DETAILED ACTION
This action is in response to papers filed 04/08/2026.
Claim 1 is pending. Claims 2-6 have been cancelled.
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 .
Withdrawn Objections and Rejections
The rejection of claim 1 under 35 U.S.C. 103 over Baek (WO 2016/209021 A1), in view of Warren (The Journal of Immunology, 1999, 162(2): 735-742), Romagne (US 2008/0063717 A1), Lee (KR 101760764), and Shin (US 2023/0203444 A1) is withdrawn in light of the amendment to claim 1 to incorporate elements of claims 3 and 5-6 into claim 1.
The cancellation of claims 2-6 render objections and rejections thereof moot. Applicant’s arguments regarding the prior art references are addressed following the rejection.
Claim Objections
Claim 1 remains objected to because of the following informalities: Claim 1, as amended, recites the limitation “T75 flask coated with the anti-CD16 antibody” in line 2 of step 2. This should be corrected to “T75 flask coated with an anti-CD16 antibody”. Appropriate correction is required.
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.
Claim 1 remains 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.
Step 2 of claim 1, as amended, recites:
“2) culturing the isolated PBMCs in a T25 flask coated with an anti-CD16 antibody
when the cell number is less than 3x107, or in a T75 flask coated with the anti-CD16 antibody when the cell number is 3x107 or more, by adding 8 ml of RPMI medium, 2 ml of plasma, IL-2 at
a concentration of 20 ng/ml, IL-15 at a concentration of 50 ng/ml, an anti-CD56 antibody at a
concentration of 5 ng/ml, an anti-NKp46 antibody at a concentration of 5 ng/ml, and an antiNKp30 antibody at a concentration of 5 ng/ml to the T25 flask or the T7 5 flask, suspending the pellet of PBMCs in 10 ml of the RPMI medium, adding the suspended pellet of PBMCs to the T25 flask or the T75 flask, and culturing in an incubator at 37°C and 5% CO2 for 3 to 4 days to obtain a cell culture suspension” (emphases added).
It is unclear whether step 2 recites two separate culturing steps, or whether step 2 refers to one culturing step. In particular, step 2 recites “suspending the pellet of PBMCs in 10 ml of the RPMI medium” (lines 6-7 of step 2), wherein “the RPMI medium” refers back to “RPMI medium” in line 3 of step 2. However, the RPMI medium recited in line 3 has a volume of 8 mL.
Step 2 of amended claim 1 incorporates limitations from cancelled claim 5, which imposed further limitations on the culturing of step 2 in claim 1, rather than introducing an additional subculturing step. Thus, for the sake of compact prosecution, step 2 of amended claim 1 is interpreted as referring to a single culturing step, wherein the limitation “10 ml of the RPMI medium” in line 7 refers to “8 ml of RPMI medium, 2 ml of plasma, IL-2 at a concentration of 20 ng/ml, IL-15 at a concentration of 50 ng/ml, an anti-CD56 antibody at a concentration of 5 ng/ml, an anti-NKp46 antibody at a concentration of 5 ng/ml, and an antiNKp30 antibody at a concentration of 5 ng/ml” as recited in lines 3-6.
To overcome the rejection, in addition to clarifying whether step 2 comprises one or two culturing steps, the volume of RPMI medium (8 mL or 10 mL) used therein must be clarified.
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.
Claim(s) 1 is rejected under 35 U.S.C. 103 as being unpatentable over Baek (WO 2016/209021 A1), in view of Warren (The Journal of Immunology, 1999, 162(2): 735-742; cited in IDS filed 08/13/2024), CN105219713A, Lee (KR 101760764, cited in IDS filed 05/11/2023), Shin (US 2023/0203444 A1), Romagne (US 2008/0063717 A1), and Arango (Autoimmunity: From Bench to Bedside, 2013, Chapter 45).
Citations to Baek and Lee are to the WIPO machine translations, which were provided in the Office Action mailed 01/12/2026.
Baek teaches a method for proliferating NK cells (para 1) comprising IL-12, IL-15, and an anti-Nik46 antibody (para 22). Baek teaches isolating PBMC from a human patient blood sample using Ficoll (reads on density gradient medium) and centrifugation (para 88), and resuspending precipitated cells (reads on the pellet) in 5 mL of RBC lysis buffer to remove red blood cells (para 90) (reads on step 1).
Baek teaches that the cell culture may be performed in steps to maximally proliferate NK cells (para 30). Baek teaches an embodiment of the method comprising three culturing steps (para 40-41). In the first culturing step, PBMCs are cultured in a medium comprising IL-2, IL-15, anti-NKp46 antibody, and plasma (para 35) (corresponds to step 2). The second step of culture comprises transferring the culture of the first step to a new container (reads on subculturing), then culturing in a medium comprising IL-2, IL-15, anti-NKp46 antibody, and plasma (reads on albumin) (para 37-38) (corresponds to step 3). The third step of culture comprises IL-2 (para 40-41) (corresponds to step 4). Baek teaches that “each culture step may have the same or different culture medium components, culture vessels, and culture periods” (para 30). Therefore, steps 2-3 of the embodiment may comprise the same culture medium as the medium used in step 1, which comprises IL-12, IL-15, anti-NKp46 antibody, and plasma. Baek further teaches that RPMI 1640 may be used as the culturing medium (para 22) (reads on RPMI culture medium of steps 2-4).
Regarding step 2:
Baek teaches that in the first culture step (corresponds to step 2 of instant claim 1), the PBMCs may be cultured for 3 days or 4 days (para 35). Baek teaches that the culture of PBMCs is carried out in a normal cell culture condition, which is at about 37℃ in a CO2 incubator (para 29) (reads on lines 8-9 of step 2).
Regarding the culture flask (lines 1-3 of step 2): Baek teaches that the total number of cells used in the initial culture was 3x107 (p 14, Table 3). Baek teaches culturing PBMCs in a T75 flask (para 101-107). Baek teaches culturing the PBMCs in a culture flask coated with fibronectin and gamma globulin (para 102-107), but does not specify that the gamma globulin is an anti-CD16 antibody.
Warren teaches that culturing NK cells on plastic coated with purified anti-CD16 antibody (i.e., CD16 ligation) stimulates NK cell activation (p 736, col 2, para 2). In contrast to NK cells grown in control cultures, NK cells growth on anti-CD16 antibody-coated plates showed a heterogeneous pattern of fluorescence indicative of asynchronous cell division (p 740, col 2, para 3). Warren teaches that “These data demonstrate unequivocally that CD16 ligation stimulates NK cell division” (p 740, col 2, para 3).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Baek by culturing the NK cells on an anti-CD16 antibody-coated incubator, as taught by Warren. One of ordinary skill in the art would have been motivated to make this modification because Warren teaches that the data taught therein “demonstrate unequivocally that CD16 ligation stimulates NK cell division” (p 740, col 2, para 3). One of ordinary skill in the art would have had a reasonable expectation of successfully making this modification because Warren teaches that NK cells can be grown on an anti-CD16 antibody-coated incubator.
Regarding the components in the culture medium (lines 3-6 of step 2):
Regarding 8 mL of RPMI: Baek teaches that RPMI 1640 may be used as the culturing medium (para 22). In a working example, Baek teaches that the amount of medium used in the culture is approximately 13.5 mL (para 111).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have optimized the amount of medium in the culturing solution based on factors such as duration of culture and desired number of cells at harvest to arrive at the claimed invention of using 8 mL of medium. See MPEP 2144.05(II)(A). As noted in In re Aller, 105 USPQ 233 at 235, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.
Regarding 2 mL of plasma: Baek teaches that the amount of plasma added to the culture is 1.5 mL (para 111).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have optimized the amount of plasma in the solution based on factors such as the total volume of culture medium to arrive at the claimed invention of using 2 mL of plasma. See MPEP 2144.05(II)(A). As noted in In re Aller, 105 USPQ 233 at 235, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.
Regarding 20 ng/mL of IL-2: Baek teaches that the concentration of IL-2 may be 500-5000 IU/mL (para 65). However, because Baek does not disclose the manufacturer or catalogue number of IL-2 used in the disclosure, the concentration of IL-2 disclosed therein cannot be reliably converted into units of ng/mL.
CN105219713A teaches a method for in vitro expansion of NK cells, comprising culturing PBMCs in a cell culture flask coated with anti-CD16 antibody in a solution comprising IL-2 with a concentration of 20-50 ng/mL and IL-15 with a concentration of 20-50 ng/mL (p 1, Summary of the Invention, steps 1-3).
It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Baek by culturing PBMCs in a solution comprising 20 ng/mL of IL-2, as taught in CN105219713A. One of ordinary skill in the art would have been motivated to make this modification because CN105219713A teaches that culturing NK cells in a solution comprising 20-50 ng/mL of IL-2 results in NK cells with high proliferation and killing force (p 1, Summary of the Invention, para 1). One of ordinary skill in the art would have had a reasonable expectation of successfully making this modification because CN105219713A teaches that IL-2 can be used at a concentration of 20-50 ng/mL to culture NK cells.
Regarding 50 ng/mL of IL-15: Baek teaches that the concentration of IL-15 (i.e., the second interleukin in the composition) may be 30 to 100 ng/mL of 50 to 100 ng/mL (para 66).
Regarding 5 ng/mL of anti-CD56 antibody: Baek teaches that the composition for proliferation taught therein may include other carriers or adjuvants for PBMC culture (para 70), but does not specify the inclusion of an anti-CD56 antibody.
Lee teaches a culture method for mass proliferation of NK cells, comprising culturing PBMCs in an anti-CD16-antibody coated vessel in a medium containing IL-2, anti-CD335 (anti-NKp46) antibody, and anti-CD56 antibody (p 1, Means for Solving the Problem, para 1).
Shin teaches that NK cells stably proliferate at a higher rate when pre-stimulated with an anti-CD56 antibody (para 58). Shin teaches that culturing lymphocytes in the presence of an anti-CD16 antibody or an anti-CD56 antibody, which performs a similar function to the anti-CD16 antibody, causes signal transduction in NK cells, resulting in the expression of transferrin receptors or the production of TNF or IFN-γ (para 72).
It would have been prima facie obvious for a person of ordinary skill before the effective filing date of the claimed invention to have modified the method of Baek by culturing NK cells in the presence of an anti-CD56 antibody, as taught by Lee. One of ordinary skill in the art would have been motivated to make this modification because Shin teaches that culturing lymphocytes in the presence of an anti-CD56 antibody causes signal transduction in NK cells, resulting in the expression of transferrin receptors or the production of TNF or IFN-γ (para 72). One of ordinary skill in the art would have had a reasonable expectation of successfully making this modification because Lee teaches that PBMCs can be cultured in the presence of an anti-CD56 antibody and IL-2.
Regarding the concentration, the instant claim recites the concentration of 5 ng/mL anti-CD56 antibody in 10 mL of solution (8 mL of RPMI medium and 2 mL of plasma), which amounts to 50 ng of anti-CD56 antibody. Lee teaches that 1 to 50,000 ng of anti-CD56 antibody can be used to culture PBMCs (p 1, Means for Solving the Problem, para 2).
Regarding 5 ng/mL of anti-NKp46 antibody: The instant claim recites the concentration of 5 ng/mL anti-NKp46 antibody in 20 mL of solution (18 mL of RPMI medium and 2 mL of plasma), which amounts to 100 ng of anti-NKp46 antibody. Lee teaches that 1 to 50,000 ng of anti-CD335 (anti-NKp46) antibody can be used to culture PBMCs (p 1, Means for Solving the Problem, para 2).
Regarding 5 ng/mL of anti-NKp30 antibody: Baek teaches that the composition for proliferation taught therein may include other carriers or adjuvants for PBMC culture and NK cell proliferation (para 70), but does not specify the inclusion of an anti-NKp30 antibody.
Romagne teaches a method for stimulating the proliferation of NK cells (Abstract). Romagne teaches resuspending PBMCs in complete medium comprising RPMI 1640 and 10% fetal calf serum (reads on plasma and albumin) (para 93), and adding interleukins and antibodies to establish a primary cell culture (para 97). The interleukins are a combination of IL-2 and IL-15 (para 94, 17), and the antibodies are anti-NKp46, anti-NKp30, or a combination of both (para 17). Romagne teaches that the combination of anti-NKp46 and anti-NKp30 gives the best enrichment of NK cells, compared to either antibody alone (para 148).
It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Baek by culturing isolated PBMCs in a medium comprising an anti-NKp30 antibody. One of ordinary skill in the art would have been motivated to make this modification because Romagne teaches that the combination of anti-NKp46 and anti-NKp30 gives the best enrichment of NK cells, compared to either antibody alone (para 148). One of ordinary skill in the art would have had a reasonable expectation of successfully making this modification because Romagne teaches that PBMCs can be cultured in a medium comprising an anti-NKp30 antibody in addition to IL-2, IL-15, and an anti- NKp46 antibody.
Regarding the concentration, Romagne discloses an experiment to establish a titration curve of anti-NKp30 antibody to evaluate the amount of anti-NKp30 antibody necessary to obtain proliferation (Example 3, para 160, Fig 5). Romagne teaches that the effect of the anti-NKp30 antibody for induction of proliferation is saturable with a plateau effect at about 1 µg/mL, and that the dose to obtain 50% of maximum effect is below 0.1 µg/mL in this particular experiment (para 160). Romagne further teaches that “It should be noted that the characteristics of the curve may depend on the particular antibody used, and particularly of its affinity. The use of humanized anti NCR antibodies may also display a different titration curve” (para 161).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have optimized the concentration of the anti-NKp30 antibody based on aspects of the particular antibody used, as taught by Romagne, to arrive at the claimed invention. See MPEP 2144.05(II)(A). As noted in In re Aller, 105 USPQ 233 at 235, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.
Regarding step 3:
Baek teaches that in the second culturing step (corresponds to step 3 of instant claim 1), the culture of the first culturing step (corresponds to step 2 of instant claim 1) may be transferred to a new container (para 38). Baek teaches that in the second culture step, the cells may be cultured for 3 days to 6 days (para 39). Baek teaches that the culture of PBMCs is carried out in a normal cell culture condition, which is at about 37℃ in a CO2 incubator (para 29) (reads on lines 8-9 of step 3).
Regarding amounts of cell culture suspension from the first culture, RPMI, and albumin:
In a working example, Baek teaches that approximately 53 mL of medium and were added to 27 mL of the cell culture suspension from the primary culture (para 123), with the final culture solution comprising 9 mL of plasma, which comprises albumin (para 123).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have optimized the amount of cell culture suspension from the first culture, the amount of medium, and the amount of albumin in the culturing solution based on factors such as the desired number of cells at harvest, to arrive at the claimed invention. See MPEP 2144.05(II)(A). As noted in In re Aller, 105 USPQ 233 at 235, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.
The limitations regarding the concentrations for IL-2, IL-15, and the anti-NKp30, anti-NKp46, and anti-CD56 antibodies are rendered obvious over Baek, in view of CN105219713A, Lee, Shin, and Romagne, as discussed for step 2 above.
Baek does not teach a secondary subculturing step to obtain a secondary subculture suspension (lines 6-9 of step 3).
Arango teaches that cells should be subcultured in a series of passages in order to keep the best condition for cell growth (p3, para 3).
It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Baek by subculturing the cells obtained in the second culturing step (corresponds to step 3 of instant claim 1). One of ordinary skill in the art would have been motivated to make this modification because Arango teaches that subculturing cells keeps the cells in the best condition for growth. One of ordinary skill in the art would have had a reasonable expectation of making this modification because Arango teaches that cells isolated from a tissue can be subcultured.
It would have been further prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have optimized the amount of cell culture suspension from the primary subculture, the amount of medium, and the amount of plasma in the culturing solution based on factors such as the desired number of cells at harvest, to arrive at the claimed invention. See MPEP 2144.05(II)(A). As noted in In re Aller, 105 USPQ 233 at 235, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.
Regarding step 4: The third step of culture taught in Baek (para 40-41) corresponds to step 4 of instant claim 1. Baek teaches that “each culture step may have the same or different culture medium components, culture vessels, and culture periods” (para 30). Therefore, the third step of culture taught in Baek may comprise may comprise the same culture medium as the medium used in previous steps. As set forth above for steps 2-3 of instant claim 1, the use of a medium comprising RPMI medium, albumin, anti-NKp30 antibody, anti-NKp46 antibody, anti-CD56 antibody, 50 ng/mL IL-2, and 50 ng/mL IL-15, is rendered obvious over Baek, in view of CN105219713A, Lee, Shin, and Romagne.
Response to Arguments
Culturing in Anti-CD16 Antibody-Coated Flask (Step 2)
Applicant argues: First, Baek does not teach the use of an anti-CD16 antibody-coated flask. Baek teaches culturing PBMCs in a culture flask coated with fibronectin and gamma globulin (paras. 102-107). However, Baek does not specify that the gamma globulin used therein is an anti-CD16 antibody. Gamma globulin is a broad class of immunoglobulins, and there is no basis in Baek for concluding that the gamma globulin disclosed therein corresponds to, or functions equivalently to, an anti-CD16 antibody.
In response: Applicant’s arguments have been fully considered, but are not persuasive. The limitation regarding the anti-CD16 antibody-coated flask is taught in Warren. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant argues: Second, Warren does not provide a stable mass culture methodology using an antiCD16 antibody-coated flask. The examiner relies on Warren for the teaching that culturing NK cells on plastic coated with purified anti-CD16 antibody stimulates NK cell activation and division. However, Warren is an experimental study analyzing the effects of CD16 ligation on human NK cells, and its teachings must be considered in their entirety.
Critically, Warren also teaches that CD16 ligation not only promotes NK cell division but simultaneously increases apoptosis in NK cells. Furthermore, Warren indicates that loss of NK progenitor cells may occur in the absence of co-stimulatory signals. Thus, the teaching of Warren, taken as a whole, would not have led a person of ordinary skill in the art to conclude that culturing PBMCs on an anti-CD16 antibody-coated flask would result in stable mass proliferation of NK cells. To the contrary, a person of ordinary skill in the art would have recognized that CD16 ligation alone carries a significant risk of increasing cell death alongside cell division, and would not have had a reasonable expectation of successfully achieving stable mass culture of NK cells using an anti-CD16 antibody-coated flask.
In response: Applicant’s arguments have been fully considered, but are not persuasive. In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, Warren teaches that culturing NK cells on plastic coated with purified anti-CD16 antibody stimulates NK cell activation (p 736, col 2, para 2). In contrast to NK cells grown in control cultures, NK cells growth on anti-CD16 antibody-coated plates showed a heterogeneous pattern of fluorescence indicative of asynchronous cell division (p 740, col 2, para 3). Despite CD16 ligation carrying a risk of increasing cell death alongside cell division, the teachings of Warren that “These data demonstrate unequivocally that CD16 ligation stimulates NK cell division” (p 740, col 2, para 3) provides motivation for an artisan to modify the method of Baek by culturing NK cells in an anti-CD16 antibody-coated incubator. Moreover, the results form Warren show that NK cells can be cultured in an anti-CD16 antibody-coated incubator.
Applicant argues: Third, neither Baek nor Warren, alone or in combination, teaches the specific culture conditions of the claimed invention.
Even assuming that a person of ordinary skill in the art would have been motivated to modify Baek by using an anti-CD16 antibody-coated flask as taught by Warren, the combination of Baek and Warren still fails to teach or suggest the specific culture conditions that are essential to the claimed invention, namely, selecting between a T25 and T75 flask based on cell count, and culturing in a medium comprising the specific combination of IL-2 at a concentration of 20 ng/ml, IL-15 at a concentration of 50 ng/ml, anti-CD56 antibody at a concentration of 5 ng/ml, anti-NKp46 antibody at a concentration of 5 ng/ml, and anti-NKp30 antibody at a concentration of 5 ng/ml. These specific culture conditions, which are essential to achieving stable mass proliferation of NK cells as demonstrated in the examples of the specification, are not taught or suggested by any of the cited references.
In response: Applicant’s arguments have been fully considered, but are not persuasive. The limitations regarding culturing in a medium comprising IL-2 at a concentration of 20 ng/ml, IL-15 at a concentration of 50 ng/ml, anti-CD56 antibody at a concentration of 5 ng/ml, anti-NKp46 antibody at a concentration of 5 ng/ml, and anti-NKp30 antibody at a concentration of 5 ng/ml, are taught in CN105219713A, Lee, Shin, and Romagne, as set forth above. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., “selecting between a T25 and T75 flask based on cell count” and “achieving stable mass proliferation of NK cells”) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Regarding the limitation “selecting between a T25 and T75 flask based on cell count,” this limitation is not recited in the claim. Claim 1, as amended, recites “culturing the isolated PBMCs in a T25 flask coated with an anti-CD16 antibody when the cell number is less than 3x107, or in a T75 flask coated with the anti-CD16 antibody when the cell number is 3x107 or more” (emphasis added). The claim sets forth the use of a T25 or T75 flask as alternative embodiments. As set forth in the rejection above, Baek teaches culturing 3x107 cells in a T75 flask, which satisfies the condition of the second embodiment.
The Specific Culture Conditions of Step 2
Applicant argues: As a preliminary matter, Applicant notes that while antibodies and cytokines known to activate NK cells have been individually identified and catalogued for decades, the development of an effective mass proliferation culture method for NK cells is far more complex than simply selecting from a known list of components. In the experimental sciences, particularly in the fields of chemistry and biology, countless variables interact simultaneously, and outcomes cannot be predicted from theory alone - experimental verification is essential. This is especially true for NK cell mass proliferation, where the goal is not merely to increase the NK cell ratio in a small-scale culture, but to simultaneously achieve a high NK cell ratio and large-scale cell proliferation. Whether mass proliferation is achievable depends critically on the specific types and concentrations of antibodies and cytokines used, as well as the manner in which they are applied. Even an identical combination of components can yield vastly different outcomes depending on the concentrations and treatment protocols employed. Accordingly, the mere fact that individual components are known in the art does not render their specific combination and use conditions obvious.
In response: Applicant’s arguments have been fully considered, but are not persuasive. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant argues: Amended Claim 1, step 2 recites the use of 8 ml of RPMI medium. Baek teaches the use of RPMI 1640 as a culture medium (see paragraph [22] of Baek), but uses approximately 13.5 ml of Alys505NK-EX medium in its working example (see paragraph [111] of Baek), an amount
that differs from the 8 ml of RPMI medium recited in the claimed invention. The examiner's contention that optimizing the amount of medium would have been a matter of routine experimentation is not persuasive, because the amount of medium is not an independently optimizable parameter. It interacts with the concentrations of all other components in the culture, and changing the volume of medium necessarily affects the effective concentrations of all components present. Accordingly, the specific amount of RPMI medium recited in the claimed invention cannot be said to be an obvious optimization.
In response: Applicant’s arguments have been fully considered, but are not persuasive. A cell culture medium interacts with other components in the culture, which is true of all components in a culture. Although changing the volume of medium affects the effective amount of other components, it does not affect the concentration of said components, which are adjusted according to the amount of medium. An ordinary artisan would understand that the amount of medium used in a cell culture can be optimized.
Applicant argues: CN105219713A teaches culturing PBMCs in a solution comprising IL-2 at a
concentration of 20-50 ng/ml and IL-15 at a concentration of 20-50 ng/ml, and reports optimal concentrations of IL-2 at 35 ng/ml and IL-15 at 35 ng/ml (see claims 1, 6, and 7 of CN105219713A).
The claimed invention, by contrast, determines the optimal concentrations to be IL-2 at 20 ng/ml and IL-15 at 50 ng/ml. These concentrations differ from the optimal values disclosed in CN105219713A. Importantly, CN105219713A employs an entirely different combination of culture components from the claimed invention, and the optimal IL-2 and IL-15 concentrations determined in CN105219713A cannot be expected to apply to the specific culture system of the claimed invention. The optimal concentrations of the claimed invention were determined through systematic experimental verification as described in Example 3 of the specification, wherein IFN-y secretion was measured across a range of IL-2 and IL-15 concentrations. These results confirm that the optimal concentrations are specific to the particular culture system of the claimed invention and cannot be predicted from CN105219713A.
In response: Applicant’s arguments have been fully considered, but are not persuasive. CN105219713A teaches culturing PBMCs in a solution comprising IL-2 at a concentration of 20-50 ng/ml and IL-15 at a concentration of 20-50 ng/ml, which encompasses the concentrations of the instant claim 1. That CN105219713A does not teach 20 ng/mL of IL-2 and 50 ng/mL of IL-15 as the optimal concentrations is immaterial in the absence of unexpected results.
Applicant argues: The examiner contends that Lee teaches the use of 1 to 50,000 ng of anti-CD56 antibody and 1 to 50,000 ng of anti-CD335 (anti-NKp46) antibody for culturing PBMCs. While this range nominally encompasses the concentrations recited in the claimed invention, the range disclosed in Lee is extraordinarily broad. A range of such breadth provides no meaningful guidance to a person of ordinary skill in the art as to what specific concentration would be effective for mass proliferation of NK cells. Identifying the optimal concentration within such a vast range would require an enormous number of experiments. Moreover, as noted above, the effective concentration of each antibody in the culture flask depends on the total volume of the culture medium, which further complicates any attempt to derive the specific concentrations of the claimed invention from the broad ranges of Lee.
In response: Applicant’s arguments have been fully considered, but are not persuasive. The claimed concentration of anti-CD56 antibody and anti-NKp46 antibody are taught in Lee. Moreover, it is noted that the features upon which applicant relies (i.e., concentration effective for mass proliferation of NK cells) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Applicant argues: Romagne teaches that the proliferation-inducing effect of anti-NKp30 antibody reaches saturation at approximately 1 μg/ml (1,000 ng/ml), and that the dose required to achieve 50% of the maximum effect is below 0.1 μg/ml (100 ng/ml) in Romagne's particular experiment (see
paragraph [0160] of Romagne). The claimed invention, by contrast, employs anti-NKp30 antibody at a concentration of only 5 ng/ml, which is a concentration that is approximately 200- fold lower than the saturation concentration disclosed in Romagne, and well below the concentration required for 50% of maximum effect as disclosed therein.
This dramatic difference in concentration is not a mere matter of routine optimization. A person of ordinary skill in the art reading Romagne would have had no basis to expect that antiNKp30 antibody at a concentration as low as 5 ng/ml would contribute meaningfully to NK cell mass proliferation. The fact that the claimed invention achieves superior NK cell mass proliferation at this dramatically lower concentration of anti-NKp30 antibody is attributable to the synergistic effect of the complete culture system of the claimed invention, specifically, the combination of the anti-CD16 antibody-coated flask, IL-2 at 20 ng/ml, IL-15 at 50 ng/ml, antiCD56 antibody, anti-NKp46 antibody, and anti-NKp30 antibody, which cannot be predicted from Romagne or any other cited reference considered alone or in combination.
In response: Applicant’s arguments have been fully considered, but are not persuasive. As set forth in the rejection, regarding the result of the titration curve experiment, Romagne teaches that “It should be noted that the characteristics of the curve may depend on the particular antibody used, and particularly of its affinity. The use of humanized anti NCR antibodies may also display a different titration curve” (para 161). Therefore, given that the claimed method does not require use of the particular antibody as the antibody used in Romagne’s experiment, an ordinary artisan would understand that the difference between the claimed concentration and the concentration taught in Romagne is a matter of routine optimization.
The Subculturing Protocol of Step 3 is Not Taught or Suggested by the Cited References, and Differs Fundamentally from Conventional Subculturing
The Subculturing Steps of the Claimed Invention is Not Conventional
Applicant argues: While subculturing generally refers to transferring cells to fresh medium at appropriate intervals to sustain cell proliferation, the subculturing steps of the claimed invention go fundamentally beyond conventional subculturing. In conventional subculturing, spent medium is entirely replaced with fresh medium. In the claimed invention, however, a portion of the previously conditioned culture medium is deliberately retained and carried forward into each subsequent subculture together with fresh medium. This is because NK cells secrete critical factors, including IFN-y, TNF-a, and Granzyme B, into the culture medium as they proliferate, and maintaining the concentrations of these secreted factors across successive subcultures is essential for sustaining NK cell proliferation and achieving mass culture. This approach is not taught or suggested by the cited references, and is a non-obvious and critical feature of the claimed invention.
In response: Applicant’s arguments have been fully considered, but are not persuasive. As set forth in the rejection above, the primary reference, Baek, teaches “a portion of the previously conditioned culture medium is deliberately retained and carried forward into each subsequent subculture together with fresh medium,” as stated by Applicant (e.g., see paragraph 32 of Baek: “In one embodiment, the method for proliferating NK cells of the present invention may comprise: a first culture step of adding and culturing peripheral blood mononuclear cells, first interleukin, second interleukin, anti-NKp46antibodies, and plasma to a culture container coated with gamma globulin and fibronectin; and a second culture step of further adding and culturing a culture solution containing first interleukin, second interleukin, anti-NKp46antibody, and plasma to the culture obtained from the first culture step” (emphasis added), and paragraphs 37-38 of Baek: “In one embodiment, the method for proliferating NK cell cells according to the present invention includes a second culture step of further adding a culture solution including first interleukin (IL -2), second interleukin (IL -12, IL -15, and IL -18), anti-NKp46 antibody, and plasma to a culture obtained from the first culture step. In the second culturing step, the culture of the first culturing step may be transferred to a new container” (emphasis added).).
Moreover, it is noted that the features upon which applicant relies (i.e., that NK cells secrete critical factors, including IFN-y, TNF-a, and Granzyme B, into the culture medium as they proliferate, and maintaining the concentrations of these secreted factors across successive subcultures is essential for sustaining NK cell proliferation and achieving mass culture) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
The Specific Compositions and Volumes Differ from the Cited References
Applicant argues: The specific compositions and volumes of the subculturing steps of the present invention are entirely different from those of any of the cited references. Specifically, the claimed invention employs 20 ml of cell culture suspension and 72 ml of RPMI medium in the primary subculture, and 50 ml of primary subculture suspension and 180 ml of RPMI medium in the secondary subculture, whereas Baek, the primary reference, teaches the use of approximately 27 ml of cell culture suspension and approximately 53 ml of medium (see paragraph [0123] of Baek). Moreover, the overall culture compositions, including the specific antibodies and their concentrations, the amount of albumin, and the culture durations, of the claimed invention are not taught or suggested by any of the cited references, individually or in combination.
In response: Applicant’s arguments have been fully considered, but are not persuasive.
CN105219713A (D6) Does Not Suggest That the Combination of Antibodies, IL-2 at 20 ng/ml, and IL-15 at 50 ng/ml is Effective for Mass Proliferation of NK Cells
Applicant argues: First, the overall culture compositions of CN105219713A and the claimed invention are entirely different. CN105219713A discloses an in vitro NK cell expansion method using aserum-free culture medium supplemented with IL-2 and IL-15. Apart from IL-2 and IL-15, the culture compositions of CN105219713A are entirely different from those of the claimed invention, which employs an anti-CD16 antibody-coated flask together with a specific combination of anti-CD56, anti-NKp46, and anti-NKp30 antibodies. The optimal concentrations of IL-2 and IL-15 are necessarily dependent on the overall culture composition in which they are used, and the optimal values determined in CN105219713A cannot be expected to apply to the fundamentally different culture system of the present invention. Indeed, CN105219713A discloses optimal concentrations of IL-2 at 35 ng/ml and IL-15 at 35 ng/ml, whereas the claimed invention determines the optimal concentrations to be IL-2 at 20 ng/ml and IL-15 at 50 ng/ml, which are concentrations that differ from those of CN105219713A and were derived through systematic experimental verification as described in Example 3 of the specification.
In response: Applicant’s arguments have been fully considered, but are not persuasive. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). The argument regarding the concentrations of IL-2 and IL-15 have been addressed above.
Applicant argues: Second, CN105219713A does not teach or suggest that the combination ofIL-2 and IL-15 with anti-CD56, anti-NKp46, and anti-NKp30 antibodies is effective for NK cell mass proliferation. As demonstrated in Tables 2 and 3 and Figures 4 and 7 of the specification, the combination of antibodies (anti-CD56, anti-NKp46, and anti-NKp30) together with IL-2 and IL-15 achieves a dramatically superior NK cell ratio and cell proliferation compared to any control group, including groups treated with IL-2 and IL-15 alone, or with antibodies alone. Specifically, the combination of antibodies with IL-2 + IL-15 achieved an NK cell ratio of 40.4% after 7 days (Table 2), compared to 18.1 % for IL-2 + IL-15 alone, and an NK cell ratio of 83.2% after 14 days of mass culture (Table 3), compared to 42.4% for IL-2 + IL-15 alone. These results demonstrate that the superior performance of the claimed invention is attributable not merely to the use of IL-2 and IL-15, but critically to their combination with the specific antibodies of the claimed invention. This synergistic effect cannot be predicted from or suggested by D6, which does not employ any of the antibodies used in the present invention.
In response: Applicant’s arguments have been fully considered, but are not persuasive. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant argues: Third, CN105219713A does not teach mass proliferation of NK cells. The claimed invention is characterized not only by an improved NK cell ratio, but by the achievement of large-scale NK cell proliferation, as demonstrated in Example 6 of the specification, wherein cells proliferate to more than 2x109 cells after 14 days of culture. CN105219713A provides no teaching or suggestion that its culture system is capable of achieving such mass proliferation. The claimed invention achieves remarkable and unexpected results in NK cell mass proliferation, including an NK cell ratio of 83.2% and a total cell count exceeding 2x109 after 14 days of culture.
In response: Applicant’s arguments have been fully considered, but are not persuasive. In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., mass proliferation of NK cells and the achievement of large-scale NK cell proliferation, including an NK cell ratio of 83.2% and a total cell count exceeding 2x109 after 14 days of culture) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Conclusion
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Risa Takenaka whose telephone number is (571)272-0149. The examiner can normally be reached M-F, 12-7 EST.
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/RISA TAKENAKA/Examiner, Art Unit 1632
/TITILAYO MOLOYE/Primary Examiner, Art Unit 1632
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