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
This application claims priority to application 63/480,434 filed 01/18/2023.
Information Disclosure Statement
The information disclosure statements filed 01/30/2024 have been considered.
Claim Objections
Claims 1, 3, 4, 8, 10, 11, 12, and 16-17 are objected to because of the following informalities:
Claim 1 recites “to control release of a drug into a cytosol of said cancerous cells”. “a cytosol” is awkward. It would be remedial to change this to recite ““to control release of a drug into the cytosol of said cancerous cells”.
Claim 3 recites “moving siRNA (siR)”. It would be remedial to change this to “releasing siRNA” to be consistent with the “releasing and anticancer drug”.
Claim 4 recites “allowing dendritic cells (DCs) to mature” and depends from the method claim 1. In context of claim 4, the method of claim 1 cannot allow a particular thing to happen. It would be remedial to change claim 4 to recites “further comprising inducing maturation of dendritic cells (DCs)”.
Claim 8 recites “wherein the T cells are CD8+ cytotoxic T cells activated with bone marrow dendritic cells (BMDCs)”. The term “with” does not express the intended relationship between the BMDCs and T cells. If it is intended that the BMDCs activate the T cells the it would be remedial to recite “wherein the T cells are CD8+ cytotoxic T cells activated by bone marrow-derived dendritic cells (BMDCs)”.
Claim 10 recites “said distant tumor”. While it is clear that this refers back to “distant/metastatic tumor”, it would be remedial to change “said distant tumor” to “the distant or metastatic tumor”.
Claim 12 recites “wherein the polymers are poly N-isopropylacrylamide copolymerized with butyl acrylate and the change of their ratios for copolymerization”. While it is clear that “the change of their ratios” is referring to the ration of poly N-isopropylacrylamide:butyl acrylate, it would be remedial to change the phrase to recite “wherein the polymers comprise poly N-isopropylacrylamide copolymerized with butyl acrylate having different ratios of poly N-isopropylacrylamide to butyl acrylate, and the change of their ratios for copolymerization yields polymers with different LCSTs”.
Claim 16 recites “green fluorescence protein (GFP)”. The conventional term is green fluorescent protein (GFP). It would be remedial to change the claim to say “green fluorescent protein”.
Claim 16 recites “GFP silencing siRNA loaded inside the CRNPs”. It would be remedial to change the phrase to “CRNPs comprising siRNA that silences expression of GFP” for cleaner way to define the relationships.
Regarding claim 17, there should be a space between “CPT” and “&”. Also “&” should be changed to “and”,
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 1-19 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 1 recites “utilizing a synthesized series of polymers that have lower critical solution temperatures (LCSTs) below positive four degrees Celsius (4 °C).” It is unclear what constitutes the recited “series of polymers” and what relationship the series has to the claimed treatment method. In particular, the claim does not make clear whether multiple different polymers must actually be utilized during performance of the method, whether the “series” refers merely to a plurality of polymers synthesized and screened before selecting a polymer for incorporation into the CRNPs, or what number or relationship among polymers is necessary to constitute the claimed “series.” Further, it is unclear whether each polymer within the recited series must have an LCST below 4 °C. Accordingly, one of ordinary skill in the art would not be reasonably apprised of the metes and bounds of the claimed “utilizing a synthesized series of polymers” limitation.
Claim 1 recites “utilizing a synthesized series of polymers … to control release of a drug into a cytosol of said cancerous cells” and “inducing cold-triggered endo/lysosomal escape of small interfering RNA … into the cytosol.” However, the claim fails to define the relationship between the CRNPs, the recited polymers, the drug, and the siRNA. It is unclear whether the polymers constitute the CRNPs, whether the drug and/or siRNA are carried by the CRNPs, and whether cold-triggered alteration of the CRNPs causes release of both the drug and siRNA. Accordingly, the scope of the method is unclear.
Claim 1 recites “inducing cold-triggered endo/lysosomal escape.” The use of “endo/lysosomal” renders the scope of the limitation unclear because it is uncertain whether the claim requires escape from an endosome, escape from a lysosome, escape from either type of compartment, or escape from both compartments. The specification does not appear to provide an objective definition resolving the alternative meanings encompassed by the slash terminology.
Claim 1 introduces singular “a drug,” whereas claim 2 characterizes “the drug” as comprising plural “chemotherapy and immunotherapy agents.” It is unclear whether the claim requires a single drug or composition comprising both a chemotherapy agent and an immunotherapy agent, multiple drugs comprising respective chemotherapy and immunotherapy agents, or an agent having both chemotherapeutic and immunotherapeutic activity. Accordingly, the metes and bounds of the claimed subject matter cannot be determined with reasonable certainty.
Claim 3 recites “an anticancer drug” and depends from claim 1. Claim 1 recites “a drug”. It is unclear whether “an anticancer drug” is the drug of claim 1.
The term “rapidly” in claim 3 is a relative term which renders the claim indefinite. The term “rapidly]” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The claim does not specify an objective temporal or release-rate boundary by which one of ordinary skill can determine whether a particular release satisfies the limitation. It is therefore unclear how rapidly the anticancer drug must be released to fall within the scope of the claim.
Claim 5 recites “wherein maturation is caused, at least in part, by enhancing production of damage-associated molecular patterns (DAMPs) so as to provoke immunogenic cell death (ICD)”. Thee causal relationship required by this limitation is unclear. The claim does not make clear whether enhanced DAMP production is required to cause ICD, whether ICD causes production or release of DAMPs which thereafter promotes dendritic-cell maturation, or whether enhanced DAMP production independently causes dendritic-cell maturation and ICD. Accordingly, the sequence and causal relationship among ICD, DAMP production, and dendritic-cell maturation required to satisfy the claim cannot be determined with reasonable certainty.
Claim 6 recites “promoting the expression of said DAMPs including HMGB1, CRT, HSP-70, and HSP-90” and does not make clear what constitutes the claimed “expression.” In the context of immunogenic cell death, the recited molecules may undergo different measurable events, including increased cellular expression, cell-surface exposure or translocation, and extracellular release. The claim does not specify whether “promoting the expression” requires increased synthesis or intracellular abundance of the recited molecules, increased cell-surface exposure, extracellular release, or some combination thereof. Accordingly, the scope of the limitation cannot be determined with reasonable certainty.
Claim 9 recites “further comprising circulating the T cells in the blood”. This limitation fails to clearly define the act required to be performed in the claimed method. It is unclear whether “circulating” requires administering the T cells into the bloodstream, inducing endogenous T cells to enter the circulation, maintaining T cells within the circulation, or merely permitting naturally occurring physiological circulation of activated T cells. Accordingly, the metes and bounds of the claimed method cannot be determined with reasonable certainty.
Regarding claim 9, the relative term “rapid” in the recitation “rapid cytotoxicity” fails to provide an objective boundary for determining the scope of the claim. Neither the claim nor, absent an express definition in the specification, the disclosure provides a temporal standard distinguishing “rapid” cytotoxicity from cytotoxicity that is not rapid.
Claim 10 recites “utilizing memory immune cells induced by combining CRNPs with freezing to kill a primary tumor and to destroy a distant/metastatic tumor without freezing said distant tumor.” It is unclear what is required by “combining CRNPs with freezing,” including whether the CRNPs are administered in conjunction with cryosurgery, whether CRNP-containing tumor cells are subjected to freezing, or whether some other relationship is intended. Further, the claim fails to clearly define the causal relationship among freezing, the CRNPs, the memory immune cells, and destruction of the respective tumors. In particular, it is unclear whether the recited memory immune cells are required to kill both the primary tumor and the distant/metastatic tumor, or whether the primary tumor is killed by CRNP-assisted freezing and the resulting memory immune cells thereafter destroy an unfrozen distant/metastatic tumor. Accordingly, the scope of the claimed method cannot be determined with reasonable certainty. The recitation “distant/metastatic tumor” further renders the scope unclear because the slash does not make clear whether the claim requires a distant tumor, a metastatic tumor, or a tumor that is both distant and metastatic.
Claim 12 recites “the polymers are poly N-isopropylacrylamide copolymerized with butyl acrylate and the change of their ratios for copolymerization yields polymers with different LCSTs.” The phrase “their ratios” lacks a clear antecedent and fails to identify the particular ratio being varied. For example, it is unclear whether the limitation refers to a molar ratio, weight ratio, monomer feed ratio, or ratio of monomers incorporated into the resulting copolymer. The claim further fails to specify whether performance of the claimed method requires changing the N-isopropylacrylamide/butyl acrylate ratio and synthesizing multiple polymers having different LCSTs, or whether the language merely characterizes previously synthesized polymers according to the process by which they were made. Accordingly, the scope of the claimed method cannot be determined with reasonable certainty.
Claim 13 recites “co-encapsulating irinotecan (CPT) and ... PD-L1 silencing siRNA ... using a double-emulsion method”. This limitation fails to identify the material or structure in which the two agents are co-encapsulated. Although claim 1 recites CRNPs, claim 13 does not expressly require that CPT and PD-L1-silencing siRNA are co-encapsulated in those CRNPs. Accordingly, the relationship between the co-encapsulated agents and the CRNPs of the claimed method is unclear.
Claim 14 recites “the resultant CPT and siR-laden CRNPs”. This limitation lacks sufficient antecedent basis and renders the scope of the claim unclear. Claim 14 depends directly from claim 1. Claim 1 does not recite CPT, PD-L1-silencing siRNA, co-encapsulation of CPT and siRNA, or resultant CPT and siRNA-laden CRNPs. Those subject matter limitations are introduced in claim 13, from which claim 14 does not depend. Accordingly, it is unclear what previously recited structure constitutes “the resultant CPT and siR-laden CRNPs.”
Claim 16 recites “the green fluorescence protein (GFP) tumor cells,” for which there is insufficient antecedent basis. Claim 1 recites “cancerous cells” but does not establish that the cancerous cells are GFP-expressing tumor cells. Further, the phrase “green fluorescence protein (GFP) tumor cells” fails to clearly define the relationship between GFP and the tumor cells. It is unclear whether the claim requires tumor cells expressing GFP or some other relationship between GFP and the tumor cells. Accordingly, the scope of the limitation cannot be determined with reasonable certainty.
Regarding claim 17, the comparative phrase “than one of the single treatment” is indefinite because the claim does not identify the treatment against which the antitumor immune response must be compared. It is unclear whether the combined treatment must produce a more potent response than cryosurgery alone, CPT alone, PD-L1-silencing siRNA alone, CRNP treatment without cryosurgery, any one of these treatments, or each treatment individually.
Claim 19 recites “further comprising attenuating a frequency of monocytic myeloid-derived suppressor cells (M-MDSCs, CD11b+Ly6C+Ly6G-), polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs, CD11b+Ly6C-Ly6G+), pro-tumorigenic tumor associated macrophages (F4/8Q+CD2Q6+CD86-), regulatory T cells (Tregs, CD4+Foxp3+) that perform immunosuppressive activities in the tumor microenvironment (TME)”. It is unclear if the claim requires all cell types or just one cell type because “and” or “or” is missing before the last cell type.
Those claims identified in the statement of rejection but not explicitly referenced in the rejection are also rejected for depending from a rejected claim but failing to remedy the indefiniteness therein.
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 1-20 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.
For claims drawn to a genus, MPEP § 2163 states 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 applicant was in possession of the claimed genus. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
Claim 1 is directed to a method for engineering an immunologically hot tumor microenvironment using cold-responsive nanomaterials and broadly encompasses the use of a “synthesized series of polymers” having LCSTs below +4 °C to control release of a drug into the cytosol and to induce cold-triggered endo/lysosomal escape of siRNA into the cytosol. Claim 1 does not limit the recited polymers to N-isopropylacrylamide/butyl acrylate (NIPAAm/BA) copolymers, does not specify a particular polymer architecture, does not limit the CRNPs to the particular nanoparticle architecture exemplified in the specification, does not require NaCl or another endo/lysosome-disrupting component, and broadly encompasses a drug, siRNA, cancerous cells, and CRNPs that perform the recited functions.
Independent claim 20 similarly encompasses the broad genus of “cold-responsive nanomaterials capable of targeting cancerous cells” comprising irinotecan and PD-L1-silencing siRNA. Claim 20 does not require NIPAAm/BA, any particular thermoresponsive polymer, any LCST, PLGA, PF127, chitosan-modified PF127, DPPC, NaCl, a particular particle architecture, a particular mechanism of cold responsiveness, or another structural feature identifying which members of the broad nanomaterial genus possess the recited functional characteristics.
The substantive disclosure, however, describes a considerably narrower class of materials. Applicant synthesized a series of closely related pNIPAAm-BA copolymers, varying the NIPAAm:BA ratio, and experimentally determined their LCSTs [0127]. The detailed synthesis describes NIPAAm:BA ratios of 81:19, 80:20, 82:18, and 83:17 rather than representative members of structurally different thermoresponsive-polymer families [0127]. Applicant ultimately selected p(NIPAAm)589-co-(BA)117, having an LCST of −4.4 ± 0.6 °C, because its LCST approximated the temperature at the outer region of the cryosurgical iceball [0101]. Moreover, the CRNP actually made and tested is not merely an unspecified cold-responsive nanomaterial having a low LCST. Applicant's working nanoparticle comprises a particular combination of pNIPAAm-BA, PLGA, PF127, chitosan-modified PF127, DPPC, NaCl, CPT, and siRNA, prepared according to a disclosed double-emulsion procedure. Applicant does not disclose representative examples from structurally distinct classes of low-LCST polymers demonstrating the complete combination of properties required by claim 1. Likewise, Applicant does not disclose representative examples from structurally distinct classes of cold-responsive nanomaterials capable of performing the functions encompassed by claim 20. Instead, the working disclosure is concentrated upon closely related NIPAAm/BA polymers incorporated into the same basic nanoparticle architecture.
Applicant's own experimental evidence further demonstrates that the functional properties defining the claimed genera cannot be attributed merely to the broadly recited property of cold responsiveness. Specifically, Applicant teaches that cold-triggered enhancement of endo/lysosomal escape of siRNA in the exemplified CRNPs results from NaCl encapsulated within the particles[0105]. Applicant reports that when CRNPs without NaCl were subjected to cold treatment, there was no evident separation of the siRNA signal from the endo/lysosomal compartment [0105]. Applicant proposes that cold-triggered release of Na+ and Cl− increases osmotic pressure, resulting in water influx and destabilization or rupture of the endo/lysosome [0105]. Thus, Applicant's own negative-control experiment demonstrates that the recited cold responsiveness does not itself identify a structure capable of providing the cold-triggered cytosolic siRNA-delivery function. A structural/compositional feature important to achieving the disclosed function—NaCl—is absent from claims 1 and 20, and the specification does not identify a broader structural class of components that can predictably substitute for NaCl to provide the disclosed endo/lysosomal-disruption mechanism.
The state of the prior art further supports the conclusion that one of ordinary skill would not have understood Applicant's disclosure of the particular pNIPAAm-BA CRNP species as evidencing possession of the materially broader functional genera claimed. For example, Zhu et al., Biomaterials 162:47–59. 2018) teaches that, notwithstanding advances in nanoparticle delivery systems addressing extracellular barriers, important intracellular barriers following internalization remained, including efficient carrier disassembly and endosomal escape [abstract]. Zhu therefore developed a specialized pH- and redox-responsive polyplex incorporating particular structural features directed to carrier disassembly and endosomal escape. Zhu therefore supports a finding that cytosolic siRNA delivery and endosomal escape were not functions that one of ordinary skill would have understood to follow generally from nanoparticle internalization or stimulus responsiveness. Rather, the carrier required appropriate structural characteristics capable of addressing these intracellular barriers. Zhu consequently does not supply a known general structure-function relationship from which the skilled artisan could extrapolate Applicant's particular cold-responsive CRNP to the unrestricted genus encompassed by claim 1. Similarly, Yin (Yin et al. Journal of Colloid and Interface Science 578 (2020) 685–697) demonstrates that thermoresponsive behavior is sensitive to polymer composition and architecture even within the comparatively narrow NIPAM/BA chemical family [abstract]. Yin reports a transition temperature of approximately 9.62 °C for a statistical copolymer containing about 80 wt.% NIPAM and 20 wt.% BA and demonstrates materially different transition behavior for related polymer architectures [pg. 690, col. 1, para 2]. Thus, the art does not establish that merely selecting NIPAM and BA, much less selecting an arbitrary thermoresponsive polymer, predictably produces the particular sub-4 °C transition behavior required by claim 1. These teachings are consistent with Applicant's own experimental approach of synthesizing a series of NIPAAm/BA polymers, empirically measuring LCST, and selecting the particular species having an LCST of −4.4 ± 0.6 °C.
Accordingly, the specification neither discloses a representative number of species commensurate with the structural diversity encompassed by the claimed genera nor identifies a common structural characteristic correlated with the complete set of claimed functions that would permit one of ordinary skill to recognize the boundaries and members of those genera. The disclosed NIPAAm/BA species may reasonably demonstrate possession of a substantially narrower genus centered upon Applicant's disclosed pNIPAAm-BA CRNP technology, but they do not demonstrate possession of the substantially broader functional polymer/CRNP genus encompassed by claim 1 or the unrestricted cold-responsive-nanomaterial genus encompassed by claim 20. The specification contains generic language corresponding to the breadth of the pending claims. However, mere recitation of the desired functional result does not necessarily establish possession of the full functionally defined genus where the substantive disclosure does not provide representative species or identifying structural characteristics sufficient to demonstrate possession thereof.
Therefore, one of ordinary skill reviewing the specification as filed would reasonably recognize Applicant as having possessed the specifically disclosed pNIPAAm-BA-based CRNP platform and reasonably related variations thereof, but would not recognize Applicant as having possessed the full structurally diverse genera encompassed by claims 1 and 20.
Claims 2-19 are rejected for the same reasons by virtue of their dependency from claim 1. The additional limitations of the respective dependent claims are acknowledged as narrowing individual aspects of the genus but do not provide the structural limitations necessary to restrict the claims to the genus shown to have been possessed.
Claims 1-20 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 the particular pNIPAAm589-BA117 polymer incorporated into PLGA/PF127/chitosan-modified PF127/DPPC/NaCl CRNP and co-encapsulating CPT and PD-L1 siRNA into the cold-responsive nanoparticle system, does not reasonably provide enablement for the full scope of the claimed invention. 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 use the invention commensurate in scope with these claims.
• Nature of the Invention
Claim 1 encompasses a synthesized series of polymers generally, provided that the polymers have LCSTs below +4 °C and operate in a CRNP system that successfully targets cancerous cells during cryosurgery, controls drug release into the cytosol, and induces cold-triggered endo/lysosomal escape of siRNA into the cytosol. Claim 20 similarly encompasses structurally diverse cold-responsive nanomaterials provided that the nanomaterials are capable of targeting cancerous cells and comprise co-encapsulated CPT and PD-L1-silencing siRNA.
• Breadth of the claims
Claim 1 does not limit the polymer chemistry, CRNP architecture, drug, siRNA target, endosomal-escape component or mechanism, or cancer type. Claim 20 does not limit the polymer or nanomaterial chemistry, LCST, nanoparticle architecture, cold-response mechanism, targeting mechanism, or other structural characteristics of the claimed CRNPs.
The breadth of these claims therefore requires substantial extrapolation beyond the specific working embodiment.
• State of the Art
Zhu et al., Biomaterials 162:47–59. 2018) teaches that, notwithstanding advances in nanoparticle delivery systems addressing extracellular barriers, important intracellular barriers following internalization remained, including efficient carrier disassembly and endosomal escape [abstract]. Zhu therefore developed a specialized pH- and redox-responsive polyplex incorporating particular structural features directed to carrier disassembly and endosomal escape. Zhu therefore supports a finding that cytosolic siRNA delivery and endosomal escape were not functions that one of ordinary skill would have understood to follow generally from nanoparticle internalization or stimulus responsiveness. Rather, the carrier required appropriate structural characteristics capable of addressing these intracellular barriers. Zhu consequently does not supply a known general structure-function relationship from which the skilled artisan could extrapolate Applicant's particular cold-responsive CRNP to the unrestricted genus encompassed by claim 1. Similarly, Yin (Yin et al. Journal of Colloid and Interface Science 578 (2020) 685–697) demonstrates that thermoresponsive behavior is sensitive to polymer composition and architecture even within the comparatively narrow NIPAM/BA chemical family [abstract]. Yin reports a transition temperature of approximately 9.62 °C for a statistical copolymer containing about 80 wt.% NIPAM and 20 wt.% BA and demonstrates materially different transition behavior for related polymer architectures [pg. 690, col. 1, para 2]. Thus, the art does not establish that merely selecting NIPAM and BA, much less selecting an arbitrary thermoresponsive polymer, predictably produces the particular sub-4 °C transition behavior required by claim 1. These teachings are consistent with Applicant's own experimental approach of synthesizing a series of NIPAAm/BA polymers, empirically measuring LCST, and selecting the particular species having an LCST of −4.4 ± 0.6 °C.
• Guidance from the Specification
The specification demonstrates the sensitivity of the system to formulation variables. The specification teaches the synthesis of multiple NIPAAm/BA compositions, experimentally determined their LCSTs, and selected the p(NIPAAm)589-co-(BA)117 species having the desired −4.4 ± 0.6 °C LCST [0101]. The specification also teaches optimized CPT loading [0102]. The specification reports changes in encapsulation efficiency with CPT feed concentration and a plateau in CPT loading, after which a particular feed concentration was selected for further experiments [0102]. This demonstrates that therapeutic cargo incorporation itself is formulation-dependent rather than necessarily interchangeable across the claimed genus. Most significantly, the specification demonstrate that the cold-triggered endo/lysosomal escape required by claim 1 does not follow merely from use of a cold-responsive nanoparticle. The specification teaches that NaCl encapsulated in the CRNP is responsible for enhancement of endo/lysosomal escape [0105]. When CRNPs lacking NaCl were subjected to cold treatment, separation of siRNA from the endo/lysosomal compartment was not observed [0105]. The successful escape in the NaCl-containing particles was attributed to cold-triggered release of Na+ and Cl−, increased osmotic pressure, water influx, and destabilization or rupture of the endo/lysosomal compartment [0105]. This negative control is particularly probative because it demonstrates that a nanoparticle closely related to the successful embodiment can fail an essential claim-1 function when a particular component is removed. Nevertheless, claim 1 does not require NaCl or identify a class of alternative structures that will predictably provide cold-triggered endo/lysosomal escape. The specification experimentally demonstrates that this specific pNIPAAm-BA platform undergoes cold-triggered nanoparticle disassembly and CPT/siRNA release; provides NaCl-dependent cold-triggered endo/lysosomal escape; achieves GFP and PD-L1 gene silencing; produces cancer-cell killing and immunogenic-cell-death-associated effects; promotes BMDC maturation and CD8+ T-cell responses; and produces local and systemic antitumor immune effects when employed with the disclosed cryosurgical treatment. The specification does not provide a general structural rule that would allow one of ordinary skill, without substantial experimentation, to determine which structurally different polymer classes can be engineered to have the claimed LCST while retaining suitable nanoparticle-forming properties. Nor does the specification provide a general rule for determining which structurally different cold-responsive nanomaterials can co-encapsulate CPT and functional PD-L1 siRNA while providing suitable stability, cancer-cell targeting, cold responsiveness, and cargo release. Likewise, the specification does not provide a general rule for determining which alternative CRNP architectures will provide the cold-triggered endo/lysosomal escape required by claim 1 or which components may replace the demonstrated NaCl-dependent osmotic mechanism. Although the specification generally identifies additional drugs that could potentially be employed, including paclitaxel and doxorubicin, the disclosure does not provide corresponding working examples establishing that substitution of those drugs into the claimed cold-responsive system preserves the relevant loading, particle stability, release kinetics, intracellular behavior, and biological response.
• Experimentation Required
Accordingly, to practice the full scope of claim 1, one of ordinary skill selecting a materially different polymer or CRNP would need to synthesize candidate polymers and/or nanoparticle formulations and determine experimentally, among other things, whether the candidate has the required temperature-responsive behavior; forms a suitable nanoparticle; encapsulates and protects the selected cargo; remains sufficiently stable; undergoes the desired cold-triggered structural change; releases the drug and siRNA under appropriate conditions; permits cancer-cell uptake; achieves endo/lysosomal escape; preserves siRNA activity; produces target-gene silencing; and ultimately provides the claimed therapeutic and immunological function.
Similarly, to practice the full scope of claim 20, the skilled artisan would need to identify materially different classes of cold-responsive nanomaterials and experimentally determine whether each candidate can successfully co-encapsulate CPT and PD-L1-silencing siRNA, maintain cargo stability and activity, target cancerous cells, and provide the required cold-responsive behavior.
The required experimentation therefore would not merely confirm predictable properties of species already taught by the specification. Instead, the skilled artisan would be required to synthesize or formulate candidate members and screen those candidates to discover which members actually possess the combination of functional characteristics defining the claims.
Here, the specification does not identify a general structural quality running throughout the entire claimed polymer/CRNP genus that predicts the complete combination of functions. To the contrary, Applicant's own LCST screening, CPT-loading optimization, and NaCl-negative-control experiments demonstrate that important properties must be determined empirically.
Considering the Wands factors as a whole, the breadth of claims 1 and 20, the multivariable nature of the claimed technology, the formulation-dependent behavior demonstrated by Applicant's own experiments, the lack of predictability demonstrated by the prior art, the concentration of Applicant's guidance and working examples upon a particular pNIPAAm-BA CRNP architecture, and the quantity of synthesis, formulation, and functional screening required to identify operable species outside that disclosed platform collectively establish that the specification does not enable the full scope of claims 1 and 20 without undue experimentation.
Claim 2 is further rejected under 35 U.S.C. 112(a) for lack of enablement because the specification does not enable the full scope of the recited genus of “chemotherapy and immunotherapy agents.” Although the specification identifies certain therapeutic agents that may be employed, the working examples are directed principally to irinotecan/CPT. Applicant's own experiments demonstrate that incorporation of CPT required empirical optimization of drug feed concentration, encapsulation efficiency, and loading. The specification does not provide a general formulation principle by which therapeutic agents having materially different physicochemical properties can be substituted into the CRNP while predictably retaining nanoparticle formation, encapsulation, cold-triggered release, intracellular delivery, and therapeutic activity. Accordingly, practicing the full therapeutic-agent genus would require empirical formulation and testing to determine which agents operate successfully in the claimed system.
Although claims 2-19 further limit the claimed method, the claims do not narrow the method enough to cure the particular scope defect as discussed above.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-4, 7, 12-16 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Wang (Wang et al. Biomaterials 180 (2018): 265-278) in view of Lee (Lee et al. Journal of Controlled Release 125.1 (2008): 25-32), Tang (Tang et al. European Journal of Pharmaceutical Sciences 127 (2019) 161–174) and Yin (Yin et al. Journal of Colloid and Interface Science 578 (2020) 685–697).
Regarding claim 1-3 and 20, Wang teaches the development of a cold-responsive nanoparticle for controlled drug release (regarding claim 3) as a result of the irreversible disassembly of the nanoparticle when cooled to below ~10°C for use in a cancer treatment method [abstract, section 3.3]. Wang explains that cryosurgery (also called cryotherapy and cryoablation), the destruction of diseased tissues by first cooling/freezing and then warming back, has been used to treat various diseases including cancer in the clinic [abstract]. Wang explains that cryosurgery generates an iceball having a substantial temperature gradient and that tumor cells near the peripheral iceball region, where the temperature is approximately 0 °C, may survive conventional cryosurgery [pg. 265, col. 2, para 2]. Wang therefore identifies a need for cold-responsive nanoparticles capable of cold-triggered drug release to improve the efficacy of cryosurgery against tissue in this inadequately treated cold-temperature region [pg. 266, col. 1, para 1]. Wang teaches that a thermoresponsive polymer becomes hydrophilic below its LCST and causes disassembly of the nanoparticle, such that, drug release can be triggered by the cold-induced disassembly of the nanoparticle [pg. 266, col. 1, para 2]. Wang further teaches targeting cancerous cells with the cold-responsive nanoparticles [see section 3.3]. Wang decorates the nanoparticle surface with hyaluronic acid because CD44 is overexpressed on many cancer cells and cancer stem-like cells, thereby providing targeted delivery of theranostic agents to cancer cells and CSCs [[pg. 266, col. 1, para 3]. Wang experimentally examines targeted nanoparticle delivery to MDA-MB-231 breast cancer cells and mammospheres and observes intracellular uptake of encapsulated irinotecan (CPT) [pg. 272, col. 1]. More particularly, Wang prepares the cold-responsive nanoparticles from poly(N-isopropylacrylamide-co-butylacrylate) (PNIPAM-B) together with PF127, chitosan-modified PF127, and HA using a double-emulsion approach [pg. 268, col. 1, para 4]. Wang teaches that polymers are hydrophobic at temperatures higher than their LCST, while they have high solubility in water at temperatures lower than their LCST; and that if the LCST of the thermoresponsive polymer is lower than room temperature, this polymer can be used as a hydrophobic polymer to prepare nanoparticles at room temperature; however once the temperature is lower than the LCST, the thermo-responsive polymer becomes hydrophilic (i.e., highly soluble in water) and can cause disassembly of the nanoparticles in aqueous solutions [pg. 266, col. 1, para 2]. Wang's PNIPAM-B has a NIPAM:BA ratio of 8:1 and an LCST of approximately 14–16°C, which Wang expressly notes is lower than that of unmodified PNIPAM pg. 268, col. 1, para 4]. Wang encapsulates hydrophobic irinotecan/CPT (i.e., chemotherapy agent) in the PNIPAM-B-containing hydrophobic phase pg. 268, col. 2, para 1]. Wang reports that more than 80% of the encapsulated CPT can be released by the cells after five minutes of ice cooling [pg. 271, col. 1, para 2]. Wang is also particularly pertinent to the claimed endo/lysosomal aspect. Wang observes multiple cold-responsive nanoparticles within endo/lysosomes following cellular uptake and reports that, after ice cooling, almost all of the nanoparticles disappeared/disassembled within those endo/lysosomes, demonstrating that the cold-responsive behavior is retained after intracellular uptake [Fig. 4; pg. 273, col. 1, para 1]. Thus, Wang teaches or strongly suggests cancer-targeting CRNPs, use of PNIPAM-BA thermoresponsive polymers, a cryosurgical/cold-treatment setting, intracellular cold-triggered drug release, and cold-triggered nanoparticle disassembly within endo/lysosomes.
Wang does not, however, expressly teach (i) carrying siRNA that undergoes cold-triggered endosomal escape into the cytosol, (ii) use of the system specifically to engineer an immunologically hot TME for cancer immunotherapy, or (iii) PNIPAM-BA polymers having LCSTs below 4°C. Wang does reports an LCST of 14–16°C.
Lee teaches thermally sensitive nanocapsules specifically adapted for intracellular siRNA delivery [abstract]. Lee explains that siRNA-mediated RNA interference occurs in the cytosol and that endocytosed siRNA should escape the endosomal compartment before trafficking to lysosomes and degradation [pg. 25, col. 1-col. , para 1]. Lee loads siRNA onto thermally responsive nanocapsules, allows cellular uptake at 37°C, and applies cold shock to cause abrupt expansion of nanocapsules trapped within endosomes, to induce an abrupt volume expansion of the NCs within an endosome compartment to physically burst out the endosomal membrane [abstract]. Lee's confocal experiments show that, before cold treatment, fluorescent siRNA-containing nanocapsules co-localize with endosome/lysosome compartments. After cold treatment, the fluorescent siRNA complexes no longer co-localize with the acidic vesicles and instead appear throughout the cytosolic region (regarding claim 3) [section 3.4-3.5; Figs. 4-5]. Lee attributes the result to cold-triggered physical destabilization of the endosomal compartment and delivering the siRNA-PEG conjugate into the cytoplasm [section 3.4-3.5; Figs. 4-5]. Lee further demonstrates functional gene silencing following the cold-triggered escape: GFP expression decreased from 68.1% without cold shock to 37.3% after cold shock in cells receiving the siRNA-loaded thermoresponsive nanocapsules [section 3.5].
Regarding claim 16, Lee teaches that GFP siRNA-PEG/PEI25K complexes were transfected to GFP expressing MDA-MB-435-GFP cells, the level of GFP expression decreased slightly from 46.9±0.3% to 42.2±3.6% after the cold shock treatment [pg. 30, col. 1].
Tang teaches combining nanoparticle-mediated chemotherapy, paclitaxel (PTX), with PD-L1-silencing siRNA (siP) to overcome tumor-mediated immunosuppression and promote antitumor T-cell immunity abstract]. Tang explains that tumor PD-L1 suppresses T-cell activation and that blocking PD-1/PD-L1 interactions in tumors can restore immune activity of the tumor microenvironment [pg. 161, col 2, para 2]. Tang co-delivers PD-L1 siRNA and paclitaxel in the same hybrid micelle (HM) and demonstrates simultaneous delivery to cancer cells, PD-L1 knockdown, and synergistic suppression of tumor growth through chemotherapy together with cytotoxic T-cell immunity [abstract]. Tang teaches that both CD8+ CTLs/Tregs ratios and CD4+ Teffs/Tregs ratios were greatly enhanced in HM-PTX/siP treatment which indicate that HM-PTX/siP could reconstruct tumor immune microenvironment and enhance tumor immunotherapy [Fig. 6C; section 3.8]. Tang teaches that the chemoimmunotherapy scheme teaches that PTX chemotherapy induces immunogenic cell death and calreticulin exposure; dendritic cells (DCs) process the resulting tumor antigen and activate tumor-specific cytotoxic T lymphocytes; PD-L1 siRNA relieves checkpoint-mediated immunosuppression; and the chemotherapeutic agent also suppresses Treg activity, which suppressed primary tumors [see conclusion; Scheme 1; pg. 162, col. 1, para 2].
Regarding claim 4, Tang teaches that HM-siP and HM-PTX exhibited a remarkable increase in the number of both CD80+ and CD86+ DCs compared, and HM-PTX/siP showed greater expression of DC maturation markers (CD80 and CD86) compared to other groups, which might be the joint action of PTX and siP [pg. 171, col. 1, para 1; Fig. 5].
Regarding claim 7, Tang teaches that PTX and siP could significantly enhance the secretion of IFN-γ (a multifunctional cytokine that was primarily secreted by activated T) by lymphocytes, and the combination of PTX and siP could enhance the secretion of IFN-γ, and increase the number of CD8+ T and CD4+ T cells [pg. 168; pg. 166, col. 1, para 2].
Yin teaches that thermosensitive systems, obtained by dissolving a polymer in aqueous solutions, have been extensively studied and present a characteristic transition temperature called cloud point temperature (Tc) at which the interactions between the polymer chains and the aqueous media dramatically changed which results in the collapse or expansion of polymer chains [pg. 686, col. 1, para 1]. Yin teaches that if a change from a two-phase to a single-phase system is observed when the temperature is increased, an upper critical solution temperature (UCST) is defined; and in the opposite case, a lower critical solution temperature can be defined (LCST) [pg. 686, col. 1, para 1 Yin expressly synthesizes three statistical P(BA-s-NIPAM) polymers having BA:NIPAM weight ratios of 0.05:0.95, 0.1:0.9, and 0.2:0.8 to investigate the effect of composition on thermoresponsive properties, thus, Yin supplies the claimed concept of a synthesized series in which the NIPAM/BA ratio is varied [pg. 687, col 2, para 3-6]. Yin teaches the direction of the relationship, explaining that “insertion of a hydrophobic moiety significantly decreases the level of hydration of polymer chain and thus decreases the cloud point temperature.” Yin experimentally reports that, at 80 wt.% PNIPAM, the statistical copolymer has a cloud-point temperature of 9.62 °C, versus 25.13°C and 27.60°C for corresponding triblock and diblock structures [pg. 690, col. 1, para 2].
Regarding claim 1 and 20, it would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to modify Wang's cancer-targeting PNIPAM-BA cold-responsive nanoparticle system, comprising the HCPN-CG nanoparticles by (a) incorporating therapeutic siRNA into the HCPN-CG nanoparticles and using cold-triggered endosomal escape according to Lee because Wang already demonstrates that its CRNPs are internalized into endo/lysosomes and disassemble there when subjected to cold treatment, Lee establishes that thermally responsive nanoparticles carrying siRNA can exploit a cold-induced structural change to disrupt the endosomal compartment and release siRNA into the cytosol and the modification would have predictably addressed the known requirement that siRNA reach the cytosol to exert RNA interference; (b) selecting PD-L1-silencing siRNA (i.e., immunotherapy) and the therapeutic siRNA together with the CPT chemotherapy according to Tang to reduce tumor immunosuppression and enhance cytotoxic T-cell immunity as Tang teaches that chemotherapy-induced tumor cell death generates antigenic/immunogenic stimulation, while PD-L1 silencing removes checkpoint-mediated inhibition of the resulting T-cell response making the expected result be an immunologically more active tumor microenvironment (i.e., hot TME); and (c) optimizing the NIPAM/BA composition and microstructure according to Yin to lower the transition temperature into the desired cryosurgical range of below 4°C because Wang identifies the transition temperature as controlling nanoparticle disassembly and release, Wang identifies the approximately 0°C peripheral cryosurgical region as the therapeutic region needing improved tumor killing, and Yin teaches that increasing the hydrophobic BA component and selecting statistical architecture substantially depresses the transition temperature. The combination of prior art elements according to known methods to yield predictable results supports can support a conclusion of obviousness. See MPEP 2143(I). This combination merely applies known cold-responsive intracellular delivery, known cold-triggered siRNA endosomal escape, known chemo/checkpoint-silencing immunotherapy, and known NIPAM/BA transition-temperature tuning to Wang's expressly disclosed cold-responsive cancer/cryosurgery platform, with each component performing its established function.
Regarding claim 12, Wang teaches poly(N-isopropylacrylamide-co-butylacrylate) [see section 3.1] and Yin synthesizes different copolymers at different temperatures and concentrations in aqueous solution in relation with their composition and microstructure [pg. 687, col. 2, para 5].
Regarding claim 13, Wang teaches the incorporation of CPT into PNIPAM-B nanoparticles using the double-emulsion method [Fig. 1; section 2.3 and 3.1]
Regarding claim 14, Wang teaches that the PPG of PF127-chitosan is integrated into the hydrophobic shell formed during the first emulsion while HA and chitosan-PEG stabilize and
decorate the surface of the resultant nanoparticles through the electrostatic interactions between HA (negatively charged) and chitosan (positively charged) during the second emulsion [pg. 268, col. 2, para 2].
Regarding claim 15, Wang teaches that the concentration of CPT was varied from 1 to 100 mg/ml for testing the cytotoxicity in vitro; therefore, it would have been routine optimization to use 10ug/ml of CPT in the method of Wang [pg. 276, col. 1, para 1].
Claims 1 and 11 are rejected under 35 U.S.C. 103 as being unpatentable over Wang (Wang et al. Biomaterials 180 (2018): 265-278) in view of Lee (Lee et al. Journal of Controlled Release 125.1 (2008): 25-32), Tang (Tang et al. European Journal of Pharmaceutical Sciences 127 (2019) 161–174) and Yin (Yin et al. Journal of Colloid and Interface Science 578 (2020) 685–697) as applied to claim 1 and further in view of Zhang (Zhang et al. International Journal of Heat and Mass Transfer 165 (2021) 120663).
The teachings of Wang, Lee, Tang, and Yin are discussed above as applied to claim 1 and similarly apply to claims 1 and 11.
Wang, Lee, Tang, and Yin do not teach wherein the lower critical solution temperatures (LCSTs) are below negative four degrees Celsius (-4°C) (i.e., below positive four degrees Celsius ( 4 °C)).
Zhang teaches the development of a new combined treatment of chemotherapy and cryotherapy (chemo-cryo) by injecting cold-responsive drug-loaded nanoparticles around the tumor before cryosurgery to enhance the treatment of deep-lying tumor because the tumors in the peripheral region near the iceball surface (0°C) may not be completely killed [abstract, pg. 1, col. 1, para 1]. Zhang teaches the establishment of a macroscopic mathematical model, covering the cryosurgery, the release of anti-tumor drug (doxorubicin) from cold-responsive nanoparticles, and the drug transport in biological tissues [abstract]. Zhang teaches that the nanoparticles are 100 nm in diameter with spherical morphology, and their component, polymer, is hydrophobic [pg. 6, col. 1, para 2]. Zhang teaches that once the temperature is lower than the lower critical solution temperature (LCST), the polymer becomes water-soluble, which causes the disassembly of drug-loaded nanoparticles, namely, drug release [pg. 6, col. 1, para 2]. Zhang illustrates drug-loaded nanoparticles and doxorubicin concentrations, and the effective killing volumes of tumor and normal tissue in the treatments using drug-carrier with the different sensitive temperatures of 268.15, 263.15, 258.15, 253.15 and 243.15 K (all less than -4°C) [pg. 6, col. 1, para 2; Fig. 5]. Thereby, Zhang evidences that the temperature at which the nanoparticles respond was recognized as a treatment-design parameter. Zhang teaches that the amount of drug released depends upon the cryoprobe holding time, nanoparticle sensitive temperature, and nanoparticle concentration, and that adjustment of nanoparticles can augment tumor killing [pg. 6; Fig. 5].
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to modify the method as taught and suggested by Wang, Lee, Tang, and Yin by using use lower critical solution temperatures (LCSTs) that are below negative four degrees Celsius (-4 °C). One of ordinary skill would be motivated to use lower temperature for the purpose of optimizing the transition temperature of the drug-loaded nanoparticles toward the temperature range encountered in the cryosurgical peripheral region according to Yin and Zhang.
Claims 5, 8-10, and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Wang (Wang et al. Biomaterials 180 (2018): 265-278) in view of Lee (Lee et al. Journal of Controlled Release 125.1 (2008): 25-32), Tang (Tang et al. European Journal of Pharmaceutical Sciences 127 (2019) 161–174) and Yin (Yin et al. Journal of Colloid and Interface Science 578 (2020) 685–697) as applied to claims 1, 4 and 7 and further in view of Yakkala (Yakkala et al. Frontiers in immunology 10 (2019): 2283) and Udagawa (Udagawa et al. Clinical Cancer Research 12.24 (2006): 7465-7475).
The teachings of Wang, Lee, Tang, and Yin are discussed above as applied to claim 1 and similarly apply to claims 5, 8-10, and 17.
Yakkala teaches that cryoablation is particularly suited to generating antitumor immune responses because freezing causes tumor cell death while leaving tumor material in situ, thereby exposing tumor antigens to the immune system. Yakkala explains that freezing the tumor locally and allowing it to remain in situ unleashes an array of tumor antigens to be exposed to the immune system, paving the way for the generation of anti-tumor immune responses, while recognizing that cryoablation alone often provides an insufficient systemic response and therefore teaching combination of cryoablation with immunotherapy [abstract].
Regarding claim 5, Wang, Lee, Tang, and Yin do not teach that DC maturation is caused by enhancing production of damage-associated molecular patterns (DAMPs) so as to provoke immunogenic cell death (ICD). Tang does teach that PTX induced ICD and CRT exposure, which was phagocytosed by DC cells and presented to unactivated T cells, and activated T cells into tumor-specific cytotoxic T lymphocytes (CTL) to kill tumor cells resulted in ICD. Yakkala teaches that cells in the core of the ablation zone die by necrosis as a result of osmotic shock or physical damage from ice crystals, thereby releasing their intracellular contents into the extracellular space, triggering an active immune response [pg. 4, col. 1, para 2]. Yakkala further teaches that the balance between immunogenic necrosis and immune-tolerant apoptosis determines the course of the immune response induced by cryoablation [pg. 4, col. 1, para 2]. Yakkala teaches that Toll-like receptors on dendritic cells, recognize … danger-associated molecular patterns (DAMPs) derived from damaged and necrotic cells, and that signaling through these pathways results in production of inflammatory mediators and initiation of an immune response [pg. 4, col. 2, para 3]. Yakkala further teaches that administration of a TLR agonist together with cryoablation results in dendritic-cell maturation by teaching that the analysis of tumor-draining lymph nodes demonstrated DC maturation with an increased expression of CD80 and CD86 co-stimulatory markers [pg. 4, col. 2, para 4]. Figure 1 of Yakkala further illustrates that antigens released from necrotic cells are taken up by dendritic cells and induce co-stimulatory signals that result in generation of antitumoral T-cell responses and effector T cells, whereas apoptotic material lacking appropriate co-stimulation may induce immune tolerance. Figure 2 of Yakkala teaches that the effector T cells that will migrate to the cryoablated tumor site encountering the tumor cells and DCs presenting tumor antigens on their surface MHC molecules where introduction of checkpoint inhibitors (for example, anti-CTLA-4 and anti-PD-1 (siP)) will allow the T-cells to execute tumor cell killing without being inhibited by the checkpoint signaling. Accordingly, it would have been obvious to one of ordinary skill in the art to carry out the cryosurgery/immunotherapy method rendered obvious by Wang, Lee, Tang, and Yin under conditions that favor immunogenic tumor-cell injury and generation/exposure of DAMPs, as taught by Yakkala, because Yakkala and Tang teaches that cryoablation-induced immunogenic necrosis releases intracellular danger signals, that DAMPs activate innate immune pathways, and that the resulting immune stimulation promotes dendritic-cell maturation and effector T-cell responses that aid in killing of the tumor cell. One of ordinary skill would have been motivated to favor this immunogenic response rather than immunologically tolerogenic apoptosis in order to enhance the antitumor immune response sought by the combined method.
Regarding claim 8, Wang, Lee, Tang, and Yin do not teach wherein the T cells are CD8+ cytotoxic T cells activated with bone marrow dendritic cells (BMDCs). Yakkala teaches that DCs are capable of presenting tumor antigens and activating anti-tumor T-cell responses and teaches that DCs are efficient in inducing the differentiation of CD8+ T-cells to cytotoxic T-cells (CTLs) by a well-described mechanism [pg. 5, col 1, para 2]. Yakkala teaches that generation of tumor-specific CTLs is the goal of most cancer immunotherapies [pg. 5, col 1, para 2]. Yakkala teaches that tumor cryoablation followed by intra-tumoral DC transfer exhibited excellent improvements in the overall survival and resistance to re-challenge known as cross-presentation and the systemic antitumor immunity that conferred protection abrogated by the depletion of CD8+ T-cells [pg. 5, col 1, para 4]. Udagawa teaches the generation of dendritic cells from bone marrow [pg. 7466, col. 1, para 4-5]. Udagawa teaches combining the bone marrow dendritic cells (BMDCs) with cryoblation [pg. 7466, col. 2, para 3]. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use BMDCs as taught by Udagawa as the DCs for activating CD8+ cytotoxic T-cell response as taught by Yakkala in the method taught and suggested by Wang, Lee, Tang, and Yin. One of ordinary skill would be motivated to make the modification for the advantage of exhibiting excellent improvements in the overall survival and resistance to re-challenge. Udagawa demonstrates that BMDCs were a known and successfully employed source of dendritic cells for immunotherapy in conjunction with tumor cryoablation, while Yakkala teaches the known function of such dendritic cells in cross-presenting tumor antigen and inducing differentiation of CD8+ T cells into cytotoxic T cells. One of ordinary skill therefore would have had reason to employ BMDCs to activate tumor-specific CD8+ CTLs in the claimed cryo-immunotherapy method, with a reasonable expectation of obtaining the known antitumor cytotoxic T-cell response.
Regarding claim 9, Wang, Lee, Tang, and Yin do not teach the method further comprising circulating the T cells in the blood so as to exert direct and rapid cytotoxicity against any existing tumors. Yakkala teaches that cryoablation generates a systemic tumor-specific T-cell response capable of attacking tumors outside the cryoablated site [Fig. 2]. Yakkala reports that, following cryoablation of mammary tumors, tumor rejection was immune-mediated and that T cells obtained from tumor-draining lymph nodes of cryoablated animals reduced lung metastases after adoptive transfer into tumor-bearing recipients, producing approximately threefold fewer lung metastases than T cells obtained from surgically treated animals [pg. 4, col. 1, para 3]. Yakkala's Figure 2 expressly illustrates systemic dissemination of activated effector T cells following cryo-immunotherapy. The Figure 2 legend teaches that mature dendritic cells present tumor antigens to naïve T cells in the tumor-draining lymph node, resulting in enhanced activation and differentiation into effector T-cells [Fig. 2]. The resulting effector T cells will migrate to the cryoablated tumor site, where they encounter tumor cells, and, following checkpoint inhibition, the effector T cells execute tumor-cell killing [Fig. 2]. Yakkala further teaches that these effector T cells will also migrate to the distant metastasized tumor sites, leading to the regression of metastases [Fig. 2]. Yakkala additionally expressly teaches that the locally initiated antitumor immune response can “disseminate systemically to regress the distant untreated tumors [pg. 8, paragraph bridging cols. 1-2]. Thus, It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that the activated cytotoxic T cells generated according to the method as taught and suggested by Wang, Lee, Tang, and Yin would circulate systemically and traffic through the blood to primary and metastatic tumor sites, where the activated effector T cells would directly exert cytotoxic activity against existing tumor cells. This is the ordinary physiological mechanism expressly depicted and described by Yakkala for producing a systemic antitumor response from a locally cryoablated tumor.
Regarding claim 10, Wang, Lee, Tang, and Yin do not teach the method further comprising utilizing memory immune cells induced by combining CRNPs with freezing to kill a primary tumor and to destroy a distant/metastatic tumor without freezing said distant tumor. Yakkala teaches that cryoablation produces durable, tumor-specific protective immunity. In a mammary-tumor model, Yakkala reports that 84% of cryoablated mice resisted tumor rechallenge compared with only 14% of surgically treated mice, and expressly notes that the resulting protection was tumor-specific because the surviving animals remained susceptible to rechallenge with an unrelated tumor cell line [pg. 4, col. 1, para 3]. Yakkala further teaches that protection against subsequent tumor rechallenge in B16-OVA tumor-bearing mice and explains that leaving the cryoablated tumor in situ makes tumor antigens available for generation of an antitumor immune response [pg. 4, col. 1, para 4]. These findings teach a durable tumor-specific immunological response consistent with antitumor immune memory.
Yakkala additionally teaches systemic effects against untreated distant tumors by teaching that patients undergoing cryoablation of hepatic tumors exhibited necrosis of distant untreated tumors accompanied by systemic Th1 immune responses [pg. 4, col. 2, para 1], and teaching that a colon-cancer model treated with cryoablation plus stimulated dendritic cells exhibited regression of distant untreated tumors mediated by tumor-specific systemic immunity dependent upon CD8+ T cells [pg. 5, col. 1, para 4]. Moreover, Yakkala expressly teaches the contemplated abscopal mechanism: locally initiated antitumor immune responses disseminate systemically to regress distant untreated tumors, and patients treated at a primary tumor with cryoablation and local immune-checkpoint inhibition showed regression of distant metastatic tumors [pg. 8, bridging paragraph of cols. 1-2; pg. 9, col. 2, para 2]. It therefore would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to utilize the durable, tumor-specific immune response produced by the cryo-immunotherapy method as taught and suggested by Wang, Lee, Tang, and Yin to attack not only the cryoablated primary tumor but also distant or metastatic tumors that themselves were not subjected to freezing. Yakkala provides both a reasonable expectation of systemic antitumor activity against untreated disease and evidence of durable tumor-specific protection upon tumor rechallenge. Such use would have predictably exploited the systemic and memory components of the antitumor immune response to obtain an abscopal therapeutic effect.
Regarding claim 17, Wang, Lee, Tang, and Yin do not teach the method further comprising combining cryosurgery with CPT & PD-L1 silencing siRNA CRNPs to induce a more potent antitumor immune response than one of the single treatment. As discussed above, Tang teaches combining chemotherapy with PD-L1-silencing siRNA to enhance antitumor T-cell immunity, while the prior combination renders obvious employing those agents in the cold-responsive nanoparticle/cryosurgery platform. Yakkala provides an express motivation and reasonable expectation that combining cryoablation with checkpoint-directed immunotherapy would produce a stronger antitumor response than either treatment alone [pg. 4, col. 2, para 2; Fig. 1]. Yakkala explains that tumor cryoablation can trigger a tumor-specific protective immune response, but that the magnitude and sustainability of this immune response may not be adequate to protect against tumor rechallenge or cause regression of distant metastases[pg. 4, col. 2, para 2]. Yakkala therefore concludes that synergy between cryoablation and immunotherapy provides an opportunity to reverse immunosuppression and treat metastatic cancer [pg. 4, col. 2, para 2]. More significantly, Yakkala teaches that in a murine prostate cancer model, all mice bearing secondary tumors died when treated with either CTLA-4 blockade alone or cryoablation alone, whereas 44% survived when the therapies were combined [pg. 6, col. 2, para 3]. Yakkala teaches that in a B16-OVA melanoma model, CTLA-4 blockade combined with cryoablation protected 80% of mice following tumor rechallenge, compared with 40% for cryoablation alone [pg. 6, col. 2, para 3]. Yakkala teaches that these pre-clinical studies unambiguously exhibit a superior therapeutic outcome when cryoablation is applied along with checkpoint inhibitors [pg. 6, col. 2, para 3], describing the comparative outcomes of the combination and monotherapies. Yakkala also identifies ongoing clinical approaches combining cryoablation with PD-1/PD-L1-directed immune checkpoint therapies, including trials of cryoablation with nivolumab, pembrolizumab, and other checkpoint inhibitors [Table 2]. Yakkala expressly teaches that checkpoint inhibitors including anti-PD-L1 and anti-PD-1 combined with cryoablation appear particularly appealing [pg. 8, col. 1, para 2]. Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the CPT and PD-L1-silencing-siRNA CRNP treatment rendered obvious by Wang, Lee, Tang, and Yin with cryosurgery, with a reasonable expectation that the combined treatment would induce a more potent antitumor immune response than cryosurgery or immunotherapy alone because Tang teaches that PD-L1 silencing relieves PD-1/PD-L1-mediated immunosuppression, while Yakkala expressly teaches that immune-checkpoint inhibition potentiates the immune response generated by cryoablation and demonstrates superior antitumor outcomes from the combination relative to individual treatment modalities.
One of ordinary skill would have had reason to combine Yakkala's teachings with the cold-responsive chemo-immunotherapy system of Wang, Lee, Tang, and Yin because Yakkala expressly identifies the limitation of cryoablation alone: although cryoablation generates tumor antigens and tumor-specific immune responses, those responses frequently are insufficient to eradicate systemic disease. Yakkala therefore teaches combining cryoablation with immunotherapy to increase dendritic-cell activation, generate cytotoxic effector T cells, overcome tumor-mediated immune suppression, generate durable tumor-specific protection, and obtain systemic effects against distant untreated tumors. The proposed combination would therefore have amounted to applying Yakkala's established cryo-immunologic principles to the cold-responsive nanoparticle chemo-immunotherapy platform of the primary references, with each element performing its known function and with a reasonable expectation of enhancing both local tumor destruction and systemic antitumor immunity.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Wang (Wang et al. Biomaterials 180 (2018): 265-278) in view of Lee (Lee et al. Journal of Controlled Release 125.1 (2008): 25-32), Tang (Tang et al. European Journal of Pharmaceutical Sciences 127 (2019) 161–174) and Yin (Yin et al. Journal of Colloid and Interface Science 578 (2020) 685–697) and Yakkala (Yakkala et al. Frontiers in immunology 10 (2019): 2283) as applied to claims 1 and 5 and further in view of and Fucikova (Fucikova et al. International journal of cancer 135.5 (2014): 1165-1177).
The teachings of Wang, Lee, Tang, Yin, and Yakkala are discussed above as applied to claims 1 and 5 and similarly apply to claim 6.
Wang, Lee, Tang, Yin, and Yakkala do not teach where the method further comprises promoting the expression of said DAMPs including HMGB1, CRT, HSP-70, and HSP-90. Fucikova expressly identifies the particular DAMPs associated with immunogenic tumor-cell death. Fucikova states that molecular events characteristic of ICD include surface exposure of calreticulin (CRT), the heat shock proteins HSP70 and HSP90, and the release of high-mobility group box protein 1 (HMGB1), as well as ATP release [abstract; pg. 1, col. 2, para 2]. Fucikova further experimentally demonstrates this complete DAMP phenotype in treated human tumor cells. Fucikova teaches that high hydrostatic pressure (HHP) treatment in tumor cells caused rapid cell-surface expression of HSP70, HSP90, and CRT and also caused release of HMGB1 and ATP [abstract]. Importantly, Fucikova does not merely identify these proteins as arbitrary biomarkers. Fucikova teaches that the interaction of dendritic cells with the treated tumor cells resulted in increased dendritic-cell phagocytosis, upregulation of the dendritic-cell maturation markers CD83, CD86, and HLA-DR, and secretion of IL-6, IL-12p70, and TNF-α; and induced high numbers of tumor-specific T cells and relatively few regulatory T cells [abstract]. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to promote and/or monitor the DAMP-associated immunogenic-cell-death phenotype taught by Fucikova, including HMGB1 release and surface exposure of CRT, HSP70, and HSP90, when carrying out the cryo-immunotherapy method as taught and suggested by Wang, Lee, Tang, Yin, and Yakkala. One of ordinary skill would have been motivated to do so because Yakkala teaches that the therapeutic immune response generated by cryoablation depends upon favoring immunogenic tumor-cell injury and release of danger signals rather than immune-tolerant apoptosis, and Fucikova identifies the particular DAMP phenotype known to characterize effective ICD and demonstrates that this phenotype promotes dendritic-cell maturation and tumor-specific T-cell immunity. Thus, selecting treatment conditions that enhance the generation, release, and/or surface exposure of HMGB1, CRT, HSP70, and HSP90 would have constituted the predictable use of known ICD-associated danger signals to obtain the already desired result of enhanced dendritic-cell activation and antitumor T-cell immunity in the cryo-immunotherapy method as taught and suggested above.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Wang (Wang et al. Biomaterials 180 (2018): 265-278) in view of Lee (Lee et al. Journal of Controlled Release 125.1 (2008): 25-32), Tang (Tang et al. European Journal of Pharmaceutical Sciences 127 (2019) 161–174) and Yin (Yin et al. Journal of Colloid and Interface Science 578 (2020) 685–697) as applied to claim 1 and further in view of and Rubinsky (US005674218A, 10/7/1997).
The teachings of Wang, Lee, Tang, and Yin are discussed above as applied to claim 1 and similarly apply to claim 18.
Wang, Lee, Tang, and Yin do not teach where the method further comprises manipulating a killing temperature of the cancerous cells by turning a cryoprobe on and off intermittently. Rubinsky teaches a cryosurgical system for destroying living tissue, including malignant or benign solid tumors, using one or more cryoprobes and further teaches controlling the operating temperature and freezing zone of the cryoprobes during cryosurgery. Rubinsky teaches that the surgeon may adjust the freezing-zone length of the cryoprobe in response to images obtained before or during cryosurgery [col.1, lines 10-30]. Rubinsky teaches controlling the cryoprobe about a selected temperature setpoint by intermittent on/off operation. Rubinsky teaches that the setpoint temperature may differ somewhat from the displayed temperature when the temperature is being controlled by the on-off switching of a valve [col. 32, lines 1-17]. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to operate the cryoprobe used in the cryosurgical method as taught and suggested by Wang, Lee, Tang, and Yin according to the intermittent/on-off temperature-control teachings of Rubinsky. One of ordinary skill would have been motivated to do so in order to regulate the cryoprobe about a desired killing temperature and control the extent of the frozen region, rather than permit uncontrolled continued cooling and enlargement of the iceball. Such operation would have amounted to use of a known cryoprobe temperature-control technique for its established purpose of controlling temperature and the extent of freezing during tumor cryosurgery. The modification also would have been particularly appropriate to the claimed method because the cold-responsive therapeutic system depends upon the temperature reached within the tumor. Controlling cryoprobe operation about a selected temperature therefore would predictably permit the practitioner to manipulate the tumor temperature and frozen region while carrying out the claimed cold-triggered therapeutic release.
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Wang (Wang et al. Biomaterials 180 (2018): 265-278) in view of Lee (Lee et al. Journal of Controlled Release 125.1 (2008): 25-32), Tang (Tang et al. European Journal of Pharmaceutical Sciences 127 (2019) 161–174) and Yin (Yin et al. Journal of Colloid and Interface Science 578 (2020) 685–697) as applied to claim 1 and further in view of and Cai (Tang and Cai et al. International Journal of Nanomedicine (2021): 5811-5829).
The teachings of Wang, Lee, Tang, and Yin are discussed above as applied to claim 1 and similarly apply to claim 19.
Wang, Lee, Tang, and Yin do not teach where the method further comprises attenuating a frequency of monocytic myeloid-derived suppressor cells (M-MDSCs, CD11b+Ly6C+Ly6G-), polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs, CD11b+Ly6C-Ly6G+), pro-tumorigenic tumor associated macrophages (F4/8Q+CD2Q6+CD86-), regulatory T cells (Tregs, CD4+Foxp3+) that perform immunosuppressive activities in the tumor microenvironment (TME). Cai teaches that the tumor microenvironment possesses immunosuppressive characteristics that interfere with effective antitumor immunity and expressly teaches nanoparticle-mediated remodeling of the TME from an immunosuppressive state to an immunostimulatory state. Cai identifies: “MDSCs, M2 TAMs, and Tregs” as three principal types of immunosuppressive cells in the tumor microenvironment [pg. 5823, col. 1, para 4]. Cai teaches that these immunosuppressive populations deactivate immunostimulatory cells and compromise tumor immunotherapy and therefore teaches that their depletion, downregulation or phenotype reversion can enhance tumor immunogenicity and circumvent immunosuppression in the TME [pg. 5823, col. 1, para 4]. Cai further teaches that MDSCs comprise two principal populations, monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs), and expressly teaches that both populations contribute to an immunosuppressive tumor microenvironment [pg. 5823, col. 2, para 1]. Cai teaches that MDSCs, M2 TAMs, and Tregs exhibit immunosuppressive properties; thus, it is essential to circumvent their immunosuppressive effects through nanotechnology in cancer therapy [pg. 5820, col. 1, para 2]. Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention seeking to convert the immunosuppressive TME of the method as taught and suggested by Wang, Lee, Tang, and Yin into an immunologically active TME to attenuate the recognized immunosuppressive populations comprising M-MDSCs, PMN-MDSCs, M2 TAMs, and Tregs. One of ordinary skill would be motivated because these populations were known to suppress effective antitumor immunity, and their depletion, downregulation, inhibition, or phenotypic reversion was known to enhance the immunogenicity of tumors and improve cancer immunotherapy. It therefore would have been obvious to carry out the chemo/cryo-immunotherapy treatment of the claimed method under conditions effective to reduce these immunosuppressive cell populations, because the desired result of the method is conversion of the TME from an immunosuppressive state to an immunologically active state, and the prior art expressly identifies attenuation of MDSCs—including M-MDSCs and PMN-MDSCs—M2 TAMs, and Tregs as known means of accomplishing that objective. With respect to the claimed F4/80+CD206+CD86− phenotype, the recited markers characterize the M2-type TAM population whose attenuation is being measured. The obviousness does not depend upon the marker nomenclature itself producing a new biological population; rather, the prior art supplies the reason to attenuate the underlying immunosuppressive M2 TAM population as part of remodeling the TME toward an immunostimulatory state.
Conclusion
No claims allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to TIFFANY N GROOMS whose telephone number is (571)272-3771. The examiner can normally be reached M-F 830-530.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Jennifer Dunston can be reached at 571-272-2916. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/TIFFANY NICOLE GROOMS/Examiner, Art Unit 1637
Prosecution Insights