DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group I (claims 1-8 and new claims 18-23) and in the reply filed on 07/06/2026 is acknowledged.
It is noted that Applicant traverses the conclusion regarding Groups I-III lacking unity of invention. However, Applicant has made an election without traverse and furthermore has not provided any specific arguments against the cited art used to demonstrate a lack of unity of invention. As such, the restriction/election requirement is deemed proper and is made FINAL.
Claim Status
Claims 10-12 have been cancelled and claims 18-23 have been newly added, as requested in the amendment filed on 07/06/2026. Following the amendment, claims 1-9 and 13-23 are pending in the instant application.
Claims 9 and 13-17 stand as withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected inventions in the Response filed 07/06/2026 there being no allowable generic or linking claim.
Claims 1-8 and 18-23 are under examination in the instant office action.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. Furthermore, it is noted that all foreign priority documents are in English, and as such the claim to foreign priority has been perfected.
Claims 1-8 and 18-23 have an effective filing date of December 02, 2020 corresponding to LU102272.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 06/01/2023 and 07/01/2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosures statement 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.
Drawings
The drawings are objected to because the figure labels (i.e., Figure 1, Figure 2, etc.) do not match the orientation of Figures 1-2, 4-7, 9-11, 13-15, 18-23, 26-36, 39-40, 43-48, 63, 65, 67-68, 70-71, 74-77, and 79-80; if the figure is in the landscape orientation, the figure label should also be in landscape orientation. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification
The disclosure is objected to because of the following informalities: there are typographical errors in Paragraph 0134. Appropriate correction is required.
The disclosure is further objected to for the use of the terms Onpattro, Hoechst, nanobodies, DART, and TandAb, for example, which are trade names or marks used in commerce, have been noted in this application. The terms should be accompanied by the generic terminology; furthermore, the terms should be capitalized wherever they appear or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the terms.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claim 20 is objected to because of the following informalities: the claim currently reads "[t]he method of claim 6, wherein nucleic acid Appropriate correction is required.
Claim Rejections - 35 USC § 112
Claims 7 and 18 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.
Claims 7 and 18 each recite the limitation "the positively charged polypeptide" at lines 1-2, 1, and 1, respectively. There is insufficient antecedent basis for this limitation in the claim because independent claim 3, from which each of claims 4, 7, and 18 depend, recites “a positively charged polypeptide” in each of steps (c) and (d) and it is unclear as to which positively charged polypeptide is being referred to or if the limitation applies to both recitations. Thus, claims 7 and 18 are considered to be indefinite.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-8 and 18-23 are rejected under 35 U.S.C. 103 as being unpatentable over non-patent literature by Baumer et. al. (Clin. Cancer Res., 2015, 21(6), 1383-1394; Non-Patent Literature Citation No. 4 on 06/01/2023 IDS; herein after referred to as "Baumer") in view of US 2003/0027773 A1 (US Patent Application Publication Citation No. 1 on 07/01/2026 IDS; herein after referred to as "Bernstiel") and non-patent literature by Liu et. al. (Nanoscale Research Letters, 2015, 10(134), 1-7; herein after referred to as “Liu”).
Baumer teaches that the coupling of siRNA against KRAS to anti-EGFR antibodies provides a novel therapy approach for KRAS-mutated ECFR-positive cancer cells in vitro and in vivo; these findings provide an innovative approach for cancer-specific siRNA application and for enhanced therapeutic potential of monoclonal antibody therapy and personalized treatment of
cancer entities (Abstract, Conclusions). More specifically, Baumer teaches that protamine sulfate (1.67 mmol/ L) was amino-terminally coupled to the bifunctional cross-linker Sulfo-SMCC in a 1:12 molar ratio in PBS buffer, then coupled to cysteine residues of anti-EGFR antibody (i.e., cetuximab) in a 5:1 molar ratio (Page 1384, Materials and Methods, Coupling of Anti-EGFR Monoclonal Antibody to Protaminesulfate). Nonreacted educts and protamine doublets were separated from the high-molcular weight anti-EGFR mAB-protamine product by gel filtration chromatography in Zeba spin desalting columns (Id.). siRNA duplexes (at 4- to 10-fold molar excess) were bound to anti-EGFR mAB-protamine (Page 1384, Materials and Methods, Coupling of siRNA to Anti-EGFR mAB-Protamine). Chemically coupled antibody-(e)siRNA complexes are a new and potentially powerful approach for targeted anticancer therapy following a simple building-block strategy; the combination of multiple siRNAs conjugated to cetuximab targeting independent oncogenes in combination underlines the flexibility and high potential of this approach (Page 1392. Column 2, Last Paragraph). With the high loading capacity of the protamine for siRNA, one even can envisage loading of siRNA molecules targeting different oncogenic molecules tailored to the individual tumor specifications within one antibody construct; in fact, this principle can contribute to a personalized anti-cancer therapeutic approach where tumors can be treated with specific siRNAs against several driver mutations and genes at the same time (Page 1393, Column 1, First Full Paragraph). Thereby, antibody-siRNA conjugates can provide a valuable alternative, especially in rare cancer entities when the cost-effective design of conventional inhibitor drugs is difficult (Id.).
However, Baumer does not explicitly teach contacting the antibody-SMCC-protamine conjugate with a negatively charged molecule (e.g., siRNA) and a positively charged polypeptide, after removal of nonreacted educts. This deficiency is remedied by Baumer.
Bernstiel teaches that antibodies which bind to a cell surface protein can be used for transporting nucleic acids into higher eukaryotic cells if they are conjugated with polycations (Paragraph 0017). It was demonstrated by means of antibody-polycation conjugates with various antibody components that, with the aid of such conjugates in cells which express the particular surface antigen against which the antibody is directed, the internalization and expression of DNA can be achieved (Paragraph 0021); the invention therefore relates to protein-polycation conjugates which are capable of forming complexes with nucleic acids, the protein component being an antibody against a cell surface protein which is capable of binding to the cell surface protein, so that the complexes formed are absorbed into the cells which express the cell surface protein (Paragraph 0022). Suitable antibodies are all those antibodies, particularly monoclonal antibodies, against cell surface antigens or the fragments thereof which bind to the cell surface antigen, e.g. Fab' fragments (Paragraph 0026). The choice of the antibody is determined particularly by the target cells, e.g. by certain surface antigens or receptors which are specific or largely specific to one type of cell and thus enable a directed introduction of nucleic acid into this type of cell (Paragraph 0028). Suitable polycations include natural DNA-binding proteins of a polycationic nature such as histones or protamines or analogues or fragments thereof (Paragraph 0037); the size and/or length of the polycations is not critical and as an example in the case of polylysine it is preferably such that the sum of the positive charges is about 20 to 1000 and is matched to the particular nucleic acid to be transported, further wherein if, for example, DNA has 6,000 bp and 12,000 negative charges, the quantity of polycation is, for example, 60 mol polylysine 200 or 30 mol polylysine 400 or 120 mol polylysine 100, etc. wherein an average person skilled in the art is also capable of choosing other combinations of polycation sizes and molar quantities by means of routine experiments (Paragraph 0038). The antibody polycation conjugates according to the invention may be prepared chemically in a method known for the coupling of peptides, and if necessary the individual components may be provided before the coupling reaction with linker substances (this measure is necessary if there is no available functional group suitable for coupling such as a mercapto or alcohol group; the linker substances are bifunctional compounds which are reacted first with functional groups of the individual components, after which the modified individual components are coupled (Paragraph 0039). The molar ratio of antibody to polycation is preferably 10:1 to 1:10, and this ratio may if necessary be within wider limits provided that the condition is met that complexing of the nucleic acid or acids takes place and it is ensured that the complex formed is bound to the cell surface protein and conveyed into the cell. This can be checked by simple tests carried out in each individual case (Paragraph 0047). The preferred nucleic acid component of the antibody- polycation-nucleic acid complexes according to the invention having an inhibiting effect on the grounds of complementarity is antisense DNA, antisense RNA or a ribozyme or the gene coding therefor (Paragraph 0053). The antibody-polycation-nucleic acid complexes which can be absorbed into higher eukaryotic cells by endocytosis may additionally contain one or more polycations in a non-covalently bound form which may be identical to the polycation in the conjugate, so as to increase the internalization and/or expression of the nucleic acid achieved by means of the conjugate (Paragraph 0062). With the aid of such measures, a smaller amount of antibody-polycation conjugate is required, based on the quantity of nucleic acid to be imported into the cell, to achieve at least the same efficiency of transfection/expression, which means on the one hand that synthesis is less costly; a smaller amount of conjugate may also be advantageous when it is desirable to avoid the effect of having several adjacent "docking sites" occupied by a large number of antibody molecules within a complex, with the consequence that they are no longer available for additional complexes and restricting the quantity of antibody contained in the complexes to the necessary minimum, i.e. keeping the quantity of conjugate as small as possible and diluting it with free polycation, is particularly advantageous when there is only a small number of cell surface proteins on the target cells to be treated (Paragraph 0063). The performance of conjugates which are not particularly efficient per se can be increased substantially and the performance of conjugates which are already highly efficient can be increased still further by using such measures (Paragraph 0064). The preparation of the antibody-polycation/nucleic acid complexes of the invention may be carried out by methods known per se for the complexing of polyionic compounds (Paragraph 0061).
However, neither Bernstiel nor Baumer explicitly teach that the second conjugate (i.e., antibody-linker-protamine) is enriched in the outer portion of the nanoparticle, the negatively charged molecules are enriched in the inner portion of the nanoparticle, nor that the nanoparticle has a mean diameter of about 0.05 um to about 10 um. These deficiencies are remedied by Liu.
Liu teaches that protamine nanoparticles were designed by encapsulating small hairpin RNA (shRNA)-expressing plasmid DNA targeting the Bcl-2 gene (shBcl-2) to silence apoptosis-related Bcl-2 protein for improving the transfection efficiency and cytotoxicity in cancer therapy; the findings demonstrated that the obtained protamine nanoparticles possessed excellent characterizations of small particle size, homogenous distribution, positive charge, and high encapsulation efficiency of gene, wherein the shBcl-2 loaded in nanoparticles (NPs) were protected effectively from the degradation of DNase I and serum and significantly improved the efficiency of transfection of shRNA in vitro in A549 cells and increased its cytotoxicity and induced more cell apoptosis by silencing Bcl-2 (Abstract). Certain amount of protamine was swelled with distilled water overnight to obtain an aqueous solution containing protamine at 1.5, 2.5, and 4 mg/mL, after which the shBcl-2 was also added into the obtained protamine solution followed by the addition of 1 mL of ethanol at 37°C under continuous stirring (1,000 rpm); the resulting white suspension was further stirred for 24 h, and then ethanol was removed with vacuum distillation succeeded by the addition of 8% glutaraldehyde in water (0.5 μL per mg of protamine) for particle cross-linking (Page 2, Column 1, The Preparation and Characterization of shBcl-2-Loaded Protamine NPs). After washing with cold PBS three times, the resulting nanoparticles with different mass ratio of protamine and shBcl-2 (50:1, 100:1, and 200:1) were purified and sterilized by filtration, the characterization of shBcl-2-loaded NPs was investigated to determine the particle size and zeta potential by using dynamic light scattering (DLS), the morphology of particles was observed by using transmission electron microscope (TEM), and the difference between the amount of the initially added shRNA and shRNA in the supernatant was measured at detecting absorbance at 260 nm using a UV/Vis spectrophotometer to determine the encapsulation efficiency (EE) of shRNA in nanoparticles (Id.). The morphology and size distribution results of the prepared nanoparticles are displayed in Table 1; with the increasing amount of protamine, the particle size was enhanced and the zeta potential also continued to rise, and in terms of encapsulation efficiency of shBcl-2 in NPs, about 85% of shBcl-2 was loaded in NPs with the mass ratio at 100:1 in contrast with the other two NPs (Page 3, Column 1, Results, The Characterization of shBcl-2-Loaded NPs; see Table 1 and Figure 1). Judging by the TEM in Figure 1, it was observed that shBcl-2-loaded NPs with different mass ratios possessed an excellent characterization of suitable particle size, homogenous size distribution, and high monodispersion, indicating that shBcl-2-loaded NPs with the mass ratio of 100:1 was a potential optimized gene carrier with good characterization and higher encapsulation efficiency (Id.). Thus, Liu demonstrates that protamine is sufficient on its own to cross-link and encapsulate siRNAs (wherein the positive charge is enriched in the outer portion of the nanoparticles and the negative charge is enhanced in the inner potion of the nanoparticles), and wherein the resultant nanoparticles are, generally, 82-298 nm in diameter (0.082-0.298 um; see Table 1 and Figure 1D) depending on the ratio of protamine:RNA.
Furthermore, as evidenced by the references, it is noted that working concentrations/molar ratios for the various steps of nanoparticle/complex formation are recognized as reaction/conjugation/nanoparticle variables which achieve a recognized result and as set forth in MPEP 2144.05: “A particular parameter must first be recognized as a result-effective variable, i.e., a variable which achieves a recognized result, before the determination of the optimum or workable ranges of said variable might be characterized as routine experimentation.” In re Antonie, 559 F.2d 618, 195 USPQ 6 (CCPA 1977). It is a common objective in the art to optimize result effective variables, so as achieve optimal effect and maximal benefit. See In re Boesch, 617 F.2d 272, 276, 205 USPQ 215, 219 (CCPA 1980) (“[D]iscovery of an optimum value of a result effective variable in a known process is ordinarily within the skill of the art.” (citations omitted)). Therefore, any optimization of reagent/component concentrations/molar ratios for nanoparticle/complex formation would be seen as routine optimization.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to modify the method of Baumer, based on the teachings of Bernstiel, to arrive at a method for making a nanoparticle comprising contacting an antibody with a first conjugate (wherein the first conjugate comprises a positively charged polypeptide conjugated to a bifunctional linker) to yield a second conjugate, and contacting the second conjugate with a positively charged polypeptide and a negatively charged molecule. One would have been motivated to modify the method of Baumer to include contacting the antibody-SMCC-protamine conjugate with, in addition to negatively charged siRNA, free protamine because Bernstiel teaches that for antibody-containing polycation-RNA conjugates/complexes, it is desirable to add free protamine when reacting the antibody-protamine complexes with RNA because (i) doing so avoids the effect of having several adjacent "docking sites" occupied by a large number of antibody molecules within a complex, with the consequence that they are no longer available for additional complexes; (ii) it is particularly advantageous to do when there is only a small number of cell surface proteins on the target cells to be treated; and (iii) it can further enhance efficiency and performance of the resulting conjugates/nanoparticles. One of ordinary skill in the art would have a reasonable expectation of success because Baumer discloses a successful method of making antibody-SMCC-protamine-siRNA conjugates/nanoparticles, wherein nonreacted educts and protamine doublets may be removed after the reaction to conjugate protamine to the SMCC linker, and Bernstiel explicitly teaches that diluting antibody-protamine conjugates, when contacting them with RNA, can improve the efficiency and performance of the resulting complexes/nanoparticles, and Liu suggests that for a protamine-based nanoparticle, it would reasonably be expected that positive charges (i.e., antibody-linker-protamine and free protamine) would be enriched in the outer portion of the nanoparticle while the negatively charged molecules would be enriched in the inner portion of the nanoparticle, and the resulting nanoparticles would reasonably be expected to have a mean diameter of about 0.05 um to about 10 um, which is also optimizable by way of the reaction conditions used as suggested by Liu.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-8 and 18-23 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-2, 5-6, 8-9, and 18-19 of copending Application No. 18/562,164 (herein after referred to as “reference application”) in view of non-patent literature by Baumer et. al. (Clin. Cancer Res., 2015, 21(6), 1383-1394; Non-Patent Literature Citation No. 4 on 06/01/2023 IDS; herein after referred to as "Baumer"), US 2003/0027773 A1 (US Patent Application Publication Citation No. 1 on 07/01/2026 IDS; herein after referred to as "Bernstiel"), and non-patent literature by Liu et. al. (Nanoscale Research Letters, 2015, 10(134), 1-7; herein after referred to as “Liu”).
Claim 1 of the reference application is drawn to a method of generating a nanoparticle comprising contacting a) a fusion protein (A), said fusion protein (A) comprising an antibody (Al) and a positively charged polypeptide (A2); b) a positively charged polypeptide (B); and c) a negatively charged molecule (C); thereby forming a nanoparticle. Reference application claim 2 further limits the method of claim 1 wherein the molar ratio between the positively charged polypeptide (B) and the fusion protein (A) is at least about 10:1. Reference application claims 5-6 further limit the method of claim 1, wherein (i) the antibody (Al) is specific for a cell surface molecule and (ii) the negatively charged molecule (C) is a nucleic acid, respectively. Claim 8 of the reference application further limits the method of claim 1 wherein the positively charged polypeptide (B) is a protamine or a histone. Claim 9 of the reference application is directed to a nanoparticle obtainable by the method of claim 1. Claims 18-19 of the reference application further limit the method of claim 1 wherein (i) the positively charged polypeptide (A2) has a net charge of at least +5 and (ii) the positively charged polypeptide (B) has a net charge of at least +5, respectively.
However, the reference application does not claim a method of generating a nanoparticle comprising contacting an antibody with a composition comprising a first conjugate to obtain a second conjugate, wherein the first conjugate comprises a positively charged polypeptide conjugated to a bifunctional linker further wherein the composition is essentially free of unconjugated bifunctional linker, and contacting the second conjugate (comprising an antibody, bifunctional linker, and positively charged polypeptide) with a positively charged polypeptide and negatively charged molecule to form a nanoparticle. The reference application also does not claim that the second conjugate is enriched in the outer portion of the nanoparticle, the negatively charged molecules are enriched in the inner portion of the nanoparticle, nor that the nanoparticle has a mean diameter of about 0.05 um to about 10 um. These deficiencies are remedied by Baumer, Bernstiel, and Liu.
Baumer teaches that the coupling of siRNA against KRAS to anti-EGFR antibodies provides a novel therapy approach for KRAS-mutated ECFR-positive cancer cells in vitro and in vivo; these findings provide an innovative approach for cancer-specific siRNA application and for enhanced therapeutic potential of monoclonal antibody therapy and personalized treatment of cancer entities (Abstract, Conclusions). More specifically, Baumer teaches that protamine sulfate (1.67 mmol/ L) was amino-terminally coupled to the bifunctional cross-linker Sulfo-SMCC in a 1:12 molar ratio in PBS buffer, then coupled to cysteine residues of anti-EGFR antibody (i.e., cetuximab) in a 5:1 molar ratio (Page 1384, Materials and Methods, Coupling of Anti-EGFR Monoclonal Antibody to Protaminesulfate). Nonreacted educts and protamine doublets were separated from the high-molcular weight anti-EGFR mAB-protamine product by gel filtration chromatography in Zeba spin desalting columns (Id.). siRNA duplexes (at 4- to 10-fold molar excess) were bound to anti-EGFR mAB-protamine (Page 1384, Materials and Methods, Coupling of siRNA to Anti-EGFR mAB-Protamine). Chemically coupled antibody-(e)siRNA complexes are a new and potentially powerful approach for targeted anticancer therapy following a simple building-block strategy; the combination of multiple siRNAs conjugated to cetuximab targeting independent oncogenes in combination underlines the flexibility and high potential of this approach (Page 1392. Column 2, Last Paragraph). With the high loading capacity of the protamine for siRNA, one even can envisage loading of siRNA molecules targeting different oncogenic molecules tailored to the individual tumor specifications within one antibody construct; in fact, this principle can contribute to a personalized anti-cancer therapeutic approach where tumors can be treated with specific siRNAs against several driver mutations and genes at the same time (Page 1393, Column 1, First Full Paragraph). Thereby, antibody-siRNA conjugates can provide a valuable alternative, especially in rare cancer entities when the cost-effective design of conventional inhibitor drugs is difficult (Id.). Liu teaches that protamine nanoparticles were designed by encapsulating small hairpin RNA (shRNA)-expressing plasmid DNA targeting the Bcl-2 gene (shBcl-2) to silence apoptosis-related Bcl-2 protein for improving the transfection efficiency and cytotoxicity in cancer therapy; the findings demonstrated that the obtained protamine nanoparticles possessed excellent characterizations of small particle size, homogenous distribution, positive charge, and high encapsulation efficiency of gene, wherein the shBcl-2 loaded in nanoparticles (NPs) were protected effectively from the degradation of DNase I and serum and significantly improved the efficiency of transfection of shRNA in vitro in A549 cells and increased its cytotoxicity and induced more cell apoptosis by silencing Bcl-2 (Abstract). Certain amount of protamine was swelled with distilled water overnight to obtain an aqueous solution containing protamine at 1.5, 2.5, and 4 mg/mL, after which the shBcl-2 was also added into the obtained protamine solution followed by the addition of 1 mL of ethanol at 37°C under continuous stirring (1,000 rpm); the resulting white suspension was further stirred for 24 h, and then ethanol was removed with vacuum distillation succeeded by the addition of 8% glutaraldehyde in water (0.5 μL per mg of protamine) for particle cross-linking (Page 2, Column 1, The Preparation and Characterization of shBcl-2-Loaded Protamine NPs). After washing with cold PBS three times, the resulting nanoparticles with different mass ratio of protamine and shBcl-2 (50:1, 100:1, and 200:1) were purified and sterilized by filtration, the characterization of shBcl-2-loaded NPs was investigated to determine the particle size and zeta potential by using dynamic light scattering (DLS), the morphology of particles was observed by using transmission electron microscope (TEM), and the difference between the amount of the initially added shRNA and shRNA in the supernatant was measured at detecting absorbance at 260 nm using a UV/Vis spectrophotometer to determine the encapsulation efficiency (EE) of shRNA in nanoparticles (Id.). The morphology and size distribution results of the prepared nanoparticles are displayed in Table 1; with the increasing amount of protamine, the particle size was enhanced and the zeta potential also continued to rise, and in terms of encapsulation efficiency of shBcl-2 in NPs, about 85% of shBcl-2 was loaded in NPs with the mass ratio at 100:1 in contrast with the other two NPs (Page 3, Column 1, Results, The Characterization of shBcl-2-Loaded NPs; see Table 1 and Figure 1). Judging by the TEM in Figure 1, it was observed that shBcl-2-loaded NPs with different mass ratios possessed an excellent characterization of suitable particle size, homogenous size distribution, and high monodispersion, indicating that shBcl-2-loaded NPs with the mass ratio of 100:1 was a potential optimized gene carrier with good characterization and higher encapsulation efficiency (Id.). Thus, Liu demonstrates that protamine is sufficient on its own to cross-link and encapsulate siRNAs (wherein the positive charge is enriched in the outer portion of the nanoparticles and the negative charge is enhanced in the inner potion of the nanoparticles), and wherein the resultant nanoparticles are, generally, 82-298 nm in diameter (0.082-0.298 um; see Table 1 and Figure 1D) depending on the ratio of protamine:RNA.
Bernstiel teaches that antibodies which bind to a cell surface protein can be used for transporting nucleic acids into higher eukaryotic cells if they are conjugated with polycations (Paragraph 0017). It was demonstrated by means of antibody-polycation conjugates with various antibody components that, with the aid of such conjugates in cells which express the particular surface antigen against which the antibody is directed, the internalization and expression of DNA can be achieved (Paragraph 0021); the invention therefore relates to protein-polycation conjugates which are capable of forming complexes with nucleic acids, the protein component being an antibody against a cell surface protein which is capable of binding to the cell surface protein, so that the complexes formed are absorbed into the cells which express the cell surface protein (Paragraph 0022). Suitable antibodies are all those antibodies, particularly monoclonal antibodies, against cell surface antigens or the fragments thereof which bind to the cell surface antigen, e.g. Fab' fragments (Paragraph 0026). The choice of the antibody is determined particularly by the target cells, e.g. by certain surface antigens or receptors which are specific or largely specific to one type of cell and thus enable a directed introduction of nucleic acid into this type of cell (Paragraph 0028). Suitable polycations include natural DNA-binding proteins of a polycationic nature such as histones or protamines or analogues or fragments thereof (Paragraph 0037); the size and/or length of the polycations is not critical and as an example in the case of polylysine it is preferably such that the sum of the positive charges is about 20 to 1000 and is matched to the particular nucleic acid to be transported, further wherein if, for example, DNA has 6,000 bp and 12,000 negative charges, the quantity of polycation is, for example, 60 mol polylysine 200 or 30 mol polylysine 400 or 120 mol polylysine 100, etc. wherein an average person skilled in the art is also capable of choosing other combinations of polycation sizes and molar quantities by means of routine experiments (Paragraph 0038). The antibody polycation conjugates according to the invention may be prepared chemically in a method known for the coupling of peptides, and if necessary the individual components may be provided before the coupling reaction with linker substances (this measure is necessary if there is no available functional group suitable for coupling such as a mercapto or alcohol group; the linker substances are bifunctional compounds which are reacted first with functional groups of the individual components, after which the modified individual components are coupled (Paragraph 0039). The molar ratio of antibody to polycation is preferably 10:1 to 1:10, and this ratio may if necessary be within wider limits provided that the condition is met that complexing of the nucleic acid or acids takes place and it is ensured that the complex formed is bound to the cell surface protein and conveyed into the cell. This can be checked by simple tests carried out in each individual case (Paragraph 0047). The preferred nucleic acid component of the antibody- polycation-nucleic acid complexes according to the invention having an inhibiting effect on the grounds of complementarity is antisense DNA, antisense RNA or a ribozyme or the gene coding therefor (Paragraph 0053). The antibody-polycation-nucleic acid complexes which can be absorbed into higher eukaryotic cells by endocytosis may additionally contain one or more polycations in a non-covalently bound form which may be identical to the polycation in the conjugate, so as to increase the internalization and/or expression of the nucleic acid achieved by means of the conjugate (Paragraph 0062). With the aid of such measures, a smaller amount of antibody-polycation conjugate is required, based on the quantity of nucleic acid to be imported into the cell, to achieve at least the same efficiency of transfection/expression, which means on the one hand that synthesis is less costly; a smaller amount of conjugate may also be advantageous when it is desirable to avoid the effect of having several adjacent "docking sites" occupied by a large number of antibody molecules within a complex, with the consequence that they are no longer available for additional complexes and restricting the quantity of antibody contained in the complexes to the necessary minimum, i.e. keeping the quantity of conjugate as small as possible and diluting it with free polycation, is particularly advantageous when there is only a small number of cell surface proteins on the target cells to be treated (Paragraph 0063). The performance of conjugates which are not particularly efficient per se can be increased substantially and the performance of conjugates which are already highly efficient can be increased still further by using such measures (Paragraph 0064). The preparation of the antibody-polycation/nucleic acid complexes of the invention may be carried out by methods known per se for the complexing of polyionic compounds (Paragraph 0061).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to modify the method of the reference application, based on the teachings of Baumer and Berntiel, to arrive at a method for making a nanoparticle comprising contacting an antibody with a first conjugate (wherein the first conjugate comprises a positively charged polypeptide conjugated to a bifunctional linker) to yield a second conjugate, and contacting the second conjugate with a positively charged polypeptide and a negatively charged molecule. One would have been motivated to modify the method of the reference application such that fusion protein of the reference application is replaced with an antibody-linker-protamine conjugate, because Baumer discloses a successful method of making antibody-SMCC-protamine-siRNA conjugates/nanoparticles comprising the use of antibody-linker-protamine conjugates, and subsequently contacting said conjugate with free protamine and negatively charged siRNA, as suggested by Bernstiel, in order to arrive at an efficient complex/nanoparticle for siRNA delivery to target cells. One of ordinary skill in the art would have a reasonable expectation of success because Baumer discloses a successful method of making antibody-SMCC-protamine-siRNA conjugates/nanoparticles, wherein nonreacted educts and protamine doublets may be removed after the reaction to conjugate protamine to the SMCC linker, and Bernstiel explicitly teaches that diluting antibody-protamine conjugates, when contacting them with RNA, can improve the efficiency and performance of the resulting complexes/nanoparticles, and Liu suggests that for a protamine-based nanoparticle, it would reasonably be expected that positive charges (i.e., antibody-linker-protamine and free protamine) would be enriched in the outer portion of the nanoparticle while the negatively charged molecules would be enriched in the inner portion of the nanoparticle, and the resulting nanoparticles would reasonably be expected to have a mean diameter of about 0.05 um to about 10 um.
This is a provisional nonstatutory double patenting rejection.
Conclusion
Claims 1-9 and 13-23 are pending. Claims 9 and 13-17 are withdrawn. Claims 1-8 and 18-23 are rejected. No claims are allowed.
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/ALYSSA RAE STONEBRAKER/Examiner, Art Unit 1642
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