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 .
The Response of 14 May 2026 has been entered.
Claims 1-3, 5 and 7-22 are currently pending.
Election/Restrictions
Applicant’s election without traverse of the invention of Group I, claims 1-3, 5 and 7-16, in the reply filed on 14 May 2026 is acknowledged.
Claims 17-22 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 14 May 2026.
Claim Objections
Claims 1, 3 and 5 are objected to because of the following informalities:
Step (ii) of claim 1 should be amended as follows: "… of the modified first (poly)peptide to yield a dually modified …".
Claim 3 should be amended as follows: "… ligated to the C-terminus of the modified first (poly)peptide …".
Claim 5 should be amended as follows: "… ligated to the N-terminus of the modified first (poly)peptide …".
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a) (scope of enablement)
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.
Claim 13 is rejected under 35 U.S.C. 112(a) because the specification, while being enabling for a tandem ligation method using first and second asparaginyl ligases that are identical and have pH-dependent activity and specificity, wherein the pH-dependent specificity/activity comprises ligation of a P1-Asp recognition site at low pH and ligation of a P1-Asn recognition site at higher pH, does not reasonably provide enablement for such a method involving any type of pH-dependent activity and specificity. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to carry out the invention commensurate in scope with these claims.
Whether a disclosure satisfies the enablement requirement is assessed with respect to the factors set forth in In re Wands, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988); MPEP 2164.01 (a). These factors include: breadth of the claims, nature of the invention, state of the prior art, level of one of ordinary skill, level of predictability in the art, amount of direction provided by the inventor, existence of working examples and quantity of experimentation needed to make or use the invention.
All of the Wands factors have been considered with respect to the instant claims. The most relevant factors are discussed in detail below.
Breadth of the claim
Claim 13 is drawn to a method for tandem ligation as set forth in claim 1, wherein each of the two ligation steps are carried out at different pH and wherein the ligation steps use the same/identical asparaginyl ligase and said asparaginyl ligase has pH-dependent activity and specificity. The claim does not specify any particular type of pH-dependent specificity/activity - e.g., towards any specific recognition sequence(s) or at any particular pH. The claim also does not require any use of the pH-dependent specificity/activity in the ligation method; i.e. the claim merely recites a property of the enzyme without requiring any pH-dependent ligation steps in the method. Thus, the claim broadly encompasses any type of usage of an asparaginyl ligase having any type of pH-dependent specificity/activity.
State/unpredictability of the prior art
The prior art discloses a pH-dependent activity of asparaginyl ligases towards Asx-Xaa-Xaa recognition sites, with broad activity towards Asn and pH-dependent activity towards Asp at low pH (e.g., Hemu-3 (cited below), 2nd ¶ under INTRODUCTION), although thus use of such selectivity for orthogonal tandem ligation does not appear to be disclosed. It is also known in the art that butelase-1 and VyPAL2 have pH-dependent activity (as would be expected for any enzyme) (see, Hemu-1 (cited below), p. 106, under 5.; Hemu-2 (cited below), Fig. 1C). However, the prior art does not disclose any known pH-dependent selectivity for butelase-1 and VyPAL2 outside of that known for Asn/Asp above.
Amount of direction provided, existence of working examples and quantity of experimentation needed
The instant specification describes the same Asn/Asp-based pH-dependent selectivity recognized in the art (Published Spec. US20240117402, Example 7 - " PALs, such as VyPAL2, butelase-1 and OaAEP1b, can all catalyze peptide cyclization and intermolecular ligation at aspartyl peptide bonds at acidic pH …. the stability of a newly formed Asp-Xaa bond towards the PALs at neutral to slightly basic pH also makes it possible to conduct a second ligation reaction on the same protein at an asparaginyl junction. This pH-controlled, PAL-catalyzed tandem ligation strategy allows protein dual labeling in either N-to-C or C-to-N direction and the reactions at the two steps can be done by using the same PAL or two different PALs."). The specification does not describe any other types of pH-dependent selectivity, and does not contain any guidance (e.g., a structure-function relationship indicating portions of the enzyme that could be altered to modulate pH-dependent selectivity) or other information that would allow one of ordinary skill in the art to practice a pH-dependent orthogonal tandem ligation on a basis other than Asn/Asp-based selectivity.
In light of the breadth of claim 13 in encompassing any type of pH-dependent usage of an asparaginyl ligase having any type of pH-dependent specificity/activity and the lack of knowledge/unpredictability in the art regarding such selectivity other than Asn/Asp-based selectivity, one of ordinary skill in the art would be forced to undertake extensive and undue experimentation to carry out the full scope of the claimed method.
Claim Rejections - 35 USC § 112(b) (indefiniteness)
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.
Claims 8-11 and 14-16 are rejected under 35 U.S.C. 112(b) 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.
Claims 8-11, 14 and 15 contain multiple recitations of "preferably", "more preferably" and "even more preferably". The term "preferably" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d). Further regarding claim 14, each of elements (1) and (2) are limited as a whole by the term "preferably", making it unclear how/whether the claim further limits claim 12.
Claim 15 recites the term “about”, which is a relative term which renders the claim indefinite. The term “about” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Thus, one of ordinary skill would be able to reasonably ascertain what range of pH values fall within/without the claim.
Claim 16 recites "the asparaginyl ligase is VyPAL2". It is unclear which asparaginyl ligase is being limited by the claim, as claim 1 (from which claim 16 indirectly depends) recites first and second asparaginyl ligases. It appears that claim 16 may have been intended to depend from claim 13 which recites that the first and second asparaginyl ligases are the same.
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.
Claims 1-3, 5, 7-12 and 14-16 are rejected under 35 U.S.C. 103 as being unpatentable over the combination of Hemu et al., Enzyme-mediated ligation methods 2019: 83-109 (referred to herein as "Hemu-1") in view of Hemu et al., Proceedings of the National Academy of Sciences 116.24 (2019): 11737-11746 (referred to herein as "Hemu-2") and Hemu et al., The Journal of organic chemistry 85.3 (2019): 1504-1512 (referred to herein as "Hemu-3"), as evidenced by Nguyen et al., Nature chemical biology 10.9 (2014): 732-738 (each cited in IDS of 3 Nov. 2023).
Regarding claim 1, Hemu 1 teaches a method for tandem polypeptide ligation, comprising i) contacting a first polypeptide having at its C-terminus a binding and ligation site for a first asparaginyl ligase (e.g., an NHV site) with a second polypeptide to be ligated and a first asparaginyl ligase (butelase-1) under conditions that allow ligation of the second polypeptide to the C-terminus of the first polypeptide to yield a modified first polypeptide; and ii) contacting the modified first polypeptide with a third polypeptide and a second asparaginyl ligase (sortase) under conditions that allow ligation of the third polypeptide to the N-terminus of the modified first polypeptide to yield a dually modified first polypeptide (p. 85, under 8.; under 3.5. Butelase 1-Mediated Generation of C-terminal Thioester Peptides for Chemoenzymatic ligation; Fig. 8). The first polypeptide comprises (in addition to the C-terminal recognition site for the first ligase) an N-terminal nucleophilic dipeptide that can be accepted by the second ligase (GG in Fig. 8). The butelase-1 has 100% identity to SEQ ID 2 (Hemu 1, p. 8, 1st full ¶ (citing Nguyen); Nguyen, Supp. Fig. 6; see attached alignment). Hemu 1 teaches that butelase-1 can ligate polypeptides having a C-terminal NHV or NGL recognition site with an N-terminal G/R/K-L/V/I dipeptide sequence (p. 85, under 2.; p. 105-106, under 3.-5.). Hemu 1 further teaches that butelase-1 can be used generally with another ligase for sequential enzymatic tandem ligations (p. 85, under 8.).
Regarding claim 5, the tandem ligation of Hemu 1 comprises ligating the second polypeptide to the C-terminus of the first polypeptide by the first asparaginyl ligase (butelase), wherein the binding and ligation site for an asparaginyl ligase at the C-terminus of the first polypeptide (NHV in Fig. 8) is for the first asparaginyl ligase, and wherein the third polypeptide has at its C-terminus a binding and ligation site for the second asparaginyl ligase (LPET sortase sequence in Fig. 8) and is ligated to the N-terminus of the modified first polypeptide by the second asparaginyl ligase (Hemu 1, Fig. 8).
Regarding claim 9, Hemu 1 teaches use of an NHV or NGL butelase recognition sequence (p. 85, under 2.; p. 105-106, under 3.-5.).
Regarding claim 11, Hemu-1 teaches that butelase-1 can ligate polypeptides having a C-terminal NHV or NGL recognition site with an N-terminal G/R/K-L/V/I dipeptide sequence (p. 85, under 2.; p. 105-106, under 3.-5.). As such, it would have been obvious to use a first polypeptide having a butelase binding site at the C-terminus and a second polypeptide having a sequence at the N-terminus as set forth in claim 11(2).
Claims 1-3, 5, 7-12 and 14-16 differ from Hemu-1 in that: the second asparaginyl ligase is vyPAL2 and has at least 80% identity to SEQ ID 1 (claims 1, 7, 16); the second (poly)peptide has at its C- terminus a binding and ligation site for the first asparaginyl ligase and is ligated to the N- terminus of the first (poly)peptide by the first asparaginyl ligase (claim 2); the binding and ligation site for an asparaginyl ligase at the C-terminus of the first (poly)peptide is for the second asparaginyl ligase, and wherein the third (poly)peptide is ligated to the C-terminus of the first (poly)peptide by the second asparaginyl ligase (claim 3); the binding and ligation site(s) for VyPAL2 are as set forth in claims 8 and 10; steps (i) and (ii) are carried out at a first and a second pH-value that are different from each other (claims 12, 14); and the first pH value is a about 6.0 or lower, and the second pH value is about 6.5 or higher; or the second pH value is about 6.0 or lower, and the first pH value is about 6.5 or higher (claim 15).
Hemu-2 teaches that the family of asparaginyl ligases that includes butelase-1 also includes VyPAL2, which has 100% identity to SEQ ID 1 (p. 11737, 1st ¶ to p. 11738, 2nd full ¶; Table 1; p. 11739-11740, under Ligase vs. Protease Activity of VyAEP1 and VyPAL1-3; Fig. 1; amino acid sequence set forth in Fig. S2, see attached alignment vs SEQ ID 1). Hemu-2 teaches that VyPAL2 catalyzes peptide ligation of a C-terminal tripeptide recognition sequence (e.g., NSL) and an N-terminal dipeptide nucleophile (e.g., GI or GL) (p. 11739-11740, under Ligase vs. Protease Activity of VyAEP1 and VyPAL1-3; Fig. S4). Hemu-2 further teaches that VyPAL2 showed good ligase activity without any hydrolytic product at mild/neutral pH (5.5.-8) (similar to butelase-1), making it valuable for biotechnological applications (p. 11742-11743, under Discovery of PALs and AEPs from Violacae).
Hemu-3 teaches that VyPAL2 can catalyze intermolecular polypeptide ligations, in addition to the cyclization reactions taught by Hemu-2 (p. 1509, 1st full ¶).
It would have been obvious to one of ordinary skill in the art at the time the invention was made to carry out a tandem ligation reaction to modify both the N- and C-termini of a polypeptide using butelase and an additional ligase, as taught by Hemu-1, wherein the second ligase is VyPAL2 as taught by Hemu-2 and Hemu-3 because it would have been obvious to combine prior art elements according to known methods to yield predictable results. One of ordinary skill would have been motivated to use VyPAL2 as the second ligase because Hemu-2 teaches that VyPAL2 good ligase activity without any hydrolytic product at mild/neutral pH (5.5.-8) (similar to butelase-1), making it valuable for biotechnological applications. Using VyPAL2 as the second ligase in the method of Hemu-1 would have led to predictable results with a reasonable expectation of success because Hemu-2 teaches that VyPAL2 is an asparaginyl ligase similar to butelase-1, and Hemu-3 teaches that VyPAL2 can catalyze intermolecular polypeptide ligation in the same manner as in the method of Hemu-1.
Regarding claims 2, 3, 7 and 10, Hemu-1 and Hemu-2 teach that each of butelase-1 and VyPAL2 catalyze ligations between a C-terminal tripeptide and an N-terminal dipeptide nucleophile, and it would have been prima facie obvious to carry out ligations wherein the tripeptide and dipeptide for each of butelase and VyPAL2 are at any position on the first, second and third polypeptides, including such that the N-terminus of the first polypeptide is modified initially (as in claims 2, 3, 7(1) and 10(1)) or wherein the C-terminus of the first polypeptide is modified initially (as in claims 5 and 11(2)) (see MPEP 2144.04, VI. - the reversal, duplication or rearrangement of parts is prima facie obvious in the absence of new or unexpected results). Moreover, Hemu-2 teaches that VyPAL2 does not have appreciable ligation activity against the preferred NHV recognition sequence of butelase-1 (Hemu-2, Fig. S4). Thus, one would have been motivated to ligate the N-terminus of a first polypeptide comprising an N-terminal VyPAL2 nucleophile (e.g., GI) and a C-terminal NHV sequence to the C-terminus of a second polypeptide comprising a C-terminal VyPAL2 recognition sequence (e.g., NSL), since the first polypeptide (e.g., GI-----NHV) would not have a competing cyclization reaction due to VyPAL2's non-recognition of NHV.
Regarding claim 8, Hemu-2 teaches that VyPAL2 recognizes a C-terminal sequence comprising N-G/S-L/I/F (p. 11740, 1st full ¶; Fig. S4).
Regarding the recitation of an XF N-terminal nucleophile sequence for VyPAL2 in claim 10, Hemu-2 teaches that the nucleophile can be hydrophobic amino acids L and I (Fig. S4), and it would have been prima facie obvious to use other hydrophobic amino acids such as F.
Regarding claims 12, 14 and 15, Hemu-1 teaches that pH 6-6.5 is optimal for butelase-1 mediated ligation (p. 106, under 5.), and it would have been obvious to carry out the butelase ligation at any pH within such range (e.g., at pH 6). Hemu-2 teaches that the optimal pH for VyPAL2 is 6.5 (Fig. 1C). Thus, it would have been obvious to carry out the two-step tandem ligation with a butelase ligation at pH 6 and a VyPAL2 ligation as pH 6.5.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ROBERT J YAMASAKI whose telephone number is (571)270-5467. The examiner can normally be reached M-F 930-6 PST.
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/ROBERT J YAMASAKI/Primary Examiner, Art Unit 1657
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