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 .
This second non-final rejection is being sent out to the applicant since Examiner issued a first action final improperly after Applicant filed an RCE dated 1/6/2026.
Applicants’ amendment to the claims filed on 4/23/2026 is acknowledged. This listing of claims replaces all prior listings of claims in the application.
Claims 9-17, 23, 27-33, 35-37 are pending.
Claim 37 is newly added.
Claims 1-8, 18-22, 24-26, 34 are canceled
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 4/23/2026 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner.
Maintained Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The rejection of claims 9-17, 23, 27-33, 35-37 under 35 U.S.C. 103 as being unpatentable over Fleishman et al. (US 2017/0032079 A1, Date Published: Feb. 2, 2017, cited on PTO-892 filed 5/7/2024) {herein Fleishman} is maintained. The rejection has been modified in view of Applicant’s remarks that Fleishman does not make obvious a ΔΔG threshold value above zero.
Claims 9-17, 23, 27-33, 35-37 are drawn to a method for producing a plurality of non-naturally occurring polypeptide variants of an original polypeptide, comprising: (i) providing a template structure that is structurally homologous to the structure of the original polypeptide and optionally subjecting said template structure to energy minimization; (ii) providing a plurality of polypeptide sequences that are each homologous to the amino- acid sequence of the original polypeptide; (iii) within an active/binding site in said template structure identifying substitutable positions and optionally identifying unsubstitutable positions in the amino-acid sequence of the original polypeptide; (iv) for at least 2 of said substitutable residues identifying mutations comprising: a position-specific scoring matrix (PSSM) value above or equal to a threshold value, wherein said PSSM threshold value is below zero; and a ΔΔG value below or equal to threshold value, wherein said ΔΔG threshold value is above zero,(v) simultaneously permuting at least two combinations of at least two of said identified mutations, thereby obtaining a list of variants; (vi) enumerating and subjecting each of the variants to energy minimization, and ranking the variants of said list of variants by a stability score based on said energy minimization; and (vii) cloning and expressing the variants with the highest stability score in a protein expression system using a protein expression vector; thereby producing the polypeptide variants in said system.
With respect to claims 9, 27-28, 30, 33, 35, 37, Fleishman teaches a method of producing a modified polypeptide chain starting from an original polypeptide chain and selecting a protein having improved specific activity (abstract and column 1, para 0012). The method requires assembling a database of qualifying homologous amino acid sequences related to the amino acid sequence of the original polypeptide chain (para 0123). Examiner is interpreting ‘a database of qualifying homologous amino acid sequences’ to be a plurality of polypeptide sequences that are each homologous to the amino acid sequence of the original polypeptide due to the teaching of ‘qualifying homologous amino acid sequences’ by Fleishman which is indicative of more than one amino acid sequence. The position-specific stability scoring is determined for each amino acid alternative, including the original amino acid at that position, by defining a weight fitting shell around the position within which all residues are subjected to a local energy minimization (weight fitting within the weight fitting shell) to determine the lowest energy arrangement for each amino acid within the shell (para 0204). Fleishman further teaches for enzymes catalyzing reactions of substrate molecules in an active site, key residues may be selected within a radius of about 5-8 A around the substrate binding site (para 0179), of which Examiner is interpreting as being within the functional area of the binding/active site. The method includes a step that determines which of the positions in the amino-acid sequence of the original polypeptide chain will be subjected to amino-acid substitution and which amino acid alternatives will be assessed (referred to herein as substitutable positions)(para 0188). Fleishman teaches at least three substitutable positions (figures 2A, 2B, 2C, 2D). At each substitutable position only amino acids having a non-negative PSSM score (i.e. equal to or greater than 0), are subjected to the single position scanning step (para 0201). In claim 9 of the instant Application, Applicant has limited the PSSM threshold value as being below zero with a position-specific scoring matrix (PS SM) value above or equal to a threshold value, of which Fleishman teaches. Applicant’s recitation of ‘a position-specific scoring matrix (PSSM) value above or equal to a threshold value , wherein said PSSM threshold value is below zero’ within claim 9 of the instant application is broad as it encompasses any PSSM threshold value below 0, of which Fleishman teaches with the teaching of at each substitutable position only amino acids having a non-negative PSSM score (i.e. equal to or greater than 0), are subjected to the single position scanning step (para 0201). Examiner is interpreting said recitation as identifying all mutations comprising a PSSM value above or equal to the claimed PSSM threshold, including those within the first shell and active/binding site as Fleishman teaches at each substitutable position, thereby indicating the all substitutable positions were identified. The method was implemented using a minimal acceptance threshold of -0.45 r.e.u (∆ ∆G); however, if an overly-permissive acceptance threshold of zero were used, the method would have correctly predicted four additional stabilizing mutations (overall 47% true positives), and would have also predicted eight additional false-positives (i.e., overall 96% true negatives) (para 0385). It is the Examiner’s position that said teaching would provide one skilled in the art motivation to try ΔΔG values of amino acids that are above 0 as doing so identified additional stabilizing mutations (para 0385) although 8 additional false-positives was also identified (para 0385). Typically, the minimal and thus most permissive (least negative ΔΔG value) acceptance threshold can be determined in a benchmark study (para 0211). As such, it is the examiner’s position that it would be obvious to one of ordinary skill in the art to modify the ‘ΔΔG threshold value’ depending on the protein being mutated. Within the instant application, Applicant recites ‘a plurality of non-naturally occurring polypeptide variants’ which is broad. Fleishman further teaches 1-10 alternatives (para 0201). Examiner is interpreting 1-10 alternatives as at least two substitutable residues. Additionally, Fleishman teaches a method for designing a stabilized protein typically results in a modified polypeptide chain having more than 6 amino-acid substitutions with respect to the original (wildtype) polypeptide chain which have diverse physiochemical properties in various combinations (para 0094). Fleishman further teaches a combinatorial design of the entire amino acid sequence of the modified polypeptide chain, wherein numerous amino acid substitutions are simultaneously introduced to the sequence of the original polypeptide chain (para 0214). Examiner is interpreting said recitation to be synonymous to ‘simultaneously permuting combinations of said identified mutations’ as the amino acids are being simultaneously ‘permuted/substituted’ or subjected to a process of alteration in combinations. At least two of the amino acids of the substituted amino acids in the designed protein interact with one another such that the interaction stabilizes the modified protein (para 0278). The combinatorial step yields a final variant with a combination of mutations that are all compatible with one another (para 0215). A position-specific stability score is given to each of the allowed amino acid alternatives at each substitutable position (para 0189). The position-specific stability scoring is determined for each amino acid alternative, including the original amino acid at that position, by defining a weight fitting shell around the position within which all residues are subjected to a local energy minimization (weight fitting within the weight fitting shell) to determine the lowest energy arrangement for each amino acid within the shell (para 0204). Examiner is interpreting the recitations of ‘determine the lowest energy arrangement for each amino acid within the shell’ to be ranking the variants by a stability score and providing a list of variants. Three exemplary variants (stabilized PTE variants) were cloned into an expression vector and cell (para 0409). Examiner is interpreting ‘exemplary variants’ to be synonymous to those variants with the highest stability score. The polypeptide variants were purified (para 0409). The PTE variants displayed increased levels of soluble, functional enzyme compared to the reference protein (para 0412). Additionally, said variants exhibited increased metal affinity (para 0412). Examiner is interpreting an increase in metal affinity to be an increase in specific activity because metal binding can facilitate enzymatic activity.
With respect to claim 10, Fleishman teaches a method wherein a variant polypeptide exhibited a marked increase in metal affinity compared to other variants (para 0412). Examiner is interpreting the binding affinity of the metal to the protein variants to be synonymous to a ligand-binding score since the metal was shown by Fleishman to bind the protein variants differently, based on their designs. Said protein variants were selected based on their metal binding affinity (table 5).
With respect to claim 11, Fleishman teaches a position-specific stability score is given to each
of the allowed amino acid alternatives at each substitutable position (para 0189). A comprehensive list of amino acid alternatives that have a position-specific stability score below -0.45 r.e.u. (i.e., are predicted to be stabilizing) is referred to herein as the "sequence space" (para 0189). In addition, the rate at which free energy is changed is correlated to a stability score, which is referred to herein as "a position-specific stability scoring (para 0197). Polypeptide variants with the highest stability scores were selected (table 4).
With respect to claim 12, Fleishman teaches redundant sequences are clustered into a single
representative sequence (para 0127). The clustering is carried out with a threshold of 0.97, meaning that all sequences that share at least 97% identity among themselves are clustered into a single representative sequence that is the average of all the sequences contributing to the cluster (para 0127).
With respect to claim 13, Fleishman teaches at each substitutable position only amino acids having a non-negative PSSM score (i.e. equal to or greater than 0), are subjected to the single position scanning step (para 0201). Examiner is interpreting said recitation as identifying all mutation comprising a PSSM value above -2, which is the claimed PSSM threshold. Furthermore, the method was implemented using a minimal acceptance threshold of -0.45 r.e.u; however, if an overly-permissive acceptance threshold of zero were used, the method would have correctly predicted four additional stabilizing mutations (overall 47% true positives), and would have also predicted eight additional
false-positives (i.e., overall 96% true negatives). Examiner is interpreting a threshold of -0.45 r.e.u and an overly-permissive acceptance threshold to be a threshold below +6 r.e.u.
With respect to claim 14, Fleishman teaches a stabilized variant (SEQ ID NO: 7), which is a template of the original polypeptide (table 5, para 0409).
With respect to claim 15, Fleishman teaches a method wherein the amino acid sequence of the original polypeptide chain has been threaded thereon and subjected to weighted fitting to afford energy minimization (para 0109). Examiner is interpreting the original polypeptide to be the template.
With respect to claims 16 and 23, Fleishman teaches a method wherein the template structure is of a homologous protein and the query sequence is first threaded on the protein's template structure using well established computational procedures (para 0120). The structural information is a set of atomic coordinates of the original polypeptide chain (para 0109). This set of atomic coordinates is referred to herein as the "template structure" (para 0109). The template structure is a computationally generated structure based on a crystal structure of a close homolog (more than 40-60% identity) of the original polypeptide chain, wherein the amino acid sequence of the original polypeptide chain has been threaded thereon and subjected to weighted fitting to afford energy minimization thereof (para 0109).
With respect to claim 17, Fleishman teaches a method wherein the energy minimization may include iterations of rotamer sampling (repacking) followed by side chain and backbone minimization. (para 0118).
With respect to claim 29, Fleishman teaches a method wherein the position-specific stability scoring is determined for each amino acid alternative, including the original amino acid at that position, by defining a weight fitting shell around the position within which all residues are subjected to a local energy minimization (weight fitting within the weight fitting shell) to determine the lowest energy arrangement for each amino acid within the shell (para 0204). Examiner is interpreting the recitations of ‘determine the lowest energy arrangement for each amino acid within the shell’ to be ranking the variants by a stability score and providing a list of variants. Three exemplary variants (stabilized PTE variants) were cloned into an expression vector and cell (para 0396). Examiner is interpreting ‘exemplary variants’ to be synonymous to those variants with the highest stability score.
With respect to claims 31-32, Fleishman teaches a method wherein combinatorial design of the entire amino acid sequence of the modified polypeptide chain, wherein numerous amino acid substitutions are simultaneously introduced to the sequence of the original polypeptide chain (para 0214). Examiner is interpreting said recitation to be synonymous to ‘simultaneously permuting combinations of said identified mutations’ as the amino acids are being simultaneously ‘permuted/substituted’ in combinations. At least two of the amino acids of the substituted amino acids in the designed protein interact with one another such that the interaction stabilizes the modified protein (para 0278). The combinatorial step yields a final variant with a combination of mutations that are all compatible with one another (para 0215). A position-specific stability score is given to each of the allowed amino acid alternatives at each substitutable position (para 0189). The position-specific stability scoring is determined for each amino acid alternative, including the original amino acid at that position, by defining a weight fitting shell around the position within which all residues are subjected to a local energy minimization (weight fitting within the weight fitting shell) to determine the lowest energy arrangement for each amino acid within the shell (para 0204). Examiner is interpreting the recitations of ‘determine the lowest energy arrangement for each amino acid within the shell’ to be ranking the variants by a stability score and providing a list of variants. Three exemplary variants (stabilized PTE variants) were cloned into an expression vector and cell (para 0409). Examiner is interpreting ‘exemplary variants’ to be synonymous to those variants with the highest stability score. All stability scores of the variants (ΔΔG) are below -0.45 (para 0189). However, if an overly-permissive acceptance threshold
of zero were used, the method would have correctly predicted four additional stabilizing mutations (overall 47% true positives), and would have also predicted eight additional false-positives (i.e., overall 96% true negatives) (para 0385). PSSM values are at least 0 (para 0024), which is above the recited PSSM threshold value of below 0. Fleishman teaches for enzymes catalyzing reactions of substrate molecules in an active site, key residues may be selected within a radius of about 5-8 A around the substrate binding site (para 0179), of which Examiner is interpreting as being within the functional area of the binding/active site. The method includes a step that determines which of the positions in the amino-acid sequence of the original polypeptide chain will be subjected to amino-acid substitution and which amino acid alternatives will be assessed (referred to herein as substitutable positions), and in which positions in the amino acid sequence of the original polypeptide chain the amino-acid will not be subjected to amino-acid substitution (referred to herein as unsubstitutable positions) (para 0188). At each substitutable position only amino acids having a non-negative PSSM score (i.e. equal to or greater than 0), are subjected to the single position scanning step (para 0201). Examiner is interpreting said recitation as identifying all mutation comprising a PSSM value above or equal to the claims PSSM threshold, including those within the first shell and active/binding site. In addition, only amino acid alternatives that have ΔΔG values lower than this acceptance threshold will be permitted into the next step of the method (para 0209). Fleishman teaches a minimal acceptance threshold of -0.45 r.e.u ∆ ∆G (para 0211) of which Examiner is interpreting as being a ΔΔG value below a threshold value that is above 0 as recited in the instant application claims 9 and 28. Since said minimal threshold value is below the claimed threshold of above 0, Examiner is interpreting said teaching to read on claims 9 and 28, ‘a ΔΔG value below or equal to said ΔΔG threshold.’ Fleishman further teaches 1-10 alternatives (para 0201). Examiner is interpreting ‘1-10 alternatives’ as at least two substitutable residues.
With respect to claim 36, Fleishman teaches specific activity of the designed protein being higher than a specific activity of the original peptide (claim 1). Fleishman further teaches a method for designing and selecting a protein having a stabilized structure compared to a corresponding wild type
protein, and proteins having at least six amino acid substitutions with respect to a corresponding wild type protein, designed for improved thermal stability, improved specific activity (abstract).
However, Fleishman does not teach wherein said ΔΔG threshold value is above zero (claim 9).
Before the effective filing date of the claimed invention, it would have been obvious to one of ordinary skill in the art to try a ΔΔG threshold value above 0 as Fleishman teaches if an overly-permissive acceptance threshold of zero were used, the method would have correctly predicted four additional stabilizing mutations (overall 47% true positives) (para 0385).
One of ordinary skill in the art would have had a reasonable expectation of success, a reasonable level of predictability, and would be motivated to try a ΔΔG threshold value above 0 as Fleishman teaches 4 additional stabilizing mutations would have been identified if the ΔΔG threshold value were changed from -0.45 r.e.u to 0 r.e.u. As such, it would be obvious to one of ordinary skill in the art to try a threshold value above 0 as doing so may result in additional stabilizing mutations being identified. Especially since Fleishman teaches the invention, is not limited to any particular minimal acceptance threshold, and other values are contemplated within the scope of the invention (para 0211). It is the Examiner position that a threshold value above 0 would be within the scope of the invention taught by Fleishman since Fleishman teaches the unexpected result of 4 additional stabilizing mutations at a threshold value above -0.45 r.e.u. Additionally, MPEP 2144.05 states"[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (MPEP 2144.05 IIA)." One of ordinary skill would desire to optimize the acceptance threshold of the protein depending on the particular application. It would be routine for one to arrive at a ΔΔG threshold value of above 0 for the application they intend on using the polypeptides. Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
RESPONSE TO REMARKS: Beginning on p. 9 of Applicant’s remarks, Applicant contends that the rejection has been addressed by amendment. In summary, Applicant contends that The rejection is based on essentially a single comment from Fleishman which states: "In the presented studies it was found that a minimal acceptance threshold of -0.45 r.e.u is permissive enough to provide sufficient substitutable positions with sufficient amino acid alternatives substantially without introducing false positive substitutions. It is noted herein that the method, according to some embodiments of the invention, is not limited to any particular minimal acceptance threshold, and other values are contemplated within the scope of the invention." The Office Action is essentially stating that this one sentence motivates any and all threshold values, even though all it really says is that thresholds other than -0.45 may be used. The Office Action is taking this statement so broadly that this reading actually contradicts several other statements of Fleishman. Applicant contends that Applicant asserts reading this statement as including positive ΔΔG threshold and negative PSSM thresholds is a mistake in light of the other sections of Fleishman explicitly forbidding such actions. Applicant contends that the Office Action also repeatedly refers to the fact that when the threshold was moved from -0.45 to 0, Fleishman discovered "four additional stabilizing mutations". Applicants fully acknowledge that loosening a threshold will discover more mutations, but it is important to note that while finding 4 additional effective mutations Fleishman also found 8 additional false positives.
The argument is not persuasive. Examiner contends that Fleishman teaches if an overly-permissive acceptance threshold of zero were used, the method would have correctly predicted four additional stabilizing mutations (overall 47% true positives), and would have also predicted eight additional false-positives (i.e., overall 96% true negatives) (para 0385). It is the Examiner’s position that said teaching would provide one skilled in the art motivation to try ΔΔG values of amino acids that are equal to or above 0 as doing so identified additional stabilizing mutations (para 0385) although 8 additional false-positives were also identified (para 0385).
Applicant contends that It is further important to note that after reporting about the fact that only 4 out of 12 of the new mutations were stabilizing Fleishman calls the threshold of 0 "overly permissive". This means that Fleishman is teaching the reader that a threshold of zero is not a good threshold; it is too (overly) permissive. If zero is too permissive, a skilled artisan would not be motivated to select an even more permissive threshold, such as one above zero. Applicant contends that while Fleishman may say that "other values are contemplated" it does not state where these other values might be; Fleishman is just referring to values other than -0.45. This statement does not change the fact that Fleishman explicitly teaches away from a threshold of zero because it is "overly permissive". If a threshold of zero is too permissive and thus to be avoided, certainly a threshold above 0 is also too permissive and thus to be avoided. Fleishman thus teaches away from a threshold above 0.
The argument is not persuasive. Examiner contends that Applicant’s claim limitations recite a ΔΔG threshold value above 0 which encompasses a ΔΔG threshold value as small is 0.1. Examiner suggests that Applicant recite a specific ΔΔG threshold value that is above 0.
Conclusion
Status of the claims:
Claims 9-17, 23, 27-33, 35-37 are pending.
Claims 1-8, 18-22, 24-26, 34 are canceled.
Claims 9-17, 23, 27-33, 35-37 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ERICA NICOLE JONES-FOSTER whose telephone number is (571)270-0360. The examiner can normally be reached mf 7:30a - 4:30p.
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/ERICA NICOLE JONES-FOSTER/Examiner, Art Unit 1656
/MANJUNATH N RAO/Supervisory Patent Examiner, Art Unit 1656
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