DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 06/02/2025 has been entered.
Priority
The present application was filed 02/05/2023 and claims benefit under 35 U.S.C. 119(3) to national stage entry of PCT/US2021/060572, 11/23/2021, which in turn claims priority to provisional application PRO 63/117,767, filed 11/24/2020.
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 63117767, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. Claim 6 recites “wherein the TILs are rapid-expansion phase (REP) TILs.” Although the prior-filed application does recite TILs as effector cells, the prior filed application does not disclose that the TILs are in rapid-expansion phase. Therefore claim 6 does not comply with one or more conditions for receiving the benefit of the earlier fling date.
The effective filing date of claim 6 is 11/23/2021.
Status of the Claims
Claims 2-14 and 16-30 are pending. Claims 2-3, 11, 23, 28, and 30 are amended. Claim 22 is withdrawn and claims 1 and 15 are cancelled. Claims 2-14, 16-21, and 23-30 are examined below.
Withdrawn Objections/Rejection
The rejection of claim 30 under 35 U.S.C. 112(b), lack of antecedent basis, has been withdrawn due to the amendment of the claim.
The objection to the specification is withdrawn due to the amendment of the specification.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 2-14, 16-21, and 23-30 is 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 is vague and indefinite because it recites “a plurality of dissociated cells”. It is unclear whether “a plurality” refers to more than one of the same cell type, for example a plurality of cancer cells, or more than one cell type, for example cancer cells and other cell types found in the tumor microenvironment. Therefore the claim is indefinite.
Claim 28 is indefinite because of the term “primary tumor tissue environment” because it is unclear what area or cell types are encompassed in the language “primary” tumor tissue environment.
In the interest of compact prosecution, the term “primary tumor tissue environment” is interpreted as non-cancerous cells in the tumor and as such are comprised in the biopsy tissue.
The following rejection of claim 28 under 35 U.S.C. 112(b) is maintained for the reasons of record reiterated below:
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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 2, 4-5, 7-13, 16-21, 23-26, and 28 are rejected under 35 U.S.C. 103 as being unpatentable over Kong et al. Tumor-infiltrating lymphocyte function predicts response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer. JCO Precision oncology. 2018 Nov;2:1-5 (see PTO-892, 05/23/2024), in view of Wu et al. Rapid microfluidic formation of uniform patient-derived breast tumor spheroids. ACS Applied Bio Materials. 2020 Aug 2;3(9):6273-83 (see PTO-892, 05/23/2024), Mayer B, US20090325178A1 (see PTO-892, 12/16/2024), and Dijkstra et al. Generation of tumor-reactive T cells by co-culture of peripheral blood lymphocytes and tumor organoids. Cell. 2018 Sep 6;174(6):1586-98. (2018) Cell (see PTO-892, 05/23/2024).
Regarding claims 2 and 28, Kong teaches a method to test tumor-infiltrating lymphocyte (TIL; effector immune cells) cytotoxicity (Kong, page 1, Abstract, lines 3-4). Kong further teaches processing fresh tumor biopsy specimen into tumoroids (forming patient derived organoids) and further teaches taking biopsy specimen for expanding tumor infiltrating lymphocytes (Kong, especially page 3, ‘Tumoroid and TIL Expansion’; online data supplement, page 3, lines 1-2). Kong teaches that tumoroids comprise approximately 500 cells (plurality of cells; Kong, page 7, Figure 1 legend, line 2). Kong further teaches that the tumoroids are embedded in Matrigel® (Kong, page 7, Legend Figure 1, lines 2-3). Kong further teaches expanding the tumoroids in 96-well plates and coculturing them with matched tumor-infiltrating lymphocytes in appropriate media (co-culturing organoids and effector immune cells) and quantifying tumoroid death by adding two fluorescent dyes to the wells, an apoptotic marker and a cell death marker, and measuring fluorescence (quantifying tumor cell killing; Kong, page 3, ‘Immune Cytotoxicity Assay Analysis’, see entire first paragraph; online data supplement, pages 2-3). Regarding the limitation of determining potency, the specification recites on page 19, paragraph [0086], a Micro-Organosphere assay for immune cytotoxicity to measure potency which can be used to quantify effector immune cell toxicity against matched tumor cells. As such the method of Kong evaluating tumor-infiltrating lymphocyte cytotoxicity (Kong, page 1, Abstract, lines 3-4) comprising the quantification of tumoroid death (Kong, page 3, ‘Immune Cytotoxicity Assay Analysis’, lines 17-19) meets said limitation of the claim.
Kong teaches tumoroids, which would be considered organoids, but fails to specifically teach “droplet” organoids. Furthermore, Kong fails to teach forming the tumoroids in a microfluidic device. Kong further fails to teach organoids from a plurality of dissociated cells from a primary tumor tissue (claim 2) where the dissociated cells are combined to include both cancer and normal tissues (claim 28). Kong further fails to teach assessing immune cells for immunotherapy in which the effector immune cells are to be administered to a patient.
Wu teaches a method of generating three-dimensional tumor spheroids (i.e., analogous to the tumoroids of Kong; droplet organoids) by employing droplet microfluidics (in a microfluidic device) and scaffold materials, encapsulating breast tumor cells from patient into a large number of Matrigel®-in-oil droplets (Wu, Abstract, lines 4-8; Fig. 1; sections 3.2-3.3). Wu further teaches suspending a cell pellet in culture medium with 50% Matrigel® and adding drops to a 24-cell culture plate (dispensing the droplet organoids into a well; Wu, page 6276, ‘2.9. Conventional Organoid Formation’, lines 12-15). Wu further teaches that the method of rapidly fabricating uniform patient-derived tumor spheroids in vitro can accurately guide clinical therapeutics because the tumor spheroids recapitulate histological, pathological, and molecular characteristics of the original patient’s cancer (Wu, page 6280, 2nd column, lines 7-13) and the method is rapid and reliable; as compared with conventional methods of tumor organoid formation, Wu’s droplet method is indicated to result in more rapid growth of tumor spheroids (section 3.3, lines 1-17; see also Fig. S4). Wu further teaches that the method can be used for screening drugs and other assays (Wu, page 6278, lines 9-10; abstract). Like Kong, Wu also envision use of the generated droplet organoid products for the purposes of toxicity testing and treatment testing (Wu, ‘Abstract’, lines 15-16).
Mayer teaches methods for testing the response of spheroids to exposure with therapeutics, where the spheroids are derived from primary isolate biological tissues (Mayer, ‘Abstract’, lines 1-4). Mayer further teaches that a spheroid refers to an aggregate of cells cultured to allow three-dimensional growth and that it may comprise a single cell type (homotypic) or more than one cell type (heterotypic; a plurality of dissociated cells; Mayer, page 2, see entire paragraph [0020]). Mayer further teaches that the tissue which may be used for spheroid preparation may be biological tissue afflicted with a disease such as a tumor tissue (Mayer, page 2, paragraph [0026]). Still further, Mayer teaches that the multicellular spheroids can be used for cytotoxicity studies (Mayer, page 5, paragraph [0061], lines 1-5). Mayer further teaches that although homotypic spheroids are able to closely mimic the in vivo morphology, some of the biological complexity is lost. Thus, by co-culture of more than one cell type in a spheroid, tumor cell interactions with other cell types reflecting natural cell interaction in vivo can be established better representing the in vivo environment (Mayer, page 5, paragraph [0055], lines 8-14). Mayer further teaches that suspensions of single cell lines may be combined with other cells, for example primary cells generated from benign and/or malignant tissues (Mayer, page 5, paragraph [0056], lines 1-4). As such, Mayer teaches combining tumor cells with other (non-tumor) cells from benign or tumor tissue and as such teaches combining both cancer and normal tissues from primary tumor tissue (claim 28).
Even though Mayer does not explicitly teach combining tumor and non-tumor tissue from the same tumor environment, it does teach that the spheroids are derived from primary isolate biological tissue and refer to an aggregate of cells that can comprise more than one cell type and it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined tumor and non-tumor tissue from the same tumor environment tissue because this would most closely model the natural tumor environment.
Dijkstra teaches that adoptive transfer of ex-vivo-expanded autologous tumor-infiltrating lymphocytes has shown impressive clinical responses in melanoma and an early clinical signal in cervical cancer but that despite these encouraging results, a large fraction of patients does not respond to current immunotherapies. Dijkstra further teaches that it is currently challenging to predict whether an individual patient will be sensitive to immunotherapy (Dijkstra, page 1586, ‘Introduction’, see entire first paragraph). Dijkstra further teaches autologous T cell-tumor organoid co-culture platform for individual patients to assess tumor cell killing of peripheral blood lymphocyte derived tumor-reactive T cells (Dijkstra, page 1587, see entire 2nd paragraph). Dijkstra further teaches the high value of a patient-specific model system for the implementation of personalized medicine, especially in immuno-oncology, given the inherent diversity in human leukocyte antigen and T cell receptor genes, the private nature of the neo-antigens expressed in human cancers, and the multifactorial nature of T cell-mediated tumor destruction (Dijkstra, page 1593, ‘Discussion’, lines 1-9). Put another way, Dijkstra teaches the importance of a patient specific model system, an autologous T cell-tumor organoid co-culture platform, which can be applied to evaluate whether an individual cancer patient responds to immunotherapy, and further teaches that immunotherapy in cancer comprises the adoptive transfer of ex-vivo-expanded autologous tumor infiltrating lymphocytes.
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Kong of testing tumor-infiltrating lymphocytes using tumoroids by first preparing the tumoroids in a microfluidic device, as taught by Wu, thereby resulting in three-dimensional tumor spheroids in droplets (i.e., droplet organoids) because of the teaching of Wu that the spheroids recapitulate histological, pathological, and molecular characteristics of the original patient’s cancer and that the method of generating the spheroids is reliable and rapid. One of ordinary skill in the art would have been motivated to perform Kong’s methods of quantifying tumor-infiltrating lymphocyte infiltration and tumor death by co-culturing cells with tumor organoids in Matrigel®, using tumor organoids prepared by the methods of Wu, so that the natural cancer would be more faithfully recapitulated in this in vitro model system allowing for drug responses to be accurately tested in Kong’s cytotoxicity assays, as well as for the stated benefits of being a rapid and reliable way to form tumoroids for testing treatments. These advantages of recapitulating the original patient’s cancer characteristics and rapidity, as taught by Wu, would have been particularly desirable as Kong indicates that current disadvantages include that their assay is time-consuming and does not fully represent the [tumor microenvironment] (online data supplement, Table S3). Accordingly, one of ordinary skill in the art would have been motivated to apply Wu’s known microfluidic technique for forming droplet organoids in order to improve the similar tumoroid-based methods of Kong.
The ordinary artisan would have a reasonable expectation of success, because Kong teaches success studying tumor cell killing by TIL in tumor organoids comprising Matrigel® and Wu teaches a method of preparing tumor cell organoids comprising Matrigel® in a rapid and reliable manner.
It would have further been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Kong in view of Wu to combine tumor with non-tumor tissues in the droplet organoid because of the teaching of Mayer that co-culturing of tumor cells with other cells more closely represent the in vivo environment and biological complexity and can be used in cell cytotoxicity assays.
The ordinary artisan would have a reasonable expectation of success in doing so because both Kong and Mayer teach three-dimensional cell spheroids derived from primary tissues that can be used in cytotoxicity assays.
It would have further been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have applied the method of Kong to assess tumor infiltrating lymphocytes for immunotherapy comprising transfer of autologous cells into patients because of the teaching of Dijkstra of the high value of a patient-specific model system for the implementation of personalized medicine, especially in immuno-oncology, given the inherent diversity in human leukocyte antigen and T cell receptor genes, the private nature of the neo-antigens expressed in human cancers, and the multifactorial nature of T cell-mediated tumor destruction and because even though adoptive transfer of ex-vivo-expanded autologous tumor-infiltrating lymphocytes has shown impressive clinical responses in melanoma, a large part of patients does not respond to current immunotherapies.
The ordinary artisan would have a reasonable expectation of success in applying the method of Kong because both Kong and Dijkstra teach assessing the cytotoxicity of tumor specific lymphocytes using co-culture of tumor specific cytotoxic T lymphocytes with an autologous tumoroid.
Regarding claims 4 and 5, Kong teaches a method to test tumor-infiltrating lymphocyte cytotoxicity (Kong, page 1, Abstract, lines 1-4).
Regarding claim 7, Kong teaches a cytotoxicity assay of tumoroids cocultured with patient-matched tumor-infiltrating lymphocytes (Kong, page 1, Abstract, ‘Patients and Methods’, lines 3-4).
Regarding claim 8, Kong teaches co-culturing the tumoroids and tumor-infiltrating lymphocytes on a microscopy-grade plate and adding two fluorescent dyes (Kong, page 3, 2nd column, lines 1-12). Kong further teaches imaging at least 10 tumoroids every 2 hours for 48 hours (in real time; Kong, page 3, 2nd column, 2nd paragraph, lines 6-9).
Regarding claim 9, Kong teaches labeling the cells with Caspase-3/7 (Kong, page 3, 2nd column, line 14).
Regarding claim 10, Kong teaches the method comprising four wells, two negative controls with tumoroids alone (absence of effector cells; Kong, page 3, 2nd column, 2nd paragraph, lines 1-4).
Regarding claim 11, the combination of Kong and Wu as applied to claim 2 also applies to claim 11.
Kong teaches that the tumoroids are embedded in Matrigel® matrix (Kong, page 7, Legend Figure 1, lines 2-3; online data supplement, page 2, ‘Passaging of tumoroids for cytotoxic assay’).
Kong does not teach polymerizing droplets.
Wu teaches dissociating a solid mouse tumor by dissecting and cutting into small pieces, washing and centrifuging the cells, and suspending the pellet in cold breast organoid culture medium with 50% Matrigel® (unpolymerized) and adding the drops of the cell suspension onto prewarmed 24-well culture plates fat 37°C for 30 min (Wu, Fig. 1, sections 2.6-2.7; page 6276, ‘2.9. Conventional Organoid Formation’, lines 12-15). Wu further teaches that Matrigel® is a temperature-sensitive material that polymerizes to produce a gel through incubation at 37°C (Wu, page 6275, lines 5-7). Wu further teaches droplets containing between 12 cells per droplet (at 1 million cells/ml) of a breast tumor cell line (Wu, page 6276, 2nd column, lines 2-3).
Accordingly, it would have been further obvious to arrive at the claimed steps when applying the microfluidic methods of Wu to prepare the droplet organoids for use in the methods of Kong.
Regarding claims 12 and 25, the combination of Kong and Wu as applied to claim 11 also applies to claim 12.
Wu teaches that tumor cells were encapsulated into Matrigel®-in-oil droplets (Wu, ‘Abstract’, lines 7-8). As such, Wu teaches that the dissociated tissue sample comprises not stem cells.
Regarding claims 13 and 26, the combination of Kong and Wu as applied to claim 11 also applies to claim 13.
Both Kong and Wu teach Matrigel®, which, as evidenced by the instant specification on page 9, is a substrate basement membrane matrix. Therefore, when performing the combined methods of Kong and Wu using Matrigel®, the recited features would necessarily flow from the reference teachings even though this matrix is not expressly described in the reference as a “basement membrane” matrix. For instance, Wu teaches suspending the pellet in cold breast organoid culture medium with 50% Matrigel® (Wu, especially section 3.2; page 6276, ‘2.9. Conventional Organoid Formation’, lines 12-15). Note that Wu does teach that Matrigel® is derived from organisms’ extracellular matrix (Wu, page 6274, 2nd column, 2nd paragraph, lines 7-9).
Regarding claims 16-19, the combination of Kong and Wu as applied to claim 11 also applies to claims 16-19.
Wu teaches a method of using a microfluidic device to prepare spheroids of breast cancer cell line cells, seeding the droplets with cell suspensions at a concentration of 1 x 106, 2.5 x 106, 5 x 106, 1x 107, and 1.5 x 107 cells/ml (Wu, page 6277, 3.2. ‘Generation of Matrigel Droplets’, 2nd paragraph, line 13). Wu further teaches preparing droplets with cell suspensions of dissociated tumor cells at a concentration of 3 x 107, (Wu, page 6277, line 13). Still further Wu teaches that by tuning flow rate and cell concentration, cell numbers per droplet can be precisely controlled from several hundred to several thousand cells (Wu, page 6277, lines 20-22). Still further Wu teaches encapsulating the tumor cells into a liquid Matrigel® droplet at 4°C (Wu, page 6275, 2nd paragraph, lines 5-6), whereby Matrigel® reads on the claimed “fluid matrix material.”
Regarding the limitations “a density of less than 1 x 107” (claim 16), “a density of less than 5 x 106” (claim 17), “a density of less than 1 x 106” (claim 18), or “a density of 5 x 105” (claim 19), as presently recited in the claims; it has been previously established by the courts that a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art, but are merely close (see MPEP 2144.05). In the present case, at less than a density of 5 x 106 (claim 17), 1 x 106 (claim 18), or 5 x 105 (claim 19) is a range that is substantially close/similar to 1 x 106, 2.5 x 106, 5 x 106, 1x 107, and 1.5 x 107 cells/ml, as taught by Wu and as such it would have been considered prima facie obvious in view of Wu to have arrived at the presently claimed cell densities out of the course of routine experimentation, in order to achieve a desired number of cells per droplet. The courts have also held that a particular parameter, when recognized as a result effective variable, i.e., a variable that achieves a recognized result, may be subject to routine optimization in order to determine optimum or workable ranges of said variable (also MPEP 2144.05). In the present case it would have been prima facie obvious to one of ordinary skill in the art to have determined the optimum tumor cell number in the cell suspension by optimizing what was known in the art to be a suitable cell concentration as taught by Wu. Wu teaches that it was routine in the art to use a cell concentration of 1 to 10 million cells/ml. It would have been obvious to have arrived at the claimed range between 0.5 and 10 million cells/ml as an obvious matter of routine experimentation because the cell number is an art recognized result effective variable (namely the variable which results in a certain number of cells per droplet). One of ordinary skill in the art would have a reasonable expectation of success because a cell number of between 1 to 10 million cells/ml is substantially similar to 0.5 and 10 million cells/ml. One of ordinary skill would expect success optimizing by routine experimentation because such optimization would allow the operator to achieve the desired cell number per drop and further because optimization is well within the skill level of the ordinary artisan.
Regarding claim 20, the combination of Kong and Wu as applied to claim 2 also applies to claim 20.
Wu teaches dissociating patient breast cancer tissue (dissociated tumor cells; Wu, page 6276, see ‘2.7. Patient Breast Cancer Dissociation”).
Regarding claim 21, the combination of Kong and Wu as applied to claim 2 also applies to claim 21.
Wu teaches dissociating a solid mouse tumor washing and centrifuging the cells and suspending the pellet in cold breast organoid culture medium with 50% Matrigel® (unpolymerized) and adding the drops of the cell suspension onto prewarmed 24-well culture plates fat 37°C for 30 min (Wu, page 6276, ‘2.9. Conventional Organoid Formation’, lines 12-15). Wu further teaches that Matrigel® is a temperature-sensitive material that polymerizes to produce a gel through incubation at 37°C (Wu, page 6275, lines 5-7). Wu further teaches generation of Matrigel® droplets by fixing the flow rate for the highest flow and that this allows fabrication of at least 1000 droplets within 1 min (continuous flow) and that the diameter of the formed spheroids was 378.13 ± 3.1 μm (less than 25% variation in size; Wu, page 6277, ‘Generation of Matrigel Droplets’, 2nd paragraph, lines 6-10).
Regarding claims 23 and 24, the combination of Kong and Wu as applied to claim 2 also applies to claims 23 and 24.
Wu teaches that microfluidic droplet diameter, generation yield, and cell number per droplet were controlled by changing the velocity ratio between the aqueous and oil phases. Thus, uniform spheroids can be fabricated with the desired size (Wu, page 6277, lines 18-24). Wu further teaches generating spheroids that are 278.13 ± 3.1 μm in diameter (Wu, page 6277, 3rd paragraph, lines 7-9). Figure 2 further shows droplets that are between about 250 and 350 μm in diameter (Wu, page 6277, figure 2). Wu further teaches that tumor spheroids formed from primary mouse tumor using the method were about 200 μm in diameter within 1 day (Wu, page 6279, 2nd column, line 3).
Regarding the limitations “between 50 μm and 400 μm” (claim 23) and “between 50 μm and 200 μm” (claim 24) as presently recited in the claims, see specifically 278 μm lies inside of, and as such addresses, the range of claim 23 (50-400 μm). Further, it has been previously established by the courts that a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art, but are merely close (see MPEP 2144.05). In the present case, 278 μm (as taught by Wu) is a value that is substantially close/similar to between 50 μm and 200 μm as claimed, and as such it would have been considered prima facie obvious in view of Wu to have arrived at the presently claimed diameters between 50 μm and 200 μm from routine optimization of experimental conditions, namely by optimizing conditions to uncover the optimum workable for tumor cell killing. The diameter of the spheroid is a result effective variable, as it is the case that the diameter of the spheroid is correlated with the number of tumor cells in the spheroid, and based on the cited art, is manipulated based on the days of formation (e.g., about 200 μm in diameter within 1 day, as taught by Wu). The courts have also held that a particular parameter, when recognized as a result effective variable, i.e., a variable that achieves a recognized result, may be subject to routine optimization in order to determine optimum or workable ranges of said variable (also MPEP 2144.05). In the present case, it would have been prima facie obvious to one of ordinary skill in the art to determine the optimum spheroid size to achieve an optimal cell number, thereby arriving at between 50 μm and 400 or 200 μm by optimizing what was known in the art to be a suitable spheroid size for executing a cell cytotoxicity assay using tumor infiltrating lymphocytes and tumor organoids, as taught by Kong and Wu.
It would have been prima facie obvious to have arrived at the claimed diameter of between 50 μm and 200 μm as an obvious matter of routine optimization of experimental conditions because the diameter is an art recognized result effective variable (namely the variable which results in a certain cell number, depends on the days formed/conditions of formation). One of ordinary skill in the art would have a reasonable expectation of success because a diameter of 278 μm is substantially close to a diameter of between 50 μm and 200 μm. One of ordinary skill in the art would expect success optimizing by routine experimentation because such optimization would allow the operator to achieve the optimum cell number and further because Wu teaches that by changing the velocity ratio between the aqueous and oil phases diameter and cell number can be controlled and further because optimization is well within the skill of the ordinary artisan.
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Kong et al. in view of Wu et al., Mayer et al. and Dijkstra et al. as applied to claim 2 above, and further in view of Galeano Niño et al. Antigen‐specific T cells fully conserve antitumour function following cryopreservation. Immunology and cell biology. 2016 Apr;94(4):411-8.
Regarding claim 3, Kong and the cited art above teach a method of determining the potency of tumor cell killing comprising a patient derived droplet organoid substantially as claimed.
Kong does not teach freezing and storing the effector or droplet organoids.
Galeano Niño teaches cryopreservation of tumor-reactive CD8+ T cells (Galeano Niño, page 411, abstract, lines 4-5). Galeano Niño further teaches that there is increasing evidence that T cell function is progressively lost during extended ex vivo culture and that multiple administrations of adoptively transferred T cells are more effective than single infusions in immunotherapies targeting cancers and that T cells isolated at early stages of the disease respond to tumors more efficiently than T cells isolated at later stages and therefore cryopreservation of ex vivo-expanded T cells at an early stage could constitute a beneficial strategy to store functional T cells for prolonged periods and would allow for their delayed or repeated use during the course of therapy (Galeano Niño, page 411, lines 3-4 and 19-35). Galeano Niño further teaches that cryopreserved lymphocytes can infiltrate tumors at the same rate as fresh lymphocytes (Galeano Niño, page 415, Figure 3 (d) and legend, lines 8-9). Galeano Niño further shows that cryopreserved T cells reject tumors with the same efficiency as freshly isolated ones (Galeano Niño, page 414, 7th paragraph, lines 28-30).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Kong of testing effector immune cells using tumoroids by cryopreserving (freezing and storing) the tumor-infiltrating lymphocytes as taught by Galeano Niño because of the teaching of Galeano Niño that T cell function is progressively lost during extended ex vivo culture and that T cells isolated in early stages of the disease respond to tumors more efficiently than T cells isolated at later stages. The ordinary artisan would have been motivated to do so because storing functional T cells for prolonged periods would allow for their delayed or repeated use during the course of therapy.
One of ordinary skill in the art would have a reasonable expectation of success modifying the method of Kong by freezing and storing effector lymphocytes because of the teaching of Galeano Niño that cryopreserved tumor specific T cells reject tumors with the same efficiency as freshly isolated ones.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Kong et al. in view of Wu et al., Mayer et al., and Dijkstra et al. as applied to claim 5 above, and further in view of and Dudley et al. Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients. Journal of immunotherapy. 2003 Jul 1;26(4):332-42. (see PTO-892, 05/23/2024).
Regarding claim 6, Kong and the cited art above teach a method of determining the potency of tumor cell killing comprising a patient derived droplet organoid substantially as claimed.
Kong fails to teach that the tumor infiltrating lymphocytes are rapid expansion phase tumor infiltrating lymphocytes.
Dijkstra teaches a method of inducing and analyzing tumor-specific T cell responses in a personalized manner which can be used to enrich tumor-reactive T cells from peripheral blood of patients (Dijkstra, page 1586, ‘Summary’, lines 6-12). Dijkstra further teaches that adoptive transfer of ex-vivo-expanded autologous tumor infiltrating lymphocytes has shown impressive clinical responses in melanoma, but that nevertheless a large fraction of patients does not respond to current immunotherapies. Dijkstra further teaches that it is presently challenging to predict whether an individual patient will be sensitive to immunotherapy (Dijkstra, page 1586, ‘Introduction’, see whole paragraph). Dijkstra further teaches a method co-culturing T cell tumor organoid for individual patients, to evaluate tumor cell killing efficiency by T cells (Dijkstra, page 1587, 2nd paragraph, lines 1-15). Dijkstra further teaches co-culturing tumor organoids with autologous tumor reactive T cell populations, which are first expanded using the rapid expansion protocol previously established to generate tumor-infiltrating lymphocyte products for adoptive cell therapy by Dudley et al., 2003. Dijkstra teaches that organoids of epithelial tumors can be used to measure the rate of destruction by autologous T cells (Dijkstra, page 1593, see ‘Tumor Organoids Are Killed by Autologous Tumor-Reactive T cells’).
Dudley teaches methods to generate tumor-infiltrating lymphocyte cultures suitable for adaptive cell therapy of melanoma patients that comprise optimizing, generating, selecting, and expanding tumor-infiltrating lymphocytes to large numbers and find that increase in cell numbers to therapeutic levels was readily achieved using a single round of a rapid expansion protocol (Dudley, page 333, see 3rd paragraph). Dudley further teaches that one cycle of rapid expansion generated large TIL numbers with high functional activity appropriate for patient treatment (Dudley, page 338, 2nd column, title of 2nd paragraph).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Kong and Wu of determining the potency of TIL tumor cell killing comprising a patient derived droplet organoid with the method of Dijkstra of using rapid expansion T cells in the assay because of the teaching of Dijkstra that adoptive transfer of ex-vivo-expanded autologous tumor infiltrating lymphocytes, expanded with a rapid expansion protocol, have shown impressive clinical responses in melanoma, but many patients do not respond. The ordinary artisan would be motivated to do so, because of the teaching of Dijkstra that the method can be used to evaluate whether T cells from individual patients can efficiently kill patient tumors. One would further be motivated because of the teaching of Dudley that using a rapid expansion protocol for TIL readily generated a large number of TILs with high functional activity in one cycle.
The ordinary artisan would have a reasonable expectation of success evaluating rapid expansion T cells in the method of Kong and Wu because Dijkstra teaches success assessing T cells generated by rapid expansion using a tumor organoid and Dudley teaches success using the rapid expansion method to expand tumor-infiltrating lymphocytes.
Claims 14 and 27 are rejected under 35 U.S.C. 103 as being unpatentable over Kong et al. in view of Wu et al., Mayer et al., and Dijkstra et al. as applied to claims 11 and 2 above, and further in view of Khoo et al. Expansion of patient-derived circulating tumor cells from liquid biopsies using a CTC microfluidic culture device. Nature protocols. 2018 Jan;13(1):34-58. (see PTO-892, 05/23/2024).
Regarding claims 14 and 27, Kong and the cited art above teaches a method of determining the potency of tumor cell killing comprising a patient derived droplet organoid substantially as claimed.
Wu fails to teach a time within which the tissue sample is removed from the patient and combined with the fluid matrix material.
Khoo teaches a method for establishing patient-derived primary cancer cell clusters with demonstrated relevance to patient response from liquid biopsies (Khoo, page 36, 3rd paragraph, lines 1-5). Khoo further teaches processing the liquid biopsy samples by lysing the red blood cells and resuspending the nucleated cell fraction in fresh supplemented medium (combining with fluid) before seeding into the assay (Khoo, page 41, ‘Processing of clinical samples for drug screening’, lines 2-11). Khoo further teaches that it is a critical step to process the samples the samples within six hours to maintain cell viability.
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Kong in view of Wu to have combined the sample with medium within six hours of sample collection because of the teaching of Khoo that this is time frame is critical in order to maintain cell viability.
The ordinary artisan would have a reasonable expectation of success, because both Kong in view of Wu, and Khoo teach a method of maintaining tumor cells in culture.
Claims 29 and 30 are rejected under 35 U.S.C. 103 as being unpatentable over Kong et al. in view of Wu et al., Mayer et al., and Dijkstra et al. as applied to claim 2 above, and further in view of Primo Ramos et al., US20210189336A1 (see PTO-892; 12/16/2024).
Regarding claims 29 and 30, Kong and the cited art above teaches a method of determining the potency of tumor cell killing comprising a patient derived droplet organoid substantially as claimed.
Kong does not teach that the effector immune cells are engineered or in vitro engineered T cells.
Primo Ramos teaches a method of producing genetically engineered T cells expressing Chimeric Antigen Receptors (engineered; CAR T cells) by providing a sample comprising at least one T cell form a subject having cancer, providing at least one cancer cell, selecting an activated T cell using a bispecific T cell engager antibody and genetically engineering the active T cell to produce Chimeric Antigen Receptors (ex vivo engineered; Primo Ramos, page 2, see entire paragraph [0017]). Primo Ramos further teaches that these CAR-T cells can provide highly effective therapies for diverse cancer types, e.g., solid cancers, hematological cancers, and metastatic forms thereof and are also suited for treating cancers that typically do not elicit a strong immune response in a subject and can be personalized to a given subject by generating CAR-T cells that selectively and effectively target the subject’s cancer (Primo Ramos, page 2, see entire paragraph [0015]). Primo Ramos further teaches methods for evaluating activated T cells or CAR-T cells by assay systems comprising 3D cell culture constructs, such as spheroids, to mimic the microenvironment architecture of solid tumors (Primo Ramos, page 62, paragraph [0770], lines 1-6).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Kong in view of Wu to comprise ex vivo engineered T cells as taught by Primo Ramos because of the teaching of Primo Ramos that engineered CAR-T cells can provide highly effective therapies for diverse cancer types which are also suited for treating cancers that typically do not elicit a strong immune response.
The ordinary artisan would have a reasonable expectation of success because Primo Ramos teaches that the engineered cells types can be evaluated in assay systems comprising 3D cell culture constructs such as spheroids and Kong as modified by Wu teaches a T cell assay comprising three-dimensional tumor spheroids.
Response to Arguments
Applicant's arguments filed 02/18/2025 have been fully considered but they are not persuasive.
Applicant argues starting on page 2 that the preamble and the steps of the claim have been amended to comprise “quantifying tumor cell killing […] so as to determine the potency for immunotherapy in which the effector immune cells are to be administered to the patient” and that therefore the claim now comprises an active method step correlating the result with immunotherapy. Applicant further argues that Kong utilizes patients’ tumor infiltrating lymphocytes as a biomarker for response to chemo and radiation to predict outcome or prognosis, but applicant’s method is directed to a therapeutic testing method, testing a patient’s effector immune cells as therapeutics.
This argument is not persuasive.
As explained previously in detail above, the recitation that the effector immune cells are to be administered does not add an active to the method of determining the potency. Rather, the potency is detected with the method as claimed.
The method as claimed requires the steps of
forming patient derived droplet organoids from a plurality of dissociated cells from a primary tumor tissue in a microfluidic device
dispensing one or more of the droplet organoids from the microfluidic device into a well of a plate
co-culturing the one or more droplet organoids and effector immune cells in a medium in the well
quantifying tumor cell killing of one or more of the droplet organoids by the effector immune cells
As discussed previously in detail above, the combination of the prior art teaches all the steps of the method, briefly, Kong teaches processing fresh tumor biopsy specimen into tumoroids (forming patient derived organoids) and coculturing them with matched tumor-infiltrating lymphocytes (co-culturing organoids and effector immune cells) and quantifying tumoroid death (quantifying tumor cell killing).Wu teaches a method of generating three-dimensional tumor spheroids by employing droplet microfluidics and Meyer teaches generating tumoroids comprising a plurality of cells.
The steps of the method would be the same whether the cells are used for diagnostics, immunotherapy, or are to be administered to the patient and as such the recitation of immunotherapy including adoptive transfer of the cells does not add a manipulative difference to the method of measuring potency. Further, Dijkstra teaches a method for and the importance of measuring the potency of tumor specific lymphocytes in immunotherapy including adoptive transfer of tumor specific lymphocytes (see rejection under 35 U.S.C. 103 above).
Applicant argues on page 3 that Kong does not disclose, hint, or suggest working with dissociated primary cells from a tumor tissue to prepare their tumoroid nor with combined primary cells from a tumor tissue and normal tissue from the primary tumor tissue environment and that the office action acknowledges this on page 21 and 22.
This argument is not persuasive.
Kong teaches processing fresh tumor biopsy specimen into tumoroids and therefore teaches forming patient-derived droplet organoids from a plurality of dissociated cells. Kong does not teach that the plurality of cells comprises normal and tumor cells. However, the combination of Kong, Wu, and Mayer does teach tumor derived organoids comprising different cell types from the primary tumor environment as recited in claim 28 and as such teaches a tumor organoid comprising combined dissociated primary cells comprising tumor and normal cells. The office action acknowledges in arguments that Kong and Wu do not teach a tumoroid with a plurality of cells if the cells comprise different cell types (a heterotypic organoid), but rather that Mayer is relied on to teach a heterotypic organoid. Kong and Wu do teach a homotypic organoid comprising a plurality of cells of the same type.
Applicant further argues on page 3 that neither Kong nor Wu recreate the authentic tumor microenvironment and that neither method replicates the specific niches that enable tumor growth in a specific patient.
This argument is not persuasive.
Kong and Wu are not relied on to recreate the authentic tumor microenvironment. Rather, the combination of Kong, Wu, and Mayer are relied on to teach a droplet organoid comprising tumor and normal cells from a patient. As such, the combination of the prior art is teaching a droplet organoid as claimed and therefore the droplet organoid would be expected to behave in the same manner and replicate the authentic tumor microenvironment.
Applicant further argues on page 3 that Wu’s use of single cells to make their spheroids is contrary to Applicant’s method because independent claim 2 recites “from a plurality of dissociated cells from a primary tumor tissue” and that Kong and Wu teach away from using a plurality of dissociated cells.
This argument is not persuasive.
The lack of teaching the use of a plurality of cell types by Kong and Wu does not equal teaching away from applying the method to comprise different cell types. Further, the combination of Kong, Wu, and Mayer supports obviousness of a plurality of dissociated cells from a primary tumor tissue comprising tumor and normal cells.
Applicant further argues (bottom of page 3) that the examiner uses improper hindsight reasoning and that neither Kong nor Wu alone or in combination suggests using a plurality of dissociated primary cells including cells from both tumor and normal tissues.
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). Kong uses a method of assessing potency of tumor infiltrating lymphocytes and Wu teaches a method of preparing droplet tumoroids with improved speed and uniformity therefore providing motivation to improve upon the tumoroids of Kong. Further, neither Wu nor Kong are relied on to teach a plurality of dissociated primary cells including tumor and normal cells but rather Mayer is relied on to establish the obviousness of tumoroids comprising both tumor and normal cells.
Therefore the argument is not persuasive.
Regarding the argument that Mayer does not disclose droplet organoids but rather multicellular spheroids, rather the combination of Kong and Wu is relied on to teach droplet organoids. As explained in detail above, Mayer teaches a spheroid which refers to an aggregate of cells cultured to allow three-dimensional growth and that may comprise more than one cell type. Regarding the argument that Mayer’s methods for testing the response of spheroids to exposure with therapeutics, which by way of non-limiting example may include small molecules or biologics and is therefore a drug screening method, applicant is reminded that a recitation of the intended use of the claimed invention must result in a manipulative difference between the claimed invention and the prior art in order to patentably distinguish the claimed invention from the prior art. Determining the potency of ex vivo engineered effective immune cells would not require a manipulative difference in the method of Kong in view of Wu and Mayer as compared to testing patient derived immune effector cells. Regarding the argument that Mayer does not exemplify any testing or any therapeutics and that it is not clear that Mayer actually made spheroids, the prior art does not have to show possession of the claimed matter, Mayer teaches a spheroid comprising aggregated cells that may comprise more than one cell type and Kong teaches a method of determining the potency of tumor infiltrating lymphocytes and as such, the combination of the art meets the limitation of the claim.
Applicant further argues on page 5 that Mayer teaches dissociated single cells.
This argument is not persuasive.
Mayer does teach dissociating single cells, not culturing single cells. Rather, Mayer teaches “an aggregate of cells cultured to allow three-dimensional growth and that it may comprise a single cell type (homotypic) or more than one cell type (heterotypic)”. Mayer is not relied on to teach a droplet organoid but rather Mayer is relied on to teach an organoid comprising different cell types from the tumor environment.
Applicant further argues on page 4 that Kong, Wu, and Mayer show disparate and contradictory elements because Kong teaches measurement of patient’s tumor-infiltrating lymphocyte function for prognosis and Wu and Mayer teach making spheroids of dissociated single cells for treatment prediction.
This argument is not persuasive.
Even though the intended use of Kong as opposed to Wu and Mayer may be different, the methods are directed to determining cytotoxicity and therefore one would have a reasonable expectation of success in combining the three.
Regarding the argument that none of the references suggest a method administered to a patient, as explained previously above, the recitation of the cells that “are to be administered to the patient” does not add an active step to the method and therefore does not patentably distinguish the method from the prior art and further Dijkstra teaches the importance of determining the potency of tumor specific cells in immunotherapy comprising adoptive transfer of tumor specific T cells.
For all the reasons above, the arguments are not persuasive.
Communication
Any inquiry concerning this communication or earlier communications from the examiner should be directed to STEFANIE J KIRWIN whose telephone number is (571)272-6574. The examiner can normally be reached Monday - Friday 7.30 - 4 pm.
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/STEFANIE J. KIRWIN/Examiner, Art Unit 1677
/ELLEN J MARCSISIN/Primary Examiner, Art Unit 1677