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 .
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 10/23/2025 has been entered.
Status of Claims
Receipt of Arguments/Remarks filed on 10/23/2025 is acknowledged. Claims 51-53, 55-62, and 65-71 are pending. Claims 51 and 71 were amended. Claims 67-70 are withdrawn as being directed to a non-elected invention.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 51-53, 55, 60-62, 65, and 71 are rejected under 35 U.S.C. 103 as being unpatentable over Sun et al. (CN 101172996A) in view of Kjeldsen et al., US 6,500,645 B1.
Regarding claims 51 and 71, Sun teaches a fusion protein comprising plurality of target protein sequences connected in series with every two adjacent target protein sequences connected by a linker, as indicated by the structure Z0-(Z-Z1)n, where Z0 is a first target polypeptide, Z is a linker, Z1 is a second target polypeptide, and n = 1-99 (Sun pg. 2 claim 5). Sun teaches that the linker sequence can be cleaved by protease to form target protein sequences in free form (Sun pg. 9 "Mode for carrying out the invention"). Sun further teaches that the target protein sequences do not contain cleavage sites for protease, so therefore are not cleaved by the protease (Sun pg. 2 claim 5). Sun teaches that the target polypeptide will not have any amino acids in the N-terminal and C-terminal of the target polypeptide after enzyme digestion (pg. 13 para. 1). Sun teaches that the N-terminal of expressed fusion proteins may be connected to an auxiliary peptide segment (Sun pg. 4 para. 3). Sun teaches fusion proteins with a plurality of target sequences connected via linker sequences (Sun pg. 2 claim 5). Sun teaches that the auxiliary peptide segment is a tag sequence (Sun pg. 4 para. 3). Sun teaches that the fusion protein is expressed in (obtained from) E. coli (Sun pg. 14 para. 1).
Regarding claim 52, Sun teaches that the linker peptide contains the sequence of an
enterokinase enzyme cleavage site, Kex2 enzyme cleavage site, peptidase B cleavage site, or recognition sites (Sun pg. 9 "Connecting peptide"). Sun teaches that the linker sequence is X-Arg-Y-Asp-Asp-Asp-Asp-Lys, with Y being 0-10 amino acids, meaning the linker sequence can be 8-10 amino acids in length (Sun pg. 9 "Connecting peptide"). Sun teaches that the fusion protein has the structure Z0-(Z-Z1)n, where Z0 is a first target polypeptide, Z is a linker, Z1 is a second target polypeptide, and n = 1-99, indicating that there are a plurality of linker sequences that can be the same or different (Sun pg. 2 claim 5).
Regarding claims 53 and 71, Sun teaches that the protease recognition site is KR (Lys-Arg) and that the protease is Kex2 protease (Sun pg. 9 "Connecting peptide").
Regarding claims 55 and 71, Sun teaches a linker sequence comprising a first and second protease recognition site, as it contains enterokinase, Kex2, and peptidase B (CPB) cleavage sites (Sun pg. 9 "Connecting peptide"). Sun teaches that the proteases are used jointly to cleave the fusion polypeptide, and that the cleaved target product does not contain amino acids of non-target polypeptide (Sun pg. 10 para. 5). Sun teaches that Kex2 recognizes the Lys-Arg or Arg-Arg at the N-terminus of the linker peptide and CPB hydrolyzes terminal Arg and Lys residues (Sun pg. 11 para. 3-4). Sun additionally teaches that the free form of the target polypeptide will not have any amino acids in the N-terminal and C-terminal of the target polypeptide after enzyme digestion (pg. 13 para. 1).
Regarding claims 60 and 71, Sun teaches a fusion protein wherein none of the target protein sequences have cleavage/recognition sites (Sun pg. 2 claim 5).
Regarding claim 61, the overlapping domain of the first and second protease
recognition site is interpreted to mean that the recognition sites share at least one amino acid in
common. Sun teaches a first protease recognition site of KR or RR, and a second recognition
site of terminal R or K (Sun pg. 11 para. 3-4). Therefore, the recognition sites as taught by Sun
have an overlapping domain, as they have one amino acid in common.
Regarding claims 62 and 71, Sun teaches that the amino acid sequence of the target protein does not have cleavage sites, i.e. consecutive KR or RR (Sun pg. 2 claim 5). Sun teaches that the first protease recognition site is a KR, the first protease is Kex2, the second protease recognition site is carboxyl terminal R or K and the second protease is CPB (Sun pg. 11 para. 3-4).
Regarding claims 65 and 71, Sun teaches that the tag sequence is a repeated His sequence (Sun pg. 4 para. 3).
Sun does not teach that the fusion protein comprises an expression promoting sequence with an amino acid sequence according to SEQ ID NOs: 20 or 21 as recited in claims 51 and 71.
Regarding claims 51 and 71, Kjeldsen teaches modifications of the N-terminal end of the heterologous polypeptide designed as extensions which can be cleaved off either by naturally occurring yeast proteases before purification from the culture media or by in vitro proteolysis during or subsequently to purification of the product from the culture media (Kjeldsen col. 2 lines 61-67). Kjeldsen teaches a polypeptide with the structure: signal peptide-leader peptide-X1-X2-X3-X4X5-X6-X7-heterologous protein (Kjeldsen col. 2 lines 1-4). X3 may be Glu (Kjeldsen col. 3 line 10). X4 is a sequence of amino acids with the structure (A-B)n, wherein n is 0-5, A may be Glu, and B may be Ala (Kjeldsen col. 3 lines 12-19). X5 is one or more amino acids which may be the same or different (Kjeldsen col. 3 lines 27-28). X6 may be Gly (Kjeldsen col. 3 lines 29-31).
If X3 is Glu, X4 is (Glu-Ala)3, X5 is one amino acid (Gly or Arg), and X6 is Gly, the sequence is EEAEAEAGG (SEQ ID NO: 20) or EEAEAEARG (SEQ ID NO: 21). Therefore, Kjeldsen teaches a sequence according to SEQ ID NOs: 20 or 21 that is in an auxiliary peptide segment connected to the N-terminus of the target protein sequence (heterologous protein). Kjeldsen teaches that the heterologous protein may be precursors of insulin and insulin like growth factors, and peptides of the proglucagon family (Kjeldsen col. 8 lines 40-48).
As instantly claimed, "expression-promoting" is a functional limitation of the sequence having a structure according to SEQ ID NOs: 20-21. While Kjeldsen does not expressly teach that this sequence is expression-promoting, it is expected that any fusion protein having the claimed structure, i.e. a sequence according to SEQ ID NOs: 20-21 as part of an auxiliary peptide segment connected to the N-terminus of the target protein, would have the claimed functional effect of promoting expression.
It would have been obvious to a skilled artisan, before the effective filing date, to incorporate a sequence according to SEQ ID NOs: 20 or 21 in the fusion protein sequence taught by Sun. Both Sun and Kjeldsen are directed to fusion proteins for production of polypeptides such as insulin or insulin precursors. Sun teaches that the fusion proteins have an auxiliary sequence at the N-terminus (Sun pg. 4 para. 3). The sequences EEAEAEAGG or EEAEAEARG taught by Kjeldsen are N-terminal sequences connected to the fusion protein, so it would have been obvious to a person having ordinary skill in the art that these sequences could be included in the fusion protein taught by Sun.
A person of ordinary skill in the art would have been motivated to combine these teachings because Kjeldsen teaches that the N-terminal extension sequences increase fermentation yield of the heterologous protein and protect against dipeptidyl aminopeptidase (DPAP A) processing, resulting in a homogenous N-terminal of the polypeptide (Kjeldsen col. 3 lines 40-50). It would therefore be considered advantageous to incorporate an amino acid sequence according to SEQ ID NO: 20 or 21 as part of an auxiliary peptide segment on a fusion protein as instantly claimed to achieve increased protein yield. A skilled artisan would have been motivated to utilize a sequence with Gly in position X5, which may be any amino acid, as a Gly residue in the N-terminal extension may result in improved protein yield due to the ability of glycine to adopt a wide range of conformations due to having only a hydrogen atom as a side chain, and thus could be considered advantageous to do so (Kjeldsen col. 4 lines 1-10).
A skilled artisan would have a reasonable expectation of success in incorporating a sequence according to SEQ ID NOs: 20 or 21 at the N-terminus of the target protein sequence in a fusion protein according to Sun, given the teachings of Kjeldsen that these amino acid sequences may be added as N-terminal extensions to a fusion protein sequence for producing heterologous proteins including insulin and insulin precursors. As Sun teaches a fusion protein sequence for production of insulin which comprises auxiliary sequences at the N-terminus of the target protein sequence, a skilled artisan could expect success in incorporating a sequence according to SEQ ID NOs: 20 or 21 in this fusion protein construct.
Claims 56, 58, and 59 are rejected under 35 U.S.C. 103 as being unpatentable over by Sun et al. in view of Kjeldsen et al. as applied to claims 51-53, 55, 60-62, and 65 above, and further in view of Suzuki et al. (US 5,891,671).
Sun and Kjeldsen teach the fusion protein according to claim 55 as set forth above. Sun teaches a first protease recognition site of KR for the Kex2 protease and a second protease recognition site of carboxyl terminal R or K for the CPB protease as recited in claim 59 (Sun pg. 11 para. 3-4).
Sun and Kjeldsen do not teach that the target protein sequence comprises an internal protease recognition site with an acidic amino acid adjacent to the internal protease site, and that the internal protease site is essentially not recognized by the protease as recited in claim 56. These references not teach that the consecutive acidic amino acid sequence is 1-2 amino acids with the acidic amino acid being Glu or Asp, as recited in claim 58. These references do not teach that the target protein sequences comprise consecutive DKR, DRR, DKK, or DRK as recited in claim 59.
Regarding claim 56, Suzuki teaches a chimeric protein (fusion protein) with the formula A-L-B, wherein A is a protective peptide, L is a linker, and B is a physiologically active target peptide (Suzuki col. 1 lines 50-59, col. 2 lines 60-65, col. 5 lines 11-14). Suzuki teaches that the target peptide is cleaved from the chimeric protein by Kex2 (Suzuki col. 2 lines 6-15). Suzuki further teaches a protective peptide with an internal protease recognition site (Suzuki col. 5 lines 43-47; claim 6).
Regarding claims 58-59, Suzuki teaches that the amino acids in the vicinity of the Kex2 protease recognition site greatly impact its activity (col. 6 lines 1-5). Suzuki teaches changing the amino acid adjacent to the protease recognition site to an acidic amino acid, either aspartic acid (Asp) or glutamic acid (Glu) (Suzuki col. 15 lines 38-45). Suzuki teaches that when the amino acid adjacent to the protease recognition site is changed to Glu, there is substantially lower cleavage efficiency (Suzuki Fig. 10). Suzuki further teaches that when the amino acid located two residues before the protease recognition site KR is an acidic amino acid Asp or Glu, there is no excision of the target protein, meaning the protease recognition site is not recognized (Suzuki col. 16 lines 28-44, Fig. 11). Suzuki additionally teaches a sequence comprising consecutive DKR, which has diminished protease recognition (Suzuki col. 15 Ex. 7).
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of Sun and Kjeldsen with the teachings of Suzuki to create a fusion protein wherein one of the target proteins has an internal recognition site with an adjacent acidic amino acid and is not recognized by the protease. Sun teaches fusion proteins with linker sequences, using Kex2 recognition sites for cleaving. Suzuki teaches that if the amino acids within 1-2 residues of the KR recognition site are acidic, there is significantly reduced or no cleaving activity. It would therefore be obvious to a person of ordinary skill in the art that a target protein that has an internal protease recognition site with an adjacent acidic amino acid would have reduced or zero recognition of the site by the protease.
A person of ordinary skill in the art would have been motivated to combine the teachings
of these references because it is preferable for the target protein or protective peptide to not have internal recognition sites, so that the protease will cleave at the linker cleavage site rather
than the internal site (Suzuki col. 5 lines 40-47). A person of ordinary skill would have been
motivated to incorporate a target protein with the internal recognition site having an adjacent acidic amino acid, such as DKR, with the expectation that there would be none or reduced recognition of this internal KR cleavage site, allowing instead for recognition of the linker cleavage site.
A skilled artisan would have a reasonable expectation of success in making this combination to achieve the predictable outcome of a fusion protein containing a target protein with a recognition site that is not cleaved by the protease, given the teachings of Suzuki that the
same protease, Kex2, exhibits diminished cleaving activity when there is an acidic amino acid
near the recognition site.
Claim 57 is rejected under 35 U.S.C. 103 as being unpatentable over by Sun et al. in view of Kjeldsen et al. and Suzuki et al. as applied to claims 56, 58, and 59 above, and further in view of Wang et al., (US 8,518,667 B2).
Sun, Kjeldsen, and Suzuki teach the fusion protein according to claim 56. Sun teaches that the first protease is Kex2, and that the first protease recognition site is KR or RR (Sun pg. 11 para. 3-4). These references do not teach an internal recognition site of KK or RK.
Regarding claim 57, Wang teaches fusion proteins that are cleaved by Kex2 to produce a desired target protein (Wang col. 2 lines 10-19). Wang additionally teaches that a Kex2 site refers to a two amino acid Kex2 cleavage motif in a protein, containing two contiguous basic amino acids in any order, including KK, RR, KR, or RK (Wang col. 8 lines 11-15).
It would have been obvious to a skilled artisan, before the effective filing date, to
combine the teachings of Sun, Kjeldsen, and Suzuki with the teachings of Wang, incorporating a target protein with an internal protease recognition site of KK or RK. All of these references teach the use of Kex2 in cleaving fusion proteins. Sun, Suzuki, and Wang all teach that KR and RR are Kex2 recognition sites. As Wang teaches that KK and RK are also cleavage sites for Kex2, it would have been obvious to a person of ordinary skill in the art to substitute one cleavage site for another, incorporating target protein with an internal cleavage site of KK or RK. Wang teaches that all of these sites are cleaved by Kex2, so a simple substitution of one known
cleavage site for another would be expected to yield predictable results.
Claim 66 is rejected under 35 U.S.C. 103 as being unpatentable over Sun et al. in view of Kjeldsen et al. as applied to claims 51-53, 55, 60-62, and 65 above, and further in view of Kim (US 11,267,863 B2).
Sun and Kjeldsen teach the fusion protein according to claim 51, as set forth above. Sun teaches that the target protein sequence has a length of 1-500 amino acids with the most preferable embodiment being 10-50 amino acids, which is within the claimed range of 1-100 (Sun pg. 11 "Objective polypeptide"). Sun additionally teaches that the fusion protein comprises 2-100 target polypeptide sequences, optimally 2-20, which overlaps with the claimed range of 4-16 (Sun pg. 12 "Fusion polypeptide"). Sun teaches that the target protein sequence is insulin, paraythroid hormone, osteogeneic growth peptide, or B-chorionic gonadotropin, but that the target peptide is not limited to these (Sun pg. 11 "Objective polypeptide"). Sun does not teach that the target protein is one of SEQ ID NO: 1-6.
Regarding claim 66, Kim teaches a fusion protein connected to an N-terminal fusion partner via a linker sequence (Kim col. 1 lines 58-63, col. 2 lines 4-10). Kim further teaches a GLP-1 K28R fusion polypeptide, wherein the sequence of GLP-1 K28R (SEQ ID NO: 341) is identical to instant SEQ ID NO: 1 (Kim col. 29 Example 7-1).
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of Sun, Kjeldsen, and Kim to create a fusion protein with a GLP-1 target protein sequence according to SEQ ID NO: 1. Both references recite fusion polypeptides for the
production of medically relevant proteins (Kim col. 1 "Background of the Invention"). While Sun
does not teach the use of GLP-1 as the target protein, Sun teaches that the target protein is not
limited to the examples presented. It would have been obvious to a person of ordinary skill in
the art that a GLP-1 sequence corresponding to instant SEQ ID NO: 1 could be used as a target
sequence in a fusion protein as taught by Sun, given the incorporation of GLP-1 in a similar
fusion protein taught by Kim.
A person of ordinary skill in the art would have been motivated to combine the teachings
of these references because a therapeutic agent for type 2 diabetes or obesity, liraglutide, can
be obtained by expressing the precursor peptide GLP-1 K28R, which has an identical sequence
to instant SEQ ID NO: 1 (Kim col. 14 lines 61-67, col. 15 lines 1-12). Given the therapeutic use
of this peptide it would have been of interest to produce it via a fusion protein system as taught
by Sun.
A skilled artisan would have a reasonable expectation of success in making this combination to achieve the predictable outcome of a fusion protein as taught by Sun with SEQ
ID NO: 1 as the target protein sequence, given the success of incorporating this same sequence
in a similar fusion protein as taught by Kim.
Response to Arguments
Applicant’s arguments, filed 10/23/2025, have been fully considered, and in light of amendments to the claims, the rejection of claims 51-53, 55-62, 65-66 and 71 under 35 U.S.C. § 103 over Sun et al. (CN 101172996A) in view of Kjeldsen et al., Gene; 170(1) :107-12, has been withdrawn. However, upon further consideration, new grounds of rejection of claims 51-53, 55-62, 65-66 and 71 are made under 35 U.S.C. § 103 in view of Sun et al. (CN 101172996A) and Kjeldsen et al., US 6,500,645 B1, as set forth above. Given these new grounds of rejection, the arguments presented regarding claims 51-53, 55-62, 65-66 and 71 rejected under 35 U.S.C. § 103 in view of Sun et al. (CN 101172996A) and Kjeldsen et al., Gene; 170(1) :107-12, are moot.
Regarding applicant’s statement: “It is noted that the examiner didn't raise any rejection or comment on the fusion protein comprising the expression promoting sequence of EEAEAEAGG (SEQ ID NO: 20) or EEAEAEARG (SEQ ID NO: 21). Applicant respectfully thanks the examiner for acknowledging the inventiveness of the fusion protein”, the fact that a rejection was not raised regarding SEQ ID NOs: 20 and 21 in the previous Office action does not indicate inventiveness. As previously claimed, the expression promoting sequence could be SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. In the previous rejection, SEQ ID NO: 19 was addressed, thus meeting the requirements of claim 51, which only required one of those sequences. Further, as SEQ ID NO: 19 has now been removed as an option from the claim, new prior art is applied in the rejection of claim 51, as discussed above.
Regarding arguments directed to a potential rejection over WO 95/35384 A1 and WO 2018/172921 A1, these references were not used in the rejection and therefore arguments regarding the teachings of these references are moot.
Conclusion
Claims 51-53, 55-62, 65-66, and 71 are rejected. No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMILY F EIX whose telephone number is (571)270-0808. The examiner can normally be reached M-F 8am-5pm ET.
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/EMILY F EIX/Examiner, Art Unit 1653
/JENNIFER M.H. TICHY/Primary Examiner, Art Unit 1653