DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of the Application
Receipt is acknowledged of Applicants’ Remarks, filed on 06/11/2026. No amendments were made. Claims 1-13 and 15-20 are pending.
In the Office Action of 03/12/2026, claim 10-13 and 15-18 were withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 1-9 and 19-20 are examined on the merits herein.
Priority
The instant application is a 371 of PCT/EP2020/087108 filed 12/18/2020, which claims foreign priority to EP 19218587.4, filed on 12/20/2019.
No rejections are withdrawn.
The following are maintained grounds of rejection.
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.
Claims 1-8 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Martos-Maldonado et al. (Nature Communications, 2018; IDS 10/03/2022) in view of Chaveriat et al. (Tetrahedron, 2006; IDS 10/03/2022).
Martos-Maldonado teaches that polyhistidine tags (His tags) are widely used for protein purification by immobilized-metal ion affinity chromatography (page 2, paragraph 3). Martos-Maldonado discloses methods for highly selective N-terminal acylation of proteins and identifies an optimized N-terminal sequence, the His tag GHHHn−, for the reaction with either D-gluconic acid δ-lactone (GDL) or a 4-methoxyphenyl ester as an acylating agent (abstract).
First, Martos-Maldonado teaches acylation using GDL as an acylating agent. His-tagged proteins are known to undergo N-terminal acylation with GDL as an inadvertent side reaction (page 2, paragraph 3). Martos-Maldonado teaches a strategy in which this undesirable side-reaction is instead used as a highly selective chemical method for modification of peptides and proteins. GHHHn is identified as an optimal N-terminal sequence for modification using GDL. The GHHHHHH tag, which can be fused to the N-terminus of any protein of interest, offers a dual functionality His tag as it can still be used for affinity purification (page 2, paragraph 4). Martos-Maldonado shows the reaction scheme for gluconoylation in Figure 1.a, which is reproduced in part below.
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Secondly, Martos-Maldonado teaches 4-methoxyphenyl esters as another, alternative acylating agent for N-terminal acylation of His-tagged proteins. One such 4-methoxyphenyl ester is shown in the reaction scheme in Figure 1.b, which is reproduced in part below.
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This azido substituted 4-methoxyphenyl ester reagent allows azido acetyl moieties to be smoothly introduced to a His tagged protein. The resulting azido substituted protein can be further transformed using strain-promoted azide–alkyne couplings (page 8, paragraph 1). As proof of concept for such a transformation, Martos-Maldonado reacts DBCO-OEG4-biotin and DBCO-PEG with the azido-functionalized N3-CH2-C(O)-GH6-EGFP obtained by N-terminal acylation of His-tagged EGFP (page 4, paragraph 6).
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Martos-Maldonado provides additional applications of the N-terminal acylation of His tagged proteins by showing the functionalization of GH6-BIR2 with an azide, a fluorophore and biotin (Figure 5c and 5d on page 9).
The teachings of Martos-Maldonado differ from that of the instantly claimed invention in that Martos-Maldonado does not disclose an N-terminally acylated protein in which at least one of R1- R4 is a handle as required by the instant claims, as the instant specification defines “handle” as a chemical moiety which provides a protein with a non-native chemical functionality.
Chaveriat teaches synthesis of 6-amino-6-deoxyaldonic acids. In the synthesis of 6-amino-6-deoxy-D-gluconic acid, Chaveriat discloses the synthesis (page 1351, paragraph 3 and scheme 6), isolation, and analysis (section 4.1.5 on page 1352), of compound 17, which is 6-azido-6-deoxy-D-glucono-1,5-lactone and is reproduced below for convenience.
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One of ordinary skill in the art would have been motivated to incorporate an azido functional group on the gluconic acid δ-lactone in the acylation of His tagged proteins in order to synthesize an azido functionalized N-terminally acylated protein because Martos-Maldonado teaches that azido functionalized His-tagged proteins have the added benefit of being capable of participating in alkyne–azide conjugation, which allows subsequent conjugation of the His tagged protein to moieties such as a fluorophore.
However, Martos-Maldonado does not provide instructions for obtaining an azido functionalized gluconic acid δ-lactone. It would have been prima facie obvious rely on the teachings of Chaveriat for the synthesis of an azido functionalized gluconic acid δ-lactone to be used as an acylating agent in the method suggested by Martos-Maldonado because Chaveriat teaches the synthesis of 6-azido-6-deoxy-D-glucono-1,5-lactone, which is a gluconic acid δ-lactone suitable for the acylation reaction of Martos-Maldonado.
One of ordinary skill in the art would have a reasonable expectation of success in using an azido functionalized gluconic acid δ-lactone as an acylating agent in the method suggested by Martos-Maldonado because Martos-Maldonado teaches acylation using D-gluconic acid δ-lactone and also teaches that His-tagged proteins can undergo N-terminal acylation with small molecules bearing an azido group. Thus the combined teachings of Martos-Maldonado and Chaveriat suggest an N-terminally acylated protein of instant formula VIII in which R1-3 and R5 are hydroxyl, R4 is an azide handle, and X comprises the N-terminal amino acid residue of the GHHHHHH tagged protein.
Regarding instant claim 8, Martos-Maldonado teaches that polyhistidine tags (His tags) are widely used for protein purification by immobilized-metal ion affinity chromatography. Thus Martos-Maldonado suggests a composition in which the N-terminally acylated protein is bound by a metal ion.
Claims 9 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Martos-Maldonado et al. (Nature Communications, 2018; IDS 10/03/2022) in view of Chaveriat et al. (Tetrahedron, 2006; IDS 10/03/2022), as applied to claims 1 and 8, further in view of Block et al. (Methods in Enzymology, 2009; PTO-892 03/12/2026).
The combined teachings of Martos-Maldonado and Chaveriat are as above. Furthermore, in teaching that polyhistidine tags are widely used for protein purification by immobilized-metal ion affinity chromatography, Martos-Maldonado cites Block et al. in footnote 29.
The combined teachings of Martos-Maldonado and Chaveriat do not expressly teach that the metal cation is from the d-block elements.
Block discusses the state of the art in immobilized-metal affinity chromatography (abstract). Block teaches that divalent Cu2+, Ni2+, Zn2+, and Co2+ ions are used for purification of His-tagged proteins. Combinations of a tetradentate ligand that ensures strong immobilization, and a metal ion that leaves two coordination sites free for interaction with biopolymers (Ni2+, Co2+) has gained most acceptance and leads to similar recovery and purity of eluted protein (page 444, paragraph 2).
It would have been prima facie obvious to substitute divalent cations of Zn, Ni, or Cu in the protein purification of the compound suggested by the combined teachings of Martos-Maldonado and Chaveriat because it is prima facie obvious to substitute the known elements of the divalent cations of Block in the protein purification by immobilized-metal ion affinity chromatography taught by Martos-Maldonado to obtain the predictable result of purification of His-tagged proteins. One of ordinary skill in the art would have had a reasonable expectation of success because Martos-Maldonado teaches that His-tagged proteins may be used in immobilized-metal ion affinity chromatography and Block teaches that divalent cations of Zn, Ni, or Cu are suitable ions for the purification of His-tagged proteins.
Response to Arguments
Applicant's arguments filed 06/11/2026 have been fully considered but they are not persuasive.
Applicant argues that one of ordinary skill in the art would not be able to synthesize 6-AGDL, let alone a protein acylated with 6-AGDL as recited in claim 1, based on the cited parts of Chaveriat because the instant specification demonstrates that the bromine oxidation of Chaveriat does not allow for "practically relevant production of suitable acylating reagent" (Remarks, page 12, paragraph 1). Applicant further argues that the product obtained in Chaveriat is different from the product obtained using the synthesis methods described (Remarks, paragraph bridging pages 12-13). This is not persuasive.
The instant claims are directed to an N-acylated protein rather than to a method of synthesizing an N-acylated protein. As discussed in the above grounds of rejection, the teachings of Martos-Maldonado would have suggested incorporation of an azido functional group on the gluconic acid δ-lactone in the acylation of His tagged proteins in order to synthesize an azido functionalized N-terminally acylated protein to one of ordinary skill in the art. One of ordinary skill in the art would have looked to Chaveriat for the synthesis of this azido gluconic acid δ-lactone. The instant specification does not indicate that the compound produced by method of Chaveriat is entirely incapable of producing the claimed N-acylated protein. Rather, the instant specification indicates that a small but detectable the amount of peptide acylation was observed upon contacting the model protein with the bromine oxidation products of 6-azido-6-deoxyglucose (Specification, page 8, lines 8-10). Thus it would nevertheless have been prima facie obvious for one of ordinary skill in the art to have synthesized the claimed compound. MPEP2121.01(II) states that even if a reference discloses an inoperative device, it is prior art for all that it teaches. Therefore, a non-enabling reference may qualify as prior art for the purpose of determining obviousness under 35 U.S.C. 103.
Applicant further argues that because Martos-Maldonado does not teach synthesis of a handle-substituted 1,5-lactone and Chaveriat does not teach that a handle-substituted lactone could be used to acylate a protein, the rationale of the instant rejection is based on improper hindsight reasoning (Remarks, page 14, paragraph 4). This is not persuasive.
As discussed in the above grounds of rejection, one of ordinary skill in the art would have been motivated to incorporate an azido functional group on the gluconic acid δ-lactone in the acylation of His tagged proteins of Martos-Maldonado in order to synthesize an azido functionalized N-terminally acylated protein because Martos-Maldonado teaches that azido functionalized His-tagged proteins have the added benefit of being capable of participating in alkyne–azide conjugation. MPEP 2145(X)(A) states that any judgment on obviousness is in a sense necessarily a reconstruction based on hindsight reasoning, but so long as it takes into account only knowledge which was within the level of ordinary skill in the art at the time the claimed invention was made and does not include knowledge gleaned only from applicant’s disclosure, such a reconstruction is proper. MPEP 2145(X)(A) further states that Applicants may also argue that the combination of two or more references is "hindsight" because "express" motivation to combine the references is lacking. However, there is no requirement that an "express, written motivation to combine must appear in prior art references before a finding of obviousness." One of ordinary skill in the art, prior to the effective filing date of the claimed invention, would have been motivated to synthesize an azido functionalized N-terminally acylated protein by incorporating an azido functional group on the gluconic acid δ-lactone of Martos-Maldonado in the acylation of His tagged proteins of Martos-Maldonado. One of ordinary skill in the art would have been motivated to do so because Martos-Maldonado teaches incorporation of azido functionalization on His-tagged proteins and teaches that this provides the added benefit of being able to undergo alkyne–azide conjugation.
Applicant further argues that the N-terminally acylated proteins recited in the present claims demonstrate unexpected results over the cited references because 6-AGDL results in a significantly higher degree of mono-acylation without a significant extent of off-target acylation when compared to use of 4-methoxyphenyl 2-azidoacetate (4MPAA) (Remarks, page 15, paragraph 3). This is not persuasive.
Table 2 in Example 13 of the instant specification (beginning on page 99) compares acylation using either 2.5 mM or 4 mM 4MPAA against acylation reactions using 100 mM 6-AGDL. Thus the comparison cannot be made between the experimental results of the acylation using 6-AGDL and that of the closest prior art in terms of 4MPAA because there is no way of knowing that any possible unexpected results are due to the structure of 6-AGDL and not to the differences in concentration. Furthermore, the acylation reactions using 100 mM of acylation reagent would generally be expected to have a higher degree of acylation as compared to a reaction using only 2.5 or 4 mM of acylation reagent. Thus Applicant does not demonstrate that a higher degree of acylation in the 100 mM 6-AGDL reaction as compared to the 2.5 mM and 4 mM 4MPAA reactions is in fact an unexpected result. Furthermore, the scope of the instant claims is drawn to compounds bearing any functional handle and which could be synthesized by a wide variety of lactones, including those of different ring sizes and substitution patterns. Thus the scope of the instant claims are not commensurate with the experimental examples. MPEP 716.02(b)(I) states that the evidence relied upon should establish unexpected results must establish that the differences in results are in fact unexpected and unobvious and of both statistical and practical significance.
Because Applicant’s arguments are not persuasive, the instant claims are rejected for the reasons of record.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the date of this final action.
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/S.G.H./Examiner, Art Unit 1693
/SCARLETT Y GOON/
Supervisory Patent Examiner
Art Unit 1693
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