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 .
Priority
This application is a National-Stage entry of PCT/EP2021/056026, filed 3/10/2021. Applicant’s claim for the benefit of a prior-filed application EP20200918.9 filed 10/8/2020, under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 4/7/2023; 12/9/2024; and 5/6/2025 are acknowledged. The submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, each of the information disclosure statements are being considered by the examiner.
Election/Restrictions
Applicant's election with traverse of Group I, claims 1-6 and 15-16 in the reply filed on 12/4/2025 is acknowledged. Election with traverse of the species of the invention as the variant containing the mutation X167T (i.e. N167T of SEQ ID NO:1) is acknowledged.
The traversal is on the grounds that there is no lack of unity due to lack of a shared special technical feature because the amended claims recite a sequence of the α/β hydrolase variant that is not taught in the prior art (i.e. having at least 82% identity to SEQ ID NO: 1). This is not found persuasive because the cited prior art (Fruhauf WO2015/027259) does teach a sequence having 81.2% identity to the sequence of SEQ ID NO:1 and having mutations at positions 182 and 183, which corresponds to mutations at 167 and 168 of the sequence claimed herein. Fruhauf also teaches additional mutations including N25D, S29P, R31A, R35Q, F46Y, R99K, V123I, L132V, P165H, L170R, I190V, A205S, G229A, E269H, H281R, R287E, Q296A, L307Q, of which the mutated residues correspond to residues in the instantly claimed SEQ ID NO:1. Fruhauf ’259 teaches embodiments having at least two of the mutations identified above and thus having 82% or more identity to the instant SEQ ID NO:1 (see the enzyme named “ZH1-A-006” in Table 4). Further, in considering the newly recited sequence percent identity, an amino acid sequence having 100% identical to the instantly recited SEQ ID NO:1, which corresponds to the instantly claimed parental α/β hydrolase, from Aeromicrobium endophyticum was known to the art prior to the effective fling date, as evidenced by "A0A371P0I7_9ACTN, Sequence Version 1, entered 11/7/2018” (ACCESSION: A0A371P0I7 from UniProtKB, entry version dated October 7, 2020, cited on IDS filed 5/6/2025). Thus, the putative shared technical feature cannot be considered a special technical feature in view of the knowledge in the prior art and the groups of the instant application lack unity of invention.
The restriction requirement is still deemed proper and is therefore made FINAL.
Claims 7-13 and 18 are herein withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected inventions, there currently being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 12/4/2025.
Applicant’s amendments to the claims filed 12/4/2025 are acknowledged. Claims 19-20 are newly added and directed to the elected invention.
Claims 1-6, 15-16, and 19-20 are examined on the merits herein.
Specification
The disclosure is objected to because of the following informalities:
In [0071], the term “patent” appears to be a typo for the term “parent”.
In [00131], in the first row heading of Table 4, the word “Identifikation” should be “Identification”
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code, see [0027], [0028], [0094], [0095], and “embodiment 29” on page 41. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-6, 15-16, and 19-20 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, 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.
Regarding claim 1, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). Claim 1 recites “preferably having EC:3.1.1- hydrolase activity” in line 8 and “preferably an EC:3.1.1- hydrolase activity” in line 12. The limitations following “preferably are being treated as optional for the purposes of compact prosecution.
Claim 2 recites “preferably” numerous times, e.g. in lines 4, 5, 6, 8, 15, 16, 18, 20, 21, 27-41. It is unclear whether the limitations following each use of “preferably” are required of the claim or if they are merely exemplary. The claim is rendered indefinite because the metes and bounds of the claimed composition cannot be determined.
In claim 3, the phrase “and/or” is used on page 5 of the claims, in between rows of the table. The resulting language is indefinite because it is impossible to determine what the phrase “and/or” is applying to. There is also no recitation of “and/or” after the penultimate Markush option and before the final one. Therefore, to overcome this rejection, it is recommended to move the use of “and/or” to after the second-to-last alternative, before the final one.
Claim 4 also recites “preferably having EC:3.1.1-hydrolase activity”, and is indefinite for the same reasons described above for claim 1.
Claim 5 recites “comprises or consists of a substitution/s selected from the group consisting of:” and then also recites “and/or” after the table of options i) before ii). This amounts to an improper Markush grouping, because although the language “comprises or consists of a substitution/s selected from the group consisting of:” the claim also recites “and/or”. A Markush group of alternatives requires the conjunction “and”. The use of “and/or” makes it impossible to determine the actual limits of the claim.
If the sequences of SEQ ID NOs: 2-35 are indeed identical to the 34 mutations recited in the table under “i)”, then it is highly recommended to amend the claim to remove the table and only recite “wherein said variant comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 2-35”. This also delineates the limitations of claim 5 from the limitations of claim 3 (noting that option “i)” is substantially the same as those of claim 3).
Claims 15 and 16 also recite the term “preferably” and are indefinite for all of the reasons described above. There is no way to determine the actual limits of claim protection that is sought, as the limitations following “preferably” are narrower than other portions of the claims. Therefore, one cannot determine if these are optional limitations are required of the claim.
Claims 15 and 16 are also rejected as being indefiniteness for reciting “for use in” or “for use as” without defining any practical steps of administration. It is unclear if the limitations following “for use...” are necessary structural features of the claimed protein of these are only intended use recitations. MPEP 2173.05(q) establishes that “[a]ttempts to claim a process without setting forth any steps involved in the process generally raises an issue of indefiniteness under 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, second paragraph”.
Claims 19 and 20 depend from the rejected claim 1 and are, therefore, also rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, for the reasons set forth above.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 4, 15, and 16 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 4 recites “wherein said parent α/β hydrolase, preferably having EC:3.1.1.- hydrolase activity, is selected from the group consisting of: i) a polypeptide having at least 60% sequence identity to the polypeptide of SEQ ID NO: 1; and ii) a fragment of the polypeptide of SEQ ID NO: 1, wherein said fragment has α/β hydrolase activity, preferably an EC:3.1.1.- hydrolase activity”. Claim 1, on which claim 4 depends, recites “wherein said parent α/β hydrolase, preferably having EC:3.1.1.- hydrolase activity, is selected from the group consisting of: (i) a polypeptide having at least 60% sequence identity to the polypeptide of SEQ ID NO: 1; and (ii) a fragment of the polypeptide of SEQ ID NO: 1, wherein said fragment has α/β hydrolase activity, preferably an EC:3.1.1.- hydrolase activity.”
Claim 4 fails to further limit the subject matter of the claim upon which it depends because claim 4 does not limit the claimed variant according to claim 1 in any way further than in the independent claim. The additional limitations of claim 4 only pertain to the identity of the parental enzyme and has no limitations on the variant. Further, the limitations in claim 4 appear to be identical to those regarding the parental enzyme in claim 1. To overcome this deficiency, Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
Claims 15 and 16 each recite “The variant and/or parent α/β hydrolase of claim 1”. Thus, the B.R.I. of the claims include the unmodified parent α/β hydrolase, without any of the structural features (i.e. mutations) and functional properties required of claim 1. Because the claims recite broader subject matter than the independent claim 1, drawn only to the variant α/β hydrolase, the claims each fail to include all of the limitations the claim upon which it depends.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
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 of carrying out his invention.
Claims 1-2, 4, 15-16, and 19-20 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for an α/β hydrolase variant capable of tetramerization wherein the variant polypeptide comprises at least one of the specific mutations embodied in Table 3 of the specification (i.e. the specific substitutions recited in claims 3 and 5 which correspond to SEQ ID NOs: 2-35), does not reasonably provide enablement for all α/β hydrolase variants comprising a substitution at one or more positions corresponding to positions 167, 168, 174, 218, 155, 175, 179, 182, 183, 186, 187, 268, 306, 10, 17, 28, 39, 47, 50, 57, 58, 59, 63, 65, 69, 126, 139, 225, 230, 245, 251, 281, 287, 289, 293 and 304, wherein the variant polypeptide has at least 82% sequence identity to the polypeptide of SEQ ID NO: 1.
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to produce the invention commensurate in scope with these claims.
“The test of enablement is not whether any experimentation is necessary, but whether, if experimentation is necessary, it is undue.” In re Angstadt, 537 F.2d 498, 504, 190 USPQ 214, 219 (CCPA 1976). Factors to be considered in determining whether undue experimentation is required are summarized in In re Wands (858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988)). The factors include, but are not limited to: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the inventor; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. See MPEP § 2164.01(a). The factors considered to be most relevant to the instant invention are addressed in detail below.
(A) The breadth of the claims and (B) nature of the invention: The broadest claims are drawn to a variant of a parent α/β hydrolase comprising a substitution at one or more positions corresponding to positions: 167 (which is elected), 168, 174, 218, 155, 175, 179, 182, 183, 186, 187, 268, 306, 10, 17, 28, 39, 47, 50, 57, 58, 59, 63, 65, 69, 126, 139, 225, 230, 245, 251, 281, 287, 289, 293 and 304 of SEQ ID NO: 1, wherein said variant has α/β hydrolase activity and wherein the variant is a polypeptide having at least 82% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1, wherein said variant having a hydrolase EC: 3.1.1.- activity and wherein said variant is capable of tetramerization, as recited in claim 1.
Claim 2 further recites additional functional aspects of the variant, including that the variant has an increased temperature stability compared to the parent α/β hydrolase (see option i) and see [0015] of the specification).
Claims 3 and 5 practically limit the mutations to be selected from to only those reduced to practice and thus demonstrated in the specification to fulfill the claimed functions.
Claim 4 does not practically limit the claimed variant, as discussed above.
Claims 15 and 16 each recite the variant and additional intended uses thereof, and also further expand the subject matter to include the parent enzyme, which is clearly demonstrated in the specification to be a dimer, not a tetramer (see Table 3). Claim 19 requires that the sequence is greater than 90% identical to SEQ ID NO:1. Claim 20 further limits the identity to 95%, however no specific mutations are required in claims 19-20.
It is noted that the claims are comprising-type claims and thus additional mutated positions are not excluded by the claim language. Further, there are no limitations in claims 1, 2, 4, 15-16, and 19-20 that limit the mutations at the positions 167, 168, 174, 218, 155, 175, 179, 182, 183, 186, 187, 268, 306, 10, 17, 28, 39, 47, 50, 57, 58, 59, 63, 65, 69, 126, 139, 225, 230, 245, 251, 281, 287, 289, 293 and 304. Thus, when just considering all of the possible variants with a single mutation at one of these positions (from the 19 other amino acids), there could be about 684 (36 x 19 = 684) singular mutations encompassed by these claims. If one was to consider all of the possible variants including distinct combinations of mutations, then the number of variant polypeptides encompassed by these claims is greater than 1.08 x 1046 (19^36, if any and all of the mutation sites can have any alternative amino acid). Further, if one was to consider the full extent of the comprising claim language, take the situation of a variant according to the instant claim having the X167T mutation and being 82% identical to the parent sequence of SEQ ID NO:1. Up to 55 other residues, anywhere in the sequence of the peptide, could be varied (316 x 0.18 - 1= ~55). This amounts to greater than 19^55 possible combinations (i.e. more than 2.145x1070). In either sense, the number of possible variants according to the structural limitations of the claims is extremely large. Even for the sequence limitations of claim 20, wherein 5% of the sequence can be any of the other amino acids, this variance amounts to about 8x1017 possible variants (e.g. 316 x 0.05 - 1 = ~14 and 19^14 = 8 x 10^17).
The specification at [0047] describes that “the term "variant" may refer to a polypeptide having specific activity as described herein comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.” Further, the specification describes that the wildtype parent α/β hydrolase of SEQ ID NO: 1 is capable of degrading ZEN to non-toxic HZEN but exhibits a low melting point (Tm =50C) and has a dimeric homo-dimeric quaternary structure ([0071]), not the claimed tetrameric structure. Further, the specification describes that “In the course of the present invention specific amino acid substitutions were identified in the parent α/β hydrolase having SEQ ID NO: 1 directing the quaternary structure formation of from homo-dimers to homo-tetramers and thus inter alia increasing temperature stability” ([0071]). These are further detailed in Table 3 and the examples of the instant specification ([00123]).
Generally, the nature of the invention is the specific mutations to the α/β hydrolase parent sequence of SEQ ID NO:1 which result in the functional limitations regarding tetramerization while maintaining specific activity of the α/β-hydrolases, especially towards zearalenone.
(C) The state of the prior art, (D) The relative skill of those in the art, and (E) the predictability or unpredictability of the art: The prior art discussed herein is for both determining the relative level of skill and knowledge in the art around the time of the invention and to provide an understanding of the unpredictability in the art of α/β hydrolase mutation.
Fruhauf (US PGPub No. 20160345606, the US National Stage (371) entry corresponding to WO2015/027259, of record) discloses polypeptides for the hydrolytic cleavage of zearalenone and/or at least one zearalenone derivative, having a sequence identity of at least 70% at least one of the amino acid sequences recited therein, including the sequence of SEQ ID NO:1 (Abstract, Claim 1). Fruhauf also states that these polypeptides are hydrolases, and in particular α,β-hydrolases, same as recited in the instant claims ([0032]). The sequence of the parental enzyme, SEQ ID NO:1 in Fruhauf, is 81.2% identical to the sequence of the instantly claimed α,β-hydrolase derived from SEQ ID NO:1. It is evident from sequence alignments that positions 4-312 of the instantly claimed sequence corresponds to positions 19-327 of the prior art sequence. Various combinations of possible mutations are disclosed in Table 5 of Fruhauf, however, there are no teachings regarding obtaining a variant that forms a tetramer and maintains α/β hydrolase activity as instantly recited. Fruhauf discloses that the polypeptide taught therein is thermally stable up to a temperature of 90° C ([0029]) but there are no instructions or guidance regarding what mutations may interfere with the stability.
Aleschko et al. (WO 2020025580 to ERBER AKTIENGESELLSCHAFT, herein termed “Aleschko”, on IDS filed 5/6/2025) discloses methods for increasing stability of an α /β-hydrolase, degrading zearalenone (ZEN) or treating or prophylaxing a disease (Abstract, [001]). Aleschko teaches a ‘hydropathy index’ also referred to as ‘hydropathy value’, of Kyte and Doolittle summarized therein in Table 1, which is used to predict hydrophobic or hydrophilic properties of the sidechain of an amino acid and methods of calculating temperature stability of enzymes ([0065]; [0083]-[0085]). Aleschko also teaches certain specific mutations and sequences, which are targeted to particular motifs and domains of an α/β-hydrolase, including the Vl-domain and a CAP-loop ([00164], Table 2). Aleschko teaches performing practical assays to determine specific activity and thermal stability of α /β-hydrolases that degrade ZEN and ZEN derivatives ([00181]-[00191]; FIGs 3A, 3B). There are less than 100 specific mutations taught in Aleschko, and not all of these possess the instantly claimed properties, while the parental sequences in Aleschko are distinct from that of the instantly recited SEQ ID NO:1. Aleschko does not provide any guidance pertaining to dimerization of α/β-hydrolases nor does it teach any general means for predicting the effects of mutations, without practical assays and experimentation.
Andersen (US PGPub No. 20190345469) discloses polypeptide variants having alpha-amylase activity and an improved property, such as improved specific activity, as compared to the parent polypeptide (Abstract). Andersen pertains to alpha-amylase enzymes, of the class EC 3.2.1.1, which is distinct from the instantly claimed class of hydrolases, however Andersen generally teaches improvements to enzyme thermostability ([0164]-[0167]). Andersen describes that in order to determine the thermostability of an enzyme one must incubate the polypeptide variant or the parent polypeptide in a composition at an elevated temperature, e.g. 60 degrees Celsius for a given period of time, e.g. 24 hours, and then determine the residual activity by use of an alpha-amylase activity assay ([0167]). Although teachings a variety of possible mutations, Andersen also teaches that to identify essential amino acids and potential enzyme-affecting mutations, one must perform “site-directed mutagenesis or alanine-scanning mutagenesis” ([0098]). There is no teaching or suggestion of rational ways to predict the effect of mutations on the thermostability or activity of an enzyme taught in Andersen.
Jones et al. (“Comparison of Five Protein Engineering Strategies for Stabilizing an α/β-Hydrolase”. Biochemistry. 2017;56(50):6521-6532. doi:10.1021/acs.biochem.7b00571) provides a comparison of five strategies to identify stabilizing mutations in a model α/β-hydrolase fold enzyme (Abstract). Jones teaches that the methods included a location agnostic approach (random mutagenesis using error-prone polymerase chain reaction), two structure-based approaches [computational design (Rosetta, FoldX) and mutation of flexible regions], and two sequence-based approaches (addition of proline at locations where a more stable homologue has proline and mutation to consensus) (Abstract). Jones concludes that the strategies identified stabilizing mutations, but the best balance of success rate, degree of stabilization, and ease of implementation was mutation to consensus (Abstract). Table 1 of Jones demonstrates a handful of single-amino acid substitutions that stabilize the model hydrolase. Jones states that “A two-step screen identified highly stable mutants... Those that had significantly higher thermal stability were expressed in a larger format, and the purified proteins were more thoroughly characterized, as described above. In total, we screened ∼500 combinations. This screen was far from complete, and many beneficial combinations were certainly missed.” (pg 6527, left hand col). Jones also states that “Most of these eight best substitutions were discovered by either the mutation to consensus or random mutagenesis. The other strategies (computational design, mutation of flexible regions, and addition of proline) each only identified zero or one of these top hits” (pg 6529, right hand col). Jones concludes that the computational methods performed poorly for α/β-hydrolase fold enzymes (pg. 6530, left hand col). Jones also states that “some combinations showed remarkable improvements not only over the wild type but also over any single substitution. There was no clear pattern of which combinations were highly stabilizing and which were not. All single mutations were able to combine with others to form highly stable combination mutants, yet none were found in all of the highly stable mutants. The effect of any particular mutation seems to be highly dependent on what other mutations are present. Because combining stabilizing mutations identified in a wild-type background one at a time can result in unpredictable results, a stepwise combination of identified stabilizing mutations is inadvisable...” (pg. 6530, right col). Thus, Jones provides evidence that the effects of mutations on the activity and stability of α/β-hydrolases are highly unpredictably, especially using computational models.
Further, Jones establish that combinations of mutations are even further unpredictable. In terms of the instantly claimed functional limitations, Jones provides no guidance on predicting mutations that will change a dimerizing protein to a tetramerizing one.
Bauer et al. (“The modular structure of α/β-hydrolases.” The FEBS journal vol. 287,5 (2020): 1035-1053. doi:10.1111/febs.15071) teaches that the α/β-hydrolase fold family is highly diverse in sequence, structure and biochemical function and includes 280,638 protein sequences and 1557 analyzed protein structures (Abstract). Further, Bauer teaches that there exist 12 different architectures: core domain only, additional lids at three different positions, three different caps, additional N- or C-terminal domains and combinations of N- and C-terminal domains with caps and lids respectively (Abstract, Figure 1, Table 2). Bauer provides additional evidence that discoveries and features of one particular α/β-hydrolase can be predictably transferred to those of a divergent sequence. Further, one having skill in the art would be aware of the unpredictability and large diversity in the α/β-hydrolase family of enzymes.
Since the ability to predict the effect of mutations of α/β-hydrolases, especially on combination of two or more mutations on enzyme stability and activity remains largely unsolved, means for predicting what substitutions at which residues will result in active tetramer-forming variants of the claimed sequence is highly unpredictable. The level of one skilled in the art is high when it comes to producing enzymes and performing experiments, however, it can be gleaned from the cited literature that, around the time of the instant invention, predicting effects of mutations, especially among thousands and thousands of possible substitutions, would have been well outside of the level of one skilled in the art. One skilled in the art would thus turn to the instant specification for further guidance.
(F) The amount of direction or guidance presented and (G) the existence of working examples:
The specification outlines knowledge in the art concerning determining sequence identity ([0030]-[0033]), and sets forth guidelines for conservative mutations ([0046]). These conservative mutations however are discussed in regards to the “highly conservative substitutions” described in certain aspects of the disclosure, and clearly do not amount to teachings regarding what mutations yield the claimed variant activities. As merely one example, the elected species of a variant comprising the N167T mutation is not a conservative mutation according to Table 1 and conventional wisdom in the art. Thus, this cannot be considered guidance regarding suitable mutations for fulfilling the claimed limitations.
Yet, when considered as a whole, there is no guidance in the specification that amounts to a prediction or teaching regarding how to predict which mutations at which residues in the parent sequence will result in a variant retaining activity and having the ability to form a tetramer.
The instant disclosure indicates that through experimentation, specific amino acid substitutions were identified in the parent α/β hydrolase having SEQ ID NO: 1 directing the quaternary structure formation of from homo-dimers to homo-tetramers ([0071]). There is no indication that other mutations will provide the same effect.
The specification provides working examples for approximately 34 mutated variants ([00106]-[00133]), those specific sequences with mutations outlined in Table 3 (these are also recited in claims 3 and 5). These 34 examples are stated to be tetramer-forming hydrolases, and have suitable hydrolase activity. These 34 examples are not representative of the enormous amount of possible mutations that fall within the claimed scope (greater than 1.08 x 1046 just considering the variation allowed in the specifically named residues of claim 1). The disclosure clearly teaches that these specific embodiments/examples were produced and tested to determine the claimed properties. Thus, there doesn’t appear to be any other method than producing and testing each and every possible variant according to the full claimed scope, to determine if they can form tetramers and still retain specific hydrolase activity. Further, the compounding effects of multiple mutations are well-known in the art to be incredibly challenging to predict in silico, and thus testing all possible combination of mutations would be necessary.
Thus, the specification does not provide suitable guidance such that one can envision and predict the effects of any possible mutation that falls within the scope of the claimed structure.
(H) The quantity of experimentation necessary: Due to the unpredictability in the art of mutations, especially considering the complexity of multiple mutations, one would have to produce and test each and every possible variant encompassed by the claims, in order to determine the mutation’s effects on tetramerization and enzymatic activity (as well as other effects such as thermostability).
Considering the state of the art as discussed by the cited references, especially from the discussions of Jones et al. and Bauer et al., and when considering the high unpredictability in the art and lack of guidance provided in the specification, one of ordinary skill in the art would be burdened with undue experimentation to produce each and every of the claimed sequences that are encompassed by the full scope of claims 1-2, 4, 15-16, and 19-20 in order to assay if the functional limitations (i.e. tetramerization and α/β-hydrolase activity) are met.
It is the Examiner’s position that one skilled in the art could not practice the invention commensurate in the scope of the claims without undue experimentation.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 15 and 16 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a natural product without significantly more.
Claims 15 and 16 each recite “The variant and/or parent α/β hydrolase of claim 1”. Thus, the B.R.I. of claims 15 and 16 encompass the unmodified parent α/β hydrolase, without any of the mutations and functional properties required of claim 1
The claims recite a parent α/β hydrolase of claim 1, which is a parent a/b hydrolase having at least 60% sequence identity to the polypeptide of SEQ ID NO:1, thus including the sequence that is 100% identical to SEQ ID NO:1.
For determining subject matter eligibility, the following analysis was considered, per MPEP § 2106.
Patent Eligibility Analysis Step 1: Step 1 of the eligibility analysis asks: is the claim to a process, machine, manufacture or composition of matter? Yes, the claims are directed to a composition (Claims 15 and 16, STEP 1: YES).
Patent Eligibility Analysis Step 2A Prong 1: Step 2A, prong 1 asks: does the claim recite an abstract idea, law of nature, or a natural phenomenon (product of nature)?
The instantly recited sequence of the parental enzyme is identical to a naturally occurring hydrolase from the bacteria Aeromicrobium endophyticum, as evidenced by the recitation of an identical sequence in the UniProt entry “A0A371P0I7_9ACTN”, (Sequence Version 1, entered 11/7/2018”, ACCESSION: A0A371P0I7 from UniProtKB, entry version dated 10/7/2020, cited on IDS filed 5/6/2025), which describes the wildtype sequence obtained naturally from Aeromicrobium endophyticum. The instant specification at ([0071]) also describes the “wildtype parent α/β hydrolase of SEQ ID NO: 1”. Claims 15 and 16 are so broad as to encompass the wildtype parent α/β hydrolase of Aeromicrobium endophyticum. Thus, the B.R.I. of claims 15 and 16 include limitations encompassing natural products.
In this case, the claim is clearly directed to a naturally occurring product, with no differences from the wildtype structure and therefore no markedly difference characteristic analysis is needed. See MPEP 2106.04(c).I : “Examiners should keep in mind that if the nature-based product limitation is naturally occurring, there is no need to perform the markedly different characteristics analysis because the limitation is by definition directed to a naturally occurring product and thus falls under the product of nature exception”. Regardless, in order to be markedly different, the inventor must have caused the claimed product to possess at least one characteristic that is different from that of the counterpart (MPEP § 2016.04(c).II.C.). In this case, there are no differences than the wild-type counterpart of the natural sequence.
Therefore, the claimed parental α/β hydrolase lacks markedly different characteristics, and is a product of nature exception (Claims 15 and 16, Step 2A, Prong 1: YES).
Patent Eligibility Analysis Step 2A Prong 2: Step 2A, prong 2 asks: does the claim recite additional elements that integrate the judicial exception into a practical application?
Claim 15 recites that the parent α/β hydrolase is “for use as a medicament and/or in therapy, preferably for use in the prophylaxis or treatment of a disease”.
Claim 16 recites that the parent α/β hydrolase is “for use in the prophylaxis or treatment of mycotoxicosis, preferably of hormonal disruption caused by zearalenone or a derivative thereof”.
These additional elements amount to a general recitation of an intended use. Such “use” claims fail to integrate the judicial exception into a practical application as these are merely intended uses that attempt to link the use of the judicial exception to a particular technological environment, as further described in MPEP § 2106.05(h).
There are no recited steps of administering the judicial exception, such as in a suitable pharmaceutical carrier or excipient, wherein the enzyme is provided a dose sufficient to cause a particular effect in an intended subject for which treatment is sought. See MPEP § 2106.04(d)(2) which describes that “Examiners should keep in mind that in order to qualify as a "treatment" or "prophylaxis" limitation for purposes of this consideration, the claim limitation in question must affirmatively recite an action that effects a particular treatment or prophylaxis for a disease or medical condition... If the limitation does not actually provide a treatment or prophylaxis, e.g., it is merely an intended use of the claimed invention or a field of use limitation, then it cannot integrate a judicial exception under the "treatment or prophylaxis" consideration”.
The “use of” the parental hydrolases in these additional elements does not require a particular application. Therefore, the judicial exception is not integrated into a practical application because the claims do not recite any additional elements other than the naturally-occurring products and general intended uses thereof (Claims 15 and 16, Step 2A, Prong 2: NO).
Patent Eligibility Analysis Step 2B: Does the claim as a whole recite additional elements that amount to significantly more than the judicial exception?
Claims 15 and 16 do not recite any particular treatment or prophylaxis steps, instead the claims only amount to high level “use of” claims, with no specific steps for administration and no recitation of a particular effect in a specific subject population. This amounts to intended uses that attempt to link the use of the judicial exception to a particular technological environment, as further described in MPEP § 2106.05(h), see e.g. the discussion of Mayo Collaborative Servs. v. Prometheus Labs. Inc., 566 U.S. 66, 78, 101 USPQ2d 1961, 1968 (2012) therein. None of the additional elements recited in claims 15 or 16 amount to significantly more than the ineligible natural product (i.e. the parental, unmodified α/β hydrolase).
An inventive concept "cannot be furnished by the unpatentable law of nature (or natural phenomenon or abstract idea) itself." Genetic Techs. Ltd. v. Merial LLC, 818 F.3d 1369, 1376, 118 USPQ2d 1541, 1546 (Fed. Cir. 2016). See also Alice Corp., 573 U.S. at 21-18, 110 USPQ2d at 1981 (citing Mayo, 566 U.S. at 78, 101 USPQ2d at 1968 (after determining that a claim is directed to a judicial exception, "we then ask, ‘[w]hat else is there in the claims before us?").
In the instant case, there is no inventive concept, improvement, or particular and specific application of the wildtype α/β hydrolases required by the claims at hand. When considered individually or in combination, the additional elements do not amount to significantly more than the judicial exception as these are merely intended uses.
Thus, the claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception (Claims 15 and 16, Step 2B: NO).
Because claims 15 and 16 are so broad as to encompass the unmodified parental α/β hydrolases, and recite only general “use of” applications, the claims are found to be directed to a natural product, and the claims do not qualify as eligible subject matter.
For these reasons, claims 15 and 16 are rejected under section 101 as being directed to non-statutory subject matter.
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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 15 and 16 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by "A0A371P0I7_9ACTN”, (Sequence Version 1, entered 11/7/2018”, ACCESSION: A0A371P0I7 from UniProtKB, entry version dated 10/7/2020, cited on IDS filed 5/6/2025).
The B.R.I. of claims 15 and 16 encompass an unmodified amino acid sequence having 100% identical to the instantly recited SEQ ID NO:1, with corresponds to the parental α/β hydrolase, from Aeromicrobium endophyticum.
The UniProt entry A0A371P0I7 discloses an α/β hydrolase from Aeromicrobium sp. having a sequence that is 100% identical to that of the instantly recited SEQ ID NO:1. A sequence alignment with SEQ ID NO:1 is included below:
A0A371P0I7_9ACTN
ID A0A371P0I7_9ACTN Unreviewed; 316 AA.
AC A0A371P0I7;
DT 07-NOV-2018, integrated into UniProtKB/TrEMBL.
DT 07-NOV-2018, sequence version 1.
DT 08-OCT-2025, entry version 19.
DE SubName: Full=Α/β hydrolase {ECO:0000313|EMBL:REK68866.1};
GN ORFNames=DX116_18555 {ECO:0000313|EMBL:REK68866.1};
OS Aeromicrobium endophyticum.
OC Bacteria; Bacillati; Actinomycetota; Actinomycetes; Propionibacteriales;
OC Nocardioidaceae; Aeromicrobium.
OX NCBI_TaxID=2292704 {ECO:0000313|EMBL:REK68866.1,
...
KW Hydrolase {ECO:0000313|EMBL:REK68866.1};
KW Reference proteome {ECO:0000313|Proteomes:UP000265581}.
FT DOMAIN 43..301
FT /note="AB hydrolase-1"
FT /evidence="ECO:0000259|Pfam:PF12697"
SQ SEQUENCE 316 AA; 34686 MW; 7D3887108EB69A02 CRC64;
Query Match 100.0%; Score 1669; Length 316;
Best Local Similarity 100.0%;
Matches 316; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MTDQSPETRDIFVPHAFEEQQVDLGEVVMNYVEAGSADHPALLLLPEQTGSWWSYEPVIG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MTDQSPETRDIFVPHAFEEQQVDLGEVVMNYVEAGSADHPALLLLPEQTGSWWSYEPVIG 60
Qy 61 LLAESFHVFAVDIRGQGRSTWTPKRYSLDNFGNDLVRFISLVVKRPVIVSGNSSGGVLAA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 LLAESFHVFAVDIRGQGRSTWTPKRYSLDNFGNDLVRFISLVVKRPVIVSGNSSGGVLAA 120
Qy 121 WLSAYAMPGQIRAALCEDTPFFASELVPAHGHSVRQAAGPAFELYRNFLGDQWSVADWDG 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 WLSAYAMPGQIRAALCEDTPFFASELVPAHGHSVRQAAGPAFELYRNFLGDQWSVADWDG 180
Qy 181 FAAAAKASPSKIMSLFPSSDEPPQNLKEYDPEWARAFFEGTVALHCPHDLMLSQVKTPIL 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 FAAAAKASPSKIMSLFPSSDEPPQNLKEYDPEWARAFFEGTVALHCPHDLMLSQVKTPIL 240
Qy 241 ITHHARAVDPHSGDLLGALSDFQAERAQAIIESAGVPVEYASFPDALHMMHQFEPERFAE 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 ITHHARAVDPHSGDLLGALSDFQAERAQAIIESAGVPVEYASFPDALHMMHQFEPERFAE 300
Qy 301 VLTSWVARLPADTPGA 316
||||||||||||||||
Db 301 VLTSWVARLPADTPGA 316
The additional limitations of claims 15 and 16 that appears after “for use” are merely functional language reciting intended uses of a polypeptide of the recited structure. A peptide having 100% sequence identity to the instantly recited “parental” hydrolase is encompassed in both claims 15 and 16 and was known to the art, as evidenced from the cited UniProt entry.
Therefore, the instantly claimed composition of claims 15 and 16 cannot be considered novel. MPEP 2112.01 describes that in the situation where the claimed and prior art products are identical or substantially identical in structure or composition, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977). "When the PTO shows a sound basis for believing that the products of the applicant and the prior art are the same, the applicant has the burden of showing that they are not." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990).
Any claimed functional properties of the enzyme having 100% sequence identity are clearly inclusive of those appreciated in the instant disclosure as being present (see MPEP 2112.02 at Ex parte Novitski, in reference to reference-silent functioning of biological materials providing anticipation of the functions based upon the material itself - noting the reference of “Dart” therein did not appreciate the claimed function but still anticipated the function based on the inherent function of the material, and that the Applicant’s disclosure appreciating the function upon usage thereof as further evidence of the presence of the function).
Thus, the claimed parental α/β hydrolase comprising the sequence of SEQ ID NO:1 and the therapeutic effects upon administration thereof, would have been anticipated by “A0A371P0I7”.
Please note, since the Office does not have the facilities for examining and comparing Applicants’ composition with the composition of the prior art, the burden is on applicant to show a novel or unobvious difference between the claimed product and the product of the prior art. See In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA 1977) and In re Fitzgerald, 619 F.2d 67, 205 USPQ 594 (CCPA 1980), and “as a practical matter, the Patent Office is not equipped to manufacture products by the myriad of processes put before it and then obtain prior art products and make physical comparisons therewith.” In re Brown, 459 F.2d 531, 535, 173 USPQ 685, 688 (CCPA 1972).
Conclusion
Claims 1-5, 15-16, and 19-20 are rejected.
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/A.T.M./Examiner, Art Unit 1655
/ANAND U DESAI/Supervisory Patent Examiner, Art Unit 1655