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 .
Applicant’s election without traverse of group I and the species wherein the first click reagent is azide and the second click reagent is DBCO; a CRISPR associated protein 9 (Cas9), and a stem cell in the reply filed on 8/1/25 is acknowledged.
Claims 14, 15, 24, 25, 38, 39, and 51 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 8/1/25.
Specification
The disclosure is objected to because of the following informalities: There are structures on pages 19 and 26 that are not fully legible.
Appropriate correction is required.
Improper Markush Rejection
Claims 10-13, 17, and 18 are rejected on the judicially-created basis that it contains an improper Markush grouping of alternatives. See In re Harnisch, 631 F.2d 716, 721-22 (CCPA 1980) and Ex parte Hozumi, 3 USPQ2d 1059, 1060 (Bd. Pat. App. & Int. 1984). The improper Markush grouping includes species of the claimed invention that do not share both a substantial structural feature and a common use that flows from the substantial structural feature. The members of the improper Markush grouping do not share a substantial feature and/or a common use that flows from the substantial structural feature for the following reasons:
With regards to the click reagents, the claims recite that the reagent is an azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine, norbornene, and any possible variants thereof. Each of the reagents have different structures and do not have identical activity.
With regards to the agents, the claims recite that the agent is selected from the group consisting of a small molecule, a nucleic acid, a protein or a peptide. The nucleic acid is a siRNA, shRNA, ribozyme RNA, iRNA, sgRNA, or miRNA. The protein or peptide is a transcriptional factor, a growth factor, a cytokine, an antibody, or a gene editing protein or peptide, wherein the gene editing protein or peptide is meganuclease, zinc finger nuclease (ZFN), transcription activator like effector-based nuclease (TALEN), or CRISPR associate protein (Cas), or wherein the protein or peptide is a CRISPR associate protein 9 (Cas9).
Each of these agents has a different structure and activity. None of the agents can be substituted for any of the other agents with expectation of identical activity. None of the agents have the same structure or function.
For example, the term “gRNA’ refers to an RNA that will bind to a Cas protein for the purpose of localizing that protein with a DNA or RNA target. In contrast, siRNAs do not bind to Cas proteins, but instead bind to Argonaute proteins and function to guide those proteins to targets. Accordingly, siRNAs and gRNAs do not have sufficiently similar structures to provide the same function because each has a structure that dictates that it must bind to a different protein effector molecule.
Therefore, the members of the Markush group lack any common substantial structural feature that provides a common use, and the group of alternatives is an improper Markush group. In response to this rejection, Applicant should either amend the claim(s) to recite only individual species or grouping of species that share a substantial structural feature as well as a common use that flows from the substantial structural feature, or present a sufficient showing that the species recited in the alternative of the claims(s) in fact share a substantial structural feature as well as a common use that flows from the substantial structural feature. This is a rejection on the merits and may be appealed to the Board of Patent Appeals and Interferences in accordance with 35 U.S.C. 134 and 37 CFR 41.31(a)(1).
When the Markush grouping is for alternatives of chemical compounds, they shall be regarded as being of a similar nature where the following criteria are fulfilled:
(A) All alternatives have a common property or activity; and
(B) (1) A common structure is present, i.e., a significant structural element is shared by all of the alternatives; or
(B) (2) In cases where the common structure cannot be the unifying criteria, all alternatives belong to a recognized class of chemical compounds in the art to which the invention pertains.
In paragraph (B)(1), above, the words “significant structural element is shared by all of the alternatives” refer to cases where the compounds share a common chemical structure which occupies a large portion of their structures, or in case the compounds have in common only a small portion of their structures, the commonly shared structure constitutes a structurally distinctive portion in view of existing prior art, and the common structure is essential to the common property or activity. The structural element may be a single component or a combination of individual components linked together.
In paragraph (B)(2), above, the words “recognized class of chemical compounds” mean that there is an expectation from the knowledge in the art that members of the class will behave in the same way in the context of the claimed invention. In other words, each member could be substituted one for the other, with the expectation that the same intended result would be achieved.
In order for the members of the Markush group to belong to “recognized class of chemical compounds” there must be an expectation that the members of the class will behave in the same way in the context of the claimed invention. In other words, each member of the class could be substituted one for the other with the expectation that the same intended result would be achieved. In the instant case, activity of any specific recited reagent or agent is dependent upon the specific structure. There is no expectation that any one of the reagents or agents as claimed can be substituted for any of the other with a completely different structure with the expectation of the same activity.
As set forth in MPEP2117, “Note that where a Markush group includes only materials from a recognized scientific class of equivalent materials or from an art-recognized class, "the mere existence of such a group in an application tend[s] to prove the equivalence of its members and when one of them [is] anticipated the group [is] therefore rendered unpatentable, in the absence of some convincing evidence of some degree of non-equivalency of one or more of the remaining members." In re Ruff, 256 F.2d 590, 598-99, 118 USPQ 340, 348 (CCPA 1958)("[A]ctual equivalence is not enough to justify refusal of a patent on one member of a group when another member is in the prior art. The equivalence must be disclosed in the prior art or be obvious within the terms of Section 103." Id. at 599, 118 USPQ at 348).”
In the instant case, art against any one of the instant click reagents or agents would not be evidence against any of the remaining members that have completely different structures, act via different mechanisms, and do not have identical activity.
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 10, 17, and 18 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 10 recites azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine, norbornene, and variants thereof. The specification does not define the term “variant”. The metes and bounds of “variant” cannot be ascertained and therefore the claims are not definite. It is unclear which compounds meet the limitation of being a variant of any one of the recited reagents.
Claim 17 is directed to the method of claim 1, comprising contacting a cell coupled to an azide with the agent coupled to dibenzocyclooctyne (DBCO), thereby delivering the agent to the cell intracellularly, wherein the agent retain its structural integrity, function and/or activity while residing within the cell.
Claim 18 is directed to the method of claim 1, comprising delivering a CRISPR associated protein 9 (Cas9) to a cell intracellularly, comprising contacting a cell coupled to an azide with the Cas9 coupled to dibenzocyclooctyne (DBCO), thereby delivering the Cas9 to the cell intracellularly, wherein the Cas9 retains its structural integrity, function and/or activity while residing within the cell.
Claims 17 and 18 do not recite that the first click reagent is an azide and the second click reagent is dibenzocyclooctyne (DBCO), but rather recite that the method comprises contacting a cell coupled with azide to the agent coupled with dibenzocyclooctyne (DBCO) and therefore it is not clear whether this is an additional limitation in addition to the click reagents or if it is limiting to the click reagents. Therefore, the claims are not definite. For purposes of the instant examination, it is interpreted as limiting each of the click reagents.
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 1-6, 8, 10, 11-13, and 16-21 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.
The claims are directed to a method of delivering an agent to a cell intracellularly, comprising contacting the cell coupled to a first click reagent with the agent coupled to a second click reagent, wherein the second click reagent selectively reacts with the first click reagent coupled to the cell, thereby delivering the agent to the cell intracellularly.
However, the specification does not adequately describe the structure required for an agent to function as a “click” reagent. Without further description of the structure required, one would not be able to readily recognize which agents are necessarily included or excluded from the recited genus and therefore would not be able to recognize that applicant was in possession of the entire genus at the time of filing. The species of the specification are not representative of the entire claimed genus of “click” reagents wherein the second click reagent has the structure to “selectively react” with the first click reagent. The specification does not adequately describe the structure required for each agent in order to result in a selective reaction between the two.
Additionally, the specification does not adequately describe the structure of each combination of first and second click reagents that have the function of delivering any possible agent, as not any click reagent has the function of delivering any possible agent.
For example, Pickens et al. (Bioconjug Chem. 2018 March 21; 29(3): 686–701) teach that some click reagents are only able to deliver agents that are able to be solubilized by a click reagent that is an organic solvent (Table 3); and that using CuAAC as a click reaction may lead to side reactions such as oxidation of amino acids or small molecules, or reduction of cysteines (Table 3).
Pickens et al. teach: As the field continues to emerge, careful attention should be given to key parameters such as solubility and stability of reactants, which are often mismatched in terms of their physiochemical properties. In addition, variations in product solubility, size, charge, and stability should be more systematically investigated, since these features directly affect pharmacokinetics, pharmacodynamics, and safety (page 14).
Pickens et al. teach: When designing bioconjugates that utilize the AAC reaction, the chemical and physical stability of the biomolecule, linker, payload, and any intermediates or catalysts must be taken into consideration and monitored appropriately (page 10).
Therefore, it was known in the art that not any combination of click reagents can deliver any agent. The specification does not adequately describe the structure required for the combinations of click reagents or for the agent to be delivered.
Claims 10 and 25 azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine, norbornene, and variants thereof. The specification does not define the term “variant” and does not adequately describe the structure required for the variant to have the required function. Without further description of the structure required, one would not be able to readily recognize which agents are necessarily included or excluded from the recited genus and therefore would not be able to recognize that applicant was in possession of the entire genus at the time of filing. The speciation does not disclose even a single species of variants of each of the recited reagents.
Even with regards to azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine, and norbornene, the claims do not specify which reagent is the first click reagent, the second click reagent, etc. The specification does not adequately describe if each reagent can be any of these reagents and function as a click reagents together. For example, if both reagents are norbornene, would the system have the structure for the required function?
The MPEP states that for a generic claim, the genus can be adequately described if the disclosure presents a sufficient number of representative species that encompass the genus. See MPEP § 2163. If the genus has a substantial variance, the disclosure must describe a sufficient variety of species to reflect the variation within that genus. See MPEP § 2163. Although the MPEP does not define what constitute a sufficient number of representative species, the courts have indicated what do not constitute a representative number of species to adequately describe a broad genus. In Gostelli, the courts determined that the disclosure of two chemical compounds within a subgenus did not describe that subgenus. In re Gostelli, 872, F.2d at 1012, 10 USPQ2d at 1618. Additionally, in Carnegie Mellon University v. Hoffman-La Roche Inc., Nos. 07-1266, -1267 (Fed. Cir. Sept. 8, 2008), the Federal Circuit affirmed that a claim to a genus described in functional terms was not supported by the specification’s disclosure of species that were not representative of the entire genus. Furthermore, for a broad generic claim, the specification must provide adequate written description to identify the genus of the claim. In Regents of the University of California v. Eli Lilly & Co. the court stated:
"A written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula, [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; In re Smythe, 480 F.2d 1376, 1383, 178 USPQ 279, 284985 (CCPA 1973) ("In other cases, particularly but not necessarily, chemical cases, where there is unpredictability in performance of certain species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus ...") Regents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
The claims are rejected under the written description requirement for failing to disclose adequate species to represent the claimed genus, the genus being agents that function as click reagents and variants thereof that have the required function.
The Guidelines for Examination of Patent Applications under the 35 USC § 112, first paragraph, “Written Description” Requirement”, published at Federal Register, Vol. 66, No. 4, pp. 1099-1111 outline the method of analysis of claims to determine whether adequate written description is present. The first step is to determine what the claim as a whole covers, i.e., discussion of the full scope of the claim. Second, the application should be fully reviewed to understand how applicant provides support for the claimed invention including each element and/or step, i.e., compare the scope of the claim with the scope of the description. Third, determine whether the applicant was in possession of the claimed invention as a whole at the time of filing.
Thus, having analyzed the claims with regard to the Written Description guidelines, it is clear that the specification does not disclose a representative number of species for click agents within the instant enormous genus that function as claimed. Thus, one skilled in the art would be led to conclude that Applicant was not in possession of the claimed invention at the time the application was filed.
Claims 1-6, 8, 10, 11-13, and 16-21 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 a method of contacting human dermal fibroblasts labeled with azide in vitro with DBCO-GFP/Cas9, does not reasonably provide enablement for a method of delivering any agent to a cell intracellularly, comprising contacting the cell coupled to any first click reagent with the agent coupled to any second click reagent, wherein the second click reagent selectively reacts with the first click reagent coupled to the cell, thereby delivering the agent to the cell intracellularly to any cell type. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
Factors to be considered in a determination of lack of enablement include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988)
The instant invention is drawn to a method of delivering any agent to a cell intracellularly, comprising contacting the cell coupled to any first click reagent with the agent coupled to any second click reagent, wherein the second click reagent selectively reacts with the first click reagent coupled to the cell, thereby delivering the agent to the cell intracellularly to any cell type.
The specification demonstrates incubation of human dermal fibroblasts labeled with azide in vitro with DBCO-GFP/Cas9 with a resultant GFP signal in almost all hDFs that were labeled with azide, which is not commensurate in scope with a method of delivering any agent to a cell intracellularly, comprising contacting the cell coupled to any first click reagent with the agent coupled to any second click reagent, wherein the second click reagent selectively reacts with the first click reagent coupled to the cell, thereby delivering the agent to the cell intracellularly to any cell type.
It was known in the art that broad delivery of any agent via any combination of click reagents was unpredictable. The claims are not enabled over the full scope.
For example, Wang et al. (Angew. Chem. Int. Ed. 2016, 55, 5452 –5456) teach: Metabolic sugar labeling followed by the use of reagent-free click chemistry is an established technique for in vitro cell targeting. However, selective metabolic labeling of the target tissues in vivo remains a challenge to overcome, which has prohibited the use of this technique for targeted in vivo applications (abstract).
Wang et al. teach: Our strategy can intrinsically enhance the tumor–organ accumulation ratio of therapeutic agents. Ac4-MB coupled with the use of targeted ultrasound could be a simple but powerful tool for in vivo cancer targeting and targeted cancer therapies (page 5455).
It is noted that Wang et al. teach a system that “could” be a powerful tool, but the instant claims do not require any specific carrier or ultrasound pulses. It is clear from the teachings of Wang et al. that broad systemic delivery of any combination of click reagents with any agent would not predictably result in intracellular delivery of the agent.
Kim et al. (Chem. Sci., 2019, 10, 7835–7851) teaches that: ‘photoclick’ reactions between electron-deficient olefins and diaryltetrazoles are also an attractive tool with special advantages. These reactions can be triggered by light of a particular wavelength, which means that two molecules are conjugated to each other under spatiotemporal control depending on the purpose. Furthermore, the nitrilimine produced by photoexcitation of diaryltetrazole is highly reactive, so that the reaction rate is very fast. A recent study of the Wagenknecht group used this reaction for facile labelling of DNA using cyanine dye showing the utility of the photoclick reaction. However, the wavelength of light used in photoclick reactions is generally in the ultraviolet (UV) range, which is a limitation in biomedical research. It is well-known that UV light is cytotoxic, so it is not suitable for cell studies. In addition, its short wavelength resulted in poor tissue penetration for application in vivo. For these reasons, it has been applied mainly for research outside of cells or animals. If a new photoclick reaction using visible or NIR light is proposed in the future, it will be more useful for biomedical applications (page 7838).
Therefore, Kim et al. is evidence that broad systemic delivery of any combination of click reagents with any agent would not predictably result in intracellular delivery of the agent. Kim et al. teaches the limitations of photoclick reagents, which fall within the instant claim scope.
Kim et al. teach: Artificial chemical reactions in vivo have been challenging for researchers because the environment in a living body has large amounts of different molecules including ions, small chemicals, nucleotides, and proteins. This means that biorthogonality is required for reactions in vivo. Furthermore, the concentration and contact time of click molecules are limited in vivo, and the second order reaction rate constant is also important (page 7842).
The instant claims are not limited to any specific concentration of any specific click reagents with any specific agent that has demonstrated successful in vivo or in vitro delivery intracellularly.
Kim et al. teach: Particularly, it is more delayed in drug delivery and therapy compared to imaging, because the required amount of molecules to be delivered for therapy is generally larger than that for imaging. Therefore, research using click chemistry in vivo for drug delivery and therapy is relatively unexplored, and more information is needed (page 7844).
Kim et al. teach: It is evident that the azide-DBCO reaction is slower than the Tz–TCO reaction. Some researchers have pointed out that it is too slow for pretargeting in vivo.
Kim et al. teach:. In addition, nanoparticles have longer circulation times than small molecules after i.v. injection. Thus, they could provide sufficient contact time among click molecules.
However, the instant claims are directed to naked delivery of any combination of click reagents to deliver any type of agent.
Kim et al. teach: Particularly, for applications in vivo, there is not sufficient time for conjugation in many situations, including i.v. injections. For these cases, the importance of reaction time should be considered in more detail (page 7848).
Kim et al. teach: In 2012, the Weissleder group pointed out that favorable pharmacokinetics and increased circulation time of injected molecules by conjugation with polymers of high molecular weight enhanced the reaction of click chemistry in vivo. After i.v. injection into mice, Tz groups showed a greater than 10-fold increase in the efficiency of click chemistry in vivo when they were attached to a 10 kDa dextran polymer compared to that of direct attachment to a fluorescent dye. Koo et al. have also shown the effects of pharmacokinetics of injected molecules on click chemistry in vivo. They conjugated Tz groups with fluorescent dyes of various chemical structures. In mice, the change of charge or hydrophobicity changed their biodistribution, circulation, and secretion. When they compared the efficiency of click chemistry in vivo using these molecules, they found that the efficiency was highly dependent upon the kinetics in vivo even though all of them contained the same Tz groups. These results suggest that we need to consider all molecules and situations carefully to use click chemistry successfully (page 7848).
Kim et al. teaches: Besides the second order reaction rate constant, the stability of click molecules under special conditions is also important to consider (page 7849).
Additionally, Pickens et al. (Bioconjug Chem. 2018 March 21; 29(3): 686–701) teach that some click reagents are only able to deliver agents that are able to be solubilized by a click reagent that is an organic solvent (Table 3); and that using CuAAC as a click reaction may lead to side reactions such as oxidation of amino acids or small molecules, or reduction of cysteines (Table 3).
Pickens et al. teach: As the field continues to emerge, careful attention should be given to key parameters such as solubility and stability of reactants, which are often mismatched in terms of their physiochemical properties. In addition, variations in product solubility, size, charge, and stability should be more systematically investigated, since these features directly affect pharmacokinetics, pharmacodynamics, and safety (page 14).
Pickens et al. teach: When designing bioconjugates that utilize the AAC reaction, the chemical and physical stability of the biomolecule, linker, payload, and any intermediates or catalysts must be taken into consideration and monitored appropriately (page 10).
Therefore, there are many considerations known in the click chemistry field that need to be considered for each combination of click reagents and the agent to be delivered. There is a large amount of unpredictably in the art for a broad systemic method of delivering any combination of click reagents to deliver any possible agent intracellularly. It was known that
As outlined above, it is well known that there is a high level of unpredictability in the click chemistry art for therapeutic in vivo applications. The scope of the claims in view of the specification as filed together do not reconcile the unpredictability in the art to enable one of skill in the art to make and/or use the claimed invention, namely a broad method of delivering any agent via any combination of click reagents encompassing in vivo effects.
MPEP 2164.01
Any analysis of whether a particular claim is supported by the disclosure in an application requires a determination of whether that disclosure, when filed, contained sufficient information regarding the subject matter of the claims as to enable one skilled in the pertinent art to make and use the claimed invention.
Also, MPEP 2164.01(a)
A conclusion of lack of enablement means that, based on the evidence regarding each of
the above factors, the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed
invention without undue experimentation. In re Wright, 999 F.2d 1557,1562, 27
USPQ2d 1510, 1513 (Fed. Cir. 1993).
Given the teachings of the specification as discussed above, one skilled in the art could not predict a priori whether introduction of any agent in combination with any possible combination of click reagents in vivo by the broadly disclosed methodologies of the instantly claimed invention, would result in successful intracellular delivery. To practice the claimed invention, one of skill in the art would have to de novo determine; the stability of the molecule in vivo, delivery of the molecule to the whole organism, solubility of the molecule, stability of the molecule, specificity to the target tissue in vivo, dosage and toxicity in vivo, and entry of the molecule into the cell in vivo and the effective action therein. Without further guidance, one of skill in the art would have to practice a substantial amount of trial and error experimentation, an amount considered undue and not routine, to practice the instantly claimed invention.
A conclusion of lack of enablement means that, based on the evidence regarding each of the above factors, the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed invention without undue experimentation (see MPEP 2164.01(a)).
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1-3, 6, 8, 10, 17, and 19-21 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Wang et al. (Angew. Chem. Int. Ed. 2016, 55, 5452 –5456).
Wang et al. teach: we report the use of targeted ultrasound pulses to induce the release of tetraacetyl Nazidoacetylmannosamine (Ac4ManAz) from microbubbles (MBs) and its metabolic expression in the cancer area. Ac4ManAz-loaded MBs showed great stability under physiological conditions, but rapidly collapsed in the presence of tumor-localized ultrasound pulses. The released Ac4ManAz from MBs was able to label 4T1 tumor cells with azido groups and significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)-Cy5 by subsequent click chemistry. We demonstrated for the first time that Ac4ManAz-loaded MBs coupled with the use of targeted ultrasound could be a simple but powerful tool for in vivo cancer-selective labeling and targeted cancer therapies (Abstract) (instant claims 10 and 21).
Therefore, Wang et al. teach a method of delivering an agent, Cy5, to a cell comprising contacting the cell coupled to a first click reagent, an azide group, with the agent coupled to a second click reagent, DBCO, thereby delivering the agent to the cell. Wang et al. teach that incorporation of liposomes allowed release of the encapsulated azido sugars through fusion with cellular or intracellular membranes or by lytic degradation in the lysosomes (page 5453) (instant claims 1, 2, 6, and 17).
Wang et al. teach: To understand how the enhanced azido expression in the right tumors would improve the tumor accumulation of DBCO-cargo through the click reaction, in a separate study, DBCO-Cy5 was intravenously (i.v.) injected via the tail vein, and its biodistribution was monitored by in vivo fluorescence imaging. At 24 or 48 h p.i. of DBCO-Cy5, a distinct difference in Cy5 FI between the right tumors treated with 100% US and left tumors treated with4%US was observed (Figures 3a and S7d), while mice injected with blank MB showed negligible difference in Cy5 FI between the right and left tumors (Figures 3a and S7d). Ex vivo fluorescence imaging showed a 1.35-fold Cy5 FI in the right tumor compared to the left tumor in Ac4-MB group (Figure 3b) (page 5453) (instant claims 19 and 20).
Wang et al. teach: The expressed azido groups were able to significantly enhance the tumor accumulation of DBCO-Cy5 through the efficient click reaction (page 5454) (instant claims 1, 2, 6, and 17).
Wang et al. teach: In comparison with the three-injection regimen, mice administered with one i.v. injection of Ac4-MB showed a 1.16-fold accumulation of DBCO-Cy5 in the left tumors with US treatment compared to the right tumors without US treatment (Figure S9), which suggested that azido labeling of tumors and subsequent tumor targeting of DBCO-cargo could (page 5455) (instant claim 3).
It is noted that instant claim 8 recites an outcome rather than a method step. Additionally, intracellular delivery is an outcome from the recited method step of contacting a cell with the instant composition. The outcomes would necessarily flow from the recited method or the enablement of the instantly claimed breadth is in question. Since Wang et al. teaches a method comprising each of the instant method steps, the method would necessarily achieve the recited outcomes, absent evidence to the contrary. As stated in the MPEP (see MPEP 2112), something that is old does not become patentable upon the discovery of a new property.
Therefore, the claims are anticipated by Wang et al.
Claim(s) 1, 2, 6, 8, 10, 17, and 19-21 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Kim et al. (Chem. Sci., 2019, 10, 7835–7851).
Kim et al. teach: Click chemistry in vitro allows specific labelling of cellular target proteins and studying of drug target engagement with drug surrogates in live cells. Furthermore, cellular membrane lipids and proteins could be selectively labelled with click chemistry in vitro and cells could be adhered together using click chemistry. Click chemistry in vivo enables efficient and effective molecular imaging and drug delivery for diagnosis and therapy (abstract).
Kim et al. teaches SPAAC click chemistry utilizing an azide and DBCO (page 7836 and Table 1).
Kim et al. teaches that TCO/Tz mediated cell adhesion has been studied (Fig. 4).73 Cells including A549 human lung cancer, Jurkat T cell lymphoma, NIH3T3 murine fibrobroblasts, and EL4 lymphoma cells were first tagged with an azido group using metabolic glycoengineering with Ac4ManNAz. Subsequently, Tz and TCO were incorporated onto azido-modified sialic acid by Tz-DBCO or TCO-DBCO treatment. After TCO modified Jurkat T cells were added to layers of TCO modified A549 cells in a micro-fluidic setting, it was confirmed that artificial adhesion between A549 and Jurkat T cells was completed within 10 min (page 7840).
Kim et al. teach: In addition, intravenously injected cell pairs were localized to lung tissues. They were trapped in lung capillary beds while maintaining their attachment, even under in vivo conditions (page 7841)
Kim et al. teach: After the generation of azide groups on tumor cells by metabolic glycoengineering, liposomes modified with DBCO groups were injected into mice intravenously. They showed that the accumulation of DBCO modified liposomes was significantly increased by SPAAC in vivo between azide and DBCO (page 7844) (instant claims 10 and 17).
Therefore, Kim et al. teaches a method of delivering an agent, a liposome, to a cell intracellularly, the method comprising contacting the cell coupled to a first click reagent (azide) with the liposome coupled to a second click reagent (DBCO) (instant claims 1, 2, 6, and 19-21).
Kim et al. teaches: In vivo tumor-targeted delivery of photosensitizers and photodynamic therapy by copper-free click chemistry. (A) Azide group labelling of tumor cells by Ac4ManNAz-loaded nanoparticles and second tumor targeting by DBCO-modified nanoparticles containing photosensitizers. Both nanoparticles were injected into mice sequentially, and tumor-targeting was enhanced by SPAAC between azide groups and DBCO (page 7844, Figure 8).
It is noted that instant claim 8 recites an outcome rather than a method step. Additionally, intracellular delivery is an outcome from the recited method step of contacting a cell with the instant composition. The outcomes would necessarily flow from the recited method or the enablement of the instantly claimed breadth is in question. Since Kim et al. teaches a method comprising each of the instant method steps, the method would necessarily achieve the recited outcomes, absent evidence to the contrary. As stated in the MPEP (see MPEP 2112), something that is old does not become patentable upon the discovery of a new property.
Therefore, the claims are anticipated by Kim et al.
Claim(s) 1, 2, 6, 8, 10, 17, and 19-21 is/are rejected under 35 U.S.C. 102(a)(1) or (a)(2) as being anticipated by Kim et al. (US 2013/0251784 A1).
Kim et al. teach: The present disclosure relates to a method for in vivo targeting of a nanoparticle via bioorthogonal copper-free click chemistry, more particularly to a method for in vivo targeting of a nanoparticle, including: injecting a precursor capable of being metabolically engineered in vivo when injected into a living system and having a first bioorthogonal functional group into the living system; and injecting a nanoparticle having a second bioorthogonal functional group which can perform a bioorthogonal copper-free click reaction with the first bioorthogonal functional group attached thereto into the living system. In accordance with the present disclosure, accumulation of nanoparticles at a target site in a living system can be increased remarkably and the biodistribution of the nanoparticles can be controlled since the nanoparticles bound to a cell surface are taken up into the cell with time (abstract) (instant claim 1).
Kim et al. teach: For example, FIG. 1 schematically illustrates a method for in vivo targeting of a nanoparticle by bioorthogonal copper-free click chemistry according to an exemplary embodiment of the present disclosure, wherein tetraacetylated N-azidoacetyl-D-mannosamine is used as the precursor having the first bioorthogonal functional group and a compound with a dibenzylcyclootyne group attached to amine-functionalized polyethylene glycol (PEG)-liposome is used as the nanoparticle having the second bioorthogonal functional group attached thereto. Referring to FIG. 1, the tetraacetylated N-azidoacetyl-D-mannosamine (Ac.sub.4ManNAz) injected into the living system as the precursor having the first bioorthogonal functional group forms an azide group attached to the cell membrane via metabolic glycoengineering in the cell. Then, when the substance with the dibenzylcyclootyne group attached to the amine-functionalized PEG-liposome is injected into the living system as the nanoparticle, copper-free click chemical reaction occurs between the azide group attached to the cell membrane and the dibenzylcyclootyne group. Accordingly, the PEG-liposome bound to the cell membrane does not remain fixed to the cell surface but is transported into the cell via the intrinsic glycan internalization process. Accordingly, the method according to the present disclosure can deliver a specific drug into the cell via the nanoparticle [0032] (instant claims 1 and 10).
Kim et al. teach: The fluorescence intensity of the cells treated with 50 .mu.M Ac.sub.4ManNAz was about 20-fold higher than that of the control cells not treated with Ac.sub.4ManNAz, indicating that the binding of the liposomes was greatly enhanced. The azide groups on the cell surface can be greatly decreased with tris(2-carboxyethyl)phosphine (TCEP) under reducing condition. When the cells were pretreated with TCEP, the amount of the liposomes decreased drastically. This shows that the enhanced binding between DBCO and the azide group results from the chemical reaction. Importantly, the bound liposomes were taken up by the cells without remaining on the cell surface, as shown in the time-lapse images. It may be due to the intrinsic glycan internalization followed by endocytosis of the nanoparticles, which is significant for the intracellular delivery of drugs (see FIGs. 2d-2e) [0065] (instant claims 2 and 17).
Kim et al. teach: Generation of azide groups, binding with nanoparticles and cellular uptake thereof were tested in A549 human lung cancer cells. After administration of Ac.sub.4ManNAz, modified azide groups could be generated on the surface of the A549 cells through metabolic glycoengineering. The correlation between the concentrations of the introduced azide groups and that of Ac.sub.4ManNAz in the cell culture medium could be inferred from Coomassie blue staining and western blot analysis of the cells (see FIG. 2b). When the cells were treated with DBCO-functionalized liposomes (DBCO-lipo), the amount of DBCO-lipo bound to the cell surface increased along with the increasing number of azide groups. This indicates the high reactivity of copper-free click chemistry. Interestingly, the fluorescence intensity was much higher than that of the DBCO-dye conjugate (DBCO-SETA). This may be attributable to the multivalent effect of the nanoparticles since one fluorescent dye in the DBCO-lipo has about 20 DBCO groups [0064] (instant claim 6).
Kim et al. teach: FIGS. 3a-3d show in vivo tumor targeting of DBCO-lipo in a tumor-bearing mouse model (FIG. 3a shows whole-body biodistribution of DBCO-lipo in a Ac.sub.4ManNAz-treated tumor-bearing mouse, FIG. 3b shows z-section images of 50 mM Ac.sub.4ManNAz-treated tumors after intravenous injection of DBCO-lipo, FIG. 3c shows ex vivo fluorescence images and fluorescence intensity of tumors in an Ac.sub.4ManNAz-treated tumor-bearing mouse after intravenous injection of DBCO-lipo, and FIG. 3d shows ex vivo fluorescence images and fluorescence intensity of organs of a 50 mM Ac.sub.4ManNAz-treated mouse after intravenous injection of DBCO-lipo) [0024] (instant claim 6).
It is noted that instant claim 8 recites an outcome rather than a method step. Additionally, intracellular delivery is an outcome from the recited method step of contacting a cell with the instant composition. The outcomes would necessarily flow from the recited method or the enablement of the instantly claimed breadth is in question. Since Kim et al. teaches a method comprising each of the instant method steps, the method would necessarily achieve the recited outcomes, absent evidence to the contrary. As stated in the MPEP (see MPEP 2112), something that is old does not become patentable upon the discovery of a new property.
Therefore, the claims are anticipated by Kim et al.
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.
Claim(s) 1-6, 8, 10, 11-13, and 16-21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (Angew. Chem. Int. Ed. 2016, 55, 5452 –5456), in view of Dudkin et al. (WO 2019/125982 A1), Kim et al. (Chem. Sci., 2019, 10, 7835–7851), Lee (WO 2018/094356 A2), and Hart et al. (WO 2019/166826 A1).
The references are considered to be as enabled as the instant claims.
Wang et al. teach: we report the use of targeted ultrasound pulses to induce the release of tetraacetyl Nazidoacetylmannosamine (Ac4ManAz) from microbubbles (MBs) and its metabolic expression in the cancer area. Ac4ManAz-loaded MBs showed great stability under physiological conditions, but rapidly collapsed in the presence of tumor-localized ultrasound pulses. The released Ac4ManAz from MBs was able to label 4T1 tumor cells with azido groups and significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)-Cy5 by subsequent click chemistry. We demonstrated for the first time that Ac4ManAz-loaded MBs coupled with the use of targeted ultrasound could be a simple but powerful tool for in vivo cancer-selective labeling and targeted cancer therapies (Abstract) (instant claims 10 and 21).
Therefore, Wang et al. teach a method of delivering an agent, Cy5, to a cell comprising contacting the cell coupled to a first click reagent, an azide group, with the agent coupled to a second click reagent, DBCO, thereby delivering the agent to the cell. Wang et al. teach that incorporation of liposomes allowed release of the encapsulated azido sugars through fusion with cellular or intracellular membranes or by lytic degradation in the lysosomes (page 5453) (instant claims 1, 2, 6, and 17).
Wang et al. teach: To understand how the enhanced azido expression in the right tumors would improve the tumor accumulation of DBCO-cargo through the click reaction, in a separate study, DBCO-Cy5 was intravenously (i.v.) injected via the tail vein, and its biodistribution was monitored by in vivo fluorescence imaging. At 24 or 48 h p.i. of DBCO-Cy5, a distinct difference in Cy5 FI between the right tumors treated with 100% US and left tumors treated with4%US was observed (Figures 3a and S7d), while mice injected with blank MB showed negligible difference in Cy5 FI between the right and left tumors (Figures 3a and S7d). Ex vivo fluorescence imaging showed a 1.35-fold Cy5 FI in the right tumor compared to the left tumor in Ac4-MB group (Figure 3b) (page 5453) (instant claims 19 and 20).
Wang et al. teach: The expressed azido groups were able to significantly enhance the tumor accumulation of DBCO-Cy5 through the efficient click reaction (page 5454) (instant claims 1, 2, 6, and 17).
Wang et al. teach: In comparison with the three-injection regimen, mice administered with one i.v. injection of Ac4-MB showed a 1.16-fold accumulation of DBCO-Cy5 in the left tumors with US treatment compared to the right tumors without US treatment (Figure S9), which suggested that azido labeling of tumors and subsequent tumor targeting of DBCO-cargo could (page 5455) (instant claim 3).
It is noted that instant claim 8 recites