Human T cell acute lymphoblastic leukemia cell lines and their use for treatment of cancer

Abstract

A novel human T cell acute lymphoblastic leukemia (T-ALL) cell line (ATCC preservation number PTA-125809), referred to as INB16, induces memory-like function thereon when contacted with natural killer cells that have been proved to be capable of identifying and killing cancer cells, including hematological tumor cells and solid tumor cells. Useful applications of the INB16 cell lines include study, administration in vivo, and cancer therapeutic agents comprising non-replication-ability INB16 cells and / or membrane portions thereof for restoring the function of a patient's own NK cells, as well as related methods of treating cancer.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of the priority of U.S. Provisional Application Serial No. 63 / 394,862, filed on August 3, 2022, and U.S. Provisional Application Serial No. 63 / 417,514, filed on October 19, 2022; the entire contents of each are incorporated herein by reference for all purposes. Technical Field

[0003] The present invention relates to the field of immortalized human T - cell acute lymphoblastic leukemia cells, as well as related compositions and methods for treating cancer. Background Art

[0004] CTV - 1 (product number ACC - 40, DSMZ), hereinafter referred to as "CTV - 1", is a human monocytoid leukemic cell line established in approximately 1982 from the peripheral blood of a 40 - year - old female patient with relapsed acute monoblastic leukemia. Peripheral blood (10 mL) was obtained from the patient by venipuncture. At this time, 15×10 4 / mm 2 and more than 90% were blast cells. The leukemic cells were first separated by Ficoll - Hypaque gradient centrifugation and then washed three times with RPMI - 1640 medium. Then they were suspended in flasks in the same medium supplemented with 20% heat - inactivated fetal bovine serum. The suspended cells were incubated at 37°C in a humid environment containing 5% CO 2 and fed twice a week. From the beginning, the cells showed slow but definite proliferation. After 4 weeks, the cells grew vigorously in suspension and were continuously transferred every 3 - 4 days. These cells grew best at a concentration of 2×10 5 cells / mL and had a doubling time of approximately 36 hours. See Chen, 1984.

[0005] Other human leukemia cell lines include K562, HL - 60, KG - 1, U - 937, PL - 21, KCL - 22, BV173, THP - 1, RC - 2A, and P31 / Fujioka.

[0006] T-cell acute lymphoblastic leukemia is an aggressive type of leukemia in which too many T-cell lymphoblasts are found in the bone marrow and blood. T-cell acute lymphoblastic leukemia is also known as precursor T-lymphoblastic leukemia and T-cell acute lymphocytic leukemia. Summary of the Invention

[0007] Technical problem

[0008] Human leukemia cells are generally difficult to proliferate in vitro. Leukemia colonies typically undergo terminal differentiation, and subculturing beyond 2 or 3 passages is usually unsuccessful.

[0009] Although there are many established leukemia cell lines available for research, there is a need for additional leukemia cell lines to enhance the understanding of various leukemias and to develop therapeutic agents and treatment strategies for treating the disease.

[0010] In addition, novel biopharmaceutical compositions that are effective in treating cancer are in continuous and high demand.

[0011] Furthermore, methods of incorporating or utilizing such novel biopharmaceutical compositions are also in continuous and high demand.

[0012] Solution to the problem

[0013] A novel human T-acute lymphoblastic leukemia cell, designated herein as "INB16" (ATCC Deposit No. PTA-125809), has been isolated and immortalized in a cell line useful for cancer research and the development of biopharmaceutical anti-cancer therapeutic agents.

[0014] Non-replicating INB16 cells, membrane fractions thereof, and combinations of non-replicating INB16 cells and membrane fractions have been prepared to form NK cell priming agents useful for treating cancer.

[0015] In various embodiments, the invention incorporates or utilizes cells of the novel INB16 cell line for the purpose of researching and developing therapeutic agents and treatment strategies for treating cancer.

[0016] Advantageous effects of the invention

[0017] Cells from the INB16 cell line and / or membrane fractions of such cells, when rendered non-replicating, can be used as agents for priming natural killer cells in a subject such that the primed NK cells can become available to monitor, recognize, and kill cancer cells. In this regard, INB16 cells have unique biological characteristics that enable in vivo NK cell priming to enhance NK cell function in patients suffering from cancer.

[0018] Surprisingly, NK cells primed by non-replicative INB16 cells exhibit a memory-like phenotype, which means that the NK cells maintain priming and therapeutic activity over an extended duration.

[0019] Priming of NK cells by non-replicative INB16 cells can be achieved without the use of cytokines, thereby alleviating the drawbacks of potential cytokine storms.

[0020] The effect of a single course can last for at least four months.

[0021] Priming of NK cells by non-replicative INB16 cells functions in the antagonistic tumor microenvironment (TME) of solid tumors. The TME is hypoxic (low oxygen levels). Immune cells generally do not function in hypoxic TME. NK cells primed by a cell preparation containing non-replicative INB16 cells function in hypoxic TME.

[0022] The affinity of NK cells for binding to tumor cells is increased in such NK cells primed by non-replicative INB16 cells. In other words, a cell preparation containing non-replicative INB16 cells shows enhanced affinity of NK cells for binding to tumor cells.

[0023] In such NK cells primed by non-replicative INB16 cells, persistence is increased.

[0024] A cell preparation containing non-replicative INB16 cells is shown to activate key genes and pathways for enhanced NK cell function and survival. INB16 upregulates 522 genes different from IL2, including CD70, CXCL10, and Stat5. Additionally, INB16 upregulates 1461 proteins different from IL15, including TIMM29 and USP20. Brief Description of the Drawings

[0026] Figure 1 A graph showing the percentage of activated NKG2D NK cells and blasts in patients treated with a cell preparation containing non-replicative INB16 cells is shown.

[0027] Figure 2 In vitro killing of NK cells obtained from patients treated with a cell preparation containing non-replicative INB16 cells is shown.

[0028] Figure 3 A table showing the effect of healthy donor NK cells on various tumor cell lines in two cases with and without INB16 treatment is shown.

[0029] Figure 4Shows a table reflecting the effect of NK cells obtained from cancer patients treated with riINB16 cells on various cancer cell lines in in vitro assays.

[0030] Figure 5 Shows a graph illustrating target cell lysis-related proteins that are altered in the responses of NK cells treated or untreated with IL-15 and INB16.

[0031] Figure 6 Shows a graph illustrating mitochondrial survival-related proteins that are altered in the responses of NK cells treated or untreated with IL-15 and INB16.

[0032] Figure 7 Shows a graph illustrating the affinity of each of rNK, NK-IL-2, NK-IL-15, and riINB16-primed NK cells for SKOV3.

[0033] Figure 8 Shows a graph representing the percentage of cell lysis of rNK and TP-NK cells in a hypoxic environment simulating the TME.

[0034] Figure 9 Shows a graph indicating that riINB16-primed NK cells from MDS patients exhibit a restored ability to kill MDS cells in vitro.

[0035] Embodiment Description

[0036] For purposes of explanation and not limitation, the following description includes certain details and descriptions of preferred embodiments of the present invention as set forth in the claims. Those skilled in the art will recognize that these details and descriptions are not exhaustive and, together with the ordinary level of those skilled in the art as of the date of this filing, will assist the examiner in understanding how to make and use the inventive concepts disclosed and claimed herein. Nothing in these details and descriptions should be construed as limiting the spirit and scope of the present invention.

[0037] Prior to the observations and findings presented herein, it had been determined that human patients suffering from various forms of cancer (e.g., leukemia) generally have natural killer (NK) cells that function less well than those of healthy individuals, which led the scientific community to hypothesize that one of the underlying problems in cancer progression is immune cell dysfunction and, more specifically, defective NK cells. For example, it was observed that NK cells in relapsed patients do not have the ability to kill residual cancer cells.

[0038] Recently, during research aimed at understanding the role of NK cells in the innate immune response against cancer, many observations and findings have been made that suggest the problem lies not with the NK cells, but with the environment in which the NK cells exist.

[0039] For example, it has been observed that, although the NK cell activity of patients with acute myeloid leukemia (AML) is lower than that of healthy individuals, as demonstrated by in vitro cell killing assays, NK cells from AML patients after chemotherapy are capable of killing AML cancer cells. Further studies have determined that no difference in NK function was observed between surviving AML patients (patients surviving for two years or longer) and those AML patients who relapsed after chemotherapy, which means that NK cells seem to have the same function between surviving and relapsing AML patients after chemotherapy. After these observations, further analysis was performed on AML tumor cells from various patients and their ability to induce NK cells to kill NK-resistant cells from the RAJI cell line, which revealed that the NK cells or their function / dysfunction were not the differences among survivors, but rather the patient's own leukemia cell variants were directly involved in whether the resulting NK cells could kill RAJI cells in vitro, or whether the patient's cancer cells could induce the patient's own NK cells to kill residual cancer in the patient after chemotherapy treatment. Thus, it was found that some cancer cells (derived from survivors) showed a biological effect on the patient's NK cells, enabling the NK cells to monitor, recognize, and kill cancer cells; while other cancer cells incubated with the same NK cells did not result in cancer cell killing.

[0040] This finding, that certain cancer cells can affect the function of a patient's NK cells, inducing or not inducing an effector state, led to a series of experiments testing whether different cancer cells have the ability to change NK cells from a resting (non-effector) state to an active (effector) state.

[0041] It was further found that CTV-1 cells unexpectedly were able to convert resting NK cells into activated NK cells in vitro in response to the NK-resistant RAJI cell line. This work led to a method of treating cancer that includes activating NK cells in vitro by CTV-1 co-cultures and subsequently infusing them into patients in need thereof. See, for example, U.S. Patent No. 8,257,970.

[0042] Although CTV-1 has been shown to successfully kill cancer in vitro and has had some success in human patients, as a treatment strategy for treating cancer, there are disadvantages such as the high costs of in vitro culture, manufacturing, and subsequent infusion.

[0043] Based on these observations, a method of priming NK cells in vivo was envisioned, including the steps of inactivating CTV-1-derived leukemia cells so that they become unable to replicate, and directly administering the non-replicating CTV-1 cells in vivo to a patient by infusion or other acceptable route of administration. See, for example, U.S. Patent No. 10,758,567.

[0044] Although the use of cells from the CTV-1 cell line to prime NK cells in vivo has been shown to be effective for treating residual cancer, it is unclear whether other leukemia cells can be used in a similar manner, i.e., rendering the cells non-replicative and administering the cells to a patient for priming the patient's own NK cells in vivo, such that the NK cells can kill residual cancer. Another leukemia cell line is needed to test this open hypothesis.

[0045] Preparation of cell line

[0046] Cell lines can be generated according to established methods known to those skilled in the art. Generally, cell lines are generated by culturing primary cells derived from a patient until immortalized cells spontaneously arise in culture.

[0047] These cells are then isolated and further cultured to produce a clonal cell population or cells that exhibit resistance to apoptosis.

[0048] For example, T-ALL cells can be isolated from peripheral blood drawn from a patient suffering from T-ALL. The cells can be washed and optionally immunotyped to determine the cell types present. Subsequently, the cells can be cultured in a medium, such as a medium containing IL-4 or other replication-stimulating factors. Advantageously, all or part of the medium is replaced one or more times during the culturing process. The cell line can thereby be isolated and identified by increased growth in culture.

[0049] In accordance with the foregoing, cells from the T cell acute lymphoblastic leukemia cell line INB16 (ATCC Deposit No. PTA-125809) are described herein.

[0050] In one useful application of the INB16 cell line, a biopharmaceutical composition is disclosed that comprises cells and / or membrane fractions of cells from the INB16 cell line (ATCC Deposit No. PTA-125809), wherein the cells and / or membrane fractions are rendered non-replicative.

[0051] Cells from the INB16 cell line can be prepared or rendered non-replicative by contacting the cells with mitomycin C.

[0052] Alternatively, cells from the INB16 cell line can be made non-replicating by first fixing the cells in a dry saccharide matrix and secondarily administering the cells while fixing with penetrating ionizing radiation. The cells are fixed in a dry saccharide matrix by preparing a solution comprising a non-reducing sugar (such as but not limited to sucralose) and suspending the cells in the solution to form a suspension, wherein after suspension, the suspension is dried according to a primary drying protocol that combines temperature and pressure regulation in a freeze dryer to effect a gradual and simultaneous evaporation, sublimation, and boiling of the suspension to remove water until there is less than 5% residual water content. An example of such a preservation technique is disclosed in U.S. Patent 9,469,835, issued October 18, 2016. In some examples, the dose can include a penetrating ionizing radiation dose and can be delivered by electron beam radiation, gamma radiation, or X-ray radiation. The dose can include at least 2 Gy and up to 20 kGy or higher.

[0053] In another aspect, a method for treating cancer is disclosed, the method comprising: administering to a patient having the cancer a therapeutically effective amount of a priming tumor cell preparation for priming the patient's natural killer cells in vivo, the priming tumor cell preparation comprising: cells and / or membrane portions thereof derived from the INB16 cell line (ATCC Deposit No. PTA-125809), wherein the cells and / or membrane portions of the priming tumor cell preparation are made non-replicating to prevent proliferation in the patient; whereby treating the patient. The cancer can include solid tumors or hematological tumors. In one embodiment, the cancer can include acute myeloid leukemia (AML). In another embodiment, the cancer can include myelodysplastic syndromes. In a preferred embodiment, the therapeutically effective amount of cells in the dose of the priming tumor cell preparation includes administering 1×10 8 cells one, two, or three times, once a week (one, two, or three weekly administrations of 1x10 8 cells), the cells being derived from the INB16 cell line and made non-replicating.

[0054] Example 1 - STR analysis of CTV-1 cells

[0055] Obtain CTV-1 cells, perform short tandem repeat (STR) analysis, and record the results of the STR analysis.

[0056] In Table 1 below, STR analysis of CTV-1 cells was performed using the AmpFISTR Identifier Plus and AmpFISTR NGM kits (Applied Biosystems) for 21 loci (including D21S11, CSF1PO, vWA, D8S1179, TH01, D18S51, D5S818, D16S539, D3S1358, D2S1338, TPOX, FGA, D7S820, D13S317, amelogenin, D19S433, D10S1248, D22S1045, D2S441, D1S1656, and D12S391) whose analysis is consistent with all major global standards. Allele detection was performed by PCR amplification and subsequent capillary sequencing using an ABIPRISM 3100-Avant genetic analyzer (Applied Biosystems). The sequencing results were analyzed using Genemapper software (Applied Biosystems).

[0057] Table 1 STR results of CTV-1

[0058] Locus Result Locus Result D8S1179 12 / 13 TPOX 8 / 11 D21S11 27 / 29 D18S51 13 / 14 D7S820 9 / 11 AMEL X / X CSF1PO 12 / 12 D5S818 12 / 12 D3S1358 16 / 16 FGA 22 / 25 TH01 6 / 9.3 D10S1248 14 / 14 D13S317 12 / 12 D22S1045 16 / 17 D16S539 12 / 12 D2S441 10 / 10 D2S1338 17 / 20 D1S1656 17.3 / 17.3 D19S433 14 / 16 D12S391 17 / 18 vWA 14 / 15

[0059] Example 2 - New INB16 cell line

[0060] INB16 is a human T-cell acute lymphoblastic leukemia (T-ALL) cell line established from the peripheral blood of a male patient with T-cell acute lymphoblastic leukemia (T-ALL).

[0061] INB16 cells were isolated according to conventional techniques and suspended in RPMI-1640 medium containing L-glutamine and 10% fetal bovine serum. The cells were inoculated at 0.5×10 6 cells / mL in static medium and harvested at (1.0 - 2.0)×10 6 cells / mL. The cells were desirably stored in gaseous nitrogen.

[0062] INB16 cells were obtained, short tandem repeat (STR) analysis was performed, and the results of the STR analysis were recorded.

[0063] In Table 2 below, STR analysis of INB16 cells was performed using the AmpFISTR Identifier Plus and AmpFISTR NGM kits (Applied Biosystems) for 21 loci (including D21S11, CSF1PO, vWA, D8S1179, TH01, D18S51, D5S818, D16S539, D3S1358, D2S1338, TPOX, FGA, D7S820, D13S317, amelogenin, D19S433, D10S1248, D22S1045, D2S441, D1S1656, and D12S391) that are consistent with all major global standards. Allele detection was performed by PCR amplification and subsequent capillary sequencing using an ABIPRISM 3100-Avant genetic analyzer (Applied Biosystems). The sequencing results were analyzed using Genemapper software (Applied Biosystems).

[0064] Table 2 STR results of INB-16

[0065] Locus Result Locus Result D8S1179 9 / 15 TPOX 8 / 8 D21S11 27 / 28 D18S51 8 / 14 D7S820 11 / 12 / 13 AMEL X / Y CSF1PO 9 / 10 / 12 D5S818 12 / 12 D3S1358 15 / 17 FGA 23 / 24 / 25 TH01 9 / 9 D10S1248 13 / 17 / 18 D13S317 11 / 12 D22S1045 16 / 17 / 18 / 19 D16S539 9 / 14 / 15 D2S441 10 / 11 / 14 D2S1338 17 / 18 D1S1656 14 / 15 / 16 / 17 D19S433 13 / 14 / 16 D12S391 19 / 20 / 21

[0066] The INB16 cell line was deposited with the ATCC Patent Depository, 10801 University Boulevard, Manassas, Virginia 20110 USA, under the Budapest Treaty with the ATCC accession number PTA-125809.

[0067] Key differential loci between CTV-1 and INB16 were noted at least at the loci AMEL, D10S1248, D16S539, and D12S391. Although both are leukemia cell lines, given the genetic differences between the CTV-1 and INB16 cell lines as demonstrated by the attached STR results, it is not clear whether the INB16 cells have the biological effects demonstrated by CTV-1, and thus additional experiments are needed.

[0068] Example 3 - In vitro tumor killing of NK cells triggered by ri-INB16

[0069] A 78-year-old male human subject with MDS was given a treatment that included three administrations of replication-incompetent INB16 (ri-INB16) cells, 3×10 8 cells for the first administration, and each of the second and third administrations contained 1×10 8cells. Blood was obtained from the patient at -1 (pre-treatment), +8 days, +15 days, +29 days, +43 days, +73 days, and +119 days relative to the first treatment and NK cells were isolated therefrom. The percentage of activated NK cells in the patient was identified as those with biomarkers CD69+ and NKG2D+. Figure 1 The results are shown, where the patient presented approximately 30% activated NK cells pre-treatment and at least 60% activated NK cells post-treatment up to at least day +119. Thus, the data indicate that the treatment regimen, including administration of non-replicating INB16 cells by infusion, provides an increase in activated NK cells in the patient's blood that begins immediately post-treatment and persists for at least +119 days. Additionally, it is also reflected in Figure 1 that the patient showed a continuous decrease in blasts from day -1 (pre-treatment, 100%) to day +29 (45%) and day +119 (65%).

[0070] Next, NK cells isolated from the patient were cultured with NK-resistant tumor cell lines K562 and RAJI to evaluate the cancer-killing effect of ri-INB16-primed NK cells. In vitro cancer cell-specific lysis % was measured at days +15, +43, +73, and +119. Figure 2 The immediate cancer killing at day +15 is shown, which shows a peak at day +43 and persists at least until day +119. These results indicate that NK cells primed in vivo with ri-INB16 cells are capable of killing NK-resistant cancer cells (K562 and RAJI) in vitro. Without ri-INB16 priming, these NK cells would not be able to kill NK-resistant cancer cells.

[0071] Example 4 - Solid tumor killing of INB16-timlNK cells compared to resting NK cells

[0072] Surprisingly, it was found that INB16 tumor-induced memory-like NK (INB16-timlNK) cells significantly improved tumor cell killing in in vitro assays. Experimental data show that INB16-timlNK cells are effective against NK-resistant cell lines, including DU145, 786O, ACHN, SKOV3, H3, and C17. Based on this data, a method for treating solid tumors in a subject is proposed, the method comprising administering to the subject a therapeutically effective amount of an NK cell inducer comprising non-replicating cells and / or membrane fractions thereof, the cells and / or membrane fractions being derived from the INB16 cell line.

[0073] Tumor-induced memory-like NK (timlNK) cells are a type of NK cell phenotype that exhibits the ability to lyse NK-resistant tumor cells and exhibits memory-like persistence. Those skilled in the art will understand that timlNK cells are different from cytokine-induced memory-like NK (cimlNK) cells, which exhibit the ability to lyse certain NK-resistant tumor cells and exhibit memory-like persistence, but with lower persistence than INB16-timlNK cells. Additionally, compared to cimlNK cells, INB16-timlNK cells exhibit different proteomic characteristics, including upregulation of at least one hundred and forty-one expressed proteins. For clarity, cimlNK cells are typically prepared by culturing resting NK cells with cytokines, particularly IL-2, IL-15, or IL-12 / 15 / 18.

[0074] INB16-timlNK cells can be generated in vitro by exposing resting NK cells to INB16, or they can be generated in vivo by exposing resting natural killer (rNK) cells to INB16 (replication-incompetent INB16) or a preparation containing membrane fragments of INB16 cells; either by contacting rNK cells with replication-incompetent INB16 cells or their membrane portions. The resulting INB16-timlNK cells are effective in killing cancer.

[0075] In this example, INB16-timlNK cells were generated in vitro by culturing human resting NK cells obtained from healthy donors with replication-deficient cells of the INB16 cell line inactivated with mitomycin C. The resulting NK cells are herein referred to as INB16-timlNK cells. Another option for generating replication-incompetent NK cells is irradiation; however, care should be taken to ensure that NK cell-activating ligands are not destroyed by the radiation dose. To achieve appropriate replication incompetence using irradiation, the cells cannot be over-stimulated, as this may disrupt epitopes useful for NK cell activation. For these reasons, mitomycin C is the preferred means for rendering tumor cells non-replicating for use in the embodiments described herein.

[0076] In another example not described herein, replication-incompetent INB16 cells can be directly administered to a patient via intravenous infusion, at a dose between 1.0×10 6 and 1.0×10 9 cells per treatment, and once weekly for three weeks. For human subjects, the preferred dose of replication-incompetent INB16 cells is 1.0×10 8 .

[0077] In this context, we turn to Figure 3The relevant embodiment herein, wherein rNK cells are obtained from healthy donors and divided into two allocations, one is retained as rNK (not exposed to INB16 cells), and the other is co-cultured with replication-deficient cells of the INB16 cell line (ri-INB16) to generate INB16 tumor-induced memory-like NK (INB16-timlNK) cells. The rNK cells or INB16-timlNK cells are introduced into solid tumor cell lines in vitro, and the NK lysis percentage is determined according to conventional techniques. The solid tumor cell lines include DU145 (prostate cancer), 786O (renal cell carcinoma), ACHN (renal cell carcinoma), SKOV3 (ovarian cancer), H3 (nasopharyngeal carcinoma), and C17 (nasopharyngeal carcinoma). As Figure 3 shown, INB16-timlNK cells perform better than rNK in each tumor cell model. Improvements of 0 - 200% (+), 201% - 500% (++), and greater than 501% (+++) are marked in the column labeled "Improvement". INB16-timlNK cells show enhanced ability to kill these tumor cell lines.

[0078] Accordingly, a method for treating cancer can include administering to a subject in need thereof a therapeutically effective amount of an NK cell inducer comprising non-replicating INB16 cells, wherein the subject is treated, and wherein the cancer includes solid tumors selected from prostate cancer, renal cell carcinoma, ovarian cancer, or nasopharyngeal carcinoma.

[0079] Figure 4 The performance of NK cells obtained from human cancer patients is shown. The NK cells are incubated with non-replicating INB16 cells and introduced into the SKOV3 (ovarian cancer patient) or 768O (renal cell carcinoma patient) cell lines. As shown, timlNK cells are significantly superior to the patients' rNK cells in killing tumor cells in vitro. The data are presented as the percentage of tumor cell lysis in each of the rNK and timlNK cells, and the improvements are Figure 2 labeled as -200% - 0 (-), 0 - 200% (+), 201% - 500% (++), and greater than 501% (+++).

[0080] Example 5 - Difference between INB16-timlNK cells and cimlNK cells

[0081] More than 1,500 proteins were identified as being upregulated in INB16-timlNK cells, and subsequent analysis compared them to NK cells primed with a cytokine mixture of IL-12, IL-15, and IL-18 (to generate cimlNK cells). Among the 250 most highly upregulated proteins, 141 were completely unique to INB16-timlNK cells and were not upregulated by the cytokines IL-12, IL-15, and IL-18. Many of these unique proteins are involved in cell survival and may protect the enhanced metabolism of INB16-timlNK cells in the TME.

[0082] Figure 5 Graphs showing target cell lysis-related proteins measured from untreated resting NK cells, IL-15 cytokine-induced memory-like NK (IL15-cimlNK) cells, and INB16-timlNK cells are presented. Here, untreated rNK cells showed negligible log2 protein fold changes, while both IL15-cimlNK and INB16-timlNK cells showed protein fold changes, specifically: S100A12, LTF, PGLYRP1, DEFA1, LYZ, ROMO1, H2BC12, DEFA3, DCD, RPL30, DEFA4, GNLY, RPS19, HMGN2, and GAPDH. These results indicate that INB16-timlNK cells are similar to IL15-cimlNK cells in terms of the expression of target cell lysis-related proteins.

[0083] Figure 6Graphs showing mitochondrial survival-related proteins measured from untreated resting NK cells, IL-15 cytokine-induced memory-like NK (IL15-cimlNK) cells, and INB16-timlNK cells are presented. Here, untreated rNK cells and IL15-cimlNK showed negligible log2 protein fold changes, while INB16-timlNK cells showed significant protein fold changes, specifically: GATB, GATC, GFM2, HARS1, HARS2, LARS2, MRPL10, MRPL16, MRPL2, MRPL23, MRPL47, MRPL51, MRPL57, MRPS11, MRPS12, MRPS15, MRPS16, MRPS17, MRPS18A, MRPS18B, MRPS18C, MRPS2, MRPS21, MRPS24, MRPS34, MRPS6, MRPS7, NDUFA7, NOA1, PTCD3, QRSL1, and RARS2. These results indicate that INB16-timlNK cells are superior to IL15-cimlNK and rNK cells in terms of the expression of mitochondrial survival-related proteins. These results are thought to be related to the improvement in NK cell persistence observed in INB16-timlNK cells compared to rNK and IL15-cimlNK.

[0084] For the purposes of this article, the terms "riINB16-primed NK cells" and "INB16-timlNK cells" are interchangeable. The former is used to indicate that non-replicating INB16 cells were used to prime the resulting NK cells, while the latter is used to indicate that INB16 was used to induce the phenotype of tumor-induced memory-like NK cells (INB16 is a tumor cell that induces the NK phenotype).

[0085] Example 6 - Replication-incompetent INB16 cell preparation for treating cancer

[0086] NK cells can have effective anti-tumor responses and the potential for memory-like functions has recently been emphasized, but the best method for generating memory-like NK (mlNK) cells has remained unclear to date. Triggering NK cells with a pharmaceutical-grade replication-incompetent tumor cell product derived from INB16 cells generates tumor-induced mlNK (TIML-NK) cells, which have enhanced cytokine production and cytotoxicity against multiple NK-resistant tumor target cell lines in vitro, similar to cytokine-triggered memory-like NK cells reported previously. Additionally, proteomic profiling of TIML-NK cells revealed differential abundances of proteins involved in promoting mitochondrial survival and function as well as key nutrient receptors, which may provide unique benefits for NK cell activation while avoiding mitochondrial damage typically associated with cytokine-mediated activation. A cell preparation containing replication-incompetent INB16 cells increased NK glycolysis and oxidative phosphorylation upon triggering of NK cells, while enhancing mitochondrial respiratory capacity and maintaining glycolytic reserve.

[0087] A cell preparation containing INB16 cells was made replication-incompetent by exposing the cells to mitomycin C in vitro.

[0088] Four human patients were each treated with 1×10 8 replication-incompetent INB16 cells in a 3-week cycle with weekly infusions. All four patients received three doses each, without incident and without side effects.

[0089] Patient 1 was a 78-year-old male with a three-year history of refractory MLD MDS, dependent on transfusions and platelets and requiring G-CSF. Within 7 days of the first infusion, more than 50% of his peripheral blood NK cells were activated, and this increased with each dose. By day +29, 72% of his NK cells were activated, and this remained above 68% at day +119 at the end of monitoring. At day +119, his ECOG status decreased from 2 before treatment to 0, and his RAD-1ctDNA decreased from 45 to 38. Analysis of systemic cytokines showed increases in MIP-1a / b, TNF-a, and sIL2R, which paralleled changes in the percentage of CD69+ NK cells. IL6 levels peaked after the second infusion, but levels remained low and there was no evidence of CRS. During one year of follow-up, he remained well, was no longer dependent on platelets and G-CSF, had reduced transfusion requirements, and resumed exercising with friends.

[0090] Patients 2, 3, and 4 were all patients with multiple relapses and refractory AML; 2 of them relapsed after HLA-mismatched HSCT. All patients showed rapid activation of peripheral NK cells after infusion and had sustained activity throughout the follow-up period. In all cases, these activated NK cells killed NK-resistant tumor cells in vitro without additional activation.

[0091] Patient 2 was a 21-year-old female with neutropenia and refractory AML (M2) after 70% mixed chimerism and VUD HSCT at the time of treatment. Within 1 month of the start of INKmune treatment, she was discharged with PMN > 500, and her mixed chimerism regressed to complete donor chimerism. On day +140 after treatment with a cell preparation containing replication-incompetent INB16 cells, her bone marrow NK cells remained highly activated (>60% CD69+, K562 and Raji lysis >70%). Her AML relapsed 4 months after infusion and she died of recurrent disease 8 months after treatment.

[0092] Patient 3 was a 21-year-old male with refractory AML (M6) after two failed HSCTs (haplo m / m and VUD). He showed rapid generation of the same activated NK cells in vivo, but to a lesser extent (maximum 35% CD69+, continuous increase in lysis of K562, but no lysis of Raji cells), and there was no evidence of clinical improvement.

[0093] Patient 4 was a patient with multiple relapses of AML who had just received treatment and showed peripheral NK proliferation and maturation on day +8. The subject had no adverse reactions after the 1st and 2nd doses.

[0094] Infusion of a cell preparation containing replication-incompetent INB16 cells was shown to be safe and led to rapid and sustained activation of peripheral NK cells, accompanied by an increase in relevant systemic cytokines. TIML-NK has unique phenotypic, proteomic, and metabolomic features that mirror in vitro and in vivo activities.

[0095] This is the first time to generate memory-like NK cells in vivo without cytokine induction or support, and these findings inform the development of more optimized NK cell-based immunotherapy strategies for cancer.

[0096] Example 7 - riINB16-triggered NK cells show increased tumor-binding affinity

[0097] A variety of NK cell preparations were obtained, including resting NK (rNK) cells, NK cells primed with non-replicating INB16 (riINB16), IL-2-primed NK (NK-IL-2) cells, and IL-15-primed NK (NK-IL-15) cells. The NK cell binding of each of these preparations relative to rForce (pN) was studied using a LUMICKS cell affinity analyzer, and the results were observed and recorded. Specifically, ultrasound was used to apply a completely contactless force to the NK cells in a microfluidic chip. This technique is based on ultrasound and is used to apply a force within the microfluidic chip. Tumor cells were seeded in the chip and anchored to the bottom. Then NK cells were introduced and allowed to interact with the tumor cells. By turning on the ultrasound, acoustic forces were applied even without contacting the NK cells, and these forces were directed upward. NK cells showing strong tumor specificity would require a high force to be displaced. In contrast, NK cells that are not specific to any tumor antigen would require a weaker force to be displaced because no immune synapses would form in this case.

[0098] Figure 7 A graph showing the percentage of NK cell binding relative to rForce (pN) indicates the order of stronger affinity for tumor cell binding (SKOV3), with riINB16-primed NK cells having the strongest affinity, followed by NK-IL-15 cells, NK-IL-2 cells, and rNK cells in sequence.

[0099] Example 8 - riINB16-triggered NK cells function in a hypoxic environment

[0100] Figure 8 A graph shows the relationship of the percentage of NK cell lysis to time (hours post-treatment) before and after treatment with a cell preparation containing riINB16 cells in a hypoxic environment. Here, untreated (unprimed) NK cells showed significantly lower percentages of lysis compared to tumor-primed NK (TP-NK) cells primed with riINB16. In addition, as the ratio of riINB16 to NK cells increased (1:1, 2:1, 5:1), the percentage of lysis also increased. This data indicates that riINB16-primed NK cells show increased lysis in a dose-dependent manner. Since the tumor microenvironment (TME) is hypoxic, this data indicates that tumor-primed NK cells generated by contact with riINB16 are functional in a hypoxic environment such as in the TME.

[0101] Example 9 - riINB16-triggered NK cells restore tumor killing of dysfunctional NK cells

[0102] NK cells from three healthy human donors and from one human patient with MDS were obtained from peripheral blood. The ability of MDS tumor cell lines and corresponding NK cells to lyse MDS cells was studied for tumor cell killing in vitro. Resting NK (rNK) cells were evaluated against tumors arising from riINB16 cells, NK (TPNK). Figure 9 It was shown that rNK cells from healthy donors were able to lyse MDS cells in vitro; however, rNK cells from MDS subjects were unable to kill MDS cells. Instead, all NK cells were contacted with replication-incompetent INB16 (riINB16) to form TPNK cells, and when presented to MDS cells in vitro, all NK cells were able to lyse MDS cells, including those associated with MDS subjects. This data indicates that priming NK cells of MDS subjects with a cell preparation containing riINB16 cells results in primed NK cells being able to contact and kill MDS cells.

[0103] Industrial Applicability

[0104] Cells from the INB16 cell line can be used in the research and development of therapeutic agents and treatment strategies for treating cancer.

[0105] Compositions comprising replication-incompetent cells and / or membrane fractions from the INB16 cell line can be used as therapeutic agents and in treatment strategies for treating cancer.

[0106] Methods of treating patients suffering from cancer incorporating or utilizing compositions comprising replication-defective cells and / or membrane fractions from the INB16 cell line can be used as part of treatment strategies for treating cancer.

[0107] These and other industrial applications will be apparent to those skilled in the art upon reading this disclosure, including the claims and documents incorporated by reference.

[0108] Reference to the Deposited Biological Material

[0109] The INB16 cell line was deposited with the ATCC Patent Depository, 10801 University Boulevard, Manassas, Virginia 20110 USA, under the Budapest Treaty with the ATCC Deposit Number PTA-125809, first deposited on June 26, 2019, and viability was confirmed on July 5, 2019.

[0110] List of References

[0111] For all purposes, the entire contents of each of the following references are hereby incorporated by reference herein:

[0112] 1. Chen, P., Chiu, C., Chiu, T., Maeda, S., Chiang, H., Tzeng, C., Sugiyama, T., & Chiang, B. N. (1984). Establishment and Characterization of a Human Monocytoid Leukemia Cell Line, CTV-1. Gann Japanese Journal of Cancer Research, 75(8), 660-664. (Chen et al., 1984).

[0113] 2. Lowdell, Mark et al., (2022). In Vivo Generation of Memory-like NK Cells for the Treatment of AML and Myelodysplastic Syndrome; Early Clinical Applications of INKmuneTM Blood. 140. 4590-4591. 10.1182 / blood-2022-166175.

[0114] 3. LOWDELL, MARK, U.S. Patent No. 8,257,970, issued September 4, 2012.

[0115] 4. TESI, RAYMOND J et al., U.S. Patent No. 10,758,567, issued September 1, 2020.

[0116] 5. BRONSHTEIN, VICTOR, U.S. Patent No. 9,469,835, issued October 18, 2016.

Claims

1. A cell, wherein the cell is from the T-cell acute lymphoblastic leukemia cell line INB16 (ATCC deposit number PTA-125809).

2. A biopharmaceutical composition comprising cells and / or membrane portions of cells from the INB16 cell line (ATCC deposit number PTA-125809), wherein the cells and / or membrane portions of the cells are rendered non-replicative.

3. The biopharmaceutical composition according to claim 2, comprising the cells from the INB16 cell line, wherein the cells are rendered non-replicative by contact with mitomycin C.

4. The biopharmaceutical composition according to claim 2, comprising the cells from the INB16 cell line, wherein the cells are rendered non-replicative by first fixing the cells in a dry saccharide matrix and second administering the cells while fixing with permeating ionizing radiation.

5. The biopharmaceutical composition according to claim 4, wherein the dose comprises at least 2 Gy and at most 20 kGy.

6. A method for treating cancer, comprising: administering to a patient suffering from the cancer a therapeutically effective amount of a priming tumor cell preparation for priming natural killer cells in the patient in vivo, the priming tumor cell preparation comprising: cells and / or membrane portions thereof from the INB16 cell line (ATCC deposit number PTA-125809), wherein the cells and / or membrane portions of the priming tumor cell preparation are rendered non-replicative to prevent proliferation in the patient; thereby treating the patient.

7. The method according to claim 6, wherein the cancer comprises solid tumors.

8. The method according to claim 6, wherein the cancer comprises acute myeloid leukemia (AML).

9. The method according to claim 6, wherein the cancer comprises myelodysplastic syndrome.

10. The method according to claim 6, wherein the therapeutically effective amount comprises administering 1×10 8 cells three times, once a week.

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