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 .
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 .
Detail Action
Claims 1-19 submitted on 6/3/2026 are pending for examination.
Applicants on 6/3/2026 with traverse elected group I Claims 1-20; directed to method of polymer engineering comprising: deploying a constitutively active dehydratase enzyme of ribosomally synthesized and post translationally modified peptide (RiiPP) in a dehydration reaction under conditions wherein the enzyme accepts a substrate comprising a reaction site comprising an amino acid that participates directly in the dehydration reaction and a non-a-amino acid monomer at a position flanking the reaction site. Applicants argument of traversal is considered and accepted. Therefore groups II-III invention will be rejoined with group I. The restriction of office action 6/3/26 is withdrawn. Claims 1-19 will be examined,
Election made final.
Claims 1--19 are for examination.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 2/1/2024 in compliance with the provisions of 37 CFR 1.97. Accordingly, the examiner has considered the IDS statement.
Claim Rejections, 35 U.S.C 112(a)
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 1-19 are rejected under 35 U.S.C. 112, first paragraph, as containing 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(s), at the time the application was filed, had possession of the claimed invention.
The claims are directed to method of polymer engineering comprising: deploying a constitutively active dehydratase enzyme of ribosomally synthesized and post translationally modified peptide (RiiPP) in a dehydration reaction under conditions wherein the enzyme accepts a substrate comprising a reaction site comprising an amino acid that participates directly in the dehydration reaction and a non-a-amino acid monomer at a position flanking the reaction site.
It is noted that MPEP 2111.01 states that "[d]uring examination, the claims must be interpreted as broadly as their terms reasonably allow." In this case, in light of the specification, the examiner has broadly interpreted the claims by reciting “
method of polymer engineering comprising: deploying a constitutively active dehydratase enzyme of ribosomally synthesized and post translationally modified peptide (RiiPP) in a dehydration reaction under conditions wherein the enzyme accepts a substrate comprising a reaction site comprising an amino acid that participates directly in the dehydration reaction and a non-a-amino acid monomer at a position flanking the reaction site” comprise use of any constitutively active dehydratase enzyme of ribosomally synthesized and post translationally modified peptide (RiiPP) having any structure from any source in a dehydration reaction under unknown conditions wherein the enzyme accepts any substrate comprising a reaction site comprising an amino acid that participates directly in the dehydration reaction and any non-a-amino acid monomer at a position flanking the reaction site to form polymer having specific reaction producing specific outcome.
Therefore in light of the above interpretation the claims are broadly use of any constitutively active dehydratase enzyme of ribosomally synthesized and post translationally modified peptide (RiiPP) any structure from any source to in a dehydration reaction under unknown conditions wherein the enzyme accepts a any substrate having any structure comprising a reaction site comprising an amino acid that participates directly in the dehydration reaction and a non-a-amino acid monomer at a position flanking the reaction site.
The Court of Appeals for the Federal Circuit has recently held that a "written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." University of California v. Eli Lilly and Co., 1997 U.S. App. LEXIS 18221, at *23, quoting Fiers v. Revel, 25 USPQ2d 1601, 1606 (Fed. Cir. 1993). To fully describe a genus of genetic material, which is a chemical compound, applicants must (1) fully describe at least one species of the claimed genus sufficient to represent said genus whereby a skilled artisan, in view of the prior art, could predict the structure of other species encompassed by the claimed genus and (2) identify the common characteristics of the claimed molecules, e.g., structure, physical and/or chemical characteristics, functional characteristics when coupled with a known or disclosed correlation between function and structure, or a combination of these (paraphrased from Enzo Biochemical).University of Rochester v. G.D. Searle & Co. (69 USPQ2d 1886 (2004)) specifically points to the applicability of both Lilly and Enzo Biochemical to methods of using products, wherein said products lack adequate written description. While in University of Rochester v. G.D. Searle & Co. the methods were held to lack written description because not a single example of the product used in the claimed methods was described, the same analysis applies wherein the product, used in the claimed methods, must have adequate written description (see Enzo paraphrased above).
In this case, there is no structure associated with function with regard to the members of the genus of constitutively active dehydratase enzyme of ribosomally synthesized and post translationally modified peptide (RiPP) having any structure and in a dehydration reaction under unknown conditions wherein the enzyme accepts any substrate comprising a reaction site comprising an amino acid that participates directly in the dehydration reaction and any non-a-amino acid monomer at a position flanking the reaction site to form polymer having specific reaction producing specific outcome.
The genus of genus constitutively active dehydratase enzyme of ribosomally synthesized and post translationally modified peptide (RiPP) and genus of substrate comprising a reaction site comprising an amino acid in the claimed invention is an extremely large structurally and functionally variable genus.
An argument can be made that the recited genus of polypeptides is adequately described by the disclosure of the structure of (RiPP) polypeptides like MicD- F having SEQ ID No; 7 or ARTGox of SEQ ID NO: 8 ( page 27-28 of the specification ) and specified substrate like, mCerry variants having SEQ ID NO: 9-14 ( page 30-31 of the specification) , one could use structural homology to isolate those polypeptides the encoding polynucleotides. However, the art clearly teaches there is a practical limits to predict function of a polypeptide based structural homology:
A. Devos et al., (Proteins: Structure, Function and Genetics, 2000, Vol. 41: 98-107), teach that the results obtained by analyzing a significant number of true sequence similarities, derived directly from structural alignments, point to the complexity of function prediction. Different aspects of protein function, including (i) enzymatic function classification, (ii) functional annotations in the form of key words, (iii) classes of cellular function, and conservation of binding sites can only be reliably transferred between similar sequences to a modest degree. The reason for this difficulty is a combination of the unavoidable database inaccuracies and plasticity of proteins (Abstract, page 98) and the analysis poses interesting questions about the reliability of current function prediction exercises and the intrinsic limitation of protein function prediction (Column 1, paragraph 3, page 99) and conclude that "Despite widespread use of database searching techniques followed by function inference as standard procedures in Bioinformatics, the results presented here illustrate that transfer of function between similar sequences involves more difficulties than commonly believed. Our data show that even true pair-wise sequence relations, identified by their structural similarity, correspond in many cases to different functions (column 2, paragraph 2, and page 105).
B. Whisstock et al., (Quarterly Reviews of Biophysics 2003, Vol. 36 (3): 307-340,) also highlight the difficulties associated with "Prediction of protein function from protein sequence and structure": "To reason from sequence and structure to function is to step onto much shakier ground", closely related proteins can change function, either through divergence to a related function or by recruitment for a very different function, in such cases, assignment of function on the basis of homology, in the absence of direct experimental evidence, will give the wrong answer (page 309, paragraph 4), it is difficult to state criteria for successful prediction of function, since function is in principle a fuzzy concept. Given three sequences, it is possible to decide which of the three possible pairs is most closely related. Given three structures, methods are also available to measure and compare similarity of the pairs. However, in many cases, given three protein functions, it would be more difficult to choose the pair with most similar function, although it is possible to define metrics for quantitative comparisons of different protein sequences and structures, this is more difficult for proteins of different functions (page 312, paragraph 5), in families of closely related proteins, mutations usually conserve function but modulate specificity i.e., mutations tend to leave the backbone conformation of the pocket unchanged but to affect the shape and charge of its lining, altering specificity (page 313, paragraph 4), although the hope is that highly similar proteins will share similar functions, substitutions of a single, critically placed amino acid in an active-site residue may be sufficient to alter a protein's role fundamentally (page 323, paragraph 1).
C. This finding is reinforced in the following scientific teachings for specific proteins in the art that suggest, even highly structurally homologous polypeptides do not necessarily share the same function and many functionally similar proteins will have little or no structural homology to disclosed proteins. For example, proteins having similar structure have different activities (structure does not always correlate to function); Witkowski et al., (Biochemistry 38:11643-11650, 1999) teaches that one conservative amino acid substitution transforms a beta -ketoacyl synthase into a malonyl decarboxylase and completely eliminates beta-ketoacyl synthase activity. The art also teaches that functionally similar molecules have different structures; Kisselev L., (Structure, 2002, Vol. 10: 8-9) teach that polypeptide release factors in prokaryotes and eukaryotes have same function but different structures.
As stated above, no information beyond the characterization of a few species; the structure of (RiPP) polypeptides like MicD- F having SEQ ID & or ARTGox of SEQ ID NO: 8 ( page 27-28 of the specification ) and specified substrate like, mCerry variants having SEQ ID NO: 9-14 ( page 30-31 of the specification) , and one could use structural homology to isolate those polypeptides the encoding polynucleotides having specific activity, has been provided by the applicants’, which would indicate that they had the possession of the claimed genus of polypeptides. The claimed genera of polypeptides and the encoding polynucleotides have widely variable structures and associated functions. As it is discussed above, a minor changes in structure may result in changes affecting function, since, the specification provided no additional information (species/variant/mutant) correlating structure with function, and one skilled in the art cannot reasonably conclude that applicant had possession of the claimed invention at the time the instant application was filed. Furthermore, "Possession may not be shown by merely describing how to obtain possession of members of the claimed, genus or how to identify their common structural features" (See University of Rochester, 358 F.3d at 927, 69 USPQ2d at 1895). A definition by function, as we have previously indicated, does not suffice to define the genus because it is only an indication of what the .gene does (function), rather what it is (structure), see University of California v. Eli Lilly & Co., 43 USPQ2d 1938, thus above claims lack adequate written description.
Applicants' are referred to the revised guidelines concerning compliance with the written description requirement of U.S.C. 112, first paragraph, published in the Official Gazette and also available at www.uspto.gov
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.
Claim(s) 1-5, 7-12 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Naismith et al. (US 20180245061, 2018, Univ of Aberdeen) .
Regarding claim 1, Naismith et al. discloses a method of polymer engineering comprising: deploying a constitutively active dehydratase enzyme of RiPP biosynthesis to accept a substrate containing a non-alpha-amino acid monomer (para [0002] "Ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by marine organisms have been shown to possess anti-tumor, anti-fungal, antibacterial and antiviral properties"; para [0013] "a method of introducing heterocyclic residues into a target molecule comprising; treating a target peptide comprising one or more heterocyclisable residues with a modified heterocyclase as described herein."; para [0020] "FIG. 3 shows LCMS analysis of heterocyclization reactions of core peptide (ITACITFCAYDG) incubated with TruD, LynD and LynD fusion." para [0034] "A heterocyclisable residue is a residue in the target molecule that the heterocyclase sequence in the modified heterocyclase is capable of converting into a heterocyclic residue. For example, modified heterocyclases comprising PatD or MicD may be used to heterocyclise Se-Cys, Cys, Thr and Ser residues in the target molecule and modified heterocyclases comprising LynD or TruD may be used to heterocyclise Cys or Se-Cys residues in the target molecule but not Thr or Ser residues."; para [0184] "Two enzymes (MicD and PatD) are capable of catalyzing the heterocyclization of Cysteine, Threonine and Serine residues. Using the fused-LynD enzyme as a template, MicD and fPatD enzymes were fused with leader peptide sequences to produce fused-MicD and fused-PatD enzymes."; Note, MicD, PatD, LynD, LynD-F, and MicD-F are constitutively active dehydratase enzymes of RiPP biosynthesis indicated by the instant application, see instant claims 8-11) at a position flanking the reaction site (para [0032] "Heterocyclisable residues may include naturally occurring and non-naturally occurring amino acids such as cysteine, homocysteine, selenocysteine, tellurocysteine, threonine, serine, homoserine, 2,3-diaminopropanoic acid, 2,4-diaminobutanoic acid, and synthetic derivatives thereof with additional R groups at the alpha, beta and/or gamma positions"; para [0036] - "The residues that are heterocyclised by the modified heterocyclase may be located at any position in the target molecule other than the C terminal. The C terminal residue of target molecule, which contains a free carboxyl group, is not heterocyclisable. The C terminal of the target molecule may comprise the sequence HtXn, where Ht is a heterocyclisable residue, X is any amino acid and n is 1-10. For example, residues that are heterocyclised may be adjacent to a cyclisation signal, if present, and/or located at other positions within the target peptide (i.e. internal residues). Preferably, the modified heterocyclase heterocyclises all of the residues in the target molecule that are potentially targeted by that heterocyclase."; para [0098] - "A cyclisation signal is the recognition site for the cyanobacterial macrocyclase and may be useful in macrocyclizing a target peptide after the introduction of heterocycles to produce a cyclic peptide. The sequence of the cyclisation signal in the target peptide may depend on the cyanobacterial macrocyclase being used. Typically, a cyclisation signal will comprise the sequence; small residue-bulky residue-acidic residue. Suitable cyclisation signals include AYD, AYE, SYD, AFD and FAG. For example, the cyclisation signal may be AYD, which allows the macrocyclization of the target peptide using a PatG macrocyclase."; para [0181] "The ability of heterocyclases to separate recognition from the catalytic site is highly desirable in biotechnology, conferring specificity by recognizing an invariant leader but tolerating a wide range of residues adjacent to the target cysteine (or serine/threonine).").
Regarding claim 2, Naismith et al. discloses the method of claim 1, and Naismith et al. further discloses wherein the polymer comprises an amino acid polymer compatible and operable with the disclosed constitutively active dehydratase enzyme of RiPP biosynthesis to accept a substrate containing a non-alpha-amino acid monomer at a position flanking the reaction site (para [0032]; para [0036]; para [0098]; para [0181]). Regarding claim 3, Naismith et al. discloses the method of claim 1, and Naismith et al. further discloses wherein the polymer comprises an antibody, cytokine, replacement enzyme, or therapeutic protein (para [0002] "Ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by marine organisms have been shown to possess anti-tumor, anti-fungal, antibacterial and antiviral properties"; para [0133] "The methods of the invention are suitable for the production of usable amounts of fully heterocyclized target molecules. Target molecules produced as described herein, such as peptides or other biomolecules, may be useful in therapeutics").
Regarding claim 4, Naismith et al. discloses wherein the method imbues a polymeric material comprising the polymer with new structures or functions (para [0013] "a method of introducing heterocyclic residues into a target molecule"). Regarding claim 5, Naismith et al. discloses wherein the polymer comprises a protein (para [0091] "Suitable target molecules may include heterocyclisable amino acids, peptides, peptide analogues and other biomolecules comprising heterocyclisable amino acids or amino acid analogues."; para [0096] "The target peptide lacks a leader sequence i.e. the target protein may be devoid of the amino acid sequence that is naturally located N terminal of the core cyanobactin sequence in the cyanobactin pre-pro-peptide."), and the method introduces a non-native backbone modification to expand protein function (para [0003] "The site-selective introduction of heterocycles into peptide backbones alters both conformation and reactivity of peptides; this tailoring of peptides is highly desirable in modifying their biological properties"; para [0123]-[0124] "The target molecule may be chemically modified or modified using one or more enzymes, for example cyanobacterial enzymes. The target molecule may be oxidized to oxidize the heterocyclic amino acids introduced into the target molecule. For example, the target molecule may be treated with a bacterial or cyanobacterial oxidase or a chemical oxidizing agent to oxidize thiazoline residues into thiazoles. Suitable cyanobacterial oxidases include PatG oxidase from Prochloron spp. Suitable bacterial oxidases are well known in the art and include BcerB oxidase from the thiazole/oxazole modified microcin cluster"; Note, introduction of thiazoline by MicD-F is a backbone modification, see instant application para [045]).
Regarding claims 7-8, Naismith et al. discloses wherein the enzyme catalyzes formation of an azole (e.g. oxazole or thiazole) ring (para [0123]-[0124] "The target molecule may be chemically modified or modified using one or more enzymes, for example cyanobacterial enzymes. The target molecule may be oxidized to oxidize the heterocyclic amino acids introduced into the target molecule. For example, the target molecule may be treated with a bacterial or cyanobacterial oxidase or a chemical oxidizing agent to oxidize thiazoline residues into thiazoles. Suitable cyanobacterial oxidases include PatG oxidase from Prochloron spp. Suitable bacterial oxidases are well known in the art and include BcerB oxidase from the thiazole/oxazole modified microcin cluster"). Regarding claims 9- 11, Naismith et al. discloses the method of claim 1, and Naismith et al. further discloses wherein the enzyme is a constitutively active heterocyclase in which the leader sequence is fused directly to the enzyme, such as LynD fusion (LynD-F) and MicD fusion (MicD-F) (para [0020]; para [0184]). and further discloses wherein the enzyme is selected from MicD-F and ArtGox (para [0034]; para [0184]). Regarding claim 11, the enzyme is selected from heterocyclases MicD, PatD, and LynD (para [0034]; para [0184]). Regarding claim 12, Naismith et al. discloses the method of claim 1 and further teach providing a protein therapeutic fused to a RiPP natural product, synthesized in a single step, without a separate chemical bio-conjugation step, without sequential or separate purification steps, and in situ (para [0091]; para [0096]; abstract produce a homogenous heterocycle-containing product."; para [0133] "The methods of the invention are suitable for the production of usable amounts of fully heterocyclized target molecules. the heterocyclized target molecule may be used without further isolation or purification. Target molecules produced as described herein, such as peptides or other biomolecules, may be useful in therapeutics").
Claim Rejections: 35 USC § 103
The following is a quotation of 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims under 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of 35 U.S.C. 103(c) and potential 35 U.S.C. 102(e), (f) or (g) prior art under 35 U.S.C. 103(a).
The factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103(a) 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.
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 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.
According to MPEP 2143:"Exemplary rationales that may support a conclusion of obviousness include:(A) Combining prior art elements according to known methods to yield predictable results;(B) Simple substitution of one known element for another to obtain predictableresults;(C) Use of known technique to improve similar devices (methods, or products) in the same way;(D) Applying a known technique to a known device (method, or product) ready for improvement to yield predictable results;(E) "Obvious to try" - choosing from a finite number of identified, predictable solutions, with a reasonable expectation of success;(F) Known work in one field of endeavor may prompt variations of it for use in either the same field or a different one based on design incentives or other market forces if the variations are predictable to one of ordinary skill in the art;(G) Some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention.Note that the list of rationales provided is not intended to be an all-inclusive list. Other rationales to support a conclusion of obviousness may be relied upon by Office personnel."
Claim(s) 6, 13-16, 17-19 under 35 U.S.C. 103 as being unpatentable over Naismith et al. (US 20180245061, 2018, Univ of Aberdeen) in view of Quels et al. Chemistry Pub soc, Chemopen 2017, 6, pp 11-14 )..
Naismith et al. discloses a method of polymer engineering comprising: deploying a constitutively active dehydratase enzyme of RiPP biosynthesis to accept a substrate containing a non-alpha-amino acid monomer (para [0002] "Ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by marine organisms have been shown to possess anti-tumor, anti-fungal, antibacterial and antiviral properties"; para [0013] "a method of introducing heterocyclic residues into a target molecule comprising; treating a target peptide comprising one or more heterocyclisable residues with a modified heterocyclase as described herein."; para [0020] "FIG. 3 shows LCMS analysis of heterocyclization reactions of core peptide (ITACITFCAYDG) incubated with TruD, LynD and LynD fusion." para [0034] "A heterocyclisable residue is a residue in the target molecule that the heterocyclase sequence in the modified heterocyclase is capable of converting into a heterocyclic residue. For example, modified heterocyclases comprising PatD or MicD may be used to heterocyclise Se-Cys, Cys, Thr and Ser residues in the target molecule and modified heterocyclases comprising LynD or TruD may be used to heterocyclise Cys or Se-Cys residues in the target molecule but not Thr or Ser residues."; para [0184] "Two enzymes (MicD and PatD) are capable of catalyzing the heterocyclization of Cysteine, Threonine and Serine residues. Using the fused-LynD enzyme as a template, MicD and fPatD enzymes were fused with leader peptide sequences to produce fused-MicD and fused-PatD enzymes."; Note, MicD, PatD, LynD, LynD-F, and MicD-F are constitutively active dehydratase enzymes of RiPP biosynthesis indicated by the instant application. ( see the 102 rejection above.).
Regarding claim 6 Naismith et al. discloses the method of claim 1, but does not specifically disclose used to engineer a therapeutic protein to express at higher levels, resist degradation, improve thermal and proteolytic stability, alter immunogenicity, antigenicity or immune reactivity, or encode a function, such as targeting the protein to distinct cells or tissues
Naismith et al. disclose further modification of a target peptide (para [0122]-[0123] - "Following the introduction of heterocycles using a modified heterocyclase, the target molecule may be further modified as required. For example, one or more of the modifications may be introduced into the target molecule. The target molecule may be chemically modified or modified using one or more enzymes, for example cyanobacterial enzymes."), and macrocycling a target (para [0130] - "The target molecule may be macrocyclised to produce a cyclic molecule. For example, the target molecule may be treated with a cyanobacterial macrocyclase. In some embodiments, a target peptide may be macrocyclised to produce a cyclic peptide. Suitable macrocyclases include PatG macrocyclase").
Ouels discloses therapeutic macrocycle peptides and where macrocycles offer improved stability (abstract - "Peptidic macrocycles have attracted attention as potential therapeutics possessing cell permeability, stability, and easy-to-control variability. Here, we show that enzymes from the patellamide biosynthetic pathway can be harnessed to make macrocycles that are hybrids of amino acids and a variety of manmade chemical building blocks, including aryl rings, polyethers, and alkyl chains."; pg 11, coll 1, para 1 col 2, para 2 "Macrocycles, in particular, whether peptidic or polyketides, are especially appealing, owing to their inherent chemical stability and structural rigidity. Several natural macrocycles possess useful biological and medicinal activities. Hybrid peptide macrocycles show increased chemical and structural diversity, for example, largazole (PKS/NRPS hybrid), zizyphines (cyclopeptide alkaloids), and maytansin (macrolide)"), and where cyclizing enzymes include PatG, PatD, and LynD (pg 12, col 1, para 1 "The synthetic utility of PatGmac derives precisely from its promiscuity towards the core sequence. The cyclic dehydratases, PatD and LynD, which make thiazoline and oxazoline rings from cysteine and serine (threonine), respectively, have also been shown to tolerate the presence of non-natural amino acid").
Since Naismith et al. discloses using various heterocyclase and macrocyclase enzymes including LynD, PatD, and PatG, macrocycling a target, and where a target substrate may be a protein, and Ouesis discloses where macrocyclizing peptides may Improve stability,
it would have been obvious to a person of ordinary skill in the art at the time the invention was effectively filed to combine the teaching of Naismith et al. and Ouels et al. and to modify the method of Naismith et al., to include or instead consider modifications which may increase stability, such as by cyclizing a portion of a target protein, thereby providing any improved structural rigidity afforded by an increased number of intramolecular bonds.
Regarding claims13-16, Naismith et al. discloses the method of claim 1, but Naismith et al. does not specifically disclose wherein the enzyme processes a substrate comprising: (a) an aromatic ring, even at the +1 position that precedes the site of cyclization, e.g. Benzoic acid, tetrafluoro-benzoic acid, 2- amino benzoic acid, 2- amino-5-methoxy benzoic acid, 2-aminopyridyl, and coumarin; (b) beta-3-amino acid, even at the +1 site, e.g. beta-3-isoleucine; or (c) an aramid monomer, even at the -1 site. Naismith et al. does disclose using various heterocyclases (para [0020]; para [0034]; para [0184]; para [0098] "For example, the cyclisation signal may be AYD, which allows the macrocyclization of the target peptide using a PatG macrocyclase."; Note, MicD tolerates aromatic rings at the +1 site, and MicD-F tolerates beta-3-isolucine at +1 site and aramid monomers at -1 site, see instant application para [038]-[043], Figures 5-10. Thus, the enzymes of Naismith et al. inherently tolerate theses substrates, as the properties of an enzyme cannot be separated from its structure), and where the target peptide may comprise non-natural amino acids (para [0093] "The target peptide may include modified amino acids, unmodified amino acids, heterocyclic amino acids, non-heterocyclic amino acids, naturally occurring amino acids and/or non-naturally occurring amino acids and amino acid analogues.").
Ouels discloses methods for heterocyclase modification of a substrate comprising non-alpha-amino acids and non-amino acid monomers, including aromatic rings, at positions near the enzyme reaction site (abstract "Peptidic macrocycles have attracted attention as potential therapeutics possessing cell permeability, stability, and easy-to-control variability. Here, we show that enzymes from the patellamide biosynthetic pathway can be harnessed to make macrocycles that are hybrids of amino acids and a variety of manmade chemical building blocks, including aryl rings, polyethers, and alkyl chains."; pg 12, col 1, para 1-2 "The synthetic utility of PatGmac derives precisely from its promiscuity towards the core sequence. The cyclic dehydratases, PatD and LynD, which make thiazoline and oxazoline rings from cysteine and serine (threonine), respectively, have also been shown to tolerate the presence of non-natural amino acids, but it is not known how they tolerate non-amino acid groups Nineteen analogues (8-26) were synthesized based on a common precursor peptide VGAGIGFP, in which one or more amino acids were substituted with non-natural, non-amino acid scaffolds all possessed a C-terminal AYD, the minimal recognition sequence for PatGmac. Peptides 8- 10 contain a one-residue substitution involving small non-natural amino acids b-Ala, GABA, and Doc (8-amino-3,6-dioxaoctanoic acid), respectively. Peptides 12-18 contain either an isomer of amino benzoic acid (Abz) at different positions or a ribose-derived sugar amino acid (Rib)."; Table 1 "Synthetic hybrid precursor peptides VGA-2-Abz- and their corresponding PatGmac macrocyclization products Peptide Sequence VGA-beta-Ala-IGWPAYD-Doc. 12. Since IGFPAYD"). Naismith et al. discloses using various heterocyclase and macrocyclase enzymes including LynD and PatG and where substrates may comprise non-naturally occurring amino acids, and Ouesis discloses testing activity of heterocyclase enzymes including PatG on substrates comprising the aromatic aminobenzoic acid nearby the enzyme reaction site,
it would have been obvious to one of ordinary skill in the art to modify the method, as disclosed by Naismith et al., to include or instead consider a substrate having a non-natural amino acid at a +1 site, such as an aromatic group, such as aminobenzoic acid, since this would simply allow the consideration, evaluation, and use of the heterocyclase enzymes of Naismith et al. on a peptide substrate comprising a particular non-native group, such as a synthesized peptide or protein having a group, such as aminobenzoic acid, which may be later functionalized, thereby increasing the applicability and therefore value of the methods and enzymes of Naismith et al.
Regarding Claims 17-19 Naismith et al. discloses the method of claim 1, but Naismith et al. does not specifically disclose wherein the method installs heterocyclic backbones within protein loops and linkers without disrupting the native tertiary fold. Naismith et al. does disclose where various target amino acids may be modified, including natural amino acids, at any internal position in an amino acid sequence (para [0034]-[0036] "Different cyanobacterial heterocyclases introduce heterocycles in different amino acids, so the amino acids in the target molecule that are heterocyclisable by a heterocyclase depend on which heterocyclase is employed. A heterocyclisable residue is a residue in the target molecule that the heterocyclase sequence in the modified heterocyclase is capable of converting into a heterocyclic residue. For example, modified heterocyclases comprising PatD or MicD may be used to heterocyclise Se- Cys, Cys, Thr and Ser residues in the target molecule and modified heterocyclases comprising LynD or TruD may be used to heterocyclise Cys or Se-Cys residues in the target molecule but not Thr or Ser residues. Different patterns of heterocyclic residues may therefore be produced in the same target molecule through the use of different heterocyclase sequences. The residues that are heterocyclised by the modified heterocyclase may be located at any position in the target molecule other than the C terminal. Preferably, the modified heterocyclase heterocyclises all of the residues in the target molecule that are potentially targeted by that heterocyclase."), where a target substrate may be a protein (para [0091] "Suitable target molecules may include heterocyclisable amino acids, peptides, peptide analogues.. and other biomolecules comprising heterocyclisable amino acids or amino acid analogues."; para [0096] "The target peptide lacks a leader sequence i.e. the target protein may be devoid of the amino acid sequence that is naturally located N terminal of the core cyanobactin sequence in the cyanobactin pre-pro-peptide."), and introducing heterocyclic backbones into peptides (para [0003]; para [0123]-[0124]; Note, introduction of thiazoline by MicD-F is a backbone modification, see instant application para [045]). Since Naismith et al. discloses post-translational modification of internal residues of peptides with various heterocyclases which may target various desired residues, where a target may be a protein, and introducing heterocyclic backbones into a target,
it would have been obvious to one of ordinary skill in the art to modify the method, as disclosed by Naismith et al., to include or instead use an enzyme for post-translational modification to modify an internal peptide sequence of a protein, such as any available or exposed protein surface loop or linker sequence, and to avoid changes which alter tertiary folding, so as to not disrupt protein form and function, since this would simply allow post-translational modification of a target protein using the enzymes of Naismith et al., thereby, for instance, introducing additional internal bonds and increasing protein stability. Regarding claim 19, Naismith et al. discloses the method of claim 1, but Naismith et al. does not specifically disclose wherein the enzyme (e.g. MicD) introduces an azoline heterocycle into one or more loops of a target protein (e.g. GFP) to generate proteins with altered and improved properties. Naismith et al. does disclose where various target animo acids may be modified, including natural amino acids, at any internal position in an amino acid sequence (para [0034]-[0036]), where a target substrate may be a protein (para [0091]; para [0096]), and introducing an azoline heterocycle into a peptide sequence (para [0184] "The ability of fused-MicD and fused-PatD to introduce thiazolines and oxazolines on leaderless substrates was demonstrated."; para [0033] "cysteine residues may be converted into thiazoline residues, selenocysteines may be converted into selenazoline residues, serine residues may be converted into oxazoline residues, threonine residues may be converted into oxazoline residues, and/or 2,3 diaminopropanoic acid residues may be converted into imadazoline residues by a modified heterocyclase described herein. "). Since Naismith et al. discloses post-translational modification of internal residues of peptides with various heterocyclases which may target various desired residues, where a target may be a protein, and introducing azoline heterocycles, such as thiazoline and oxazoline, into a target peptide sequence, it would have been obvious to one of ordinary skill in the art to modify the method, as disclosed by Naismith et al., to include or instead use an enzyme, such as MicD, for post-translational modification to introduce an azoline into an internal peptide sequence of a protein, such as any available or exposed protein surface loop or linker sequence, since this would simply allow post-translational modification of a target protein using the enzymes of Naismith et al. thereby, for instance, introducing additional internal bonds and increasing protein stability.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art at the time the invention was made.
Conclusion
Claims 1-19 are rejected and no claim is allowable.
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