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 1-32 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Election was made without traverse of Group I, claims 1-17, in the reply filed on Aug. 14, 2025, and claims 18-32 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected subject matter, there being no allowable generic or linking claim.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 1-17 have been considered on the merits.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Objections
Claim 9 is objected to because of the following informalities:
Claim 9 recites the abbreviation “DMSO”, which is later spelled out as dimethyl sulfoxide in claim 13. Appropriate correction is request such that the first instance of the abbreviation in the claim set is spelled out.
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 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 7, the term “antigen presenting cells” is interpreted contrary to the standard prior art meaning (in view of claim 16 and instant pg. 4, lines 1-5; pg. 5, lines 16-20) as any cell (e.g., any PBMCs) containing 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. In the field of immunology, antigen presenting cells are typically those presenting T cell antigenic peptides bound to major histocompatibility complex (MHC) proteins, such as certain cancer cells, virus-infected cells, macrophages, dendritic cells, and B cells. Certain cancer cells lack this MHC I and/or MHC II feature, including HeLa cells and 1106mel cells, as well as certain white blood cell lines, including 721.221 cells and K562 cancer cells (see e.g., Imai et al., Blood 106: 376-83 (2005) at pg. 378, left col., 2nd para.; Denman et al., PLoS One 7: e30264 (2012) at pg. 7, left col.). More particularly in the subfield of expanding immune cells using immunostimulatory feeder cells, the term antigen presenting cells is used to refer to tumor cell line cells, that although having tumor-associated cell surface antigens, are not necessarily “antigen presenting” via any MHC. Thus, not all these cell types included are typically considered “antigen presenting cells” by one of ordinary skill in the art of immunology, in particular because claim 16 recites the “antigen presenting cells are or are engineered from K562 cells, PBMC, . . . 1106mel cells.”
Claim 7 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 7 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 the claims, the modifier “artificial” regarding the term “antigen present cells” is interpreted as meaning the antigen present cells are intentionally modified or engineered in some way compared to naturally occurring antigen presenting cells, such as via immortalization and/or genetic engineering as are the transduced K562 “aAPC” in instant Example 1 (see also instant pg. 4, lines 1-5; pg. 5, lines 16-20).
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
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 1-17 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 culturing a population of NK cells with a population of internally gelated cells, each such cell comprising a fluid cell membrane containing one or more membrane-bound proteins selected from the group comprising: sialic acid, an agonist of TLR-1, and an NLRP3 agonist. It must be emphasized that the scope of the claimed invention of claims 1-17 encompasses membrane-bound proteins which are sialic acid, a TLR-1 agonist, 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 and 5-17 encompasses any membrane-bound protein or proteins 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. 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.
While it is clear from the instant specification that the membrane associated proteins recited in claim 3 are included in this genus 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 and 5-17.
35 USC § 112(a), Scope of Enablement
Claims 1-4 and 6-17 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 internally gelated cells display non-self antigens and/or “missing-self” recognition and the membrane-bound protein is selected from those bona fide ones recited in dependent claim 3, 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).
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. 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 et al., PLoS One 7: e30264 (2012) 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 stimulating protein or antigen is not exposed on the cell surface or 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 tumor cells, 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 (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. The science of immune cell stimulation 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 of the internally gelated cell such that the NK cells can contact it and respond. A nexus was demonstrated in the instant application only using a single combination of specific proteins and single cell types; however applicant is invited to furnish evidence to the contrary.
Claim Rejections - 35 USC § 112(b)
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 2-4 and 6-16 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 2 recites the modifier term “enriched” regarding the subset of NK cells in a population of cells; however, enriched is a relative term which renders the claim indefinite without further definition. Neither the specification nor the prior art provides a standard definition or recognized standard for measuring the degree for “enriched NK cells.” See MPEP 2173.05(b) (I). Thus, a person of ordinary skill in the art would not be appraised of the metes and bounds of the scope of the claim with regard to this term as it is not clear how the degree of “enriched” affects the invention, if at all. Claim 4 is included in this rejection for being dependent from indefinite claim 2.
Claim 3 recites the phrase or term “MICA/MICB”, which is not clear if what is before and after the slash forms a single limitation, recites an optional feature, or merely alternative combinations.
In claim 6, the phrase “ratio of the number of NK cells to the number of internally gelated cells in the culturing step” is incoherent or otherwise unclear when both the preamble and phrase “under conditions that allow expansion of NK cells” of claim 1 implies the limitation that the NK cells expand (increase in number) during the culture step. As the internally gelated cells are not changing in number, the ratio recited in claim 6 is dynamic and not static. Thus, the ratio must be limited to a specific point in time regarding the culturing step.
Claim 7 recites “DMEM”, which appears to be an abbreviation that needs to be spelled out at least once in the claim set for clarity. Claims 8-16 are included in this rejection for being dependent from indefinite claim 7.
Claim 12 recites wherein the optional photo-initiator ranges from 0.01 to 1 wt%, which is ambiguous and unclear as to if this is throughout the method or specifically in the gelation solution, the second cell suspension, the third cell suspension, and/or the population of internally gelated cells. Claim 13 is included in this rejection for being dependent from indefinite claim 2.
Claim 16 recites the relative term “engineered from” regarding artificial cells relative to a specifically recited cell types, which is indefinite because neither the claim nor the specification provides a standard for ascertaining the requisite degree of the “engineering” and, thus, one of ordinary skill in the art would not be reasonably apprised of the scope of this claim limitation.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 8 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 8 recites the osmotic concentration is alternatively any value lower than 290 mOsmol, any value from 290-320 mOsmol, or any value greater than 320 mOsmol. Thus, claim 8 encompasses any osmotic concentration without limitation. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Hu (WO2018026644A1) in view of Imai (Imai et al., Blood 106: 376-83 (2005)).
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). Hu teaches internally fixing antigen-presenting cells (APC) to deprive proliferative activity while maintain antigen presentation for stimulating immune responses, (pg. 9, 1st para.) and also teaches using artificial APCs to expand immune cells (pg. 8 lines 21-22). Hu teaches that internally gelated cells are more stable and less susceptible to damage during storage (pg. 14, lines 31, to pg. 15, line 8; FIG. 5).
Regarding claim 1, Hu does not teach wherein the target cell is an NK cell population and the immune response comprises stimulation of NK cell expansion.
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.).
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.
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 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.).
Thus, the claimed invention as a whole is prima facie obvious before the effective filing date in the absence of evidence to the contrary.
Claims 1-8, 10-12, and 14-17 are rejected under 35 U.S.C. 103 as being unpatentable over Hu in view of Imai as applied above, and further in view of Lin (Lin et al., Nat Commun 10: 1057 (2019), Rosen (US20190125795A1) and Morgan (Morgan et al., Chem Biol Interact 310: 108739 (2019)).
Regarding claim 7, 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 and Imai does not teach whereby the transient permeabilization step comprises using a medium comprising phenol-red free DMEM and a protease inhibitor cocktail or whereby the washing step comprises centrifuging the crosslinker loaded cell composition to obtain a cell pellet and resuspending the pellet in a medium comprising phenol-red free DMEM.
However 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 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.
Claim 8, as noted above, fails to further limit the subject matter of claim 7 and, thus, Hu in view of Imai, Lin, Rosen, and Morgan renders obvious claim 8 by virtue of teaching the subject matter of claim 7 as set forth fully above.
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, the combination of Hu and Imai 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 15, 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).
Regarding claims 15-16, 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).
Thus, the claimed invention as a whole is prima facie obvious before the effective filing date in the absence of evidence to the contrary.
Claims 1-17 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 (Chan et al., Lab Chip 10: 2062-70 (2010)).
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.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-6 and 17 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 32-33 of copending Application No. 16/322014 (the reference application) in view of Imai (Imai et al., Blood 106: 376-83 (2005)).
Although the claims at issue are not identical, they are not patentably distinct from each other because claims 32-33 of the reference application teaches stimulating in co-culture a target cell with an internally fixed cell (claim 2) wherein the interior is gelated and wherein the outer lipid membrane is fluid, preserves membrane contents, and biological functions (claim 1). While the reference claim does not teach wherein the target cells are specifically NK cells and the composition comprising the internally gelated cells and the target cells are cultured to allow expansion of the target cells, it would have been prima facie obvious to one of ordinary skill in the art to modify the reference claims in view of Imai.
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 adoptive NK cell immunotherapy (pg. 382, last para.).
It would have been prima facie obvious to one of ordinary skill in the art to perform the method of the reference claims of stimulating immune cells in culture wherein the internally-fixed (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 use the internal fixation method of the refence claim to preserve membrane contents and biological functions, namely the NK-expanding stimulatory ability of the 4-1BBL and/or mbIL-15 membrane proteins are demonstrated as effective on non-fixed cells.
Regarding instant 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 instant 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 instant 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 instant claim 6, Imai teaches culturing at a starting ratio of 1:1 NK cells to K562 aAPC cells (Fig. 1). Regarding instant 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.).
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 1-16 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 32-33 of copending Application No. 16/322014 (the reference application) in view of Imai as applied above, and further in view of Lin (Lin et al., Nat Commun 10: 1057 (2019), Chan (Chan et al., Lab Chip 10: 2062-70 (2010)), Rosen (US20190125795A1), and Morgan (Morgan et al., Chem Biol Interact 310: 108739 (2019)).
Regarding instant claims 7 and 15-16, Lin teaches adding a gelation solution to a cell suspension of antigen presenting cells (HeLa or dendritic cells (JAWSII)) for cell entry of the photo-reactive crosslinker PEG-DA (Mn 700 Da) and the photo-initiator I2959 via membrane poration, washing via centrifugation to remove extracellular PEG-DA and I2959, irradiating with UV light to cause intracellular hydrogelation and generate internally gelated cells and washing the resulting cells (Fig. 1a; pg. 2, left col., last para.; pg. 10, left col., 3rd para.). Lin teaches including various protease inhibitors in the gelation buffer (pg. 9, left col., 3rd para.).
Chan teaches a gelation process comprising PEGDA and I2959 wherein gelation occurs in the presence of cells are in suspension comprising DMEM without phenol red (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 to perform a method of the reference claims comprising stimulating immune cells in culture wherein the internally-fixed (gelated) cell type is a 4-1BBL+ mbIL-15+ K562 cell already taught by Imai wherein the gelated cells are prepared by suspending the K562 cells in a solution comprising phenol-red free DMEM, a cocktail of protease inhibitors, the photo-reactive crosslinker PEG-DA and the photo-initiator I2959 in order to get the crosslinker and initiator inside the cells via a membrane poration method comprising incubation at room temperature, then centrifuging and resuspending the cells in phenol-red free DMEM and applying UV light to crosslink the crosslinker thereby creating a population of internally gelated aAPC K562 cells, followed by washing and collected the internally gelated K562 cells. As noted above, instant claim 8 fails to further limit the subject matter of claim 7 and, thus, claim 8 is rendered obvious by reference claims 30-32 in view of Imai, Lin, and Chan by virtue of teaching the subject matter of claim 7 as set forth fully above.
Regarding instant claim 9, Lin teaches performing internal cell gelation with 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) at 1 wt% with PEG-DA at 4-40 wt% (pg. 9, left col., 3rd para.). Chan teaches I2959 (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.) and this I2959 stock solution is then diluted to 1 wt% when forming a gelation solution (pre-pol