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 .
Claim Status
Claims 1-6, 8-14, and 16-32 are currently pending in this application.
Election/Restrictions
Election was made without traverse of Group I, claims 1-17, in the reply filed on Aug. 14, 2025, and claims 18-32 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected subject matter, there being no allowable generic or linking claim. Claims 1-6, 8-14, and 16-17 have been considered on the merits.
Previous Rejections
Status of the rejections: previous rejections pursuant to 112(b), 112(d) and double patenting are withdrawn in view of the claim amendments. Previous rejections pursuant to section 103 is withdrawn in view of the claim amendments except as maintained below.
Claim Interpretation
In claim 1, the “culturing” of the cells is interpreted as occurring in vitro. In the claims, the term “membrane-bound” with regard to “membrane-bound proteins” is interpreted to encompass integral, peripheral, and lipid-anchored proteins. Also in claim 1, the phrase “capable of responding to the one or more membrane-bound proteins” is interpreted as meaning capable of having proliferation be stimulated by the one or more membrane-bound proteins as the response.
In claim 1, the term “artificial antigen presenting cells” is interpreted as any cell, whether naturally occurring (e.g., K562, HeLa, or any cell type of PBMCs) or engineered (e.g., derived from any of the aforementioned), that contains one or more membrane-bound proteins capable of stimulating expansion of NK cells, such as healthy mononuclear cells, cancer cells, or transformed B cells as in claim 16 ([0019], [0029], Example 1). This is contrary to the more common technical term “antigen presenting cell” but yet “artificial antigen presenting cell” or “aAPC” has been used for this meaning occasionally in the prior art (see e.g., Denman et al., PLoS One 7: e30264 (2012) at abstract, Table 1). In this usage, the term “antigen” is not used in strict accordance with its customary meaning of a molecule bound by an antibody or immune receptor (e.g., TCR, BCR, NKG2D), but instead includes natural receptor ligands, like cytokines and other immunostimulatory proteins (e.g., wild-type human IL-1, IL-2, IL-12, IL-15, IL-18, IL-21, 41BBL, etc.), as well as in combination with a lack of MHC I.
Claim 1 recites a gelation solution and second cell suspension optionally containing a photo-initiator termed the “optional photo-initiator” as well as a light applying step requiring the cross-linking of the photo-reactive cross-linker in the third cell suspension, thus claim 1 is interpreted only requiring the photo-initiator as recited when necessary to effectively generate internally gelated cells capable of stimulating NK expansion during the light applying cross-linking step, which in that case would be required not only in both the gelation solution and second cell suspension but also required to enter the antigen presenting cells during the incubating step. In alternative cases, photo-reactive crosslinkers which effectively crosslink during the light applying step without any photo-initiator render the presence of a photo-initiator optional to produce internally gelated antigen presenting cells capable of stimulating NK expansion.
In claim 5, the phrase “performed in the presence of IL-21 or IL-2” is interpreted as meaning the culturing of the population of cells containing NK cells with the population of internally gelated cells is “performed in the presence of IL-21 or IL-2” having access to contact both populations of cells but not met due to the IL-21 and/or IL-2 being the one or more membrane-bound proteins of the fluid cell membrane of the internally gelated cells, as in dependent claim 3.
In claim 17, the term “isolating” or “isolated” is interpreted as meaning subjecting or subjected to some purification process to increase the purity of a cell type(s) in a cell population via a technique known in the art, e.g., using the “NK isolation kit” commercially sold by Miltenyi Biotec referred to in instant Example 6.
Claim Rejections - 35 USC § 112(a), Written Description (modified)
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, 8-14, and 16-17 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 pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
When the claims are analyzed in light of the specification, the instant invention is broadly directed to a method comprising both (1) a method making a population of internally gelated cells and (2) a method of culturing a population of natural killer (NK) cells with a population of internally gelated (i.e., chemically cross-linked) artificial antigen presenting cells (aAPC) capable of stimulating expansion of NK cells, each such cell comprising a fluid cell membrane containing one or more membrane-bound proteins.
Membrane-Bound Protein (presented antigen(s)) Capable of Expanding NK cells
In view of dependent claim 3, all the claims encompasses wherein the membrane-bound proteins are sialic acid, an agonist of TLR-1, or an NLRP3 agonist.
M.P.E.P. §2163 states “To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventors had possession of the claimed invention. See, e.g., Moba, B.V. v. Diamond Automation, Inc., 325 F.3d 1306, 1319, 66 USPQ2d 1429, 1438 (Fed. Cir. 2003); Vas-Cath, Inc. v. Mahurkar, 935 F.2d at 1563, 19 USPQ2d at 1116.”
Firstly, the prior art is silent as to any protein consisting of sialic acid or any membrane protein functioning as a TLR-1 or NLRP3 agonist. Second, there is a lack of evidence in the instant specification as filed that the inventors were in possession of a TLR-1 agonist or NLRP3 agonist that is a membrane-bound protein of the fluid cell membrane of an internally gelated cell.
The written description requirement may be satisfied through actual reduction to practice or by disclosure of relevant identifying characteristics, i.e. structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between structure and function, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of such a broad genus of membrane-bound proteins. In the instant case, the specification fails to provide sufficient descriptive information. Therefore, the skilled artisan cannot envision the membrane-bound protein genus comprising sialic acid, a TLR-1 agonist, or an NLRP3 agonist.
In addition, when analyzing claim 1 in light of the specification, the instant invention is broadly directed to a method using a population of internally gelated cells, each such cell comprising a fluid cell membrane containing one or more membrane-bound proteins capable of stimulating expansion of natural kill (NK) cells, either alone or collectively. Thus, it is emphasized that the scope of the claimed invention of claims 1-2, 5-6, 8-14, and 16-17 encompasses any membrane-bound protein or proteins defined functionally that are capable alone or collectively of stimulating expansion of NK cells.
As this subset of membrane-bound proteins is merely defined functionally, whether any given membrane-bound protein falls within or outside this functional definition is unclear without a standard method of determining this ability either disclosed in the instant application or the prior art. The instant application is silent to any such standard. Rather in instant specification provides representative species of .
The prior art teaches for the human species alone there are over 4,000 unique transmembrane proteins as well as an estimated 20,000 across all mammalian species (Cunha et al., PNAS 106: 16752-7 (2009) at Fig. 1) and, yet, this genus encompasses engineered proteins, e.g., fusions comprising tethered cytokine or immunomodulatory polypeptide sequences, thus making the genus virtually unlimited structurally due to permutations of transmembrane anchors and cytokine antigens (see e.g., Nelson, Oncoimmunology 2: e26619 (2013) at Fig. 1). The prior art teaches that NK cell expansion is stimulated by specific membrane proteins (including IL-15, IL-18, MICA, MICB, ULBP, NKG2D, NKp30, NKp44, NKp46, and DNAM-1) as well as non-membrane, soluble proteins (including IL-2, IL-12, IL-21, and interferons). The prior art teaches engineered transmembrane fusion proteins displayed by cells function as NK stimulating/activating factors, such as a recombinant membrane bound form of IL-21 (see Denman, of record, at pg. 2, left col., Table 1; Imai et al., Blood 106: 376-83 (2005)).
Further as the instant invention is directed to a method of expanding NK cells by culturing NK cells capable of responding to the membrane-bound protein(s), each or collectively capable of stimulating expansion of the NK cells, these functional limitations (in the preamble limiting the method as a whole and limiting the membrane-bound protein(s) and NK cells) represent a circular definition lacking sufficient structural detail in the disclosure when the response is due to a collective effect of a plurality of membrane bound proteins.
While it is clear from the instant specification that the membrane associated proteins recited in claim 3 are included in this genus as claimed and others may be known in the prior art, the skilled artisan cannot envision the full genus of any membrane-bound protein having this ability without more specific written disclosure of structure or other physical and/or chemical properties correlated with this functional limitation of being capable of stimulating expansion of NK cells. Therefore, the skilled artisan cannot envision the full scope of the membrane-bound protein genus limited to those membrane-bound proteins functional capable of stimulating expansion of NK cells in claims 1-2, 5-6, 8-14, and 16-17.
Genus of Engineered Artificial Antigen Presenting Cells
The term “artificial antigen presenting cells” as used in the claims encompasses modified or engineered cells having one or more surface membrane-bound proteins capable of stimulating expansion of at least one type of NK cell thereby meeting the limitation of being “artificial” (instant [0019], [0029], Example 1 and 5, claim 16). However how such cells are “modified” or “engineered” is unlimited without any formal definition in the instant application or as ordinarily used in the prior art. This engineering could mean the expression of the membrane protein or “antigen” is due to a recombinant transgene or mRNA as well as wherein the “antigen” is endogenous but the cell is modified in another manner, e.g., immortalized via c-Myc or SV40 T antigen.
In analyzing whether the written description requirement is met for genus claims, it is first determined whether a representative number of species have been described. In the instant case, the specification describes modified forms of aAPCs that are genetically modified (Example 5, e.g., for forced expression of both 41BBL and membrane-bound IL-15 known in the prior art ) or possibly artificially transformed/immortalized (e.g., via intentional EBV infection at [0019], [0029]; Fujisaki et al., Cancer Res. 69, 4010-7 (2009). The prior art teaches various other genetically engineered aAPCs involving membrane bound recombinant cytokines, such as noted above citing Denman and Imai, of record.
The instant specification fails to provide a single working example wherein the modification is any other than genetically engineering the expression of a membrane tethered cytokine already known to stimulate NK cells by empirical data. To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, or any combination thereof. Here, the broad genus of any artificial antigen presenting cell (i.e., encompassing both engineered cells and non-engineered) lack sufficient guidance as to which of these, or which features/characteristics of these, reasonably and predictably result in stimulating expansion of NK cells, e.g., in vitro after internally gelation. Thus, the skilled artisan cannot envision which, or even how to identify which, “antigen presenting cells” satisfy the claim limitations of claim 1. Therefore, there is a lack of evidence in the instant specification as filed that the inventors were in possession of the entire scope of any instant claim, including claim 16 due to use of undefined and nebulous term “modified.”
As there is no established correlation between all cell types predicted to stimulate NK cell expansion, whether internally gelated or not, the instant specification in view of the prior art fails to provide any additional species beyond those already empirical shown to do so in the prior art. However Applicant is invited to provide evidence to the contrary.
Adequate written description of a method requires more than statements of broad steps recited at a high level of generality yet purporting to achieve specific results from practicing the invention, e.g., stimulating expansion of NK cells or providing a fluid cell membrane containing a membrane bound protein or wherein the membrane-bound protein encompasses any artificial antigen presenting cell, e.g., as recited in claim 3 as having a membrane bound form of 41BBL, IL-15, IL-21, B7-H6, BAT3, HLA-DP, HLA-E, HLA-C2, HLA-A, HLA-C, HLA-G, HLA-F, HLA-C, MICA/MICB, MICA, MICB,ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, AICL, CD48, NTB-A, 2B4, CD2, CD58, CD1la, ICAM1, CRACC, OX40L, CD137L, Nectin-1, Nectin-2, Nectin-3, Nectin- 4, nel-1, necl-2, necl-3, necl-4, necl-5, PCNA, AICL, IgG, CD27L, CD72, CEACAM-1, CEACAM-5, OCIL, N-Cadherin, E-Cadherin, R-CadherinIL-1, IL-2, IL-4, IL-7, IL-9, IL-12, IL-18, IL-27, IL-33, IL-6, IL-li, CNTF, LIF, OSM, CT-1, CLC, IFN-a, INF-b, CCL-5, TLR-2,TLR-3, TLR-5, TLR-6, TLR-9, NOD-1, NOD-2, or NOD-3.
Response to arguments
Applicant’s arguments filed 4/9/26 regarding the previous 112(a) WD rejections have been fully considered but not found persuasive. Applicant traverses arguing the internally gelated aAPC can reasonably be made from virtually any cell type with a cell surface protein antigen known in the art to possess an NK-cell-stimulating capability (pg. 9-10). As noted above, the genus of protein antigen of the internally gelated aAPC is merely functionally defined, which leads to a lack of possession of the extreme breadth of any membrane protein possessing an NK-cell-stimulating capability when there is no clear way to determining this capability nor predict its retention throughout the aAPC internal gelating process (as now recited in detail in independent claim 1).
Applicant’s remarks are silent as to any evidence of a sialic acid that is a protein or any membrane protein which functions as a TLR-1 or NLRP3 agonist. Thus, the instant application is still found to lack sufficient description so as to reasonably convey to one skilled in the relevant art that the inventors, at the time the application was filed, had possession of the full scope of the claimed invention (e.g., wherein the membrane bound protein is sialic acid).
35 USC § 112(a), Scope of Enablement (modified)
Claims 1-6, 8-14, and 16-17 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because while the claims are enabled wherein the membrane-bound protein is selected from those bona fide ones recited in dependent claim 3 (i.e., all except sialic acid, TLR-1 agonist, or NLRP3 agonist), the specification does not enable any person skilled in the art to which it pertains or with which it is most nearly connected to expand an NK cell population with the combination of any internally gelated cells and any membrane-bound protein(s) having undergone the process of the amended language of claim 1.
Enablement is considered in view of the Wands factors (MPEP 2164.01 (a)). The court in Wands states that "Enablement is not precluded by the necessity for some experimentation such as routine screening. However, experimentation needed to practice the invention must not be undue or unreasonable experimentation. The key word is 'undue.' Not 'experimentation;" (Wands, 8 USPQ2d 104). Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. "Whether undue experimentation is needed is not a single, simple factual determination, but rather is a conclusion reached by weighting many factual considerations." (Wands, 8 USPQ2d 1404).
The factors to be considered when determining whether there is sufficient evidence to support a determination that a disclosure does not satisfy the enablement requirement and whether any necessary experimentation required is “undue” or unreasonable 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.
Furthermore, the USPTO does not have laboratory facilities to test if an invention will function as claimed when working examples are not disclosed in the specification. Therefore, enablement issues are raised and discussed based on the state of knowledge pertinent to an art at the time of the invention. And thus, skepticism raised in the enablement rejections are those raised in the art by artisans of expertise.
All of the Wands factors have been considered with regard to the instant claims, with the most relevant factors discussed below.
Nature and Breadth of the invention:
The claims are directed to a method comprising culturing a population of NK cells with a population of internally gelated cells comprising a fluid cell membrane that contains a membrane-bound protein (as an engineered antigen material). Thus, the scope of the claimed invention encompasses any such internally gelated cell type comprising any cell membrane-bound protein(s) that are capable alone or collectively of stimulating expansion of NK cells.
The state of the art:
The prior art teaches NK cells can be expanded in an in vitro cell culture in the absence of any other cell type, gelated cell, or membrane-bound protein (see e.g., Cella et al., Proc Natl Acad Sci 107: 10961-6 (2010) at Fig. 1, 4). The prior art also teaches that NK cell expansion can be improved by including a second cell type, e.g., a prokaryotic pathogen, immune cells secreting pro-inflammatory cytokines, or vertebrate tumor cell lacking MHC I expression engineered to display a membrane bound immunostimulatory protein, such as via K562-based aAPCs (Denman, of record, at pg. 3, right col., Fig. 2-3; Guma et al., Blood 107: 3624-31 (2006) at Fig. 1-2 and 5). However the prior art is silent as to wherein the non-immune cell type is not an infected and/or tumor/immortalized cell.
As the prior art does not teach working examples of methods of stimulating NK cell expansion using internally gelated cells displaying membrane-bound proteins, these aspects must be shown to a reasonable extent so that one of the ordinary skill in the art would be able to practice the invention without any undue burden being on such an artisan, such as to determine which one or membrane bound proteins each or collectively stimulate expansion in combination with which internally gelated cell types, if possible at all. Similarly, because the prior art does not disclose methods using cells not selected from either tumor cells and/or virally-infected cells or hematopoietic stem cell-lineage immune cells, these aspects must be shown to a reasonable extent so that one of the ordinary skills in the art would be able to practice the invention over the full scope of the claims without any undue burden being on such an artisan, such as wherein the internally gelated cell is a prokaryotic cell comprising a cell wall.
The amount of direction and guidance and working examples provided by Applicant:
The instant application provides generic guidance by describing methods of expanding NK cells using any membrane-bound protein(s) capable of stimulating NK cell expansion and any type of cell for the internally gelated cell population as the carrier of the protein(s). However in the working examples, the internally gelated cell type is always the artificial antigen presenting cell (aAPC) K562 genetically engineered to express a membrane bound signals via viral vector (Examples 1-6; FIG. 3, 5, 7).
In all the working embodiments, the NK cell population is either within PBMCs or enriched therefrom, the internally gelated cell (GC) is always a genetically modified APC cell type (irradiated K562-41BBL-mb15 feeder cells), and the membrane bound proteins are always the combination of 4-1BBL (41BBL) and membrane bound IL-15 (mb15) (Examples 1-6; FIG. 3, 5, 7).
Thus, there is no evidence provided in the application that the claimed method could be predictably used to expand any NK cells with any APC and other membrane-bound proteins beyond the combination of 41BBL and mbIL-15, such as wherein the NK cells are primary tissue resident NK cells or within a splenocyte cell population. Instead, the instant specification merely describes prophetic methods without empirical evidence beyond a single working embodiment representing the combination of the limitation of dependent claim 4 and using internally gelated K562 cells expressing both 41BBL and mbIL-15.
The quantity of experimentation needed to make and/or use the invention:
Extensive experimentation would be required to determine how to test and use each membrane-bound protein for the requisite capability either alone or in combination with any other membrane-bound protein or the context of a particular cell type engineered to express said membrane-bound protein The science of NK cell stimulation and internally gelation of any cell has not evolved such that, without guidance or working examples in the specification regarding a nexus between specific membrane-bound protein structures and NK proliferation responses, one can perform the full scope of the claimed method without undue and unreasonable experimentation, which may never be achieved across the entire scope of any internally gelated cell type and possibly infinite combinations of membrane-bound proteins collectively. In particular, extensive experimentation would be required to determine how to use membrane bound proteins not exposed on the cell surface in high quantities/densities of the internally gelated cell or with access to the “antigen” such that the NK cells can contact it and respond. A nexus was demonstrated in the instant application only using 41BBL and mbIL-15; however, applicant is invited to furnish evidence to the contrary, such as teachings in the prior art regarding any of the membrane proteins recited in claim 3, such as by any of the cell types recited in claim 16.
Response to arguments
Applicant’s arguments filed 4/9/26 regarding the previous 112(a) rejections have been fully considered but not found persuasive. Applicant traverses arguing the internally gelated aAPC can reasonably be made from virtually any cell type with a cell surface protein antigen known in the art to possess an NK-cell-stimulating capability and that it reasonably predictable said protein would remain within a fluid cell membrane by avoiding internal cross-linking (“non-interference”) (pg. 9-10). As noted above, the single working embodiment of membrane protein antigen of a single internally gelated aAPC cell type is not representative of the full genus, especially when there is no clear way to determining this NK stimulating capability without experimentation nor predict its retention throughout the aAPC internal gelating process (as now recited in detail in independent claim 1).
Applicant argues that the method is generalizable across different system without undue experimentation without providing any evidence or relationship to the single embodiment of K562 expressing 41BBL and mbIL-15. Such response is mere attorney argument without specific support, e.g., regarding other cell types and membrane bound proteins known in the prior art.
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 1-6, 8, 10-12, 14, and 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Hu (of record) in view of Imai (of record), Lin (of record), Rosen (of record), and Morgan (of record).
Regarding claim 1, Hu teaches methods for producing and using internally gelated (fixed) cells, such as cells having an engineered cell-surface protein, wherein the uses comprise culturing target cells with the internally fixed cells, including wherein the target cells are immune cells for eliciting an immune response (claims 2-4, 10-11, 13, and 18-19; Examples 2-3; pg. 2, lines 7-11; pg. 3, lines 5-18; pg. 18, lines 25-26). Further, Hu teaches that internally gelated aAPC are more stable and less susceptible to damage during storage and have uses in expanding immune cells (pg. 14, lines 31, to pg. 15, line 8; FIG. 5; pg. 8 lines 21-22).
Hu teaches a process of internally fixing artificial antigen-presenting cells (transfected cancer cell, e.g., displaying CD80) to deprive proliferative activity while maintain antigen presentation for stimulating immune responses, (pg. 9, 1st para.) by a method comprising adding a gelation solution to a first cell suspension in a medium to generate a second/third cell suspension containing a photo-reactive crosslinker (pg. 2, line 13 to pg. 3, line 18; e.g., UV activated PEGDA paired with photoinitiator I-2959 at room temperature (Example 3)) that enters the cells, wherein the gelation solution is crosslinked such that membrane permeability of the artificial antigen presenting cells is increased (pg. 9, last para., to pg. 10). More particularly, Hu teaches a method of generating internally gelated cells (vesicle), the method comprising adding a gelation solution which increases membrane permeability (transiently) to a photo-reactive crosslinker (e.g., PEGDA) and allowing entry of the crosslinker into the cells (permeabilization), optionally in the presence of a photo-initiator (e.g., I-2959) and then restoring non-permeability, washing to remove any free crosslinker, activating the crosslinker using light exposure (e.g., UV irradiation), and finally collecting and washing the resulting internally gelated cells (pg. 1, line 29, to pg. 2, line 28; pg. 6, line 2, to pg. 7, line 19; pg. 7, line 30, to pg. 8, line 5; Examples 1 and 4).
Hu does not teach wherein the target cell is an NK cell population and the immune response comprises stimulation of NK cell expansion nor wherein the internal gelating process uses a phenol-red free Dulbecco's modified eagle medium (DMEM) containing a protease inhibitor cocktail, centrifuging the second cell suspension to generate a cell pellet, resuspending the cell pellet in phenol-red free DMEM to generate a third cell suspension for the photo-crosslinking reaction.
However Imai teaches expanding NK cells in an in vitro culture using NK-stimulating cells engineered to display stimulating membrane proteins, more specifically using artificial APC (aAPC) (K562 leukemia cells) expressing membrane bound (mb) IL-15 (IL-15–CD8α) and 4-1BB ligand (CD137) in the presence of IL-2 produces a significant NK cell yield of all NK subsets with more than 96% purity and high expression of activating immune receptors (pg. 378, left col., to right col., Fig. 1-2). Imai also teaches administering the expanded NK cells to patients as an NK cell therapy (pg. 382, last para.).
Additionally Lin teaches methods for making intracellularly gelated cells comprising a washing step after permeabilizing entry of the crosslinker PEG-DA wherein the wash step comprises centrifugation to remove extracellular PEG-DA and photo-initiator to minimize extracellular crosslinking during the UV light irradiation step and preserve the outer membrane by minimizing disruption (pg. 2, left col., last para.). Lin teaches including various protease inhibitors in the gelation buffer (pg. 9, left col., 3rd para.). Rosen teaches culturing NK cells in a cell culture medium comprising DMEM without expressly indicating the presence of any phenol-red ([0223]). Furthermore, Morgan teaches while phenol-red is a commonly used as a visual pH indicator in media, it could also unwantedly function as a crosslinker upon exposure to UV light (Abstract).
It would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to perform a method of Hu of stimulating immune cells in culture wherein the gelated cell type chosen in view of Imai is a tumor cell type already known to stimulate NK expansion in culture and further engineered to have induce superior expansion by expressing two additional stimulatory outer cell membrane proteins in view of Imai, such as specifically using the 4-1BBL+ mbIL-15+ K562 cells already taught by Imai. One of ordinary skill in the art with the goal of increasing NK cell expansion for use in adoptive cell therapies would be motivated use the internal fixation method of Hu to prevent contamination with viable tumor cells while maintaining cell surface immunostimulatory protein(s) as taught by Hu, such as wherein the immunostimulatory protein(s) include 4-1BBL and/or mbIL-15 as already validated by Imai. Furthermore, one of ordinary skill in the art would be motivated to use internally gelated cells for convenience of their mass production and better storage than living cells as taught by Hu as a resource material for use in NK stimulation and activation during NK in vitro culture and/or expansion.
It would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to further perform a method of Hu of stimulating immune cells in culture wherein the gelated cells are K562 tumor cells engineered to express 4-1BBL and/or mbIL-15 as taught by Imai wherein the method of making the gelated K562 cells comprises using a cell culture medium comprising phenol-red free DMEM as taught by Rosen and Morgan, the permeabilization step comprises adding multiple protease inhibitors (cocktail) as taught by Lin, and the washing step comprises centrifuging the crosslinker loaded cell composition to obtain a cell pellet and resuspending the pellet in the same medium. One of ordinary skill in the art with the goal of increasing NK cell expansion for use in adoptive cell therapies would be motivated to use various equivalent media known in the prior art, such as DMEM taught by Rosen, to exclude phenol red as taught by Morgan to avoid uncontrolled bystander crosslinking, to include a cocktail of protease inhibitors as taught by Lin to avoid membrane-bound protein degradation during the permeabilization step, and to use centrifugation pelleting and resuspension to wash the cells as taught by Lin to remove unincorporated (extracellular) crosslinker and photo-initiator prior to proceeding to the photo-crosslinking step.
Regarding claim 2, Imai teaches wherein the NK cells are peripheral blood NK cells and this NK cell population is obtained within a PBMC population (pg. 377, left col., 2nd to last para.).
Regarding claims 3-4, Imai teaches wherein the membrane-bound proteins are IL-15 and 41BBL (id.; Fig. 1, pg. 378, right col., 2nd para.).
Regarding claim 5, Imai teaches the culturing in the presence of IL-2 (pg. 377, left col., 2nd to last para.; pg. 378, right col., 2nd para.).
Regarding claim 6, Imai teaches culturing at a starting ratio of 1:1 NK cells to K562 aAPC cells (Fig. 1).
Regarding claim 8, Hu teaches transiently permeabilize the cell membrane using techniques comprising any one of sonoporation, electroporation, laser, or surfactant-based permeabilization (pg. 6, lines 20-31, pg. 2, lines 25-26, claim 6), all of which are ordinarily performed at osmolality around 300 mOsmol. Note, a prima facie case of obviousness exists where claimed ranges overlap prior art disclosed values or ranges (MPEP 2144.05).
Regarding claim 10, Hu teaches gelation conditions comprising 10, 15, 20, 25, 30, 35, 40, 45 or 50 wt% photo-reactive crosslinker PEG-DA (polyethyleneglycol diacrylate (PEGDA)) or in the range of 1-70 wt% (pg. 2, lines 13-21; FIG. 4), which provides a prima facie case of obviousness because the claimed range overlaps the range disclosed in the prior art (see MPEP 2144.05). Furthermore, Hu teaches using the combination of the photo-reactive crosslinker PEG-DA as above with the photo-initiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I-2959) at 1 wt%, such as wherein the PEG-DA has a molecular weight specifically of 700 Da (pg. 7, lines 16-19; pg. 7, line 30, to pg. 8, line 5; pg. 2, lines 14-24).
Regarding claims 11-12 and 14, Hu does not teach wherein the photoactivation of polymerization uses 365 nm blue light.
However Lin teaches using photoactivating light of a wavelength of 365 nm (blue light) (pg. 2, left col., last para.; Fig. 1b; pg. 9, left col., 3rd para.; Fig. 5) in methods of preparing internally cells also using the combination of the photo-reactive crosslinker PEG-DA with the photo-initiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) at wt% 1 wherein the PEG-DA ranges from 4-40 wt% and the average molecular weight is 700 Da (Mn 700) (pg. 2, left col., last para.; Fig. 1b.; pg. 6, right col.; pg. 10, right col., 3rd para.; Fig. 5; Suppl. Fig. 16), preferably wherein the PEG-DA in the second cell suspension is at 4-20 wt% for products better mimicking of living cells’ outer membrane fluidity and membrane protein mobility (pg. 6, left col., 1st para.; Fig. 3-4 and 6-7; Fig. 2).
It would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to perform a method taught by Hu, Imai, Lin, Rosen, and Morgan of culturing NK immune cells with gelated K562 cells wherein the gelated K562 cells are first prepared by a gelation method using 4-20 wt% PEG-DA having an average molecular weight of 700 Da (e.g., 10-20 wt% PEG-DA) in the gelation solution and 1 wt% of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (photo-initiator) in the gelation solution to generate the second cell suspension and then applying 365 nm blue light to the third cell suspension as taught by Lin. One of ordinary skill in the art would be motivated by Lin demonstrating the effectiveness of using 365 nm blue light for making internally gelated cells that mimic living cells’ outer membrane fluidity, membrane protein mobility, and cell-surface antigen presentation sufficient for immune cell stimulation (Fig. 3-4 and 6-7).
Regarding claim 16, Hu teaches using gelated cells that are artificial antigen presenting cells (pg. 6, lines 13-18; pg. 2, lines 7-12; pg. 8, lines 20-22) or Hela cells (Example 1; FIG. 5). Further, Imai teaches using cells that are K562 cells engineered to express 4-1BBL and mbIL-15 (K562-mb15-41BBL), i.e., a type of artificial antigen presenting cell, and that these artificial antigen presenting cells exhibit improved NK expansion ability compared to either protein alone (pg. 378, left col., to right col., Fig. 1-3). Thus as mentioned above, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to perform a method taught by Hu, Imai, Lin, Rosen, and Morgan of culturing NK immune cells with gelated K562 cells wherein the gelated K562 cells are engineered to express 4-1BBL and/or mbIL-15 as taught by Imai. One of ordinary skill in the art with the goal of increasing NK cell expansion for use in adoptive cell therapies would be motivated to use improved artificial antigen presenting cells already demonstrated in the art by Imai (pg. 378, left col., to right col., Fig. 1-3).
Regarding claim 17, Imai teaches isolating the expanded NK cells from the culture and administering them to subjects as an adoptive NK-cell immunotherapy (pg. 382, last para.).
Claims 1-6, 8-14, and 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Hu in view of Imai, Lin, Rosen, and Morgan as applied above, and further in view of Chan (of record).
Regarding claims 9 and 13, the combination of Hu, Imai, Lin, Rosen, and Morgan does not teach wherein the second cell suspension comprising the photo-reactive crosslinker, antigen presenting cells, and optional photo-initiator also comprises 01.-5 wt% DMSO, such as wherein the light is 365 nm blue light, the crosslinker is specifically PEG-DA having an average molecular weight within the range 200-5000 Da and at 10-40 wt%, and the 0.1-1 wt% photo-initiator is specifically 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) dissolved in DMSO.
However Chan teaches 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959)) is typically dissolved in DMSO as a higher concentration stock solution prior to use in gelation, such as 50 wt% (w/v) (pg. 2063, right col., last para.). This I2959 stock solution is then diluted to 1 wt% when forming a gelation solution (pre-polymer solution) comprising PEGDA used to form “a second cell suspension” comprising diluted I2959, cells, DMEM without phenol red, and 20 wt% PEGDA, such as wherein the PEGDA molecular weight is 700 Da (pg. 2063, right col., last para., to pg. 2064, left col., 1st para.).
It would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to perform a method taught by Hu, Imai, Lin, Rosen, and Morgan of preparing internally gelated K562 aAPC wherein the photo-crosslinker (PEGDA) and photo-initiator (I2959) are incubated with the cells in a suspension comprising no more than 2 wt% DMSO in view of Chan teaching a DMSO stock solution of I2959 at 50 wt% for dilution to use at 1 wt% as taught by Hu or Lin (DMSO dilution of 1:50 or more).
Regarding claim 13, it also would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to perform a method taught by Hu, Imai, Lin, Rosen, and Morgan of preparing internally gelated K562 aAPC wherein the photo-crosslinker is PEG-DA having an average molecular weight of 700 Da and at 4-20 wt% as set forth fully above wherein the photo-initiator I2959 is initially dissolved in a DMSO solution and then this solution is combined with the PEG-DA creating a gelation solution comprising at most 2 wt% DMSO using the stock concentration (50 wt%) as taught by Chan and more than 4-20% PEG-DA for use in forming the second cell suspension with PEG-DA at 4-20 wt% after further dilution. One of ordinary skill in the art would be motivated by convenience to use a high concentration stock solution of the photo-initiator I2959, which is not miscible in aqueous solutions without an organic solvent, in DMSO solvent as taught by Chan, e.g., DMSO at 50 wt% as taught by Chan. In the gelation solution, this would account for a DMSO carryover amount of at most 2 wt% DMSO when using the stock concentration (50 wt%) taught by Chan, e.g., 2 wt% DMSO after a single dilution. Therefore the claimed invention as a whole is prima facie obvious before the effective filing date in the absence of evidence to the contrary.
Response to arguments
Applicant’s arguments filed 4/9/26 regarding the 103 rejections (pg. 13-14) have been fully considered but not found completely persuasive.
Applicant traverses arguing because Imai teaches irradiation that one of ordinary skill in the art would not choose an internally gelation method taught by Hu lacking irradiation. However as set forth above, motivations to use teachings of Hu are provided, including providing surface protein fluidity, preserving natural membrane bilayers, and not disrupting antigen presentation (pg. 9, 1st para.; pg. 10 last para.). Hu notes at pg. 6, lines 27-31, that a skilled person can determine how to temporarily create pores in the membrane without permanent damage, i.e., accomplish a transient poration technique not resulting in protein denaturation.
Applicant traverses arguing unexpected results based on special features of the invention that are not commensurate with the claim limitations, such as avoiding irradiation, freeze-thaw and osmotic shock of the aAPC in the process. Only claim 8 provides the purported unexpected effect might possibly be present but does not ensure it. Furthermore, the claims do not require any minimum level of expansion, rather a culturing step is required and an ability to stimulate expansion.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/ERIC J ROGERS/
Examiner, Art Unit 1638
/KEVIN K HILL/Primary Examiner, Art Unit 1638
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