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 1-12 and 16-27), in the reply filed on 11/6/2025 is acknowledged. Claim 15 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Priority
This application is a National-Stage entry of PCT EP2021/082980, filed 11/25/2021. Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). The certified copy has been filed in the instant application, filed on May 31, 2023. The priority date is December 1, 2020.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 8/31/2023 and 8/1/2024 are acknowledged. The submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is 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.
Response to Amendment
Applicant’s amendments to the claims filed 2/8/2024 are acknowledged and have been entered. This listing of the claims replaces all prior versions and listings of the claims.
Claims 1-12 and 16-27 are pending and have been examined on the merits.
Specification
The disclosure is objected to because of the following informalities:
ABSTRACT: Applicant is reminded of the proper content of an abstract of the disclosure.
A patent abstract is a concise statement of the technical disclosure of the patent and should include that which is new in the art to which the invention pertains. The abstract should not refer to purported merits or speculative applications of the invention and should not compare the invention with the prior art.
If the patent is of a basic nature, the entire technical disclosure may be new in the art, and the abstract should be directed to the entire disclosure. If the patent is in the nature of an improvement in an old apparatus, process, product, or composition, the abstract should include the technical disclosure of the improvement. The abstract should also mention by way of example any preferred modifications or alternatives.
Where applicable, the abstract should include the following: (1) if a machine or apparatus, its organization and operation; (2) if an article, its method of making; (3) if a chemical compound, its identity and use; (4) if a mixture, its ingredients; (5) if a process, the steps.
Extensive mechanical and design details of an apparatus should not be included in the abstract. The abstract should be in narrative form and generally limited to a single paragraph within the range of 50 to 150 words in length.
See MPEP § 608.01(b) for guidelines for the preparation of patent abstracts.
The abstract of the disclosure is objected to because the abstract is greater than 150 words. It is noted that the presented abstract has redundant words (“Specifically”, “Furthermore”, “In addition”, et cetera) and begins by repeating the title of the invention. A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
TRADE NAMES, TRADEMARKS, AND OTHER MARKS USED IN COMMERCE:
The use of the terms Triton™ X-100, pg. 59; Milli-Q™ , pg. 61; and ENDONEXT™ EndoZyme®, pg. 64 - which are each a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the terms.
Applicant’s attention is requested to ensure that all trade names and marks used in commerce are properly identified in the specification. 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 (see MPEP 608.01(v) and 608.01(u)).
Appropriate correction is required.
Claim Objections
Claims 1 and 11 are objected to because of the following informalities:
In claim 1, line 4, the article “the” has been deleted in the amended claim set but should be there, so that the phrase reads “the superparamagnetic iron-based particles...” (note that this is how the similar phrase is presented in claim 10).
In claim 11, the phrase “still viable” should be “viable”. The word “still” is unnecessary and may cause confusion as to what the previous state of the pathogens is.
Appropriate correction is required.
Claim Interpretation
Regarding the sequences and motifs recited in claims, the specification, pg. 12 states:
“various pathogens have been shown to bind to a 16-mer peptide (QGRVEVLYRGSWGTVC, SEQ ID NO: 19) located within the SRCR domains of e.g. DMBT1. An exemplary SRCR domain is disclosed herein as SEQ ID NO: 11. For different bacteria, there was a highly significant correlation between adhesion to DMBT1 and adhesion to SRCR peptide 2 suggesting that SRCR peptide 2 is the major bacteria binding site. Leito et al. (2008) also disclose that 8 amino acids were involved in binding (xRVEVLYxxSWxxxx) by performing an alanine substitution scan. Said motif is derived from DMBT1 (SEQ ID NO: 4), which is a member of the scavenger receptor cysteine-rich (SRCR) superfamily. DMBT1 is secreted protein mainly expressed by epithelia and glands. DMBT1 is also known as salivary agglutinin or glycoprotein-340 (gp340). DMBT1 is a member of the scavenger receptor cysteine-rich (SRCR) superfamily, i.e. proteins that have one or more SRCR domains”.
Thus, the “target binding peptide” of the invention, and particularly in regards to the peptides having the specific motifs recited in claims 7, 12, and 24, is being interpreted as an SRCR peptide, derived from the DMBT1 family of proteins, also known in the art as salivary agglutinin (DMBT1SAG) and gp-340, as evidenced in Leito et al. (2008, Biol. Chem., Vol. 389, pp. 1193-1200; cited in IDS filed 8/1/2024).
In claims 1, and 10, the phrase “wherein the magnetic attractability is a reduction of the superparamagnetic iron-based particle concentration in Ringer solution by 65% to 99.95% when applying a magnetic field of 0.31 Tesla for three minutes in static condition” is being interpreted under the broadest reasonable interpretation (B.R.I). of the terms, in light of the specification, as a functional property of the particles, or a desired result, that may be achieved when tested under the specified conditions (i.e. “when applying a magnetic field... ”). It is noted that this does not practically limit the structure of the superparamagnetic iron-based particles, nor does it require that a magnetic field having exactly these conditions (0.31 Tesla for 3 minutes) is the field used for the separation/immobilizing step (c). This limitation has been afforded the proper patentable weight, in accordance with MPEP § 2173.05(g) (“without reciting the particular structure, materials or steps that accomplish the function or achieve the result, all means or methods of resolving the problem may be encompassed by the claim. Ariad Pharmaceuticals., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1353, 94 USPQ2d 1161, 1173 (Fed. Cir. 2010) (en banc).”) and see also MPEP § 2112.02 at Ex parte Novitski, in reference to reference-silent functioning of biological materials providing anticipation of the functions based upon the material itself.
Therefore, without further specific structural limitations, the claims are interpreted broadly as being drawn to a separation method using any superparamagnetic iron-based particles that could fulfill the functional limitation, if so tested.
Claim Rejections - 35 USC § 112(a)- Written Description
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 1-12 and 16-27 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 contains subject matter which was 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 1 and 10 each recite “wherein the superparamagnetic iron-based particles are magnetically attractable, and wherein the magnetic attractability is a reduction of the superparamagnetic iron-based particle concentration in Ringer solution by 65% to 99.95% when applying a magnetic field of 0.31 Tesla for three minutes in static condition.”, which is a functional limitation that is directed to a desired result arising from the properties of the materials used. The claims do not practically limit the structure of the superparamagnetic iron-based particles to any particular structure or species thereof. Under the B.R.I. of the claim terms, the full genus of the particles include any and all which achieve this functional result, if so tested in the manner described in the specification. The dependent claims are thus drawn to a broad genus of functionalized magnetic particles, but do not practically limit the encompassed structure of these to only those described in the claims.
Further, claims 9 and 27 requires that “the target binding peptide are capable of reducing the concentration of lipopolysaccharides (LPS) in (c) by at least 70% compared to the sample of (a) comprising a LPS concentration of 10 EU/ml, wherein the reduction is determined after incubating the SPIONs linked to the target binding peptide at a concentration of 1 mg Fe/ml in Ringer solution followed by immobilising the SPIONs linked to a target binding peptide in a magnetic field of 0.31 Tesla for a time period of 30 seconds by using an assay based on the detection of recombinant factor C”.
MPEP § 2163.03.(V) establishes that an original claim may lack written description support when (1) the claim defines the invention in functional language specifying a desired result but the disclosure fails to sufficiently identify how the function is performed or the result is achieved or (2) a broad genus claim is presented but the disclosure only describes a narrow species with no evidence that the genus is contemplated. See Ariad Pharms., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1349-50 (Fed. Cir. 2010) (en banc). See also Ariad, 593 F.3d at 1349, 94 USPQ at 1171: "The problem is especially acute with genus claims that use functional language to define the boundaries of a claimed genus. In such a case, the functional claim may simply claim a desired result, and may do so without describing species that achieve that result. But the specification must demonstrate that the applicant [inventor] has made a generic invention that achieves the claimed result and do so by showing that the applicant [inventor] has invented species sufficient to support a claim to the functionally-defined genus."
According to the full scope of the instant claims, viewed in light of the specification, there exists a very large number of structural variation within the claimed scope, having various combinations of iron-based cores, linker molecules, binding agents, and functional peptides which are provided as the target peptide. Comparatively, the specification only recites a small number of species of the broad genus that fulfill the recited functional limitations.
MPEP § 2163.(II)(A)(3)(a) 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 inventor was in possession of the claimed genus. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
The specification fails to reasonably describe the full genus of the claimed invention by providing identifying characteristics or predictable structural properties of the iron particles, the linker chemicals, and the functional target-binding peptides, which amount to a known or disclosed correlation between function and structure.
There is no manner in the specification, except for performing the experimental assay described on page 63, lines 23-37, of the specification, to determine or predict if any species comprising a particular structure of a superparamagnetic particle, anchor, linker, and functional peptide will achieve the claimed function, such that the entirety of the claimed genus of iron-based superparamagnetic particles is encompassed by the description in the disclosure.
The only examples in the specification (on pages 48-65) that are demonstrated to fulfill the claimed functional properties and which are capable of binding pathogens or pathogen components comprise the two peptide-functionalized superparamagnetic iron oxide nanoparticles (SPION) referred to as SPIONAPTES-SBA-SPP04 and SPIONAPTES-SBA-SPP19, which are bound with peptides containing the specific sequences of SEQ ID NO:1 (RKQGRVEVLYRASWGTVC) and SEQ ID NO:2 (RKQGRVEILYRGSWGTVC), each derived from the motif of SRCR2 of DMBT1, and possess all of the resulting properties thereof, as demonstrated in Examples 2-5.
In addition, non-functionalized particles, including those referred to as SPIONAPTES (coated only with the anchor compound 3-aminopropyltriethoxysilan); SPIONAPTES-SBA (coated with 3-aminopropyltriethoxysilan and with the linker N-succinimidyl bromoacetate); and SPIONHAp (coated with calcium phosphate), were also produced and tested (see FIGs. 6-10), however these clearly are not encompassed by the claimed scope as no target-binding peptide is bound to these particles, and instead are tested and discussed as controls.
Thus, the specification provides only a few examples of the claimed genus (only with iron oxide particles using the combination of the anchor of 3-aminopropyltriethoxysilan and the linker N-succinimidyl bromoacetate to peptides having the motifs of SEQ ID NO:5 or SEQ ID NO:6), which cannot be considered a sufficient description of a representative number of species by actual reduction to practice of the full breadth of the vast genus. There is no evidence on the record that, at the time of filing, the Applicant possessed additional representative species of the genus recited in the claims beyond those species provided in the working examples that fulfill the claimed functional limitations.
For these reasons, the disclosure fails to provide adequate written description to support the entirety of the broad genus claim to methods using any and all superparamagnetic iron-based particles having a functionalized target-binding peptide.
Claims 1 and 10 are thus rejected under 35 U.S.C. § 112(a) because the claimed subject matter is not described in the specification in such a way as to reasonably convey to a skilled artisan that the inventor, or a joint inventor, had possession of the claimed genus.
Dependent claims 2-9, 11-12, and 16-27 are also rejected under 35 U.S.C. § 112(a), because these claims require all of the limitations of claims 1 or 10, and fail to further limit the claimed invention to only those the species of the superparamagnetic particles that fulfill all of the claimed functional limitations, evidenced by the experimental results herein. None of the dependent claims limit the species of the SPION particle to only one of the demonstrated species that are reduced to practice herein, SPIONAPTES-SBA-SPP04 or SPIONAPTES-SBA-SPP19.
Claim Rejections - 35 USC § 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 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 1-12, 16-17, and 26-27 are rejected under 35 U.S.C. 103 as being unpatentable over Karawacka et al. ("SPIONs functionalized with small peptides for binding of lipopolysaccharide, a pathophysiologically relevant microbial product." Colloids and Surfaces B: Biointerfaces 174 (2019): 95-102, on IDS filed 8/31/2023) in view of Dryga et al. (US PGPub No. 2013316355).
Karawacka, disclosed in the instant specification, teaches functionalized magnetic nanoparticles with specific peptides, derived from the binding motives of agglutinating salivary proteins, that allow immobilization of pathogens (Title, Abstract). Karawacka teaches superparamagnetic iron oxide nanoparticles with stable polycondensed aminoalkylsilane layers were developed, to which the heterobifunctional linkers N-succinimidyl 3-(2-pyridyldithio)-propanoate (SDPD) or N-succinimidyl bromoacetate (SBA) were bound (Abstract). Karawacka demonstrates that the particle-bound peptide species succeeded in capturing about 90% of the toxin lipopolysaccharide (LPS) (Abstract, pg. 101, Fig. 7). Karawacka also teaches that “the two peptides (A and B) were conjugated to Fe3O4@APTES through SBA linker, due to its better peptide binding properties” (pg. 101, right col).
Specifically, Karawacka teaches producing and testing of Fe3O4 superparamagnetic iron oxide nanoparticles coated with aminopropyl triethoxysilane (APTES) and then either of the linkers SBA or SDPD (pg. 96, Materials and methods). The particles were then functionalized with one of two different peptides: “peptide A”: CVTGWSGRYLVEVRGQ and “peptide B”: RKQGRVEVLYRASWGTVC (pg. 96, right col). It is noted that in reverse amino acid sequence, peptide A is QGRVEVLYRGSWGTVC. Thus both peptides and A and B contain the motif of SEQ ID NO:5 (VEVLxxxxW) as instantly claimed. The data in Figure 7 of Karawacka shows that the Fe3O4@APTES@SBA@A (panel c) and Fe3O4@APTES@SBA@B (panel d) could bind, in a dose-dependent manner, approximately 95% or greater of LPS provided at a concentration of 10 EU/mL. Karawacka teaches that “For the future use in sepsis diagnosis and treatment, the designed nanoparticles should efficiently bind LPS and other bacterial toxins in order to remove them from the bloodstream” (pg. 101, right col). Furthermore, the Supplementary Data (provided herein) demonstrates that Fe3O4@APTES causes no apparent haemolysis (Figure S8) and has very minimal effects on blood coagulation, suggesting that the APTES coated SPIONs are compatible with blood, serum, or plasma.
In regards to the magnetic attractability property, Karawacka teaches that “A useful intrinsic property of superparamagnetic iron oxide nanoparticles (SPIONs) is their magnetization by external magnetic fields... The magnetic properties of SPIONs enable magnetic separation of pathogens bound to particles” (pg. 96, left col). Karawacka did not explicitly disclose separation of the SPION particles for removing LPS (or other pathogen components) but instead states: “Because the free peptides could not be magnetically separated, the samples were centrifuged to provide the same conditions” (pg. 101, right col, Fig. 7). Thus, although the authors choose not to magnetically separate the experimental particles, so that the binding properties could be fairly compared to free peptide controls in the experiments illustrated in Fig. 7. Regardless, the teachings of Karawacka suggest that the particles are magnetically attractable.
However, Karawacka does not explicitly teach that the particles have a reduction of the superparamagnetic iron-based particle concentration in Ringer solution by 65% to 99.95% when applying a magnetic field of 0.31 Tesla for three minutes in static condition.
Dryga et al. teaches magnetic particles, members of each set being conjugated to capture moieties (i.e. binding elements) for pathogens, and using magnets to isolate pathogens from a body fluid sample (Abstract; claim 1; [0006]). Dryga teaches that such magnetic-based isolation tools and methods result in improved detection and identification of blood-borne infections ([0006]-[0007]). Dryga teaches that the capture moieties include oligonucleotides (including nucleic acid probes), proteins, ligands, lectins, antibodies, aptamers, bacteriophages, host innate immunity biomarkers (e.g., CD14), host defense peptides (e.g., defensins), bacteriocins (e.g., pyocins), and receptors. ([0028]). Dryga teaches that nanometal materials such as Fe3O4 are superparamagnetic and provide advantageous properties, such as being capable of being magnetized by an external magnetic field ([0024]). Dryga further teaches optimization of the superparamagnetic particle properties ([0024]-[0027]: “While maximizing is referenced by example above, other optimizations or alterations are contemplated, such as minimizing or otherwise desirably affecting conditions.”) Regarding the applied magnetic field, Dryga teaches applying a magnetic field of about 0.3 Tesla ([0025]: “In order to maximize the moment provided by a magnetizable nanometal, a certain saturation field may be provided. For example, for Fe3O4 superparamagnetic particles, this field may be on the order of about 0.3 T.”). Dryga also teaches that superparamagnetic particles were synthesized by encapsulating iron oxide nanoparticles and used to label and capture bacteria bound to the particles ([0104]-[0109]).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the instant invention, to perform a method for binding (i.e. including separating, isolating, and/or identifying) pathogens and/or pathogen components in an aqueous or body fluidic sample using superparamagnetic iron oxide nanoparticles (SPIONs) linked to a pathogen component binding peptide according to the teachings of Karawacka, wherein the nanoparticles have been optimized for improved magnetic attractability in a magnetic field of about 0.3 T (300mT), according to the combined teachings of Dryga.
One having ordinary skill in the art, and knowledge of the teachings of Karawacka and Dryga, would be motivated to optimize the properties (i.e. optimize the production methods) and predictably, arrive at a functionalized target-binding SPION that fulfills the claimed functional properties (i.e. a particle SPIONAPTES-SBA linked to a peptide having the motif of SEQ ID NO:5), for the predictable benefits of improved target binding and detection capabilities.
The teachings of Karawacka and Dryga both pertain to the production and use of superparamagnetic nanoparticles, including those of iron oxide, for binding to target pathogens (via pathogen components). The particles taught in Karawacka fulfill all of the structural limitations of the instant claims. The optimization of producing the particles, according to methods known in the art, as taught in both Karawacka and Dryga, would have been a matter of routine optimization to one having ordinary skill. Further, Dryga directly teaches that particles are to be optimized for magnetic susceptibility and size. Because a magnetic field strength of 0.3 Tesla is taught in Dryga for use with nearly identical iron oxide (Fe3O4) particles, one having ordinary skill would have been adequately motivated to apply a field of this magnitude for testing such particles or for the capturing step.
The instant disclosure discusses testing the magnetic properties of the superparamagnetic particles taught in Karawacka and this is demonstrated in the instant Figure 7. However, these are not the functionalized particles with a bound peptide used in Karawacka. Instead the tested particles appear to be only the SPIONAPTES particles, and thus, any assay of their function cannot be compared directly to that of the fully functionalized particles, which would have an increased diameter and mass. Regardless, although the instant specification claims improvements over the particles used in Karawacka, the claimed scope does not limit the structure of particles to only those tested in the instant specification (e.g. those made by the processes described therein). The claimed improvement amounts to a desired result to be achieved, which would be predictable to one of ordinary skill in the art, for the reasons described above. Thus, any improvements on the magnetic attractability are due to predictable optimizations, within the ordinary level of skill in the art. There is no convincing evidence, on the record, of the criticality of amount of magnetic attractability for performing the pathogen binding and separation method, as claimed.
In regards to claim 2, Karawacka and Dryga both teach using particles comprising iron oxide, including Fe3O4. Claim 16 further recites that the particles are superparamagnetic iron oxide nanoparticles (SPIONs), which are taught in both Karawacka and Dryga.
Regarding claims 3 and 17, Karawacka (and its supporting data) teach that the SPION particles taught therein are to be used with patient blood samples, including serum or plasma. One having ordinary skill in the art would be motivated to optimize the particles for use in any of instantly claimed samples, as these are all well-known sources for pathogens.
Claim 4 recites that the concentration of pathogens and/or pathogen components in the sample of step (c) is reduced by at least 20%. This claim limitation has been afforded the B.R.I. in view of the specification and the plain meaning of the terms. Since no particular structure has been specified that comprises the nanoparticles required to obtain the functional characteristics, the claims are found to be obvious over the combined teachings of Karawacka and Dryga. Karawacka teaches that about 95% of the pathogen component LPS could be bound to the particles. It would have thus been obvious over the cited teachings that 20% of the sample or more could be removed when a magnetic field is applied to the bound particles.
Regarding claims 5 and 6, Karawacka teaches that the particles were tested for their ability to bind to LPS, and LPS is a pathogenic cell wall component and endotoxin from bacteria. Further, Karawacka states that “the designed nanoparticles should efficiently bind LPS and other bacterial toxins”. Dryga also teaches that the targeted magnetic particles therein could be used to isolate and detect various bacteria and/or fungi (see Tables 2 and 3 on pg. 6). Thus, the targets recited in claims 5 and 6 would be obvious binding targets to one of ordinary skill.
Regarding claims 7-8 and 26, the targeting peptides used in Karawacka (peptides A and B) have a sequence comprising 12 to 30 amino acids and have the motif of SEQ ID NO: 5 (VEVLxxxxW). The peptides of Karawacka are taught to have connecting chemicals of an aminoalkylsilane - (3-aminopropyl)-triethoxysilane, or APTES - with the linkers N-succinimidyl 3-(2-pyridyldithio)-propanoate (SDPD) or N-succinimidyl bromoacetate (SBA) used to attach the peptide. Thus, Karawacka teaches at least one if not more of the superparamagnetic particles and the alternative compounds used to provide the anchor unit and optionally the linker unit. These would have been obvious selections to one having ordinary skill to make the SPION particles for binding pathogens or pathogenic components.
Claims 9 and 27 each recite further functional limitations regarding the desired result to be achieved (claim 9: “...are capable of reducing the concentration of lipopolysaccharides (LPS) in step (c) by at least 70%... compared to the sample of step (a) comprising a LPS concentration of 10 EU/ml”; claim 27: “...by at least 95%...”). These claim limitations have been afforded the B.R.I. in view of the specification and the plain meaning of the terms, as explained previously. Further, Karawacka teaches the use of an assay with recombinant Factor C to detect the amount of pathogen (Endotoxin-binding studies were performed with EndoZyme Recombinant Factor C Assay kit (Hyglos, Germany), on pg. 97, right col, section 2.5), which is the same as that used in the experiments of the instant disclosure. Since no particular structure has been specified that comprises the nanoparticles required to obtain the functional characteristics, these claim limitations are found to be obvious over the combined teachings of Karawacka and Dryga. Karawacka teaches that about 95% of the pathogen component LPS could be bound to the particles. It would have thus been obvious over the cited teachings that 20% of the sample or more could be removed when a magnetic field is applied to the bound particles.
In regards to the detection methods of claims 10 and 11, Dryga clearly teaches that similar target-binding nanoparticles, including superparamagnetic nanoparticles, could be used for the method therein, which is directed to the detection of pathogens and/or pathogen compounds. Thus, to one having ordinary skill in the art, there would have been sufficient motivation to use the SPIONAPTES-SBA linked to a binding peptide having the motif of SEQ ID NO:5, taught in Karawacka, for a binding and detection method, because Dryga teaches that similar magnetic binding assays allows detection of a pathogen in a heterogeneous biological sample at lower levels. Dryga teaches the labeling and monitoring of viable bacteria ([0047]). It would have been common knowledge to one of ordinary skill that bacteria must be viable, in order to perform plating and a colony forming assay, a routine quantitative assay in the art.
Regarding claim 12, the combination of Karawacka and Dryga make obvious all of the limitations therein, for the reasons set forth above. Arrival at a method with any of these samples, and any of the target pathogens using a SPION bound to the motif of SEQ ID NO:5 would have been a matter of judicious selection and routine optimization to one of ordinary skill in the art, for all of the reasons described above.
Generally, MPEP § 2144.05 describes that the determination of suitable or effective concentrations and conditions for performing a known method can be determined by one of ordinary skill in the art through the use of routine or manipulative experimentation to obtain optimal results, as these are variable parameters attainable within the art. “Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). In the instant case, the optimization of the particles for improved binding and magnetic capture would have been obvious to one of ordinary skill, in view of all of the teachings of Dryga and Karawacka. It is predictable that one would have arrived at the instantly claimed methods if the samples were tested in the manner of the instant claims.
As discussed above, the evidence presented in the specification regarding the improvements of specific nanoparticles over those of Karawacka has been considered, however the claims are so broad that they encompass more than just these specific embodiments. Limitations from the specification are not imported into the claims. Further, the claiming of the particles by functional aspects, instead of by specific structural features, does not practically limit the claims to only the material in which the Inventors demonstrated improved properties.
From the teachings of the cited references, it is apparent that there would have been a reasonable expectation of success in combining the teachings therein to arrive at the claimed invention because both of the references are drawn to capturing and/or labeling pathogen and pathogen components with superparamagnetic nanoparticles and each demonstrate successfully, the ability of these functionalized nanoparticles to bind to target compounds.
Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the cited references, especially in the absence of convincing evidence to the contrary.
Claims 1-12, 16-24, and 26-27 are rejected under 35 U.S.C. 103 as being unpatentable over Karawacka et al. (Colloids and Surfaces B: Biointerfaces 174) and Dryga et al. (US PGPub No. 2013316355) as applied to claims 1-12, 16-17, and 26-27 above, and further in view of Mollenhauer US Pat. No. US 8,017,124, with supporting evidence from Product Specification: L-3012 (Escherichia coli LPS, Sigma-Aldrich) to include the rejections of claims 18-24.
The combined teachings of Karawacka and Dryga make obvious a method for using a superparamagnetic iron oxide nanoparticle (e.g. SPIONAPTES-SBA), linked to a binding peptide having the motif of SEQ ID NO:5, as taught in Karawacka, for binding to target endotoxins and/or cell wall components of bacteria pathogens, such as lipopolysaccharide.
However, the combination of Karawacka and Dryga do not explicitly teach that the targeted pathogen is one of the bacteria recited in claims 18 and 19, nor that the lipopolysaccharide is one of the species derived from E. coli as in claims 20-21, nor that the pathogenic component is lipoteichoic acid (LTA), including one from the species in claim 23. Further, claim 2 recites that the peptide has a sequence with the motif of SEQ ID NO:5 and an N-terminal sequence of RCQGR, which is not explicitly taught in Karawacka.
Mollenhauer is drawn to the use of DMBT1 for capturing sulphate and phosphate group exposing agents (Title, Abstract). Mollenhauer teaches that polypeptides from DMBT1, a scavenger receptor cysteine-rich (SRCR), can be used for the manufacture of a medicament for the prophylaxis and/or treatment of a disease caused by an agent, wherein the agent possesses at least one accessible sulphate and/or at least one accessible phosphate group (Col 1, lines 30-65). Mollenhauer teaches that the pattern recognition of DMBT1 is mediated via an 11 amino acid motif and teaches a polypeptide comprising the amino acids GRVEVLYRGSW (SEQ ID NO: 9), which is present several times within the sequence of DMBT1, and multiples thereof, and which represents the 11 amino acid motif that binds sulfate and phosphate groups (Col 3, lines 10-18). Mollenhauer also teaches the sequences of GRVEILYRGSW (SEQ ID NO: 10) and/or GRVEVLYQGSW (SEQ ID NO: 11) for binding target pathogens/agents (Col 3, lines 15-18). These sequences comprise one or more of the claimed motifs, and are homologous to the exemplified binding peptides used in the instant invention. Further, from Figure 1a, one can determine that the repeated motif of VEVLxxxxW in the DMBT1 sequence is preceded immediately by the sequence RCQGR, and this portion is included in at least one of the tested peptides (i.e. SRCRP2+3N, “e”), which had strong binding affinity demonstrated in Figure 1b.
Mollenhauer teaches that, preferably, the targeted microorganism is a bacterium or a virus, the bacteria including the genera Streptococcus, Staphylococcus, Escherichia, Helicobacter, Salmonella and Bacillus. (Col 6, lines 1-6). Mollenhauer teaches that bacterial surface structures, lipoteichoic acid (LTA) of Gram-positive and lipopolysaccharide (LPS) of Gram-negative bacteria, bind to DMBT1 via the SRCR domain, the same as the instantly claimed binding properties (FIG. 2c,d; Col 23, lines 27-46).
Mollenhauer teaches that compounds tested included, inter alia, Staphylococcus aureus LTA (L-2515), Escherichia coli LPS (L-3012), and Klebsiella pneumoniae LPS (L-1770) (Col 31, lines 33-42, Table 1). Further, from the Product Specification sheet for Escherichia coli LPS (L-3012) from Sigma-Aldrich, it is evident that the LPS tested in Mollenhauer is LPS O111:B4, one of the alternatives of claim 21.
Thus, to one of ordinary skill in the art before the effective filing date of the instant invention, it would have been prima facie obvious, when the method for binding pathogens and/or pathogen components made obvious by the combination of Karawacka in view of Dryga, that the targeted pathogen and/or pathogen-derived component would be one or more of the pathogens that bind to the fragment of SRCR motif from DMBT1, as taught in Mollenhauer and used in the SPION particles of Karawacka. Further, it would have been prima facie obvious that the motif for the binding peptide may have the N-terminal sequence RCQGR, its natural sequence according to Mollenhauer.
One would have been motivated by the combined teachings of Mollenhauer and Karawacka to use the claimed motif from DMBT1 to bind to cell wall components (i.e. LPS or LTA) of known pathogens including E. coli, a Klebsiella spp., Staphylococcus aureus, or a Streptococcus spp., because Mollenhauer teaches these are highly phosphorylated binding targets which the DMBT1-derived peptides can bind. Further, Mollenhauer teaches that DMBT1-derived peptides can bind to specific cell wall components including LPS O111:B4 from E. coli and lipoteichoic acid from Staphylococcus aureus. These are known pathogenic components in the art, and one would have been motivated to use these for capturing and/or identifying pathogenic bacteria from which they are derived.
Regarding claim 24, the peptides used in Karawacka and Mollenhauer are homologous to one another, and comprise a sequence having the claimed motif of SEQ ID NO: 5. Further, the N-terminal motif of SEQ ID NO:9 is taught to be part of the endogenous (i.e. wild-type) sequence in DMBT1 (in Mollenhauer, Figure 1). Because Mollenhauer teaches testing polypeptide sequence of different lengths and locations near the binding motif, arriving at the instantly claimed sequence of 17-19 amino acids and having SEQ ID NO:9 would have been a matter of routine optimization to one of ordinary skill in the art. Further, a peptide with the claimed N-terminus portion and the binding motif was demonstrated to having binding affinity (e.g. SRCRP2+3N, “e” of Mollenhauer), so there would have been a reasonable prediction that lengthening the sequences of Karawacka would have been successful. The instant disclosure does not demonstrate that the N-terminal portion (i.e. RCQ) is critical for binding, and instead the evidence in the cited art teaches the criticality of the core motif of VE[V/I][L/Y]xxxxW.
There would have existed a reasonable expectation of success in combining the teachings of the cited reference to arrive at the claimed invention, because the Mollenhauer is drawn to applications of the same pathogen-binding sequence as Karawacka, which is analogous to that of the instant invention. The cited references are drawn to binding and/or labeling pathogens and pathogen components by targeting the cell wall components including LPS and LTA, well-known endotoxins for gram-negative and gram-positive bacteria, respectively.
Therefore, the invention of claims would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the cited references, especially in the absence of convincing evidence to the contrary.
Claims 1-12, 16-17, and 25-27 are rejected under 35 U.S.C. 103 as being unpatentable over Karawacka et al. (Colloids and Surfaces B: Biointerfaces 174) and Dryga et al. (US PGPub No. 2013316355) as applied to claims 1-12, 16-17, and 26-27 above, and further in view of Mondal et al. (“Magnetic hydroxyapatite: a promising multifunctional platform for nanomedicine application.” International journal of nanomedicine vol. 12 8389-8410. (2017)), to include the rejections of claim 25.
The combined teachings of Karawacka and Dryga make obvious a method for using a superparamagnetic iron oxide nanoparticle (e.g. SPIONAPTES-SBA), linked to a binding peptide having the motif of SEQ ID NO:5, as taught in Karawacka, for binding to target endotoxins and/or cell wall components of bacteria pathogens, such as lipopolysaccharide.
However, the combination of Karawacka and Dryga do not explicitly teach that the linking material or connecting module can be hydroxyapatite covalently linked to the target binding peptide.
Mondal et al., a review article, teaches that target proteins can be adsorbed and released by particles of nanostructured magnetic hydroxyapatite (MHAp) (see Figures 6 and 7; pg. 8402). Mondal teaches that such coated magnetic nanoparticles can bind to proteins, enzymes, nucleotides, drugs, and antibodies, and Mondal teaches that hydroxyapatite has the advantages of being biocompatible, stable, and osteoconductive (pg. 8391). Mondal teaches that “HAp is a well-recognized nontoxic biocompatible material that has good stability and a flexible structure that allows easy assimilation of magnetic ions” (pg. 8393, right col.).
Thus, to one of ordinary skill in the art before the effective filing date of the instant invention, it would have been prima facie obvious to modify the method of binding pathogens and/or pathogen components made obvious by the combination of Karawacka in view of Dryga, by substituting a hydroxyapatite based linker for the aminosilane linker taught in Karawacka, for the predictable benefits taught in Mondal of stability, low toxicity, and protein binding ability.
One of ordinary skill in the art would have been motivated to do so by the general teachings of Mondal regarding selection of appropriate bio-based linker materials. Further, MPEP§ 2143.I.(B). describes that the simple substitution of one known element for another to obtain predictable results, when the known elements perform equivalent functions. In this case, the hydroxyapatite and the aminosilane are both known linkers that can bind metal oxide materials to polypeptides. The chemistry of these linkers would be known to one of ordinary skill in the art, as evidenced by the cited references. Thus, the substitution of one for another would be predictable and obvious variants to one of ordinary skill.
From the teachings of the cited references, it is apparent that there would have been a reasonable expectation of success in combining the teachings to arrive at the claimed invention because Karawacka and Dryga are drawn to capturing and/or labeling pathogen and pathogen components with superparamagnetic nanoparticles, including Fe oxides, while Mondal describes the general level of knowledge in the art concerning the selection and use of chemical linkers for functionalizing such nanoparticles.
Therefore, the invention of claim 25 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the cited references, especially in the absence of convincing evidence to the contrary.
Conclusion
No claims are allowable.
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/A.T.M./Examiner, Art Unit 1655
/ANAND U DESAI/Supervisory Patent Examiner, Art Unit 1655