Notice of Pre-AIA or AIA Status
The present application, filed on or after 16 Mar 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 1-10 filed 27 Sep 2024 are originally presented and considered on the merits.
Specification
Use of the terms G-Rex®, Takara (which appears to refer to the company Takara Bio USA®), Origen (which appears to refer to the company OriGen Biomedical®), ImmunoCult™-XF, TexMACS™, and XVIVO™, Miltenyi® (which appears to refer to the company Miltenyi Biotec B.V. & Co. KG), GE (which appears to refer to the company General Electric®), and Ficoll-Paque®, which are a trade names or marks used in commerce, has been noted in this application. The terms should be accompanied by the generic terminology, be capitalized wherever they appear, and, where appropriate, include a proper symbol indicating use in commerce, such as ™, SM , or ® following the term. Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claim 1 is objected to because it includes Arabic numerals for steps of the claimed method. Arabic numerals should be reserved for numbering of claims. The steps should be renumbered using letters or Roman numerals.
Claims 1, 6-7, and 9 are objected to because of the following informalities:
Regarding claim 1 step 4, "obtain" should be amended to recite "obtaining".
Regarding claims 6-7, proper scientific notation and units are required for numeric values.
Regarding claim 6, the recitation "on a day D0" should be changed to "on day 0".
Regarding claim 7, the following corrections are required:
The recitation "up to a day D12" should be changed to "up to 12 days".
The recitation "on a day D2" should be changed to "on day 2".
The recitation "on a day D4" should be changed to "on day 4".
The recitation "on a day D6" should be changed to "on day 6".
The recitation "on a day D8" should be changed to "on day 8".
The recitation "on a day D10" should be changed to "on day 10".
Regarding claim 9, the first instance of any abbreviation recited in the claims should be accompanied by the full, written out term, then the abbreviation may be used alone thereafter.
Appropriate correction is required.
Claim Interpretation
The following interpretations are made to promote compact prosecution:
The recitation, "on a day d0, and cell density is adjusted to 1 to 4E6/mL" in claim 6 is interpreted as "on day 0, and cell density is adjusted to 1 cell/mL to 4·106 cells/mL".
The recitation, "is up to a day D12, 2 to 3 times the volume of the second solution is added on a day D2, and the cell density is adjusted to 5E4 to 1E6/mL on D4, 6E4 to 1.2E6/mL on a day D6, to 7E4 to 1.4E6/mL on a day D8, and 8E4 to 1.6E6/mL on a day D10" in claim 7 is interpreted as "is up to 12 days, 2 to 3 times the volume of the second solution is added on day 2, and the cell density is adjusted to 5·104 to 1·106 cells/mL on day 4, 6·104 to 1.2·106 cells/mL on day 6, to 7·104 to 1.4·106 cells/mL on day 8, and 8·104 to 1.6·106 cells/mL on day 10".
The recitations "Origen culture bags" and "Takara culture bags" in claims 8 and 10 are interpreted as "cell culture bags".
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.
Claims 1, 3, 5, and 8-10 are rejected under 35 U.S.C. § 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claim 1 is rejected under 35 U.S.C. 112(b) as being incomplete for omitting essential steps, such omission amounting to a gap between the steps. See MPEP § 2172.01. Claim 1 step 4 is drawn to the use of different culture containers to determine an optimal culture container. However, no active steps are recited connecting the use of the culture containers to the determining step. The intervening step of comparing cell expansion folds and phenotypes is insufficiently detailed for a skilled artisan to understand how an optimal culture container is determined.
The term “optimal” recited in claim 1 step 4 is a relative term that renders the claim indefinite. The term “optimal” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Claim 1 step 4 includes the step of comparing cell expansion folds and phenotypes to determine the optimal container. However, it is unclear 1) whether cell expansion and phenotype are the only considerations for which culture container is optimal, and 2) the degree of cell expansion, which aspect of cell phenotype, and the requisite degree of the phenotype are considered optimal. Therefore, the metes and bounds of the claim are indefinite because a skilled artisan would be unable to recognize what culture vessel the inventor regards as optimal.
Claims 3 and 5 contain the trademark/trade names ImmunoCult™-XF, TexMACS™, and XVIVO™. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b). See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe cell culture media and, accordingly, the identification/description is indefinite.
Claim 8 contains the trademark/trade name G-Rex®. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b). See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name appears to be used to identify/describe numerous cell culture vessels, accordingly, the identification/description is indefinite.
Claims 8 and 10 contain the trademark/trade name Origen. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b). See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). Furthermore, it appears that the trademark/trade name refers to OriGen Biomedical®, which is a company, not a cell culture vessel. The term "Origen culture bag" is not a term of art. The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name appears to be used to identify/describe the cell culture bags and, accordingly, the identification/description is indefinite.
Claims 8 and 10 contain the trademark/trade name Takara. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b). See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). Furthermore, it appears that the trademark/trade name refers to Takara Bio USA®, which is a company, not a cell culture vessel. The term "Takara culture bag" is not a term of art. The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name appears to be used to identify/describe cell culture bags and, accordingly, the identification/description is indefinite.
Claim 9 is rejected under 35 U.S.C. 112(b) as being incomplete for omitting essential elements, such omission amounting to a gap between the elements. See MPEP § 2172.01. Blood samples and aqueous solutions used in centrifugation to isolate blood cells have densities of about 1 g/mL (the density of water) to 1.1 g/mL. It is, therefore, unclear how a 50 mL centrifugal tube can contain 500 to 1000 g of such a solution, which would require that the density of the solution to be about 9 to 18 times denser than blood samples or solutions used in the art to isolate blood cells. Furthermore, white blood cells only comprise about 1% of the volume of blood and have a density of about that of water. Centrifuging 500 to 1000 g of blood, even without dilution with PBS and a separation solution as claimed, would result in separation of about 5 to about 10 g of white blood cells; therefore, it is unclear how mixing the resulting separated white blood cell layer with 10 mL PBS (about 10 g) can result in 250 g of solution for the second centrifugation step. No elements are recited in the claims or in the specification to explain the discrepancy between the density of the recited solutions and the mass of solution centrifuged. To promote compact prosecution, the claim is interpreted to mean that the solutions centrifuged are prepared in multiple 50 mL centrifuge tubes and 1) pooled to centrifuge large volumes at once or 2) numerous individual 50 mL centrifuge tubes are centrifuged at once.
Claim Rejections - 35 USC § 101
35 U.S.C. § 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1 and 6 are rejected under 35 U.S.C. § 101 because the claimed invention is directed to an abstract idea without significantly more. Claim 1 step 4 recites, "using different culture containers and comparing cell expansion folds and phenotypes to determine an optimal culture container and obtain the memory immune cells". The steps of comparing cell expansion folds and phenotypes and determining an optimal container are mental processes, which fall into the abstract idea grouping of judicial exception. These judicial exceptions are not integrated into a practical application because the comparing step and the determining step are not integrated into practical application. The claims do not include additional elements sufficient to amount to significantly more than the judicial exceptions because the additional elements merely amount to instructions to apply the judicial exceptions, are recited at a high level of generality, and are well, understood, routine, and conventional in the art.
The Office published the guidance document entitled 2014 Interim Guidance on Patent Subject Matter Eligibility (Interim Eligibility Guidance), published 16 Dec 2014. Step 2A was revised to include two prongs (Federal Register / Vol. 84, No. 4 / 07 Jan 2019). Analysis is as follows:
Step 1: Is the claim drawn to one or more of the statutory categories of invention (a process, a machine, a manufacture, or a composition of matter)?
Step 1: The claim is directed to a process (an in vitro expansion method).
Step 1: Yes.
Step 2A, Prong One: Does the claim recite a judicial exception (a law of nature, a natural product/phenomenon, or an abstract idea)?
Step 2A, Prong One: The process (an in vitro expansion method) recites steps to 1) compare cell expansion folds and phenotypes and 2) determine an optimal culture container, both of which mental processes, which follows into the group of abstract ideas. Comparing and determining are concepts performed in the human mind.
Step 2A, Prong One: Yes.
Step 2A, Prong Two: Does the claim recite additional elements that integrate the judicial exception into practical application of the exception?
Step 2A, Prong Two: These exceptions are not integrated into practical application because the claim does not recite additional elements that integrate the judicial exceptions into practical application. Steps 1-3 of claim 1 and the recitations, "using different culture containers" and "obtain the memory immune cells" in step 4 amount to mere instructions to apply the judicial exception. The additional elements of the claim only recite the idea of the solution of determining the optimal container rather than reciting details for how the solution is accomplished. Furthermore, both the comparing step and the determining step are recited at a high level of generality, as no criteria are recited for how the in vitro expansion method steps 1-3 or the criteria for expansion folds and phenotypes result in a choice of optimal container. No recitations are provided for what cell expansion folds are compared, what amount of cell expansion fold is considered optimal, what phenotypes are being compared, and what requisite degree of change in one or more phenotypes is considered optimal. The recitation of claim limitations that attempt to cover any solution to an identified problem with no restriction on how the result is accomplished and no description of the mechanism for accomplishing the result, does not integrate a judicial exception into a practical application or provide significantly more because this type of recitation is equivalent to the words "apply it". See MPEP § 2106.05(f).
Step 2A, Prong Two: No.
Step 2B: Does the claim recite additional elements that individually or in combination amount to significantly more than the judicial exception (i.e., whether the additional elements provide an inventive concept)?
Step 2B: The additional elements recited in the claim do not add an inventive step to add significantly more than mere instructions to apply the judicial exceptions when considered alone or in combination. The preamble to claim 1 is generic to the type of memory immune cell cultivated. Step 1 is generic to the lymphocyte separation solution and the source of memory immune cells. Step 2 is generic to the method of activating the immune cells and the culture medium. Step 3 is generic the second solution, the density of cell culture, and interval of adjustment. Step 4 is generic to the type and number of culture containers, and how the memory immune cells are obtained. Baudequin (T. Baudequin, et al., Biotechnol Adv, 2021) teaches that "expansion procedures developed over the last decades have led to excellent result for phase I/II developmental studies" for antigen-specific lymphocytes (in vitro expansion method for cultivating memory immune cells) (Introduction pp. 1-2). Turner (R.J. Turner, et al., Purinergic Signal, 2020) teaches that isolation of human peripheral blood mononuclear cells (PBMC, which comprise several type of memory immune cells) by Ficoll-Paque® density gradient centrifugation is a routine isolation procedure that dates back the 1960s, and further teaches that "a number of modern refinements to improve ease of isolation and reduce time of processing" have been developed (using a lymphocyte separation solution to separate immune cells from peripheral blood) (p. 390). Baudequin further teaches that there are several standardized procedures for activating lymphocytes, which include additions of activators at the start of cell culture (activating immune cells on day 0, claim 6) and adding additional activators at different steps of expansion (activating the immune cells, step 2, and adding a second solution, step 3) (§ 3, pp. 2-3). Baudequin further teaches that multiple groups have determined that the ideal range for maintaining culture density is 1·106 to 3·106 cells/mL (within the claimed range in claim 6) and multiple approaches have been found to successfully maintain an optimal density (adjusting cell culture density at intervals, step 3). Baudequin further teaches that while research is ongoing, several gold standard culture vessels have been reported and compared against each other and to older methods of cultivating lymphocytes (using different culture vessels and obtaining memory immune cells, step 4) (Introduction p. 2).
Taken together, each additional element recited in the claim is recited at a high level of generality, and each element is described in the art as well understood, conventional, or routine such that the claim does not recite an unconventional step that confines the invention to a particular useful application of the judicial exception or amount to an inventive concept. Furthermore, Baudequin teaches each of the elements in the same context of reviewing the art of expanding lymphocytes for clinical use, and thus the additional elements are also considered well understood, conventional, and routine when considered in combination.
Step 2B: No.
In the instant case, the limitations of the claims do not impose limits on the scope of the claim such that the steps of comparing cell expansion folds and phenotype and determining an optimal container are integrated into practical application of the judicial exceptions. Accordingly, based on analysis of the claim as a whole, the claims do not recite additional elements adding significantly more than the judicial exception and are thus rejected under 35 U.S.C. § 101 because the claimed invention is not directed to patent eligible subject matter.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. § 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1 and 6 are rejected under 35 U.S.C. § 102(a)(1) as being anticipated by Palmerini (P. Palmerini, Cytotherapy, 2020).
Regarding claim 1, Palmerini discloses a method for expanding T cells in G-Rex® culture flasks or tissue culture flasks (in vitro expansion, using different culture containers) comprising the following steps: (1) isolation of peripheral blood mononuclear cells (PBMCs, immune cells from peripheral blood) by density gradient centrifugation in Lymphoprep™ solution (lymphocyte separation solution) from individual donor buffy coats in parallel (single blood collection) (step 1), (2) plating the cells in G-Rex® flasks or tissue culture flasks in X-VIVO® 10 medium supplemented with 1% penicillin/streptomycin (a first solution in complete culture medium) or RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 1% L-glutamine, and 1% HEPES (a first solution in complete culture medium), each medium comprising interferon γ (IFNγ), to activate the cultured cells (steps 2 and 4), (3) swapping cells in the G-Rex®6 culture flasks to G Rex®6M culture flasks on day 7 with fresh media supplemented with IFNγ and recombinant human interleukin-2 (rhIL-2) (second solution) and adjusting the cell density to 2.5·106 cells/mL (adjusting cell culture density at intervals) (step 3), and (4) comparing "growth, viability, phenotypic profile, and cytotoxic activity of cells" and fold expansion (cell expansion folds and phenotype) cultured in different conditions and between vessel types (using different culture containers) and determined that G-Rex containers preferentially expand cells with a naive phenotype compared to tissue culture flasks (determine an optimal culture container) (step 4) (Abstract, Methods pp. 512-513, Discussion pp. 514 and 516, and Fig. 1). The PBMC expanded by Palmerini comprise CD8+ T cells comprising central memory, effector memory, and effector memory RA+ populations (memory immune cells) (Results p. 514).
Regarding claim 6, Palmerini disclosing plating cells G-Rex®6 culture flasks at an initial density of 2.5·106 cells/well in 40 mL or 100 mL medium (6.25·104 cells/mL and 2.50·104 cells/mL, respectively, within the claimed range of 1 to 4·106 cells/mL) (Methods p. 512). The medium used for the initial plating comprises IFNγ to activate the cells (Methods p. 512 and Fig. 1).
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.
Claims 1-6 are rejected under 35 U.S.C. § 103 as being unpatentable over Palmerini (P. Palmerini, Cytotherapy, 2020) in view of Pan (C. Pan, et al., Chin J Cell Mol Immunol, 2021, cited in the IDS filed 27 Sep 2024) and as evidenced by Gibco (Human IL-7 Recombinant Protein, PeproTech® [Product data sheet], 2026).
Palmerini discloses a method of expanding cytotoxic T cell populations comprising memory immune cells in vitro as discussed in the rejection of claims 1 and 6 under 35 U.S.C. § 102(a)(1) above.
Palmerini teaches applying 500 IU/mL recombinant human interleukin-2 (rhIL-2) starting one day after plating peripheral blood mononuclear cells (PBMCs) to activate immune cells (claims 4-5) (Methods p. 512). Palmerini further teaches culturing immune memory cells in X-VIVO® 10 medium, which is a complete, serum-free medium (comprises a serum substitute) (claim 4) (Methods p. 512).
Palmerini further teaches that while production of cytotoxic T cells has been studied since the 1990s and combinations of interleukins, including IL-1, IL-6, IL-7, IL-12, and IL-15, have been added in culture, a standardized protocol has not yet been developed to produce the cells (Discussion p. 514). Palmerini further teaches that a higher proportion of naïve and central memory cells in the final population are desired as that subset has higher proliferative potential, thus increasing cell yield, and contribute to higher survival rates and long-term clinical responses when delivered in vivo (Discussion p. 517).
Palmerini does not teach the combination of IL-2, IL-21, IL-7, and activated magnetic beads in the first solution as required by claims 2-3 or the combination of IL-2, IL-21, IL-7, and a serum substitute in the first solution as required by claims 4-5.
However, Pan teaches comparisons of interleukin cocktails to promote expansion of CD8+ central memory T cells (memory immune cells) from PBMC (immune cells from peripheral blood) (Abstract). Pan teaches that IL-7 stimulates T memory cell activation, survival, and proliferation and is part of standard cytokine cocktails for inducing T memory cells (Discussion). Pan further teaches that IL-21 can synergistically, in a manner similar to IL-15) stimulate the same receptor as IL-7 to induce proliferation in CD8+ T memory cells (Discussion). Pan teaches addition of anti-CD3/anti-CD28 magnetic beads to stimulate and activate T cells in vitro (activated magnetic beads) with 300 IU/mL rhIL-2 or 25 ng/mL each IL-7, IL-15, and IL-21 (Materials §§ 1.1.1 and 1.2.2). 300 IU/mL IL-2 is within each of the claimed ranges of claims 2-3 (10 to 300 IU/mL) and 4-5 (50 to 500 IU/mL). Pan uses rhIL-7 from PeproTech®, which is sold with a specific activity of 2·106 international units (IU)/mL (Gibco). Therefore, Pan teaches 50 IU/mL IL-7, which is within each of the claimed ranges of claims 2-3 (20 to 250 IU/mL) and 4-5 (50 to 300 IU/mL). 25 ng/mL IL-21 is within each of the claimed ranges of claims 2-3 (5 to 500 ng/mL) and 4-5 (10 to 1000 IU/mL). Pan further teaches that the combination of IL-7/IL-15/IL-21 more potently stimulates proliferation of CD8+ T cells than IL-2 or IL-7/IL-15 (Results § 2.1.2 and Fig. 2), better inhibits CD8+ T cell apoptosis (Results § 2.1.3 and Fig. 3), and induces preferred T cell function (Results § 2.3 and Fig. 6).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to combine the method of in vitro expansion of memory immune cells in medium comprising a serum substitute comprising addition of IL-2 to a first and a second solution to activate the immune cells as taught by Palmerini with addition of IL-7, IL-15, IL-21, and activated magnetic beads as taught by Pan to arrive a to arrive at the claimed invention. One would be motivated to make such a combination as Palmerini teaches that a standardized T cell expansion protocol is desired and that preferential expansion of central memory T cells allows for greater cell yield, greater cell survival in vivo, and long-term clinical responses and as Pan teaches that IL-7 is a standard cytokine in the art for the expansion of memory T cells and IL-21 can synergistically activate the same receptor as IL-7 to further induce memory T cell proliferation. One would have a reasonable expectation of success in making the combination as Pan demonstrates that addition of IL-7, IL-15, and IL-21 to medium comprising activated magnetic beads improves expansion of memory T cells.
Claims 1, 6-8, and 10 are rejected under 35 U.S.C. § 103 as being unpatentable over Palmerini (P. Palmerini, Cytotherapy, 2020) in view of Baudequin (T. Baudequin, et al., Biotechnol Adv, 2021).
Palmerini discloses a method of expanding cytotoxic T cell populations comprising memory immune cells in vitro as discussed in the rejection of claims 1 and 6 under 35 U.S.C. § 102(a)(1) above.
Regarding claim 7, Palmerini teaches plating cells at an initial density of 6.25·104 cells/mL or 2.5·104 cells/mL on day 0 in G-Rex®6 or G-Rex®6M culture flasks or at an initial density of 5·106 cells/mL in T-flasks (traditional tissue culture flasks) (Methods p. 512). Palmerini further teaches adjusting the cell density in tissue culture flasks to 1.5·106 cells/mL every three to four days (Methods p. 512). Palmerini further teaches culturing cells for up to 28 days (Methods p. 512).
Regarding claims 8 and 10, Palmerini teaches comparison of in vitro expansion of memory T cells between G-Rex® culture flasks and tissue culture flasks (traditional culture flasks) (Abstract). Palmerini further teaches that tissue culture flasks have limitations when expanding sufficient numbers of T cells for clinical use and that gas-permeable culture bags (plastic culture bags) require large amounts of fresh medium and frequent cell density adjustments (Introduction p. 512). Palmerini further teaches G-Rex® culture flasks utilize convection rather than diffusion, which improves rate of gas and nutrient exchange, allows for increased medium depth of culture, which dilutes cell waste, and uses a semi-permeable silicone membrane in contact with the medium, improving O2 and CO2 exchange (Introduction p. 512). Palmerini demonstrates that compared to tissue culture flasks, G-Rex® culture flasks yielded more cells with a more favorable phenotype, with preferential expansion of immune cells with memory functions (Results pp. 513-514 and Fig. 2-3).
Palmerini does not teach culturing up to 12 days, addition of a specific volume, or adjustment of the cell density to the specific cell densities at specific timing as required by claim 7. Palmerini does not teach direct comparison of growth between G-Rex® culture flasks, plastic culture bags, and traditional culture flasks as required by claim 8 or between plastic culture bags and traditional culture flasks as required by claim 10.
However, regarding claim 7, Baudequin teaches that under classic culture conditions, T cell expansion (memory immune cells) "usually lasts for 14 days in classic conditions" (§ 4.2, p. 4). Baudequin further teaches that optimization of T cell bioreactor design and feeding schedules is desired to balance "nutrient support, waste removal, and culture medium costs" (§ 4.2, p. 4). Baudequin further teaches that maintaining a density of 106 to 3·106 cells/mL throughout T cell expansion is ideal to balance cell-cell communication and nutrient availability (§ 5, p. 4). Baudequin further teaches doubling the cell culture volume to decrease the cell density from 106 to 5·105 cells/mL (adding 2 to 3 times the volume of the second solution, within each of the claimed ranges on days 2, 4, 6, 8, and 10).
Thus Baudequin teaches that optimization of cell culture volume, cell density, and timing of cell culture volume adjustments are routine in the art. While Palmerini and Baudequin do not teach the exact cell density adjustment schedule of claim 7, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985) (Court held as proper a rejection of a claim directed to an alloy "having 0.8% nickel, 0.3% molybdenum, up to 0.1% iron, balance titanium" as obvious over a reference disclosing alloys of 0.75% nickel, 0.25% molybdenum, balance titanium and 0.94% nickel, 0.31% molybdenum, balance titanium. "The proportions are so close that prima facie one skilled in the art would have expected them to have the same properties."). It is routine procedure to optimize component amounts to arrive at an optimal product that is superior for its intended use, since it has been held where the general conditions of a claim are disclosed in the prior art, discovering the optimum or workable ranges involves only routine skill in the art. See MPEP § 2144.05.
Furthermore, regarding claims 8 and 10, Baudequin teaches comparisons between numerous types of culture vessels for T cell expansion, including "standard plastic culture flasks" (traditional culture flask), gas permeable culture bags (plastic culture bags), hollow fiber membrane-based bioreactors, improved culture flasks, such as G-Rex® culture flasks, perfusion-based culture bags, and spinner flasks (§ 7, pp. 5-10 and Table 1). Baudequin more specifically teaches that the G-Rex® system was developed "as an alternative to T-flask, spinner flasks, and semi-permeable bags" (§ 7.2, p. 6).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to combine the method of expanding CD8+ T memory immune cells and comparison of expansion in between traditional culture flasks and G-Rex® culture flasks as taught by Palmerini with cell culture medium volume additions to adjust cell density and comparison between traditional culture flasks, G-Rex® culture flasks, and plastic culture bags as taught by Baudequin to arrive at the claimed invention. One would be motivated to improve the adjustment of cell density and timing as Baudequin teaches that more rapid expansion of T cells (reduce culture to fewer than 12 days) is desirable to reduce cost, improve nutrient support, and improve waste removal, as Baudequin teaches that adjustment of cell density within the claimed ranges balances the density required for adequate cell-cell communication and nutrient availability. One would be motivated to compare cell culture vessels to determine an optimal culture container as Baudequin teaches that direct comparisons between the claimed culture vessel types is routine in the art, and as Palmerini teaches known differences in the operation and outcomes of traditional culture flasks, culture bags, and G-Rex® flasks. One would have a reasonable expectation of success in improving the adjustment of cell density and timing as Baudequin teaches that optimization of cell density and culture medium feeding schedules is routine in the art. One would have a reasonable expectation of success in comparing cell culture vessels as Baudequin and teaches that the G-Rex® system was developed expressly by comparison to traditional culture flasks and semi-permeable culture bags and as Palmerini demonstrates comparisons between G-Rex® flasks and tissue culture flasks.
Claims 1, 6, and 9 are rejected under 35 U.S.C. § 103 as being unpatentable over Wei (CN 109402054 A, 2019) in view of Palmerini (P. Palmerini, Cytotherapy, 2020) and Turner (R.J. Turner, et al., Purinergic Signal, 2020).
Wei teaches a method for expanding CD4+ memory T lymphocytes (memory immune cell) by culturing in a solution comprising interleukin (IL)-2, IL-7, and IL-21 (Abstract).
Regarding claims 1 and 9, Wei teaches a method to expand CD4+ memory T lymphocytes (memory immune cells, also referred to as T cells) in vitro comprising the following steps. First, Wei teaches collection of peripheral blood from healthy volunteers (separate immune cells from peripheral blood, claim 1 step 1) (Example 1). After separation of plasma from the blood, the cellular portion of the blood (blood sample) is diluted in an equal volume saline, which is then slowly added to Ficoll® (separation solution, claim 1 step 1 and claim 9) at a 2:1 ratio (which is an equal volume blood sample and Ficoll®) by pipetting (Example 1). The blood-separation solution is then centrifuged at 700 xG for 20 min (within the claimed range of 20 to 30 min). After centrifugation, the white film layer of PBMC is pipetted and transferred to a new 50 mL centrifuge tube (sucking a second layer of annular milky white lymphocytes into a new centrifuge tube) and saline (PBS) is added (Example 1). This solution is then washed and centrifuged twice to obtain peripheral blood mononuclear cells (PBMC) for culture (discarding the supernatant to obtain the cells) (Example 1). Wei further teaches activating CD4+ T cells isolated from the PBMC (immune cells obtained in step 1) in culture flasks in T lymphocyte culture medium coated with anti-CD3 and anti-CD28 antibodies to induce CD4+ memory T cell culture (activate the immune cells with a first solution in complete culture medium) (step 2) (Example 3). Wei further teaches culturing the CD4+ T cells for 24 h, then adding a solution comprising IL-2, IL-7, and IL-21 and adding culture medium every 2 days to keep the density of cells at 0.2·106 to 0.2·106 cells/mL (adding a second solution during culture and adjusting cell culture density at intervals) (step 3).
Wei further teaches that memory T cells are a rare population in the blood, only comprising about 0.4% of cells in the PBMC population (Background of the Invention).
Wei does not teach comparison between culture containers as required by claim 1 step 4 or a specific amount of diluted blood sample to centrifuge, a volume of saline to wash cells after the first centrifugation step, or timing for the second centrifugation step as required by claim 9.
However, Palmerini discloses a method of expanding cytotoxic T cell populations comprising memory immune cells in vitro as discussed in the rejection of claims 1 and 6 under 35 U.S.C. § 102(a)(1) above.
Regarding claim 1 step 4, Palmerini teaches comparison of in vitro expansion of memory T cells between G-Rex® culture flasks and tissue culture flasks (traditional culture flasks) (Abstract). Palmerini teaches that for clinical use of memory T cells, large numbers of cells are required for infusion, thereby limiting utility of traditional culture vessels. Palmerini further teaches that while production of cytotoxic T cells has been studied since the 1990s, a standardized protocol has not yet been developed to produce the cells (Discussion p. 514). Palmerini teaches that tissue culture flasks have limitations when expanding sufficient numbers of T cells for clinical use and that gas-permeable culture bags (plastic culture bags) require large amounts of fresh medium and frequent cell density adjustments (Introduction p. 512). Palmerini further teaches G-Rex® culture flasks utilize convection rather than diffusion, which improves rate of gas and nutrient exchange, allows for increased medium depth of culture, which dilutes cell waste, and uses a semi-permeable silicone membrane in contact with the medium, improving O2 and CO2 exchange (Introduction p. 512). Palmerini demonstrates that compared to tissue culture flasks, G-Rex® culture flasks yielded more cells with a more favorable phenotype, with preferential expansion of immune cells with memory functions (Results pp. 513-514 and Fig. 2-3).
Regarding claim 9, Turner teaches that Ficoll-Paque® density gradient centrifugation (the method taught by Wei) is a routine isolation technique in the art for isolation of human PBMC (Introduction p. 390). Turner further teaches centrifuging for 10 min during the second centrifugation step when using Ficoll-Paque® method for isolating PBMC (Materials and Methods p. 391). Furthermore, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%). It is routine procedure to optimize component amounts to arrive at an optimal product that is superior for its intended use, since it has been held where the general conditions of a claim are disclosed in the prior art, discovering the optimum or workable ranges involves only routine skill in the art. See MPEP § 2144.05. Wei and Turner are silent with regard to amount of solution used in the wash step before the second centrifugation step; however, both references teach performing the method in 50 mL centrifugation tubes and therefore, the volume of saline used to wash must be less than 50 mL. Turner further teaches that long centrifugation times are undesirable (Introduction p. 390); therefore, lower volume of wash solution is desirable to minimize centrifugation time.
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to combine the method of in vitro expansion of memory immune cells taught by Wei with comparison between culture containers to determine an optimal culture container and isolation of a large amount of PBMC as taught by Palmerini and optimization of the PBMC isolation method as taught by Turner to arrive at the claimed invention. One would be motivated to combine the method taught by Wei with comparison between culture containers as taught by Palmerini as Palmerini teaches that traditional culture flasks are limited when expanding large numbers of memory T cells required for clinical utility and that standardized protocol such expansion have not been developed. One would have a reasonable expectation of success in making the combination as Palmerini demonstrates comparison between G-Rex® flasks and tissue culture flasks and demonstrates that G-Rex® flasks yield greater cell fold expansion and more desirable phenotypes in the expanded cells compared to traditional culture flasks. One would be motivated to improve the method of isolating PBMC taught by Wei as Wei teaches that memory T cells are a rare blood cell population, thereby requiring a large blood volume, and as Turner teaches that the method used by Wei is routine in the art. One would have a reasonable expectation in making the improvement as Turner teaches that isolation of PBMC by Ficoll-Paque® density gradient centrifugation is routine in the art.
Conclusion
No claim is allowed.
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Eric B Wright, PhD
Examiner
Art Unit 1632
/Eric B Wright/Examiner, Art Unit 1632
/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632
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