Guiding and augmenting NK cell-based therapies in high HLA-e expressing human cancers such as leukaemia

Abstract

Disclosed herein are methods of enhancing the abundance of NKG2C+ adaptive NK cells in a population of NK cells and the use of NK cells expressing NKG2C derived from such methods for treatment of cancers, including leukemia such as chronic myeloid leukemia with enhanced expression of HLA-E molecule. In one embodiment, the method is a biphasic method comprising a selection phase and an expansion phase. In one embodiment, the selection phase comprises use of a first culture comprising IL-2 and feeder cells expressing HLA-E. In one embodiment, the expansion phase comprises use of a second culture comprising IL-2, IL-15 and feeder cells expressing HLA-E, IL-21 and 4-1 BBL. Also disclosed are methods of stratifying chronic myeloid leukemia patients into responders or non-responders to tyrosine kinase inhibitor to allow a suitable treatment for each group with tyrosine kinase inhibitor or NK cells expressing NKG2C derived from the described methods. In one embodiment, the stratification method comprises determining a percentage of NKG2A+ cells in the patients' NK cell population.

Description

DESCRIPTIONTITLE OF INVENTION: [Guiding and Augmenting NK Cell-Based Therapies In High HLA-E Expressing Human Cancers such as Leukaemia]REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority to Singapore patent application No. 10202402771V, filed 6 September 2024, the contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] The present disclosure relates generally to detection and selection of High HLA- E Expressing Human Cancers, particularly leukemia for treatment with NK cells and compositions containing the NK cells and methods of making the same.BACKGROUND

[0003] The following discussion of the background to the invention is intended to facilitate an understanding of the present invention only. It should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was published, known or part of the common general knowledge of the person skilled in the art in any jurisdiction as at the priority date of the invention.

[0004] Cancer is a group of diseases that are a serious problem in society. Cancer can start in almost any organ or tissue of the body when abnormal cells grow uncontrollably, go beyond their usual boundaries to invade adjoining parts of the body and / or spread to other organs. In 2019, annual cancer cases grew and there were about 10 million deaths worldwide (Kocarnik et al. 2022, JAMA Oncology. 8(3): 420-444). A group of cancers has been identified where overexpression of the HLA-E molecule has been observed, including in cervical cancer (Ferns et al. J immunother Cancer 2016;4:78), breast cancer (de Kruijf et al. J Immunol 2010;185:7452-9), non-small cell lung carcinoma (NSCLC) (Talebian et al. Oncotarget 2016;7:3477-88), liver, pancreas, kidney (Seliger et al. Ocotarget 2016;7:67360-72), melanoma, prostate, head and neck, stomach, rectal, colorectal cancer (Levey et al. Int J Oncol 2008;32:633-41), and leukemia including chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) (T-ALL, B-ALL, Ph+ ALLPh- ALL) or diffuse large B-cell lymphoma (DLBCL). HLA class I histocompatibility antigen, alpha chain E (HLA-E) is a human protein encoded by the HLA-E gene. It is sometimes also referred to as MHC class I antigen E and is a non-classical monomorphic-like MCH class Imolecule that presents a limited set of conserved signal peptides. HLA-E is characterized by a lower cell surface expression than other classical polymorphic MCH class I paralogues. HLA-E has a very specialized role in cell recognition by natural Killer cells.

[0005] Leukemia is a group of blood cancers. There are four main types of leukemia, CML, ALL, AML, and CLL. Less common types such as MM, DLBCL or plasma cell leukemia (PCL) are also included in the group of blood cancers. While a wide variety of treatments such as chemotherapy, radiation therapy, targeted immune therapy, bone marrow transplant or any combinations of these are known, which have greatly increased the 5-year survival rate of leukemia patients in recent years, more than 300,000 people died of leukemia in 2021 (Global Burden of Disease the Lancet 2024). This is partially due to relapse of leukemia whereby the patients become resistant to treatment. While there has been a lot of research on the likely metabolic reasons for such relapses and resistance it is still unclear what causes such relapses and resistance. For a large part there is no treatment available for treatment resistant leukemia. One of the more treatable leukemias is CML where tyrosine kinase inhibitors (TKI) such as imatinib are used. However, there is still a population of TKI-resistant patients that will progress through the same disease phases as untreated CML. In the absence of intervention CML is often divided into three phases which typically begins in the chronic phase, and over the course of several years progresses to an accelerated phase and ultimately to a blast crisis which has low survival rates.

[0006] Natural killer (NK) cells are innate immune cells with the capacity to effectively target both virally infected and malignant cells (Myers and Miller, Nat Rev Clin Oncol. 2021 ;18(2):85-100). NK cell-based cancer immunotherapy has been suggested because such therapy are considered to offer a much safer alternative to T cell-based therapies, mitigating risks associated with graft vs. host disease (GvHD) and cytokine storm complications. Furthermore, their MHC-unrestricted target recognition and the fact that they do not require antigen-specific priming makes them suitable for therapeutic use (Laskowski et aL, Nat Rev Cancer. 2022;22(10):557-75). Mechanistically, NK cells express a diverse array of cell surface activating and inhibitory receptors. Upon engagement with ligands on the target cell, NK cell receptors transmit signals within NK cells, the balance of which decides whether the NK cell becomes activated or is inhibited. Once activated NK cells can directly kill tumour cells, and by producing cytokines, induce killing by the adaptive immune system.

[0007] In an attempt to provide better customized treatments to treatment resistant leukemias some have genetically modified cells in attempts to have targeted NK cells. Other technology involves the expansion of NK cells derived from healthy peripheral blood or cord blood, using external feeder cells expressing membrane-bound-interleukins or expressing HLA-E. These expanded NK cells may undergo subsequent genetic modification, such as protein expression blocker modification to prevent inhibitory cell surface expression of NKG2A. These technologies expand the entire NK cell population including both activated and inhibited NK cells. New methods are needed that can stably result in a higher amount of adaptive NK cells.

[0008] Adaptive NK cells constitute an epigenetically distinct subset naturally accumulating only in some individuals with prior cytomegalovirus (CMV) infection (Liu et al. Mol Oncol. 2015;9(10): 1904-17. and Schlums et al. Immunity. 2015;42(3):443-56). Adaptive NK cells, like memory T-cells, persist beyond the usual lifespan of classical NK cells and are endowed with robust metabolic capacity, resistance to oxidative stress, excellent cytokine secretion, superior antibody-dependent cellular cytotoxicity (ADCC) and tumor cell killing (Woan et al. Cell Stem Cell. 2021 ;28(12):2062-75 e5). Methods have been developed to expand human NK cells using genetically modified human immortalized myelogenous leukemia cell line (Somanchi, and Lee, Methods Mol Biol. 2016:1441 :175-93).

[0009] There exists a need to accurately determine and / or find alternative treatments for treatment-resistant cancers with enhanced expression of HLA-E molecule such as leukemias or other cancers in this group to alleviate at least one of the aforementioned problems.SUMMARY

[0010] Methods of detection and selection of High HLA-E Expressing Human Cancers particularly leukemia for treatment with NK cells having enhanced NKG2C expression, or compositions containing the NK cells and methods of making the same is envisaged.

[0011] Accordingly, an aspect of the invention refers to a method of enhancing an amount or abundance of NKG2C+ adaptive NK cells in a population of NK cells, the method comprising: a) co-culturing peripheral blood mononuclear cells (PBMCs) in a first culture the first culture comprising: (i) first immortalized cell line genetically modified to express HLA-E; and (ii) interleukin 2, b) isolating the cultured PBMCs; c) co-culturing the isolated cultured PBMCs in a second culture, the second culture comprising: (i) second immortalized cell line genetically modified to express amino acid sequence of HLA-E and IL21 and 4-1 BBL (ii) interleukin 2, and (iii) interleukin 15 d) isolating NK cells from the second culture wherein the NK cell population includes cells that express NKG2C.

[0012] According to another aspect of the invention there is a method of determining a treatment outcome in patient who has been diagnosed with chronic myeloid leukemia and providing an appropriate treatment regimen, the method comprising: isolating an NK cell population from the bone marrow of the patient who has been diagnosed with chronic myeloidleukemia; determining percentage of NKG2A+ cells in the NK cell population wherein expression of NKG2A+ below a predetermined percentage of the NK cell population is indicative that the patient will be responsive to a treatment regimen with a tyrosine kinase inhibitor and the patient is administered the tyrosine kinase inhibitor and expression of NKG2A+ above a predetermined percentage of the NK cell population is indicative that the patient will become unresponsive to a treatment regimen with a tyrosine kinase inhibitor and the patient is administered NK cells expressing NKG2C+ isolated by the method of enhancing the amount or abundance of NKG2C+ adaptive NK cells isolated by an expansion method comprising: a) coculturing peripheral blood mononuclear cells (PBMCs) in a first culture the first culture comprising: (i) first immortalized cell line genetically modified to express amino acid sequence of HLA-E; and (ii) interleukin 2, b) isolating the cultured PBMCs; c) co-culturing the isolated cultured PBMCs in a second culture, the second culture comprising: (i) second immortalized cell line genetically modified to express amino acid sequence of HLA-E and IL21 and 4-1 BBL (ii) interleukin 2, and (iii) interleukin 15 d) isolating the population of NK cells from the second culture wherein the NK cell population include cells expressing NKG2C.

[0013] According to another aspect of the invention there are NK cells wherein at least 20% or 21% of the NK cells abundant in or expressing NKG2C for use in the treatment of cancer wherein expression of HLA-E molecule on cells in a cancer sample from the cancer is more than the expression of HLA-E molecule on cells in a control sample from a healthy individual.

[0014] According to another aspect of the invention there is a use of NK cells wherein at least 20% or 21 % of the NK cells abundant in or expressing NKG2C in the manufacture of a medicament for treatment of cancer wherein a cell sample from the cancer expresses HLA-E molecule on cells at a higher amount than the expression of HLA-E molecule on cells in a control sample from a healthy individual.

[0015] According to another aspect of the invention there is a composition comprising NK cells derived by the method of enhancing the amount or abundance of NKG2C+ adaptive NK cells described herein above and a tyrosine kinase inhibitor.

[0016] According to another aspect of the invention there is a composition comprising NK cells derived by the method of enhancing the amount or abundance of NKG2C+ adaptive NK cells described herein above and a therapeutic cytotoxic antibody.

[0017] According to another aspect of the invention there is a composition NK cells derived by the method of enhancing the amount or abundance of NKG2C+ adaptive NK cells described herein above and a tyrosine kinase inhibitor dasatinib or a composition for use in the treatment of cancer wherein expression of HLA-E molecule on cells in a cancer sample is more than theexpression of HLA-E molecule on cells in a control sample from a healthy individual.

[0018] According to another aspect of the invention there is use of a composition of NK cells derived by the method of enhancing the amount or abundance of NKG2C+ adaptive NK cells described herein above and a tyrosine kinase inhibitor, dasatinib or a composition in the manufacture of a medicament for treating cancer wherein a cell sample from the cancer expresses HLA-E molecule on CD34+ cells at a higher amount than the expression of HLA-E molecule on CD34+ cells in a control sample from a healthy individual.

[0019] According to another aspect of the invention there is an in vitro method for predicting subgroups of chronic myeloid leukemia comprising isolating an NK cell population from the bone marrow of a patient who has been diagnosed with chronic myeloid leukemia; determining percentage of NKG2A+ cells in the NK cell population wherein when expression or abundance of NKG2A+ below a first predetermined percentage of the NK cell population is indicative of a first subgroup that will be responsive to treatment with a tyrosine kinase, and expression or abundance of NKG2A+ above a first predetermined percentage of the NK cell population is indicative of a second subgroup that will be unresponsive to treatment with a tyrosine kinase inhibitor.

[0020] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In the figures, which illustrate, by way of non-limiting examples only, embodiments of the present invention,

[0022] [Fig. 1]: Stratification of CML patient responses to adaptive NK cell therapy based on HLA-E status. Bone marrow-derived mononuclear cells (BM-MNCs) from CML prognostic Groups A, B and C, collected at the time of diagnosis were subjected to flow cytometry. Populations are plotted as a proportion of NK cells. A. NKG2C+ adaptive NK cell abundance. B. NKG2A+ NK cell abundance. C. scRNA-seq derived HLA-E gene expression on CD34+ HSPCs within healthy and CML prognostic groups D. CD34+ HSPCs isolated from healthy control (n=2), MBC (n=3) and LBC (n=4) were stained with anti-HUX-E antibody. M.F.I (Mean fluorescence intensity) values after flow cytometry are shown. E. Longitudinal in vivo bioluminescence imaging on days D7, D21 , D28, D35, and D42 for four groups: Neg (no treatment), K562-HLA-E, K562-HLA-E+non-aNK, and K562-HLA-E+aNK. Pseudo-colour scale reflects tumor burden. The K562-HLA-E+aNK group maintains the lowest signals over time. F.Percentage of human NK cells in peripheral blood (PB) by group: Negative, K562-HLA-E only, K562-HLA-E+non-aNK, K562-HLA-E+aNK. Points represent individual mice; bars summarize the cohort. Significance is indicated by asterisks (* p<0.05, ** p<0.01). G. Kaplan-Meier survival curves for K562-HLA-E, K562-HLA-E+non-aNK, K562-HLA-E+aNK, and Negative control (no tumor). Therapy with aNK significantly prolongs survival versus others (log-rank p=0.001 . NK cell donors: one non-aNK donor (HSA42; n=4 mice) and one aNK donor (HSA39; n=7 mice).H. Populations of NKG2C+ adaptive NK cell abundance and NKG2A+ NK cell abundance plotted as a proportion of NK cells at the time of diagnosis (DayO) when adaptive NK cell therapy was stated, and 5, 7 and 14 days after therapy.

[0023] [Fig. 2]: Biphasic expansion protocol. A schematic of the steps in the biphasic expansion protocol.

[0024] [Fig. 3]: A biphasic adaptive NK cell expansion protocol. Adaptive NK cells were expanded from healthy donors by culturing PBMCs on 2 in-house generated genetically- modified K562-based feeder lines. A. Feeder#1 comprised of K562 cells with ectopic expression of HLA-E*. Feeder#2 comprised of K562 cells with ectopic expression of membranebound IL21 (mb IL-21), 4-1BBL and HLA-E*. B. Schematic illustration of the biphasic ex vivo expansion of adaptive NK cells. The biphasic protocol optimised comprises of 2 phases.Selection phase: PBMCs (1-2 million cells) were co-cultured with irradiated feeder#1 (100 Gy) at effector: target cell ratio of 1 :2 in NK cell expansion medium for 7 days in the presence of IL2 (50 lU / ml). Expansion phase: On day 7, culture was spun down and cells were co-cultured with irradiated feeder#2 (100 Gy) at effector E:T (effector: target) cell ratio of 1 :1 in NK cell expansion medium in the presence of IL2 100 lU / ml + IL15 10ng / ml. C. The plot illustrates the fold increase in total Natural Killer (NK) cells and NKG2C+ subset of NK cells (aNKE21) following ex vivo expansion for 14 days (grey dots), compared to their respective unexpanded counterparts (black dots). D. Fold expansion of NKG2C+ adaptive NK aNKE21following ex vivo expansion for 14 days. E. the number of NKG2C+CD56dim NK cells following ex vivo expansion for 14 days. F. the biphasic ex vivo expansion protocol for adaptive NK cells aNKE21was compared with a known monophasic protocol, where PBMCs are cultured continuously with irradiated K562-mlL21-41 BBL feeder cells. Our head-to-head analysis of the two protocols, based on a sample size of n=2, demonstrated the superior enrichment of adaptive NK cells by the biphasic approach and higher expression of NK cell activation pathways including MTOR signaling and IFN-gamma response following gene expression analysis. G. PBMCs were stained with markers specific to adaptive NK cells and categorized based on their proportions into two groups: low adaptive NK cells (<20%) and high adaptive NK cells (>20%). The total NK cell yield from healthy PBMC donors with greater than 20% adaptive NK cells was significantly higher compared to donors with low initial proportions of adaptive NK cells (<20%). H. PBMCswere stained with markers specific to adaptive NK. The quadrant representing NKG2C- / NKG2A- (DN) was flow sorted. The DN population of NK cells were subjected to the biphasic ex vivo adaptive NK cell expansion protocol. At the end of 14 days, NKG2C+ cells aNKE21were enriched in the final cultures. I. NKG2A+ and NKG2C+ population NK cell subsets were sorted using flow cytometry. The plot shows cytotoxicity of these populations against K562-HLA-E, MM1S-HLA-E and RPMI-8226-HLA-E as the target. J. Target cell cytotoxicity assay (4 hours) on Day 0 (DO) or after 14 days of ex vivo expansion (D14) at the indicated ET ratio. K. CML patient derived CD34+ HSPCs were incubated alone (No NK) or exposed to ex vivo expanded NK cells at the indicated E:T ratios for 4.5 hours. The surviving cells were subjected to colony formation assays in methylcellulose media for 14 days. Surviving colonies are shown relative to no NK cell controls. L. 5 X 10sK562-HLAE-LucGFP cells were injected into sub lethally irradiated (2.5 Gy) NSG mice via caudal artery. Two doses of 10 x 106ex vivo expanded NK cells aNKE21were injected via tail vein on day1 and day7. Human recombinant IL15 0.5 pg / mouse were injected intraperitoneally on day1 , day7, and day14. On day14, MS imaging was performed 15min after injecting D-luciferin 150 mg / kg intraperitoneally.

[0025] [Fig. 4]: Schematic illustration of the ‘Biphasic adaptive NK expansion protocol V2.0’. A. The protocol described in [Fig. 3B] was modified as follows. Dasatinib (100 nM) was added to the expansion medium from day 7 to day 14. B. Increase in the number of CD56dim NK cells and fold expansion of NKG2C+ adaptive (V20NK cells comparing 14 days of standard biphasic expansion protocol from [Fig. 3B] and V2 protocol (with Dasatinib). C. targeted screen of FDA-approved agents, dasatinib uniquely enhanced aNK expansion (~1 ,000-fold increase; =2x10' cells from 1 .5 mL PBMCs) compared with other TKIs (imatinib, ponatinib, nilotinib; in comparison with standard-expanded aNKE21(aNKE21) FDA-approved agents e.g., dasatinib was added at a concentration of 100 nM only during the expansion phase (day 7-14) and washed off prior to harvest on day 14. D. Total NK yield at Day 14 across independent donors. E. Representative flow cytometry plots showing CD56dim NKG2C+ KIR+ NKG2Alow adaptive NK phenotype in Day 14 products. F. Quantification of adaptive NK frequency in paired donors expanded using the biphasic protocol in the presence or absence of dasatinib (100nM). G-H. Viability analysis showing reduced Annexin V+ frequency and improved survival from Day 9 onwards in dasatinib-treated cultures.

[0026] [Fig. 5]: CML NK cell exhaustion can be overcome by expanding cells ex vivo using the biphasic adaptive NK cell expansion protocol. MNCs derived from the bone marrow of healthy individuals or CML patients were isolated and either cultured overnight in medium or subjected to ex vivo expansion using the biphasic feeder system. The functionality of NK cells was assessed through various assays across healthy and CML samples under two conditions: unexpanded and after ex vivo expansion. For panels A-E: A. Scatter plots 1 and 2represent unexpanded CML-NK cells, while scatter plots 3 and 4 represent ex vivo expanded cells, showing NK cell degranulation in the presence of K562. B. Similarly, IFN-gamma (IFNg) expression in the presence of K562 is shown. C. NK cell degranulation after CD16 crosslinking is depicted. D. IFN-gamma expression after CD18 crosslinking is illustrated. E. IFN-gamma expression following cytokine exposure is demonstrated. F. Target cell cytotoxicity is compared between healthy and CML groups for unexpanded samples. G. Similar comparison is shown for samples following ex vivo expansion. H. Healthy and CML MNCs, after 14 days of ex vivo expansion in the biphasic feeder, were assessed for their ability to kill target K562-HLAE expressing cells at indicated ratios.

[0027] [Fig. 6]: A multiple myeloma cell line (MM1 S) and plasmacytoma cell line (RPMI8226), with and without HLA-E overexpression, and two AML cell lines (THP-1 , HL-60), were cultured in the absence or presence of ex vivo expanded NK cells for 4 hours. Cells were subjected to Annexin V staining -Hive cell / dead cell discriminator to measure apoptotic rate. A. Flow cytometry plots showing Annexin V staining of MM 1 S cells, with and without HLA-E overexpression, in the presence or absence of NK cells. B. Percent apoptosis of MM1 S and RPMI8226 cells, with and without HLA-E overexpression, in the presence of NK cells at the indicated E:T ratios. C. Flow cytometry plots showing Annexin V staining of THP-1 cells in the presence or absence of NK cells D. Percent apoptosis of THP-1 and HL-60 cells in the presence of NK cells at the indicated E:T ratios. E. 1 X 106MM1 S-HLAE-LucGFP cells were injected into sub-lethally irradiated (2.5 Gy) NSG mice via caudal artery. Two doses of 2 x 10sex vivo expanded NK cells were injected via tail vein on day 1 and day 7. Human recombinant IL15 0.5 pg / mouse were injected intraperitoneally on day 1 , day 7, day 14, day 21 and day 28. MS imaging was performed weekly by injecting D-luciferin 150 mg / kg intraperitoneally.

[0028] [Fig. 7]: Single-cell RNA-seq of Day-14 products [baseline(day 0), day 14 eNKE21 and Das-eNKE21] A-C. NK cells expanded in the presence of dasatinib had a unique cluster composition, as compared to both unexpanded (Day 0) and aNK expanded in the absence of dasatinib D. Differential gene expression (DEG) analysis followed by gene ontology studies revealed that dasatinib-cultured aNKE21 cells displayed broadly suppressed type-l / l I interferon- response programs, elevated cytokine-cytokine receptors interactions E. and a significantly reduced composite exhaustion signature. F. Orthogonal protein-level validation confirmed decreased surface expression of canonical dysfunction markers, including KLRG 1 , CD161 (KLRB1), and CD69 G. Dasatinib significantly downregulated PRDM1 (encoding BLIMP1), pronounced at the protein level (flow cytometry across paired donors.

[0029] [Fig. 8]: Dasatinib-expanded aNK cells exhibit superior in vivo persistence and enhanced tumor control. A. In vitro cytotoxicity of Das-eNKE21versus standard eNKE21cellsagainst primary BC-CML SPCs across increasing effector-to-target ratios (2:1-10:1). Das- eNKE21cells show significantly greater killing in selected patient-derived lines (P670, P687, P681), while no difference is observed in samples highly sensitive to standard eNKE21. B. In vivo persistence of Das-eNKE21versus eNKE21in NSG mice with low-dose IL-15 support. Peripheral blood human NK chimerism measured weekly by flow cytometry demonstrates significantly higher early engraftment at Week 1 and sustained trends toward higher frequencies at Weeks 2-4, C. Persistence advantage of Das-eNKE21cells in K562-HLA-E xenografts and a primary MBC patient-derived xenograft (P681). Das-eNKE21frequencies are significantly higher in both models. D. Donor reproducibility of Das-eNKE21persistence advantage. Peripheral blood NK chimerism over time in NSG mice receiving Das-eNKE21or eNKE21cells from independent donors (HSA39, HSA43) with low-dose IL-15 support. Both donors exhibited consistently higher chimerism with Das-eNKE21, with significant differences for Donor HSA39 and strong positive trends for Donor HSA43. E. Tumor control in the K562-HLA-E model. Bioluminescence imaging shows delayed leukemia progression in Das-eNKE21-treated cohorts.

[0030] [Fig. 9]: Enhanced killing of head and neck cancer lines by Dasatinib-expanded NK cells. Dasatinib-expanded NK cells were tested against a panel of head and neck cancer cells lines and demonstrated higher cytotoxicity.

[0031] [Fig. 10]: Adaptive NK cell percentages correlate with Antibody-dependent cell cytotoxicity (ADCC) proficiency A. Adaptive NK cell abundance in each eNKE21feeder cell formed with the biphasic method. B. The proficiency of the expanded adaptive NK cells were tested in mediating ADCC in the presence of cetuximab, ADCC proficiency was proportional to the percent aNK population in the final expanded NK product.

[0032] [Fig. 11]: Augmenting killing of CML targets overexpressing HLA-E by the expanded NK cell products; A. HLA-E expression on the cell surface of CML CD34+ was assessed after incubating cells with control TH9 peptide or viral VL9 peptides (1 and 2) for 16 hours. Median Fluorescence intensity for each condition is shown. B. Percentage of Annexin V+ apoptotic cells across 4 conditions are shown: untreated, TH9-treated, VL9 peptide 1 and 2 in the presence or absence of the expanded NK cells at the indicated E:T ratios for 24 hours. Data is plotted as a line graph (left) and bar graph (right). C. Experiment was done as described in B, and the clonogenic potential of the surviving cells after a 24 hour incubated with NK cells was determined by plating cells on methocellulose for colony forming assays. Colonies were counted after 14 days and the data was plotted was a percentage of untreated cells. D. NKG2C- HLA-E interaction dependency of the killing was shown by incubating TH9 or VL9 treated cultures with a blocking isotype control or a NKG2C-specific blocking antibody.DETAILED DESCRIPTION

[0033] Throughout this document, unless otherwise indicated to the contrary, the terms “comprising”, “consisting of’, “having” and the like, are to be construed as non-exhaustive, or in other words, as meaning “including, but not limited to”.

[0034] Furthermore, throughout the document, unless the context requires otherwise, the word “include” or variations such as “includes” or “including” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0035] Unless defined otherwise, all other technical and scientific terms used herein have the same meaning as is commonly understood by a skilled person to which the subject matter herein belongs.

[0036] According to various embodiments, there is a method of enhancing an abundance of NKG2C+ adaptive NK cells in a population of NK cells, the method comprising: a) co-culturing peripheral blood mononuclear cells (PBMCs) in a first culture the first culture comprising: (i) immortalized cell lines genetically modified to express amino acid sequence of HLA-E; and (ii) interleukin 2, b) isolating the cultured PBMCs; c) co-culturing the isolated cultured PBMCs in a second culture, the second culture comprising: (i) second immortalized cell lines genetically modified to express amino acid sequence of HLA-E and IL21 and 4-1 BBL (II) interleukin 2, and (iii) interleukin 15 d) isolating a population of NK cells from the second culture wherein the NK cell population include cells expressing NKG2C.

[0037] In various embodiments, the amino acid sequence of HLA-E in either the first or the second immortalized cell line comprises a chimeric construct of a HLA-E peptide wherein a signal peptide of the HLA-E is replaced with a signal peptide of HLA-G.

[0038] In various embodiments, the immortalized cell line is a cell line created from a cancer for which the cells are destined to be used as a treatment. For example, where the cancer is cervical cancer an immortalized cell line such as HeLa cells may be used, or where the cancer is leukemia including chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) (T-ALL, B-ALL, Ph+ ALLPh- ALL) or diffuse large B-cell lymphoma (DLBCL) an immortalized cell line such as K562 cells may be used. There is a wide array of immortalized cell lines created from cancer cells that may be used. In various embodiments, any immortalized cell line may be used even where it is not created from the cancer for which the cells are destined to be used as a treatment. For example, an immortalized cell line such as K562 cells may be used.

[0039] In various embodiments, the immortalized cell line is genetically modified to express a construct encoding a modified HLA-E of SEQ ID NO. 1, wherein the HLA-E signal peptide (SEQ ID NO. 2: MVDGTLLLLLSEALALTQTWA) is replaced by the HLA-G signal peptide (SEQ ID NO. 3: MWMAPRTLFLLLSGALTLTETWA). In various embodiments, the immortalized cell line comprises K562 cells that are genetically modified to express a construct encoding a modified HLA-E of SEQ ID NO. 1. In various embodiments, the genetic modification may be achieved by any means known in the art that introduces recombinant nucleic acid into cells that may include, heat stress, electroporation, microinjection, vectors such as viral vectors, retroviral vectors such as pBABE-puromycin vector or any other suitable method that permits expression of transgenic nucleic acid in cells. In various embodiments, the genetically modified immortalized cell line such as K562 is deactivated to exclude its cancerous pathogenic ability before being used as the first feeder layer. In various embodiments, the genetically modified immortalized cell line such as K562 may be deactivated via gamma irradiation. In various embodiments, the genetically modified immortalized cell line such as K562 may be deactivated via the use of mitomycin C. In various embodiments, the genetically modified immortalized cell line such as K562 may be deactivated via X-Ray irradiation at 50 grey or more. In various embodiments, the genetically modified immortalized cell line such as K562 may be deactivated via X-Ray irradiation at 75 grey or more. In various embodiments, the genetically modified immortalized cell line such as K562 may be deactivated via X-Ray irradiation at 100 grey or more. In various embodiments, any method of deactivating the genetically modified immortalized cell line such as K562 may be suitable provided it produces cells with non-toxic features and satisfy the criteria to be used as a stimulatory factor.

[0040] In various embodiments, the second immortalized cell line comprises the same cell line as describe above used to create the first feeder layer with the exception that in addition to being genetically modified to express a construct encoding a modified HLA-E of SEQ ID NO. 1 , wherein the HLA-E signal peptide (SEQ ID NO. 2: MVDGTLLLLLSEALALTQTWA) is replaced by the HLA- G signal peptide (SEQ ID NO. 3: MWMAPRTLFLLLSGALTLTETWA) the second immortalized cell line is additionally modified to overexpress IL21 and 4-1 BBL. In various embodiments, the second immortalized cell line comprises K562 cells that are genetically modified to express a construct encoding a modified HLA-E of SEQ ID NO. 1 and to overexpress IL21 and 4-1 BBL. In various embodiments, genetic modification may be achieved as described above and the overexpression may be achieved by any means known in the art that results in increased expression of IL21 and 4-1 BBL. For Example, overexpression may be achieved by cloning of a gene under a strong promoter and introduction of a vector into the cell or any other suitable method that permits overexpression of IL21 and 4-1 BBL in cells would also be suitable. In various embodiments, the second immortalized cell line is genetically modified sequentially, whereby a modified Murine Stem Cell Virus (MSCV) is used to overexpress the exogenous sequences. Invarious embodiments, the second genetically modified immortalized cell line such as K562 is deactivated to exclude its cancerous pathogenic ability before being used as the second feeder layer. In various embodiments, the second genetically modified immortalized cell line such as K562 may be deactivated via gamma irradiation. In various embodiments, the second genetically modified immortalized cell line such as K562 may be deactivated via the use of mitomycin C. In various embodiments, the second genetically modified immortalized cell line such as K562 may be deactivated via X-Ray irradiation at 50 grey or more. In various embodiments, the second genetically modified immortalized cell line such as K562 may be deactivated via X-Ray irradiation at 75 grey or more. In various embodiments, the second genetically modified immortalized cell line such as K562 may be deactivated via X-Ray irradiation at 100 grey or more. In various embodiments, any method of deactivating the second genetically modified immortalized cell line such as K562 may be suitable provided it produced cells with non-toxic features and satisfy the criteria to be used as a stimulatory factor.

[0041] In various embodiments, the population of NK cells from the second culture comprises NK cells wherein at least 21% of NK cell population express NKG2C and may comprise 21% or more, or at least 22%, or at least 23%, or at least 24%, or at least 25%, or at least 26%, or at least 27%, or at least 28%, or at least 29%, or at least 30%, or at least 31%, or at least 32%, or at least 33%, or at least 34%, or at least 35%, or at least 36%, or at least 37%, or at least 38%, or at least 39%, or at least 40%, or at least 41%, or at least 42%, or at least 43%, or at least 44%, or at least 45%, or 25-45%, or 26-45%, or 27-45% or 28-45%, or 29-45%, or 30-45%, or 31-45%, or 32-45%, or 33-45%, or 34-45%, or 35-45%, or 36-45%, or 37-45% or 38-45%, or 39-45% or 40-45%, or 41-45%, or 42-45%, or 43-45% or 44-45% or 50% or 60% or 70% or 80% of NK cell population express NKG2C.

[0042] In various embodiments, the second culture further comprises addition of a Bcr-Abl allosteric inhibitor. In various embodiments, the Bcr-Abl allosteric inhibitor comprises a tyrosine kinase inhibitor. In various embodiments, the tyrosine kinase inhibitor comprises dasatinib. The use of dasatinib added to the second culture results in a fold expansion of NKG2C+ adaptive cells enhancing the biphasic method significantly.

[0043] In various embodiments, the first or second culture further comprises addition of an actin remodeler. In various embodiments the actin remodeler comprises lenalidomide. In various embodiments, the second culture further comprises addition of the actin remodeler, lenalidomide. The use of lenalidomide to the second culture increases cytotoxicity of the expanded NKG2C+ adaptive cells.

[0044] In various embodiments, the first or second culture may further comprise other additives which may include: 1) tyrosine kinase inhibitor, dasatinib; or 2) Addition of small molecule inhibitors of BCL2 antiapoptotic proteins to prevent cell death and enhance expansion; or 3) Addition of actin remodelers such as lenalidomide to improve cytotoxicity; or 4) Addition of metabolic modulators (glucose, amino acids, mTOR inhibitors), ROS quenchers to improve in vivo persistence and cytotoxic potential; or 5) Addition of epigenetic modifiers such as HDACs and DNMTi to promote adaptive phenotype of NK cells; or 6) any combination thereof, which are each additive is expected to result in an increase in expansion of NKG2C+ adaptive cells or an increase in the cytotoxicity of the resulting expanded NKG2C+ adaptive cells or to promote adaptive phenotype of NK cells. All of which enhance the biphasic method significantly.

[0045] In various embodiments, the PBMC’s are isolated from a person with at least 15% of the NK cell population identified as a CD56+CD16+CD57+NKG2C+ subset whereby each of CD56, CD16, CD57 and NKG2C are expressed in 15% of the cells in an NK cell population. In various embodiments, a person with at least 15% of the NK cell population identified as a CD56+CD16+CD57+NKG2C+ subset may be considered a super donor and may be found in approximately 40% of the Singapore population. The high proportion of Singapore super donors may be as a result of high prevalence of cytomegalovirus (CMV) infection. In various embodiments, selection of PBMC’s isolated from a super donor with at least 15% of the NK cell population identified as a CD56+CD16+CD57+NKG2C+ subset has the advantage of enhancing adaptive NK cells expressing NKG2C resulting from the disclosed method. In various embodiments, selection of PBMC’s isolated from a super donor with at least 15% of the NK cell population identified as a CD56+CD16+CD57+NKG2C+ subset exhibit greater effectiveness at ex vivo adaptive NK cell expansion. In various embodiments, the PBMC’s are isolated from a healthy person. In various embodiments, the PBMC’s are isolated from a healthy person with at least 15% of the NK cell population identified as a CD56+CD16+CD57+NKG2C+ subset. In various embodiments, the PBMC’s are isolated from a person with a prior history of CMV. In various embodiments, the PBMC’s are isolated from a person with a prior history of cancer. In various embodiments, the person with a prior history of cancer is in remission and may be considered a healthy person. In various embodiments, the person with a prior history of cancer comprises a person with a prior history of a cancer that exhibits overexpression of HLA-E. In various embodiments, cancers that exhibits overexpression of the HLA-E molecule may include any one of cervical cancer, breast cancer, non-small cell lung carcinoma (NSCLC), liver cancer, pancreatic cancer, kidney cancer, melanoma, prostate cancer, head and neck cancer, stomach cancer, rectal cancer, colorectal cancer, leukemia’s such as chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) CT- ALL, B-ALL, Ph+ ALLPh- ALL) or diffuse large B-cell lymphoma (DLBCL).

[0046] In various embodiments, the person with at least 15% of the NK cell population isolated from PBMC’s, identified as a CD56+CD16+CD57+NKG2C+ subset may comprise at least 16%, or at least 17%, or at least 18%, or at least 19%, or at least 20%, or at least 21%, of the NK cell population identified as a CD56+CD16+CD57+NKG2C+ subset whereby each of CD56, CD16, CD57 and NKG2C are expressed in at least 16%, or 17%, 18%, 19%, 20% or 21% or more of the cells in an NK cell population.

[0047] The methods provide the advantage of a stable repeatable way of arriving at NK cells population expressing NKG2C at a high percentage. In various embodiments, a high percentage of an NK cells population expressing NKG2C may comprise at least 21% of NK cell population express NKG2C and may comprise 21% or more, or at least 22%, or at least 23%, or at least24%, or at least 25%, or at least 26%, or at least 27%, or at least 28%, or at least 29%, or at least30%, or at least 31%, or at least 32%, or at least 33%, or at least 34%, or at least 35%, or at least36%, or at least 37%, or at least 38%, or at least 39%, or at least 40%, or at least 41%, or at least42%, or at least 43%, or at least 44%, or at least 45%, or 25-45%, or 26-45%, or 27-45% or 28- 45%, or 29-45%, or 30-45%, or 31-45%, or 32-45%, or 33-45%, or 34-45%, or 35-45%, or 36- 45%, or 37-45% or 38-45%, or 39-45% or 40-45%, or 41-45%, or 42-45%, or 43-45% or 44-45%, or 50%, or 60%, or 70%, or 80% of NK cell population express NKG2C.

[0048] According to various embodiments, there is a method of determining a treatment outcome in patient who has been diagnosed with chronic myeloid leukemia and providing an appropriate treatment regimen, the method comprising: isolating an NK cell population from the bone marrow of the patient who has been diagnosed with chronic myeloid leukemia; determining percentage of NKG2A+ cells in the NK cell population wherein expression of NKG2A+ below a predetermined percentage of the NK cell population is indicative that the patient will be responsive to a treatment regimen with a tyrosine kinase inhibitor and the patient is administered the tyrosine kinase inhibitor and expression of NKG2A+ above a predetermined percentage of the NK cell population is indicative that the patient will become unresponsive to a treatment regimen with a tyrosine kinase inhibitor and patient is administered NK cells expressing NKG2C+ isolated by the method of enhancing the abundance of NKG2C+ adaptive NK cells isolated by an expansion method comprising: a) co-culturing peripheral blood mononuclear cells (PBMCs) in a first culture the first culture comprising: (i) first immortalized cell line genetically modified to express amino acid sequence of HLA-E; and (ii) interleukin 2, b) isolating the cultured PBMCs; c) co-culturing the isolated cultured PBMCs in a second culture, the second culture comprising: (i) second immortalized cell line genetically modified to express amino acid sequence of HLA-E and IL21 and 4-1 BBL (ii) interleukin 2, and (iii) interleukin 15 d) isolating the population of NK cells from the second culture wherein the NK cell population include cells expressing NKG2C.

[0049] In various embodiments, the predetermined percentage of NKG2A+ cells in the NK cell population may be anywhere from 9 to 12%. In various embodiments the predetermined percentage of NKG2A+ cells in the NK cell population may be 9 %. In various embodiments the predetermined percentage of NKG2A+ cells in the NK cell population may be 10 %. In various embodiments the predetermined percentage of NKG2A+ cells in the NK cell population may be 11 %. In various embodiments the predetermined percentage of NKG2A+ cells in the NK cell population may be 12 %. In various embodiments the predetermined percentage of NKG2A+ cells in the NK cell population may be recalculated based on evaluating the bone marrow of patients as described in the Examples to determine the cutoff value for patients that respond to TKI.

[0050] According to various embodiments, there is a method of determining a treatment outcome in patient who has been diagnosed with chronic myeloid leukemia and providing an appropriate treatment regimen, the method comprising: isolating an NK cell population from the bone marrow of the patient who has been diagnosed with chronic myeloid leukemia; determining percentage of NKG2A+ cells in the NK cell population wherein when 10% or less of the NK cell population express NKG2A+ the patient will be responsive to a treatment regimen with a Bcr-Abl allosteric inhibitor and the patient is administered the Bcr-Abl allosteric inhibitor and when 11 % or more of the NK cell population express NKG2A+ the patient will become unresponsive to a treatment regimen with a Bcr-Abl allosteric inhibitor and patient is administered NK cells expressing NKG2C+ isolated by the method of enhancing the abundance of NKG2C+ adaptive NK cells described herein above.

[0051] In various embodiments, this method may provide rational selection of chronic myeloid leukemia (CML) patients who will benefit from NK cell-based therapies and / or Bcr-Abl allosteric inhibitor therapies such as tyrosine kinase inhibitor (TKI) therapies to provide alternative treatment that may minimize relapses and resistance.

[0052] In various embodiments, the Bcr-Abl allosteric inhibitor comprises tyrosine kinases inhibitors such as any one of imatinib, dasatinib, nilotinib, radotinib, bosutinib, asciminib or a combination thereof. In various embodiments, the tyrosine kinase inhibitor may be selected from the any one of the group consisting of imatinib, dasatinib, nilotinib, radotinib, bosutinib, asciminib and a combination thereof.

[0053] In various embodiments, the method of determining a treatment outcome in patient who has been diagnosed with chronic myeloid leukemia and providing an appropriate treatment regimen may provide rational selection of CML patients who will benefit from NK cell-based therapies and those that may be treated with a Bcr-Abl allosteric inhibitor. The method of enhancing the abundance of NKG2C+ adaptive NK cells provide an Off-the-shelf NK cell-basedtherapy for patients who a likely to become TKI-resistant based on the NKG2A+ expression in NK cell population from the chronic phase-CML (CP-CML) patients. Without the method it would not be possible to know whether a patient with chronic phase-CML (CP-CML) would be resistant or unresponsive to Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor (TKI) until they have been treated for quite some time at which point treatment may take much longer and the patient may end up suffering more or in severe cases may not survive.

[0054] In various embodiments, the method further comprises determining percentage of NKG2C+ cells in the NK cell population wherein expression NKG2A+ below a first predetermined percentage of the NK cell population and expression of NKG2C+ above a second predetermined percentage of the NK cell population is indicative that the patient will be responsive to a treatment regimen with a tyrosine kinase inhibitor and the patient is administered a tyrosine kinase inhibitor and wherein expression NKG2A+ above the first predetermined percentage of the NK cell population and expression of NKG2C+ below the second predetermined percentage of the NK cell population is indicative that the patient will become unresponsive to a treatment regimen with a tyrosine kinase inhibitor and the patient is administered NK cells expressing NKG2C+ isolated by the method of enhancing the abundance of NKG2C+ adaptive NK cells described herein above.

[0055] In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be anywhere from 9 to 21%. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 9 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 10 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 11 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 12 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 13%. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 14 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 15 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 16 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 17 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 18 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 19 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 20 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 21 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be recalculated basedon evaluating the bone marrow of patients as described in the Examples to determine the cutoff value for patients that respond to TKL

[0056] In various embodiments, the second predetermined percentage of NKG2C+ cells in the NK cell population may be anywhere from 13 to 16%. In various embodiments the second predetermined percentage of NKG2C+ cells in the NK cell population may be anywhere from 13%. In various embodiments the second predetermined percentage of NKG2C+ cells in the NK cell population may be anywhere from 14%. In various embodiments the second predetermined percentage of NKG2C+ cells in the NK cell population may be anywhere from 15%. In various embodiments the second predetermined percentage of NKG2C+ cells in the NK cell population may be anywhere from 16%. In various embodiments the second predetermined percentage of NKG2C+ cells in the NK cell population may be recalculated based on evaluating the bone marrow of patients as described in the Examples to determine the cutoff value for patients that do not respond to TKI.

[0057] In various embodiments, the method further comprises determining percentage of NKG2C+ cells in the NK cell population wherein when 10% or less of the NK cell population express NKG2A+ and 15 % or more of the NK cell population express NKG2C+ the patient will be responsive to a treatment regimen with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor and the patient is administered a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor and when 21% or more of the NK cell population express NKG2A+ and 14 % or less of the NK cell population express NKG2C+ the patient will become unresponsive to a treatment regimen with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor and the patient is administered NK cells expressing NKG2C+ isolated by the method of enhancing the abundance of NKG2C+ adaptive NK cells described herein above.

[0058] In various embodiments, the method of determining a treatment outcome in patient who has been diagnosed with chronic myeloid leukemia and providing an appropriate treatment regimen may provide rational selection of CML patients who will benefit from NK cell-based therapies and those that may be treated with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor. The method of enhancing the abundance of NKG2C+ adaptive NK cells provide an Off-the-shelf NK cell-based therapy for patients who a likely to become TKI-resistant based on the NKG2A+ and NKG2C+ expression in NK cell population from the chronic phase-CML (CP- CML) patients. Without the method it would not be possible to know whether a patient with chronic phase-CML (CP-CML) would be resistant or unresponsive to Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor (TKI) until they have been treated for quite some time at which point treatment may take much longer and the patient may end up suffering more or in severe cases may not survive. In various embodiments, the method of determining a treatment outcome inpatient who has been diagnosed with chronic myeloid leukemia based on two markers and providing an appropriate treatment regimen may provide an even stronger rational selection of CML patients who will benefit from NK cell-based therapies and those that may be treated with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor.

[0059] In various embodiments, the method further comprising determining expression of HLA-E molecule on CD34+ cells from the bone marrow of the patient who has been diagnosed with chronic myeloid leukemia referred to as CML sample; measuring the expression of HLA-E molecule on CD34+ cells from bone marrow of a donor that does not have chronic myeloid leukemia referred to as control sample, comparing the expression of HLA-E molecule on CD34+ cells in the CML sample to expression of HLA-E molecule on CD34+ cells in the control sample wherein when the expression of HLA-E molecule on CD34+ cells in the CML sample is more than the expression of HLA-E molecule on CD34+ cells in the control sample the patient will progress to blast crisis CML and the patient is administered a combination of a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor and NK cells expressing NKG2C+ isolated by the method of enhancing the abundance of NKG2C+ adaptive NK cells described herein above.

[0060] In various embodiments, the method of determining a treatment outcome in patient who has been diagnosed with chronic myeloid leukemia and providing an appropriate treatment regimen may provide an additional cross reference for an even stronger rational selection of CML patients who will benefit from NK cell-based therapies and those that may be treated with a Bcr- Abl allosteric inhibitor such as a tyrosine kinase inhibitor. It also provides an Off-the-shelf NK cellbased therapy for TKI-resistant chronic phase-CML (CP-CML) patients. Without the method it would not be possible to know whether a patient with chronic phase-CML (CP-CML) would be resistant or unresponsive to a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor (TKI) until they have been treated for quite some time at which point treatment may take much longer and the patient may end up suffering more or possibly not survive at all.

[0061] In various embodiments, the NK cells expressing NKG2C+ isolated by the method of enhancing the abundance of NKG2C+ adaptive NK cells described herein above are from PBMC’s taken from the patient who has been diagnosed with chronic myeloid leukemia.

[0062] In various embodiments, the NK cells expressing NKG2C+ isolated by the method of enhancing the abundance of NKG2C+ adaptive NK cells described herein above are from PBMC’s taken from a healthy person.

[0063] In various embodiments, the NK cells expressing NKG2C+ isolated by the method of enhancing the abundance of NKG2C+ adaptive NK cells as disclosed herein above are from PBMC’s taken from the patient who has been diagnosed with chronic myeloid leukemia or ahealthy person with at least 20% of the NK cell population identified as a CD56+CD16+CD57+NKG2C+ subset.

[0064] According to various embodiments, there are NK cells wherein at least 21% of NK cell population express NKG2C for use in the treatment of cancer wherein expression of HLA-E molecule on cells in a cancer sample is more than the expression of HLA-E molecule on cells in a control sample from a healthy individual.

[0065] In various embodiments, NK cells wherein at least 21% of NK cell population express NKG2C are derived from or derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above.

[0066] In various embodiments, NK cells wherein at least 21% of NK cell population express NKG2C may comprise 21% or more, at least 22%, at least 23%, at least 24%, at least 25%, or at least 26%, or at least 27%, or at least 28%, or at least 29%, or at least 30%, or at least 31%, or at least 32%, or at least 33%, or at least 34%, or at least 35%, or at least 36%, or at least 37%, or at least 38%, or at least 39%, or at least 40%, or at least 41%, or at least 42%, or at least 43%, or at least 44%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70% or at least 75%, or at least 80%, or at least 85%, or at least 90% or at least 95%, or 21-45%, or 25-45%, or 27-45% or 28-45%, or 29-45%, or 30-45%, or 31-45%, or 32-45%, or 33-45%, or 34-45%, or 35-45%, or 36-45%, or 37-45% or 38-45%, or 39-45% or 40-45%, or 41- 45%, or 42-45%, or 43-45% or 44-45% of NK cell population express NKG2C.

[0067] Off-the-shelf NK cell-based therapies may provide treatment for cancers that overexpress HLA-E molecule that may be resistant to other treatments. In various embodiments, the cancer that exhibits overexpression of HLA-E molecule may include any one of cervical cancer, breast cancer, non-small cell lung carcinoma (NSCLC), liver cancer, pancreatic cancer, kidney cancer, melanoma, prostate cancer, head and neck cancer such as head and neck squamous cell carcinoma (HNSCC), stomach cancer, rectal cancer, colorectal cancer, leukemia’s such as chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) (T-ALL, B-ALL, Ph+ ALLPh- ALL) or diffuse large B-cell lymphoma (DLBCL).

[0068] In various embodiments, the cancer is selected from group comprising or consisting of any one of cervical cancer, breast cancer, non-small cell lung carcinoma (NSCLC), liver cancer, pancreatic cancer, kidney cancer, melanoma, prostate cancer, head and neck cancer, stomach cancer, rectal cancer, colorectal cancer, leukemia.

[0069] In various embodiments, the cancer is leukemia. In various embodiments, the leukemia is selected from group comprising or consisting of any one of chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) CT- ALL, B-ALL, Ph+ ALLPh- ALL) or diffuse large B-cell lymphoma (DLBCL).

[0070] In various embodiments, the cancer is head and neck cancer. In various embodiments, the head and neck cancer comprises head and neck squamous cell carcinoma (HNSCC).

[0071] In various embodiments, the cancer is chronic myeloid leukemia. In various embodiments, the chronic myeloid leukemia may have been classified by any of the methods described in the various embodiments to be indicative of a first subgroup that will be responsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor, a second subgroup that will be unresponsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor and / or a third subgroup will progress to blast crisis CML.

[0072] According to various embodiments, there is a use of NK cells wherein at least 21% of NK cell population express NKG2C in the manufacture of a medicament for treatment of cancer wherein a cell sample from the cancer expresses HLA-E molecule on cells at a higher abundance than the expression of HLA-E molecule on cells in a control sample from a healthy individual.

[0073] In various embodiments, NK cells wherein at least 21% of NK cell population express NKG2C may comprise 21% or more, at least 22%, at least 23%, at least 24%, at least 25%, or at least 26%, or at least 27%, or at least 28%, or at least 29%, or at least 30%, or at least 31%, or at least 32%, or at least 33%, or at least 34%, or at least 35%, or at least 36%, or at least 37%, or at least 38%, or at least 39%, or at least 40%, or at least 41%, or at least 42%, or at least 43%, or at least 44%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70% or at least 75%, or at least 80%, or at least 85%, or at least 90% or at least 95%, or 21-45%, or 25-45%, or 27-45% or 28-45%, or 29-45%, or 30-45%, or 31-45%, or 32-45%, or 33-45%, or 34-45%, or 35-45%, or 36-45%, or 37-45% or 38-45%, or 39-45% or 40-45%, or 41- 45%, or 42-45%, or 43-45% or 44-45% of NK cell population express NKG2C.

[0074] In various embodiments, NK cells wherein at least 21% of NK cell population express NKG2C may be derived according to any one of the methods of enhancing the abundance of NKG2C+ adaptive NK cells described herein above.

[0075] In various embodiments, the cancer is selected from group comprising or consisting of any one of cervical cancer, breast cancer, non-small cell lung carcinoma (NSCLC), liver cancer, pancreatic cancer, kidney cancer, melanoma, prostate cancer, head and neck cancer, stomach cancer, rectal cancer, colorectal cancer, leukemia.

[0076] In various embodiments, the cancer is leukemia. In various embodiments, the leukemia is selected from group comprising or consisting of any one of chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) CT- ALL, B-ALL, Ph+ ALLPh- ALL) or diffuse large B-cell lymphoma (DLBCL).

[0077] In various embodiments, the cancer is chronic myeloid leukemia. In various embodiments, the chronic myeloid leukemia may have been classified by any of the methods described in the various embodiments to be indicative of a first subgroup that will be responsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor, a second subgroup that will be unresponsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor and / or a third subgroup will progress to blast crisis CML.

[0078] According to various embodiments, there is a composition comprising NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above and a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor.

[0079] In various embodiments, NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above in the composition with the Bcr-Abl allosteric inhibitor such as the tyrosine kinase inhibitor may comprise at least 20% of NK cell population expressing NKG2C or at least 21%, or at least 22%, or at least 23% or at least24%, or at least 25% or at least 26%, or at least 27%, or at least 28%, or at least 29%, or at least30%, or at least 31%, or at least 32%, or at least 33%, or at least 34%, or at least 35%, or at least36%, or at least 37%, or at least 38%, or at least 39%, or at least 40%, or at least 41%, or at least42%, or at least 43%, or at least 44%, or at least 45%, or 25-45%, or 26-45%, or 27-45% or 28- 45%, or 29-45%, or 30-45%, or 31-45%, or 32-45%, or 33-45%, or 34-45%, or 35-45%, or 36- 45%, or 37-45% or 38-45%, or 39-45% or 40-45%, or 41-45%, or 42-45%, or 43-45% or 44-45% of NK cell population express NKG2C.

[0080] In various embodiments, NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells described herein above in the composition with the Bcr- Abl allosteric inhibitor such as the tyrosine kinase inhibitor may comprise at least 21% or more of NK cell population expressing NKG2C+ or at least 22%, or at least 23% or at least 24%, or at least 25% or at least 26%, or at least 27%, or at least 28%, or at least 29%, or at least 30%, or at least 31 %, or at least 32%, or at least 33%, or at least 34%, or at least 35%, or at least 36%, or at least 37%, or at least 38%, or at least 39%, or at least 40%, or at least 41%, or at least 42%, or at least 43%, or at least 44%, or at least 45%, or 25-45%, or 26-45%, or 27-45% or 28-45%, or 29-45%, or 30-45%, or 31-45%, or 32-45%, or 33-45%, or 34-45%, or 35-45%, or 36-45%, or37-45% or 38-45%, or 39-45% or 40-45%, or 41-45%, or 42-45%, or 43-45% or 44-45% of NK cell population expressing NKG2C.

[0081] In various embodiments, the tyrosine kinase inhibitor in the composition with NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above may comprise any one of imatinib, dasatinib, nilotinib, radotinib, bosutinib, asciminib or a combination thereof. In various embodiments, the tyrosine kinase inhibitor in the composition with NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above may be selected from the any one of the group consisting of imatinib, dasatinib, nilotinib, radotinib, bosutinib, asciminib and a combination thereof.

[0082] According to various embodiments, there is a composition comprising NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above and a therapeutic cytotoxic antibody.

[0083] In various embodiments, NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above in the composition with the therapeutic cytotoxic antibody may comprise at least 21%, at least 22%, at least 23%, at least 24%, at least 25% or more, or at least 26%, or at least 27%, or at least 28%, or at least 29%, or at least 30%, or at least 31%, or at least 32%, or at least 33%, or at least 34%, or at least 35%, or at least 36%, or at least 37%, or at least 38%, or at least 39%, or at least 40%, or at least 41%, or at least 42%, or at least 43%, or at least 44%, or at least 45%, or 25-45%, or 26-45%, or 27- 45% or 28-45%, or 29-45%, or 30-45%, or 31-45%, or 32-45%, or 33-45%, or 34-45%, or 35- 45%, or 36-45%, or 37-45% or 38-45%, or 39-45% or 40-45%, or 41-45%, or 42-45%, or 43-45% or 44-45% of NK cell population expressing NKG2C.

[0084] In various embodiments, NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above in the composition with the therapeutic cytotoxic antibody may comprise at least 20% or more of NK cell population expressing NKG2C+ or at least 21%, or at least 22%, or at least 23% or at least 24%, or at least 25% or at least 26%, or at least 27%, or at least 28%, or at least 29%, or at least 30%, or at least 31%, or at least 32%, or at least 33%, or at least 34%, or at least 35%, or at least 36%, or at least 37%, or at least 38%, or at least 39%, or at least 40%, or at least 41%, or at least 42%, or at least 43%, or at least 44%, or at least 45%, or 25-45%, or 26-45%, or 27-45% or 28-45%, or 29-45%, or 30-45%, or 31-45%, or 32-45%, or 33-45%, or 34-45%, or 35-45%, or 36-45%, or 37-45% or 38-45%, or 39-45% or 40-45%, or 41 -45%, or 42-45%, or 43-45% or 44-45% of NK cell population expressing NKG2C.

[0085] In various embodiments, the therapeutic cytotoxic antibody in the composition with NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above may be selected from alemtuzumab, atezolizumab, bevacizumab, brentuximab-vedotin, blinatumomab, cetuximab, daratumumab, elotuzumab, gemtuzumab- ozogamicin, ipilimumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, panitumumab, pembrolizumab, pertuzumab, rituximab, rituximab-hyaluronidase, trastuzumab, trastuzumab- dkst, or any known therapeutic cytotoxic antibody.

[0086] According to various embodiments, there is a composition NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above and a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor or a composition comprising NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above and a therapeutic cytotoxic antibody for use in the treatment of cancer wherein expression of HLA-E molecule on cells in a cancer sample from the cancer is more than the expression of HLA-E molecule on cells in a control sample from a healthy individual.

[0087] In various embodiments, NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above in the composition with the tyrosine kinase inhibitor for use in the treatment of cancer may comprise at least 20%, or at least 21%, or at least 22%, or at least 23%, or at least 24% or at least 25% or more, or at least 26%, or at least 27%, or at least 28%, or at least 29%, or at least 30%, or at least 31%, or at least 32%, or at least 33%, or at least 34%, or at least 35%, or at least 36%, or at least 37%, or at least 38%, or at least 39%, or at least 40%, or at least 41%, or at least 42%, or at least 43%, or at least 44%, or at least 45%, or 25-45%, or 26-45%, or 27-45% or 28-45%, or 29-45%, or 30-45%, or 31-45%, or 32-45%, or 33-45%, or 34-45%, or 35-45%, or 36-45%, or 37-45% or 38-45%, or 39-45% or 40-45%, or 41-45%, or 42-45%, or 43-45% or 44-45% of NK cell population expressing NKG2C.

[0088] In various embodiments, NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above in the composition with the Bcr-Abl allosteric inhibitor such as the tyrosine kinase inhibitor for use in the treatment of cancer may be derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells described herein above

[0089] In various embodiments, the tyrosine kinase inhibitor in the composition with NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above for use in the treatment of cancer may comprise any one of imatinib, dasatinib, nilotinib, radotinib, bosutinib, asciminib or a combination thereof. In variousembodiments, the tyrosine kinase inhibitor in the composition with NK cells wherein at least 25% of NK cell population express NKG2C may be selected from the any one of the group consisting of imatinib, dasatinib, nilotinib, radotinib, bosutinib, asciminib and a combination thereof.

[0090] In various embodiments, NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above in the composition with the therapeutic cytotoxic antibody for use in the treatment of cancer may comprise at least 20%, or at least 21%, or at least 22%, or at least 23%, or at least 24% or at least 25% or more, or at least26%, or at least 27%, or at least 28%, or at least 29%, or at least 30%, or at least 31%, or at least32%, or at least 33%, or at least 34%, or at least 35%, or at least 36%, or at least 37%, or at least38%, or at least 39%, or at least 40%, or at least 41 %, or at least 42%, or at least 43%, or at least44%, or at least 45%, or 25-45%, or 26-45%, or 27-45% or 28-45%, or 29-45%, or 30-45%, or 31-45%, or 32-45%, or 33-45%, or 34-45%, or 35-45%, or 36-45%, or 37-45% or 38-45%, or 39- 45% or 40-45%, or 41-45%, or 42-45%, or 43-45% or 44-45% of NK cell population expressing NKG2C.

[0091] In various embodiments, NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above in the composition with the therapeutic cytotoxic antibody for use in the treatment of cancer may be derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells described herein above

[0092] In various embodiments, the therapeutic cytotoxic antibody in the composition with NK cells derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells as described herein above for use in the treatment of cancer may be selected from alemtuzumab, atezolizumab, bevacizumab, brentuximab-vedotin, blinatumomab, cetuximab, daratumumab, elotuzumab, gemtuzumab-ozogamicin, ipilimumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, panitumumab, pembrolizumab, pertuzumab, rituximab, rituximabhyaluronidase, trastuzumab, trastuzumab-dkst, or any known therapeutic cytotoxic antibody.

[0093] In various embodiments, the cancer is selected from group comprising or consisting of any one of cervical cancer, breast cancer, non-small cell lung carcinoma (NSCLC), liver cancer, pancreatic cancer, kidney cancer, melanoma, prostate cancer, head and neck cancer, stomach cancer, rectal cancer, colorectal cancer, leukemia.

[0094] In various embodiments, the cancer is leukemia. In various embodiments, the leukemia is selected from group comprises or consisting of any one of chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) CT- ALL, B-ALL, Ph+ ALLPh- ALL) or diffuse large B-cell lymphoma (DLBCL).

[0095] In various embodiments, the cancer is chronic myeloid leukemia. In various embodiments, the chronic myeloid leukemia may have been classified by any of the methods described in the various embodiments to be indicative of a first subgroup that will be responsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor, a second subgroup that will be unresponsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor and / or a third subgroup will progress to blast crisis CML.

[0096] According to various embodiments, there is a use of a composition NK cells expressing NKG2C derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells described herein above and a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor or a composition comprising NK cells expressing NKG2C derived according to the method of enhancing the abundance of NKG2C+ adaptive NK cells described herein above and a therapeutic cytotoxic antibody in the manufacture of a medicament for treating cancer wherein a cell sample from the cancer expresses HLA-E molecule on CD34+ cells at a higher abundance than the expression of HLA-E molecule on CD34+ cells in a control sample from a healthy individual.

[0097] In various embodiments, use of a composition the in the manufacture of a medicament for treating cancer may refer to cancer selected from group comprising or consisting of any one of cervical cancer, breast cancer, non-small cell lung carcinoma (NSCLC), liver cancer, pancreatic cancer, kidney cancer, melanoma, prostate cancer, head and neck cancer, stomach cancer, rectal cancer, colorectal cancer, leukemia.

[0098] In various embodiments, use of a composition the in the manufacture of a medicament for treating cancer may refer to leukemia. In various embodiments, the leukemia is selected from group comprises or consisting of any one of chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) (T-ALL, B-ALL, Ph+ ALLPh- ALL) or diffuse large B-cell lymphoma (DLBCL).

[0099] In various embodiments, use of a composition in the manufacture of a medicament for treating cancer may refer to chronic myeloid leukemia. In various embodiments, the chronic myeloid leukemia may have been classified by any of the methods described in the various embodiments to be indicative of a first subgroup that will be responsive to treatment with a Bcr- Abl allosteric inhibitor such as a tyrosine kinase inhibitor, a second subgroup that will be unresponsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor and / or a third subgroup will progress to blast crisis CML.

[0100] According to various embodiments, there is an in vitro method for predicting subgroups of chronic myeloid leukemia comprising isolating an NK cell population from the bonemarrow of a patient who has been diagnosed with chronic myeloid leukemia; determining percentage of NKG2A+ cells in the NK cell population wherein expression of NKG2A+ below a first predetermined percentage of the NK cell population is indicative of a first subgroup that will be responsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor, and expression of NKG2A+ above the first predetermined percentage of the NK cell population express NKG2A+ it is indicative of a second subgroup that will be unresponsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor.

[0101] In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be anywhere from 9 to 21%. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 9 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 10 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 11 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 12 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 13%. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 14 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 15 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 16 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 17 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 18 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 19 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 20 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be 21 %. In various embodiments the first predetermined percentage of NKG2A+ cells in the NK cell population may be recalculated based on evaluating the bone marrow of patients as described in the Examples to determine the cutoff value for patients that respond to TKI.

[0102] According to various embodiments, there is an in vitro method for predicting subgroups of chronic myeloid leukemia comprising isolating an NK cell population from the bone marrow of a patient who has been diagnosed with chronic myeloid leukemia; determining percentage of NKG2A+ cells in the NK cell population wherein when 10% or less of the NK cell population express NKG2A+ it is indicative of a first subgroup that will be responsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor, and when 11% or more of the NK cell population express NKG2A+ it is indicative of a second subgroup that will be unresponsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor.

[0103] In various embodiments, the in vitro method may further comprising determining percentage of NKG2C+ cells in the NK cell population wherein when 10% or less of the NK cell population express NKG2A+ and 15 % or more of the NK cell population express NKG2C+ it is indicative of the first subgroup that will be responsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor, and when 21% or more of the NK cell population express NKG2A+ and 14 % or less of the NK cell population express NKG2C+ it is indicative of a second subgroup that will become unresponsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor.

[0104] In various embodiments the second predetermined percentage of NKG2C+ cells in the NK cell population may be anywhere from 13 to 16%. In various embodiments the second predetermined percentage of NKG2C+ cells in the NK cell population may be anywhere from 13%. In various embodiments the second predetermined percentage of NKG2C+ cells in the NK cell population may be anywhere from 14%. In various embodiments the second predetermined percentage of NKG2C+ cells in the NK cell population may be anywhere from 15%. In various embodiments the second predetermined percentage of NKG2C+ cells in the NK cell population may be anywhere from 16%. In various embodiments the second predetermined percentage of NKG2C+ cells in the NK cell population may be recalculated based on evaluating the bone marrow of patients as described in the Examples to determine the cutoff value for patients that do not respond to TKI.

[0105] In various embodiments, the in vitro method may further comprising determining percentage of NKG2C+ cells in the NK cell population wherein expression of NKG2A+ below a first predetermined percentage of the NK cell population and expression of NKG2C+ above a second predetermined percentage of the NK cell population is indicative of the first subgroup that will be responsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor, and expression of NKG2A+ above the first predetermined percentage of the NK cell population and expression of NKG2C+ below the second predetermined percentage is indicative of a second subgroup that will become unresponsive to treatment with a Bcr-Abl allosteric inhibitor such as a tyrosine kinase inhibitor.

[0106] In various embodiments, the in vitro method may further comprising: determining expression of HLA-E molecule on CD34+ cells from bone marrow of the patient who has been diagnosed with chronic myeloid leukemia in a CML sample; determining expression of HLA-E molecule on CD34+ cells from bone marrow of a donor that does not have chronic myeloid leukemia in a control sample, comparing the expression of HLA-E molecule on CD34+ cells in the CML sample to expression of HLA-E molecule on CD34+ cells in the control sample wherein when the expression of HLA-E molecule on CD34+ cells in the CML sample is more than theexpression of HLA-E molecule on CD34+ cells in the control sample it is indicative of a third subgroup that will progress to blast crisis CML.

[0107] In various embodiments, the in vitro method may provide rational selection of CML patients who will benefit from NK cell-based therapies and / or Bcr-Abl allosteric inhibitor therapies such as tyrosine kinase inhibitor (TKI) therapies to provide alternative treatment that may minimize relapses and resistance.

[0108] In various embodiments, the biphasic expansion protocol is able to convert non- adaptive NK cells from pre-selected healthy donors to adaptive NK cells. Use of these adaptive NK cells represents the first adaptive NK cell-based immunotherapy targeting Bcr-Abl allosteric inhibitor resistant CML such as TKI-resistant CML. By culturing exhausted CML-NK cells in the biphasic feeder, we are able to rejuvenate NK cells and enhance their cytotoxicity. This novel approach offers an innovative therapeutic strategy of autologous NK cell transfer for CML patients, enhancing the depth of the patient’s molecular response to Bcr-Abl allosteric inhibitor therapy such as TKI therapy.

[0109] In various embodiments, the biphasic expansion protocol is able to increase the natural cytotoxicity of both adaptive NK and non-adaptive NK cells from healthy human donors

[0110] In various embodiments, the pre-selection criteria of healthy donors facilitates adaptive NK expansion. In various embodiments, the pre-selection harnesses the natural adaptive properties of NK cells, which are present at elevated levels in approximately 20-30% of healthy donors, likely due to prior exposure to CMV infection.

[0111] In various embodiments, the rational selection of CML patients who are amenable to adaptive NK cell-based therapies may result in better treatment.

[0112] In various embodiments, the method or use may result in safe and effective elimination of CML tumor in vivo with ex vivo expanded adaptive NK cells

[0113] In various embodiments, the ex vivo expanded adaptive NK cells may be used to effectively kill CML-blast crisis stem and progenitor cells or other HLA-E presenting progenitor cells.

[0114] In various embodiments, the biphasic expansion protocol is able to restore cytotoxicity of exhausted NK cells from CML patients.

[0115] In various embodiments, the rational selection of malignant cancer cells may ensure the leukemia is amenable to adaptive NK cell-based therapy.

[0116] In various embodiments, the adaptive NK cell expansion and cytotoxicity may be further improved through the addition of modulating compound ex vivo.

[0117] In various embodiments, antibody-dependent cellular cytotoxicity (ADCC) Referring to [Fig. 2], [more for applications with mechanical features]

[0118] In various embodiments, SEQ ID NO. 1 is set forth in amino acid sequence MWMAPRTLFLLLSGALTLTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDND AASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMH GCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLE DTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGH TQDTELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGII AGLVLLGSWSGAWAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL.

[0119] The following are non-limiting examples.

[0120] Examples

[0121] In a study focusing on chronic myeloid leukemia (CML) patients undergoing treatment with tyrosine kinase inhibitors (TKIs), it was noted that there was distinct NK cell subset dynamics across responder categories. Optimal responders to tyrosine kinase inhibitor’s (TKIs) exhibited increased NK cell counts and augmented numbers of a specific NK cell subset called as adaptive NK cells expressing the activating receptor NKG2C. In contrast, TKI-resistant patients progressing to blast crisis (BC) accumulated a NK cell subset expressing the inhibitory receptor NKG2A. These results reveal the promising role of NK cells as both a biomarker for therapeutic response and a new avenue for immunotherapy in CML patients resistant to TKIs.

[0122] 1. Rational selection of TKI-resistant CML patients who will benefit from NK cell-based therapies

[0123] Rationale and preliminary data: NK cell-based biomarkers have been identified that predict the response to first line TKI therapy in chronic phase (CP) CML patients.

[0124] This allows suitable treatment to be streamlined by selecting TKI-resistant CML patients suitable for NK cell-based therapies.

[0125] Specifically, patients are determined on the basis of the following biomarkers: An increased proportion of CD56+CD16+CD57+NKG2C+ adaptive NK cells in bone marrow of patients with major molecular remissions [Fig. 1A]; an increased proportion of CD56+CD16+NKG2A+ NK cells in patients who are destined to transform to blast crisis (BC) CML[Fig. 1 B]; patients with high NKG2A+ NK cells, we have also determined that the target cell population, CD34+ CML stem and progenitor cells (SPC), express high levels of HLA-E [Fig. 1 C]; BC-CML patients, SPCs express high levels of HLA-E as compared to control SPCs [Fig. 1 D].

[0126] 2. Off-the-Shelf adaptive NK Cell-Based Therapies for Patients

[0127] The cells were formed by taking 1 non-NK donor (HSA42) and 1 aNK donor (HSA39). The non aNK cells were used in 4 replicate mice and the aNK cells were used in 7 replicate mice administered via caudal artery. Across three readouts, aNK cells outperformed non-aNK cells. In longitudinal bioluminescence imaging (see [Fig. 1 E]), the +aNK group maintains minimal tumor signal from day 21 through day 42, whereas the non-aNK group shows recurrent and higher signals. PB monitoring of NK cell levels revealed significantly higher human NK-cell persistence of aNK cells as compared to the non aNK cells. Survival analysis [Fig. 1 F] showed markedly improved survival (log-rank p=0.001) of the aNK-injected cohort, while the non-aNK group did not improve significantly over K562 only controls (p=0.28). Together, aNK cells demonstrate superior in vivo persistence, tumor control, and survival benefit in vivo. Expanded adaptive NK cells are superior to non-adaptive NK cells in terms of percistance, their ability to reduce tumor burden and improve survival.

[0128] The Biphasic method of [Fig. 2] results in higher concentrations of adaptive NK cells that express NKG2C at much higher percentages than with just a single phase method as depicted in [Fig. 3F] where the biphasic method results in more than double the percentage of NKG2C in the total NK cell population.

[0129] A cell therapy platform specifically tailored to enrich and expand adaptive NK cells was engineered from healthy human donors. This involved expanding adaptive NK cells. The resulting cells will be tested as an adoptive immunotherapy product against TKI resistant CML SPCs.

[0130] Specifically, two genetically modified feeder lines were designed to allow the expansion of relatively pure NK cells from PBMCs procured from healthy donors [Fig. 3A], construct. This was further developed into a biphasic protocol wherein healthy PBMCs are sequentially co-cultured with the two feeder cells for 14 days [Fig. 3B]. At the end of 14 days, this process yields a cell therapy product exhibiting a significant increase of many fold, in this case up to 2700-fold enrichment for the NKG2C+ adaptive NK subset [Fig. 3D].

[0131] Genetically modified feeder lines and a novel method of ex vivo adaptive NK cell expansion:

[0132] Healthy adult donors were selected based on the percentage of NKG2C+ adaptive NK in their peripheral blood. High NKG2C+ NK% (>20%) can be observed in approximate 40% of Singaporean population [Fig. 3C], and this frequency varies with the local prevalence of CMV infection. Suitable PBMCs (1-2 million) are then co-cultured with feeder#1 at a ratio of 1 PBMC: 2 feeder#1 for 7 days in a humidified 5% CO2 incubator. Feeder#1 consists of K562 cells that have been genetically modified to express HLA- E along with the leader peptide of HLA-G. Briefly, a construct encoding HLA-E with its signal peptide (MVDGTLLLLLSEALALTQTWA) replaced by the HLA-G signal peptide (MWMAPRTLFLLLSGALTLTETWA) was designed. The construct was custom-synthesized and transduced into K562 cells. Stable transfectants expressing cell surface associated HLA-E were irradiated (100 Gy) and used as the feeder#1. The culture in feeder#1 was designed for 7-day incubation referred to as the “selection phase” which refers to the selective survival that NKG2C+ adaptive NK cells obtained at this stage of co-culture, and the depletion of the non-desired NKG2A+ subset via the inhibitory effect of HLA-E-NKG2A engagement.

[0133] Expansion medium:[Table 1]: components and preparation of the expansion medium.

[0134] The cells were harvested on day 7 and sequential plating of the cells onto a second novel triple genetically modified feeder#2 for an additional 7 days was initiated. Feeder#2 consists of K562 cells that were engineered to express the following 3 proteins: 1. 41 -BBL; 2. Membranebound IL-21 (mb-IL-21); and 3. HLA-E expressing the leader peptide of HLA-G. This phase of the 7-day NK cell culture was referred to as the “expansion phase”. Here, proliferation signals from mb-IL21 and 41-BBL signaling collaborate with HLA-E-NKG2C engagement to preferentially stimulate NKG2C+ adaptive NK proliferation compared to NKG2A+ NK cells. Overall, from 5 ml of PBMCs, it was possible to obtain 20 x 109adaptive- enriched NK cell product after ex vivo expansion. At the dose of 107 / kg for immunotherapy, these cells are sufficient for 20 cancerpatients. The fold expansion and absolute number of NKG2C+ cells using the biphasic protocol as shown in [Fig. 3D] and [Fig. 3E],

[0135] Comparing an earlier used single-phase protocol to the current biphasic protocol it was found that the biphasic protocol yielded a higher percentage of NKG2C+ enrichment as compared to a single-phase protocol(n=2) [Fig. 3F],

[0136] Additionally, careful selection of human donor permits better selection for adaptive NK enrichment. It was found that 1) patients with over 20% adaptive NK cells during PBMC collection exhibit greater effectiveness at ex vivo adaptive NK cell expansion [Fig. 3G]; and 2) the non-adaptive NK cells from these patients could be converted to adaptive NK cells through the biphasic protocol for expansion technology [Fig. 3H].

[0137] This coupled with careful selection of the patient involves selecting HLA-E-high CML patients for adaptive NK cell-based immunotherapy. It was found that adaptive NK cells are more efficient in eliminating CML cells expressing higher levels of HLA-E than those with low HLA-E expression [Fig. 3I], It is demonstrated that HLA-E overexpression in CML stem cells results in augmented cytotoxicity by the expanded NK cells (see [Table 2] and [Fig. 11]). In [Table 2] GSEA comparison of NK cells expanded in the biphasic protocol as compared to the monophasic protocol is displayed. Thus, CP-CML destined to progress to LBC or CML-LBC patients are anticipated to derive greater benefits from adaptive NK cell- based therapy.

[0138] [Table 2]: NK cells were expanded in the biphasic platform (HLA-E +ILA21 / mmlL-21 feeders) or in HLA-E only expressing feeders for 14 days. Expanded NK cells were subjected to bulk RNA-seq and geneset enrichment analysis (GSEA).

[0139] One of the advantages of the cells produced via the biphasic protocol is the resulting NK cells gain higher cytotoxicity upon ex vivo culture than cell resulting from other methods [Fig. 3J], Such expanded NK cells are proficient in killing BC-CML cells both in vitro [Fig. 3K] and data not shown) and in vivo [Fig. 3L].

[0140] To advance the cell therapy product development, continuous development and improvement of the original biphasic protocol described in [Fig. 3B] is ongoing. Specifically, adding compounds to the feeders to enhance the expansion and function of adaptive NK cells. One such modification to the original protocol has been the addition of the tyrosine kinase inhibitor, dasatinib [Fig. 4A], The addition of dasatinib (100nM) enhances the yield of NK cells to up to 2x107cells from 1.5 ml starting abundance of healthy PBMC (Fig. 4B). Studies are in progress to understand if the receptor profile of dastinib-expanded NK cells are different / superior as compared to the regular biphasic protocol.

[0141] Dasatinib co-culture augments aNK yield, enriches for adaptive NK, and attenuates exhaustion via the downregulation of several exhaustion factors, including BLIMP1

[0142] A targeted screen of FDA-approved agents identified dasatinib as a potent amplifier of aNK expansion, uniquely boosting cell yield by approximately 1 ,000-fold (=2*107cells from 1.5 mL PBMCs) compared to imatinib, ponatinib, or nilotinib (Fig. 4C). We therefore integrated dasatinib into our two-phase feeder protocol, supplementing it at a 100nM concentration only at the expansion phase, washed the drug off and systematically compared such aNK cells, henceforth referred to as Das-eNKE21 to aNK cells expanded without dasatinib (eNKE21) Dasatinib supplementation consistently augmented total NK cell output by Day 14 across independent donors (Fig. 40 & Fig. 4D). Phenotypically, it skewed the aNK product toward anadaptive state (CD56dim NKG2C+ KIR+ NKG2Alow), as confirmed by flow cytometry gating and paired quantification (Fig. 4E & Fig. 4F). Kinetic analyses revealed that this yield enhancement stemmed primarily from improved survival, not hyper-proliferation since dasatinib-treated cultures exhibited significantly reduced Annexin V+ staining and enhanced viability from Day 9 onward (Fig. 4G & Fig. 4H), while proliferation markers like Ki-67 remained unchanged or trended lower (data not shown).

[0143] The biphasic protocol focuses on and permits the development of off-the-shelf adaptive NK cell-based therapies for TKI resistant CML patients and BC-CML patients. By harnessing the innate cytotoxic capabilities of NK cells, this approach offers a novel avenue for overcoming TKI resistance and improving treatment efficacy in this patient population.

[0144] 3. Off-the-Shelf adaptive NK Cell-Based Therapies for enhancing TKI responses and facilitating earlier treatment-free remission attempts

[0145] Based on preliminary data it has been observed that CML patients who are optimal responders to TKI accumulate adaptive NK that are functionally exhausted [Fig. 5A-F], These exhausted NK cells demonstrate impaired cytotoxicity and deficient degranulation and cytokine secretion in response to target tumour cells or antibody-dependent stimulation mimicking ADCC. Surprisingly, following expansion with the biphasic protocol feeders described above, the cytotoxicity of such exhausted CML-derived NK cells was not only rescued but further enhanced compared to NK cells from healthy donors prior to expansion [Fig. 5G-H]. The above data suggests that NK cells from CML patients can be expanded ex vivo using the biphasic protocol and re-administered to patients as a novel autologous cell therapy protocol. Thus, autologous adaptive NK cell therapy can be combined with TKI treatment to achieve greater depth of molecular remission in CML patients.

[0146] One of the main goals of any CML patients who respond optimally to TKI therapy is for them to eventually reach treatment-free remission (TFR), i.e. be able to stop taking TKIs without disease recurrence. Currently, patients have had a stable 4.0-log reduction in BCR::ABL1 transcripts (MR4.0) for at least 2 years can be considered for a trial of TKI cessation. Among these patients, roughly half will experience disease recurrence, while the remainder enjoy prolonged TFR. Autologous adaptive NK cell therapy together with conventional TKI treatment may induce deeper responses more quickly, allowing patients to reach MR4.0 faster, thereby facilitating earlier TKI cessation attempts. This will be considered as a major advancement in the CML field.

[0147] 4. Screening CML patients to determine the most appropriate treatment.

[0148] By leveraging specific biomarkers on NK and CML SPCs, a rational approach to identify patients who will benefit from NK-cell based immunotherapy is developed. These are to be developed into flow panels for clinical prediction and treatment.

[0149] 5. Off-the-shelf adaptive NK cell-based therapies at the time of TKI cessation to enhance TFR rates.

[0150] Based on preliminary data it is observed that adaptive NK counts at the time of TKI cessation strongly correlate with the clinical outcome, i.e. patients experience a durable remission or a molecular relapse (Myers, and Miller Nat Rev Clin Oncol. 2021 ;18(2):85-100). The expanded adaptive NK cells from the biphasic protocol can be administered as “off-the-shelf infusions to CML patients at the point of TKI cessation to potentially enhance treatment-free remission rates.

[0151] Allogeneic adaptive NK cell therapy post-TKI cessation offers a promising, safe route to enhancing TFR rates, and could mark a major advance in the field.

[0152] 6. Off-the-shelf adaptive NK cells to induce ADCC of antibody-coated CML leukemic stem cells

[0153] Based on preliminary data a new set of leukemic stem cell (LSC) antigens unique to CML patients facing molecular relapse after TKI discontinuation was identified. Among them, PRSS21 stands out as a cell surface protein, making it an ideal target for NK cell-mediated ADCC. Prospective experiments with antibodies that can recognize PRSS21 are underway. Pending the suitability of the antibody for ADCC, there is the potential for expanded adaptive NK cells to target relapse-associated LSCs through ADCC. As mentioned earlier, adaptive NK cells demonstrate superior ADCC capabilities compared to conventional NK cells.

[0154] Targeting relapse-associated LSCs via adaptive NK cell-mediated ADCC offers a direct approach to deplete CML-driving stem cells, potentially leading to permanent disease remission.

[0155] 7. Off-the-Shelf adaptive NK Cell-Based Therapies for HLA-E Expressing Cancers: Multiple myeloma and Acute lymphoblastic leukemia

[0156] Based on preliminary data many malignant cancers were found to express elevated levels of HLA-E. These include multiple myeloma (MM) and acute myeloid leukemia (AML) [Fig. 6A],

[0157] Additionally, it has been shown that CML-LBC SPCs have elevated levels of HLA-E. Thus, it is speculated that subset of drug resistant lymphoblastic leukemias such as T-ALL, B- ALL, Ph+ ALL and Ph- ALL might also express higher levels of HLA-E.

[0158] As shown in [Fig. 6A], when the multiple myeloma cell line MM1S was exposed to the expanded adaptive NK cells at the effector: target cell ratio of 1 :1 , the cell death rate was elevated from 30% (no NK) to 80.5% (+ NK at 1 :1 ratio). Moreover, as a proof-of-concept, HLA-E was overexpressed in MM1S and where it was found that exposure to adaptive NK cells, increased cell death by -15% to 95.4% [Fig. 6B],

[0159] Similarly, the expanded NK cells showed potent cell killing activities against the AML lines THP-1 and HL-60 [Fig. 6C] and [Fig. 6D], These results are currently being expanded upon through the inclusion of more cancer cell lines and potentially primary samples in future. Lastly, recently it has been established a MM mouse model in which MM 1S cells expressing HLA-E were engrafted and mice were exposed to 2 rounds of adaptive NK cell infusions. As shown in [Fig. 6E], a remarkable suppression of MM signals was observed in the mice infused with adaptive NK cells.

[0160] Human cancers characterized by high levels of cell surface-associated HLA-E expression may be a suitable target for treatment with NK cell-based therapies for these difficult to treat cancers. Continuing tests of the potential of the expanded adaptive NK cells to kill MM, AML and ALL cells both in vitro and in vivo studies are underway and / or in planning process.

[0161] Reduced expression of exhaustion markers on Dasatinib-expanded adaptive NK cells

[0162] Single-cell RNA-seq of Day-14 products [baseline(day 0), day 14 eNKE21and Das- eNKE21] provided further mechanistic insights (Figure 7A-2C). NK cells expanded in the presence of dasatinib had a unique cluster composition, as compared to both unexpanded (Day 0) and aNK expanded in the absence of dasatinib (Figure 7C). Differential gene expression (DEG) analysis followed by gene ontology studies revealed that dasatinib-cultured aNKE21cells displayed broadly suppressed type-l / ll interferon-response programs, elevated cytokine-cytokine receptors interactions (Figure 7D) and a significantly reduced composite exhaustion signature (Figure 7E). Orthogonal protein-level validation confirmed decreased surface expression of canonical dysfunction markers, including KLRG1, CD161 (KLRB1), and CD69 (Figure 7F). Crucially, dasatinib significantly downregulated PRDM1 (encoding BLIMP1), a master transcriptional regulator of terminal differentiation and exhaustion to some extent at the transcript level, but more pronounced at the protein level (flow cytometry across paired donors; Figure 7G). Overall, these results suggest that Dasatinib-expanded NK cells are likely to be more potent in cytotoxic killing, especially when NK cell exhaustion blunts their function.

[0163] Dasatinib-expanded aNK cells show improved in vivo persistence, translating into improved tumor control

[0164] The optimized Das-eNKE21platform performance was tested against primary BC-CML SPCs than cells expanded without dasatinib. Strikingly, Das-eNKE21cells demonstrated significantly augmented cytotoxicity compared to standard eNKE21cells across increasing effector-to-target ratios (2: 1-10: 1). This enhanced killing was most pronounced against a subset of patient-derived lines (P670, P687, P681), while samples inherently more sensitive to standard eNKE21-mediated cytotoxicity showed no differential activity (Figure 8A). Together with the reduced exhaustion signature of Das-eNKE21cells, these in vitro results were then evaluated for its in vivo performance across three key parameters: engraftment, persistence, and anti-leukemic efficacy. In NSG mice receiving adoptive transfer with low-dose weekly IL-15 support, Das-eNKE21achieved markedly higher early engraftment by Week 1 (Welch’s t-test, P<0.05). Critically, this advantage persisted with consistent trends toward superior Das-eNKE21frequencies at Weeks 2- 4 (P=0.09, 0.06, and 0.14, respectively; Figure 8B).

[0165] This persistence advantage was then tested to determine whether it translates across distinct tumor microenvironments. In both K562-HLA-E xenografts and a MBC patient-derived xenograft model (P681), Das-eNKE21consistently outcompeted standard eNKE21, yielding significantly higher NK chimerism (K562-HLA-E: **P<0.01 ; primary MBC: **P<0.0001 ; Figure 8C). The persistence benefit was donor-reproducible, as independent donors exhibited significantly or substantially higher peripheral blood human NK chimerism with Das-eNKE21(Donor HSA39: P<0.05; Donor HSA43: P=0.17; Figure 8D). Functionally, enhanced persistence directly correlated with improved tumor control. Bioluminescence imaging revealed delayed leukemia progression in Das-eNKE21-treated cohorts. While the survival benefit over standard eNKE21showed a positive trend (P=0.07), both NK products significantly outperformed tumor-only controls (Figure 8E). Collectively, these results establish that dasatinib-expanded aNK cells not only retain but contextually augment cytotoxic potency in vitro, and critically, exhibit cryoresilient, donor-reproducible persistence in vivo.

[0166] Expanded adaptive NK cells are superior to non-adaptive NK cells in persistence, tumor control, and survival.

[0167] Therapies for HLA-E Expressing Cancers: head and neck cancer

[0168] Enhanced killing of head and neck cancer lines (TM49 cells) by Dasatinib-expanded NK cells was observed. Both standard eNKE21and Das-eNKE21demonstrated cytotoxicity of cancer cells in head and neck cancer lines (TM49 cells). Standard eNKE21demonstrated 63%cytotoxicity of the head and neck cancer lines while dasatinib-expanded NK cells Das-eNKE21demonstrated a much higher cytotoxicity of the head and neck cancer lines of 94% [Fig. 9].

[0169] Adaptive NK cell percentages correlate with Antibody-dependent cell cytotoxicity (ADCC) proficiency. Adaptive NK cells were formed using the biphasic method. The abundance in each eNKE21feeder cell formed was above 40% [Fig. 10A], The proficiency of the expanded adaptive NK cells were tested for the ability to mediate ADCC in the presence of cetuximab, ADCC proficiency was proportional to the percent of aNK population in the final expanded NK product [Fig. 10B],

[0170] The following steps can be taken to refine the technology:

[0171] Enhancing the yield and enrichment of adaptive NK cells post ex vivo expansion through the incorporation of NK cell modulators: Further improve expansion efficiency and cytotoxicity of adaptive NK cells through the addition of modulating compounds during ex vivo expansion including but not limited to

[0172] Addition of actin remodelers such as lenalidomide to improve cytotoxicity. Refine donor selection criteria by examining the effect of killer I g- like receptor (KIR) profile on the in vitro and in vivo efficiency of expanded adaptive NK cells. We will examine scRNA-seq data of Singaporean healthy donor derived NK cells.

[0173] Evaluating the efficacy of ex vivo expanded adaptive NK cells in combating BC-CML in vivo.

[0174] Broaden the application of adaptive NK cell therapy to various in vitro and in vivo models of human malignancies including but not limited to MM, AML, T-ALL, B-ALL, Ph+ ALL, DLBCL, Ph- ALL and solid cancers with high HLA-E such as head and neck cancers.

[0175] Leveraging the higher antibody-dependent cellular cytotoxicity (ADCC) capacity of adaptive natural killer (NK) cells, we will combine the expanded adaptive NK cells with therapeutic antibodies such as Daratumumab for drug resistant MM.

[0176] It should be further appreciated by the person skilled in the art that variations and combinations of features described above, not being alternatives or substitutes, may be combined to form yet further embodiments falling within the intended scope of the invention.

[0177] As would be understood by a person skilled in the art, each embodiment, may be used in combination with other embodiment or several embodiments.

Claims

ClaimsClaim 1 . A method of enhancing an abundance of NKG2C+ adaptive NK cells in a population of NK cells, the method comprising: a) co-culturing peripheral blood mononuclear cells (PBMCs) in a first culture the first culture comprising:(I) first immortalized cell line genetically modified to express amino acid sequence of HLA-E; and(ii) interleukin 2, b) isolating the cultured PBMCs; c) co-culturing the isolated cultured PBMCs in a second culture, the second culture comprising:(i) second immortalized cell line genetically modified to express amino acid sequence of HLA-E and IL21 and 4-1 BBL(ii) interleukin 2, and(iii) interleukin 15 d) isolating the population of NK cells from the second culture wherein the NK cell population include cells expressing NKG2C.Claim 2. The method according to claim 1 , wherein the amino acid sequence of HLA-E in the first and / or the second immortalized cell line comprises a chimeric construct of a peptide of HLA-E wherein the signal peptide of HLA-E is replaced with a signal peptide of HLA-G.Claim 3. The method according to claim 1 or 2, wherein the second culture further comprises a Bcr-Abl allosteric inhibitor.Claim 4. The method according to claim 3, wherein the Bcr-Abl allosteric inhibitor comprises a tyrosine kinase inhibitor.Claim 5. The method according to claim 4, wherein the tyrosine kinase inhibitor comprises dasatinib.Claim 6. The method according to claim 1 or 2, wherein the first or second culture further comprises an actin remodeler comprising Lenalidomide.Claim 7. The method according to any one of claims 1 to 6, wherein the PBMCs are isolated from a person with at least 15% of the NK cell population identified as a CD56+CD16+CD57+NKG2C+ subset.Claim 8. The method according to any one of claims 1 to 7, wherein the PBMC’s are isolated from a healthy person.Claim 9. The method according to any one of claims 1 to 8, wherein the PBMC’s are isolated from a person with a prior history of CMV infection.Claim 10. A method of determining a treatment outcome in patient who has been diagnosed with chronic myeloid leukemia and providing an appropriate treatment regimen, the method comprising: isolating an NK cell population from the bone marrow of the patient who has been diagnosed with chronic myeloid leukemia; determining percentage of NKG2A+ cells in the NK cell population wherein expression of NKG2A+ below a predetermined percentage of the NK cell population is indicative that the patient will be responsive to a treatment regimen with a tyrosine kinase inhibitor and the patient is administered the tyrosine kinase inhibitor or expression of NKG2A+ above the predetermined percentage of the NK cell population is indicative that the patient will become unresponsive to a treatment regimen with a tyrosine kinase inhibitor and patient is administered NK cells expressing NKG2C+ isolated by an expansion method comprising: a) co-culturing peripheral blood mononuclear cells (PBMCs) in a first culture the first culture comprising:(i) first immortalized cell line genetically modified to express amino acid sequence of HLA-E; and(ii) interleukin 2, b) isolating the cultured PBMCs; c) co-culturing the isolated cultured PBMCs in a second culture, the second culture comprising:(i) second immortalized cell line genetically modified to express amino acid sequence of HLA-E and IL21 and 4-1 BBL(ii) interleukin 2, and(iii) interleukin 15 d) isolating the population of NK cells from the second culture wherein the NK cell population include cells expressing NKG2C.Claim 11. The method according to claim 10, further comprising: determining percentage of NKG2C+ cells in the NK cell population wherein expression of NKG2A+ below a first predetermined percentage of the NK cell population and expression of NKG2C+ above a second predetermined percentage of the NK cell population is indicative that the patient will be responsive to a treatment regimen with a tyrosine kinase inhibitor and the patient is administered a tyrosine kinase inhibitor or expression of NKG2A+ above the first predetermined percentage of the NK cell population and expression of NKG2C+ below the second predetermined percentage of the NK cell population the patient will becomeunresponsive to a treatment regimen with a tyrosine kinase inhibitor and the patient is administered NK cells expressing NKG2C+ isolated by the expansion method.Claim 12. The method according to claim 10, further comprising: determining expression of HLA-E molecule on CD34+ cells from the bone marrow of the patient who has been diagnosed with chronic myeloid leukemia referred to as CML sample; measuring the expression of HLA-E molecule on CD34+ cells from bone marrow of a donor that does not have chronic myeloid leukemia referred to as control sample, comparing the expression of HLA-E molecule on CD34+ cells in the CML sample to expression of HLA-E molecule on CD34+ cells in the control sample wherein when the expression of HLA-E molecule on CD34+ cells in the CML sample is more than the expression of HLA-E molecule on CD34+ cells in the control sample the patient will progress to blast crisis CML and the patient is administered a combination of a tyrosine kinase inhibitor and NK cells expressing NKG2C+ isolated by the expansion method.Claim 13. The method according to claim 10, wherein the NK cells expressing NKG2C+ isolated by the isolation method are from PBMC’s taken from the patient who has been diagnosed with chronic myeloid leukemia.Claim 14. The method according to claim 10, wherein the NK cells expressing NKG2C+ isolated by the expansion method are from PBMC’s taken from a healthy person.Claim 15. The method according to claim 10, wherein the NK cells expressing NKG2C+ isolated by the expansion method are from PBMC’s taken from the patient who has been diagnosed with chronic myeloid leukemia or a healthy person with at least 15% of the NK cell population identified as a CD56+CD16+CD57+NKG2C+ subset.Claim 16. NK cells wherein at least 21% of the NK cells express NKG2C for use in the treatment of cancer wherein expression of HLA-E molecule on CD34+ cells in a cancer sample from the cancer is more than the expression of HLA-E molecule on CD34+ cells in a control sample from a healthy individual.Claim 17. The NK cells for use according to claim 16, wherein the NK cells are derived according to the method of any one of claims 1 to 9.Claim 18. The NK cells for use according to claim 16 or 17, wherein the cancer is leukemia.Claim 19. The NK cells for use according to claim 16 or 17, wherein the cancer is chronic myeloid leukemia.Claim 20. Use of NK cells wherein at least 21% of the NK cells express NKG2C in the manufacture of a medicament for treating cancer wherein a cell sample from the cancer expresses HLA-E molecule on CD34+ cells at a higher abundance than the expression of HLA- E molecule on CD34+ cells in a control sample from a healthy individual.Claim 21 . The use according to claim 20, wherein the NK cells are derived according to the method of any one of claims 1 to 9.Claim 22. The use according to claim 20 or 21 , wherein the cancer is leukemia.Claim 23. The use according to any one of claims 20 to 22, wherein the cancer is chronic myeloid leukemia.Claim 24. A composition comprising NK cells derived according to the method of claim 1 and a tyrosine kinase inhibitor.Claim 25. The composition according to claim 24, wherein the tyrosine kinase inhibitor comprises dasatinib.Claim 26. A composition comprising NK cells derived according to the method of claim 1 and a therapeutic cytotoxic antibody.Claim 27. The composition according to any one of claims 24 to 26, wherein the NK cells are derived from the method of any one of claims 1 to 9.Claim 28. A composition according to any one of claims 24 to 27, for use in the treatment of cancer wherein expression of HLA-E molecule on CD34+ cells in a cancer sample from the cancer is more than the expression of HLA-E molecule on CD34+ cells in a control sample from a healthy individual.Claim 29. The composition for use according to claim 28, wherein the cancer is leukemia.Claim 30. The composition for use according to claim 28, wherein the cancer is chronic myeloid leukemia.Claim 31 . Use of the composition according to any one of claim 26 to 29 in the manufacture of a medicament for treating cancer wherein a cell sample from the cancer expresses HLA-Emolecule on CD34+ cells at a higher abundance than the expression of HLA-E molecule on CD34+ cells in a control sample from a healthy individual.Claim 32. The use according to claim 31 , wherein the cancer is leukemia.Claim 33. The use according to claim 31 or 32, wherein the cancer is chronic myeloid leukemia.Claim 34. An in vitro method for predicting subgroups of chronic myeloid leukemia comprising: isolating an NK cell population from the bone marrow sample of a patient who has been diagnosed with chronic myeloid leukemia; determining percentage of NKG2A+ cells in the NK cell population wherein expression of NKG2A+ below a first predetermined value of the NK cell population is indicative of a first subgroup that will be responsive to treatment with a tyrosine kinase, and expression of NKG2A+ above the first predetermined value of the NK cell population is indicative of a second subgroup that will be unresponsive to treatment with a tyrosine kinase inhibitor.Claim 35. The in vitro method according to claim 34, further comprising: determining percentage of NKG2C+ cells in the NK cell population wherein expression of NKG2A+ below the first predetermined value of the NK cell population and expression of NKG2C+ above a second predetermined value of the NK cell population is indicative of the first subgroup that will be responsive to treatment with a tyrosine kinase inhibitor, and expression of NKG2A+ above the first predetermined value of the NK cell population and expression of NKG2C+ below the second predetermined value of the NK cell population is indicative of the second subgroup that will become unresponsive to treatment with a tyrosine kinase inhibitor.Claim 36. The in vitro method according to claim 34 or 35, further comprising: determining expression of HLA-E molecule on CD34+ cells from bone marrow of the patient who has been diagnosed with chronic myeloid leukemia in a CML sample; determining expression of HLA-E molecule on CD34+ cells from bone marrow of a donor that does not have chronic myeloid leukemia in a control sample, comparing the expression of HLA-E molecule on CD34+ cells in the CML sample to expression of HLA-E molecule on CD34+ cells in the control sample wherein when the expression of HLA-E molecule on CD34+ cells in the CML sample is more than the expression of HLA-E molecule on CD34+ cells in the control sample it is indicative of a third subgroup that will progress to blast crisis CML.

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