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
Priority to US 63/477,225, filed 12/27/2022 is acknowledged.
Information Disclosure Statement
No IDS is available as of 6/4/2026.
Claim Status
Claims 1-10, filed 12/26/2023, are pending. Claims 1-10 are under examination.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the specification are not identified by sequence identifiers in accordance with 37 CFR 1.821(d).
Specifically, the specification recites amino acid sequences that require sequence identifiers at page 15, para. [0065]; page 15, para. [0067]; page 15, para. [0068]; page 15, para. [0069]; page 17, para. [0073]; page 15, para. [0068]; page 29 para. [0110] (these are incorrectly labeled); page 30, para. [0116]; page 31, para. [0118]; page 35, para. [0138].
Additionally, claim 8 also lacks required sequence identifiers.
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Claim Interpretation
General note:
The specification recites: “As used herein, “a,” “an,” “the,” “at least one,” and “one or more” are used interchangeably.” in para. [0032].
The specification recites: “The term “or” as used herein may mean “and/or.”” in para. [0034]. There is no guidance on when this is to be the case, just that it is allowed.
While the Applicant is free to be their own lexicographer, these particular definitions will cause certain sentence structures to be indefinite as described below.
In order to advance compact prosecution, these terms will be given their ordinary meaning when determining claim scope for searching purposes so that a search can be conducted.
Regarding claim 1, claim 1 recites “An activatable polypeptide…”. The term “activatable” is not formally defined in the specification. Therefore, the term “activatable” is interpreted to refer to the induction of protein transcription via an inducible promoter. This interpretation is supported by para. [0111] from the specification:
” The plasmids harboring the designs of cyclized version of IL-2 were transformed into BL21(DE3) competent Cells. The intein chosen for preparing cyclized IL-2 was Npu DnaE intein. The protein expression was induced by adding 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside). After induction for 6-10 hours at 37℃, the present design brought significant expression that IL-2 was readily excised from Npu DnaE intein after expression. The present invention confirmed that the IL-2 was already cyclized through mass spectrum and NMR methods.” (Specification, para. [0111]).
The specification gives no other steps that could be fairly interpreted as “activation”.
Claim Objections
Claim 8 is objected to because of the following informalities. Claim 8 recites peptide sequences that require sequence identifier as described above in sequence deficiencies. Appropriate correction is required.
Claim Rejections - 35 USC § 112
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.
Claim 1-10 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, claim 1 recites: “An activatable polypeptide which comprises, in the N-terminal to C-terminal direction: (1) a C-intein; (2) a C-interleukin; (3) a linker; (4) a N-interleukin and (5) a N-intein, wherein the C-interleukin and the N-interleukin are obtained by splitting a target interleukin, the splitting site is a site of an amino acid with a side-chain nucleophile in the peptide sequence of the target interleukin, and the N-terminus of the C-interleukin contains the amino acid with the side-chain nucleophile.”
Because the article “a” is interchangeable with “one or more”, multiple C-interleukins or multiple N-interleukins may be present. It is not clear to which of these C-interleukins or N-interleukins to which “the C-interleukin” or “the N-interleukin” refers.
Also, “a target interleukin” may refer to multiple interleukins, so it is not clear in which interleukin the splitting site would be located.
Consequently, claim 1 is rejected.
Regarding claim 2, claim 2 recites: “The activatable polypeptide of claim 1, wherein the target protein comprises interleukin-2, interleukin-15 or interleukin-6.”
The specification recites: “The term “or” as used herein may mean “and/or.” (Specification, para. [0034]),
Because no guidance is provided on when this substitution is permitted, the broadest reasonable interpretation of this becomes “… wherein the target protein comprises interleukin-2, interleukin-15, and/or interleukin-6. “
It is not clear how the target protein can comprise all three recited interleukins at the same time.
Also, claim 2 recites the limitation "target protein" in line 1. There is insufficient antecedent basis for this limitation in the claim. It is not clear which target protein this refers to.
Lastly claim 2 does not resolve the indefiniteness of claim 1 as described above.
Consequently, claim 2 is rejected.
Regarding claim 3, claim 3 recites: “The activatable polypeptide of claim 1, wherein the target interleukin comprises the peptide sequence of SEQ ID No: 3, 4, 19 or 28.”
The specification recites: “The term “or” as used herein may mean “and/or.” (Specification, para. [0034]),
It is not clear how the target interleukin can comprise SEQ ID No: 3, 4, 19, and 28 all at the same time. If this were interpreted to mean all the amino acids of 3, 4, 19, and 28, it is not clear what order they would be in.
Also, claim 3 does not resolve the indefiniteness of claim 1 as described above.
Consequently, claim 3 is rejected.
Regarding claim 4, claim 4 recites: “The activatable polypeptide of claim 1, wherein the C-interleukin comprises the peptide sequence of SEQ ID NO: 5, and the N-interleukin comprises the peptide sequence of SEQ ID NO: 6.”
Because the article “a” is interchangeable with “one or more”, multiple C-interleukins or multiple N-interleukins may be present in claim 1. It is not clear to which of these C-interleukins or N-interleukins to which “the C-interleukin” or “the N-interleukin” in this claim refers.
Also, claim 4 does not resolve the indefiniteness of claim 1 as described above.
Consequently, claim 4 is rejected.
Regarding claim 5, claim 5 recites: “The activatable polypeptide of claim 1, wherein the C-interleukin comprises the peptide sequence of SEQ ID NO: 20, and the N-interleukin comprises the peptide sequence of SEQ ID NO: 21.”
Because the article “a” is interchangeable with “one or more”, multiple C-interleukins or multiple N-interleukins may be present in claim 1. It is not clear to which of these C-interleukins or N-interleukins to which “the C-interleukin” or “the N-interleukin” in this claim refers.
Also, claim 5 does not resolve the indefiniteness of claim 1 as described above.
Consequently, claim 5 is rejected.
Regarding claim 6, claim 6 recites: “The activatable polypeptide of claim 1, wherein the C-interleukin comprises the peptide sequence of SEQ ID NO: 29 or 31, and the N-interleukin comprises the peptide sequence of SEQ ID NO: 30 or 32.”
The specification recites: “The term “or” as used herein may mean “and/or.” (Specification, para. [0034]),
It is not clear how the C-interleukin can comprise SEQ ID No: 29 and 31 at the same time. If this were interpreted to mean both 29 and 31, it is not clear what order they would be in.
Also, claim 6 does not resolve the indefiniteness of claim 1 as described above.
Consequently, claim 6 is rejected.
Regarding claim 7, claim 7 recites: “The activatable polypeptide of claim 1, wherein the amino acid with the side-chain nucleophile comprises cysteine, serine or threonine.”
Because the article “a” is interchangeable with “one or more”, multiple amino acids with side-chain nucleophiles may be present in claim 1. It is not clear to which of these amino acids to which “the amino acid” in this claim refers.
Also, claim 7 does not resolve the indefiniteness of claim 1 as described above.
Consequently, claim 7 is rejected.
Regarding claim 8, claim 8 recites: “The activatable polypeptide of claim 1, wherein the peptide sequence of the linker comprises GSGSGS, GSGSG, GGGGG, GSGS, or GSG.”
Because the article “a” is interchangeable with “one or more”, multiple linkers may be present in claim 1. It is not clear to which of these linkers to which “the linker” in this claim refers.
Also, claim 8 does not resolve the indefiniteness of claim 1 as described above.
Consequently, claim 8 is rejected.
Regarding claim 9, claim 9 recites: “The activatable polypeptide of claim 1, which comprises the peptide sequence of SEQ ID NO: 11 to 14, 22 to 24, 33 or 34.”
The specification recites: “The term “or” as used herein may mean “and/or.” (Specification, para. [0034]),
It is not clear how the activatable polypeptide can comprise SEQ ID No: 11-14, 22-24, 33 and 34 at the same time. If this were interpreted to mean all the amino acids of 11-14, 22-24, 33 and 34, it is not clear what order they would be in.
Also, claim 9 does not resolve the indefiniteness of claim 1 as described above.
Consequently, claim 9 is rejected.
Regarding claim 10, claim 10 recites: “A cyclized polypeptide which is in a circular form comprising the peptide sequence of SEQ ID NO: 9, 10, 15 to 18, 25 to 27, 35 or 36.”
The specification recites: “The term “or” as used herein may mean “and/or.” (Specification, para. [0034]),
It is not clear how the activatable polypeptide can comprise SEQ ID No: 9, 10, 15 to 18, 25 to 27, 35 and 36 at the same time. If this were interpreted to mean all the amino acids of 9, 10, 15 to 18, 25 to 27, 35 or 36, it is not clear what order they would be in.
Also, claim 10 does not resolve the indefiniteness of claim 1 as described above.
Consequently, claim 10 is rejected.
Claim Rejections - 35 USC § 112
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 1-10 rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claims contain subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Regarding claim 1, claim 1 recites: “An activatable polypeptide which comprises, in the N-terminal to C-terminal direction: (1) a C-intein; (2) a C-interleukin; (3) a linker; (4) a N-interleukin and (5) a N-intein, wherein the C-interleukin and the N-interleukin are obtained by splitting a target interleukin, the splitting site is a site of an amino acid with a side-chain nucleophile in the peptide sequence of the target interleukin, and the N-terminus of the C-interleukin contains the amino acid with the side-chain nucleophile.”
Because the article “a” is interchangeable with “one or more”, multiple C-interleukins or multiple N-interleukins may be present.
The specification does not disclose any constructs in which multiple interleukins are expressed and/or cyclized.
In this case, the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the inventor was in possession of the claimed genus. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406. (MPEP § 2163 (II.A.3.a.ii.))
According to MPEP § 2163 (II.A.3.a.ii.), a "representative number of species" means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. See AbbVie Deutschland GmbH & Co., KG v. Janssen Biotech, Inc., 759 F.3d 1285, 1300, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014).
MPEP § 2163 (II.A.3.a.ii.) states that “for inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus. See, e.g., Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406. Instead, the disclosure must adequately reflect the structural diversity of the claimed genus, either through the disclosure of sufficient species that are ‘representative of the full variety or scope of the genus,’ or by the establishment of ‘a reasonable structure-function correlation.’”
Even when several species are disclosed, these are not necessarily representative of the entire genus. AbbVie Deutschland GMBH v. Janssen Biotech, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014) (“The ’128 and ’485 patents, however, only describe species of structurally similar antibodies that were derived from Joe-9. Although the number of the described species appears high quantitatively, the described species are all of the similar type and do not qualitatively represent other types of antibodies encompassed by the genus.”). Thus, when there is substantial variation within the genus, as here, one must describe a sufficient variety of species to reflect the variation within the genus to provide a "representative number” of species. Since each genus recited in the instant claims is large, it would be very challenging to describe sufficient species to cover the structures of the entire genus.
Applicant only discloses constructs with single interleukin expression.
At the time the invention was made, the level of skill for preparing peptides with desired functional properties was high. However, even if a synthesis and selection procedure was, at the time of the invention, sufficient to enable the skilled artisan to identify peptides that yield polypeptides with the recited properties, the written description provision of 35 U.S.C § 112 is severable from its enablement provision. Ariad Pharm., Inc. v. Eli Lilly & Co., 598 F.3d 1336 (Fed. Cir. 2010); see also Centocor Ortho Biotech Inc. v. Abbott Labs., 97 USPQ2d 1870, 1876 (Fed. Cir. 2011) (“The fact that a fully-human antibody could be made does not suffice to show that the inventors of the '775 patent possessed such an antibody.”) Absent the conserved structure (length) provided by the provided species, the skilled artisan generally would not be able to visualize or otherwise predict, a priori, what peptide with a particular set of properties would look like structurally.
Applicant discloses three constructs with single interleukins expressed.
Since only a limited number of species of peptides are taught within the claimed genus above, the instant claim above fails the written description requirement. A representative number of species has not been taught to describe this genus. Regarding the peptides, a single point mutation can change the biophysical properties of a peptide: “In summary, we have shown that the structural changes in the fibrillar state of the Aβ42 peptide that are observed to occur upon introduction of single point mutations can be accompanied by changes in the dominance of the microscopic processes by which these aggregates are themselves formed.” (Bolognesi et al. ACS Chem Bio 9:2 (2013) page 381 col. 2 para. 3) and “In summary, while ovispirin-1 and novispirin G-10 both had solution structures that were helical and amphipathic in the presence of TFE, a relatively simple change in their primary structure (a single glycine–isoleucine exchange) had profound effects on their respective toxicities for human erythrocytes and epithelial cells.” (Sawai et al. Protein Eng. 15:3 (2002) page 232 col. 1 para. 3).
Furthermore, many sequences allowed by the current scope of the claims, result in non-functional aggregates. Wang (Wang, et al. MAbs. Vol. 1. No. 3. Taylor & Francis, (2009)) discloses a variety of aggregation prone motifs that occur in commercial antibodies (Wang, page 262, Table 2). The scope of the claims currently may incorporate such motifs and result in non-functional aggregates.
Given this unpredictability of protein design, the skilled artisan would not have been in possession of the substantial repertoire of peptide species encompassed by the claimed invention; one of skill in the art would conclude that applicant was not in possession of the structural attributes of a representative number of species possessed by the members of the genus of every polynucleotide molecule recited by claim 1.
Regarding claim 2, claim 1 is rejected as described above. Claim 2 further recites: “The activatable polypeptide of claim 1, wherein the target protein comprises interleukin-2, interleukin-15 or interleukin-6.”
The specification recites: “The term “or” as used herein may mean “and/or.” (Specification, para. [0034]),
Because no guidance is provided on when this substitution is permitted, the broadest reasonable interpretation of this becomes “… wherein the target protein comprises interleukin-2, interleukin-15, and/or interleukin-6. “
For the same reasons as described above, the specification does not disclose sufficient representative examples for the case wherein multiple interleukins are expressed at once.
Additionally, this claim does not provide sufficient additional examples to resolve the rejection of claim 1.
Consequently, claim 2 is rejected.
Regarding claims 3-6, claim 1 is rejected as described above. These claims also refer to potential possible multiple interleukins. These claims are rejected for the same reasons as claims 1 and 2 as described above.
Additionally, these claims do not provide sufficient additional examples to resolve the rejection of claim 1.
Consequently, claims 3-6 are rejected.
Regarding claim 7, claim 1 is rejected as described above. Claim 7 further recites: “The activatable polypeptide of claim 1, wherein the amino acid with the side-chain nucleophile comprises cysteine, serine or threonine.”
Claim 7
Because the article “a” is interchangeable with “one or more”, multiple amino acids with side-chain nucleophiles may be present in claim 1.
Similar to the situation with multiple interleukins described above, the specification does not disclose representative examples of multiple side-chain nucleophiles.
Additionally, this claim does not provide sufficient additional examples to resolve the rejection of claim 1.
Consequently, claim 7 is rejected.
Regarding claim 8, claim 8 recites: “The activatable polypeptide of claim 1, wherein the peptide sequence of the linker comprises GSGSGS, GSGSG, GGGGG, GSGS, or GSG.”
Because the article “a” is interchangeable with “one or more”, multiple linkers may be present in claim 1.
Similar to the situation with multiple interleukins described above, the specification does not disclose representative examples of multiple linkers.
Additionally, this claim does not provide sufficient additional examples to resolve the rejection of claim 1.
Consequently, claim 8 is rejected.
Regarding claim 9, claim 9 recites: “The activatable polypeptide of claim 1, which comprises the peptide sequence of SEQ ID NO: 11 to 14, 22 to 24, 33 or 34.”
The specification recites: “The term “or” as used herein may mean “and/or.” (Specification, para. [0034]),
Similar to the situation with multiple interleukins described above, the specification does not disclose representative examples of multiple polypeptides.
Additionally, this claim does not provide sufficient additional examples to resolve the rejection of claim 1.
Consequently, claim 9 is rejected.
Regarding claim 10, claim 10 recites: “A cyclized polypeptide which is in a circular form comprising the peptide sequence of SEQ ID NO: 9, 10, 15 to 18, 25 to 27, 35 or 36.”
The specification recites: “The term “or” as used herein may mean “and/or.” (Specification, para. [0034]),
Similar to the situation with multiple interleukins described above, the specification does not disclose representative examples of multiple polypeptides.
Additionally, this claim does not provide sufficient additional examples to resolve the rejection of claim 1.
Consequently, claim 10 is rejected.
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 factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-4 and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Hayes et al. (Hayes, et al. Organic & biomolecular chemistry 19.18: 3983-4001 (2021)) in view of Scott, et al. (Scott, et al., Proceedings of the National Academy of Sciences 96.24: 13638-13643. (1999)), Briukhovetska et al., (Briukhovetska, et al. Nature Reviews Cancer 21.8: 481-499 (2021)), and Uniprot (Accession No. P60568, uploaded 3/15/2004, accessed 6/6/2026).
Regarding claim 1, claim 1 recites: “An activatable polypeptide which comprises, in the N-terminal to C-terminal direction: (1) a C-intein; (2) a C-interleukin; (3) a linker; (4) a N-interleukin and (5) a N-intein, wherein the C-interleukin and the N-interleukin are obtained by splitting a target interleukin, the splitting site is a site of an amino acid with a side-chain nucleophile in the peptide sequence of the target interleukin, and the N-terminus of the C-interleukin contains the amino acid with the side-chain nucleophile.”
Hayes discloses this scheme as a variant of the SICLOPPS cyclization scheme:
“While the majority of inteins exist in a single contiguous form, such as those employed for EPL, some inteins naturally exist as two separate polypeptide chains. These split inteins undergo a trans-splicing mechanism, in which the N- and C-intein fragments first associate to form the active complex in the correct conformation before the splicing takes place (Fig. 8c).143 A prominent example is found in the DnaE gene of cyanobacterium Nostoc punctiforme PCC73102. This split intein is known as Npu DnaE. It exhibits fast splicing kinetics (k = 3.7 × 10−2 s−1)144 and good tolerance towards extein sequence variations,145 compared to another commonly used DnaE split intein from Synechocystis sp. PCC6803 (Ssp DnaE) which is more sensitive to variation in the extein sequence around the splice junction.144,146 By correctly positioning the two fragments of a split intein at the two ends of a peptide, a cyclic peptide is generated upon intein splicing. This strategy, commonly referred to as split-intein circular ligation of peptides and proteins (SICLOPPS), has been employed for backbone cyclisation of peptides and proteins in E. coli, yeast and mammalian cells.” (Hayes et al., page 3991, col. 2, para. 4).
This is also illustrated in Fig. 8c of Hayes, albeit in the mirror image of the normal order:
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134
1174
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(Hayes et al., page 3992, Fig. 8c)
In this figure, the darker blue residues have no limitations because they are not involved in the splicing event that occurs at the intersection of the C-Extein and the N-Extein.
Additional aspects of this scheme is disclosed by Scott, upon which the Scheme of Hayes is based. Scot discloses an activatable polypeptide under the control of the arabinose promoter AraB in Fig. 2a:
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469
382
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(Scott et al., page 13639, Fig. 2).
Also note that Scott discloses presence of a side chain nucleophile immediately following the C-intein (Scott uses the traditional order C-intein -> target -> N-intein).
Briukhovetska discloses the therapeutic usage of IL-2 in cancer therapy: “IL-2 was the first interleukin to be approved for cancer treatment, although its use entails major safety concerns180. Recent advances focusing on modifying interleukins to reduce toxicity and increase the efficacy of treatment with recombinant and/or engineered interleukins might help to overcome this issue.
Interleukin-2
Although high-dose therapy with IL-2 induced durable complete responses in some patients with melanoma and RCC, it comes with potentially life-threatening adverse events. Therefore, low-dose IL-2 schedules were evaluated in several trials. However, in low abundance, IL-2 binds preferentially to its high-affinity receptor expressed on Treg cells, potentially leading to immune evasion. Thus, several IL-2 variants with increased affinity for the IL-2Rβ–γc complex expressed by CTLs are in development.
Bempegaldesleukin, a pegylated IL-2 variant, is a non-α IL-2 variant that is already in clinical trials. In addition to increasing its half-life, the polyethylene glycol (PEG) residues coupled to IL-2 block its binding site for the high-affinity IL-2Rα subunit. Over time, the PEG groups are lost and continually release the active form of bempegaldesleukin, thereby minimizing the risk of overdosing181” (Briukhovetska et al., page 492, col. 1, para. 4).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use the cyclization method disclosed by Hayes and Scott to cyclize an interleukin protein as suggested by Briukhovetska to arrive at the claimed invention.
A person of ordinary skill in the art would be motivated to create this construct in three aspects. First, Hayes discloses the well-known phenomenon of cyclization improving protein stability and resistance to proteolysis: “The cyclisation of polypeptides can play a crucial role in exerting biological functions, maintaining stability under harsh conditions and conferring proteolytic resistance, as demonstrated both in nature and in the laboratory. To date, various approaches have been reported for polypeptide cyclisation.” (Hayes, Abstract).
The second aspect is disclosed by Scott. Scott was aware that depending on the exact construction of the plasmid, any linker that was between the functional nucleophile or the functional asparagine/glutamine would be cyclized into the target sequence:
” The methotrexate-agarose eluant was fractionated by FPLC, allowing purification of 5 mg of the cyclic product per liter of culture (Fig. 3a, lane 5). Western blotting (not shown) with an
anti-His antibody indicated the presence of the polyhistidine linker sequence (Fig. 2d) in the FPLC-purified protein. The protein migrated more rapidly in SDS-PAGE analyses than did
recombinant DHFR (Fig. 3a, lane 6), despite the extra 11-amino acid linker sequence (Fig. 2b), implying an additional topological constraint. Furthermore, no reaction was detected when the
FPLC-purified protein was reacted with phenylisothiocyanate (27), suggesting that the amino terminus was unavailable.” (Scott et al., page 13461, col. 1 para. 2).
This outcome is also predictable from Fig. 1 of Scott:
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(Scott et al., page 13639m, Fig. 1)
The scheme of Hayes above would allow a person of ordinary skill in the art to always avoid the incorporation of unwanted amino acids so long as the appropriate nucleophile/asparagine junction is present in the target protein.
The junction is predictable by the understood mechanism in play: “Mechanistically, the splicing begins with N–S or N–O acyl shift leading to the formation of a (thio)ester intermediate, followed by trans(thio)esterification between N- and C-exteins resulting in a branched intermediate. Next, intein excision proceeds through asparagine (or sometimes glutamine) cyclisation before S–N or O–N acyl shift take place to form the peptide bond between the exteins. Inteins can be used to generate recombinant peptidyl fragments with a C-terminal thioester (Fig. 8b), which is required in native chemical ligation. This concept, known as expressed protein ligation (EPL), has greatly expanded the scope of native chemical ligation and has also been applied to cyclise large recombinantly produced proteins (e.g. β-lactamase).22” (Hayes et al., page 3991, col. 1, para 2).
Third, a person of ordinary skill in the art would be motivated to cyclize an interleukin with such a scheme because Briukhovetska discloses the usage of IL-2 as a therapeutic. Briukhovetska also discloses PEGylated forms and cyclization combined with PEGylation would have a reason chance of creating an even longer half-life for the IL-2 therapeutic.
A person of ordinary skill in the art would have a reasonable expectation of success because the scheme of Hayes is a modification of the well-known SICLOPPS scheme described above. Scott discloses: “The trans-splicing ability of the naturally occurring DnaE split
intein from Ssp (18) has been harnessed to create a general method for in vivo peptide and protein backbone cyclization. Expression from a construct in which the gene for any target
sequence is inserted in-frame between genes encoding the two components of the Ssp split intein (Fig. 2) results in the production of a fusion protein that spontaneously cyclizes the
target in vivo (Fig. 1). The cyclization vector is compatible with both large and small target sequences and has been optimized for versatility and yield.” (Scott et al., page 13641, col. 2, para. 2).
A person of ordinary skill in the art would expect the scheme disclosed by Hayes to proceed in the normal SICLOPPS fashion to produce the result depicted in Hayes. (Hayes et al., page 3992, Fig. 8c). IL-2 has an appropriate junction to utilize the scheme of Hayes:
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (Uniprot Accession No. P60568, uploaded 3/15/2004, accessed 6/6/2026, emphasis added).
Consequently, claim 1 is obvious over Hayes et al. in view of Scott et al., Briukhovetska et al., and Uniprot and rejected.
Regarding claim 2, claim 1 is obvious as described above. Claim 2 further recites the case wherein the target protein comprises interleukin-2, interleukin-15, or interleukin-6.
Briukhovetska discloses the usage of interleukin-2 as a therapeutic.
Consequently, claim 2 is obvious over Hayes et al. in view of Scott et al., Briukhovetska et al., and Uniprot and rejected.
Regarding claim 3, claim 1 is obvious as described above. Claim 3 further recites the case wherein the target interleukin comprises the peptide sequence of SEQ ID No: 3, 4, 19, or 28.
Applicant SEQ ID NO: 3 is aligned against human IL-2 below:
#=======================================
#
# Aligned_sequences: 2
# 1:
# 2:
# Matrix: EBLOSUM62
# Gap_penalty: 10.0
# Extend_penalty: 0.5
#
# Length: 133
# Identity: 133/133 (100.0%)
# Similarity: 133/133 (100.0%)
# Gaps: 0/133 ( 0.0%)
# Score: 680.0
#
#
#=======================================
1 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA 50
||||||||||||||||||||||||||||||||||||||||||||||||||
1 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA 50
51 TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE 100
||||||||||||||||||||||||||||||||||||||||||||||||||
51 TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE 100
101 TTFMCEYADETATIVEFLNRWITFCQSIISTLT 133
|||||||||||||||||||||||||||||||||
101 TTFMCEYADETATIVEFLNRWITFCQSIISTLT 133
(Uniprot Accession No. P60568, uploaded 3/15/2004, accessed 6/6/2026).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use the sequence disclosed by UniProt to implement the scheme of Hayes, Scott, and Briukhovetska to arrive at the claimed invention.
A person of ordinary skill in the art would be motivated to use this sequence because it is the sequence of IL-2 which is established as a therapeutic by Briukhovetska.
A person of ordinary skill in the art would have a reasonable expectation of success because as described above, the sequence of IL-2 contains at least one sequence junction that would allow the scheme of Hayes to operate correctly.
Consequently, claim 3 is obvious over Hayes et al. in view of Scott et al., Briukhovetska et al., and Uniprot and rejected.
Regarding claim 4, claim 1 is obvious as described above. Claim 4 further recites the case wherein the C-interleukin comprises the peptide sequence of SEQ ID NO: 5, and the N-interleukin comprises the peptide sequence of SEQ ID NO: 6.
SEQ ID NO: 5 is aligned against human IL-2 below:
#=======================================
#
# Aligned_sequences: 2
# 1:
# 2:
# Matrix: EBLOSUM62
# Gap_penalty: 10.0
# Extend_penalty: 0.5
#
# Length: 133
# Identity: 76/133 (57.1%)
# Similarity: 76/133 (57.1%)
# Gaps: 57/133 (42.9%)
# Score: 389.0
#
#
#=======================================
1 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA 50
0 -------------------------------------------------- 0
51 TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE 100
|||||||||||||||||||||||||||||||||||||||||||
1 -------CLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE 43
101 TTFMCEYADETATIVEFLNRWITFCQSIISTLT 133
|||||||||||||||||||||||||||||||||
44 TTFMCEYADETATIVEFLNRWITFCQSIISTLT 76
#---------------------------------------
#---------------------------------------
(Uniprot Accession No. P60568, uploaded 3/15/2004, accessed 6/6/2026).
SEQ ID NO: 6 is aligned against human IL-2 below:
#=======================================
#
# Aligned_sequences: 2
# 1:
# 2:
# Matrix: EBLOSUM62
# Gap_penalty: 10.0
# Extend_penalty: 0.5
#
# Length: 133
# Identity: 57/133 (42.9%)
# Similarity: 57/133 (42.9%)
# Gaps: 76/133 (57.1%)
# Score: 291.0
#
#
#=======================================
1 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA 50
||||||||||||||||||||||||||||||||||||||||||||||||||
1 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA 50
51 TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE 100
|||||||
51 TELKHLQ------------------------------------------- 57
101 TTFMCEYADETATIVEFLNRWITFCQSIISTLT 133
57 --------------------------------- 57
#---------------------------------------
#---------------------------------------
As described above, cutting the target protein at this location is predictable by the understood mechanism in play: “Mechanistically, the splicing begins with N–S or N–O acyl shift leading to the formation of a (thio)ester intermediate, followed by trans(thio)esterification between N- and C-exteins resulting in a branched intermediate. Next, intein excision proceeds through asparagine (or sometimes glutamine) cyclisation before S–N or O–N acyl shift take place to form the peptide bond between the exteins. Inteins can be used to generate recombinant peptidyl fragments with a C-terminal thioester (Fig. 8b), which is required in native chemical ligation. This concept, known as expressed protein ligation (EPL), has greatly expanded the scope of native chemical ligation and has also been applied to cyclise large recombinantly produced proteins (e.g. β-lactamase).22” (Hayes et al., page 3991, col. 1, para 2).
A person of ordinary skill in the art would be motivated to use these sequences because they form the sequence of IL-2 which is established as a therapeutic by Briukhovetska and these two fragments form the needed junction described by Hayes.
A person of ordinary skill in the art would have a reasonable expectation of success because as described above, these fragments are cut at a junction that would allow the scheme of Hayes to operate correctly.
Consequently, claim 4 is obvious over Hayes et al. in view of Scott et al., Briukhovetska et al., and Uniprot and rejected.
Regarding claim 7, claim 1 is obvious as described above. Claim 7 further recites the case wherein the amino acid with the side-chain nucleophile comprises cysteine, serine, or threonine.
Hayes discloses that the side nucleophile can be cysteine or serine:
PNG
media_image4.png
101
365
media_image4.png
Greyscale
PNG
media_image5.png
23
279
media_image5.png
Greyscale
(Hayes et al., page 3992, Fig. 8a)
Consequently, claim 7 is obvious over Hayes et al. in view of Scott et al., Briukhovetska et al., and Uniprot and rejected.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Hayes et al. (Hayes, et al. Organic & biomolecular chemistry 19.18: 3983-4001 (2021)) in view of Scott, et al. (Scott, et al., Proceedings of the National Academy of Sciences 96.24: 13638-13643. (1999)), Briukhovetska et al., (Briukhovetska, et al. Nature Reviews Cancer 21.8: 481-499 (2021)), and Uniprot (Accession No. P60568, uploaded 3/15/2004, accessed 6/6/2026) as applied to claim 1 above, further in view of Chichili et al. (Chichili, et al. Protein science 22.2: 153-167 (2013)).
Regarding claim 8, claim 1 is obvious as described above. Claim 8 further recites the case wherein the peptide sequence of the linker comprises GSGSGS, GSGSG, GGGGG, GSGS, or GSG.
Hayes, Scott, Briukhovetska, and Uniprot do not disclose a specific linker.
However, Chichili discloses a GGGGG linker and the general usage of poly-Gly linkers:
“Poly-Gly and Gly-rich linkers can be considered as independent units and do not affect the function of the individual proteins to which they attach. The fused proteins behave independently, such that the single chained proteins can perform the combined function of fused partners.” (Chichili et al., page 154, col. 2, para. 2).
“To facilitate the co-crystallization of the CaM-CBD peptide complex, the C-terminus of CaM was linked to the N-terminus of the CBD peptide using a five-amino acid Gly linker (GGGGG).61 Later, the same group determined the crystal structure of CaM-CBD peptide by co-crystallization in the absence of a linker (PDB: 2R28) and found that the structures of CaM-CBD peptide with and without the linker were same (Fig. 6); this result proved that the presence of the flexible Gly-rich linker did not restrain the interactions between CaM and CBD peptide.” (Chichili et al., page 162, col. 1, para. 1).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use the linker of Chichili in the activatable polypeptide construct of Hayes, Scott, Briukhovetska, and Uniprot to arrive at the claimed invention.
A person of ordinary skill in the art would be motivated to use this linker because it is extremely flexible and allows the attached protein to assume normal conformation and have a reasonable expectation of success because it has been successfully employed as a linker as disclosed by Chichili and the construct of Hayes, Scott, Briukhovetska, and Uniprot does not require any specific residues in that region (the dark blue dots from above).
Consequently, claim 8 is obvious over Hayes et al. in view of Scott et al., Briukhovetska et al., and Uniprot as applied to claim 1 above, further in view of Chichili et al. and rejected.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Hayes et al. (Hayes, et al. Organic & biomolecular chemistry 19.18: 3983-4001 (2021)) in view of Scott, et al. (Scott, et al., Proceedings of the National Academy of Sciences 96.24: 13638-13643. (1999)), Briukhovetska et al., (Briukhovetska, et al. Nature Reviews Cancer 21.8: 481-499 (2021)), and Uniprot (Accession No. P60568, uploaded 3/15/2004, accessed 6/6/2026) as applied to claim 1 above, further in view of Chichili et al. (Chichili, et al. Protein science 22.2: 153-167 (2013)), Seker et al. (Seker, et al. Langmuir 30.8: 2137-2143 (2014)), Lee et al. (Lee, et al. Protein Expression and Purification 135: 37-44 (2017)) and Iwai et al., (Iwai et al., FEBS letters 580.7: 1853-1858 (2006)).
Regarding claim 9, claim 1 is obvious as described above. Claim 9 further recites the case wherein the activatable polypeptide of claim 1 comprises the peptide sequence of SEQ ID NO: 11 to 14, 22 to 24, 33, or 34.
The polypeptide in question has five regions: (1) a C-intein; (2) a C-interleukin; (3) a linker; (4) a N-interleukin and (5) a N-intein.
For brevity, the nature of the C-interleukin region and N-interleukin region was discussed in the rejection of claim 4 above.
Regarding C-intein and N-intein, Iwai discloses the following sequence for Npu DNAe split inteins, which Hayes acknowledges as a prominently used intein (Hayes et al., page 3991, col. 2, para. 4):
PNG
media_image6.png
233
660
media_image6.png
Greyscale
(Iwai et al., page 1855, Fig. 1).
Furthermore, Seker discloses that the GSGSG linker a common flexible linker used in protein engineering: “ GSGSG peptide was used as a universal flexible linker for our designs because it is also widely used in peptide display libraries as well as in protein engineering studies.” (Seker et al., page 2138, col. 2, para. 1). This is expected, because of the properties of such linkers disclosed by Chichili: “Poly-Gly and Gly-rich linkers can be considered as independent units and do not affect the function of the individual proteins to which they attach. The fused proteins behave independently, such that the single chained proteins can perform the combined function of fused partners.” (Chichili et al., page 154, col. 2, para. 2).
Regarding the final LEHHHHHH sequence, this is just a C-terminal His-tag where an XhoI site occurs at the end of the construct: “The amino acid residues leucine and glutamate are encoded by the recognition site of XhoI in front of a repeat of six histidines, therefore an additional eight amino acid residues (LEHHHHHH) were detected at the C-terminal end of the target protein (Fig. 1, b). For modification of the His-tag, two vectors were used for expression.” (Lee et al., page 39, col. 2, para. 1).
The alignment of these individual elements is aligned against Applicant SEQ ID NO: 11 below:
#=======================================
#
# Aligned_sequences: 2
# 1:
# 2:
# Matrix: EBLOSUM62
# Gap_penalty: 10.0
# Extend_penalty: 0.5
#
# Length: 284
# Identity: 284/284 (100.0%)
# Similarity: 284/284 (100.0%)
# Gaps: 0/284 ( 0.0%)
# Score: 1492.0
#
#
#=======================================
1 MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCLEEELKPLEEVLN 50
||||||||||||||||||||||||||||||||||||||||||||||||||
1 MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCLEEELKPLEEVLN 50
51 LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRW 100
||||||||||||||||||||||||||||||||||||||||||||||||||
51 LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRW 100
101 ITFCQSIISTLTGSGSGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKN 150
||||||||||||||||||||||||||||||||||||||||||||||||||
101 ITFCQSIISTLTGSGSGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKN 150
151 PKLTRMLTFKFYMPKKATELKHLQCLSYETEILTVEYGLLPIGKIVEKRI 200
||||||||||||||||||||||||||||||||||||||||||||||||||
151 PKLTRMLTFKFYMPKKATELKHLQCLSYETEILTVEYGLLPIGKIVEKRI 200
201 ECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMT 250
||||||||||||||||||||||||||||||||||||||||||||||||||
201 ECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMT 250
251 VDGQMLPIDEIFERELDLMRVDNLPNLEHHHHHH 284
||||||||||||||||||||||||||||||||||
251 VDGQMLPIDEIFERELDLMRVDNLPNLEHHHHHH 284
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to implement the scheme of Hayes, Scott, Briukhovetska, and UniProt with the intein of Iwai, the interleukin of Briukhovetska and UniProt, using a junction suggested by Hayes, with the linker Seker (the linker position can quite literally be anything in the scheme of Hayes), and the His-tag of Lee.
A person of ordinary skill in the art would be motivated to select the Npu DnaE intein because Hayes describes its superior properties: “A prominent example is found in the DnaE gene of cyanobacterium Nostoc punctiforme PCC73102. This split intein is known as Npu DnaE. It exhibits fast splicing kinetics (k = 3.7 × 10−2 s−1)144 and good tolerance towards extein sequence variations, 145 compared to another commonly used DnaE split intein from Synechocystis sp. PCC6803 (Ssp DnaE) which is more sensitive to variation in the extein sequence around the splice junction.” (Hayes et al., page 3991, col. 2, para. 4). Iwai shows the common functional split of said intein.
Regarding the C-interleukin and N-interleukin, a person of ordinary skill in the art would use the sequence of Uniprot and select a junction compatible with the split intein system, such as QC as was the case here.
Regarding the linker, the scheme of Hayes makes it so the linker region needs no particular property. However, Seker describes the GSGSG linker as a common, “universal” flexible linker used in protein engineering: “ GSGSG peptide was used as a universal flexible linker for our designs because it is also widely used in peptide display libraries as well as in protein engineering studies.” (Seker et al., page 2138, col. 2, para. 1).
Regarding the His-tag, a person of ordinary skill in the art would include this to be able to purify the unwanted intein remains out of the reaction solution. The TEHHHHHH sequence is well-known as a C-terminal His-tag as described by Lee.
A person of ordinary skill in the art would have a reasonable expectation of success because each of these components are well-known in the art as described above and each fit into the scheme depicted by Hayes. The seemingly most critical aspect is the junction selection of QC to ensure the formation of the lactam occurs correctly as described by Scott:
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362
404
media_image7.png
Greyscale
(Scott et al., page 13369, Fig. 1).
A person of ordinary skill in the art would expect the above mechanism to proceed as normal in the scheme of Hayes, Scott, Briukhovetska, and UniProt because the reaction conditions would be present, namely a nucleophile side chain and an amide side chain.
Consequently, claim 9 is obvious over Hayes et al., Scott et al., Briukhovetska et al., and UniProt as applied to claim 1, further in view of Chichili et al., Seker et al., Lee et al. and Iwai et al. and rejected.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Hayes et al. (Hayes, et al. Organic & biomolecular chemistry 19.18: 3983-4001 (2021)) in view of Scott, et al. (Scott, et al., Proceedings of the National Academy of Sciences 96.24: 13638-13643. (1999)), Briukhovetska et al., (Briukhovetska, et al. Nature Reviews Cancer 21.8: 481-499 (2021)), Uniprot (Accession No. P60568, uploaded 3/15/2004, accessed 6/6/2026), Chichili et al. (Chichili, et al. Protein science 22.2: 153-167 (2013)), Seker et al. (Seker, et al. Langmuir 30.8: 2137-2143 (2014)), Lee et al. (Lee, et al. Protein Expression and Purification 135: 37-44 (2017)) and Iwai et al., (Iwai et al., FEBS letters 580.7: 1853-1858 (2006)).
Regarding claim 10, claim 10 recites a cyclized polypeptide which is in a circular form comprising the peptide sequence of SEQ ID NO: 9, 10, 15 to 18, 25 to 27, 35 or 36.
Applicant SEQ ID NO: 15 is aligned against the obvious construct of SEQ ID NO: 9 above.
#=======================================
#
# Aligned_sequences: 2
# 1:
# 2:
# Matrix: EBLOSUM62
# Gap_penalty: 10.0
# Extend_penalty: 0.5
#
# Length: 284
# Identity: 138/284 (48.6%)
# Similarity: 138/284 (48.6%)
# Gaps: 146/284 (51.4%)
# Score: 706.0
#
#
#=======================================
1 ------------------------------------CLEEELKPLEEVLN 14
||||||||||||||
1 MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCLEEELKPLEEVLN 50
15 LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRW 64
||||||||||||||||||||||||||||||||||||||||||||||||||
51 LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRW 100
65 ITFCQSIISTLTGSGSGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKN 114
||||||||||||||||||||||||||||||||||||||||||||||||||
101 ITFCQSIISTLTGSGSGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKN 150
115 PKLTRMLTFKFYMPKKATELKHLQ-------------------------- 138
||||||||||||||||||||||||
151 PKLTRMLTFKFYMPKKATELKHLQCLSYETEILTVEYGLLPIGKIVEKRI 200
138 -------------------------------------------------- 138
201 ECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMT 250
138 ---------------------------------- 138
251 VDGQMLPIDEIFERELDLMRVDNLPNLEHHHHHH 284
Note the insertion of the linker GSGSG as predicted by the scheme of Hayes, Scott, Briukhovetska, and UniProt:
CLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGSGSGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ
It would have been obvious to a person of ordinary skill in the art before the effective filing date to expect SEQ ID NO: 15 to be the logical result of the cyclization of the obvious polypeptide of claim 9 as described above.
A person of ordinary skill in the art would utilize the construct of claim 9 to self-cyclize (as is the entire purpose of inteins) to arrive at the sequence of claim 10 in order to introduce extras stability to the resultant polypeptide: : “The cyclisation of polypeptides can play a crucial role in exerting biological functions, maintaining stability under harsh conditions and conferring proteolytic resistance, as demonstrated both in nature and in the laboratory. To date, various approaches have been reported for polypeptide cyclisation.” (Hayes, Abstract).
A person of ordinary skill in the art would have a reasonable expectation of success because this is the logical outcome of the self-cyclization of the construct of claim 9 as described in the rejection of claim 9.
Consequently, claim 10 is obvious over Hayes et al. in view of Scott et al., Briukhovetska et al., UniProt, Chichili et al., Seker et al., Lee et al. and Iwai et al. and rejected.
Free of the Prior Art
It is the opinion of the Examiner that claims 5 and 6 are free of the prior art for the following reasons.
In terms of motivation to construct cyclized IL-15 and IL-6, Briukhovetska discloses that IL-15 is potentially therapeutic: “Preclinical studies showed that the expression of IL-15 in NKT cells led to a decreased expression of exhaustion markers, enhanced in vivo persistence, increased localization at the tumour site and improved tumour control. “ (Briukhovetska et al, page 494, col. 2, para. 3) and Johnson et al. (Johnson, et al. Nature reviews Clinical oncology 15.4: 234-248. (2018)) discloses that IL-6 is a target against which compounds would be tested: “The IL-6/JAK/STAT3 pathway is aberrantly hyperactivated in many types of cancer, and such hyperactivation is generally associated with a poor clinical prognosis. In the tumour microenvironment, IL-6/JAK/STAT3 signalling acts to drive the proliferation, survival, invasiveness, and metastasis of tumour cells, while strongly suppressing the antitumour immune response. Thus, treatments that target the IL-6/JAK/STAT3 pathway in patients with cancer are poised to provide therapeutic benefit by directly inhibiting tumour cell growth and by stimulating antitumour immunity.” (Johnson et al., Abstract).
Therefore, motivation is present to create these constructs.
However, upon examination of claim 5 sequences SEQ ID NOs: 20 and 21, and claim 6 sequences SEQ ID NOs: 29/30 and 31/32, it was found that these junctions lack the nucleophile/amide side chain combinations required by the scheme disclosed in the prior art.
Specifically, SEQ ID NOs: 20 and 21 have a cysteine-lysine junction, SEQ ID NOs: 29 and 30 have a cysteine-glycine junction and SEQ ID NOs: 31 and 32 have a cysteine-threonine junction.
Applicant shows data that these circularization reactions work in the specification in Tables 5-8. Consequently, it is the opinion of the Examiner that these particular sequence combinations represent an unexpected result and therefore are non-obvious.
Conclusion
No claim is allowed.
Claims 1-10 are rejected.
Claim 8 is objected to.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to David Paul Bowles whose telephone number is (571)272-0919. The examiner can normally be reached Monday-Friday 8:30-5:00.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Lianko Garyu can be reached on (571) 270-7367. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/DAVID PAUL BOWLES/ Examiner, Art Unit 1654
/LIANKO G GARYU/ Supervisory Patent Examiner, Art Unit 1654
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