Notice of Pre-AIA or AIA Status
The present application is being examined under the first-inventor-to-file provisions of the AIA .
DETAILED ACTION
Election Restrictions
In responding to the February 26, 2026 Office action, “Applicant elects Group II, the leader protein of EMCV (SEQ ID NO: 2) as the NCT inhibitor protein, and the SARS-CoV-2 spike protein (SEQ ID NO: 13) as the viral antigen, without traverse. Claims 36, 39, 46 and 60-76 encompass the elected group and species” (signed and filed 05/20/2026).
Applicant’s election without traverse of Group II invention in the reply filed on May 20, 2026 is acknowledged.
Status of Claims
Claims 1-35 and 37-38, 40-45, 47 and 48-59 are cancelled.
Claims 60-76 are new.
Thus, claims 36, 39, 46 and 60-76 are under examination for their merits.
Information Disclosure Statement
Three information disclosure statements (IDS) were submitted on 02/01/2024, 08/05/2025 and 05/20/2026. These submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, all three sets of IDS are being considered by the examiner.
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Priority
This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/162,496, filed March 17, 2021. Based on its disclosure, claims 36, 39, 46 and 60-76 are deemed eligible for receiving the earliest possible effective filing date, as the 116 embodiments in the provisional filing provide adequate specification for the claimed invention.
Objections to Specification
The disclosure as filed on 09/13/2023 for this application is objected to because of the following informalities:
The word “flu” as cited in ¶[0003] (page 1, Substitute Specification – Clean Copy) is a colloquial version of “influenza.”
“SARS CoV 2” in ¶[0004] (page 1) needs a full spelling, and the abbreviation should be consistent with the broadly accepted version – SARS-CoV-2 (as cited in ¶[0007], ¶[0035] and FIG. 4).
“SARS CoV2” in Table 2 (pages 42-44 and 46-47) should be SARS-CoV-2.
The term “viral-like particles (VLPs)” cited in ¶¶[0016]-[0017] and ¶[0023] is inconsistent with “virus-like particle (VLP)” cited in ¶[0036] and “virus like particle (VLP)” in ¶[0045].
“L1 protein” cited in ¶[0019] is inconsistent with the L protein cited elsewhere (e.g., ¶[0034]).
“S, N, M, and E proteins” and “S, N, M, and E protein bands” in ¶[0008] need full spelling.
The terms “L enhancer protein” (e.g., ¶[0011]), “enhancer protein” (¶[0019] and ¶¶[0031]-[0032]) and “enhancer L protein” (e.g., ¶¶[0058]-[0059]) are inconsistent, and they further contradicts the leader peptide’s intrinsic function as “a nucleocytoplasmic transport (NCT) inhibitor” (e.g., in claims 36, 39 and 60).
Applicant is advised to cite biological terms consistently and accurately to avoid ambiguities and/or confusions. For compact prosecution, examiner accepts “L leader peptide” as the EMCV version (instant SEQ ID NO: 2), which is an NCT inhibitor that is used to enhance VLP production when a polynucleotide encoding a SARS-CoV-2 structural protein and the EMCV leader peptide transfects an eukaryotic cell.
Claim Rejection under 35 U.S.C. §112(b)
The following is a quotation of 35 U.S.C. §112(b), which forms the basis for indefiniteness rejection set forth in this Office action:
(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.
Claims 61, 67, 69 and 75 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.
Specifically, claim 61 recites “the method of claim 36, wherein the NCT inhibitor protein comprises a sequence of SEQ ID NO: 2.” Similarly, claim 69 recites “the method of claim 39, wherein the NCT inhibitor protein comprises a sequence of SEQ ID NO: 2.” The citation of “a sequence of SEQ ID NO: 2” renders claims 61 and 69 indefinite as it is not clear if the claim requires the full sequence of SEQ ID NO: 2 or just any subsequence therein. The presence of multiple structural interpretations renders the claims indefinite.
Claim 67 recites “the method of claim 36, wherein the vector causes one or more of (i) expression of the viral antigen protein at a higher expression level, (ii) expression of the viral antigen protein for a longer period of time, or (iii) expression of the viral antigen protein with better protein quality, as compared to a vector lacking the NCT inhibitor protein.” Similarly, claim 75 recites “the method of claim 39, wherein co-expression of the NCT inhibitor protein with the viral antigen protein causes one or more of (i) expression of the viral antigen protein at a higher expression level, (ii) expression of the viral antigen protein for a longer period of time, or (iii) expression of the viral antigen protein with better protein quality, as compared to expression of the viral antigen protein without the NCT inhibitor protein.” In both claims, embodiments (i) and (ii) could be interpreted to lack a proper reference group for comparison, with said control applying to only (iii). Alternatively, the control of (iii) could apply to all of (i)-(iii). The presence of multiple interpretations renders claims 67 and 75 indefinite.
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 60, 62, 68 and 70 are 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 is 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.
Claims 60 and 68, which are drawn to “the method of 36” and “the method of 39”, respectively, extend their base claims with a limitation that “wherein the NCT inhibitor protein comprises a sequence having at least 70% sequence identity to SEQ ID NO: 2.” Claims 62 and 70 extend their base claims (claim 36 and claim 39, respectively) with another limitation that “wherein the NCT inhibitor protein is the leader (L) protein of Encephalomyocarditis virus (EMCV) or a functional variant thereof.” The disclosure is limited to SEQ ID NO: 2 itself (e.g., page 174, ¶[0371]-¶[0372]), so each genus claim fails to provide sufficient disclosure of representative species to adequately describe the breadth claimed for all variants recited.
According to MPEP §2163.04, “[t]he purpose of the written description requirement is to ‘ensure that the scope of the right to exclude, as set forth in the claims, does not overreach the scope of the inventor’s contribution to the field of art as described in the patent specification.’” Ariad Pharm., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1353-54 (Fed. Cir. 2010) (en banc) (quoting Univ. of Rochester v. G.D. Searle & Co., 358 F.3d 916, 920 (Fed. Cir. 2004)). To satisfy the written description requirement, the specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. Vas-Cath, Inc. v. Mahurkar, 935 F.2d 1555, 1562-63, 19 USPQ2d 1111 (Fed. Cir. 1991).
MPEP §2163 also states that 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, 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 applicant was in possession of the claimed genus. A “representative number of species” means that the species which are adequately described are representative of the entire genus. The disclosure of only one species encompassed within a genus adequately describes a claim directed to that genus only if the disclosure “indicates that the patentee has invented species sufficient to constitute the gen¶[us].” See Enzo Biochem, 323 F.3d at 966, 63 USPQ2d at 1615. “A patentee will not be deemed to have invented species sufficient to constitute the genus by virtue of having disclosed a single species when … the evidence indicates ordinary artisans could not predict the operability in the invention of any species other than the one disclosed.” Given the substantial (up to 30%) variation within the genus of SEQ ID NO: 2 (EMCV L protein), one must describe a sufficient variety of species to reflect the variation within the genus. However, one of ordinary skill in this art cannot envision the structure of any NCT inhibitor protein with the required or preferred function other than SEQ ID NO: 2 as provided in by the Applicant or other relevant ones from prior art. Therefore, without any other representative sequence variants in the form of Tables/Figures or GenBank accession numbers, SEQ ID NO: 2 is not sufficient enough to represent its genus, so the claims encompassing 70% identity to SEQ ID NO: 2 clearly fail the written description requirement.
Functionally defined genus claims can be inherently vulnerable to invalidity challenge for lack of written description support, especially in technology fields that are highly unpredictable, where it is difficult to establish a correlation between structure and function for the whole genus or to predict what would be covered by the functionally claimed genus. See ABBVIE DEUTSCHLAND GMBH & 2 CO. v. JANSSEN BIOTECH, INC., Appeals from the United States District Court for the District of Massachusetts in Nos. 09-CV-11340-FDS, 10-CV-40003-FDS, and 10-CV-40004-FDS, Judge F. Dennis Saylor, IV. See also Ariad, 598 F.3d at 1351 (“¶¶[T]he level of detail required to satisfy the written description requirement varies depending on the nature and scope of the claims and on the complexity and predictability of the relevant technology.”)
Even when several related species are disclosed by prior art (e.g., FIG. 6 in Porter et al., 2007. US PGPub 2007/0243169 A1, published 10/18/2007), these are not necessarily representative of the entire genus of functional equivalents for SEQ ID NO: 2 in the instant application, as Porter also taught that EMCV L peptide loses its bioactivity when its “Zn-finger” subdomain is mutated (¶[0087]). In 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 L protein, as 70% sequence identity to SEQ ID NO: 2 entails, one must describe a sufficient variety of each anticipated variant species to reflect the spectrum of variation within the genus to provide a "representative number” of species with comparable structure and/or function for a practical application (e.g., manipulation of nuclear trafficking). Since the genus recited in the instant claims is large and unpredictable at the 70% sequence identity level, it would be very challenging to describe sufficient species to cover the structures/functions of the entire genus. A single sequence for the EMCV L protein is certainly inadequate.
Overall, at the time of the claimed invention, the level of skill for obtaining variant forms of EMCV L protein for in vitro testing and in vivo function and then adding the corresponding nucleotide version to a polynucleotide encoding viral antigens for VLP production toward an effective vaccine was high. And even if a selection procedure was, at the time of the invention, sufficient to enable the skilled artisan to identify such variants with the recited functional properties (as eluded to by Porter et al., 2007), 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). Thus, without details for mapping out functionally variations within the EMCV L protein, a skilled artisan generally would not be able to visualize or otherwise predict, a priori, what amino acid residues with a particular set of functional properties would look like structurally in the particular case of EMCV L protein.
Applicant may show that the invention of claims 60, 62, 68 and 70 is complete by (i) sufficiently disclosing details beyond the listing of SEQ ID NO: 2 that is expected to serve the same function as a nucleocytoplasmic transport (NCT) inhibitor protein, (ii) providing relevant identifying characteristics to illustrate that applicant was in possession of the claimed invention, i.e., complete or partial sequence with expected physical and/or biochemical properties, and (iii) revealing functional characteristics when coupled with a known or disclosed correlation between structure and function, or some combination of such characteristics. (see Enzo Biochem, 323 F.3d at 964, 63 USPQ2d at 1613.)
Claims 60, 62, 68 and 70 are further rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while enabling the use of the 67-amino acid peptide leader (L protein) of EMCV as an NCT inhibitor protein that comprises SEQ ID NO: 2 for enhancing VLP production, does not reasonably provide enablement for its variants that bear “70% identity” (cited in claims 60 and 68) to SEQ ID NO: 2 or “other functional variants thereof” (cited in claims 62 and 70). The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the various species of EMCV L protein toward enhancing VLP production and/or VLP immunogenicity in the invention commensurate in scope with these claims.
As discussed supra, claims 60 and 68 are drawn to “the method of 36” and “the method of 39”, respectively, “wherein the NCT inhibitor protein comprises a sequence having at least 70% sequence identity to SEQ ID NO: 2.” Claims 62 and 70 extend their base claims (claim 36 and claim 39, respectively) with another limitation that “wherein the NCT inhibitor protein is the leader (L) protein of Encephalomyocarditis virus (EMCV) or a functional variant thereof.” The disclosure is limited to a single EMCV L protein (instant SEQ ID NO: 2, as cited in FIG. 6 and ¶[0371]-¶[0372]). Neither the range of other EMCV L protein variants nor their expected function in enhancing VLP production (by manipulating/blocking nuclear transport) is enabled by any evidence of record.
Protein chemistry is probably one of the most unpredictable areas of biotechnology. One prior art (Porter et al., 2007. US PGPub 2007/0243169 A1, published 10/18/2007) teaches that EMCV L peptide loses its bioactivity when its “Zn-finger” subdomain is mutated (¶[0087]). In another example, replacement of a single “lysine” residue at position 118 of acidic fibroblast growth factor by “glutamic acid” led to the substantial loss of heparin binding, receptor binding and biological activity of the protein (Burgess et al., Journal of Cell Biology 111: 2129-2138, 1990). In transforming growth factor alpha, replacement of aspartic acid at position 47 with alanine or asparagine did not affect biological activity, while replacement with serine or glutamic acid sharply reduced the biological activity of the mitogen (Lazar et al. Molecular and Cellular Biology 8:1247-1252, 1988). As these references illustrate, it is unpredictable that a polypeptide variant of a known target protein binder will also bind said target. It is also unpredictable that they would bind said target in the same way, having the same effect on the target (i.e. inhibition or activation). Ju et al. (Proceedings of the National Academy of Sciences, U.S.A., Vol. 88, pp. 2658-2662, 1991) teaches that the interleukin 1 receptor (IL-1R) antagonist IL-1ra is a naturally occurring protein with no agonist activity in vitro or in vivo (Abstract). However, substitution of a single amino acid lysine145 to aspartic acid changes the property of this peptide to a partial agonist of IL-1R (Abstract). Thus, even a single substitution can change the biological property of a peptide.
Truncation of proteins can also lead to adverse effects on protein structure and thus protein function. Martindale et al. (Nature Genetics, Vol. 18, pp. 150-154, 1998) teaches that truncation of huntingtin leads to aggregate development which compromises cell viability (Abstract). Nonaka et al. (Human Molecular Genetics, Vol. 18, No. 18, pp. 3353-3364, 2009) teaches that truncation of TDP-43 to its C-terminal fragments causes abnormally phosphorylated and ubiquitinated inclusions of the protein (Abstract). Taken together, not just any truncation of a protein will yield a soluble, functional, protein fragment.
In summary, the examples as discussed supra all teach that the biological function of peptide variants is unpredictable because even a single mutation can abolish activity or give a different function: even agonist and antagonist peptides can be interconverted through conjugation or mutagenesis. Importantly, binding can still occur after mutation or conjugation in the examples provided above, illustrating that a simple demonstration of binding is not predictive of the nature of a peptide’s biological activity. This point is underlined by Montrose-Rafizadeh et al. (Journal of Biological Chemistry, Vol. 272, pp. 21201-21206, 1997), who teaches that receptor binding does not predict agonist or antagonist activity (page 21205, column 2, first full paragraph).
Given that protein/peptide variants often have highly unpredictable function when compared with their wild-type version and that even a known function in vitro may not be maintained in vivo, while the specification in the instant application does nothing to ameliorate these concerns about polypeptide/fusion peptide variants, one would be burdened with undue experimentation to use the EMCV L protein variants of instant claims as broadly as they are currently claimed (70% sequence identity or a functional variant thereof). Thus, the VLP with an EMCV leader protein variant in claims 60, 62, 68 and 70 fall beyond the scope of the claimed invention.
Claim Rejections under 35 U.S.C. §103
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 the following:
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 36, 39, 46, 60-65, 67-73 and 75-76 are rejected under USC §103 as being unpatentable over Xu et al., 2020 (Front. Bioeng. Biotechnol. 8: 862) and Lu et al. 2020 (Cell Research 30: 936-939) in view of Zhang et al. 2020 (Signal Transduction and Targeted Therapy 5: 269), Lei et al., 2020 (Expert Review of Vaccines. 19: 235-246) and Porter et al., 2007 (US PGPub 2007/0243169 A1, published 10/18/2007).
Xu et al., 2020 taught the “construction of SARS-CoV-2 virus-like particles (VLPs) by mammalian expression system” (title), with the following disclosure:
Four plasmid vectors (FIGURE 1 on page on 3; also pasted below) encoding “major structural S glycoprotein (GenBank: QHD43416.1), E protein (GenBank: QHD43418.1), M protein (GenBank: QHD43419.1) and N protein (GenBank: QHD43423.2) (page 2, third paragraph on the right; FIGURE 1 on page 3 and pasted below) – transfection of “HEK-293T human embryonic kidney cell line and Vero E6 African green Monkey kidney cell line” (page 2, second last paragraph on the right) led to gene expression patterns dictated by single, double or quadruple vectors (FIGURE 2 on page 4);
SARS-CoV-2 VLPs assembly – “M and E are basically required for efficient assembly and release of SARS-CoV-2 VLPs ” (page 5, “Conclusion”);
Choice of eukaryotic cells for gene expression – “The corona-like structure presented in SARS-CoV-2 VLPs from Vero E6 cells is more stable and unified, as compared to those from HEK-293 T cells” (abstract);
Verification of VLP morphology – “The average diameter of SARS-CoV-2 VLPs from HEK-293T cells falls around 90.33 ± 32.45 nm, whereas those assembled in Vero E6 cells were smaller, showing about 71.02 ± 21.98 nm” (page 5, last paragraph on the left).
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FIGURE 1 of Xu et al., 2020. Schematic outline of SARS-CoV-2 VLPs constructions in mammalian expression system.
SARS-CoV-2 spike protein sequence – GenBank sequence QHD43416.1 cited by Xu is identical (a 100% match) to the instant SEQ ID NO: 13 (1273 amino acids for SARS-CoV-2 spike protein);
Intended application – VLP can be used as a promising vaccine candidate” (abstract).
Overall, Xu et al. taught that two types of mammalian (eukaryotic) cells transfected with plasmid vectors encoding SASR-CoV-2 structural proteins can produce “SARS-CoV-2 VLPs” as vaccine candidates, and their VLPs “possess molecular and morphological properties of native virion particles” (abstract). Thus, they teach a method of expressing more than one viral protein in two eukaryotic cell lines comprising contacting the cells with a vector comprising a polynucleotide encoding a viral antigen protein, which includes all the viral proteins used to construct the particles above. Transfection of cells with a vector/plasmid is contacting the cells with said nucleic acids.
Lu et al. 2020 taught the design, preparation and application of “COVID-19 mRNA vaccine encoding SARS-CoV-2 virus-like particles” for “inducing a strong antiviral-like immune response in mice” (title). Their disclosure included the following:
Choice of SARS-CoV-2 structural proteins as the basis for VLP production and vaccination – their mRNA compositions encode spike (S) protein (RQ3012-Spike), receptor-binding domain (RBD) of S protein (RQ3011-RBD), and a cocktail of S + M (membrane protein) + E (envelope protein)” (RQ3013-VLP) (Fig. 1a on page 937 and pasted below);
mRNA optimization strategies – “The final mRNAs in vaccines have an optimal combination of codon and modified nucleotides” (page 936, second paragraph on the left);
“Well-established lipid nanoparticles (LNPs) to package mRNAs” – “mRNA encapsulation efficiency of all three LNP vaccine candidates was greater than 98%” (page 936, last paragraph on the left);
Authentication of protein identity/integrity – In cell culture supernatants, “all three proteins can be detected by western blotting” (page 936, second paragraph on the left);
Production of virus-like particles – After transfection in HEK 293A cells (of human origin) (Figure S1 in Supplemental Information), RQ3013-VLP encoding S, M and E antigens allowed a proper formation of SARS-CoV-2 virus-like particles (VLPs) (Fig. 1b and pasted below);
Verification of virus-like particles – Electron microscopy confirmed that “VLPs have an average diameter of 100 nm, with the spike protein densely decorating the surface” (page 936, second last paragraph on the left; also shown in Fig. 1b and pasted below);
Immunization procedures – BALB/c mice were immunized intramuscularly on day 0 and day 21 with three mRNA-VLP compositions (page 936, second paragraph on the right);
Assessment of virus-specific immune responses – mRNA generates viral proteins in cytoplasm after cell transfection in vivo (page 936, first paragraph on the right), and the resulting VLPs (as secreted vesicles, page 939, last paragraph on the left) elicit immune responses: “RQ3013-VLP had the strongest immune response and developed significantly higher titers of S-specific binding antibody than mice receiving RQ3012-Spike” (page 936, second paragraph on the right). Indeed, “RQ3013-VLP elicited both humoral and T cell immune responses” (page 938, second paragraph on the left).
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Fig. 1a and 1b from Lu et al., 2020. 1a. “RQ3013-VLP contains three mRNAs encoding S, M, and E proteins that can assemble into VLPs” (Fig. 1 caption). 1b. “Electron microscopy images of VLPs produced by RQ3013-VLP.”
Overall, Lu’s teachings provide a roadmap for (i) preparing mRNA-LNP compositions for intramuscular immunization in mice (for in vivo assessment); (ii) generating VLP consisting of three SARS-CoV-2 structural proteins capable of eliciting humoral and T cell immune responses in vivo. An enhanced immunogenicity associated with Lu’s mRNA-LNP vaccination was attributed to VLP vesicles: “when S is presented in secreted vesicles as VLPs, it induces a more robust immune response than when it is displayed on the cell membrane” (page 939, last paragraph on the left).
Further consistent with Xu, Lu used DNA vectors (e.g., pCDNA3.1) for cloning SARS-CoV-2 cDNA encoding structural proteins or a subunit (RBD) of S protein, and these vectors were used for (i) in vitro mRNA transcription; (ii) in vitro transfection of a human kidney cell line (which is eukaryotic) for the expression of individual antigens (for ELISA and Western blot) (page 3 in “Supplementary information”); and (iii) in vitro production of VLPs (page 936, first paragraph on the right).
SARS-CoV-2 E protein in Xu and Lu’s VLPs was known to have both cytoplasmic and nuclear distribution (Zhang et al., 2020, page 1, third paragraph on the left), although the exact nuclear localization signal (NLS) was not defined at the time. Zhang also cautioned that “viral proteins from transfection may behave differently than that from viral infection” (page 2, last paragraph on the right). Without eliminating the potential nuclear targeting signal (preventing nuclear trafficking) in VLP components (such as the E protein required for VLP assembly, as taught by Xu), an alternative solution would be adding an NCT inhibitor protein to enhance VLP assembly efficiency in vitro and in vitro, as taught by Lei and Porter.
Lei et al. taught the use of “vaccine compositions” (abstract) comprising chimeric VLPs (chi-VLPs) (Figure 1 and pasted below) and provided five motivations for VLP-based vaccine formulations: (i) “VLPs possess highly ordered repetitive structure that makes them efficient nanoparticles to present antigens to the immune system” (page 242, last paragraph on the left); (ii) post-translational modification (PTM) in eukaryotic expression system ensures proper VLP maturity and bioactivities (page 242, first paragraph in section 4.4); (iii) VLPs ranging from 20 nm to 200 nm efficiently enter the lymphatic system through diffusion and drainage (page 245, second paragraph under section 4.3); (iv) in antigen-presenting cells, the VLPs are processed into small epitopes that could be presented by major histocompatibility class (MHC) II or cross-presented by the MHC I for the stimulation of both CD4+ and CD8+ T cell-mediated immunity” (page 235, first paragraph in section 1.2); and (v) VLPs have an inherent advantage of “high biosafety” when compared with vaccines using “live attenuated virus” (section 1.1 on page 235). Their disclosure regarding chimeric VLPs and vaccination includes the following:
“Introducing foreign peptides to the intact or truncated N-terminus/C-terminus (N/C-ter)… is the most applicable strategy to design and construct chi-VLPs, since this modification usually do not disturb the main structure of VLP and compromise the immunogenicity of chi-VLPs” (page 236, first paragraph in section 2.1; also in Figure 1 and pasted below);
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Figure 1 of Lei et al., 2020. 3D structures of four representative chimeric VLPs (chi-VLPs). A “foreign peptide” can be fused to either the N-terminus or C-terminus of a capsid protein to avoid disrupting the structure and/or function of the native viral capsid protein (page 236, first paragraph in section 2.1). PCV2 has a nuclear localizing signal (NLS)” (amino acid residues 1-41) that can be truncated to enhance VLP production efficiency (page 236, second paragraph in section 2.1).
In one example, VLP comprising “Cap” protein of “porcine circovirus type 2 (PCV2)” – truncation of an “N-terminal nuclear localizing signal (NLS)” (amino acid residues 1-41) was advantageous for VLP expression and assembly efficiency (page 236, second paragraph in section 2.1);
Functional RNAs can be incorporated into VLPs (page 236, second paragraph under “Article Highlights”), which requires a viral packaging signal;
Two “molecular adjuvants”, “flagellin” and “E.coli heat-labile enterotoxin B subunit”, were considered effective for “enhancing the capability of chimeric VLPs to induce… immune response” (page 240, end of first paragraph on the left).
For the case of chimeric VLP using modified PCV2 structural protein, two evidential references (cited by Lei as references 44 and 46, page 244) disclosed further details. First, Zhang et al., 2014 (Viruses 6: 4839-4855) discovered that “the nuclear localization signal (NLS; at 1–39 aa) of PCV2 capsid protein (Cap)” can be replaced by a “classical swine fever virus (CSFV) T-cell epitope” to produce a chimeric VLP without affecting VLP assembly (abstract). Second, Xiao et al., 2018 (Biologicals 51: 18–24) confirmed that N-terminus truncation (ΔCP) “didn’t affect the formation of VLPs,” as “VLPs formed by ΔCP had a morphology and size consistent with that of the VLPs assembled by CP” (page 23, second paragraph on the right). “NLS-deleted capsid protein (ΔCP) of PCV2b was expressed in Hansenula polymorpha in a high level and demonstrated with potent immunogenicity in mice” (page 22, first paragraph in section 4. Discussion).
A skilled artisan could follow the collective teachings of Xu, Lu, Zhang and Lei to arrive at a plasmid vector or mRNA-LNP composition toward a SARS-CoV-2 VLP-based vaccine, with selective NLS removal to enhance VLP efficiency and immunogenicity. When the NLS in structural proteins is not fully defined (as taught by Zhang in 2020), an alternative solution would be to apply the method of Porter et al., as they taught the use of encephalomyocarditis virus (EMCV) leader protein and several variants that block nucleocytoplasmic trafficking:
“Cardioviruses like EMCV… abrogate nucleocytoplasmic trafficking” through a leader “at the amino terminus of the viral polyprotein”, which is a “67 aa long” peptide “with a novel CHCC zinc-finger motif, a highly acidic carboxyl domain (protein pI: 3.8), and no known homologs” (¶[0005]);
“Relatively short EMCV leader proteins” (corresponding to SEQ ID NOs: 1-8) (¶[0032]) can “be manipulated for therapeutic, preventative, research and diagnostic purposes” (¶[0028]);
EMCV leader protein sequences can remain functional after “some internal deletions or external truncations” (SEQ. ID NOs: 1-13) (¶[0034]);
When “Fusion proteins” with wild type and mutant EMCV L protein (¶[0071]) are used in “In-Vitro Nuclear Import Assay” (¶[0073]-¶[0079]), “L blocks nuclear localization of fluorescent import cargo” (¶[0081]). Thus, attachment of said L protein to a heterologous protein creates a fusion protein that does not traffic into the nucleus.
In other words, Porter et al. 2007 taught the use of EMCV leader protein and several variants for manipulating/blocking nucleocytoplasmic trafficking toward therapeutic applications. In addition, their SEQ ID NO: 1 is a 100% match with instant SEQ ID NO: 2 (L protein with 67 amino acids).
Given the teaching of Lei about the advantage of eliminating nuclear localizing signal (NLS), which equates to reducing nuclear import of viral proteins and enhancing chimeric VLP production (by restricting VLP-related foreign products in the cytoplasm) in eukaryotic cells, Porter’s therapeutic use of EMCV leader protein as an NCT inhibitor stands out as a straightforward method for blocking nuclear trafficking without picking and eliminating specific signaling motifs within a viral structural protein. A skilled artisan would be prompted to adopt Porter’s EMCV leader protein and modify Xu’s design of SARS-CoV-2 vector compositions before arriving at the invention of claims 36, 60-65 and 67 toward enhancing VLP production/secretion efficiency:
A plasmid vector encoding a SARS-CoV-2 structural protein and an EMCV leader protein – through N- or C-terminus modification (as taught by Lei), Porter’s EMCV leader protein (a nucleocytoplasmic transport inhibitor) can be added to any of Xu’s SARS-CoV-2 structural proteins, and the resulting plasmid vector would be capable of expressing authentic SARS-CoV-2 antigen after transfecting (contacting) a human or monkey (eukaryotic) cell, meeting the limitations of claims 36 and 63;
EMCV-specific leader protein – Porter’s SEQ ID NO: 1 is a 100% match with SEQ ID NO: 2 of the instant application, meeting the limitations of claims 60-62;
Choice of viral antigens – Xu’s immunogens include four SARS-CoV-2 structural proteins (S, M, E or N in FIGURE 1) that can form VLP, meeting the limitations of claims 64-65;
Regarding claim 67, it is drawn to an intended use of methods in claim 36, with a recitation of three advantages inherent with the elements of VLP assembly when an NCT inhibitor is added to a VLP without interrupting its native structure (through N- or C-terminus modification), as taught by Lei and Porter. The vector in claim 36 is capable of performing the intended results in claim 67, as co-expression of NCT inhibitor would apply to any viral antigen/immunogen in the VLP composition, meeting the limitations of claim 67. Said another way, since the active steps of claim 67 are met, the intended results will necessarily occur. Expression of the viral antigen protein at a higher cytosolic level will certainly occur for example. The same arguments are made for claim 75.
For in vivo application, Porter’s therapeutic use of EMCV leader protein as an NCT inhibitor would also prompt a skilled artisan to adopt Porter’s EMCV leader protein and modify Lu’s design of mRNA encoding SARS-CoV-2 structural protein to enhance in vivo VLP production and immunogenicity (as taught by Lei), before arriving at the invention of claims 39, 68-73 and 75-76 in terms of immunization and virus-specific immune responses:
An mRNA-LNP composition that not only allows the co-expression of a SARS-CoV-2 structural protein and an EMCV leader protein (the same NCT inhibitor as in claim 36) but also elicits virus-specific immune responses – mRNA-LNP formulations are capable of inducing SARS-CoV-2-specific immune responses in mice (as taught by Lu), meeting the limitations of claims 39 and 71;
EMCV-specific leader protein sequence – the same rationale as discussed supra for claims 60-62, meeting the limitations of claims 68-70;
Choice of SARS-CoV-2 structural proteins as the target immunogen – Lu’s immunogens include three SARS-CoV-2 structural proteins (S, M and E in Figure 1a) that can form VLP and released/secreted by transfected cells, meeting the limitations of claims 72-73;
Delivery vehicle – Lu’s LNP is a well-known delivery vehicle for packaging mRNAs and for delivering mRNA into a target cell (including antigen-presenting cells taught by Lei), meeting the limitations of claim 76.
Claim 75, on the other hand, is an intended use of methods in claim 39, with a recitation of three advantages inherent for the immunogenic properties of VLP: enhancement of VLP production efficiency by an NCT inhibitor would result in improved immunogenicity after immunization in a subject, as long as the addition of an NCT inhibitor does not interrupt VLP’s native structure (as taught by Lei and Porter). In other words, the polynucleotide composition in claim 39 is capable of performing the intended use cited in claim 75 when SARS-CoV-2-specific antigenic repertoire is not altered by the EMCV-derived NCT inhibitor.
Thus, the invention of claims 36, 39, 46, 60-65, 67-73 and 75-76 as a whole was rendered prima facie obvious by five references at the time of invention, as an example of Rationale (G) of MPEP 2143: Some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. A skilled artisan would have a reasonable expectation of success because Porter’s EMCV leader protein has been shown to block “fluorescent import cargo” (¶[0081]). The motivations are also obvious, as taught by Lei regarding VLP and chimeric VLP (discussed supra).
Claims 36, 39, 66 and 74 are rejected under USC §103 as being unpatentable over Xu et al. 2020 (supra) and Lu et al. 2020 (supra) in view of Zhang et al. 2020 (supra), Lei et al., 2020 (supra) and Porter et al., 2007 (supra), as applied to claims 36, 39, 46, 60-65, 67-73 and 75-76, further in view of Hsieh et al. 2005 (Journal of Virology 79: 13848-13855).
As discussed supra, the collective teachings of Xu, Lu, Zhang, Lei and Porter render the invention of claims 36, 39, 46, 60-65, 67-73 and 75-76 as a whole obvious, in terms of a plasmid vector or an mRNA-LNP composition encoding a SARS-CoV-2 structural protein (S, M, N or E in Xu’s FIGURE 1 or S, M or E in Lu’s Fig. 1a), with an EMCV-derived NCT inhibitor (taught by Porter) that blocks nuclear transport to enhance VLP production in vitro and in vivo, and the mRNA can be formulated as a vaccine: with LNP encapsulation for delivery and the optional use of immune adjuvants for in vivo applications (as taught by Lu), and the mRNA-LNP is suitable for parenteral immunization and elicitation of SARS-CoV-2-specific immune responses (also taught by Lu). Through N- or C-terminus modification (as taught by Lei and two evidential references), the short NCT inhibitor (EMCV L protein with 67 amino acids, as defined by Porter’s SEQ ID NO: 1) is not expected to disrupt the SARS-CoV-2 VLP structure (as taught by Porter). The obvious methods above must rely on VLP production and secretion in vivo (as taught by Lu), so a packaging signal is indispensable in this process. However, these references still fall short of teaching the use of “a viral packaging signal” as part of the vector or polynucleotide encoding a viral structural protein and an EMCV-derived NCT inhibitor. This deficiency is overcome by Hsieh et al. 2005, as they taught the “assembly of severe acute respiratory syndrome coronavirus (SARS-CoV) RNA packaging signal into virus-like particles” (title). According to their disclosure, “recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA" (abstract). Through analysis of a SARS-CoV reference strain (TW1, GenBank accession number AY291451) (page 13894, third paragraph in MATERIALS AND METHODS), “the putative package signal of SARS-CoV genomic RNA” is mapped to “nucleotides 19888 to 19950 and nucleotides 19715 to 20294 (page 13894, fifth paragraph in MATERIALS AND METHODS). Given the 89% sequence homology between SARS-CoV (GenBank sequence AY291451) and SARS-CoV-2 (GenBank sequence MN908947.3, which has an S protein identical to instant SEQ ID NO: 13), a skilled artisan could follow Hsieh’s teaching to either use the SARS-CoV packaging signal or the SARS-CoV-2 version to arrive at the invention of a plasmid vector or mRNA-LNP composition cited in claims 36 and 39. The resulting vector and mRNA-LNP formulation would allow VLP assembly and packaging of SARS-CoV-2 RNA (partial viral genome) into the VLP structure after uptake by an eukaryotic cell (in vitro and in vivo), as taught by Xu, Lei, Hsieh and Lu. The VLP-RNA complex would mimic a replication deficient virion, with another inherent advantage: viral RNA is translated into viral protein antigens inside an antigen-presenting cell (APC) to boost VLP assembly and immunogenicity, as taught by Lei (one of the five motivations for VLP-base vaccines, as discussed supra). Plasmid vectors taught by Xu (FIGURE 1) already have the sequence encoding N protein, which is required for the recognition of packaging signal, as taught by Hsieh, so the inclusion of a packaging signal in a plasmid vector or mRNA construct would have a reasonable expectation of success in achieving the intended function. Thus, the invention of claims 36, 39, 66 and 74 as a whole was rendered prima facie obvious by six references at the time of invention, as another example of Rationale (G) of MPEP 2143: Some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. The motivation for retaining a viral packaging signal is also obvious – packaging signal is essential to the assembly of viral RNA into VLP (Hsieh, page 13848, paragraph on the right), which is part of the plasmid vector/mRNA → in vitro/in vivo VLP assembly process, as taught by Xu and Lu. VLP has an inherent advantage in immunogenicity (as taught by Xu, Lu and Lei), so a skilled artisan would be motivated to further modify the plasmid vector and polynucleotide product in claims 36 and 39 with Hsieh’s packaging signal toward an enhanced VLP production efficiency (in vitro and in vivo) and improved immunogenicity (in vivo).
Conclusion
No claims are allowed.
Additional Prior Art Cited but Not Applied
Kushnir et al., 2012. Virus-like particles as a highly efficient vaccine platform: Diversity of targets and production systems and advances in clinical development. Vaccine 31: 58–83.
This prior art teaches the use of virus-like particles (VLPs) as a “highly efficient vaccine platform” (title). “VLPs, non-enveloped and enveloped, have been produced for a number of targets using mammalian, plant, insect, yeast or bacterial cells, and cell-free platforms. Additionally, vaccine antigens can be produced as genetic fusions or chemical conjugates to viral structural proteins, resulting in chimeric VLPs” (page 59, second last paragraph in section 1/Introduction). “Both enveloped and non-enveloped VLPs are efficient in generating humoral and cell-mediated immune responses” (page 61, first paragraph in section 3). Among “VLP vaccines on the market and in the clinical development” (Table 2, pages 62-64), “human severe acute respiratory syndrome coronavirus (SARS CoV)-like particles” produced by “insect cells” (being eukaryotic) have “a very efficient formation of VLPs that morphologically mimic native SARS-CoV virions” (page 60, second last paragraph on the right). For immunization, route of administration includes IN (intranasal), IM (intramuscular) and SC (subcutaneous), often in the presence of immune adjuvants like aluminum hydroxide, aluminum phosphate and monophosphoryl lipid A. Moreover, “VLPs produced in different expression systems are often contaminated with residual host cell components such as lipids, nucleic acids and proteins that may stimulate the innate immunity and augment the adaptive immune response” (page 74, second paragraph on the right). Thus, before the emergence of SARS-CoV-2, VLPs for a closely related coronavirus (SARS-CoV) have been used as a subunit vaccine to elicit “protective immunogenicity associated with the VLP structure” (abstract) when coupled with various adjuvants and injection methods.
Spencer et al. 2016 (US PGPub 2016/0166613 A1, published 06/16/2016). Methods for controlled elimination of therapeutic cells.
This prior art taught the use of “a nucleic acid vaccines, such as DNA vaccines, wherein the vaccine comprises a nucleic acid comprising a polynucleotide… The vaccine may be administered to a subject, thereby transforming or transducing target cells in vivo” (¶[0029]). “Nucleic acid vaccines may include… non-viral DNA vectors, "naked" DNA and RNA, and viral vectors. Methods of transforming cells with these vaccines, and for optimizing the expression of genes included in these vaccines are known” (¶[0230]). “A nucleic acid or viral vector may be delivered to an organelle, a cell, a tissue or an organism via one or more injections (i.e., a needle injection), such as… subcutaneous, intradermal, intramuscular, intravenous, intraprostatic, intratumor, intraperitoneal, etc.”, and “the amount of the expression vector used may vary upon the nature of the antigen as well as the organelle, cell, tissue or organism used” (¶[0236]). “The cell… may be any type of eukaryotic cell, for example a mammalian cell…or human cell” (¶[0021]). “Upon introduction into a subject, the vaccine is able to provoke an immune response including, but not limited to, the production of antibodies, cytokines and/or other cellular responses” (¶[0101]), because “immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both” (¶[0047]). These teachings are pertinent to claims 36, 39 and their dependent claims in the instant application.
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/JIANMING TANG/
Examiner, Art Unit 1671
/Michael Allen/ Supervisory Patent Examiner, Art Unit 1671
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