-authored by four joint inventors and five non-inventors.
Regarding claim 81, Swaminathan discloses a method of determining information regarding a post-translational modification of one or more amino acid residues within a peptide, wherein said post-translational modification comprises phosphorylation (phosphoserines, abstract, Fig. 6), the method comprising:
(a) treating the peptide with a labeling reagent, thereby covalently coupling a label to the one or more amino acid residues to yield a labeled peptide (Fig. 6a; page 1081, third para.);
(b) immobilizing said labeled peptide to a support to obtain an immobilized peptide (abstract; Fig. 6b; page 1081, third para.);
(c) detecting said label (ibid.);
(d) removing an amino acid residue from said immobilized peptide (ibid.) by treating the N-terminal amino acid residue with an acid (trifluoroacetic acid, Fig. 1b) or heat (40° C, Fig. 1b); and
(e) repeating (c) and (d) at least one time, thereby generating information regarding said post-translational modification (ibid.), wherein said post-translational modification is phosphorylation (phosphoserines, abstract, Fig. 6) and (1) the labeling reagent comprises a thiol group (Fig. 6a; page 1081, third para.).
Regarding claim 83, Swaminathan discloses that said detecting is at a single molecule level (title, abstract).
Regarding claim 85, Swaminathan discloses that said information regarding said post-translational modification comprises an identity of said post-translational modification ("we distinguished specific phosphoserine post-translational modifications by sequencing," page 1077, right col, first sentence).
Regarding claim 86, Swaminathan discloses that said information regarding said post-translational modification comprises one or more positions of said one or more amino acid residues within said peptide ("We demonstrated identification of the specific amino acid positions of phosphoserine residues at single-molecule sensitivity," page 1081, third para.).
Regarding claim 87, Swaminathan discloses that said information regarding said post-translational modification comprises information of an absence of a second post-translational modification in said peptide ("Analysis of the peptides YpS°PTSPSK and YSPTpS°PSK (where ° indicates Atto647N coupled at phosphoserine residues) clearly discriminated serine phosphorylation sites," where both of these peptides include non-phosphorylated serine residues that are not labeled; page 1081, third para.)
Regarding claim 88, Swaminathan discloses that said information of said absence of said second post-translational modification comprises a position of said absence of said second post-translational modification (see claim 87, where the cycle number of the sequencing method indicates position).
Regarding claim 89, Swaminathan discloses that (e) further comprises identifying a sequence of said peptide (page 1081, second para.).
Regarding claims 90-92, Swaminathan discloses that said treating comprises chemically converting said post-translational modification into a reactive group, and coupling said labeling reagent with said reactive group, where chemically converting comprises eliminating said post-translational modification from said one or more amino acid residues, thereby generating said reactive group that comprises an olefin (Fig. 6a).
Regarding claim 100, Swaminathan discloses further comprising labeling at least one amino acid residue of the peptide that is not modified by a post-translational modification with a second labeling reagent (labeling cysteine and lysine, abstract).
Regarding claim 101, Swaminathan discloses that an initial position of the label is not at the N-terminus (Fig. 6b).
Regarding claim 102, Swaminathan discloses that the acid comprises trifluoroacetic acid (Fig. 1b).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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 81-83, 85-92, 99, and 100-102 are rejected under 35 U.S.C. 103 as being unpatentable over Hesselberth (US 2015/0087526; IDS of 09/07/2022; previously relied upon) in view of Stevens (US 2006/0099604; previously relied upon).
Regarding claim 81, Hesselberth discloses a method of determining information regarding a post-translational modification of one or more amino acid residues within a peptide, wherein said post-translational modification comprises phosphorylation ([0118], [0133], [0134], [0152]), the method comprising:
(a) treating the peptide with a labeling reagent, thereby covalently coupling a label to the one or more amino acid residues to yield a labeled peptide ("differential labeling of amino acids of a peptide," [0024], [0083], claim 43 step c);
(b) immobilizing said labeled peptide to a support to obtain an immobilized peptide ("attachment of a peptide to a surface," [0024], [0061], [0083], claim 43 step d, Fig. 8C);
(c) detecting said label ("imaging of a peptide by single molecule detection," [0024], [0083], claim 43 steps e and g);
(d) removing an N-terminal amino acid residue from said immobilized peptide (cleavage of a peptide by Edman degradation, which results in the lost of at least one labeled amino acid residue from the N-terminus of each peptide per cycle, [0024], [0083], [0088], claim 43 step f) by treating the N-terminal amino acid residue with an acid (strong acid, e.g. 25% trifluoroacetic acid, [0015]) or heat (50° C, [0015], 75° C, [0158], 70° C, [0174]); and
(e) repeating (c) and (d) at least one time (Fig. 3B, [0083], [0092], claim 43 step h), thereby generating information regarding said post-translational modification (claim 43 step m) wherein said post-translational modification is selected from phosphorylation and glycosylation phosphorylation ([0010], [0118], [0134], [0152]).
Hesselberth does not disclose that (1) the labeling reagent comprises a thiol group [or (2) treating the peptide comprises using a cycloaddition reaction], instead disclosing a different means of labeling the position of a phosphoryl group with a fluorescent label: reaction of a phosphoryl group with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, imidazole, and an amine-containing fluorophore ([0134], [0152], claim 45). In the case of the PTM being glycosylation, Hesselberth discloses that the sugar is oxidized and reacted with a hydrazide fluorophore ([0118], [0134], [0152], claim 44), which would be a condensation reaction to form a hydrazone.
In the analogous prior art of identifying phosphoryl groups in peptides (abstract), Stevens discloses labeling the position of a phosphoryl group using a tag having a thiol group (cysteamine moiety) attached to a variety of fluorescent moieties, including rhodamine ([0014], [0026]-[0028]). Stevens teaches that modifying phosphoserine and phosphothreonine residues by beta-elimination followed by nucleophilic attack is well known in the art, and that thiols are frequently the preferred nucleophile ([0009]). The thiol tags of Stevens are introduced by beta-elimination of the phosphate followed by Michael addition of the thiol tag ([0033], [0034]).
The selection of a known material, which is based upon its suitability for the intended use, is within the ambit of one of ordinary skill in the art. See In re Leshin, 125 USPQ 416 (CCPA 1960) (see MPEP § 2144.07). Applying a known technique to a known method ready for improvement to yield predictable results is likely to be obvious. See KSR International Co. v. Teleflex Inc., 550 U.S. __,__, 82 USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, D.).
For the benefit of using a known chemical derivatization approach for phosphorylation mapping that is flexible for use with a variety of fluorescent labeling reagents, it would have been obvious to one of ordinary skill in the art before the time of filing to modify the method of Hesselberth by replacing the EDC/imidazole method of derivatizing phosphorylation with the beta-elimination/Michael addition method of derivatizing phosphorylation disclosed by Stevens.
Regarding claim 83, Hesselberth discloses that said detecting is at a single molecule level (Fig. 3A, [0024], [0083]).
Regarding claim 85, Hesselberth discloses that said information regarding said post-translational modification comprises an identity of said post-translational modification (implicit to disclosed selective modification of phosphorylation, [0134], [0152]).
Regarding claim 86, Hesselberth discloses generating information regarding a position of a labeled amino acid residue ([0089], [0090], [0092]) and discloses labeling post-translational modifications ([0134], [0152]). Hesselberth does not explicitly disclose that the position refers to one of said one or more amino acid residues that have the post-translational modification. Given that Hesselberth discloses "(c) labeling said peptide such that a post-translational modification, if present, is labeled in a manner distinct from the labeling of any amino acid side chain" ([0133]), the type of detectable moiety that labels the post-translational modification is capable of being used to determine residue position, as set forth for the disclosed first detectable moiety and second detectable moiety (Fig. 3B). For the benefit of more fully characterizing the peptide, it would have been obvious to one of ordinary skill in the art before the time of filing to modify the method of Hesselberth such that that said information regarding said post-translational modification comprises a position of said amino acid residue comprising said post-translational modification within said peptide.
Regarding claims 87 and 88, Hesselberth discloses that said information regarding said post-translational modification comprises information of an absence ([0133]) of a second post- translational modification (glycosylation, [0134], [0152]) in said peptide, and said information of said absence of said second post-translational modification comprises a position of said absence of said second post-translational modification ([0133], where step m of "determining the … absence of a post-translational modification of said peptide" is a determination of said absence at every position of the peptide).
Regarding claim 89, Hesselberth discloses that (e) further comprises identifying a sequence of said peptide ([0089]).
Regarding claims 90-92, Stevens discloses labeling the position of a phosphoryl group via a beta-elimination/Michael addition procedure that eliminates said post-translational modification from said amino acid (beta-elimination, [0033]), thereby generating said reactive group, wherein said reactive group comprises an olefin (as required for Michael addition, [0033]).
Regarding claim 99, Hesselberth discloses that said immobilizing said peptide is subsequent to said coupling said treating ([0094]).
Regarding claim 100, Hesselberth further discloses labeling at least one amino acid residue of a peptide that is not modified by post-translational modification (the method performed on a plurality of peptides, [0135], and post translational modifications can be determined to be either present or absent, [0133]) with a second labeling reagent (the second detectable moiety, [0135]).
Regarding claim 101, Hesselberth discloses that an initial position of the label is not at the N-terminus ([0092]).
Regarding claim 102, Hesselberth discloses that the acid comprises trifluoroacetic acid (25% trifluoroacetic acid, [0015]).
Response to Arguments
Applicant's arguments filed on 31 July 2025 have been considered and are not fully persuasive and/or are moot in view of the new grounds of rejection.
A misstatement is noted on page 3 of the final office action regarding the now-canceled claim 82. The following statement is incorrect: "The disclosure of 62/702318 does not support a PTM of nitrosylation, citrullination, sulfenylation..." In [0037], page 14 of the specification of 62/702318, nitrosylation and sulfenylation are in fact mentioned as PTMs. However, the disclosure of 62/702318 does not mention citrullination, so the effective filing date of now-canceled claim 82 set forth in the final office action is accurate. Moreover, the disclosure of 62/702318 does not provide written description support for the combination of nitrosylation or sulfenylation with either limitation (1) or limitation (2) recited in claim 81. [The disclosure of PCT/US2019/042998 provides sufficient written description support for the combination of nitrosylation or sulfenylation with limitation (2) but does not provide written description support for the combination of nitrosylation or sulfenylation with limitation (1). The rejections under 35 USC 112(a) cite passages of the specification that support combining the choice of nitrosylation or sulfenylation with "using a cycloaddition reaction."]
Regarding claim 84, which recites "wherein said information regarding said post-translational modification comprises an identity of said amino acid residue," Applicant states that this reads upon the elected species and requests that claim 84 be examined on the merits.
In response, claim 84 recites "wherein said information regarding said post-translational modification comprises an identity of said one or more amino acid residues," where said one or more amino acid residues of claim 81 has a post-translational modification. The original disclosure does explicitly describe obtaining an identify of an amino acid residue that has a post-translational modification. However, sufficient written description support for claim 84 is found in at least the embodiment of identification of lysine trimethylation. The disclosed method using Hofmann elimination chemistry identifies trimethylated lysines and therefore generates "lysine" as an identify of an amino acid residue that comprises a post-translational modification. This embodiment is drawn to non-elected species F of trimethylation.
Regarding the elected species A of phosphorylation, the original disclosure provides a working example of "identifying phosphorylated residues" in a method by which both phosphorylated serines and threonines in solution are labeled after the phosphorylated residues are converted to the beta-eliminated variants using Ba(OH)2 ([0127] of published application). This disclosed technique implicitly provides information about an identify of the amino acid residue by limiting its possible identities (phosphorylated serine residue or phosphorylated threonine residue). However, providing information about an identity is broader than providing an identity itself. The disclosed technique is not an implicit disclosure of generating "an identity of said one or more amino acid residues" because the it does not distinguish between modified serine and threonine residues. Accordingly, claim 84 is not drawn to the elected species.
Applicant traverses the rejections for obviousness over Hesselberth in view of Stevens. Noting the claim 81 limitation that step (d) of claim 81 is performed with an acid, microwave irradiation, or heat, Applicant argues the following (bolding added):
[…] a person of ordinary skill in the art would not have expected to succeed in developing the claimed methods, based on the disclosures of Hesselberth and Stevens 2006 […] A person of ordinary skill in the art could not assume that labels introduced via thiol-conjugation as described in Stevens 2006 would be stable enough to withstand conditions used to remove amino acid residues repeatedly and to allow detection in subsequent rounds of detecting the label […] Stevens 2006 provides no indication that using thiol-containing reagents as nucleophiles in a beta-elimination/Michael addition reaction would ultimately result in a label which is sufficiently stable enough to be suitable for a method which requires repeatedly detecting a label and removing an N-terminal amino acid residue from an immobilized peptide by treating with an acid, microwave irradiation, or heat.
This argument is not persuasive.
First, the thiol-conjugation step (beta-elimination/Michael addition of thiol nucleophile) itself corresponds to step (a) of claim 81 and must be performed before step (d), which uses an acid, microwave irradiation, or heat, and before at least one iteration of step (c). What is important for stability concerns is not the labeling method per se, but the nature of the resulting product (the labeled peptide).
Second, the broadly recited step (c) of "detecting said label," which specifies neither a detection method nor a label type, does not invoke any stability concerns for a reasonable expectation of success. Applicant has not specifically argued that the detecting conditions of Hesselberth would have been expected by one of ordinary skill to be unsuccessful when combined with the labeling method of Stevens.
Third, while the product of the thiol conjugation of Stevens (the labeled peptide) must have a reasonable expectation to be compatible with the heat or acid disclosed by Hesselberth as conditions for Edman degradation, this product would be predicted by one of ordinary skill to be sufficiently stable. The product of the thiol conjugation of Stevens is a thioether, which is the same functional group found in the side chain of the amino acid methionine. Peptides containing methionine residue(s) are compatible with the heat and acid of Edman degradation, and in fact Hesselberth discloses methionine as a potential amino acid. Applicant has not cited any evidence or scientific reasoning regarding why one of ordinary skill in the art would doubt the stability of the product of the thiol conjugation (i.e., a thioether) under either the Edman degradation conditions or the detection conditions of Hesselberth. Regarding the remaining non-thioether portion of the label (the detectable fluorophore), Hesselberth teaches the necessity of fluorophore stability through multiple cycles of Edman chemistry ([0101]-[0104]).
Applicant further presents arguments regarding the related prior art disclosure of Stevens 2005 ("Enhancement of phosphoprotein analysis using a fluorescent affinity tag and mass spectrometry," Rapid Commun Mass Spectrom. 2005; IDS) (bolding added):
Stevens 2005 also discusses the same labeling strategy as disclosed in Stevens 2006 (use of cysteamines as the nucleophile in a beta-elimination/Michael addition reaction) and cautions that the labeling strategy could fail when phosphoresidues are only a few amino acids apart, such as may often occur within amino acid residues of the same peptide:
"There are some limitations associated with this labeling strategy... We have also found that the labeling efficiency of phosphorylated peptides in which the phosphoresidues are in close proximity is affected due to chemical factors such as steric hindrance."
Given what Stevens 2005 described as labeling efficiency issues in conditions of closely spaced phosphoresidues (such as may occur within the same peptide), a person of ordinary skill would not have expected a beta-elimination/Michael addition reaction with a cysteamine as the nucleophile would be suitable for "a method of determining information regarding a post-translational modification of one or more amino acid residues within a peptide," as recited by the present claims.
First, the rejections do not rely upon Stevens 2005.
Second, the next sentence in the passage of Stevens 2005 quoted by Applicant is "Future work will include enhancements to the FAT-labeling method to address these issues."
Third, Stevens 2005 itself is an enabled disclosure of "a method of determining information regarding a post-translational modification of one or more amino acid residues within a peptide." The enablement of the disclosure of Stevens 2005, as well as the enablement of the claimed invention, does not require a particular minimum labeling efficiency for every possible peptide sequence. A method comprising a labeling method can be enabled even if the labeling step proceeds with less than 100% yield. Stevens 2005 merely teaches that the labeling efficiency of phosphorylated peptides in which the phosphoresidues are in close proximity "is affected" and does not teach the failure or unsuitability of the labeling strategy for such peptides. Moreover, the claimed invention does not require that the peptide has plural PTM amino acid residues, let alone two PTM amino acid residues within a given position of each other.
The steric hindrance affect discussed by Stevens 2005 for the special case of phosphoresidues in close proximity to each other would have been expected by one of ordinary skill to be a potential occurrence for any labeling method that uses sterically large labels. The claimed invention does not limit the recited "label" in any way, including steric factors. Stevens 2005 does not compare the labeling efficiency of (i) beta-elimination and coupling with thiol-containing fluorophores to (ii) EDC activation and coupling with amine-containing fluorophores, as disclosed by Hesselberth. Applicant has not provided a reasoned argument or evidence to suggest that one of ordinary skill in the art would have expected, for a given fluorophore, that Steven's labeling method of beta-elimination and coupling with a thiol-containing fluorophore would have a worse labeling efficiency for closely spaced phosphoresidues than Hesselberth's labeling method of EDC activation and coupling with an amine-containing fluorophore.
As further shown by the prior art of Peters (US 2006/0263886; newly cited), both methods of (i) beta-elimination and coupling with a thiol-containing label (Figs. 2A and 2B; [0021], [0161]) and (ii) EDC activation and coupling with an amine-containing label (Fig. 5, [0022], [0234]) are known to be suitable for labeling phosphorylated amino acid residues in a method of determining information regarding a post-translational modification of one or more amino acid residues within a peptide. Peters lists the beta-elimination/Michael addition method first and describes the EDC-mediated coupling as an "Alternative Approach" (Example 17).
Applicant further argues that the "present application has overcome challenges of the prior art." However, Applicant does not clearly identify the "challenges of the prior art."1 Establishing long-felt need requires objective evidence that an art-recognized problem existed in the art for a long period of time without solution. See MPEP 716.04.
Applicant further argues that "the present application demonstrates that phosphoresidues separated by only a few positions can be labeled and accurately detected," citing a working example of labeling and fluorosequencing the peptide YpSPTpSPS. However, the instant specification makes no comment regarding the efficiency of labeling this peptide, and "labeling efficiency" is what Steven 2005 teaches is "affected." Moreover, it is not clear whether this peptide, which has phosphoserine residues separated from each other by two non-phosphorylated residues, would qualify as having phosphoresidues in "close proximity" as the phrase is used by Stevens 2005. The claimed invention itself does not require that the peptide has plural PTM amino acid residues, let alone two PTM amino acid residues within a given position of each other.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to MICHELLE ADAMS whose telephone number is (571)270-5043. The examiner can normally be reached M, T, Th, and F, 12-4 P.M.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Lyle Alexander can be reached on (571) 272-1254. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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