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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 08/28/2025 has been entered.
Priority
The present application was filed 05/20/2021. Acknowledgement is made of the present application as a proper National Stage (371) entry of PCT Application number PCT/EP2019/082672, filed 11/27/2019, which in turn claims foreign priority to EP18306576.2, filed 11/28/2018 filed with the European Patent Office.
Status of the Claims
Claims 2-4, 8, 11-13, 18-19, and 21-25 are pending. Claims 19 and 24 are amended, claim 25 is new and claims 1, 5-7, 9-10, 14-17, and 20 are cancelled. Claims 2-4, 8, 11-13, 18-19, and 21-25 are examined below.
Withdrawn Rejections
The rejection under 35 U.S.C. 103 has been withdrawn due to applicant’s arguments with respect to the primary art (Sarin et al.) being directed to studying apoptotic cell death rather than reparative membrane repair.
See new grounds of rejection 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 19, 2-4, 8, 12-13, 21 and 24 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, 11/01/2024), Keefe et al. Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis. Immunity. 2005 Sep 1;23(3):249-62, in view of Tam et al. Live imaging assay for assessing the roles of Ca2+ and sphingomyelinase in the repair of pore-forming toxin wounds. Journal of visualized experiments: JoVE. 2013 Aug 25(78):50531, , and Jacquelot et al. Predictors of responses to immune checkpoint blockade in advanced melanoma. Nature communications. 2017 Sep 19;8(1):592 (see PTO-892, 11/01/2024).
Regarding claim 19, Kong teaches an assay measuring the cytotoxicity of tumor-infiltrating lymphocytes (Kong, page 1, ‘Purpose’, line 3). Kong further teaches that the assay comprises coculture of tumoroids and tumor infiltrating lymphocytes and quantification of target death by propidium iodide staining under a microscope (contacting immune effector and target cells in the presence of dye; Kong, page 3, ‘Immune Cytotoxicity Assay Analysis”, lines 8-18). Kong further teaches that the tumor infiltrating lymphocytes, i.e., the effector cells where isolated from fresh tumor biopsy specimen (effector cells from a biological sample obtained from the patient; Kong page 3, see ‘Tumoroid and TIL Expansion’). Kong further teaches assessing cytotoxicity in the presence and absence of anti-PD-1 antibody treatment (claim 16-17; Kong, page 7, ‘Assay Evaluation of Checkpoint Inhibition’, lines 5-8). Kong further teaches that the assay can be used to screen for efficacy of a checkpoint inhibitor (determining whether a patient will achieve a response with an immune checkpoint inhibitor; Kong, page 2, 2nd column, lines 4-6). Kong further teaches that in one patient anti-PD-1 antibody treatment resulted in an increase in cytokine production which tracks with improved cytotoxicity of the increased PD-1-expressing TILs (increased cytotoxic response in the presence of the immune checkpoint inhibitor; Kong, page 7, ‘Assay Evaluation of Checkpoint Inhibition’, lines 8-11).
Kong fails to teach measuring the reparative membrane turnover level occurring at the lytic synapse of live target cells by measuring the uptake of a fluorescent lipophilic dye by the target cells and determining that the reparative membrane turnover level is higher in the presence of an immune checkpoint inhibitor. Kong further fails to teach treating a patient with an immune checkpoint inhibitor.
Keefe teaches that during cell mediated lysis perforin creates pores in the target-cell membrane, transiently allowing Ca2+ and small dyes into the cell and that the Ca2+ flux triggers a wounded membrane-repair response (reparative membrane turnover). Keefe further teaches that the restoration of target-cell membrane integrity by triggering the repair response is necessary for target cells subjected to cytotoxic T lymphocyte attack to avoid necrosis and undergo the slower process of programmed cell death (Keefe, page 249, see entire ‘Abstract’). Keefe further teaches that apoptotic target-cell death is triggered by release of cytotoxic granule contents into the immunological synapse formed by CTL binding to its target cell (Keefe, page 249, ‘Introduction’, lines 4-7). Keefe teaches subjecting target cells to activated human cytotoxic T lymphocytes and that within a few minutes of forming a firm synapse between the CTL and its target a transient target-cell Ca2+ flux was seen (sufficient period of time and under conditions suitable to induce cytotoxic response) and further teaches that the target cells also exhibit the hallmark feature of the plasma membrane-repair response, cell surface Lamp-1 staining (measuring reparative membrane turnover; Keefe, page 259, 2nd paragraph, lines 9-26). Keefe teaches visualizing the cells and Lamp-1 by fluorescence microscopy and further teaches no Lamp-1 staining and as such no membrane repair in the absence of cytotoxic T lymphocytes (Keefe, page 259, 2nd paragraph, lines 12-14, and figure 7 c). Put another way, Keefe teaches a method of detecting membrane repair in response to damage caused by perforin release at the synapse between the target and effector cells (reparative membrane turnover at the lytic synapse of live target cells) and further teaches that the absence of cytolytic activity results in a lack of membrane turnover.
Tam teaches that a frequent form of cell injury is permeabilization by pore-forming toxins produced by bacteria or immune cells and that mammalian cells have an efficient mechanism to repair their plasma membrane after permeabilization with pore-forming proteins (Tam, page 1 of 8, ‘Introduction’, 2nd paragraph, lines 2-5). Tam further teaches a method to determine the kinetics of cell resealing after permeabilization with pore-forming proteins using the lipophilic dye FM1-43 (measuring reparative membrane turnover by measuring uptake of fluorescent lipophilic dye), which stably intercalates into the lipid bilayers increasing in fluorescence intensity and when the plasma membrane bilayer is disrupted extracellular dye gains access to intracellular membranes providing a sensitive assay to detect plasma membrane injury and repair. Tam teaches treating cells with the pore-forming bacterial toxin streptolysin O and measuring the fluorescent dye by microscopy (Tam, page 2 of 8, lines 3-11). Put another way, the assay of Tam allows for direct evaluation of membrane repair after injury by measuring the uptake of a fluorescent lipophilic dye.
Jacquelot teaches that immunotherapy, including immune checkpoint blockers, has only been shown to provide durable clinical benefit in a fraction of patients (Jacquelot, page 2, lines 7-8). Jacquelot further teaches that for example combination therapy of anti-CTLA-4 + anti-PD-1 mAbs is potentially toxic (Jacquelot, page 9, lines 1-4). Jacquelot further teaches that each immune checkpoint/co-stimulatory pathway displays an independent mechanism of action and therefore will require a comprehensive analysis of their mode of action in any given patient to design appropriate combinatorial approaches (Jacquelot, page 2, 2nd column, lines 3-7). Jacquelot further teaches that 54% of patients with grade 3 or 4 melanoma have drug-related adverse events when receiving ipilimumab/nivolumab combination therapy, compared with 24% receiving ipilimumab monotherapy and that given the efficacy and relative safety of nivolumab alone, finding a predictor of response to such potentially toxic combinations is an urgent unmet clinical need (Jacquelot, page 9, 2nd column, 4th paragraph, lines 12-19). Jacquelot further teaches that predictive immune profiles or biomarkers are needed to be validated prospectively to guide immunotherapy utilization in a personalized manner (Jacquelot, page 2, lines 26-28). Put another way, Jacquelot teaches a bioassay is necessary to test a patient sample for its response to immunotherapy to personalize treatment, i.e., treat the patient with the treatment predicted to be effective. Jacquelot further teaches that immune checkpoint blockers have become pivotal therapies against metastatic melanoma (Jacquelot, ‘Abstract’, lines 1-2).
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 measuring T cell cytotoxicity by measuring cell death with the method of Keefe of measuring reparative membrane repair because of the teaching of Keefe that cytotoxic T lymphocytes trigger target cell death by apoptosis and that reparative membrane repair in response to T cell mediated cytotoxicity results in cell death by apoptosis rather than necrosis. One of ordinary skill in the art would be motivated to do so to only measure cell death caused by cytotoxic T lymphocytes in the sample. It would have further been obvious to one of ordinary skill in the art that the increase of reparative membrane repair, i.e. increased dye uptake, in the presence of an immune checkpoint inhibitor (compared to the lack of membrane repair in the absence of a checkpoint inhibitor) indicates increased cytotoxic T cell activity because of the teaching of Keefe that in the absence of cell cytotoxicity no reparative membrane turnover is detected.
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 Keefe of detecting membrane repair (by detecting Lamp-1 staining) at the synapse between target cell and cytotoxic T lymphocytes with the method of Tam using a lipophilic dye to evaluate kinetics of cell resealing because of the teaching of Tam that this is a sensitive assay to detect plasma membrane injury and repair. One of ordinary skill in the art would have been motivated to do so because the method of Tam allows for direct evaluation of plasma membrane injury and repair as opposed to an indirect evaluation of measuring a protein that is a hallmark or membrane repair (Lamp-1) as taught by Keefe.
It would further be obvious to modify the method of Kong in view of Keefe to measure the lytic potential of tumor infiltrating cells in a patient in order to predict treatment response to immune checkpoint inhibitor therapy and treat patients only when they are predicted to benefit from the treatment because of the teaching of Jacquelot that immunotherapy has a durable effect in only a fraction of patients and can be toxic. It would have further been obvious to treat patients that benefit from the therapy because of the teaching of Jacquelot that immune checkpoint blockers have become pivotal therapies against melanoma.
The ordinary artisan would have a reasonable expectation of success, because Kong, as well as Keefe and Tam use in vitro assays comprising microscopy to evaluate the effect of pore forming peptides, either by adding the toxin directly or contacting the target cell with a cytotoxic T lymphocyte. Adding a lipid dye as taught by Tam instead of measuring Lamp-1 expression by target cells would not be expected to interfere with the method of Keefe, nor would replacing propidium iodide as taught by Kong with the lipophilic dye of Tam.
Regarding claims 2 and 3, Kong teaches patient derived tumor infiltrating lymphocytes comprising CD8+ cytotoxic T lymphocytes (Kong, page 3, ‘Multiplex IHC’, lines 6-8).
Regarding claims 4, 8, and 21 Kong teaches coculturing rectal cancer tumoroids with patient-matched tumor infiltrating lymphocytes.
Regarding claims 12 and 13, Kong and the cited art above as applied to claim 19 also applies to claims 12 and 13.
Tam teaches a method comprising FM1-43 (n-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide) and recording the fluorescence with a confocal microscope (Tam, page 2 of 8, ‘3. Live cell Imaging of FM1-43 Influx’, step 4) and measuring the intracellular fluorescence intensity of FM1-43 using image analysis software (Tam, page 3 of 8, ‘4. Quantification of FM1-43 Influx Into Cells’, step 1). Tam further teaches FM1-43 that is a lipophilic dye that increases in fluorescence intensity when intercalated into the lipid bilayers (Tam, page 2 of 8, line 5).
Regarding claim 24, Kong and the cited art above teaches a method substantially as claimed.
Kong does not teach that effector and target cells are contacted for a total period of time of 2-60 min.
Keefe teaches that cells need to be able to repair plasma membrane damage and that this occurs by a rapid damaged membrane response in which intracellular vesicles, including endosomes, lysosomes, and multivesicular bodies are mobilized within seconds to donate their membranes to reseal the damaged plasma membrane (Keefe, page 250, 2nd paragraph, lines 5-13). Keefe further teaches measuring cytotoxic T lymphocyte:target cells preloaded with calcium indicator dye. Keefe further teaches detecting dye emission at regular intervals using a microscope (Keefe, supplemental data, page 1 of 2, see entire paragraph: ‘Intracellular Calcium Measurements’). Keefe further teaches that cytotoxic T lymphocytes induce a transient calcium flux detected by Fura-2 fluorescence by showing images of cytotoxic T lymphocyte:target cell interaction with approximately 30 second time lapses between each image (Keefe, page 258, Figure legend, Figure 7(a), lines 1-3). Figure 7(A) further shows calcium influx at the 5th image, i.e. 5 x 30 seconds (or 2.5 minutes) after the start of cytotoxic T lymphocyte:target cell interaction (Keefe, page 258, figure 7(A)).
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 measure the reparative membrane turnover due to the impact of an immune effector on a target cells after said cells were contacted for 2-60 minutes because of the teaching of Keefe that interaction between said cells results in a transient Ca2+ influx into the target cells within 2-3 minutes of such an interaction and that this Ca2+ influx triggers rapid membrane repair. One of ordinary skill in the art would be motivated to measure the membrane repair within 2-60 minutes of effector:target cell interaction in order to measure the reparative membrane turnover before the process is completed.
Claim22 is rejected under 35 U.S.C. 103 as being unpatentable over Kong et al., in view of Keefe et al., Tam et al., and Jacquelot et al. as applied to claim 21 above and further in view of Kobayashi et al., WO2009110614A1 (see PTO-892, 11/01/2024).
Regarding claims 22, Kong and the cited art above teach a method of determining whether a patient will achieve a response with an immune checkpoint inhibitor substantially as claimed.
Kong does not teach that the biological sample is blood (claim 22).
Kobayashi teaches a method for measuring the functions of cytotoxic T lymphocytes (Kobayashi, page 3, paragraph [0001], lines 1-2). Kobayashi further teaches mixing and culturing effector cells and target cells for a period of time, labeling a plurality of molecules with fluorescence-labeled antibody or a fluorescent substance and measuring the expression of a substance and qualitatively and quantitatively analyzing the results (Kobayashi, page 8, see paragraph [0028]). Kobayashi further teaches detection of Mage-3-specific cytotoxic T lymphocytes in a cancer patient from peripheral blood (Kobayashi, page 21, paragraph [0075], lines 1-2 and 12-13). As such, Kobayashi teaches a blood sample (claim 22). Kobayashi further discloses that activated cytotoxic T cells can be taken out of the body to enhance target cell-killing ability by activating them ex vivo (Kobayashi, page 4, see entire paragraph [0006]). Kobayashi further teaches that it is important to confirm the function of the therapeutic cell, whether the cell actually has a cytotoxic activity to kill a target cancer cell (Kobayashi, page 4, see entire paragraph [0007]).
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 applied the method of Kong and the prior art to assess cytotoxic T lymphocytes isolated from a patient tissue that is blood, because of the teaching of Kobayashi that such cells can be activated ex vivo in order to enhance their killer cell activity and that it is important to confirm their activity.
The ordinary artisan would have a reasonable expectation of success, because Kobayashi teaches testing cytotoxic T lymphocytes by mixing effector and target cells, labelling the cells and measuring the presence of a substance in the cell, as does the cytotoxic T lymphocyte assay of Kong.
Claims 11 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Kong et al., in view of Keefe et al., Tam et al., and Jacquelot et al. as applied to claim 19 above and further in view of Fomina et al. Regulation of membrane trafficking and subcellular organization of endocytic compartments revealed with FM1-43 in resting and activated human T cells. Experimental cell research. 2003 Nov 15;291(1):150-66 (see PTO-892, 11/01/2024).
Regarding claim 11, Kong and the cited art above teach a method of determining whether a patient will achieve a response with an immune checkpoint inhibitor substantially as claimed.
Kong and the cited art above does not teach measuring the uptake of lipophilic dye in the immune effector cells.
Fomina teaches a method of investigating kinetics of constitutive endocytosis using FM1-43 (Fomina, page 150, ‘Abstract’, lines 1-2). Fomina further teaches visualizing endocytosis in resting and activated human T lymphocytes by exposing the cells to FM1-43 (Fomina, page 154, ‘Results’, lines 1-3). Fomina further teaches that real-time monitoring of FM1-43 fluorescence indicates that the rate of constitutive membrane trafficking is about 10-fold higher in activated T cells compared to resting T cells.
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 to also measure membrane recycling in immune effector cells as taught by Fomina to measure membrane trafficking in T cells, because it allows to identify activated versus resting T cells.
The ordinary artisan would have a reasonable expectation of success, because the combination of Kong and the cited art above and Fomina use an assay comprising contacting human T lymphocytes with FM1-43 and both apply the method in order to measure membrane turnover of cells in culture.
Regarding claim 18, Kong and the cited art above teach a method of determining whether a patient will achieve a response with an immune checkpoint inhibitor substantially as claimed.
Kong does not teach that the method as claimed measures T cell exhaustion in a patient.
As discussed previously above, Fomina teaches a method of investigating kinetics of constitutive endocytosis (membrane turnover) using FM1-43 (Fomina, page 150, ‘Abstract’, lines 1-2). Fomina further teaches that T cell receptors undergo continuous recycling, resulting in a steady state level of receptor expression at the plasma membrane and that this steady state can be affected by T cell receptor ligation by antigen/MHC complexes, which reduces T cell receptor expression on the T cell surface and that long-lasting down-regulation of TCR expression is thought to be critical for termination of the immune response (T cell exhaustion; Fomina, page 150, see entire first paragraph). Fomina further teaches that some viruses alter receptor internalization/recycling mechanisms in order to evade immune surveillance (Fomina, page 150, 2nd column, lines 5-8). Fomina further teaches that tracking the fate of membrane-associated FM1-43 within the cell revealed a significant change in the organization of endocytic compartments as T cells become activated (Fomina, page 163, 2nd column, lines 1-4). Fomina further teaches that there is a difference in FM1-43 accumulation in resting and activated T cells (Fomina, page 153, Figure 1., see figure legend, line 1 and Figure 1, B, E, K, and N).
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 apply the method of Kong and the cited art above in view of Fomina to identify T cells with reduced T cell receptor expression (T cell exhaustion), because this can be critical for the termination of the immune response or a sign of evasion of immune surveillance.
The ordinary artisan would have a reasonable expectation of success, because both the combination of Kong and the cited art above and Fomina use an assay comprising contacting human T lymphocytes with FM1-43 and both apply the method in order to measure membrane turnover of cells in culture.
Claim 23 is rejected under 35 U.S.C. 103 as being unpatentable over Kong et al., in view of Keefe et al., Tam et al., and Jacquelot et al. as applied to claim 19 above and further in view of Sarin et al. Caspase dependence of target cell damage induced by cytotoxic lymphocytes. The Journal of Immunology. 1998 Sep 15;161(6):2810-6 (see PTO-892, 11/01/2024) and Zaritskaya et al. New flow cytometric assays for monitoring cell-mediated cytotoxicity. Expert review of vaccines. 2010 Jun 1;9(6):601-16.
Regarding claim 23, Kong and the cited art above teach a method of determining whether a patient will achieve a response with an immune checkpoint inhibitor substantially as claimed.
Kong does not teach measuring cell fluorescence by a flow cytometer.
Sarin teaches a method of measuring Target Cell Damage Induced by Cytotoxic Lymphocytes (Sarin, see title and page 2813, ‘Caspase inhibitors block the CTL-induced increase[…]”, lines 1-8), comprising target cells (Sarin, page 2811, ‘Target and effector cells’, lines 1-2) and cytotoxic T lymphocytes as effector cells (Sarin, page 2811, ‘Assays of target damage’, lines 2-8). Sarin further teaches measuring plasma membrane recycling by incubation of the cell suspension with FM1-43 before analysis of target cells on the flow cytometer (Sarin, page 2812, 2nd column, 2nd paragraph, lines 1-6).
Zaritskaya teaches using flow cytometry-based assays to detect and enumerate tumor-specific cytotoxic T lymphocytes and their specific effector functions and correlation with clinical responses (Zaritskaya, Abstract, lines 16-19). Zaritskaya further teaches that assays that allow for the simultaneous measurement of several parameters may be more advantageous for clinical monitoring and that in this respect the flow cytometric assays that will enable detection and enumeration of tumor-specific cytotoxic T lymphocytes and their specific effector functions provide new insights on the mechanism of cell-mediated killing and will help to improve cancer vaccine design and evaluation in the future. Zaritskaya further teaches that flow cytometry assays are a powerful tool for analyzing antigen specific T cell responses in a quantitative manner and that suspension cell systems such as blood and bone marrow cells and tumor single-cell suspension are ideal for flow cytometric analysis and it is easy to obtain high-quality information by labeling cells with antibodies against surface antigens or receptors. Zaritskaya further teaches that the biggest advantage of flow cytometry is the combination of multiparameter measurements and high-speed analysis (up to 18 colors at analysis rates of more than 20,000 events per second) allowing for analysis of such rare cells as antigen-specific T lymphocytes, including their phenotype and functional activity (Zaritskaya, page 603, 2nd paragraph, line 16- page 604, line 2).
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 the cited art above in order to detect cells comprising fluorescent lipophilic dye by flow cytometry as taught by Sarin because of the teaching of Zaritskaya that the biggest advantage of flow cytometry assays is the combination of multiparameter measurements and high-speed analysis. One of ordinary skill in the art would be motivated to do so because of the teaching of Zaritskaya that said combination makes for a powerful tool for analyzing rare cells such as antigen specific T cells in a quantitative manner which will help to improve cancer vaccine design and evaluation in the future.
One of ordinary skill in the art would have a reasonable expectation of success because the combination of Kong and the cited art above teaches an assay to assess reparative membrane turnover comprising the detection of a fluorescent lipophilic dye, such as FM1-43, in a cytotoxicity assay and Sarin teaches success detecting FM1-43 in a cell cytotoxicity assay by flow cytometry.
Claim 25 is rejected under 35 U.S.C. 103 as being unpatentable over Kong et al., in view of Keefe et al., Tam et al., and Jacquelot et al. as applied to claim 19 above and further in view of Cerignoli et al. In vitro immunotherapy potency assays using real-time cell analysis. PloS one. 2018 Mar 2;13(3):e0193498.
Regarding claim 25, Kong and the cited art above teach a method of determining whether a patient will achieve a response with an immune checkpoint inhibitor substantially as claimed.
Keefe teaches measuring cytotoxic T lymphocyte:target cells preloaded with calcium indicator dye, Fura-1-AM, at an effector:target ratio of 3:1. Keefe further teaches detecting Fura-2 emission at regular intervals using a microscope (Keefe, supplemental data, page 1 of 2, see entire paragraph: ‘Intracellular Calcium Measurements’).
Keefe does not teach that the ratio between immune effector and target cells is 2:1.
Cerignoli teaches a real-time cytolytic in vitro potency assay that monitors viability of target tumor cells using cellular impedance, to test the efficacy of different immunotherapy approaches. Cerignoli teaches testing several effector cells, i.e., PBMC, NK, and CAR-T cells as well as biological molecules such as Bi-specific T cell Engagers and blocking antibodies against the immune checkpoint inhibitor PD-1(Cerignoli, ‘Abstract’, lines 8-21). Cerignoli further teaches that a key question is how well the impedance readout matches the results of other techniques that monitor marker expression and membrane integrity. To address this Cerignoli teaches assessing the extent of apoptosis of target cells using flow cytometry by staining for annexin V and DPI (Cerignoli, page 9 or 21, 3rd paragraph, lines 1-6). Cerignoli further teaches that there is very good correlation between flow cytometry and the impedance assay (Cerignoli, page 17 of 21, lines 1-4). Cerignoli further teaches that percent cytolysis increases in a time and E:T ratio-dependent manner and for several E:T ratios tested the % cytolysis reach a plateau after 50 hours most likely because at low E:T ratios the effector cells are a limiting factor (Cerignoli, page 9 of 21, lines 5-9). Cerignoli further teaches monitoring CAR-T cell potency by treating target cells CAR-T or mock T cells. Cerignoli teaches approximately 50% cytolysis after 40h at a 2:1 effector to target ratio (see Figure 6 b). Cerignoli further teaches monitoring potency at different ratios (E:T=6:1, 5:1, 4:1, 3:1, and 2:1) and shows that at all E:T ratios tested, both CAR-T cells and mock-T cells display considerable cytolytic activity, though the difference between these effectors increases at lower E:T ratios (Cerignoli, page 14/21, see entire 3rd paragraph, and Figure 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 and the cited art above by using an effector to target cell ratio of 2:1 as taught by Cerignoli because of the teaching of Cerignoli that at higher effector to target ratios there is considerable background cytolytic activity but the difference increases at lower E:T ratios and that, on the other hand, at low effector to target ratios the % cytolysis reaches a plateau that is most likely due to the effector cells being the limiting factor.
One having ordinary skill in the art would have a reasonable expectation of success because Cerignoli teaches that cytotoxic T lymphocytes have considerable cytolytic ability at all E:T ratios, the ratios including 3:1 as taught by Keefe and 2:1 as claimed, in an assay to measure cell cytotoxicity in the context of immunotherapy and because Cerignoli teaches that the assay has good correlation with a flow cytometry based assay which is a common assay to measure membrane integrity.
Response to Arguments
Applicant’s arguments, see page 7, 5th paragraph, filed 08/28/2025, with respect to the rejection(s) of claims 2-4, 8, 11-13, 18-19, and 21-25 under 35 U.S.C. 103 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of Kong, Keefe, Tam, and Jacquelot.
Regarding the argument on page 8, 1st paragraph, that only the inventors have demonstrated that triggering of membrane reparation is directly related to the effector cell lytic potential, as discussed previously in detail above, Keefe teaches that during cell mediated lysis perforin creates pores in the target-cell membrane, transiently allowing Ca2+ and small dyes into the cell and that the Ca2+ flux triggers a wounded membrane-repair response in target cells subjected to cytotoxic T lymphocyte attack (Keefe, page 249, see entire ‘Abstract’).
Therefore the argument is not persuasive.
Communication
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/STEFANIE J. KIRWIN/Examiner, Art Unit 1677
/Soren Harward/Primary Examiner, TC 1600
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