DETAILED ACTION
The present application is being examined under the pre-AIA first to invent provisions. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Applicant’s Request for Continued Examination, Amendment and Arguments/Remarks received on 07 July 2025 have been entered. No claims have been cancelled and no new claims have been added by Applicant. Claims 1, 7, 9, 14, 17, 18, 20, 25, and 26 are independent claims.
Claims 1, 5-9, 14-20, and 24-28 are currently pending and under examination in the instant application. An action on the merits follows.
Specification
The disclosure is objected to because of the following informalities: the Brief Description of the Drawings does not include a description of each panel. Specifically, Figure 1 does not include individual descriptions of panels A-C, Figure 2 does not include individual descriptions of panels A-C, Figure 7 does not include individual descriptions of panels A-B, and Figure 10 does not include individual descriptions of panels A-B. See MPEP 608.01(f), which states, “When there are drawings, there shall be a brief description of the several views of the drawings and the detailed description of the invention shall refer to the different views by specifying the numbers of the figures, and to the different parts by use of reference letters or numerals”. Further, MPEP 608.01(f) instructs Examiners such that “If the drawings show Figures 1A, 1B, and 1C and the brief description of the refers only to Figure 1, the examiner should object to the brief description, and require applicant to provide a brief description of Figures 1A, 1B, and 1C.” Appropriate correction is required.
Claim Objections
Currently amended and previously presented independent claims 1, 7, 18, and 20 are objected to because of the following informalities: claim 1 recites, “consisting” in lines 3, 5, 3, and 2, respectively, which appears to be a typographical misspelling of “consist” such that the claims should read “wherein the protamine-RNA nanoparticles consist of protamine and RNA” (claims 1, 7, and 18) or “wherein the nanoparticles consist of protamine, RNA, and polyethylene glycol” (claim 20). Appropriate correction is required.
Claim Rejections - 35 USC § 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph
The rejection of amended claim 19 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as failing to particularly point out and distinctly claim the subject matter which the inventor(s) regards as the invention for reciting, “stimulates a strong interferon-α (IFN-α) production” is maintained in view of Applicant’s amendments to the claims such that claim 18 now recites, “wherein the immunostimulant composition produces immune stimulation in an endosome of a cell” and claim 19 now recites, “wherein the immunostimulant composition stimulates a stronger interferon-α (IFN-α) production than production of tumor necrosis factor-α (TNF-α)”. The amendments to the claim 18 provide context for the stimulation to be a functional limitation and to occur within an endosome of a cell. The amendments to claim 18 additionally provide that the stimulation of IFN-α is stronger than the production of TNF-α.
However, the claim is still indefinite in that it is unclear what production of TNF-α is being referenced, in that it is unclear whether the comparison is to production of TNF-α within the full cell (e.g., all TNF-α produced by the cell, including secreted TNF-α), to production of TNF-α just within the endosome of the cell, only a secreted level of TNF-α, or some other measure of TNF-α production. Additionally, it is unclear whether the IFN-α production is meant to encompass only IFN-α within the endosome of the cell, IFN-α secreted by the cell, all IFN-α produced by the cell (both intracellular and secreted), or some other measure of IFN-α production.
Further, it is unclear whether “stronger” is meant to refer to the amount of IFN-α compared to the amount of TNF-α or whether it is meant to refer to specific activities of IFN-α and TNF-α. It is also unclear whether the comparing of IFN-α production to TNF-α production is meant to be a relative induction level, absolute level in mass, absolute level in mols, or some other unit of measuring production of the factors.
Additionally, it is unclear what cell type(s) is/are meant to be encompassed by the comparing of production for the TNF-α and IFN-α, such that it is unclear whether the comparison needs be for the production of the TNF-α and IFN-α within the same cell, within the same cell type, or within any cell in vitro, ex vivo, or in vivo of any organism such that the production of IFN-α in one cell is compared to the production of TNF-α in another cell.
As such, the metes and bounds of the claim cannot be determined.
The rejection of amended and previously presented claims 14-17 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as failing to particularly point out and distinctly claim the subject matter which the inventor(s) regards as the invention for:
claim 14 reciting, “providing an aqueous solution of RNA at 1 mg/ml or less” on line 3 and “providing an aqueous solution of protamine at 1 mg/ml or less” on line 5;
Claim 17 reciting, “RNA at less than 2 mg/ml” in line 2 and “protamine at less than 2 mg/ml” in line 4;
is withdrawn over amended claim 17 and maintained over amended and previously presented claims 14-16 in view of Applicant’s amendments to the claims such that each claim now recites, “nanoparticles comprising protamine and RNA”, and such that claim 17 has been rewritten in independent form so as not to depend on claim 15.
Applicant’s amendment to claim 14 such that it now recites, “nanoparticles comprising protamine and RNA” in lines 1-2 and arguments have clarified the requirement to for both protamine and RNA within the nanoparticles produced by the method. However, recitation of “nanoparticles comprising protamine and RNA” in lines 1-2 conflicts with the limitation recited in lines 7-8 that the method results in “nanoparticles consisting of protamine and RNA”. As such, the metes and bounds of the claim cannot be determined.
Additionally, claim 14 further recites “using” in lines 3 and 5 for steps (a) and (b), respectively, which is indefinite because it is unclear whether the solution containing 0 to 25 mM electrolytes is the solution being provided in each step or whether the providing step is a step of modifying the solution containing 0 to 25 mM electrolytes to arrive at the aqueous solution of RNA at 1 mg/ml or the aqueous solution of protamine at 1 mg/ml, such as by using the solution containing 0 to 25 mM electrolytes to dilute a more concentration solution of RNA or protamine. As such, the metes and bounds of the claim cannot be determined.
Previously presented claims 15-16 are included in this rejection due to their dependence on independent claim 14.
Amended and previously presented claims 1, 5-6, 8-9, 16, 17, and 24-28 are newly 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.
Amended independent claim 1 recites, “originates” in line 5, which is indefinite because it is unclear what is encompassed by “immune stimulation that originates in an endosome” in that it is unclear what is considered the origin of the immune stimulation. For example, it is unclear whether the origin of the immune stimulation is the contacting of the cell with the immunostimulant composition, such that all the downstream processes of immune stimulation result from that original contacting, or whether the immune stimulation is referring specifically to a downstream consequence of the contacting, such as a specific binding interaction. As such, the metes and bounds of the claim cannot be determined. Claims 5-6, 8-9, and 24-28 are included in this rejection due to their dependence on or encompassing of claim 1.
The term “isotonic” in claims 16 and 17 lines 2 and 7, respectively, is a relative term which renders the claim indefinite. The term “isotonic” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Therefore, it is unclear what the solutions are “isotonic” relative to. For example, it is unclear whether the various solutions recited in the claims are isotonic with each other, isotonic to human blood, or some other solution. Further, since the electrolyte concentrations in the starting solutions prior to dilution are unspecified/unknown, it is unclear what final concentrations of electrolytes would be obtained by dilution with an aqueous solution containing 0-25 mM electrolytes. As such, the metes and bounds of the claims cannot be determined.
Claims 27 and 28 each recites “selected from the group consisting of… or…”, which is indefinite because it is unclear what the groups consists of. As such, the metes and bounds of the claim cannot be determined. Examiner suggests replacing “or” with “and” to clarify the metes and bounds of the group.
Claim Interpretation
Note that claims 1, 7, 14, 18, and 20 recite a protamine-RNA nanoparticles “consisting of” protamine and RNA (and polyethylene glycol in claim 20), which has been interpreted by Examiner to allow the additional inclusion of solvent (e.g., water) and salt atoms given the recitation in claims 14 and 17 that nanoparticles of the invention are produced in aqueous solution in the presence of electrolytes.
Previously presented claims 7 and 24 each recite, “RNA is an oligonucleotide having a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 5, 10, 11, 12 and 13”, which is have been interpreted such that “having a sequence” indicates that the oligonucleotide comprises any at least 2 consecutive nucleic acids of the sequences as set forth in SEQ ID NO: 1, 2, 3, 5, 10, 11, 12 and 13.
Amended independent claim 14 recites, “providing an aqueous solution of RNA at 1 mg/ml or less” on line 3 and “providing an aqueous solution of protamine at 1 mg/ml or less” on line 5; and claim 17 recites, “RNA at less than 2 mg/ml” in line 2 and “protamine at less than 2 mg/ml” in line 4. These limitations have been interpreted in view of the additional limitations within the claims requiring the presence of protamine and RNA in the compositions made by each method. As such, “less than” and “or less” have been interpreted to be any non-zero amount of the component which meets the upper limits specified by the claims (e.g., 1 mg/ml or less or less than 2 mg/ml).
Amended and previously presented claims 16 and 17 each recite “isotonic” stock solutions without indicating with respect to what the solutions are isotonic. Therefore, claims 16 and 17 have been interpreted such that “isotonic” solutions comprise any amount of electrolytes.
Claim Rejections - 35 USC § 103
The rejection of amended and previously presented claims 1, 5-9, 14-20, and 24-28 under 35 U.S.C. 103 as being unpatentable over Jung [EP1083232A1, published 14 March 2001 (previously cited)]; in view of Parker & Seymour [2002, Journal of Bioactive and Compatible Polymers, 17, 229-238]; Scheel et al. [2004, Eur. J. Immunol., 34, 537–547 (previously cited)]; Kikuchi [WO2008056623A1, published 15 May 2008 (previously cited)]; McSwiggen [WO2008115851A2, published 25 September 2008 (previously cited)]; Li et al. [2004, Molecular Pharmaceutics, 3(5), 579-255, (previously cited, IDS)]; Yanni [WO2007121347A2, published 25 October 2007 (previously cited, IDS)]; Hoerr [US20120009221A1, published 12 January 2012 (previously cited, IDS)]; Probst [WO2009046739A1, published 16 April 2009 (previously cited)]; Cui et al. [2004, Molecular Pharmaceutics, 2(1), 22-28 (previously cited)]; and Loeffler et al. [2005, Cancer Research, 65(12), 5027-5030]; is withdrawn.
Amended and previously presented claims 1, 5-9, 14-20, and 24-28 are newly rejected under 35 U.S.C. 103 as being unpatentable over Parker & Seymour [2002, Journal of Bioactive and Compatible Polymers, 17, 229-238 (previously cited)]; in view of Scheel et al. [2004, Eur. J. Immunol., 34, 537–547 (previously cited)]; Sioud [2006, TRENDS in Molecular Medicine, 12(4), 167-176]; Li et al. [2004, Molecular Pharmaceutics, 3(5), 579-255 (previously cited, IDS)]; McSwiggen [WO2008115851A2, published 25 September 2008 (previously cited)]; Shipman [1969, Proceedings of the Society for Experimental Biology and Medicine, 130(1), 305-310]; Scheel et al. [2005, European Journal of Immunology, 35, 1557-1566]; and Loeffler et al. [2005, Cancer Research, 65(12), 5027-5030] (previously cited).
Regarding claim 1, Parker teaches a composition consisting of a pharmaceutically acceptable carrier (e.g., HEPES buffer in water) and one or more protamine-RNA nanoparticle, wherein the nanoparticles consisting of protamine and RNA, wherein the protamine-RNA nanoparticles have an average diameter of 85.8 nm [page 231 ¶ 2, 5, page 232 ¶ 3, Table 1].
Parker does not teach that the composition is an immunostimulant composition that produces immune stimulation that originates in an endosome of a cell.
However, Scheel (2004) teaches a composition consisting of a pharmaceutically acceptable carrier (e.g., water) and one or more protamine-RNA complexes [column 16 ¶ 6- column 17 ¶ 1]. Scheel (2004) additionally teaches that RNA molecules (e.g., mRNA encoding an antigen) protected against RNase-mediated degradation by association with a cationic peptide (e.g., protamine) are very potent immunostimulatory molecules useful as natural danger signals with potential for treatment and vaccine development [column 3 ¶ 2-column 4 ¶ 1, Table 1], such that the method of Scheel (2004) successfully generated protamine-RNA nanoparticles able to activate dendritic cells (DC) [Figure 1].
Scheel (2004) further teaches that the activation of DCs by stabilized RNAs is MyD88-dependent, and that MyD88 is required for toll-like receptor (TLR)-mediated danger signal recognition and downstream cellular response which consist of the activation of different signal transduction cascades inducing the secretion of cytokines (e.g., IFN-α) [column 2 ¶ 2, Figure 2]. Scheel (2004) also teaches that MyD88 is the primary intracellular adaptor molecule that mediates the signaling cascade and which leads to the activation of DC by pathogen-associated molecular patterns (PAMP), and that the dependence on MyD88 indicates that TLR are responsible for the recognition of stabilized RNA molecules by DC [column 7 ¶ 1]. Scheel (2004) also teaches that TLR7 and TLR8 are very promising candidates as the specific TLRs responsible for the recognition of stabilized RNAs [column 12 ¶ 3]. Therefore, the dependence on MyD88 taught by Scheel (2004) indicates that the stabilized RNAs are recognized by TLRs.
Sioud teaches that RNA activates cells that express the appropriate TLRs, such that the RNAs are delivered to the endosomes to signal via TLR7, TLR8, or TLR9; that RNAs then activate TLRs within the endosomes to promote immune effects mediated by the adaptor protein MyD88; and that such immune effects include activation of NF-κB and transcription of proinflammatory cytokine genes as well as activation of the interferon regulatory factor (IRF7) which is responsible for IFN-α induction [Figure 1].
Therefore, given the teachings of Scheel (2004) and Sioud, the protamine-RNA nanoparticle composition taught by Parker is an immunostimulant composition wherein the immunostimulant composition produces immune stimulation that originates in an endosome of a cell.
Regarding claim 5, Parker teaches wherein the RNA contains an 894 nt mRNA encoding GFP which was transcribed with a cap analog at a ratio of 5:1 cap analog:GTP, thereby teaching that the RNA contains at least one G nucleotide [page 230 ¶ 5- page 231 ¶ 1].
Regarding claim 6, Parker does not teach that the RNA is an oligonucleotide of from 6 to 100 nucleotides.
However, Li teaches that selective gene inhibition by small interference RNA (siRNA) therapeutics promises the treatment of diseases that cannot be cured by conventional drugs, and that sterically stabilized nanoparticle delivery addresses the problems of antisense therapy being hindered due to poor stability in physiological fluids and limited intracellular uptake [abstract]. Therefore, an ordinarily skilled artisan would have been motivated to use RNA oligonucleotides, such as siRNA, in a protamine-RNA nanoparticle to stabilize the RNA and promote cellular uptake and to deliver the siRNA as a therapeutic for the treatment of disease.
Additionally, Scheel (2004) teaches the administration of phosphorothioate RNA oligonucleotides are immunostimulating molecules able to trigger maturation of DC as or more efficiently than CpG-containing DNA or double stranded RNA [column 6 ¶ 2]. Accordingly, an ordinarily skilled artisan would have been motivated to use a stabilized RNA oligonucleotide, such as a phosphorothioate RNA oligonucleotides, to enhance the immunostimulating effects of the protamine-RNA nanoparticle.
Regarding claims 7 and 24, Parker does not teach wherein the RNA is an oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 5, 10, 11, 12, and 13 of the instant application.
However, McSwiggen teaches siRNA constructs which include sequences which are 100% identical to sequences as claimed in claims 7 and 24 of the instant application. Specifically, SEQ ID NO: 2 of the instant application is a 21 nt siRNA which is identical to nucleotides 4-24 of the 25 nt SEQ ID NO: 3408 and nucleotides 5-25 of SEQ ID NO: 3409 of McSwiggen. McSwiggen additionally teaches that siRNAs mediate RNA interference (RNAi) for sequence-specific post-transcriptional gene silencing; that when an siRNA is introduced into a cell, it binds to the endogenous RNAi machinery to disrupt the expression of mRNA containing complementary sequences with high specificity; that any disease-causing gene and any cell type or tissue can potentially be targeted; and that one way to carry out RNAi is to introduce or express a siRNA in cells [page 1 ¶ 3-page 2 ¶ 3]. McSwiggen also teaches that RNAi can be used to specifically disrupt cellular signaling pathways, such as disrupting the viral signaling pathway that controls virus production. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to use an siRNA, such as an siRNA having the sequence of SEQ ID NO: 2 of the instant application, to target genes of interest, particularly those involved in disease processes, such as genes involved in viral signaling pathways, and to deliver the siRNA to cells to facilitate RNAi knockdown of target genes.
Regarding claim 8, Parker teaches wherein the RNA is an mRNA having 894 nt [page 230 ¶ 5- page 231 ¶ 1].
Regarding claim 9, Scheel (2004) teaches that stabilized RNA is a natural danger signal with a potential for treatment and vaccine development, in that danger signals stimulate an adaptive immune response and are used in vaccines, wherein the danger signals act as adjuvants [column 2 ¶ 1- column 3 ¶ 1, column 4 ¶ 1]. Scheel (2004) also teaches that stabilized DNA can be used as an adjuvant, but that the utilization of DNA as an adjuvant or as a vaccine delivery vehicle might face safety issues, and that mRNA-based vaccination is a newer, safer genetic vaccination strategy [column 3 ¶ 1-2].
Scheel (2004) also teaches that the mRNA-based vaccine comprises an mRNA which encodes an antigen [column 3 ¶ 2]. Scheel (2004) also teaches the administration of stabilized RNA with an β-Gal antigen, wherein antigen-specific antibodies were produced, both for phosphorothioate RNA oligos and for trans-stabilized RNA, indicating that both phosphorothioate RNA oligos and trans-stabilized RNA can act as an adjuvant to promote the induction of antigen-specific immunity [column 11 ¶ 1].
Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to use the immunostimulatory composition as taught by Parker in an mRNA-based vaccine comprising an antigen for the induction of antigen-specific immunity.
Regarding claims 14-17, Parker teaches a method for preparation of nanoparticles comprising protamine and RNA having an average size of 86 nm, the method comprising:
resuspending precipitated RNA in water (e.g., a solution containing 0 mM electrolytes);
providing an aqueous stock solution of 10 mM protamine, [page 231 ¶ 5], which is about 50 mg/ml or about 5000 heparin-neutralizing units of protamine per ml; and
combining the solutions of (a) and (b) by adding an appropriate amount of RNA to the desired amount of polycation (e.g., protamine) to achieve an amine to phosphate (N:P) ratio of 4.0 wherein the nanoparticle complexes were formed in 10 mM HEPES buffer at pH 7.4 [page 231 ¶ 2, 5, page 232 ¶ 3, Table 1].
Parker also teaches that the desired amount of polycation (e.g., protamine) is 9 µl of 10 mM stock solution for an N/P ratio of 10 [page 3 ¶ 5], and therefore, the N/P ratio of 4.0 taught specifically for protamine [page 232 ¶ 2, Table 1], would call for 3.6 µl of a 10 mM protamine stock solution, which corresponds to 180 µg of protamine. Achieving an N/P of 4 with 180 µg of protamine would require about 112 µg of mRNA.
Parker teaches that 30 µl of the protamine-RNA nanoparticle mixture were added to each well [page 231 ¶ 5]. Note that an ordinarily skilled artisan at the time of filing would have recognized the need to perform such experiments in replicates, such that an ordinarily skilled artisan would expect that the experiment was run at least in triplicate, indicating a minimum total batch volume of 100 µl to account for pipetting errors. For 100 µl, the nanoparticle solution of Parker added to the wells would be 180 µg /100 µl protamine (e.g., 1.8 mg/ml) and 112 µg mRNA/ 100 µl (e.g., 1.1 mg/ml) maximally, which are both less than 2 mg/ml.
Additionally, Scheel (2004) teaches a method of producing protamine-RNA complexes comprising (a) providing an aqueous solution of RNA at 1 mg/ml by resuspending RNA in pure water (e.g., 0 mM electrolytes) and (c) mixing the 1 mg/ml RNA solution at a 1:1 ratio with an equal volume of (b) a 1 mg/ml solution of protamine sulfate to obtain nanoparticles consisting protamine and RNA [column 16 ¶ 6- column 17 ¶ 1]. Scheel (2004) additionally teaches that that their method successfully generated protamine-RNA complexes able to activate dendritic cells (DC) [Figure 1].
Neither Parker nor Scheel (2004) teach that the protamine stock solution contains 0 to 25 mM electrolytes nor that the protamine stock solution is isotonic. Note that Applicant has not provided a reference state for “isotonic” and as such “isotonic” solutions have been interpreted to encompass solutions having any amount of electrolytes.
However, Parker teaches components of the solution, such as water, protamine, and HEPES buffer without reciting the inclusion of electrolytes. Therefore, the lack of a disclosure of electrolytes in the solution is presumed to indicate a lack of electrolytes in the solution (e.g., 0 mM electrolytes) in the absence of evidence to the contrary. Further, to the extent that sodium hydroxide (NaOH) may have been used to adjust the pH of the HEPES buffer, the amount of NaOH needed to reach a pH of 7.4 would be approximately half of the molar amount of HEPES since the target pH is close to the pKa of the HEPES acid (e.g., 7.31 at 37oC as taught by Shipman [column 1 ¶ 3]). Therefore, approximately 5 mM sodium would be present in a 10 mM HEPES buffer solution with a pH of 7.4 as taught by Parker.
Regarding claim 18, note that independent claim 18 differs from independent claim 1 only in reciting that the immunostimulant composition produces “immune stimulation in an endosome of a cell” (claim 18) instead of “immune stimulation that originates in an endosome of a cell”. Given the teachings above for the limitations of claim 1, including the teachings of Sioud that RNA activates TLRs within the endosomes to promote immune effects, the teachings above address both immune stimulation originating in an endosome of a cell as well as immune stimulation in an endosome of a cell.
Regarding claim 19, as discussed above, Scheel (2004) teaches that the activation of DCs by stabilized RNAs is MyD88-dependent, and that MyD88 is required for toll-like receptor (TLR)-mediated danger signal recognition and downstream cellular response which consist of the activation of different signal transduction cascades inducing the secretion of cytokines (e.g., IFN-α) [column 2 ¶ 2, Figure 2]. Sioud teaches that RNA activates TLRs within the endosomes to promote immune effects mediated by the adaptor protein MyD88; and that such immune effects include activation of NF-κB and transcription of proinflammatory cytokine genes as well as activation of the interferon regulatory factor (IRF7) which is responsible for IFN-α induction [Figure 1].
Further, as discussed above, it is unclear what production of TNF-α the production of IFN-α is being compared to.
Scheel (2005) teaches an immunostimulant composition consisting of a pharmaceutically acceptable carrier (e.g., water) and one or more protamine-RNA nanoparticles (produced simply by mixing together a 1 mg/ml RNA with an equal volume of a 1 mg/ml solution of protamine sulfate), wherein the immunostimulant composition stimulates a stronger IFN-α production than production of TNF-α in the tested cell types, wherein IFN-α production is measured by ELISA to have an OD405 value of about 2.5 and TNF-α production is measured by ELISA to have an OD405 value of about 0.25 in pDC and Mo-DC, respectively [column 16 ¶ 2, Figure 4]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would expect a protamine-RNA nanoparticle as taught by Parker to exhibit a stronger IFN-α production than production of TNF-α.
Regarding claim 20, Li additionally teaches that DSPE-PEG can impart a steric hindrance for lipid-based formulations and prevent particle aggregation, which appears to explain the particle size reduction they observed after PEGylation and the observed stabilization of the particles (reduced aggregation and precipitation) in the presence of FBS [column 20 ¶ 1, Figure 3]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to include PEG in a protamine-RNA nanoparticle to reduce/ prevent aggregation of the particles in solution/ media.
Regarding claim 25, as discussed above, Scheel (2004) teaches that danger signals are used in vaccines to act as adjuvants to stimulate an adaptive immune response [column 2 ¶ 1- column 3 ¶ 1, column 4 ¶ 1]. Scheel (2004) also teaches that the mRNA-based vaccine comprises an mRNA which encodes an antigen [column 3 ¶ 2]. Scheel (2004) also teaches the administration of stabilized RNA with a β-Gal antigen, wherein antigen-specific antibodies were produced, both for phosphorothioate RNA oligos and for trans-stabilized RNA, indicating that both phosphorothioate RNA oligos and trans-stabilized RNA can act as an adjuvant to promote the induction of antigen-specific immunity [column 11 ¶ 1].
Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to use the immunostimulatory composition as taught by Parker in an mRNA-based vaccine comprising an antigen and an adjuvant for the induction of antigen-specific immunity.
Regarding claims 26-28, Loeffler teaches that cyclophosphamide is an immunostimulating agent used in combination with cancer immunotherapies which has been shown to augment the efficacy of antitumor immune responses by depleting CD4+/CD25+ T regulatory cells, increasing both T-lymphocyte proliferation and T memory cells, and mediating killing of circulating endothelial progenitors [abstract]. Loeffler also teaches the combined application of cyclophosphamide with an oral DNA vaccine targeting PDGF-B completely inhibited the growth of different tumor types and also led to tumor rejections in mice [abstract]. Loeffler also teaches that cyclophosphamide induces type I interferon [title of reference 4]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to incorporated the immunostimulating/immunomodulating agent cyclophosphamide into a nucleic-acid based vaccine, such as the protamine-RNA-based vaccine as taught by references described above, to increase efficacy of anti-tumor vaccines.
Given the motivation taught by Li to use RNA oligonucleotides, such as siRNA, in a protamine-RNA nanoparticle to stabilize the RNA and promote cellular uptake and to deliver the siRNA as a therapeutic for the treatment of disease; the motivation taught by Scheel (2004) to use a stabilized RNA oligonucleotide, such as a phosphorothioate RNA oligonucleotides, to enhance the immunostimulating effects of the protamine-RNA nanoparticle; the motivation taught by McSwiggen to use an siRNA, such as an siRNA having the sequence of SEQ ID NO: 2 of the instant application, to target genes of interest, particularly those involved in disease processes, such as genes involved in viral signaling pathways, and to deliver the siRNA to cells to facilitate RNAi knockdown of target genes; the additional motivation taught by Scheel (2004) to use the immunostimulatory composition as taught by Parker in an mRNA-based vaccine comprising an antigen and an adjuvant for the induction of antigen-specific immunity; the teaching of Scheel (2004) that that their method successfully generated protamine-RNA complexes able to activate dendritic cells (DC); the additional motivation taught by Li to include PEG in a protamine-RNA nanoparticle to reduce/ prevent aggregation of the particles in solution/ media; and the motivation taught by Loeffler to incorporated the immunostimulating/immunomodulating agent cyclophosphamide into a nucleic-acid based vaccine, such as the protamine-RNA-based vaccine as taught by references described above, to increase efficacy of anti-tumor vaccines; it would have been prima facie obvious to an ordinarily skilled artisan at the time of filing the instant application to modify the composition and method of Parker to use RNA oligonucleotides, including siRNAs having the sequence of SEQ ID NO: 2, to use the composition in a vaccine comprising an antigen and/or an adjuvant and/or the immunomodulating agent cyclophosphamide, and/or to include PEG in the nanoparticles, wherein the nanoparticles are generated by combining an 1 mg/ml RNA solution having 0-25 mM electrolytes with a 1 mg/ml protamine solution having 0-25 mM electrolytes, with a reasonable expectation of success.
Insofar as applicant’s arguments apply to this new grounds of rejection, Applicant argues that:
the disclosure of Parker is obscure as regards the factual amount of protamine and mRNA used for preparing the alleged nanoparticles, such Applicant provides an extrapolated, estimated calculation of the amounts of mRNA expected to have been used, based on which Applicant then argues would have been a prohibitive expense which would have suggested to one having ordinary skill in the art to look for other methods/ratios;
also based on the extrapolated, estimated calculations, Parker must have used concentrations of protamine and RNA which are much higher than that recited in the instant claims;
Parker’s higher concentrations of RNA and protamine should have produced particles greater than 575 nm, especially since Parker teaches using 10 mM HEPES, which necessarily is a disodium salt and therefore contributes at least 20 mM sodium ion as electrolytes to the solution;
Parker’s GFP mRNA must enter the cytosol to be translated, and so Parker is not teaching endosomal immunostimulation, and an ordinarily skilled artisan would not expect to stimulate TLRs in the endosome of cells which is required for producing IFN-α;
The Declaration submitted 07 March 2013 includes an affidavit from Dr. Pascolo clearly stating that in their lab in Tubingen, they were not able to control the size of the particles as taught in the present application; and further that even though the methods in Scheel don’t disclose the presence of electrolytes, conclusions that they used a solution containing no electrolytes represents overinterpretation of the disclosure of Scheel, supported in fact by Dr. Pascolo’s attestation that they always produced the RNA/protamine formulations and received structures seen in Scheel et al. 2005.
However, this is not agreed.
In response to applicant’s arguments against the references individually, it is noted that the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). 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). Further, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In addition, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Specifically, regarding Applicant’s argument 1), note that an N/P ratio of 4 is taught for the protamine/RNA complexes, as taught in Parker in Table 1. Parker teaches using 9 µl of a 10 mM amine nitrogen stock solution for N/P = 10 [page 231 ¶ 4]. Therefore, as indicated by Parker, an N/P ratio of 4 would call for 3.6 µl of a 10 mM protamine solution, which corresponds to 180 µg of protamine. Achieving an N/P of 4 with 180 µg of protamine would require about 112 µg of mRNA, which can be prepared in as little as 25-50 µl of a T7 transcription reaction using the RiboMAXTM (Promega) kit supplemented with a 5:1 cap analog/GTP ratio, which Parker teaches to have used [page 230 ¶ 4- page 231 ¶ 1]. Such reaction volumes and reagent usage would not have been prohibitively expensive such that an ordinarily skilled artisan would not have performed the experiments as disclosed to have been performed by Parker.
Regarding Applicant’s argument 2), Applicant is assuming that the 30 µl volume is the total volume prepared, which is not taught by Parker. Parker teaches that 30 µl of the protamine-RNA nanoparticle mixture were added to each well [page 231 ¶ 5]. Note that an ordinarily skilled artisan at the time of filing would have recognized the need to perform such experiments in replicates, such that an ordinarily skilled artisan would expect that the experiment was run at least in triplicate. As such, a minimum total batch volume of 100 µl to account for pipetting errors would be a more reasonable expectation based on the teachings of Parker. For 100 µl, the solution of Parker would be 180 µg /100 µl protamine (e.g., 1.8 mg/ml) and 112 µg mRNA/ 100 µl (e.g., 1.1 mg/ml) maximally, which are both less than 2 mg/ml.
Note also that Parker was not relied on for teaching the concentrations of RNA and protamine in the solutions which are combined to obtain nanoparticles as recited in steps 14 and 17.
Scheel (2004) was cited for teaching a method of producing protamine-RNA complexes comprising providing an aqueous solution of RNA at 1 mg/ml by resuspending RNA in pure water and mixing the 1 mg/ml RNA solution at a 1:1 ratio with an equal volume of a 1 mg/ml solution of protamine sulfate [column 16 ¶ 6- column 17 ¶ 1]. Scheel (2004) was additionally cited for teaching that RNA molecules (e.g., mRNA encoding an antigen) protected against RNase-mediated degradation by association with a cationic peptide (e.g., protamine) are very potent immunostimulatory molecules useful as natural danger signals with potential for treatment and vaccine development [column 3 ¶ 2-column 4 ¶ 1, Table 1], such that the method of Scheel (2004) successfully generated protamine-RNA nanoparticles able to activate dendritic cells (DC) [Figure 1].
Regarding argument 3), note that the estimates of concentrations proposed by Applicant for the RNA and protamine concentrations used to form the nanoparticles cannot contradict that actual, specific disclosure by Parker of measurements teaching that the protamine-RNA nanoparticles obtained by their method have an average diameter of 85.8 +/- 1.5 nm [Table 1]. Therefore, if Applicant’s assertion that such nanoparticle sizes necessarily only form in solutions comprising lower concentrations of RNA (e.g., < 2 mg/ml), protamine (e.g., <2 mg/ml), and salt (e.g., 25 mM or less) is accurate, then the better presumption is that Applicant’s estimate of the concentrations used by Parker are inaccurate.
Additionally, with respect to Applicant’s assertion that 10 mM HEPES necessarily is a disodium salt and therefore contributes at least 20 mM sodium ion as electrolytes to the solution, note that while a disodium salt is one option for buying/ preparing a HEPES reagent, it is not necessarily so. HEPES is routinely sold/ prepared using the acid form which is free of sodium salts.
Further, to the extent that sodium hydroxide may have been used to adjust the pH of the HEPES buffer, the amount of NaOH needed to reach a pH of 7.4 would be approximately half of the molar amount of HEPES since the target pH is close to the pKa of the HEPES acid. Therefore, approximately 5 mM sodium would be present in a 10 mM HEPES buffer solution with a pH of 7.4 as taught by Parker.
Regarding Applicant’s argument 4), note that Parker was not relied on for teaching that the protamine-RNA nanoparticles are taken up into endosomes, where they produce immune stimulation. However, given that the structure of the protamine-RNA nanoparticles meet the claim limitations, the function of the nanoparticles are expected to be the same, including the mechanism of endosomal uptake by the cell.
Further, as discussed above, Scheel (2004) was cited for teaching a method of producing protamine-RNA complexes comprising providing an aqueous solution of RNA at 1 mg/ml by resuspending RNA in pure water and mixing the 1 mg/ml RNA solution at a 1:1 ratio with an equal volume of a 1 mg/ml solution of protamine sulfate [column 16 ¶ 6- column 17 ¶ 1]. Scheel (2004) was additionally cited for teaching that RNA molecules (e.g., mRNA encoding an antigen) protected against RNase-mediated degradation by association with a cationic peptide (e.g., protamine) are very potent immunostimulatory molecules useful as natural danger signals with potential for treatment and vaccine development [column 3 ¶ 2-column 4 ¶ 1, Table 1], such that the method of Scheel (2004) successfully generated protamine-RNA nanoparticles able to activate dendritic cells (DC) [Figure 1].
Scheel (2004) further teaches that the activation of DCs by stabilized RNAs is MyD88-dependent, and that MyD88 is required for toll-like receptor (TLR)-mediated danger signal recognition and downstream cellular response which consist of the activation of different signal transduction cascades inducing the secretion of cytokines (e.g., IFN-α) [column 2 ¶ 2, Figure 2]. Scheel (2004) also teaches that MyD88 is the primary intracellular adaptor molecule that mediates the signaling cascade and which leads to the activation of DC by pathogen-associated molecular patterns (PAMP), and that the dependence on MyD88 indicates that TLR are responsible for the recognition of stabilized RNA molecules by DC [column 7 ¶ 1]. Scheel (2004) also teaches that TLR7 and TLR8 are very promising candidates as the specific TLRs responsible for the recognition of stabilized RNAs [column 12 ¶ 3].
Additionally, Sioud teaches that RNA activates cells that express the appropriate TLRs, such that the RNAs are delivered to the endosomes to signal via TLR7, TLR8, or TLR9; that RNAs then activate TLRs within the endosomes to promote immune effects mediated by the adaptor protein MyD88; and that such immune effects include activation of NF-κB and transcription of proinflammatory cytokine genes as well as activation of the interferon regulatory factor (IRF7) which is responsible for IFN-α induction [Figure 1].
Therefore, the dependence on MyD88 taught by Scheel (2004) indicates that the stabilized RNAs are recognized by TLRs, and therefore are taken up through TLR-mediated endocytosis into endosomes such that MyD88 is activated downstream of the TLRs to induce production and secretion of cytokines such as IFN-α.
Regarding Applicant’s argument 5), note that neither Scheel (2004) nor Scheel (2005) were relied on for teaching the absence of electrolytes in the protamine solution. Additionally, the Declaration submitted 07 March 20013 indicates that the specific salt concentrations were not discovered by Dr. Pascolo and his group by the time of the experiments disclosed in Scheel (2005). However, the affidavit does not indicate the presence of absence of salt within the protamine solution used in the experiments disclosed in Scheel (2004) nor Scheel (2005). Additionally, the disclosure that Dr. Pascolo’s lab did not produce nanoparticle-sized protamine-RNA complexes prior to the optimization of salt, RNA, and protamine concentrations does not support a finding of unexpected results in that Parker discloses nanoparticle-sized (e.g., 86 nm) protamine-RNA complexes.
Further, the assertion that only the optimized nano-scale protamine-RNA nanoparticles preferably induce a strong IFN-α response, as disclosed in Figures 5C and 5D of the instant application is also not found persuasive. Figure 5C shows IFN-α induction data for administration of mRNA NY-ESO-1 + Protamine in water or mRNA NY-ESO-1 + Protamine in Ringer lactate, wherein the IFN-α induction is greater for the water condition than for the Ringer condition. Figure 5D shows TNF-α induction data for administration of mRNA NY-ESO-1 + Protamine in water or mRNA NY-ESO-1 + Protamine in Ringer lactate to PBMCs, wherein the TFN-α induction is greater for the Ringer condition than for the water condition.
However, comparing the IFN-α induction to the TNF-α induction for the water solution shows that the IFN-α induction is about 2200 pg/ml and the TNF-α induction is about 2500 pg/ml. Additionally, the data in Figures 5A and 5B for administration of RNA18UR to fresh PBMCs from 3 different donors, wherein the IFN-α induction ranges from about 1500 pg/ml to about 3000 pg/ml in water (Figure 5A) and the TNF-α induction range from about 1500 pg/ml to about 2500 pg/ml in water. Additionally, comparing the values for protamine in 25 mM electrolytes, administration of RNA18UR + protamine results in about 13k-21k pg/ml of TNF-α and about 1500-2200 pg/ml of IFN-α, which is substantially more TNF-α than IFN-α. Therefore, the data do not support an unexpected result of a stronger production of IFN-α compared to production of TNF-α as recited in claim 19.
Note that any evidence of unexpected results must be commensurate in scope with the claimed invention, and that a greater, or greater than additive, effect is not necessarily sufficient to overcome a prima facie case of obviousness because such an effect can either be expected or unexpected MPEP 716.02 (a) and (d). Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. In re Clemens, 622 F.2d 1029, 1036, 206 USPQ 289, 296 (CCPA 1980).
Specifically, amended claim 19 recites wherein the immunostimulant composition stimulates a stronger a stronger IFN-α production than production of TNF-α, which is not supported by the data presented in Figure 5 of the instant application, as discussed above. Additionally, the data presented represent specific cell types being stimulated to produce the IFN-α and TNF-α (e.g., specific cell types within a PBMC cell population, namely monocytes and dendritic cells), whereas the claims as written do not indicate any specific cell type which is stimulated by the immunostimulant composition.
Note also that the claims as written do not require any specific ratio of protamine:RNA within the protamine-RNA nanoparticles which would correspond to the ratio used in generation of the results presented in Figure 5 (e.g., equal volumes of 1 mg/ml RNA and 1 mg/ml protamine).
As such, the claims as written are not commensurate in scope with the provided evidence of unexpected results.
Therefore, Applicant’s arguments do not overcome a finding of obviousness over Parker, Scheel (2004), Sioud, Li, McSwiggen, Shipman, Scheel (2005), and Loeffler under 35 U.S.C. 103.
Conclusion
No claim is allowed.
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DR. KATIE L. PENNINGTON
Examiner
Art Unit 1634
/KATIE L PENNINGTON/Examiner, Art Unit 1634
Dr. A.M.S. Wehbé
/ANNE MARIE S WEHBE/Primary Examiner, Art Unit 1634