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 .
DETAILED ACTION
Status of Objections and Rejections
1. Applicant’s response filed December 29, 2025 is acknowledged. Claims 22, 24, 25-34, 36, 37 and newly added claims 41-44 are pending. Claims 1-21, 23, 35 and 38-40 are cancelled. Claims 25-34 and 37 are previously withdrawn as directed to non-elected inventions. Newly added claims 41-44 fall within the scope of elected invention. Accordingly, claims 22, 24, 36 and newly added claims 41-44 are examined on merits in the present Office action.
2. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim Objections
3. Newly added claim 44 is objected to because of the following informalities:
Newly added claim 44 is objected for reciting “consists of an amino acid sequence and a His-tag, wherein the amino acid sequence has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1”. The recitation “consists” is a closed translational phrase. This implies the single amino acid sequence. However, 95% would encompass variant sequences other than SEQ ID NO: 1. There is an issue of closed composition with broad sequence variability.
Appropriate correction is required.
Claim Rejections - 35 USC § 101
4. Claims 22, 24 and 36 remain, and newly added claims 41-44 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter. The claimed invention is directed to a judicial exception (i.e., a law of nature, a natural phenomenon, or an abstract idea) without significantly more for the reasons of record stated in the Office action mailed October 3, 2025.
Applicant traverses the rejection in the papers filed December 29, 2025.
Applicant primarily argues that (i) fungal proteins would not naturally express in bacteria, (ii) His-tag unexpectedly increases expression and insecticidal activity, and (iii) recombinant protein has different origin than fungi (pages 7-9).
Applicant’s arguments are carefully considered but are deemed to be unpersusaive. These arguments fail to overcome the §101 rejection for the following legally and technically grounded reasons:
(A) A claim is not eligible merely because expression occurs in a heterologous host
While Applicant states that “fungal proteins would not naturally express in bacteria”, the eligibility analysis does not turn on the host cell, but on whether the claimed product itself has markedly different structural or functional characteristics from the naturally occurring molecule. In the instant case, the claims still encompass the native ageritin sequence (SEQ ID NO:1 / SEQ ID NO:2) fused to a routine purification tag, without any required structural re-engineering that alters folding, active domain configuration, cleavage pattern, or molecular mechanism of action. As discussed and established in previous Office action that Applicant has not rebutted with intrinsic evidence from the specification that: (i) The specification does not define “tag” structurally (Gupta et al. could still fall within scope); (ii) The recombinant ageritin remains structurally and functionally identical to the native fungal toxin; (iii) Any activity increase is explained by purification and expression concentration, not a new mechanism introduced by the tag (Applicant admitted this); and (iv)
His-tags are routine fusion elements used for purification, not structural modifiers.
Under In re Roslin and Myriad, a recombinant protein remains a product of nature exception when it retains native sequence + native biological function, even if synthetically produced. The origin (bacterial vs fungal) is irrelevant for §101, as held in In re Roslin Institute. Thus, Step 2A = YES (judicial exception) and Step 2B = NO (no significantly more) remain unchanged.
(B) Applicant’s own specification admits His-tag does NOT change structure or mechanism
Applicant relies on expression improvement as “unexpected”, but the claims do not require any structural change, nor do they claim a new biochemical function beyond the native toxin activity. Critically, Applicant previously acknowledged: “The weaker entomotoxic activity of the untagged versus a His-tagged version can be explained by significantly lower expression in E. coli.” See specification at page 24, last two lines of paragraph 5. This is a binding admission that the His-tag did not create a new functional property, but only increased yield, which is a routine result of purification. A concentration-based improvement in a native biological assay is not a “markedly different characteristic” under §101, because the purified protein still operates exactly like the natural toxin once isolated.
(C) His-tag expression improvement is not a new functional attribute
Applicant argues that His-tag “unexpectedly increases expression”, but: (i) The claims do not require a modified expression system, codon optimization, secretion signal, chaperone co-expression, inducible cassette, or engineered promoter-tag synergy; (ii) His-tag does not change ribotoxin enzymatic function, target binding site, or mode of insecticidal action; and (iii) Expression variability from a routine tag is a host-dependent experimental observation, not a claimed functional transformation of the molecule itself. Functional changes must arise from molecular design, not experimental purification side effects.
In conclusion, Applicant has not demonstrated any claim-mandated structural modification that creates a new molecular function, nor any new mechanism beyond the natural entomotoxic activity.
Accordingly, the §101 rejection is maintained.
Claim Rejections - 35 USC § 103
5A. Claims 22, 24 and 36 remain, and newly added claims 41-44 are rejected under 35 U.S.C. 103 as being unpatentable over Gupta et al. (BMC Genomics 19:1-13, January 15, 2018), in view of Bornhorst et al. (Methods in Enzymology, 326:246-254, 2000), and further in view of Landi et al. (Biochimica et Biophysica, 1861(5), (Part A): 113-1121, May 2017) and Tegel et al. (FEBS Journal, 278:729-739, 2011) for the reasons of record stated in the Office action mailed October 3, 2025.
Applicant traverses the rejection in the papers filed December 29, 2025.
Gupta et al. teach a nucleotide sequence and encoded protein from Agrocybe aegerita having 100% amino acid sequence identity to instant SEQ ID NO: 1, and wherein the nucleotide sequence has 100% nucleotide sequence identity to instant SEQ ID NO: 2. The nucleotide sequence (ds DNA, cDNA, mRNA) disclose in the reference is a part of large BAC clone (same as vector) having native promoter and native terminator sequence (regulatory sequences) operably linked to it and cloned into a bacterial (E.coli) host. See in particular, abstract, methods, discussion and conclusions at pages 2-13; Figures 1-3, Tables 1-3. The complete nucleotide sequence is available as stated following under “availability of data and materials” at the bottom of left column at page 11:
“Availability of data and materials The nucleotide sequences of all scaffolds of all three genome-sequenced strains of A aegerita supporting the conclusions of this article are available in the European Nucleotide Archive (ENA) repository under the BioProject accession number PRJEB21917 and are also available through our A aegerita Genome Browser: http://www.thines-lab.senckenberg.de/agrocybe_genome Phylogeny data including alignments have been deposited in the TreeBASE repository under the submission ID 22045: http://purl.org/phylo/treebase/ phylows/study/TB2:S22045”
Sequence homology results are as follows:
Query Match 100.0%; Score 794; Length 156;
Best Local Similarity 100.0%;
Matches 156; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MSESSTFTTAVVPEGEGVAPMAETVQYYNSYSDASIASCAFVDSGKDKIDKTKLVTYTSR 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MSESSTFTTAVVPEGEGVAPMAETVQYYNSYSDASIASCAFVDSGKDKIDKTKLVTYTSR 60
Qy 61 LAASPAYQKVVGVGLKTAAGSIVPYVRLDMDNTGKGIHFNATKLSDSSAKLAAVLKTTVS 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 LAASPAYQKVVGVGLKTAAGSIVPYVRLDMDNTGKGIHFNATKLSDSSAKLAAVLKTTVS 120
Qy 121 MTEAQRTQLYMEYIKGIENRSAQFIWDWWRTGKAPA 156
||||||||||||||||||||||||||||||||||||
Db 121 MTEAQRTQLYMEYIKGIENRSAQFIWDWWRTGKAPA 156
Gupta et al. do no teach that their disclosed protein comprises His-tag. Gupta et al. also do not teach ageritin toxin activity of the protein having 100% identity to instant SEQ ID NO: 1.
Bornhorst et al. clearly teach that attaching affinity tags, such as polyhistidine affinity tags were well known, and routinely used in in the art prior to earliest filing date of the claimed invention to purify a protein of interest by making translational fusion with said protein of interest. As shown in the Fig. 2; a protein of interest (ERK2 protein) tagged with six N-terminal histidine residues is overexpressed in a host (E.coli), and subsequently purified by utilizing a Ni2+-NTA resin under nondenaturing conditions. See in particular, page 246 through first paragraph of page 247; Figure 2 at page 252.
Landi et al. isolation and purification of the protein taught by Gupta et al. having 100% identity to instant SEQ ID NO: 1. Gupta et al. further teach ribotoxin activity of the same protein. Landi et al. further teaches that said protein with ribotoxin activity significantly inhibited tumor growth in animal system, including humans. See in particular, abstract, results & discussion and figures 1-5).
Tegel et al. teach expressing and enhancing protein production levels in bacteria using strong promoters. See in particular, abstract, introduction, Tables 1-3, Figures 1-3, materials & methods, results and discussion.
Bornhorst et al. (see in particular pp. 246–247 and Fig. 2 at p. 252) clearly teach that affinity tags, including polyhistidine (His) tags, were well known in the art prior to the earliest effective filing date of the claimed invention. Bornhorst et al. further teach that a protein of interest may be expressed as a translational fusion with a His-tag and subsequently purified using Ni²⁺-NTA resin under non-denaturing conditions. It would have been understood by a person of ordinary skill in the art that purification of a protein obviously increases its activity in assays due to the increased concentration of the purified protein relative to crude extracts.
Accordingly, one of ordinary skill in the art would have found it obvious to express the Gupta et al. protein fused to a poly-His tag for the purpose of purification using the well-established method of Bornhorst et al. Applicant’s own specification supports this reasoning, acknowledging at page 24, paragraph 5, lines 2–3 from the bottom, that: “The weaker entomotoxic activity of the untagged versus a His-tagged version can be explained by its significantly lower expression in E. coli (Figure 4A).” This admission confirms that any observed differences in activity are attributable to expression levels rather than an unexpected property of the His-tagged protein.
Furthermore, Landi et al. teach ribotoxins and their use in treating tumor cells. Tegel et al. teach expression of ribotoxin proteins in bacterial cells under the control of strong heterologous promoters to achieve overproduction. It would have been obvious to one of ordinary skill in the art to combine these teachings with Gupta et al., by overexpressing the Gupta ribotoxin protein in a bacterial system under the control of a strong heterologous promoter, with or without a His-tag, in order to obtain high yields of protein suitable for therapeutic applications.
Thus, one of ordinary skill in the art, motivated by the therapeutic applications described by Landi et al., and employing routine techniques for expression and purification as taught by Bornhorst et al. and Tegel et al., would have arrived at the claimed invention with a reasonable expectation of success. No evidence of unexpected results is present, since the increased activity of the tagged protein is explained by increased expression and concentration, as admitted in the specification.
Accordingly, the claimed invention would have been obvious to a person of ordinary skill in the art in view of the combined teachings of Bornhorst et al., Landi et al., and Tegel et al.
It may be noted that claim 42 has been included in this rejection because claim 42 reads on any type of substitution, including conserved substitutions.
5B. Response to Applicant’s arguments:
Applicant asserts that the combination of references fails due to “unexpected expression increase”, “no motivation to combine”, “teaching away”, and “Gupta contains an intron leading to non-functional protein.” These arguments are not persuasive for the following reasons:
(i) Motivation to combine exists because protein tagging is a predictable and routine optimization
Bornhorst et al. explicitly establishes that His-tag fusion to enable Ni-NTA purification was standard practice in bacterial expression systems prior to filing. A POSITA would have been motivated to tag the Gupta protein to purify it, especially when Landi et al. and Tegel et al. demonstrate therapeutic interest in ribotoxins and bacterial overexpression systems. The combination follows the predictable workflow: (a) obtain gene sequence from fungus (Gupta et al.); (b) clone into bacterial system (Tegel et al./Tegel et al. + ERK2 example); (c) fuse His-tag (Bornhorst); (d) purify by Ni-NTA (Bornhorst et al.); and (e) test toxin activity (Landi et al. shows ribotoxin biological relevance). This is exactly the type of routine optimization KSR deems obvious, where techniques are well-known and applied for their expected purpose.
(ii) Applicant’s “unexpected result” is not commensurate with claim scope
Even if Applicant observed expression increase in one construct, POSITA would still reasonably expect successful expression, because: (i) Bornhorst et al. shows His-tag does not prevent bacterial expression, proving feasibility; (ii) Applicant admitted activity increase was due to expression level, not a new molecular property; (iii) The claims cover any ≥95% identity ageritin + His-tag, not a specific expression cassette; and (iv) No evidence shows that His-tag fusion produces an unpredictable structural change or new toxin mechanism. At best, Applicant has shown degree of expression improvement, but not failure of the prior art combination, nor any result that falls outside predictable optimization.
(iii) “Teaching away” does not apply because Bornhorst et al. do not discourage His-tags—it only states alternatives may also work
It is important to note that a reference teaches away only when it criticizes, discredits, or discourages the claimed path. Bornhorst et al. states that other tags may yield higher enrichment, but this is not a criticism of His-tags, nor a statement that His-tags are unsuitable for ribotoxins or Agrocybe proteins. It merely expresses multiple known options, which under KSR actually supports obviousness, since a POSITA would select among finite known choices. Thus, teaching away is not established.
(iv) Gupta et al. sequence containing an intron does not defeat obviousness
Applicant argues Gupta would lead to a non-functional protein, but the rejection is based on Gupta’s disclosure of the protein sequence itself (100% identical to SEQ ID NO:1), not the unspliced genomic context. A POSITA would obviously use the encoded protein sequence or spliced cDNA for expression, especially when Gupta et al. make the sequence publicly available and identifies it as a coding scaffold. There is no requirement that POSITA would naively express an unspliced genomic BAC when the reference clearly identifies the coding region. Thus, the intron argument does not negate reasonable expectation of success.
(v) Combining routine expression + purification methods is exactly what Alice/KSR/IVAX/Pfizer precedent considers obvious
Contrary to Applicant’s arguments, it is noted that Applicant has not provided evidence of: (a)structural change altering mechanism; (b) new binding domain created by tag; (iii) loss of activity in tagged version; (iv) or any unexpected result that is outside routine optimization effects.
Accordingly, the §103 rejection is fully maintained.
Conclusions
6. Claims 22, 24 and 36 remain, and newly added claims 41-44 are rejected.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Vinod Kumar whose telephone number is (571)272-4445. The examiner can normally be reached on 8:30 am - 5.00 pm.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Amjad A. Abraham can be reached on (571) 270-7058 The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/VINOD KUMAR/ Primary Examiner, Art Unit 1663