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’ preliminary amendment, filed on 10/23/2025, in which claim 14 is cancelled.
Claims 1-13 and 15-20 are pending.
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.
Election/Restrictions
Applicant’s election of Group I, drawn to a drawn to an N-terminally acylated protein, wherein the N-terminally acylated protein comprises formula (VIII), formula (VII) or formula (IX), encompassing claims 1-9 and 19-20 in the reply filed on 10/23/2025 is acknowledged. Election was made without traverse in the reply filed on 10/23/2025. Similarly, Applicant’s election of a species of N-terminally acylated protein comprising the formula (IV), wherein R4 is an azide, R3 is a hydroxyl, R2 is a hydroxyl and R1 is a hydroxyl, and wherein X is Gly-His6 encompassing claims 1-9 and 19-20 in the reply filed on 10/23/2025 is acknowledged.
Claims 10-13 and 15-18 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 10/23/2025.
Claims 1-9 and 19-20 are examined on the merits herein.
Information Disclosure Statement
The information disclosure statement (IDS) dated 10/08/2025 and 10/03/2022 comply with the provisions of 37 CFR 1.97, 1.98 and MPEP § 609. Accordingly, the information disclosure statements have been considered by the examiner.
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 8 and 1, further in view of Block et al. (Methods in Enzymology, 2009; PTO-892).
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.
Conclusion
No claims are allowed.
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/S.G.H./Examiner, Art Unit 1693
/SCARLETT Y GOON/Supervisory Patent Examiner, Art Unit 1693