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 .
Status of Claims
Claims 1, 3-7 and 11-27 are pending following the Reply filed 05/12/2026. Claims 1, 3, 16 and 18 have been amended without adding new matter. Claims 23-27 are withdrawn. Claims 1, 3-7 and 11-22 have been examined on the merits.
Withdrawn
The objection to claim 1 is withdrawn in light of the amendments.
The rejection of claim 1 under 35 U.S.C. 112(b) is withdrawn in light of the amendments.
Maintained Rejections and New Rejections Necessitated by Amendment
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 6 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 4 recites the fusion protein of claim 1, wherein the peptide linker 1 is a peptide consisting of 3 to 24 amino acids. This fails to further limit the subject matter of claim 1, because claim 1 recites the peptide linker 1 is a polypeptide represented by one of the amino acid sequences selected from SEQ ID NOs 6 to 11. These sequences are, respectively, 20, 18, 16, 14, 12, and 10 amino acids in length, while the claim broadens this scope to sequences that are up to 24 amino acids in length.
Claim 6 recites the fusion protein of claim 1, wherein the peptide linker 1 is a polypeptide represented by one of the amino acid sequences selected from SEQ ID NOs 5 to 11. This fails to further limit the subject matter of claim 1, because claim 1 recites the peptide linker 1 is a polypeptide represented by one of the amino acid sequences selected from SEQ ID NOs 6 to 11.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
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, 3-7 and 13-22 are rejected under 35 U.S.C. 103 as being unpatentable over Park (US 9,498,519 B2; previously cited), and further in view of Yan (US 20030017991 A1; previously cited).
Regarding claim 1, Park teaches a pharmaceutical composition for preventing or treating an amyloid β-caused disease, which comprises as an active ingredient an NIa (nuclear inclusion a) protease, effective to treat a variety of diseases or disorders including Alzheimer’s disease (see Abstract), wherein the NIa protease may be fused to a protein transduction domain (PTD) for effectively penetrating cells, which may include a growth factor signal peptide sequence (see col. 5, lines 26-28 and 31-33). Hence, Park teaches a fusion protein comprising an NIa protease (A). Additionally, Park teaches that accumulating evidence suggests that intracellular amyloid β (Aβ) is critical for the development of Alzheimer’s disease, as it has been found that Aβ is present in a diverse set of subcellular organelles, including early endosomes, the trans-Golgi network, the rough endoplasmic reticulum, the outer mitochondrial membrane, and the nuclear envelope (see col. 1, lines 45-46 and 50-54). Park teaches the NIa protease cleaves intracellular or extracellular amyloid β (see claim 1), therefore having “an activity to degrade amyloid β”.
Regarding the limitation, wherein “the NIa protease has any one of the amino acid sequences represented by SEQ ID NOs: 13 and 15”, Park teaches the NIa protease derived from TuMV consists of the amino acid sequence of SEQ ID NO: 1 (see claim 1). As shown in the following alignment, Park’s SEQ ID NO: 1 (bottom) comprises (“has”) the full-length of instant SEQ ID NO: 13 (top):
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Park does not teach the fusion protein comprising a signal sequence (X), a first peptide linker (L1), a transmembrane domain (Y), a second peptide linker (L2), and an intracellular domain (Z) (i.e., X-L1-A-L2-Y-Z).
Yan teaches novel compositions for monitoring the β-secretase activity of human Asp2 protease, useful in the identification of agents that modulate β-secretase activity and the therapeutic intervention of disorders characterized by the presence of amyloid plaques (see pg. 1, para. [0002]), including Alzheimer’s disease (see pg. 1, para. [0003]). It is understood in view of Yan’s disclosure that “Asp2 protease” is a “β-secretase” which Yan teaches is also a membrane-bound aspartyl protease (pg. 1, para. [0006]). Yan teaches that amyloid beta (Aβ) is the primary component of amyloid plaques (see pg. 1, para. [0003]) and is produced from the cleavage of amyloid protein precursor (APP) at the β-secretase cleavage site by human aspartyl protease (Hu-Asp2) (see pg. 1, col. 2, para. [0006]). Yan teaches that the amyloid protein precursor (APP) localizes to the secretory membrane structure including the cell surface (see pg. 1, para [0004]), and its β-secretase cleavage site (an Asp2 target) is located near the plasma membrane luminal surface and as such is a favored therapeutic target (see pg. 1, para. [0005]). Yan also teaches that the processing of the beta-secretase site can occur in both the endoplasmic reticulum (in neurons) and in the endosomal/lysosomal pathway after re-internalization of cell surface APP (in all cells) (see pg. 1, para. [0005]).
Yan teaches the Asp2 amino acid sequence (beta-secretase) comprises a putative signal peptide comprising residues 1 to 21 (i.e., a signal sequence (X) at the N-terminus), a transmembrane domain (Y) comprising residues 455-477, and a “cytoplasmic domain” comprising residues 478-501, (i.e., an intracellular domain (Z) at the C-terminus) (see pg. 14, para. [0110]).
Yan also teaches the design of fusion proteins, wherein such fusions include the linking of functional domains, such as the active sites from enzymes, cellular targeting signals or transmembrane regions (see pg. 11, para. [0085]). Yan teaches the transmembrane domain (Y) component of the fusion polypeptide can target and locate to the Golgi or endoplasmic reticulum of a given cell (see pg. 11, para. [0087]).
Therefore, a person of skill would have recognized from Yan that fusion proteins containing the active site of other enzymes can be constructed to localize their catalytic activity to desired therapeutic targets and would have envisaged simply replacing the active site of a beta-secretase with another enzyme, such as the NIa protease taught by park, to localize the NIa to the secretory membrane structures of a cell (Golgi network, the rough endoplasmic reticulum). Both Park and Yan teaches these structures to harbor amyloid proteins, including amyloid-beta, which Park teaches can be degraded by NIa. The obtained fusion protein would necessarily have a signal sequence (X), transmembrane domain (Y) and an intracellular domain (Z) derived from beta-secretase.
Regarding the peptide linkers L1 and L2, Yan teaches fusion polypeptides further comprising a transmembrane domain which may be separated by a linker (see pg. 3, para. [0023]). Yan teaches that the transmembrane domain anchors the polypeptide to an intracellular membrane, such as the Golgi or the endoplasmic reticulum (see pg. 3, para. [0023]). This reads on the limitation of “L2”. Regarding the limitation of a first peptide linker (L1) represented by one of the amino acid sequences selected from SEQ ID NOs 6 to 11, Yan suggests peptide linkers may be used to fuse separate elements of a fusion polypeptide, as discussed above. As shown in the following alignment, instant SEQ ID NO: 6 (top) is identical to Yan’s SEQ ID NO: 2 from residues 144 to 163 (bottom):
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156
624
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In view of the instant specification: “The linker merely has a role of linking these monomers, and it has a very limited effect on the structure and impact” (see pg. 7, lines 20-22). The Examples of Applicant’s disclosure do not state which linker regions were used, only that “SAS was prepared by fusing the N-terminus and C-terminus of NIa with the pro domain and the transmembrane domain of β-secretase” (see pg. 25, lines 1-2). Therefore, there is no evidence that the structure of said element was critical to the results achieved by the inventors. As the linker merely serves the purpose of attaching different segments of a fusion protein, and is not intended to have any impact on the structure or function of the protein, it would have been obvious to have simply included this segment of the β-secretase, or any other segment of the β-secretase, so long as it did not impact protein function.
It would have been obvious at the time of filing for a person of ordinary skill in the art to have combined the teachings of Yan and Park to make an NIa fusion protein comprising the functional N-terminal and C-terminal regions of a β-secretase, because Yan teaches Asp2 to localize to regions containing amyloid proteins which Park teaches can be degraded by NIa proteases. One would have recognized that Park teaches signal peptide sequences can be useful for effectively penetrating into cells by NIa protease to cleave amyloid proteins, which Yan teaches are effectively targeted by Asp2 (i.e., a beta-secretase) in secretory structures of the cell. As both references teach amyloid-beta to be present in the secretory structures of the cell (i.e., endoplasmic reticulum and Golgi apparatus), one would have been particularly motivated to combine these known features in order to further Park’s endeavor to provide treatments against Alzheimer’s disease using NIa. Furthermore, one would have recognized from Yan that the construction of fusion proteins containing the active site of other enzymes are known in the art, and that it would have been possible to simply replace the active site of a beta-secretase with that of an NIa protease, or an active fragment thereof, rendering a fusion protein that may localize in vivo to the desired therapeutic target.
Such a protein would have necessarily comprised a signal domain (X), N-terminal to the NIa protease (A), as well as transmembrane (Y) and intracellular domains, C-terminal to the NIa protease (A). Further, Yang teaches the active site (“A”) can be separated from the transmembrane domain (“Y”) by a linker (“L2”) and it is well within the ordinary skill in the art to generate linkers to separate each segment. Because each reference teaches these respective elements can be used in constructing fusion proteins, with each element (i.e., NIa, transmembrane domain) expected to perform the same function as it does separately (i.e., cleave amyloid beta, localize to plasma membranes), the results of the combination would have been predictable with a reasonable expectation of success. Hence, the combination would have been readily apparent and deemed to be a mere (A) combining of prior art elements according to known methods to yield predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103).
Regarding claim 3, Yan teaches the putative signal peptide of Asp2 to comprise residues 1 to 21 of SEQ ID NO: 2. As shown in the following alignment, instant SEQ ID NO: 3 (top) is identical to Yan’s SEQ ID NO: 2 (bottom) from residues 1 to 21:
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137
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Regarding claim 4, Yan teaches fusion polypeptides comprising peptide linkers comprising 20 to about 40 amino acids. This range overlaps with the claimed range from 20 to 24 amino acids. In view of the instant specification, there is no apparent support showing the criticality of the claimed range. In fact, the specification states, “[t]he linker merely has a role of linking these monomers, and it has a very limited effect on the structure and impact” (see pg. 7, lines 21-22). Applicant also discloses an embodiment wherein L1 comprises instant SEQ ID NO: 4 (see pg. 7, lines 23-25) which is 34 amino acids in length and outside the claimed range. Further, the Examples in the specification do not specify the length of the linker regions used, only that “SAS was prepared by fusing the N-terminus and C-terminus of NIa with the pro domain and the transmembrane domain of β-secretase” (see pg. 25, lines 1-2; Emphasis added). Hence, the overlapping range of 20-24 amino acids is sufficient to support a prima facie case of obviousness, particularly when there is no showing of criticality of the claimed range or evidence of unexpected results using the claimed range. See MPEP 2144.05(I).
Regarding claim 5, it would have been obvious to have derived the peptide linker from beta-secretase for the same reasons discussed regarding claim 1.
Regarding claim 6, it would have been obvious to have selected a peptide linker according to SEQ ID NO: 6 for the same reasons discussed regarding claim 1.
Regarding claim 7, Park teaches the NIa protease is derived from Turnip mosaic virus (TuMV) (see claim 1) which is disclosed as a Potyvirus (see Sequence Listing, SEQ ID NO: 1, “Organism”).
Regarding claim 13, Yan teaches that human aspartyl protease (Hu-Asp2) has an activity responsible for the processing of APP at the beta-secretase cleavage site (see pg. 1, para. [0006]), and is therefore understood to be a beta-site APP cleaving enzyme, i.e., a “BACE”. Yan teaches that Asp2 is also a membrane-bound protease (pg. 1, para. [0006]) that features a transmembrane domain near the C-terminus comprising residues 455-477 (see pg. 14, para. [110]) which anchors the protease to the membrane and is essential for the enzyme to function in cells (see pg. 1, para. [0006]). Yan also teaches the transmembrane domain anchors the polypeptide to intracellular membranes, such as the Golgi or the endoplasmic reticulum (see, e.g., claim 32).
Hence, it would have been obvious in view of Park and Yan to have included the transmembrane domain of Asp2 in the NIa fusion protein, because this element helps localize the enzyme to regions containing amyloid proteins. Furthermore, as previously discussed regarding instant claim 1, one of ordinary skill would have recognized that an NIa protease, or an active fragment thereof, could simply be fused within a β-secretase resulting in a fusion protein which would have necessarily included this region near the C-terminus.
Regarding claim 14, as shown in the following alignment, the full length of instant SEQ ID NO: 29 (top) is identical to the transmembrane domain of Yan’s SEQ ID NO: 2 from residues 458-478 (bottom):
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142
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Regarding claim 15, Yan teaches that Asp2, a β-secretase, has a cytoplasmic domain immediately following the transmembrane domain comprising residues 478-501 comprising the C-terminus of the protein (see pg. 14, para. [0110]). As discussed regarding instant claims 1 and 13, one would have recognized that an NIa protease, or an active fragment thereof, could simply be fused within a β-secretase that includes the transmembrane domain. Hence, it would have been obvious to include the C-terminal portion of the protein that includes both transmembrane and cytoplasmic domains.
Regarding claim 16, as shown in the following alignment, the full length of instant SEQ ID NO: 30 (top) is identical to the cytoplasmic domain of Yan’s SEQ ID NO: 2 from residues 479 to 501 (bottom):
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Regarding claim 17, Park teaches a pharmaceutical composition comprising an NIa protease, or alternatively, a gene carrier containing a nucleotide sequence encoding the synthesized NIa protease (see claim 1), while Yan teaches it may be necessary to express the fusion proteins of the disclosure by employing vectors comprising polynucleotide molecules which encode the proteins (see pg. 18, para. [0152]). Hence, it would have been obvious to have provided a polynucleotide that encodes the fusion NIa protein.
Regarding claim 18, instant SEQ ID NO: 42 translates to the following amino acid sequence:
1 MAQALPWLLL WMGAGVLPAH GTQHGIRLPL RSGLGGAPLG LRLPRETDEE
51 PEEPGRDYNP ISNNICHLTN VSDGASNSLY GVGFGPLILT NRHLFERNNG
101 ELVIKSRHGE FVIKNTTQLH LLPIPDRDLL LIRLPKDIPP FPQKLGFRQP
151 EKGERICMVG SNFQTKSITS VVSETSTIMP VENSQFWKHW ISTKDGQCGS
201 PMVSTKDGKI LGLHSLANFQ NSINYFAAFP DDFAEKYLHT IEAHEWVKHW
251 KYNTSAISWG SLNIQASQPN IPQTDESTLM TIAYVMAAIC ALFMLPLCLM
301 VCQWRCLRCL RQQHDDFADD ISLLKEQKLI SEEDL
As discussed above, Park teaches the TuMV NIa polypeptide represented by SEQ ID NO: 1 and a polynucleotide sequence encoding it, while Yan teaches the beta-secretase polypeptide represented by SEQ ID NO: 2. As shown in the following alignments, the full length of the translated amino acid sequence (top) encoded by instant SEQ ID NO: 42 is identical to specific regions of the sequences taught by Yan and Park.
First, amino acids 1-56 of the translated polypeptide (top) are identical to the first 56 amino acids of the beta-secretase, SEQ ID NO: 2, taught by Yan (bottom):
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Examiner notes that Yan teaches the propeptide region of Asp2, which follows the leading signal sequence, to extend to about residue 57 based on the GRR-GS sequence which has the characteristics of a protease recognition site (see pg. 14, para. [0110]).
Next, amino acids 56-269 of the translated polypeptide (top) are identical to amino acids 9-222 of the TuMV NIa, SEQ ID NO: 1, taught by Park (bottom):
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Finally, amino acids 270-335 of the translated polypeptide (top) are identical to amino acids 446-501 of the beta-secretase, SEQ ID NO: 2, taught by Yan (bottom):
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For clarity, the translated polypeptide is shown below, with Yan’s SEQ ID NO:2 in bold and Park’s SEQ ID NO: 1 underlined:
1 MAQALPWLLL WMGAGVLPAH GTQHGIRLPL RSGLGGAPLG LRLPRETDEE
51 PEEPGRDYNP ISNNICHLTN VSDGASNSLY GVGFGPLILT NRHLFERNNG
101 ELVIKSRHGE FVIKNTTQLH LLPIPDRDLL LIRLPKDIPP FPQKLGFRQP
151 EKGERICMVG SNFQTKSITS VVSETSTIMP VENSQFWKHW ISTKDGQCGS
201 PMVSTKDGKI LGLHSLANFQ NSINYFAAFP DDFAEKYLHT IEAHEWVKHW
251 KYNTSAISWG SLNIQASQPN IPQTDESTLM TIAYVMAAIC ALFMLPLCLM
301 VCQWRCLRCL RQQHDDFADD ISLLKEQKLI SEEDL
Hence, the claimed instant SEQ ID NO: 42 encodes a polypeptide that consists of the N-terminus of a beta-secretase (including signal sequence and inactive prodomain), the active site of an NIa protease, and the C-terminus of beta-secretase (including the transmembrane and cytoplasmic domains). Accordingly, for the reasons discussed regarding instant claims 1, 13 and 15, it would have been obvious to have fused an NIa protease, or an active fragment thereof, within a β-secretase to arrive at the claimed fusion protein.
Regarding claim 19, Park teaches that the gene carrier containing the NIa protease-encoding nucleotide is a suitable “expression construct” (see col. 5, lines 44-46) and may be a “viral vector” (see claim 1). Yan also teaches vectors comprising polynucleotide molecules for encoding the fusion proteins (see pg. 18, [0152]). Therefore, it would have been obvious to have made an expression vector to produce the protein.
Regarding claim 20, Park teaches the viral vector is a recombinant AAV virus (see col. 7, lines 39-43). Yan also teaches the vectors employed may be viral vectors (see pg. 19, para. 0166).
Regarding claim 21, Park teaches that the NIa gene was cloned into the expression vector, pTYB12, and transformed into a host cell to produce recombinant NIa protein (see col. 9, lines 26-30). Yan also teaches the vector comprising the polynucleotide encoding the fusion protein is expressed in a host cell transfected with said vector (see claims 36, 38 and 42).
Regarding claim 22, Park teaches that the pTYB12-NIa vector was transformed into the E. coli strain BL21 (DE3) (see col. 9, lines 28-30). Yan also teaches that mammalian cells useful in recombinant protein production include Chinese hamster ovary (CHO) cell lines (see pg. 15, para. [0123]), and useful prokaryotic cells include E. coli (see pg. 15, para. [0124]).
Claims 11-12 are rejected under 35 U.S.C. 103 as being unpatentable over Park and Yan as applied to claims 1, 3-7 and 13-22 above, and further in view of Cesaratto 2015 (previously cited), and as further evidenced by NP734212.1 (previously cited).
Regarding claim 11, the claim is directed to the “variant” of NIa protease recited in claim 1, wherein the variant of the NIa protease is a polypeptide comprising any one of the amino acid sequences represented by SEQ ID NOs: 19 to 21, in which one of the N-glycosylation sites of the NIa protease is substituted with another amino acid.
As shown in the following alignment, Park’s SEQ ID NO: 1 (bottom) comprises the full length of instant SEQ ID NO: 19 (top) with only one mismatch, as annotated below:
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Note that the mismatch above is at position 15 (Q15) in instant SEQ ID NO: 19, which corresponds to position 23 (N23) in Park’s SEQ ID NO: 1.
Park does not explicitly teach a variant of the NIa protease in which one of the N-glycosylation sites of the NIa protease is substituted with another amino acid.
Cesaratto 2015 teaches the tobacco etch virus Nuclear Inclusion a (NIa) gene which encodes a protease named tobacco etch virus protease (TEVp) (see pg. 159, col. 1, para. 1) which is widely used as a purified protein for in vitro applications and as a biological tool that that can be directly expressed in living cells (see Abstract). Cesaratto 2015 teaches that TEVp mutants with different stabilities and enzymatic properties have been reported in adapting the protease to a diverse range of applications (see Abstract). With the aim of targeting TEVp to the endoplasmic reticulum (ER) for biotechnological applications, Cesaratto 2015 engineered a mutated TEVp that showed strong cleavage activity on substrates localized to the ER lumen (see pg. 160, col. 1, para. 3). Cesaratto 2015 teaches that while the wild type TEVp targeted to the secretory pathway of mammalian cells is synthetized as an N-glycosylated and catalytically inactive enzyme, a TEVp mutant with selected mutations at two verified N-glycosylation sites was highly efficient and very active in the ER and can be used as a biotechnological tool to cleave proteins within the secretory pathway (see Abstract).
Examiner notes that Applicant appears to acknowledge Cesaratto 2015’s teachings on pg. 28, lines 6-10 of the instant specification: “It has been reported that when TEV NIa, which is an intracellular protease similar to TuMV, was secreted out of the cell, glycosylation, which did not occur in the original cell, proceeded, and as a result it affected the activity of TEV Nia (Cesaratto et al., 2015).”
Cesaratto 2015 teaches the substitution of N23Q in TEVp NIa, which NP734212.1 is relied upon to represent. Cesaratto 2015 teaches that the N23Q mutation was much more active and similarly resistant to auto-cleavage in cells, compared to the S219P mutant, previously claimed to have higher stability (see pg. 165, col. 1, para. 3).
The GenBank entry for NP734212.1 identifies this sequence as a “NIa-Pro protein” from the “Tobacco etch virus” (see “DEFINITION”). Hence, this sequence represents the TEV NIa of Cesaratto 2015’s disclosure. As shown in the following alignment, position N23 of the TEV NIa protease (bottom) corresponds to position Q15 of instant SEQ ID NO: 19 (top):
[AltContent: arrow]
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Hence, Cesaratto 2015 teaches a variant NIa protease in which one of the N-glycosylation sites of the NIa protease is substituted with another amino acid, wherein the another amino acid is a glutamine (Gln), and the resulting substitution in Park’s SEQ ID NO: 1 renders a sequence that is identical to instant SEQ ID NO: 19.
It would have been obvious at the time of filing for a person of ordinary skill in the art to have arrived at the claimed invention by combining the teachings of Park, Yan and Cesaratto 2015 by modifying Park’s NIa protease by the removal of an N-glycosylation site via an amino acid substitution, because Cesaratto 2015 teaches that doing so may increase the cleavage activity of the protease in the ER lumen which, as taught by Park, may be a critical site for targeting intracellular Aβ. One would have recognized that both Park and Cesaratto 2015 teach the use of NIa proteases in biotechnology applications for its ability to cleave substrates, while Cesaratto 2015 teaches substitutions at specific sites that enhance the protease’s activity. Furthermore, one would have recognized that the N23Q mutation in the NIa protease taught by Cesaratto 2015 corresponds to the N15 position in Park’s NIa protease, and there would have been a reasonable expectation that this substitution would have resulted in higher activity and stability. One would have also recognized that the TEVp NIa taught by Cesaratto 2015 has a publicly available amino acid sequence which could be readily used for reference when applying this substitution in the NIa protease taught by Park. Hence, the combination would have been readily apparent and deemed to be a mere (A) combining of prior art elements according to known methods to yield predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103).
Regarding claim 12, the examiner notes that SEQ ID NO: 31 is the translated product of SEQ ID NO: 42 (see, e.g., instant specification at pg. 15, lines 21-25), previously discussed regarding claim 18.
Cesaratto 2015 teaches the substitution N23Q in TEVp NIa, which NP734212.1 is relied upon to represent. Cesaratto 2015 teaches that the N23Q mutation was much more active and similarly resistant to auto-cleavage in cells, compared to the S219P mutant, previously claimed to have higher stability (see pg. 165, col. 1, para. 3).
The GenBank entry for NP734212.1 identifies this sequence as a “NIa-Pro protein” from the “Tobacco etch virus” (see “DEFINITION”). Hence, this sequence represents the TEV NIa of Cesaratto 2015’s disclosure. As shown in the following alignment, position N23 of the TEV NIa protease (bottom) corresponds to position N70 of instant SEQ ID NO: 31 (top):
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Hence, it would have been obvious to have made this substitution in the NIa fusion protein at the position corresponding to N70 in instant SEQ ID NO: 31.
Response to Arguments
Regarding the rejections under 35 U.S.C. 103, Applicant argues that there is a “fundamental conceptual distinction” between the NIa protease taught by Park and the synthetic alpha-secretase of the present application. Park's entire therapeutic strategy is based on cytosolic degradation of intracellular amyloid-beta, and the present invention is not a cytoplasmic protein, but a membrane protein designed to act in the protein secretion pathway.
Applicant’s arguments have been full considered but they are not persuasive.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). As discussed in the rejection: Yan teaches that amyloid proteins localize to secretory membrane structures including the cell surface (see pg. 1, para. [0004]); Hu-Asp2 (beta-secretase) is a membrane-bound protease which also localizes to the membrane (see pg. 1, para. [0006]); Yan teaches fusion polypeptides that include a transmembrane domain that cause the peptide to localize to the ER or Golgi (see pg. 22, para. [0195]); and both references teach the presence of amyloid-beta in the trans-Golgi network and the rough endoplasmic reticulum. Therefore, one would have recognized the advantage of combining the activities of NIa with a beta-secretase, as discussed in the rejection.
Applicant further argues that Yan explicitly states the invention relates to "methods and compositions for identification of modulators of beta-secretase activity." See Yan, paragraph [0002]. Yan's fusion proteins comprise a beta-secretase cleavage site, a transmembrane domain, and a reporter protein (such as SEAP) for monitoring beta-secretase cleavage. See Yan, paragraph [0086]. There is no teaching or suggestion in Yan to replace any enzymatic active site with a different protease.
Applicant’s arguments have been full considered but they are not persuasive.
Applicant is reminded that “[t]he use of patents as references is not limited to what the patentees describe as their own inventions or to the problems with which they are concerned. They are part of the literature of the art, relevant for all they contain." In re Heck, 699 F.2d 1331, 1332-33, 216 USPQ 1038, 1039 (Fed. Cir. 1983) (quoting In re Lemelson, 397 F.2d 1006, 1009, 158 USPQ 275, 277 (CCPA 1968)). Further, “[a] reference may be relied upon for all that it would have reasonably suggested to one having ordinary skill in the art, including nonpreferred embodiments. Merck & Co. v. Biocraft Labs., Inc. 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir. 1989), cert. denied, 493 U.S. 975 (1989). See MPEP 2123. In the instant case, Yan also teaches the structure-function of beta-secretase (Asp2) including its functional domains, including how these structures relate to the localization of the protein in vivo. Yan is also utilized to show that methods of making fusion proteins, including those made to localize proteases to particular regions of a cell, are known in the art, and Yan teaches such methods using domains from beta-secretase to localize proteins to regions comprising amyloid-beta.
Furthermore, while Yan does not explicitly teach replacing an “enzymatic active site with a different protease”, Yan reasonably suggests replacing enzymatic active sites with other enzymes: “Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions (see pg. 11, para. [0085]).”
Applicant further argues that a person of ordinary skill would have lacked motivation to combine the references, because they would not have recognized the advantage of replacing the active site of beta-secretase with Nia protease. However, Yan's fusion proteins are designed specifically as substrates and assay tools for beta-secretase activity, and they are not therapeutic proteins. The Nia protease, when converted from a cytoplasmic protein to a membrane-anchored protein in the secretory pathway, performs a fundamentally new function by acting as a synthetic a-secretase that cleaves APP at a site similar to alpha-secretase. This is a new and unexpected function that is not taught or suggested by any of the cited references.
Applicant’s arguments have been full considered but they are not persuasive.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
In the instant case, Park clearly teaches the NIa protease as a therapeutic protein and also discloses NIa to cleave amyloid-beta in a manner that is analogous to alpha-secretase:
We have previously reported that NIa possesses a highly strict substrate specificity, with its cleavage sites defined by the conserved sequence motif Val-Xaa-His-Gln↓ in which the scissile bond is located after Gln. Interestingly, the sequence Val-His-His-Gln is present in Aβ in the vicinity of the presumed α-secretase cleavage site (see col. 12, lines 36-41).
The presence of the same consensus sequence, Val12-His-His-Gln15, near the presumptive a-secretase cleavage site of the amyloid-β (Aβ) peptide led us to hypothesize that NIa could possess activity against Aβ (see col. 4, lines 52-55).
This appears to be the same cleavage site targeted by the claimed fusion protein in the instant specification (“VXHQ”) (see instant specification at pg. 7, line 30 and pg. 26, line 22). Therefore, this does not appear to be a “new and unexpected” function in view of the facts of record.
Applicant also argues that the Office's combination requires multiple conceptual leaps that are not supported by the references: (a) recognizing that a cytoplasmic plant viral protease could function as a membrane protein in the secretory pathway; (b) selecting specific structural elements from beta-secretase to create a chimeric protein with alpha-secretase-like activity; and (c) expecting that such a chimeric protein would retain protease activity when expressed in a completely different cellular compartment than its native environment.
Applicant’s arguments have been full considered but they are not persuasive.
First, Park already demonstrates that the “cytoplasmic plant viral protease” (NIa) can effectively penetrate cell membranes and cleave amyloid-beta intracellularly and cleave amyloid-beta in a manner analogous to alpha-secretase, as previously discussed.
Second, the “specific structural elements from beta-secretase” are known structures with known functions, as evidenced by Yan. Furthermore, replacing the catalytic site of beta-secretase with an NIa only requires knowing which region of the beta-secretase is the catalytic region, which can be easily inferred from Yan’s disclosure, and knowing which other regions comprise functional domains, which Yan expressly teaches (see pg. 14, para. [0110]).
Finally, it is clear that the prior art has already demonstrated that NIa can retain its protease activity in vivo and outside its native environment, e.g., in human 293T cells and mice (see Park at col. 14, lines 14-30). Applicant’s argument fails to provide sufficient evidence to support a conclusion that the retention of NIa’s function (i.e., to cleave substrates having a specifically known motif) would have been unexpected.
Applicant further argues that claim 1, as amended, now requires that the peptide linker 1 (L1) is a polypeptide represented by one of the amino acid sequences selected from SEQ ID NOs: 6 to 11, which are specific truncated sequences derived from a particular region of beta-secretase, and Yan does not teach or suggest using these specific truncated subsequences as linkers. Yan teaches linkers of "about 20 to about 40 amino acids." See Yan, paragraph [0023]. In contrast, SEQ ID NOs: 6 to 11 range from 10 to 19 amino acids which is outside Yan's taught range.
Applicant’s arguments have been full considered but they are not persuasive.
First, Applicant’s argument appears to be erroneous regarding the length of SEQ ID NOs 6 to 11, because in the sequence listing filed 05/06/2022, SEQ ID NO: 6 appears to comprise 20 amino acids.
Nonetheless, there is no apparent support showing the criticality of the claimed linkers or their length. The specification even states, “[t]he linker merely has a role of linking these monomers, and it has a very limited effect on the structure and impact” (see pg. 7, lines 21-22). Applicant also discloses an embodiment wherein L1 comprises instant SEQ ID NO: 4 (see pg. 7, lines 23-25) which is 34 amino acids in length. Further, claims 4 and 6 are still directed to linkers comprising 20 amino acids or more (e.g., SEQ ID NOs 5 and 6). Moreover, the Examples in the specification do not specify the length of the linker regions used, only that “SAS was prepared by fusing the N-terminus and C-terminus of NIa with the pro domain and the transmembrane domain of β-secretase” (see pg. 25, lines 1-2). Therefore, there is no evidence to support that the linkers themselves or their specific lengths were critical to the claimed invention. See MPEP 2144.05(I).
Applicant argues that the specification demonstrates that among the various versions of SAS tested, specific structural combinations resulted in superior activity. For example, the specification states that "among the various versions of SAS, the SASd20 activity was the most outstanding." As-Filed Specification, page 25, lines 12-13. This demonstrates that the specific structural requirements of the claimed fusion protein are critical to achieving the desired alpha-secretase activity, and such results were not predictable from the cited references.
Applicant’s arguments have been full considered but they are not persuasive.
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., “SASd20”) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
In view of the instant specification (Example 1), “SASd20 may be a polypeptide represented by the amino acid sequence of SEQ ID NO: 36” (see pg. 25, lines 20-21). However, neither “SASd20” or “SEQ ID NO: 36” are recited in the claims. Furthermore, Example 1 also states that “the SAS structures are shown in FIG. 6” (see pg. 25, lines 12-13). However, the specific structures of these fusion proteins cannot be discerned from this figure. Therefore, there is insufficient evidence to support Applicant’s argument of criticality, and it is further unclear what “specific structural requirements” the Applicant considers to have been critical.
Applicant further argues that the specification further demonstrates unexpected results in the in vivo 5XFAD mouse model. Expression of SAS using AAV9 restored memory ability in transgenic Alzheimer's disease mice and significantly reduced amyloid plaques in the hippocampus. See As-Filed Specification, pages 27-28, Experimental Examples 6-7. These therapeutic results demonstrate that the claimed fusion protein achieves a level of efficacy that was not predictable from the teachings of Park or Yan alone.
Applicant’s arguments have been full considered but they are not persuasive.
It is not clear from Applicant’s brief discussion citing pages 27-28 and Examples 6-7 what “level of efficacy” would have been unpredictable in view of the applied references. This argument also fails to specifically point out the alleged deficiencies of the applied references or how the results achieved by the inventors patentably distinguishes the claims from the prior art combination set forth by the examiner. Therefore, the evidence submitted by Applicant is insufficient to permit any conclusion regarding unexpected results. Applicant is reminded that mere allegation of “unexpected results” is not sufficient to overcome a rejection under 35 U.S.C. 103.
Applicant further argues that specification also shows that the topology of SAS is critical, because SAS4, which was designed to have the N-terminus positioned inside the cell, did not increase sAPPα. See As-Filed Specification, page 26, lines 13-14. This confirms that the specific structural arrangement recited in claim 1, with the signal sequence and linker N-terminal to the Nia protease and the transmembrane and intracellular domains C-terminal, is essential for activity and was not predictable.
Applicant’s arguments have been full considered but they are not persuasive.
It is unclear what “topology” distinguishes SAS4 from the claimed invention or the prior art combination or how this relates to the predictability of the invention. The specification states that “SAS4, which is a type2 transmembrane domain, was prepared by fusing the N-terminus of US9 with NIa” (see pg. 25, lines 11-12).This structure does not appear to relate to the basis of the rejection, which was that it would have been obvious to have replaced the catalytic domain of a beta-secretase with NIa, not to have fused the N-terminus of a “US9” with NIa. It should be noted that neither Park nor Yan even mention “US9” and this feature is only briefly mentioned in Applicant’s disclosure. Therefore, the evidence submitted by Applicant is insufficient to permit any conclusion regarding predictability.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/DENNIS IGNATIUS ARMATO JR/Examiner, Art Unit 1651
/MELENIE L GORDON/Supervisory Patent Examiner, Art Unit 1651
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