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 .
Priority
Priority to US 63/142,402, filed 1/27/2021, is acknowledged.
Information Disclosure Statement
The information disclosure statement (IDS) was submitted on 3/11/2025, before the mailing of a first office action. The submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Status
Claims 1-8, 13, 22, 25-28, 33-34, 36, and 38-40, filed 5/31/2024, are pending. Claims 1-8, 13, 22, 25-28, 33-34, 36, and 38-40 are under examination.
Claim Interpretation
For purposes of compact prosecution, claim 2 will be interpreted such that the first GAG-binding peptide preferentially binds to CS as compared to the second GAG-binding peptide and the second GAG-binding peptide preferentially binds to HS as compared to the first GAG-binding peptide. This is in light of the properties the specification is exploiting to gain a desirable effect: “Notably, the first GAG-binding peptide preferably binds to a GAG type that is predominantly found on a first alpha granule type (i.e., a P-selectin type granule) and the second GAG-binding peptide preferably binds to a GAG type that is predominantly found on a second alpha granule type (i.e., a vWF type granule).
By engineering first compounds with a first GAG-binding peptide and second compounds with a second GAG-binding peptide, a platelet can be loaded with two active agent and into two distinct granule types. Of importance, each granule type has a separate release profile, based in part on the identity of the thrombin receptor (i.e., proteinase activated receptor 1 (PAR1) and PAR4) that is associated with the granule type.
PAR1 is a high affinity thrombin receptor which is triggered first (in low thrombin conditions) and releases P-selectin type a-granules (here referred to as a first a-granule type) whereas PAR4 is the low affinity receptor which is triggered only when sufficient amounts of thrombin has accumulated to release the vWF a-granules.” (Specification, page 11, line 21).
Regarding claim 3, claim 3 will be interpreted the same way as claim 2 above for the same reasons.
Specification
The disclosure is objected to because of the following informalities. Page 47 line 26 discloses that: “…PAL1 binding tighter to CSA than PAL 1…”. It is likely this is a typo and is meant to recite “…PAL1 binding tighter to CSA than PAL 2…” based off the data in Fig. 4B.
Appropriate correction is required.
Claim Objections
Claim 26 is objected to because of the following informalities. The phrase “a N-terminal” is better written “an N-terminal”. Appropriate correction is required.
Claim 33 is objected to because of the following informalities. The phrase “another conjunction systems that does not affect” should read “another conjunction system that does not affect”. The other members of the Markush group are singular, so this change maintains consistency. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 5 and 34 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.
Regarding claim 5, the phrase "such as" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Regarding claim 34, the usage of parenthetical examples “(agonist or inhibitor)” renders the claim indefinite because it is unclear whether the claim is limited to the examples in the parentheses or if other elements are claimed. See MPEP § 2173.05(d).
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 1-8, 13, 25-28, 33-34, 36, and 38-40 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 claim(s) 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.
Claim 1 recites two polypeptides both of which possess glycosaminoglycan binding activity. These polypeptides have no defined structure, only a function, so therefore the sequence space claimed is infinite for both polypeptides.
In this case, 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, reduction to drawings, 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. (MPEP § 2163 (II.A.3.a.ii.))
According to MPEP § 2163 (II.A.3.a.ii.), a "representative number of species" means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. See AbbVie Deutschland GmbH & Co., KG v. Janssen Biotech, Inc., 759 F.3d 1285, 1300, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014).
As described above, claim 1 recites two extremely large genera of polypeptides.
MPEP § 2163 (II.A.3.a.ii.) states that “for inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus. See, e.g., Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406. Instead, the disclosure must adequately reflect the structural diversity of the claimed genus, either through the disclosure of sufficient species that are ‘representative of the full variety or scope of the genus,’ or by the establishment of ‘a reasonable structure-function correlation.’”
Even when several species are disclosed, these are not necessarily representative of the entire genus. AbbVie Deutschland GMBH v. Janssen Biotech, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014) (“The ’128 and ’485 patents, however, only describe species of structurally similar antibodies that were derived from Joe-9. Although the number of the described species appears high quantitatively, the described species are all of the similar type and do not qualitatively represent other types of antibodies encompassed by the genus.”). Thus, when there is substantial variation within the genus, as here, one must describe a sufficient variety of species to reflect the variation within the genus to provide a "representative number” of species. Since each genus recited in the instant claims is large, it would be very challenging to describe sufficient species to cover the structures of the entire genus.
Applicant discloses two primary examples in the specification and then several alanine scans of one of the two primary examples. The polypeptides PAL-1 and PAL-2 were reduced to practice.
At the time the invention was made, the level of skill for preparing polypeptides with desired functional properties was high. However, even if a synthesis and selection procedure was, at the time of the invention, sufficient to enable the skilled artisan to identify peptides that yield polypeptides with the recited properties, the written description provision of 35 U.S.C § 112 is severable from its enablement provision. Ariad Pharm., Inc. v. Eli Lilly & Co., 598 F.3d 1336 (Fed. Cir. 2010); see also Centocor Ortho Biotech Inc. v. Abbott Labs., 97 USPQ2d 1870, 1876 (Fed. Cir. 2011) (“The fact that a fully-human antibody could be made does not suffice to show that the inventors of the '775 patent possessed such an antibody.”) Absent the conserved structure provided by the provided species, the skilled artisan generally would not be able to visualize or otherwise predict, a priori, what peptide with a particular set of properties would look like structurally.
Two examples are reduced to practice and the other disclosed examples are alanine scans. Therefore, the provided examples only represent a limited structural diversity.
Since only a limited number of species of peptides are taught within the claimed genus above, the instant claim above fails the written description requirement. A representative number of species has not been taught to describe this genus. Regarding the peptides, a single point mutation can change the biophysical properties of a peptide: “In summary, we have shown that the structural changes in the fibrillar state of the Aβ42 peptide that are observed to occur upon introduction of single point mutations can be accompanied by changes in the dominance of the microscopic processes by which these aggregates are themselves formed.” (Bolognesi et al. ACS Chem Bio 9:2 (2013) page 381 col. 2 para. 3) and “In summary, while ovispirin-1 and novispirin G-10 both had solution structures that were helical and amphipathic in the presence of TFE, a relatively simple change in their primary structure (a single glycine–isoleucine exchange) had profound effects on their respective toxicities for human erythrocytes and epithelial cells.” (Sawai et al. Protein Eng. 15:3 (2002) page 232 col. 1 para. 3).
Furthermore, many sequences allowed by the current scope of the claims, result in non-functional aggregates. Wang (Wang, et al. MAbs. Vol. 1. No. 3. Taylor & Francis, (2009)) discloses a variety of aggregation prone motifs that occur in commercial antibodies (Wang, page 262, Table 2). The scope of the claims currently may incorporate such motifs and result in non-functional aggregates.
Given this unpredictability of protein design, the skilled artisan would not have been in possession of the substantial repertoire of polypeptide species encompassed by the claimed invention; one of skill in the art would conclude that applicant was not in possession of the structural attributes of a representative number of species possessed by the members of the genus of every polynucleotide molecule recited by claim 1.
Claim 1 is rejected.
Regarding claims 2-7, claim 1 is rejected as described above. These claims do not reduce the size of the claimed genera of claim 1. One of skill in the art would conclude that applicant was not in possession of the structural attributes of a representative number of species possessed by the members of the genus of every polynucleotide molecule recited by claims 2-7.
Consequently, claims 2-7 are rejected.
Regarding claim 8, claim 1 is rejected as described above. Claim 8 significantly reduces the sequence space of the claimed polypeptides. However, the claimed polypeptides still encompass a sequence space of 20^14 = 1.64 X 10^18 because the claimed 14-mers have no other sequence constraints. One of skill in the art would conclude that applicant was not in possession of the structural attributes of a representative number of species possessed by the members of the genus of every polynucleotide molecule recited by claim 8.
Consequently, claim 8 is rejected.
Regarding claim 13, claim 1 is rejected as described above. Claim 13 reduces the size of the claimed genera to 70% identity to SEQ ID NOs 1-13. 70% identity allows for up to three substitutions of any kind. This yields a sequence space of at least 20^3 = 8,000. The variants disclosed by the specification are single-substitution alanine scans only. Therefore, one of skill in the art would conclude that applicant was not in possession of the structural attributes of a representative number of species possessed by the members of the genus of every polynucleotide molecule recited by claim 13.
Consequently, claim 13 is rejected.
Regarding claims 25-28, 33-34, 36, and 38-39, claim 1 is rejected as described above. These claims do not reduce the size of the claimed genera of claim 1. One of skill in the art would conclude that applicant was not in possession of the structural attributes of a representative number of species possessed by the members of the genus of every polynucleotide molecule recited by claims 25-28, 33-34, 36, and 38-39.
Consequently, claims 25-28, 33-34, 36, and 38-39 are rejected.
Regarding claim 40, claim 40 has the same sequence space as claim 1 and no further structural information. One of skill in the art would conclude that applicant was not in possession of the structural attributes of a representative number of species possessed by the members of the genus of every polynucleotide molecule recited by claim 40.
Consequently, claim 40 is rejected.
Claims 1-8, 13, 22, 25-28, 33-34, 36, and 38-40 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 1 with a single conservative substitution or a single alanine substitution binding to chondroitin sulfate or heparan sulfate, does not reasonably provide enablement for any possible polypeptide binding to any possible GAG in an alpha granule of a platelet. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
In order to determine compliance with the enablement requirement of 35 U.S.C. 112(a), the Federal Circuit developed a framework of factors in In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988), referred to as the Wands factors to assess whether any necessary experimentation required by the specification is "reasonable" or is "undue." Consistent with Amgen Inc. et al. v. Sanofi et al., 598 U.S. 594, 2023 USPQ2d 602 (2023), the Wands factors continue to provide a framework for assessing enablement in a utility application or patent, regardless of technology area. Guidelines for Assessing Enablement in Utility Applications and Patents in View of the Supreme Court Decision in Amgen Inc. et al. v. Sanofi et al., 89 FR 1563 (January 10, 2024). These factors include, but are not limited to:
The breadth of the claims;
The breadth of claim 1 encompasses two peptides each capable of binding to glycosaminoglycans of alpha granule platelets.
The nature of the invention;
The nature of the invention is a composition made up of a first compound with an agent and a polypeptide and a second compound with an agent and a polypeptide. This composition is intended to traffic cargo to a platelet and then release that cargo in a time dependent manner in a region of the body where platelets would aggregate. This is a cargo-delivery technology.
The state of the prior art;
Etulain (Etulain, et al. Glycobiology 24.12: 1252-1259. (2014)) discloses three main types of GAGs: “GAGs are long, unbranched polysaccharides consisting of a repeating disaccharide unit. Based on core disaccharide structures, GAGs are classified into three groups: (i) heparin/heparan sulfate (HS), (ii) chondroitin/dermatan sulfate and (iii) keratan sulfate (Varki et al. 2009). Of particular relevance to cell transport, HS is strongly expressed by vascular endothelial cells and is found throughout basement membranes. Endothelial HS has long been thought to have a role in maintaining blood vessel patency through the presentation of its non-thrombogenic surface to the circulating blood. This effect is known to depend on the high net negative charge of the HS molecule. Under normal conditions, the endothelium does not support the adhesion of platelets or inflammatory cells to itself, and it is possible that this lack of non-specific adhesion is also an effect of the HS-derived charge, given that circulating cells also express this molecule. The role of GAG-binding proteins in platelet–endothelium interactions is largely associated with chemokines, cytokines and growth factors released from platelet alpha granules.” (Etulain et al., page 1255, col. 2, para. 4).
(D) The level of one of ordinary skill;
A person of ordinary skill in the art in the field of fusion proteins is usually at least a Master’s level education.
(E) The level of predictability in the art;
Protein-protein interactions are generally unpredictable. A single point mutation can change the biophysical properties of a peptide: “In summary, we have shown that the structural changes in the fibrillar state of the Aβ42 peptide that are observed to occur upon introduction of single point mutations can be accompanied by changes in the dominance of the microscopic processes by which these aggregates are themselves formed.” (Bolognesi et al. ACS Chem Bio 9:2 (2013) page 381 col. 2 para. 3) and “In summary, while ovispirin-1 and novispirin G-10 both had solution structures that were helical and amphipathic in the presence of TFE, a relatively simple change in their primary structure (a single glycine–isoleucine exchange) had profound effects on their respective toxicities for human erythrocytes and epithelial cells.” (Sawai et al. Protein Eng. 15:3 (2002) page 232 col. 1 para. 3).
Furthermore, many sequences allowed by the current scope of the claims, result in non-functional aggregates. Wang 2 (Wang, et al. MAbs. Vol. 1. No. 3. Taylor & Francis, (2009)) discloses a variety of aggregation prone motifs that occur in commercial antibodies (Wang, page 262, Table 2). The scope of the claims currently may incorporate such motifs and result in non-functional aggregates.
(F) The amount of direction provided by the inventor and the existence of working examples and the quantity of experimentation needed to make or use the invention based on the content of the disclosure.
Applicant provides two primary peptides, SEQ ID NO: 1 and SEQ ID NO: 2, but also provides alanine scans of SEQ ID NO: 1. These peptides are bound to chondroitin sulfate and heparin sulfate.
Regarding claim 1, no structural information has been provided for the polypeptides, only a function. MPEP 2164.01 states: “In Amgen Inc. et al. v. Sanofi et al., 598 U.S. 594, 2023 USPQ2d 602 (2023), the Supreme Court, held that claims drawn to a genus of monoclonal antibodies, which were functionally claimed by their ability to bind to a specific protein, PCSK9, were invalid due to lack of enablement. The claims at issue were functional, in that they defined the genus by its function (the ability to bind to specific residues of PCSK9) as opposed to reciting a specific structure (the amino acid sequence of the antibodies in the genus). The Supreme Court concluded that the patents at issue failed to adequately enable the full scope of the genus of antibodies that performed the function of binding to specific amino acid residues on PCSK9 and blocking the binding of PCSK9 to a particular cholesterol receptor, LDLR.”
Furthermore, it would require an undue amount of experimentation to test every possible amino acid sequence for GAG binding activity. Protein-protein interactions are unpredictable as described above.
Lastly, no information is provided regarding keratan sulfate binding, the third kind of GAG associated with platelet activity as described by Etulain above.
Therefore, the specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims and claim 1 is rejected.
Regarding claim 2, claim 1 is rejected as described above. Claim 2 eliminates the GAG-related issue, but provides no additional polypeptide structure.
Consequently, claim 2 is rejected.
Regarding claim 3, claim 2 is rejected as described above. Claim 3 eliminates the GAG-related issue, but provides no additional polypeptide structure.
Consequently, claim 3 is rejected.
Regarding claim 4, claim 1 is rejected as described above. Claim 4 eliminates the GAG-related issue (these granule types have glycosaminoglycans associated with them), but provides no additional polypeptide structure.
Consequently, claim 4 is rejected.
Regarding claims 5-7, claim 1 is rejected as described above. Claims 5-7 provide no additional polypeptide structure.
Consequently, claims 5-7 are rejected.
Regarding claim 8, claim 1 is rejected as described above. Claim 8 provides a limitation to the polypeptide length, but no additional structural elements. As described above, this is still an enormous sequence space.
Consequently, claim 8 is rejected.
Regarding claim 13, claim 1 is rejected as described above. Claim 13 requires 70% identity to SEQ ID NOs: 1-13. 70% identity allows for up to three substitutions of any kind. This yields a sequence space of at least 20^3 = 8,000. Testing this many variants against the target GAGs would require undue experimentation on the part of a person of ordinary skill in the art.
Consequently, claim 13 is rejected.
Regarding claim 22, claim 13 is rejected as described above. Claim 22 recites 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2. The alanine scans of SEQ ID NO: 1 enable those substitutions as well as any single conservative substitutions. However, SEQ ID NO: 2 have no such alanine scans disclosed. It is unpredictable how SEQ ID NO: 2 will tolerate mutations and a scan with canonical amino acids yields 11X20 = 210 variants to test against every GAG target.
Consequently, claim 22 is rejected.
Regarding claims 25-28, 33, 34, 36, 38, and 39, claim 1 is rejected as described above. Claims 25-28, 33, 34, 36, 38, and 39 provide no additional polypeptide structure.
Consequently, claims 25-28, 33, 34, 36, 38, and 39 are rejected.
Regarding claim 40, no structural information has been provided for the polypeptides, only a function. MPEP 2164.01 states: “In Amgen Inc. et al. v. Sanofi et al., 598 U.S. 594, 2023 USPQ2d 602 (2023), the Supreme Court, held that claims drawn to a genus of monoclonal antibodies, which were functionally claimed by their ability to bind to a specific protein, PCSK9, were invalid due to lack of enablement. The claims at issue were functional, in that they defined the genus by its function (the ability to bind to specific residues of PCSK9) as opposed to reciting a specific structure (the amino acid sequence of the antibodies in the genus). The Supreme Court concluded that the patents at issue failed to adequately enable the full scope of the genus of antibodies that performed the function of binding to specific amino acid residues on PCSK9 and blocking the binding of PCSK9 to a particular cholesterol receptor, LDLR.”
Furthermore, it would require an undue amount of experimentation to test every possible amino acid sequence for GAG binding activity. Protein-protein interactions are unpredictable as described above.
Lastly, no information is provided regarding keratan sulfate binding, the third kind of GAG associated with platelet activity as described by Etulain above.
Therefore, the specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims and claim 40 is rejected.
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, 25-28, 33, 34, and 40 are rejected under 35 U.S.C. 103 as being unpatentable over Niessen, et al. (Niessen, et al. Journal of immunological methods 328.1-2: 89-96 (2007)) in view of Loers et al. (Loers, Gabriele, et al. Scientific reports 9.1: 1064 (2019)), Pannerden et al. (Pannerden, et al. Blood, The Journal of the American Society of Hematology 116.7: 1147-1156 (2010)), Kawashima et al. (Kawashima, et al. Journal of Biological Chemistry 275.45: 35448-35456. (2000)), Dixon et al. US 20170080101, published 3/23/2017, Capila et al. (Capila, et al. Angewandte Chemie International Edition 41.3: 390-412. (2002)), Pazos et al. (Pazos, et al. Chemical Society Reviews 38.12: 3348-3359. (2009)), and Sobel et al. (Sobel et al. J Biol Chem. May 5;267(13):8857-62. PMID: 1577724. (1992)).
Regarding claim 1, claim 1 recites a composition comprising: a first compound comprising a first agent and a first polypeptide, wherein the first polypeptide comprises a first glycosaminoglycan (GAG)-binding peptide which is capable of binding a GAG in a first alpha granule type of a platelet; and a second compound comprising a second agent and a second polypeptide, wherein the second polypeptide comprises a second glycosaminoglycan (GAG)-binding peptide which is capable of binding a GAG in a second alpha granule type of the platelet.
Niessen discloses that alpha granules contain both P-Selection and von Willebrand factor: “We were able to get highly purified subcellular fractions of human platelets using several antibodies against specific markers for dense granules (LAMP2), alpha granules (P-selectin) and the plasma membrane (GPIIb/IIIa) in combination with antibody-coated magnetic beads. In the respective fractions the marker proteins used for isolation as well as further independent, structure specific markers (for example MRP4 for dense granules, von Willebrand factor (vWF) for alpha granules and protein disulfide isomerase, PDI and GPIbβ, for plasma membrane) could be detected by Western blotting.” (Nissen et al., page 89, Abstract).
Regarding the first polypeptide:
Loers et al. discloses a peptide capable of targeting chondroitin sulfate: “ With the aim to find molecules that neutralize the inhibitory functions of C4S, we screened a phage display library for peptides binding to C4S. From the phage clones binding to C4S we selected three peptides for further analysis.” (Loers et al., page 1, Abstract).
Kawashima et al. further discloses that P-selectin has chondroitin sulfate binding sites: “Here we show that a large chondroitin sulfate proteoglycan, versican, derived from a renal adenocarcinoma cell line ACHN, binds L-selectin, P-selectin, and CD44. The binding was mediated by the interaction of the chondroitin sulfate (CS) chain of versican with the carbohydrate-binding domain of L- and P-selectin and CD44.” (Kawashima et al., page 35448, Abstract).
Pazos et al. discloses the fusion of fluorophores (an agent) to peptides: “Given the polymeric nature of peptides, it is possible to select specific amino acid sequences with high affinity for a particular target by combinatorial optimization of peptide libraries. Moreover, there is a wealth of knowledge regarding small peptide sequences that are specific binding partners for important biomolecules, and in many cases there is structural information available in the form of X-ray or NMR structures that can be used for the rational design of a specific peptidic sensor. Sometimes even in the absence of detailed structural information it is possible to use known peptide sequences as the starting point for the development of a probe.4Peptide scaffolds, being intrinsically modular, also allow the introduction of multiple fluorophores in their structure while retaining biological activity, which increases the possibilities for the design of fluorescent sensors that utilize the properties resulting from the interaction between two fluorophores.” (Pazos et al., page 3349, col. 1., para. 3).
Regarding the second polypeptide:
Pannerden et al. discloses that von Willebrand factor is specific to spherical α granules:
“Thus, VWF and β-TG are selectively excluded from tubular α-granules. To obtain detailed information on the spatial protein segregation, we next combined serial cryosectioning with immunogold labeling. Using this approach, we found that connections exist between fibrinogen-positive tubules and VWF-positive spherical granules (supplemental Figure 3). Furthermore, subsequent cross sections of the same granule exposed different subpopulations (ie, dense core or multivesicular granules; supplemental Figure 1A-B). Together, these observations demonstrate that, besides different morphologies, high spatial protein segregation also exists within individual α-granule subtypes.” (Pannerden et al., page 1152, col. 1, para. 1).
Dixon et al. discloses a peptide that targets heparan sulfate:
“The HS-GAG binding element may comprise one or more of the heparan binding sulphate domains of any FGF protein (e.g. domains A, B or C). The HS-GAG binding element may comprise FGF4. The HS-GAG binding element may comprise FGF1 HBD A (heparan sulphate binding domain A (the first HBD domain of FGF1)), FGF2 HBD A (heparan sulphate binding domain A), FGF4 HBD A (heparan sulphate binding domain A), FGF1 HBD C (heparan sulphate binding domain C), FGF2 HBD B (heparan sulphate binding domain B), FGF2 HBD C (heparan sulphate binding domain C), FGF4 HBD C (heparan sulphate binding domain C), FGF7 HBD B (heparan sulphate binding domain B), FGF7 HBD C (heparan sulphate binding domain C), antithrombin, such as ATIII, VEGF, or PDGF, or variants thereof.” (Dixon et al., para. [0026]).
Sobel et al. discloses that von Willebrand factor binds heparin: “Human von Willebrand factor, a plasma glycoprotein which plays a critical role in regulating hemostasis, binds heparin, but the physiological importance and mode of this interaction is poorly understood. Using the motif of an amino acid sequence of a consensus heparin binding synthetic peptide, a 23-residue sequence (Tyr565-Ala587) of human von Willebrand factor was identified that retains the consensus motif and binds heparin with affinity comparable with native von Willebrand factor and the consensus peptide.”
Capila et al. discloses that heparin and heparan sulfate have nearly identical properties: “Although structurally similar, heparin and heparan sulfate GAGs can often be structurally distinguished through their different sensitivity towards a family of GAG-degrading, microbial enzymes, the heparin lyases.22 In the following sections, most of the properties that we discuss with respect to heparin are also applicable, except where specified, to heparan sulfate.” (Capila et al, page 393, col. 2, para. 2).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to create a first polypeptide as disclosed by Loers capable of binding chondroitin sulfate as disclosed by Kawashima fused to a fluorophore agent as disclosed by Pazos and a second polypeptide as disclosed by Dixon capable of binding heparan sulfate as disclosed by Sobel and Capila fused to a different fluorophore agent as disclosed by Pazos in the same composition to arrive at the claimed invention.
A person of ordinary skill in the art would be motivated to create such a composition to deliver two different fluorophores as disclosed by Pazos to measure the degree of von Willebrand factor segregation as disclosed by Pannerden.
A person of ordinary skill in the art would have a reasonable expectation of success because each polypeptide is confirmed to bind to the desired target by the prior art and alpha granules possess both P-selectin and von Willebrand factor as disclosed by Niessen. Regarding heparan sulfate, Capila discloses that it has extremely similar properties to heparin, so the heparin binding site in von Willebrand factor would be reasonably expected to also have heparan sulfate bound in that location. Pazos describes the common usage of fluorophores attached to peptide targeting moieties.
Consequently, claim 1 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Regarding claim 2, claim 1 is obvious as described above. Claim 2 further recites the case wherein the first GAG-binding peptide preferentially binds to chondroitin sulfate (CS) and the second GAG-binding peptide preferentially binds to heparan sulfate (HS).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use one GAG-binding protein that binds significantly better to CS and another protein that binds significantly better to HS in order to better measure the presence of P-selectin and von Willebrand factor via the attached fluorophores. The better the specificity, the more clean the resulting data would be. A person of ordinary skill in the art would have a reasonable expectation of success because more binding specificity would result in fewer off target fluorophore labeling events.
Consequently, claim 2 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Regarding claim 3, claim 2 is obvious as described above. Claim 3 further recites the case wherein the first GAG-binding peptide preferentially binds to chondroitin sulfate A (CSA) and does not preferably bind to heparan sulfate (HS).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use one GAG-binding protein that binds significantly better to CS and another protein that binds significantly better to HS in order to better measure the presence of P-selectin and von Willebrand factor via the attached fluorophores. The better the specificity, the more clean the resulting data would be. A person of ordinary skill in the art would have a reasonable expectation of success because more binding specificity would result in fewer off target fluorophore labeling events.
Consequently, claim 3 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Regarding claim 4, claim 1 is obvious as described above. Claim 4 further recites the case wherein the first alpha granule type is a P-selectin associated granule and the second alpha granule type von Willebrand factor associated granule.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to create a first polypeptide as disclosed by Loers capable of binding chondroitin sulfate as disclosed by Kawashima fused to a fluorophore agent as disclosed by Pazos and a second polypeptide as disclosed by Dixon capable of binding heparan sulfate as disclosed by Sobel and Capila fused to a different fluorophore agent as disclosed by Pazos in the same composition to arrive at the claimed invention.
A person of ordinary skill in the art would be motivated to create such a composition to deliver two different fluorophores to measure the degree of von Willebrand factor segregation as disclosed by Pannerden.
A person of ordinary skill in the art would have a reasonable expectation of success because each polypeptide is confirmed to bind to the desired target by the prior art and alpha granules possess both P-selectin and von Willebrand factor as disclosed by Niessen. Regarding heparan sulfate, Capila discloses that it has extremely similar properties to heparin, so the heparin binding site in von Willebrand factor would be reasonably expected to also have heparan sulfate bound in that location. Pazos describes the common usage of fluorophores attached to peptide targeting moieties.
Consequently, claim 4 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Regarding claim 5, claim 1 is obvious as described above. Claim 5 further recites the case wherein contents of the first alpha granule type are released via the high-affinity thrombin receptor PAR1 and contents of the second alpha granule type are released via the low-affinity thrombin receptor PAR4, optionally, the contents of an alpha granule may be released in response to contact with a matrix metalloproteinase (MMP), peroxidase, phosphohydrolase, plasmin, or a plasmin derivative such as tissue plasminogen activator (tPA).
MPEP 2112.01(II) states: “"Products of identical chemical composition can not have mutually exclusive properties." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). A chemical composition and its properties are inseparable. Therefore, if the prior art teaches the identical chemical structure, the properties applicant discloses and/or claims are necessarily present. Id.
The obvious composition of claim 1 would necessarily have this property since all structural limitations are met in the rejection above.
Consequently, claim 5 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Regarding claim 6, claim 1 is obvious as described above. Claim 6 further recites the case wherein the contents of the first alpha granule type are released at a lower concentration of thrombin than the concentration of thrombin needed to provide release of the contents of the second alpha granule type.
MPEP 2112.01(II) states: “"Products of identical chemical composition can not have mutually exclusive properties." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). A chemical composition and its properties are inseparable. Therefore, if the prior art teaches the identical chemical structure, the properties applicant discloses and/or claims are necessarily present. Id.
The obvious composition of claim 1 would necessarily have this property since all structural limitations are met in the rejection above.
Consequently, claim 6 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Regarding claim 7, claim 1 is obvious as described above. Claim 7 further recites the case wherein the contents first alpha granule type is released before the contents of the second alpha granule type are released.
MPEP 2112.01(II) states: “"Products of identical chemical composition can not have mutually exclusive properties." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). A chemical composition and its properties are inseparable. Therefore, if the prior art teaches the identical chemical structure, the properties applicant discloses and/or claims are necessarily present. Id.
The obvious composition of claim 1 would necessarily have this property since all structural limitations are met in the rejection above.
Consequently, claim 7 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Regarding claim 8, claim 1 is obvious as described above. Claim 8 further recites the case wherein the first and the second GAG-binding peptides are each between about 8 amino acids and about 14 amino acids in length.
Dixon discloses a sequence: “The HS-GAG binding element may comprise the amino acid sequence PRESGKKRKRKRLKPT (PDGF, SEQ ID NO. 3). The HS-GAG binding element may comprise a sequence having at least 80% identity to SEQ ID NO. 3.” (Dixon et al., para. [0030]).
A peptide that is 80% identical to Dixon SEQ ID NO: 3 may be 0.8*16= 13 amino acids in length.
Consequently, claim 8 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Regarding claim 25, claim 1 is obvious as described above. Claim 25 further recites the case wherein the first polypeptide consists of the first GAG-binding peptide and the second polypeptide consists of the second GAG-binding peptide.
Changing the transitional phrase from “comprising” to “consists of” does not change any part of the analysis of claim 1 above.
Consequently, claim 25 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Regarding claim 26, claim 1 is obvious as described above. Claim 26 further recites the case wherein a N-terminal of the first polypeptide is directly or indirectly linked to the first agent and/or the N-terminal of the second polypeptide is directly or indirectly linked to second first agent or wherein a C-terminal of the first polypeptide is directly or indirectly linked to the first agent and/or the C-terminal of the second polypeptide is directly or indirectly linked to second first agent.
Pazos et al. discloses multiple examples of fluorophores attached at the N-terminus or C-terminus. One such example is a peptide beacon (Pazos et al., page 3354, Fig 10).
Consequently, claim 26 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Regarding claim 27, claim 26 is obvious as described above. Claim 27 further recites the case wherein the C-terminal of the first polypeptide is directly or indirectly linked to the first agent and/or the C-terminal of the second polypeptide is directly or indirectly linked to second first agent.
Pazos et al. discloses multiple examples of fluorophores attached at the C-terminus. One such example is a peptide beacon (Pazos et al., page 3354, Fig 10).
Consequently, claim 27 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Regarding claim 28, claim 1 is obvious as described above. Claim 28 further recites the case wherein the first agent is indirectly linked to the first polypeptide via a first linker and/or the second agent is indirectly linked to the second polypeptide via a second linker.
Pazos et al. discloses the usage of linkers to attach fluorophores: “This imaging strategy could also be applied for studying other enzymes by attaching the fluorochrome or quencher units to the carrier backbone through specific linkers that could be targeted by those selected enzymes.” (Pazos et al., page 3355, col. 1, para. 5).
Consequently, claim 28 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Regarding claim 33, claim 1 is obvious as described above. Claim 33 further recites the case wherein the first agent is directly or indirectly linked to the first polypeptide and/or the second agent is directly or indirectly linked to the second polypeptide using a maleimide reaction, succinimidyl ester reaction, an enzymatic reaction, or another conjugation systems that does not affect protein structure or activity.
In the peptide beacon example in Pazos et al., the peptide necessarily retains its binding activity (Pazos et al., page 3354, Fig 10). The binding scheme is a result of a conjugation reaction outlined in Fig. 6c (Pazos et al., page 3352, Fig. 6c).
Consequently, claim 33 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Regarding claim 34, claim 1 is obvious as described above. Pazos et al. discloses the conjugation of fluorescent moieties.
Consequently, claim 34 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Regarding claim 40, claim 40 recites an isolated platelet comprising: at least one copy of a first compound comprising a first agent and a first polypeptide, wherein the first polypeptide comprises a first glycosaminoglycan (GAG)-binding peptide which is capable of binding a GAG in a first alpha granule type of a platelet; and at least one copy of a second compound comprising a second agent and a second polypeptide, wherein the second polypeptide comprises a second glycosaminoglycan (GAG)-binding peptide which is capable of binding a GAG in a second alpha granule type of the platelet.
This claim has the same minimal elements as claim 1. The difference is that the composition in this case is an isolated platelet.
Niessen discloses that alpha granules contain both P-Selection and von Willebrand factor: “We were able to get highly purified subcellular fractions of human platelets using several antibodies against specific markers for dense granules (LAMP2), alpha granules (P-selectin) and the plasma membrane (GPIIb/IIIa) in combination with antibody-coated magnetic beads. In the respective fractions the marker proteins used for isolation as well as further independent, structure specific markers (for example MRP4 for dense granules, von Willebrand factor (vWF) for alpha granules and protein disulfide isomerase, PDI and GPIbβ, for plasma membrane) could be detected by Western blotting.” (Nissen et al., page 89, Abstract).
Regarding the first polypeptide:
Loers et al. discloses a peptide capable of targeting chondroitin sulfate: “ With the aim to find molecules that neutralize the inhibitory functions of C4S, we screened a phage display library for peptides binding to C4S. From the phage clones binding to C4S we selected three peptides for further analysis.” (Loers et al., page 1, Abstract).
Kawashima et al. further discloses that P-selectin has chondroitin sulfate binding sites: “Here we show that a large chondroitin sulfate proteoglycan, versican, derived from a renal adenocarcinoma cell line ACHN, binds L-selectin, P-selectin, and CD44. The binding was mediated by the interaction of the chondroitin sulfate (CS) chain of versican with the carbohydrate-binding domain of L- and P-selectin and CD44.” (Kawashima et al., page 35448, Abstract).
Pazos et al. discloses the fusion of fluorophores (an agent) to peptides: “Given the polymeric nature of peptides, it is possible to select specific amino acid sequences with high affinity for a particular target by combinatorial optimization of peptide libraries. Moreover, there is a wealth of knowledge regarding small peptide sequences that are specific binding partners for important biomolecules, and in many cases there is structural information available in the form of X-ray or NMR structures that can be used for the rational design of a specific peptidic sensor. Sometimes even in the absence of detailed structural information it is possible to use known peptide sequences as the starting point for the development of a probe.4Peptide scaffolds, being intrinsically modular, also allow the introduction of multiple fluorophores in their structure while retaining biological activity, which increases the possibilities for the design of fluorescent sensors that utilize the properties resulting from the interaction between two fluorophores.” (Pazos et al., page 3349, col. 1., para. 3).
Regarding the second polypeptide:
Pannerden et al. discloses that von Willebrand factor is specific to spherical α granules:
“Thus, VWF and β-TG are selectively excluded from tubular α-granules. To obtain detailed information on the spatial protein segregation, we next combined serial cryosectioning with immunogold labeling. Using this approach, we found that connections exist between fibrinogen-positive tubules and VWF-positive spherical granules (supplemental Figure 3). Furthermore, subsequent cross sections of the same granule exposed different subpopulations (ie, dense core or multivesicular granules; supplemental Figure 1A-B). Together, these observations demonstrate that, besides different morphologies, high spatial protein segregation also exists within individual α-granule subtypes.” (Pannerden et al., page 1152, col. 1, para. 1).
Dixon et al. discloses a peptide that targets heparan sulfate:
“The HS-GAG binding element may comprise one or more of the heparan binding sulphate domains of any FGF protein (e.g. domains A, B or C). The HS-GAG binding element may comprise FGF4. The HS-GAG binding element may comprise FGF1 HBD A (heparan sulphate binding domain A (the first HBD domain of FGF1)), FGF2 HBD A (heparan sulphate binding domain A), FGF4 HBD A (heparan sulphate binding domain A), FGF1 HBD C (heparan sulphate binding domain C), FGF2 HBD B (heparan sulphate binding domain B), FGF2 HBD C (heparan sulphate binding domain C), FGF4 HBD C (heparan sulphate binding domain C), FGF7 HBD B (heparan sulphate binding domain B), FGF7 HBD C (heparan sulphate binding domain C), antithrombin, such as ATIII, VEGF, or PDGF, or variants thereof.” (Dixon et al., para. [0026]).
Sobel et al. discloses that von Willebrand factor binds heparin: “Human von Willebrand factor, a plasma glycoprotein which plays a critical role in regulating hemostasis, binds heparin, but the physiological importance and mode of this interaction is poorly understood. Using the motif of an amino acid sequence of a consensus heparin binding synthetic peptide, a 23-residue sequence (Tyr565-Ala587) of human von Willebrand factor was identified that retains the consensus motif and binds heparin with affinity comparable with native von Willebrand factor and the consensus peptide.”
Capila et al. discloses that heparin and heparan sulfate have nearly identical properties: “Although structurally similar, heparin and heparan sulfate GAGs can often be structurally distinguished through their different sensitivity towards a family of GAG-degrading, microbial enzymes, the heparin lyases.22 In the following sections, most of the properties that we discuss with respect to heparin are also applicable, except where specified, to heparan sulfate.” (Capila et al, page 393, col. 2, para. 2).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to create an isolated platelet comprising a first polypeptide as disclosed by Loers capable of binding chondroitin sulfate as disclosed by Kawashima fused to a fluorophore agent as disclosed by Pazos and a second polypeptide as disclosed by Dixon capable of binding heparan sulfate as disclosed by Sobel and Capila fused to a different fluorophore agent as disclosed by Pazos in the same composition to arrive at the claimed invention.
A person of ordinary skill in the art would be motivated to create such a platelet to deliver two different fluorophores as disclosed by Pazos to measure the degree of von Willebrand factor segregation as disclosed by Pannerden and then measure the resulting fluorescent signal in the platelet.
A person of ordinary skill in the art would have a reasonable expectation of success because each polypeptide is confirmed to bind to the desired target by the prior art and alpha granules possess both P-selectin and von Willebrand factor as disclosed by Niessen. Regarding heparan sulfate, Capila discloses that it has extremely similar properties to heparin, so the heparin binding site in von Willebrand factor would be reasonably expected to also have heparan sulfate bound in that location. Pazos describes the common usage of fluorophores attached to peptide targeting moieties.
Consequently, claim 40 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. and rejected.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Niessen, et al. (Niessen, et al. Journal of immunological methods 328.1-2: 89-96 (2007)) in view of Loers et al. (Loers, Gabriele, et al. Scientific reports 9.1: 1064 (2019)), Pannerden et al. (Pannerden, et al. Blood, The Journal of the American Society of Hematology 116.7: 1147-1156 (2010)), Kawashima et al. (Kawashima, et al. Journal of Biological Chemistry 275.45: 35448-35456. (2000)), Dixon et al. US 20170080101, published 3/23/2017, Capila et al. (Capila, et al. Angewandte Chemie International Edition 41.3: 390-412. (2002)), Pazos et al. (Pazos, et al. Chemical Society Reviews 38.12: 3348-3359. (2009)), and Sobel et al. (Sobel et al. J Biol Chem. May 5;267(13):8857-62. PMID: 1577724. (1992)) as applied to claim 1 above, further in view of LaRosa et al. US2004123343, published 6/24/2004.
Regarding claim 13, LaRosa et al. US2004123343 discloses the following protein aligned against Applicant SEQ ID NO: 4 below:
SQ Sequence 538 AA;
Query Match 70.8%; Score 46; Length 538;
Best Local Similarity 87.5%;
Matches 7; Conservative 1; Mismatches 0; Indels 0; Gaps 0;
Qy 1 EARIWFPY 8
:|||||||
Db 209 DARIWFPY 216
The sequence of LaRosa would necessarily possess the required binding activity. MPEP 2112.01(II) states: “Products of identical chemical composition can not have mutually exclusive properties." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). A chemical composition and its properties are inseparable. Therefore, if the prior art teaches the identical chemical structure, the properties applicant discloses and/or claims are necessarily present. Id. “
Consequently, claim 36 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. as applied to claim 1, further in view of LaRosa et al. and rejected.
Claim 36 is rejected under 35 U.S.C. 103 as being unpatentable over Niessen, et al. (Niessen, et al. Journal of immunological methods 328.1-2: 89-96 (2007)) in view of Loers et al. (Loers, Gabriele, et al. Scientific reports 9.1: 1064 (2019)), Pannerden et al. (Pannerden, et al. Blood, The Journal of the American Society of Hematology 116.7: 1147-1156 (2010)), Kawashima et al. (Kawashima, et al. Journal of Biological Chemistry 275.45: 35448-35456. (2000)), Dixon et al. US 20170080101, published 3/23/2017, Capila et al. (Capila, et al. Angewandte Chemie International Edition 41.3: 390-412. (2002)), Pazos et al. (Pazos, et al. Chemical Society Reviews 38.12: 3348-3359. (2009)), and Sobel et al. (Sobel et al. J Biol Chem. May 5;267(13):8857-62. PMID: 1577724. (1992)) as applied to claim 1 above, further in view of Olsson et al. (Olsson, et al. Platelets 29.6: 569-573. (2018)), Marqus et al. (Marqus, et al. Journal of biomedical science 24.1: 21. (2017)) , Pollaro et al. (Pollaro et al. MedChemComm 1.5: 319-324. (2010)), and Böttger et al. (Böttger, et al., PloS one 12.6: e0178943. (2017)).
Regarding claim 36, claim 1 is obvious as described above. Claim 36 further recites the case wherein the first agent and/or the second agent is harmful to mammalian cells and/or is toxic to a subject and/or the first agent and/or the second agent is susceptible to degradation when administered directly into the bloodstream of a subject.
Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. do not describe such an agent.
Olsson et al. describes the relationship between platelets and cancer: “Individuals with cancer have an increased risk for thrombotic events, and thrombosis is a major contributor to morbidity and mortality in cancer patients. Several factors contribute to the pro-thrombotic state associated with malignant disease. The leaky tumor vasculature enables fibrinogen leakage into the extravascular space, which in the presence of thrombin, expressed for instance by tumor cells, can be cleaved to form a fibrin clot. Moreover, clotting factor expression is often altered in the presence of a tumor. Expression of pro-coagulant factors such as tissue factor (TF), the inducer of the extrinsic coagulation pathway, and cancer procoagulant (CP), is frequently enhanced, and levels of anticoagulant factors such as tissue plasminogen activator are reduced. In addition to this, platelet activation and turnover is often increased in individuals with cancer. The tumor vessel leakiness contributes to platelet activation in the tumor due to exposure of subendothelial factors such as collagen and TF. Furthermore, aberrant expression of TF and CP by tumor cells and the tumor microenvironment contributes to activation of platelets. High platelet counts are considered a poor prognostic factor in many cancer types, and a high platelet-lymphocyte ratio has been suggested to predict poor patient outcome.” (Olsson et al., page 569, col. 1, para. 3).
Marqus et al. discloses peptides that are cytotoxic to human cancer cells: “These pore-forming peptides target cancer cell membranes, and can induce cell death either by necrosis or apoptosis. In necrosis, the AMPs target the negatively-charged molecules on the cancer cell membrane and cause cell lysis; while in apoptosis, they cause disruption of the mitochondrial membrane [21]. Another AMP is magainin, which is derived from the skin of the African clawed frog Xenopus laevis [31]. Lehmann et al. [32] observed that margainin II was cytotoxic to human bladder cancer cells but not human or murine fibroblasts. Magainin killed the bladder cancer cells by inducing pores in the plasma membrane [32]. Pleurocidin is isolated from the winter flounder, Pleuronectes americanus [33] and members of this family of cationic peptides (such as NRC-3 and NRC-7) were cytotoxic against human breast cancer cells and mouse mammary carcinoma cells but not human dermal fibroblasts [34]. These two peptides were shown to disrupt the integrity of the cell membrane [34]. The pre-treatment of human breast cancer cells (MDA-MB-231) with NRC-3 or NRC-7 and cisplatin enhanced the latter’s cytotoxic effect (EC50) by 5.5- and 1.7-fold, respectively [34].” (Marqus et al., page 2, col. 2, para. 2).
Böttger et al. discloses that such peptides are subject to degradation in the blood stream: “Proteolytic degradation of peptide-based drugs is often considered as major weakness limiting systemic therapeutic applications. Therefore, huge efforts are typically devoted to stabilize sequences against proteases present in serum or plasma, obtained as supernatants after complete blood coagulation or centrifugation of blood supplemented with anticoagulants, respectively.” (Böttger et al., page 1, Abstract).
It would have been obvious to a person of ordinary skill before the effective filing date of the claimed invention to use the peptide therapeutics of Marqus as an agent in place of the fluorophore in the composition of Niessen, et al., Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. to arrive at the claimed invention.
A person of ordinary skill in the art would be motivated to deliver an anti-cancer peptide as disclosed by Marqus via a platelet because Olsson discloses that platelets are recruited to the sites of tumors. Böttger discloses that peptides are subject to proteolysis in the blood stream, which would serve to mitigate off-target effects. Any peptides that do not make it to the desired tumor are then degraded in the blood stream as described by Böttger.
A person of ordinary skill in the would have a reasonable expectation of success because Pollaro et al. discloses that peptide half-lives are increased when associated to larger molecules: “Peptides are an attractive class of molecules for the development of therapeutics because they combine unique properties such as high binding affinity, excellent target specificity, low toxicity and a relatively small mass. However, the short in vivo half-life of peptides which is typically only a few minutes had hampered the development of a larger number of peptide leads into drugs. The main reasons for the fast elimination of peptides from the circulation are enzymatic degradation and/or fast renal clearance. To prolong the half-life of peptides, their proteolytic stability can be improved by chemical modification strategies and the rate of clearance can be reduced by conjugating the peptides to molecules that prevent their elimination through the kidney. In this article we review the latter class of strategies that aims at prolonging the in vivo plasma residence time of peptides. Techniques including peptide-drug linkage to large polymers, fusion to long-lived proteins such as albumin or the Fc fragment of immunoglobulin and conjugation to small molecule albumin-binding tags are discussed and the peptide-conjugate half-lives achieved are compared.” (Pollaro et al., page 319, Abstract).
Consequently, claim 36 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. as applied to claim 1, further in view of Olsson et al., Pollaro et al., Marqus et al., and Böttger et al. and rejected.
Claim 38 is rejected under 35 U.S.C. 103 as being unpatentable over Niessen, et al. (Niessen, et al. Journal of immunological methods 328.1-2: 89-96 (2007)) in view of Loers et al. (Loers, Gabriele, et al. Scientific reports 9.1: 1064 (2019)), Pannerden et al. (Pannerden, et al. Blood, The Journal of the American Society of Hematology 116.7: 1147-1156 (2010)), Kawashima et al. (Kawashima, et al. Journal of Biological Chemistry 275.45: 35448-35456. (2000)), Dixon et al. US 20170080101, published 3/23/2017, Capila et al. (Capila, et al. Angewandte Chemie International Edition 41.3: 390-412. (2002)), Pazos et al. (Pazos, et al. Chemical Society Reviews 38.12: 3348-3359. (2009)), and Sobel et al. (Sobel et al. J Biol Chem. May 5;267(13):8857-62. PMID: 1577724. (1992)) as applied to claim 1 above, further in view of Kvist et al. (Kvist, Alexander J., et al. Journal of Biological Chemistry 281.44: 33127-33139. (2006)).
Regarding claim 38, claim 1 is obvious as described above. Claim 38 further recites the case wherein the first GAG-binding peptide and/or the second GAG-binding peptide also preferentially binds serglycin, perlecan, dermatan sulfate, keratan sulfate, and/or GPIIb/IIIa.
Kvist et al. discloses that perlecan is a form of heparan sulfate and can also be substituted with chondroitin sulfate:
“The basement membrane heparan sulfate (HS)5 proteoglycan perlecan was first isolated from the murine Engelbrecht-Holm-Swarm tumor. Perlecan binds to extracellular matrix components integral to basement membranes, such as collagen type IV, nidogen, laminin, and fibronectin (1), as well as to extracellular matrix components outside the basement membrane (e.g. PRELP (proline/arginine-rich end leucine-rich repeat protein) and collagen type I) (2). Perlecan supports cell attachment by both binding and clustering integrins (3). Binding to growth factors has been shown for both the HS side chains (fibroblast growth factor-2) (4) and the core protein (progranulin) (5). Based on these interactions, perlecan is believed to have a role in basement membrane integrity. In addition, perlecan is expressed outside basement membranes (e.g. in cartilage) (6–9). Although identified as an HS proteoglycan, perlecan can also be partially substituted with chondroitin sulfate (CS).” (Kvist et al., page 33127, col. 1, para. 1).
Consequently, a person of ordinary skill in the art would have a reasonable expectation that the peptide of Loers or the peptide of Dixon would also bind perlecan.
Claim 38 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. as applied to claim 1, further in view of Kvist et al. and rejected.
Claim 39 is rejected under 35 U.S.C. 103 as being unpatentable over Niessen, et al. (Niessen, et al. Journal of immunological methods 328.1-2: 89-96 (2007)) in view of Loers et al. (Loers, Gabriele, et al. Scientific reports 9.1: 1064 (2019)), Pannerden et al. (Pannerden, et al. Blood, The Journal of the American Society of Hematology 116.7: 1147-1156 (2010)), Kawashima et al. (Kawashima, et al. Journal of Biological Chemistry 275.45: 35448-35456. (2000)), Dixon et al. US 20170080101, published 3/23/2017, Capila et al. (Capila, et al. Angewandte Chemie International Edition 41.3: 390-412. (2002)), Pazos et al. (Pazos, et al. Chemical Society Reviews 38.12: 3348-3359. (2009)), and Sobel et al. (Sobel et al. J Biol Chem. May 5;267(13):8857-62. PMID: 1577724. (1992)) as applied to claim 1 above, further in view of Toyama-Sorimachi et al. (Toyama-Sorimachi, et al. Journal of Biological Chemistry 270.13: 7437-7444 (1995)).
Regarding claim 39, claim 1 is obvious as described above. Claim 39 further comprising a third compound comprising a third agent and a third polypeptide, wherein the third polypeptide comprises a third glycosaminoglycan (GAG)-binding peptide which is capable of binding a GAG in a third alpha granule type of a platelet; and wherein the third GAG-binding peptide preferentially binds serglycin,perlecan, dermatan sulfate, keratan sulfate, and/or GPIIb/IIIa.
Toyama-Sorimachi discloses a molecule that can target serglycin:
“The lymphocyte adhesion molecule CD44 recognizes a non-hyaluronate proteoglycan, gp600, secreted by mouse T cell line CTLL2. We now demonstrate that gp600 is identical to serglycin, a member of the small proteoglycan family stored in intracellular secretory granules of lymphoid, myeloid, and some tumor cells. Purified gp600 has the ability to bind specifically to CD44, and the binding is dependent on activation of CD44.” (Toyama-Sorimachi et al., page 7437, Abstract).
Lord et al. discloses that serglycin is present in alpha granules: “Serglycin is an heparin/HS/CS PG that is produced by many different cell types including endothelial cells [191], neutrophils and mast cells [192], and has been shown to be important for binding and packaging proteases into intracytoplasmic secretory granules. It is also present in the α-granules of platelets where it binds and packages platelet factor 4 and the serglycin in mast cells is thought to be the major source of commercial heparin manufactured from porcine intestinal mucosa or bovine lungs.” (Lord et al., page 2501, col. 2, para. 3).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use the targeting molecule of Toyama-Sorimachi to target serglycin in alpha granules as disclosed by Lord to deliver a fluorophore as disclosed by Pazos to arrive at the claimed invention.
A person of ordinary skill in the art would be motivated to include a third polypeptide to target a third GAG with a third fluorophore to further elucidate the locations of these GAGs within the overall structure of platelets.
A person of ordinary skill in the art would have a reasonable expectation of success because Lord discloses that serglycin is associated with alpha granules in platelets and Toyama-Sorimachi discloses a molecule that can target serglycin. Pazos describes the common usage of fluorophores attached to peptide targeting moieties.
Consequently, claim 39 is obvious over Niessen, et al. in view of Loers et al., Pannerden et al., Kawashima et al., Dixon et al., Capila et al., Pazos et al., and Sobel et al. as applied to claim 1, further in view of Lord et al. and Toyama-Sorimachi et al. and rejected.
Free of the Prior Art
Regarding claim 22, no prior art was found that meets the sequence requirements recited by claim 22. Therefore, claim 22 is free of the prior art.
Conclusion
No claim is allowed.
Claims 1-8, 13, 22, 25-28, 33-34, 36, and 38-40 are rejected.
Claims 26 and 33 are objected to.
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/DAVID PAUL BOWLES/ Examiner, Art Unit 1654
/LIANKO G GARYU/Supervisory Patent Examiner, Art Unit 1654
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