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 .
Continued Examination Under 37 CFR 1.114
1. A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on June 1, 2026 has been entered.
Status of Objections and Rejections
2. Claims 22, 28, 29, 30, 31, 36, 37, 41, 42, 43 and newly added claim 45 are pending. Claims 1-21, 23-27, 32-35, 38-40 and 44 are cancelled. Claims 28-31 and 37 are previously withdrawn as directed to non-elected inventions. Newly added claim 45 falls within the scope of non-elected group IX (see restriction/election mailed June 27,2024), and is thus excluded from the present examination. Accordingly, claims 22, 36 and 41-43 are examined on merits in the present Office action.
3. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
4. Rejection of claims 22, 36 and 41-43 under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter is withdrawn in light of claim amendments and arguments filed in the papers of June 1, 2026, and upon further consideration.
Claim Rejections - 35 USC § 103
5A. Claims 22, 36 and 41-43 remain 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’s arguments have been fully considered but are not persuasive.
Applicant argues that the cited references fail to teach or suggest a bacterial host cell containing a recombinant nucleic acid molecule encoding a His-tagged entomotoxic ageritin toxin protein and further argues that the claimed host cell exhibits unexpected expression, solubility, and insecticidal activity.
These arguments are not persuasive.
Gupta et al. disclose the ageritin gene from Agrocybe aegerita and disclose the nucleotide sequence encoding ageritin as well as the encoded protein corresponding to Applicant’s SEQ ID NO:1 and SEQ ID NO:2. Gupta further identify ageritin as a ribotoxin-like protein and provide the sequence information necessary to obtain the encoded protein.
Applicant appears to argue that Gupta does not expressly disclose an intron-free bacterial expression construct and therefore fails to teach the claimed recombinant nucleic acid molecule.
Even assuming, arguendo, that Gupta’s disclosure originates from genomic DNA containing introns, Gupta et al. nevertheless expressly disclose the complete amino acid sequence of ageritin. Once a protein sequence is known, generation of a nucleic acid encoding that protein through routine molecular biology techniques, including cDNA cloning, gene synthesis, codon optimization, or back-translation of the known amino acid sequence, constituted ordinary skill in the art. Additionally, for example, in response to Applicant’s arguments, see Alberts et al. (Isolating, Cloning , and sequencing DNA; Cell, 4th edition, 2002; see the entire text, pages 491-513, Figures 8-21 to 8-44; Tables 8-7; copy enclosed) who teach that bacterial hosts such as Escherichia coli do not process eukaryotic introns and that expression of eukaryotic proteins in bacteria routinely employs intron-free complementary DNA (cDNA) prepared from mature mRNA transcripts.
Accordingly, one of ordinary skill in the art, upon identifying the ageritin gene disclosed by Gupta et al., would have recognized that bacterial expression requires an intron-free coding sequence and would have routinely generated a cDNA corresponding to the mature ageritin transcript using well-established molecular biology techniques.
Bornhorst et al. teach expressing proteins as translational fusions with polyhistidine affinity tags and further teach recombinant expression of His-tagged proteins in E. coli followed by purification using Ni2+-NTA affinity chromatography. See Bornhorst et al., pp. 246-247 and Figure 2.
Tegel et al. teach recombinant bacterial expression systems employing heterologous promoters and expression vectors for efficient overproduction of recombinant proteins in E. coli. See Tegel et al., Abstract, Introduction, Tables 1-3, and Results.
Landi et al. teach purification and biological activity of ageritin and further demonstrate that ageritin possesses ribotoxin activity. See Landi et al., Abstract, Results and Discussion, and Figures 1-5.
In view of these teachings, one of ordinary skill in the art would have been motivated to obtain an intron-free ageritin coding sequence based on the sequence information provided by Gupta et al, clone the coding sequence into a conventional bacterial expression vector under the control of a promoter and terminator as taught by Tegel et al., and express the protein as a His-tag fusion using the routine expression and purification techniques taught by Bornhorst et al.
The Supreme Court has explained that when there are a finite number of identified, predictable solutions, pursuing known options may be obvious to try and therefore obvious under §103. KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 421 (2007).
Here, once Gupta et al. disclosed the ageritin gene and encoded protein, only a limited number of conventional approaches were available for bacterial expression. These included preparing an intron-free cDNA, placing the coding sequence into a bacterial expression vector, and optionally incorporating a His-tag for purification. Each of these techniques was well established in the art and routinely used for expression of eukaryotic proteins in bacterial hosts. Accordingly, one of ordinary skill in the art would have possessed a reasonable expectation of success.
Applicant further relies upon allegedly unexpected increases in expression, solubility, and insecticidal activity.
The evidence is not persuasive because it is not commensurate in scope with the claims. Claim 22 encompasses any bacterial host cell, any promoter sequence, any terminator sequence, any recombinant construct encoding SEQ ID NO:2 or a sequence having at least 95% identity thereto, and any protein comprising SEQ ID NO:1 or a sequence having at least 95% identity thereto. Applicant has not established that all embodiments encompassed by the claims exhibit the alleged advantages.
Moreover, Applicant’s own specification states: “The weaker entomotoxic activity of the untagged versus a His-tagged version can be explained by significantly lower expression in E. coli.” This statement attributes the observed activity differences to expression level differences rather than to any newly discovered property imparted by the His-tag itself. Increased activity resulting from increased recombinant protein production would have been expected upon successful overexpression and therefore does not establish unexpected results sufficient to outweigh the strong evidence of obviousness.
Applicant additionally argues that the cited references fail to teach an insecticidal bacterial host cell.
This argument is not persuasive. The claimed insecticidal property is an inherent consequence of expressing an entomotoxic protein within a bacterial host. Where the prior art teaches the same protein and teaches routine methods for expressing that protein in bacteria, the resulting biological activity would have been expected by one of ordinary skill in the art.
Accordingly, one of ordinary skill in the art would have found it obvious to prepare an intron-free ageritin coding sequence based on Gupta et al., express the protein in a bacterial host using conventional expression systems as taught by Tegel et al. and Sambrook & Russell, and employ a His-tag fusion as taught by Bornhorst et al., with a reasonable expectation of success. Landi et al. further provide motivation to obtain recombinant ageritin because of its known biological activities and potential utility.
Conclusions
6. Claims 22, 36 and 41-43 remain rejected.
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
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