Compositions comprising NKG2d, cxcr2 and dap10 / dap12 fusion polypeptides and methods of use thereof

Abstract

The present invention relates to a method for preparing immune response cells, immune response cells thereof, pharmaceutical compositions, kits, uses thereof and methods of treatment thereof. Specifically, the invention provides a method for preparing an immune response cell, and the method comprises the following steps: (a) carrying out gene modification on an immune cell to express an NKG2D polypeptide; wherein the immune cells have been activated by anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix; or (b) activating an immune cell wherein the immune cell is genetically modified to express an NKG2D polypeptide, and wherein activation is performed by anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix.

Description

Technical Field

[0001] The present invention relates to methods for preparing immune response cells, their immune response cells, pharmaceutical compositions, kits, their uses and treatment methods. Background Art

[0002] Immunotherapies using chimeric antigen receptor (CAR)-modified T cells have proven transformative for the treatment of B cell malignancies and multiple myeloma. However, due to the lack of tumor-selective targets, the application of this technology in solid tumor immunotherapy is hindered. Most tumor antigens are intracellular and thus not readily recognized by CAR T cells. Therefore, most CARs currently being developed for solid tumors bind to targets that are upregulated in tumor cells but have low expression levels in normal tissues.

[0003] NKG2D ligands are one of the few groups of highly tumor-selective targets. In humans, these ligands include a group of 8 stress-induced proteins (MICA, MICB, ULBP1-6) that are aberrantly expressed in almost all tumor cell types. In addition, NKG2D ligands are also present on tumor-associated stromal components, such as endothelial cells, regulatory T cells, and myeloid-derived suppressor cells (Parihar, R., et al., 2019, Cancer Immunol. Res. 7(3):363-375; Schmiedel & Mandelboim, 2018, Front. Immunol. (9) 2040). Mice deficient in the NKG2D gene exhibit impaired immune surveillance against epithelial and lymphoid malignancies. Evidence for NKG2D ligands as safe therapeutic targets stems from the fact that they are not found in healthy tissues. Clinical trials using autologous NKG2D-targeted CARs have not found any major safety issues (see https: / / pubmed.ncbi.nlm.nih.gov / 30396908 / ).

[0004] The NKG2D receptor is naturally expressed by natural killer (NK) cells and some subsets of T cells. Each NKG2D homodimer binds to two homodimeric DAP10 adapter molecules through complementary charged amino acids within the plasma membrane. This interaction is essential for the cell surface expression and function of NKG2D. DAP10, similar to CD28, is capable of providing co-stimulatory signals through phosphatidylinositol 3-kinase, but importantly, DAP10 lacks the p56lck binding motif that promotes the unnecessary recruitment of regulatory T cells (Kofler, et al., 2011, Mol. Ther. 19:760-767). The potency of DAP10 co-stimulation is highlighted by its ability to continue signaling even after internalization. However, due to the lack of an immunoreceptor tyrosine-based activation motif (ITAM) in DAP10, engagement of NKG2D does not lead to complete activation of T cells.

[0005] A variety of CARs have been developed, which adopt different methods to provide ITAM-dependent signal 1 in addition to co-stimulatory signals (also known as signal 2), as both of these signal types are required to trigger full T cell activation. The first CAR targeting NKG2D was developed by Sentman et al. and consists of a fusion of NKG2D and CD3ζ (Zhang et al., 2005, Blood 106:1544-1551). Although nominally a first-generation CAR, it binds to endogenous DAP10 in T cells, meaning that both signal 1 and signal 2 are provided simultaneously. This CAR is currently in clinical development by Celyad Oncology under the name CYAD-01. Recently, Chang et al. (2013, Cancer Res. 73:1777-1786) engineered NK cells to co-express an identical CAR while expressing exogenous DAP10. In addition, two other NKG2D CARs have been described that incorporate 4-1BB (Song et al., 2013, Hum. Gene Ther. 24:295-305) or CD28 (Lehner et al., 2012, PLoS One 7:e31210) to provide co-stimulation in place of that provided by DAP10. All of these CARs enable T cell-mediated tumor cell killing while producing cytokines, and the CAR described by Chang et al. also exhibits transient in vivo anti-tumor activity.

[0006] The ligands for the CXCR2 receptor include chemokines of the cysteine-X-cysteine (CXC) family containing the (Glu-Leu-Arg) ELR motif, namely CXCL1-3 and CXCL5-8. These chemokines within tumors are produced not only by malignant cells but also by stromal components such as fibroblasts and macrophages (Thuwajit et al., 2018, Med Res Rev. 38:1235-54; Thongchot et al., 2021, Int J Oncol. 58:14). In addition, tumor cells can condition stromal cells to produce these factors, thereby promoting disease progression and conferring resistance to cytotoxic chemotherapy (Le Naour, 2020, J Mol Cell Biol. 12:202-15).

[0007] The most well - studied member of the CXC family is CXCL8, also known as interleukin (IL) - 8. Circulating CXCL8 levels are elevated in some cancer patients, including ovarian tumors (Zhang, et al., 2019, Oncol Lett. 17:2365 - 9), malignant mesothelioma (Judge, et al., 2016, Ann Surg Oncol. 23:1496 - 500), pancreatic cancer (Hou, et al., 2018, J Clin Med. 7:502), breast cancer (Milovanovic, et al., 2019, Cytokine 118:93 - 98; Autenshlyus, et al., 2021, 35:20587384211034089), esophageal cancer (Huang, et al., Cancer Biomark. 29:139 - 149) and head and neck cancer (Rezaei, et al., 2019, 39:727 - 739). In addition to CXCL8, high - level expression of CXCR2 ligands CXCL1, CXCL3 and CXCL5 has also been described in several cancers. Summary of the Invention

[0008] The present invention provides a method for preparing immune - responsive cells. The method comprises:

[0009] (a) genetically modifying immune cells to express NKG2D polypeptide; wherein the immune cells have been activated by anti - CD3 and anti - CD28 antibodies or fragments thereof conjugated to a nanomatrix; or

[0010] (b) activating immune cells, wherein the immune cells have been genetically modified to express NKG2D polypeptide, and wherein activation is carried out by anti - CD3 and anti - CD28 antibodies or fragments thereof conjugated to a nanomatrix.

[0011] The present invention also provides a method for preparing immune - responsive cells. The method comprises:

[0012] (a) activating immune cells, wherein the activation does not include phorbol 12 - myristate 13 - acetate (PMA), phytohemagglutinin (PHA) or magnetic beads; and

[0013] (b) genetically modifying the immune cells to express the NKG2D polypeptide.

[0014] The present invention further provides an immune - responsive cell obtainable by the method of any of the above aspects.

[0015] There is also provided an immune response cell genetically modified to express an NKG2D polypeptide, wherein the immune response cell has been activated by anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix.

[0016] Furthermore, the present invention provides a pharmaceutical composition comprising the immune response cell of the present invention and a pharmaceutically or physiologically acceptable diluent and / or carrier.

[0017] The present invention also provides a kit comprising the immune response cell or the pharmaceutical composition of the present invention.

[0018] There is also provided the immune response cell or the pharmaceutical composition of the present invention for treating or preventing a disease, optionally wherein the disease is cancer.

[0019] On the other hand, there is provided the use of the immune response cell or the pharmaceutical composition of the present invention in (i) therapy or (ii) cancer treatment.

[0020] The present invention also provides a method for treating or preventing cancer in a subject, wherein the method comprises administering to the subject the immune response cell or the pharmaceutical composition of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] One or more embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings, in which:

[0022] Figure 1 Schematic diagrams of CAR constructs N1012, N1012_CXCR2, NKG2D, and CYAD-01_10 are shown. In CYAD-01_10, the CYAD-01 replicon is co-expressed with additional DAP10.

[0023] Figure 2 Shown is the development over time of subcutaneous CFPac-1 pancreatic tumor xenografts in mice treated with PBS, high-dose (1×10 7 ) CAR T cells (N1012, N1012_CXCR2, CYAD-01 replicon, or untransduced) or low-dose (4×10 6 ) CAR T cells (N1012 or N1012_CXCR2) 28 days after tumor inoculation. A shows the mean tumor volume of each treatment group. B shows the survival rate of the mice. C shows an individual mouse illustration of the mean data in A.

[0024] Figure 3Shows that compared with the control group, the ability of N1012 and N1012_CXCR2 T cells to persist and proliferate was enhanced after multiple rounds of stimulation with triple-negative breast cancer (TNBC) cell lines MDA-MB_468 and MDA-MB_231.

[0025] Figure 4 Shows the bioluminescence emission of BxPC3 tumor xenografts expressing firefly luciferase (ffLuc) in mice treated with N1012_CXCR2, N1012, or untransduced T cells, which were activated in vitro by different activating stimuli (PHA, TransAct, or immobilized anti-CD3 and anti-CD28 antibodies). T cells were injected intraperitoneally on day 12 after tumor inoculation (marked by the vertical dashed line). Tumor development was measured by total flux (photons / second).

[0026] Figure 5 Shows Figure 4 The bioluminescence results shown, but directly compares the anti-tumor activities of N1012, N1012_CXCR2, and untransduced T cells activated under the same stimulating conditions (PHA, TransAct, or immobilized anti-CD3 and anti-CD28 antibodies) through graphs.

[0027] Figure 6 Shows Figure 4 and Figure 5 Separate illustrations of the results shown. Additional rechallenge tests were performed on mice that achieved complete remission on day 64. This rechallenge is marked by the second vertical dashed line.

[0028] Figure 7 Shows the CD4 to CD8 ratio (measured by flow cytometry) of N1012, N1012_CXCR2, untransduced, CYAD-01, and CYAD-01_10 T cells at the end of the culture period (days 10 to 12) after stimulation with 5 μg / mL phytohemagglutinin-L (PHA-L) or addition of 10 μL TransAct reagent per 1 × 10 6 peripheral blood mononuclear cells. Activation with TransAct resulted in a higher CD4 to CD8 ratio for all T cells. This shift was most pronounced in N1012 and N1012_CXCR2 T cells.

[0029] Figure 8 Shows (A) the transduction efficiency of T cells evaluated by flow cytometry at the end of the ex vivo culture period (days 10 to 12) after activation with PHA or TransAct. The transduction efficiency was evaluated for any cells positive for NKG2D or CXCR2 staining, and due to the lack of endogenous NKG2D expression in CD4 +Evaluated in T cells; (B) At the end of the ex vivo culture period, the median fluorescence intensity (MFI) of NKG2D in CD4 + T cells was evaluated by flow cytometry. The expression intensities of each construct activated by PHA or TransAct reagent were similar; (C) At the end of the ex vivo culture period, the median fluorescence intensity of CXCR2 in CD4 + T cells was evaluated by flow cytometry. The expression intensity of N1012_CXCR2 T cells was similar whether activated by PHA or TransAct reagent.

[0030] Figure 9 Shows the fold expansion of T cells activated by 5 μg / mL phytohemagglutinin-L (PHA-L) or 10 μL TransAct reagent added per 1 × 10 6 peripheral blood mononuclear cells. This value was calculated by dividing the number of T cells present at the end of the ex vivo culture period by the number of initially transduced T cells.

[0031] Figure 10 Shows the median fluorescence intensity (MFI, A) and transduction percentage (B) of cell surface NKG2D expression in pan-γδ TCR + CD3 + cells transduced with a retroviral vector encoding N1012 or N1012_CXCR2 and expanded for 21 days. Results for untransduced T cells are shown for comparison.

[0032] Figure 11 Shows (A) the fold expansion of γδ T cells calculated based on the percentage of pan-γδ TCR + CD3 + cells on day 0 (when cells were transduced with a retroviral vector encoding N1012 or N1012_CXCR2), day 7, day 14, and day 21. (B) The percentage of pan-γδ TCR + CD3 + cells on day 0, day 7, day 14, and day 21 (n = 11).

[0033] Figure 12 Shows the results of cytotoxicity dose-response assays of untransduced (UT) and CAR + (N1012 or N1012_CXCR2) γδ T cells against the triple-negative breast cancer cell line MDA-MB-468 (A) and the pancreatic cancer cell line BxPC-3 (B) at different effector-to-target ratios (n = 3). The results were compared with untransduced cells.

[0034] Figure 13 Shows (A) untransduced (UT) versus CAR +(N1012 or N1012_CXCR2) γδ T cell cytotoxicity restimulation assay against acute myeloid leukemia (AML) cells THP-1-LT (n = 2); (B) Untransduced (UT) vs. CAR + γδ T cell cytotoxicity restimulation assay against MDA-MB-468 cells (n = 9); (C) Untransduced (UT) vs. CAR + γδ T cell cytotoxicity restimulation assay against BxPC-3 cells (n = 8).

[0035] Figure 14 Shows the purity of CAR + γδ T cells after restimulation on MDA-MB-468 cells; (B) The purity of CAR + γδ T cells after restimulation on BxPC-3 cells; (C) The percentage of αβ T cells after restimulation on MDA-MB-468 cells; and (D) The percentage of αβ T cells after restimulation on BxPC-3 cells. Percentages were calculated from flow cytometry dot plots to indicate marker expression.

[0036] Figure 15 Shows the anti-tumor efficacy of N1012 or N1012_CXCR2 γδ T cells in NSG mice with intraperitoneal transplantation of BxPC-3-LT tumors. The cryopreserved γδ T cells were thawed and intraperitoneally injected at a dose of 10 million units on the 11th day after tumor transplantation. Untransduced γδ T cells and PBS were used as controls.

[0037] Figure 16 For survival curves of BxPC3 tumor intraperitoneal xenograft mice treated with PBS or 1 × 10 7 CAR T cells (N1012, N1012_CXCR2 or untransduced).

[0038] Figure 17 For survival curves showing pooled data from in vivo experiments of CFPac1, BxPC3, Kuramochi, Ovsaho, mesothelioma, triple-negative breast cancer or SKOV3 tumor xenograft mice treated with PBS or 1 × 10 7 CAR T cells (N1012, N1012_CXCR2 or untransduced).

[0039] Figure 18 Shows the transduction (A to B) and expansion (C) of NKG2D_CXCR2 into primary human T cells after activation with different concentrations of TransAct TM After three and ten days of transduction, CD4 was evaluated by flow cytometry +The expression level of NKG2D in T cells was used as an indicator of transduction efficiency. Untransduced (UT) CD4 + T cells were used as a negative control for transduction.

[0040] Figure 19 The anti-tumor efficacy of N1012 and N1012_CXCR2 T cells in NSG mice subcutaneously transplanted with LS180 or SW620 metastatic colorectal cancer (mCRC) cells was shown. T cells were intravenously injected at a dose of 1×10 7 and tumor growth was monitored by caliper measurement. The average growth of each treatment group (A to B) or the tumor growth of each mouse in the SW620 model (C) was compared with untransduced (UT) T cells or CYAD-01 T cells. DETAILED DESCRIPTION

[0041] The present invention provides a method for preparing immune response cells. The method comprises:

[0042] (a) genetically modifying immune cells to express NKG2D polypeptide; wherein the immune cells have been activated by anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix; or

[0043] (b) activating immune cells, wherein the immune cells have been genetically modified to express NKG2D polypeptide, and wherein activation is carried out by anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix.

[0044] The inventors have found that immune response cells with unexpectedly enhanced function can be obtained by activating immune cells with anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix and genetically modifying the immune cells to express NKG2D polypeptide. Specifically, the resulting immune response cells have unexpectedly enhanced anti-tumor activity.

[0045] In some embodiments, the method comprises:

[0046] (i) activating the immune cells with anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix; and

[0047] (ii) genetically modifying the immune cells to express the NKG2D polypeptide.

[0048] Alternatively, the method may comprise:

[0049] (i) genetically modifying the immune cells to express the NKG2D polypeptide; and

[0050] (ii) activating the immune cells with anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix.

[0051] In embodiments that include steps (i) and (ii), it is understood that step (i) occurs before step (ii).

[0052] In some embodiments, the method includes activating immune cells by simultaneously utilizing anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix, and genetically modifying the immune cells to express an NKG2D polypeptide.

[0053] The duration of activating immune cells by utilizing anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix can be from about 6 hours to about 504 hours. In some embodiments, the duration of activating immune cells by utilizing anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix is at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours, or at least about 168 hours. In some embodiments, the duration of activating immune cells by utilizing anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix does not exceed about 504 hours, does not exceed about 480 hours, does not exceed about 456 hours, does not exceed about 432 hours, does not exceed about 408 hours, does not exceed about 384 hours, does not exceed about 360 hours, does not exceed about 336 hours, does not exceed about 312 hours, does not exceed about 288 hours, does not exceed about 264 hours, does not exceed about 240 hours, does not exceed about 216 hours, does not exceed about 192 hours, does not exceed about 168 hours, does not exceed about 144 hours, does not exceed about 120 hours, or does not exceed about 96 hours.

[0054] Preferably, the duration of activating immune cells by utilizing anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix is at least about 12 hours. More preferably, the duration of activating immune cells by utilizing anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix is at least about 24 hours. Most preferably, the duration of activating immune cells by utilizing anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix is at least about 48 hours.

[0055] Preferably, the duration of activating immune cells by utilizing anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix does not exceed about 120 hours. More preferably, the duration of activating immune cells by utilizing anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix does not exceed about 96 hours. Most preferably, the duration of activating immune cells by utilizing anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix does not exceed about 72 hours.

[0056] In some embodiments, the duration of activating immune cells using anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix is from about 12 hours to about 72 hours.

[0057] In some embodiments, the duration of activating immune cells using anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix is about 12 hours. In other embodiments, the duration of activating immune cells using anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix is about 24 hours. In some embodiments, the duration of activating immune cells using anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix is about 48 hours.

[0058] In some embodiments, the method includes step (iii), which includes culturing the immune cells in vitro. In some embodiments, the method includes:

[0059] (i) activating the immune cells using anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix;

[0060] (ii) genetically modifying the immune cells to express the NKG2D polypeptide; and

[0061] (iii) culturing the immune cells in vitro.

[0062] The duration of culturing immune cells in vitro can be from about 24 hours to about 504 hours. In some embodiments, the duration of culturing immune cells in vitro is at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours, or at least about 168 hours. In some embodiments, the duration of culturing immune cells in vitro does not exceed about 504 hours, does not exceed about 480 hours, does not exceed about 456 hours, does not exceed about 432 hours, does not exceed about 408 hours, does not exceed about 384 hours, or does not exceed about 360 hours.

[0063] Preferably, the duration of culturing immune cells in vitro is at least about 48 hours. More preferably, the duration of culturing immune cells in vitro is at least about 72 hours. Most preferably, the duration of culturing immune cells in vitro is at least about 96 hours.

[0064] Preferably, the duration of culturing immune cells in vitro does not exceed about 336 hours. More preferably, the duration of culturing immune cells in vitro does not exceed about 312 hours. Most preferably, the duration of culturing immune cells in vitro does not exceed about 288 hours.

[0065] In some embodiments, the duration of in vitro culturing of immune cells is about 168 hours. In other embodiments, the duration of in vitro culturing of immune cells is about 192 hours. In some embodiments, the duration of in vitro culturing of immune cells is about 216 hours.

[0066] Preferably, the immune cells are cultured in vitro in a suitable culture medium. The culture medium may include RPMI-1640 medium or DMEM high-glucose medium. Those skilled in the art should be aware of other suitable culture media. The culture medium may be supplemented with, for example, antibiotics, IL-2, human AB serum, FBS (fetal bovine serum) or FCS (fetal calf serum) and / or amino acids. Preferably, the culture medium contains IL-2. The IL-2 may be recombinant IL-2. The IL-2 may be human IL-2. In some embodiments, the concentration of IL-2 in the culture medium is about 20 U / mL.

[0067] In some embodiments, the in vitro culturing of immune cells includes reactivating the immune cells in vitro. The reactivation may include activating the immune cells using anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix. Alternatively, the reactivation may include activating with phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA) or anti-CD3 and anti-CD28 antibodies conjugated to magnetic beads.

[0068] Those skilled in the art should be familiar with the term "magnetic beads" used herein. Magnetic beads conjugated with anti-CD3 and anti-CD28 antibodies are generally inert and have a uniform diameter, similar to that of antigen-presenting cells. Thus, the diameter of the magnetic beads is typically about 1 μm to about 10 μm. The magnetic beads are also generally spherical and have a solid, non-porous surface area. Such magnetic beads have previously been used in in vitro cell culture to activate and expand T cells. A variety of such magnetic beads are commercially available, including but not limited to (Thermo Fisher Scientific, UK).

[0069] In some embodiments, the method does not include phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA) or magnetic beads.

[0070] In embodiments including steps (i) and (ii), steps (i) and (ii) may not include phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA) or magnetic beads.

[0071] Nanomatrix

[0072] As used herein, "matrix" refers to a 3D structure comprising multiple polymer chains. The polymer chains may be crosslinked with each other and / or arranged in a cross-cross structure.

[0073] In the context of the present invention, the nano - matrix is a flexible nano - scale polymer matrix. The nano - matrix has a non - solid surface area. Generally, due to the inclusion of multiple polymer chains, the nano - matrix is porous. "Flexible" is to be understood as the nano - matrix being movable and thus capable of bending. This means that the nano - matrix does not have a solid - phase surface; its shape can vary according to the surrounding environment. Thus, the nano - matrix of the present invention actually has a grid - like or network - like structure.

[0074] Preferably, the nano - matrix comprises a movable polymer - chain matrix conjugated with anti - CD3 and anti - CD28 antibodies or fragments thereof. In some embodiments, the nano - matrix comprises at least two nano - matrices, a first nano - matrix conjugated with an anti - CD3 antibody or fragment thereof, and a second nano - matrix conjugated with an anti - CD28 antibody or fragment thereof.

[0075] The polymer chains may comprise collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions.

[0076] In some embodiments, the polymer chains comprise polysaccharides. Exemplary polysaccharides may include, but are not necessarily limited to, cellulose, agarose, dextran, chitosan, hyaluronic acid, and alginate. In some embodiments, the polysaccharide comprises dextran. Thus, in some embodiments, the nano - matrix comprises dextran chains.

[0077] Other polymers may include polyesters, polyethers, polyanhydrides, polyalkyl cyanoacrylates, polyacrylamides, polyorthoesters, polyphosphazenes, polyvinyl acetates, block copolymers, polypropylenes, polytetrafluoroethylene (PTFE), or polyurethanes. The polymer may be lactic acid or a copolymer. The copolymer may comprise lactic acid and glycolic acid (PLGA).

[0078] The polymer chains may be hydrophilic.

[0079] In some embodiments, the diameter of the nano - matrix is from about 1 nm to about 500 nm.

[0080] In some embodiments, the diameter of the nano - matrix is at least about 1 nm, at least about 10 nm, at least about 20 nm, at least about 50 nm, or at least about 70 nm. In some embodiments, the diameter of the nano - matrix does not exceed about 1000 nm, does not exceed about 500 nm, or does not exceed about 200 nm.

[0081] Preferably, the diameter of the nano - matrix is from about 50 nm to about 200 nm. More preferably, the diameter of the nano - matrix is from about 10 nm to about 200 nm. Most preferably, the diameter of the nano - matrix is about 100 nm.

[0082] Preferably, the nano - matrix is biodegradable. Also preferably, the nano - matrix is non - toxic to living cells. The non - toxicity to living cells should be understood as that the nano - matrix does not have an adverse effect on cell viability.

[0083] In some embodiments, the nano - matrix is in a soluble form or a colloidal form. In the context of the present invention, the term "soluble" should be understood as that the nano - matrix can be dissolved in a solvent. The solvent can be an aqueous solution, such as the above - mentioned culture medium. Those skilled in the art should understand that the term "colloidal" means that the nano - matrix is insoluble but suspended in the solvent, forming a mixture composed of the nano - matrix and the solvent.

[0084] As used herein, the term "antibody" refers to polyclonal antibodies or monoclonal antibodies. Antibody fragments can include Fab, Fab’, F(ab’)2, Fv, and single - chain antibodies. These antibodies and antibody fragments can be easily generated by various methods known in the art. The antibodies can be from any species source, such as murine, ovine, or human.

[0085] A variety of anti - CD3 antibodies, anti - CD28 antibodies, and their fragments are known and readily available in the art. In the context of the present invention, any fragment of an anti - CD3 antibody or an anti - CD28 antibody is a functional fragment because it retains the functional activity of the anti - CD3 antibody or the anti - CD28 antibody.

[0086] In some embodiments, the anti - CD3 antibody or its fragment is humanized. In some embodiments, the anti - CD28 antibody or its fragment is humanized. In some embodiments, the anti - CD3 antibody or its fragment and the anti - CD28 antibody or its fragment are humanized. Those skilled in the art should understand that a humanized antibody is an antibody from a non - human species, in which the amino acid sequence of the antibody is genetically modified to increase its similarity to a human antibody, thereby reducing immunogenicity. Methods for humanizing antibodies are well - known in the art.

[0087] "Conjugation" or "coupling" should be understood as the connection or binding of an antibody (or other agent) to the nano - matrix. The connection or binding can be covalent or non - covalent. Covalent bonds can include connections to carboxyl groups on the polymer chain. Non - covalent bonds can include biotin - avidin interactions.

[0088] The nano - matrix can be conjugated with additives of anti - CD3 and anti - CD28 antibodies or their fragments. Those skilled in the art should be aware of various suitable additives. Additives can include, but are not necessarily limited to, polynucleotides and / or proteins. Proteins can include antibodies, their fragments and derivatives, fusion proteins, and genetically modified proteins. Preferably, the additives include co - stimulatory molecules. Exemplary co - stimulatory molecules include, but are not necessarily limited to, anti - CD5, anti - CD4, anti - CD8, anti - MHC I, anti - MHC II, anti - CTLA - 4, anti - ICOS, anti - PD - 1, anti - OX40, anti - CD27L (CD70), anti - 4 - 1BBL, anti - CD30L, and anti - LIGHT antibodies, their fragments or derivatives.

[0089] Other additives can include cytokines, chemokines, cytokine receptors, and / or chemokine receptors. Suitable cytokines can include IL - 2, IFN - γ, IL - 12, IL - 17, IL - 1, IL - 15, IL - 4, IL - 10, and TNF - α. Exemplary chemokine receptors include, but are not necessarily limited to, CCR1, CCR2, CCR3, CCR4, CCR5, and CXCR3.

[0090] In some embodiments, the additives can include any agent capable of binding to cell adhesion molecules on T cells, such as monoclonal antibodies, fusion proteins, and their ligands or fragments corresponding to the following classes of adhesion molecules: cadherins, intercellular adhesion molecules, integrins, and selectins. Examples of adhesion molecules on T cells include CD44, CD31, CD18 / CD11a (LFA - 1), CD29, CD54 (ICAM - 1), CD62L (L - selectin), and CD29 / CD49d (VLA - 4).

[0091] In some embodiments, other agents can be embedded in the nano - matrix. For example, other agents can be embedded in the polymer chains. Such other agents can include magnetic agents or fluorescent agents. For example, metal oxide crystals can be embedded in the polymer chains. Preferably, iron oxide crystals are embedded in the polymer chains. Various other suitable magnetic agents and fluorescent agents are known to those skilled in the art.

[0092] "Embedded" should be understood that the agent is located within one or more polymer chains of the nano - matrix. Thus, in such embodiments, the polymer chains contain the other agent.

[0093] In some embodiments, the nano - matrix contains iron oxide crystals with a diameter of about 100 nm embedded in a biocompatible polysaccharide matrix. In some embodiments, the nano - matrix is in colloidal form and contains iron oxide crystals with a diameter of about 100 nm embedded in a biocompatible polysaccharide matrix.

[0094] In some embodiments, the anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to the nanomatrix are the nanomatrices disclosed in WO2014 / 048920A1, the entire content of which is incorporated herein by reference. This nanomatrix may be referred to as the MACS GMPTransAct CD3 / CD28 Kit (Miltenyi Biotec GmbH, order number: 170-076-140).

[0095] In some embodiments, the concentration of the anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to the nanomatrix is at least about 0.1 μL per 1 × 10 6 cells, at least about 0.5 μL per 1 × 10 6 cells, at least about 1 μL per 1 × 10 6 immune cells, at least about 2 μL per 1 × 10 6 immune cells, at least about 5 μL per 1 × 10 6 immune cells or at least about 10 μL per 1 × 10 6 immune cells.

[0096] In some embodiments, the concentration of the anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to the nanomatrix is less than about 50 μL per 1 × 10 6 immune cells, less than about 40 μL per 1 × 10 6 immune cells, less than about 30 μL per 1 × 10 6 immune cells or less than about 20 μL per 1 × 10 6 immune cells.

[0097] Preferably, the concentration of the anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to the nanomatrix is about 10 μL per 1 × 10 6 immune cells.

[0098] In some embodiments, the concentration of the anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to the nanomatrix is about 5 μL per 1 × 10 6 immune cells.

[0099] In some embodiments, the concentration of the anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to the nanomatrix is about 1 μL per 1 × 10 6 immune cells.

[0100] In some embodiments, the concentration of the anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to the nanomatrix is about 1 μL to about 5 μL per 1 × 10 6 immune cells.

[0101] In some embodiments, one or more steps of the method are automated. As used herein, the term "automated" refers to the automation of a method by using equipment and / or computers and computer software such that once the automated step / method is initiated, it requires no human intervention. In some embodiments, the method is automated.

[0102] In some embodiments, one or more steps of the method are performed in a closed sterile system. In some embodiments, the method is performed in a closed sterile system. A "closed sterile system" should be understood as a closed cell sample processing system that complies with international clinical cell processing standards.

[0103] In some embodiments, the immune cells are genetically modified to further express a DAP10 / DAP12 fusion polypeptide. The genetic modification to further express the DAP10 / DAP12 fusion polypeptide can be performed simultaneously with the genetic modification to express the NKG2D polypeptide, or before or after that.

[0104] In some embodiments, the method comprises:

[0105] (a) genetically modifying immune cells to express an NKG2D polypeptide and a DAP10 / DAP12 fusion polypeptide; wherein the immune cells have been activated by anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix; or

[0106] (b) activating immune cells, wherein the immune cells are genetically modified to express an NKG2D polypeptide and a DAP10 / DAP12 fusion polypeptide, and wherein activation is performed by anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix.

[0107] In some embodiments, the method comprises:

[0108] (i) genetically modifying the immune cells to express an NKG2D polypeptide and a DAP10 / DAP12 fusion polypeptide; and

[0109] (ii) activating the immune cells by using anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix.

[0110] Preferably, the method comprises:

[0111] (i) activating the immune cells by using anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix;

[0112] (ii) genetically modifying the immune cells to express an NKG2D polypeptide and a DAP10 / DAP12 fusion polypeptide.

[0113] The fusion polypeptide may have the following structural formula from the N-terminus to the C-terminus:

[0114] A - B - C - D - E,

[0115] wherein

[0116] A is an optional N - terminal sequence;

[0117] B is a DAP10 polypeptide;

[0118] C is an optional linker sequence;

[0119] D is a DAP12 polypeptide; and

[0120] E is an optional C - terminal sequence.

[0121] In some embodiments, the fusion polypeptide does not contain SEQ ID NO:84. In some embodiments, the fusion polypeptide does not contain an anti - EpCAM peptide. In some embodiments, the fusion polypeptide does not contain SEQ ID NO:85. In some embodiments, the fusion polypeptide does not contain SEQ ID NO:86. In some embodiments, the fusion polypeptide does not contain SEQ ID NO:85 and SEQ ID NO:86.

[0122] DAP10 polypeptide and its functional variants

[0123] The DAP10 polypeptide can be a mammalian polypeptide, such as a human polypeptide. Wild - type human DAP10 is encoded by the amino acid sequence (SEQ ID NO:1) of UniProt accession number: Q9UBK5. The polypeptide consists of 93 amino acids. The first 18 amino acids are regarded as the signal / leader sequence, the 19th to 48th amino acids are the extracellular domain, the 49th to 69th amino acids are the transmembrane domain, and the 70th to 93rd amino acids are the cytoplasmic / intracellular domain.

[0124] As used herein, the term "mammal" includes humans and non - humans, including but not limited to humans, non - human primates, canines, felines, murine, bovines, equines, and porcines.

[0125] In some embodiments, the DAP10 polypeptide is a functional variant of DAP10, which comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the DAP10 polypeptide shown in SEQ ID NO:1. In some embodiments, the DAP10 polypeptide comprises or consists of an amino acid sequence having the sequence shown in SEQ ID NO:1.

[0126] In the context of two or more nucleic acid or polypeptide sequences, the term "identity" percentage refers to two or more sequences or subsequences having a specified percentage of identical nucleotide or amino acid residues when compared and aligned for maximum correspondence using one of the following sequence comparison algorithms (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, the "identity" percentage may exist over a region of the sequences being compared, such as over a functional domain, or over the full length of the two sequences being compared.

[0127] When performing a sequence alignment, one sequence is typically designated as the reference sequence to which the test sequence is aligned. When using a sequence alignment algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. The sequence alignment algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the designated program parameters.

[0128] For purposes herein, the BLAST algorithm is used to calculate percent identity and sequence similarity, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0129] Truncated forms of the DAP10 polypeptide can also be used in the fusion polypeptides of the present invention. In some embodiments, the DAP10 polypeptide is a functional variant of the DAP10 polypeptide, which is a truncated form of the polypeptide having the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the truncated form of DAP10 contains only amino acids 19 to 93 of SEQ ID NO:1 (i.e., amino acids 1 to 18 are deleted, i.e., the signal / leader sequence). This sequence is referred to herein as SEQ ID NO:2. Other truncated forms may contain amino acids 19 to 69 of SEQ ID NO:1, which contains only the extracellular domain and transmembrane domain of DAP10, and is referred to herein as SEQ ID NO:3. Another truncated form of DAP10 for use in the present invention may contain amino acids 1 to 71 of SEQ ID NO:1 (i.e., the signal / leader sequence, extracellular domain, transmembrane domain, and 2 amino acids from the cytoplasmic / intracellular domain), and is referred to herein as SEQ ID NO:4. Another truncated form of DAP10 for use in the present invention may contain amino acids 19 to 71 of SEQ ID NO:1 (i.e., the extracellular domain, transmembrane domain, and 2 amino acids from the cytoplasmic / intracellular domain), and is referred to herein as SEQ ID NO:5. Another truncated form of DAP10 for use in the present invention may contain amino acids 70 to 93 of SEQ ID NO:1 (i.e., the intracellular domain), and is referred to herein as SEQ ID NO:6. Yet another truncated form of DAP10 for use in the present invention may contain amino acids 49 to 93 of SEQ ID NO:1 (i.e., the transmembrane domain and cytoplasmic / intracellular domain), and is referred to herein as SEQ ID NO:7. Yet another truncated form of DAP10 for use in the present invention may contain amino acids 49 to 69 of SEQ ID NO:1 (i.e., the transmembrane domain), and is referred to herein as SEQ ID NO:8.

[0130] In some embodiments, the truncated form of the DAP10 comprises or consists of amino acids 19 to 93, 19 to 69, 1 to 71, 19 to 71, 19 to 48, 49 to 69, 49 to 93, or 70 to 93 of SEQ ID NO:1.

[0131] Thus, in some embodiments, the DAP10 polypeptide comprises or consists of any one of the sequences of SEQ ID NOs: 1 to 8.

[0132] Those skilled in the art should understand that a functional variant is a variant of a wild-type protein that retains the functional activity of the wild-type protein. The variant should be understood as having an amino acid sequence that is altered from the amino acid sequence of the wild-type protein. Relative to the wild-type protein sequence, the variant may have at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more point mutations. The point mutations may include substitutions, deletions, or additions of amino acids.

[0133] In some embodiments, the functional variant of the DAP10 polypeptide retains at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or higher) of the activity of the wild-type polypeptide shown in SEQ ID NO:1. The activity can be measured by assessing tyrosine phosphorylation of DAP10 and / or the p85 subunit of phosphatidylinositol 3-kinase and the recruitment and activation of the downstream anti-apoptotic kinase AKT.

[0134] DAP12 polypeptide and its functional variants

[0135] The DAP12 polypeptide may be a mammalian polypeptide, such as a human polypeptide. The wild-type human DAP12 is encoded by the amino acid sequence (SEQ ID NO:9) of UniProt accession number: O43914. The first 21 amino acids are regarded as the signal / leader sequence, the amino acids at positions 22 to 40 are the extracellular domain, the amino acids at positions 41 to 61 are the transmembrane domain, and the amino acids at positions 62 to 113 are the cytoplasmic / intracellular domain.

[0136] In some embodiments, the DAP12 polypeptide is a functional variant of DAP12 that comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the DAP12 polypeptide having the sequence shown in SEQ ID NO:9. In some embodiments, the DAP12 polypeptide comprises or consists of an amino acid sequence having the sequence shown in SEQ ID NO:9.

[0137] Truncated forms of the DAP12 polypeptide can also be used in the fusion polypeptides of the present invention. Thus, in some embodiments, the DAP12 polypeptide is a truncated form of the DAP12 polypeptide having the amino acid sequence shown in SEQ ID NO:9. An exemplary truncated form of DAP12 contains only amino acids 22 to 113 of SEQ ID NO:9 (i.e., amino acids 1 to 21 are deleted, i.e., the signal / leader sequence). This sequence is referred to herein as SEQ ID NO:10. Other truncated forms may contain amino acids 62 to 113 of SEQ ID NO:9, which sequence contains only the cytoplasmic / intracellular domain of DAP12 and is referred to herein as SEQ ID NO:11. Other truncated forms may contain amino acids 41 to 61 of SEQ ID NO:9 (i.e., the transmembrane domain) and are referred to herein as SEQ ID NO:12. Another truncated form may contain amino acids 22 to 61 of SEQ ID NO:9 (i.e., the extracellular domain and the transmembrane domain) and is referred to herein as SEQ ID NO:13.

[0138] In some embodiments, the truncated form of DAP12 comprises or consists of amino acids 22 to 113, 62 to 113, 22 to 61, or 41 to 61 of SEQ ID NO:9.

[0139] In some embodiments, the DAP12 polypeptide comprises or consists of any one of the sequences of SEQ ID NOs:9 to 13.

[0140] Functional variants of the DAP12 polypeptide can retain at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or higher) of the activity of the wild-type polypeptide shown in SEQ ID NO:9. In some embodiments, functional assays can be used to measure the activity, such as the MTT assay and measurement of cytokine secretion by ELISA.

[0141] In some embodiments, the fusion polypeptide comprises or consists of full-length human DAP10, the C-terminus of which is fused to the intracellular domain of the human DAP12 polypeptide, wherein the intracellular domain is encoded by amino acids 62 to 113 of human DAP12. In some embodiments, the fusion polypeptide has the sequence shown in SEQ ID NO:60.

[0142] NKG2D polypeptide and its functional variants

[0143] The NKG2D polypeptide can be a mammalian polypeptide. In some embodiments, the NKG2D polypeptide is a human polypeptide. Wild-type human NKG2D is encoded by the amino acid sequence (SEQ ID NO:14) with UniProt accession number: P26718. The wild-type NKG2D polypeptide is considered to comprise a cytoplasmic domain (amino acids 1 to 51), a transmembrane domain (amino acids 52 to 72), and an extracellular domain (amino acids 73 to 216).

[0144] In some embodiments, the NKG2D polypeptide is a functional variant of NKG2D. The functional variant of NKG2D can comprise an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the NKG2D polypeptide having the sequence shown in SEQ ID NO:14. In some embodiments, the NKG2D polypeptide comprises or consists of SEQ ID NO:14.

[0145] The functional variant of NKG2D can comprise or consist of a truncated form of NKG2D. In some embodiments, the NKG2D polypeptide can be a truncated form of NKG2D that only comprises amino acids 73 to 216 of SEQ ID NO:14 (i.e., the extracellular domain). This sequence is referred to herein as SEQ ID NO:15. Other truncated forms can comprise amino acids 82 to 216 of SEQ ID NO:14, which sequence comprises a partial extracellular domain of NKG2D and is referred to herein as SEQ ID NO:16. Another truncated form comprises amino acids 52 to 216 of SEQ ID NO:14 (i.e., the transmembrane domain and the extracellular domain), which is referred to herein as SEQ ID NO:17.

[0146] The functional variant of the NKG2D polypeptide can retain at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or higher) of the activity of the wild-type polypeptide shown in SEQ ID NO:14. Activity can be measured by flow cytometry to confirm sustained binding to the NKG2D ligand, and can also be measured by a variety of cell culture assays (such as the MTT assay and the ELISA assay) to verify targeted T cell lysis, cytokine secretion, and co-stimulation.

[0147] In some embodiments, the functional variant of the NKG2D polypeptide is a chimeric NKG2D polypeptide. The chimeric NKG2D polypeptide can be a human-mouse chimeric polypeptide. The term "mouse" refers to rodents of the subfamily Murinae. The term "mouse" includes rats and mice.

[0148] The term "chimeric NKG2D polypeptide" refers to an NKG2D receptor composed of domains from two or more different organisms. The chimeric NKG2D polypeptide is described in more detail in WO2021 / 234163, the disclosure of which is incorporated herein by reference in its entirety.

[0149] In some embodiments, the chimeric NKG2D polypeptide comprises, in the N-terminal to C-terminal direction, a murine NKG2D transmembrane domain or a variant thereof and a human NKG2D extracellular domain or a variant thereof.

[0150] CXCR2 polypeptide and its functional variants

[0151] In some embodiments, immune cells are genetically modified to express a CXCR2 polypeptide. The genetic modification to express the CXCR2 polypeptide can be carried out simultaneously with the genetic modification to express the NKG2D polypeptide, or before or after that.

[0152] In some embodiments, the method comprises:

[0153] (a) genetically modifying immune cells to express an NKG2D polypeptide and a CXCR2 polypeptide; wherein the immune cells have been activated by anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix; or

[0154] (b) activating immune cells, wherein the immune cells are genetically modified to express an NKG2D polypeptide and a CXCR2 polypeptide, and wherein activation is carried out by anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix.

[0155] In some embodiments, the method comprises:

[0156] (i) genetically modifying the immune cells to express an NKG2D polypeptide and a CXCR2 polypeptide; and

[0157] (ii) activating the immune cells with anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix.

[0158] Preferably, the method comprises:

[0159] (i) activating the immune cells with anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix;

[0160] (ii) genetically modifying the immune cells to express an NKG2D polypeptide and a CXCR2 polypeptide.

[0161] In some embodiments, the method comprises:

[0162] (a) Genetically modifying immune cells to express NKG2D polypeptide, DAP10 / DAP12 fusion polypeptide, and CXCR2 polypeptide; wherein the immune cells have been activated by anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix; or

[0163] (b) Activating immune cells, wherein the immune cells have been genetically modified to express NKG2D polypeptide, DAP10 / DAP12 fusion polypeptide, and CXCR2 polypeptide, and wherein activation is carried out by anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix.

[0164] In some embodiments, the method comprises:

[0165] (i) Genetically modifying the immune cells to express NKG2D polypeptide, DAP10 / DAP12 fusion polypeptide, and CXCR2 polypeptide; and

[0166] (ii) Activating the immune cells using anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix.

[0167] Preferably, the method comprises:

[0168] (i) Activating the immune cells using anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix;

[0169] (ii) Genetically modifying the immune cells to express NKG2D polypeptide, DAP10 / DAP12 fusion polypeptide, and CXCR2 polypeptide.

[0170] Preferably, the CXCR2 polypeptide is a mammalian polypeptide. More preferably, the CXCR2 polypeptide is a human polypeptide. Wild-type human CXCR2 is encoded by the amino acid sequence (SEQ ID NO:87) of UniProt accession number: P25025.

[0171] In some embodiments, the CXCR2 polypeptide comprises a functional variant of the wild-type CXCR2 polypeptide. Thus, the CXCR2 polypeptide can be a functional variant of the CXCR2 polypeptide of SEQ ID NO:87. The functional variant of the CXCR2 polypeptide can comprise an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the CXCR2 polypeptide having the sequence shown in SEQ ID NO:87. In some embodiments, the CXCR2 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:87.

[0172] Functional variants of the CXCR2 polypeptide can retain at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or higher) of the activity of wild-type human CXCR2 (SEQ ID NO:87). The activity of the polypeptide can be measured by flow cytometry to confirm the binding of CXCR2 to a ligand (e.g., IL-8). In some embodiments, the activity of the polypeptide is measured by cell culture assays known in the art.

[0173] Linker

[0174] The DAP10 and DAP12 polypeptides in the fusion polypeptides described herein can bind directly to each other in a continuous polypeptide chain or indirectly to each other through a suitable linker. Thus, the DAP10 / 12 fusion polypeptide can contain a linker sequence.

[0175] The linker can be a peptide linker. Peptide linkers are commonly used in fusion polypeptides, and methods for selecting or designing linkers are well known in the art (see, e.g., Chen X et al., 2013, Adv. Drug Deliv. Rev. 65(10):1357–1369 and Wriggers W et al., 2005, Biopolymers 80:736–746). The linker can also be used to link the fusion polypeptide of the invention to another polypeptide (e.g., an NKG2D polypeptide and / or a CXCR2 polypeptide) in a chimeric construct as further described below.

[0176] Peptide linkers are generally classified into i) flexible linkers, ii) helix-forming linkers, and iii) cleavable linkers, and examples of each type are known in the art. In one example, a flexible linker is included in the fusion polypeptides described herein. Flexible linkers can contain mostly sterically unhindered amino acids such as glycine and alanine. The hydrophilic amino acid Ser is also commonly used in flexible linkers. Examples of flexible linkers include, but are not limited to: polyglycine (e.g., (Gly)4 and (Gly)5), polyalanine, poly(Gly-Ala), and poly(Gly-Ser) (e.g., (Gly n -Ser n ) n or (Ser n -Gly n ) n , where each n is independently an integer equal to or greater than 1).

[0177] The peptide linker can be of a suitable length. The length of the peptide linker sequence can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more amino acid residues. For example, the length of the peptide linker can be about 5 to about 50 amino acids; about 10 to about 40 amino acids; about 15 to about 30 amino acids; or about 15 to about 20 amino acids. Altering the length of the peptide linker can preserve or enhance activity, thus resulting in better efficacy in activity studies. The peptide linker sequence can consist of natural or unnatural amino acids, or a mixture of natural and unnatural amino acids.

[0178] In some embodiments, the linker sequence comprises or consists of glycine and serine. For example, the linker region can comprise a set of glycine repeats (GSG3) n (SEQ ID NO:18), where n is a positive integer equal to or greater than 1 (e.g., 1 to about 20). More specifically, the linker sequence can be GSGGG (SEQ ID NO:19). The linker sequence can be GSGG (SEQ ID NO:20). In certain other embodiments, the linker region direction comprises a set of glycine repeats (SerGly3) n (SEQ ID NO:21), where n is a positive integer equal to or greater than 1 (e.g., 1 to about 20).

[0179] In other embodiments, the linker can comprise glycine (G) and serine (S) arranged in a random or repeating pattern. For example, the linker can be (GGGGS) n (SEQ ID NO:22), where n is an integer from 1 to 20, such as 1 to 4. In one specific instance, n is 4 and the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:23). In another specific instance, n is 3 and the linker is GGGGSGGGGSGGGGS (SEQ ID NO:24).

[0180] In other embodiments, the linker can comprise glycine (G), serine (S) and proline (P) arranged in a random or repeating pattern. For example, the linker can be (GPPGS) n , where n is an integer from 1 to 20, such as 1 to 4. In one specific instance, n is 1 and the linker is GPPGS (SEQ ID NO:25).

[0181] Generally, when administered to a patient (e.g., a human), the linker does not produce immunogenicity. Thus, a linker with low immunogenicity or considered to have low immunogenicity can be selected.

[0182] The linkers described herein are exemplary and, if desired, the linker may further comprise other amino acids such as Glu and Lys. If desired, the peptide linker may comprise multiple repeats, such as (G3S) (SEQ ID NO:26), (G4S) (SEQ ID NO:27), (GYS) (SEQ ID NO:28), and / or (GlySer) (SEQ ID NO:29). The peptide linker may comprise multiple repeats such as (SG4) (SEQ ID NO:30), (SG3) (SEQ ID NO:31), (SG2) (SEQ ID NO:32), (SG)2 (SEQ ID NO:33), or (SerGly) (SEQ ID NO:34).

[0183] The peptide linker may comprise combinations and multiples of repeating amino acid sequence units, such as (G3S)+(G4S)+(GlySer) (SEQ ID NO:26+SEQ ID NO:27+SEQ ID NO:29). Alternatively, Ser may be replaced with Ala, such as (G4A) (SEQ ID NO:35) or (G3A) (SEQ ID NO:36). In other embodiments, the linker comprises the motif (EAAAK) n (SEQ ID NO:37), where n is a positive integer equal to or greater than 1, such as 1 to about 20. In certain embodiments, the peptide linker further comprises a cleavable linker.

[0184] The linker may comprise other domains and / or features, such as a furin cleavage site (RRKR) (SEQ ID NO:38), a P2A ribosome skipping peptide (ATNFSLLKQAGDVEENPGP) (SEQ ID NO:39), and / or a T2A ribosome skipping peptide (EGRGSLLTCGDVEENPGP) (SEQ ID NO:40). Examples of linkers comprising these domains include SGSG + P2A ribosome skipping peptide (SGSGATNFSLLKQAGDVEENPGP) (SEQ ID NO:41), SGSG + T2A ribosome skipping peptide (SGSGEGRGSLLTCGDVEENPGP) (SEQ ID NO:42), and forms of the furin cleavage site, namely furin cleavage site + SGSG + P2A ribosome skipping peptide (RRKRSGSGATNFSLLKQAGDVEENPGP) (SEQ ID NO:43) and furin cleavage site + SGSG + T2A ribosome skipping peptide (RRKRSGSGEGRGSLLTCGDVEENPGP) (SEQ ID NO:44). Alternative ribosome skipping peptides that can be used in the present invention include F2A (VKQTLNFDLLKLAGDVESNPGP) (SEQ ID NO:45) and E2A (QCTNYALLKLAGDVESNPGP) (SEQ ID NO:46).

[0185] N-terminal sequence and C-terminal sequence

[0186] A variety of sequences can be linked to the N-terminus or C-terminus of the fusion polypeptides, NKG2D polypeptides, and / or CXCR2 polypeptides disclosed herein. These sequences can be functional, such as signal peptides, purification tags / sequences, or half-life extension moieties, or can simply comprise spacer sequences. Alternatively, these sequences can comprise another function, such as T cell stimulation function.

[0187] Purification tag and marker

[0188] A variety of tags or markers can be linked to the N-terminus or C-terminus of the fusion polypeptide, NKG2D polypeptide, and / or CXCR2 polypeptide to assist in purification. For example, the tag can include an affinity tag. Examples of such affinity tags include His tag, FLAG tag, Arg tag, T7 tag, Strep tag, S tag, aptamer tag, V5 tag, AviTagTM, myc epitope tag, or any combination of these tags. In some embodiments, the affinity tag is a His tag (usually containing 5 to 10 histidine residues), such as a 6His tag (i.e., HHHHHH) (SEQ ID NO:47). In some embodiments, the affinity tag is a FLAG tag (i.e., DYKDDDDK) (SEQ ID NO:48). In some embodiments, the affinity tag is AviTagTM (i.e., GLNDIFEAQKIEWHE) (SEQ ID NO:49). In some embodiments, the affinity tag is a V5 tag (GKPIPNPLLGLDST) (SEQ ID NO:50) or (IPNPLLGLD) (SEQ ID NO:51). In some embodiments, the affinity tag is a myc epitope tag (EQKLISEEDL) (SEQ ID NO:52) recognized by the 9e10 antibody. A variety of other tags are suitable for use and are well known in the art.

[0189] Combinations of such affinity tags can also be used, which may contain one or more tags at the N-terminus, one or more tags at the C-terminus, or one or more tags at both the N-terminus and C-terminus. Examples of such combinations include the combination of His tag (H) and AviTag (A), or the combination of His tag (H), AviTag (A), and FLAG tag (F). These tags can be in any orientation, so the orientation of AviTag / His tag can be N-AH-C or N-HA-C, and the orientation of Avi / His / FLAG tag can be N-AHF-C, N-FHA-C, etc.

[0190] For example, the fusion polypeptide can contain an "AHF" tag with the sequence "GLNDIFEAQKIEWHEGGHHHHHHDYKDDDDK" (SEQ ID NO:53). Alternatively, the fusion polypeptide can contain an "FHA" tag with the sequence "DYKDDDDKHHHHHHGGGLNDIFEAQKIEWHE" (SEQ ID NO:54).

[0191] The CD8α leader sequence (amino acids 1 to 21 of UniProt accession number P01732 or a truncated derivative containing amino acids 1 to 18) is a commonly used T cell sequence, referred to herein as SEQ ID NO:55. Thus, the fusion polypeptide, NKG2D polypeptide, and / or CXCR2 polypeptide may comprise SEQ ID NO:55. In some embodiments, the N-terminus of the fusion polypeptide, NKG2D polypeptide, and / or CXCR2 polypeptide comprises SEQ ID NO:55.

[0192] Costimulatory sequence

[0193] It is known from previous studies that various T cell co-stimulatory activation sequences can be used to design CAR-T cells. The fusion polypeptide, NKG2D polypeptide, and / or CXCR2 polypeptide may also comprise or be fused with a T cell co-stimulatory activation sequence.

[0194] The 4-1BB intracellular domain (amino acids 214 to 255 of UniProt accession number Q07011) can also be used as an N-terminal or C-terminal sequence. The 4-1BB intracellular domain is referred to herein as SEQ ID NO:56. The 4-1BB intracellular domain can serve as a co-stimulatory domain.

[0195] The CD27 intracellular domain (amino acids 213 to 260 of UniProt accession number P26842) can also be used as an N-terminal or C-terminal sequence. The CD27 intracellular domain is referred to herein as SEQ ID NO:57. The CD27 intracellular domain can serve as a co-stimulatory domain.

[0196] The human IgG1 hinge region (amino acids 218 to 229 or 218 to 232 of UniProt accession number P0DOX5) can also be used as an N-terminal or C-terminal sequence. The human IgG1 hinge region is referred to as SEQ ID NO:58 or SEQ ID NO:104.

[0197] The truncated CD8α hinge region (amino acids 138 to 182 of UniProt accession number P01732) can also be used as an N-terminal or C-terminal sequence. The truncated CD8α hinge region is referred to as SEQ ID NO:59.

[0198] In embodiments where the cell is genetically modified to further express a DAP10 / 12 fusion polypeptide, the NKG2D polypeptide and the DAP10 / 12 fusion polypeptide can be genetically encoded as part of a contiguous chimeric construct. The fusion polypeptide and the NKG2D polypeptide can then be separated during translation (e.g., using a ribosome skipping peptide) or by post-translational cleavage (e.g., using a furin protease cleavage site). Accordingly, the fusion polypeptide and the NKG2D polypeptide can be linked by an optional linker. Such a linker can contain a cleavage site to facilitate cleavage.

[0199] In embodiments where the cell is genetically modified to further express a CXCR2 polypeptide, the NKG2D polypeptide and the CXCR2 polypeptide can be genetically encoded as part of a contiguous chimeric construct. The CXCR2 polypeptide and the NKG2D polypeptide can then be separated during translation (e.g., using a ribosome skipping peptide) or by post-translational cleavage (e.g., using a furin protease cleavage site). Accordingly, the CXCR2 polypeptide and the NKG2D polypeptide can be linked by an optional linker. Such a linker can contain a cleavage site to facilitate cleavage.

[0200] In embodiments where the cell is genetically modified to further express a CXCR2 polypeptide and a DAP10 / 12 fusion polypeptide, the NKG2D polypeptide, the DAP10 / 12 fusion polypeptide, and the CXCR2 polypeptide can be genetically encoded as part of a contiguous chimeric construct. The CXCR2 polypeptide, the DAP10 / 12 fusion polypeptide, and the NKG2D polypeptide can then be separated during translation (e.g., using a ribosome skipping peptide) or by post-translational cleavage (e.g., using a furin protease cleavage site).

[0201] Exemplary construct

[0202] The present invention provides the following exemplary fusion polypeptide constructs in Table 1:

[0203] Table 1: Exemplary DAP10 / DAP12 Fusion Polypeptide Constructs

[0204]

[0205] In embodiments comprising a fusion polypeptide, as described above, the fusion polypeptide can be expressed as a single chimeric construct with NKG2D and optionally CXCR2 polypeptides for translational or post-translational cleavage. In such constructs, after expression, the translated polypeptide is cleaved to produce separate polypeptides which then self-assemble to form a CAR. In some embodiments, the fusion polypeptide is cleaved from the NKG2D polypeptide. Examples of such constructs are shown in Table 2:

[0206] Table 2: Exemplary Chimeric Constructs

[0207]

[0208]

[0209] Gene modification

[0210] As used herein, "gene modification" shall be understood to mean the introduction of an exogenous polynucleotide into a cell or an organism such that the protein encoded by the exogenous polynucleotide can be expressed in the cell.

[0211] Gene modification may include or consist of transfection or transduction. As used herein, the term "transfection" includes a variety of techniques commonly used to introduce exogenous DNA into cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like.

[0212] The exogenous polynucleotide may be DNA or RNA. The polynucleotide may encode a polypeptide sequence shown in any one or more of SEQ ID NOs: 60 to 69, 87, 90, and 102.

[0213] In some embodiments, the polynucleotide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity with any polynucleotide sequence encoding SEQ ID NOs: 60 to 69, 87, 90, and 102. Sequence identity is typically determined along the full length of the reference sequence.

[0214] The polynucleotide may have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity with any sequence of SEQ ID NOs: 70 to 79, 88, 91, and 103. In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence shown in any one of SEQ ID NOs: 70 to 79, 88, 91, and 103.

[0215] Unless otherwise specifically defined herein, the term "polynucleotide" encompasses polynucleotides containing known analogs of natural nucleotides which have similar properties to the reference polynucleotide and are metabolized in a manner similar to natural nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequences explicitly recited. Specifically, as described below, degenerate codon substitutions may be achieved by generating sequences in which one or more selected (or all) codons' third positions are substituted with mixed bases and / or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985, J. Biol. Chem. 260:2605-2608; and Rossolini et al., 1994, Mol. Cell. Probes 8:91-98).

[0216] Polynucleotide sequences can be generated by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences (such as those described in the examples below). The direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68:109; the diethylphosphoramidite method of Beaucage et al., 1981, Tetra. Lett., 22:1859; and the solid-phase support method of U.S. Patent No. 4,458,066. Introduction of mutations into polynucleotide sequences by PCR can be carried out according to the methods described in the following references, such as PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif, 1990; Mattila et al., 1991, Nucleic Acids Res. 19:967; and Eckert et al., 1991, PCR Methods and Applications 1:17.

[0217] Preferably, the genetic modification comprises or consists of transduction. Generally, transduction involves the introduction of an exogenous polynucleotide into a cell by a virus or viral vector. Thus, the genetic modification can include delivery of a vector encoding an NKG2D polypeptide (and optionally other polypeptides disclosed) into the cell.

[0218] A variety of vectors can be used to express the polynucleotides encoding the polypeptides of the invention. Both viral expression vectors and non-viral expression vectors can be used to produce polypeptides in immune cells. Non-viral vectors and systems include plasmids, episomal vectors (usually with an expression cassette for expressing proteins or RNAs), and human artificial chromosomes (see, e.g., Harrington et al., 1997, Nat Genet. 15:345). For example, useful non-viral vectors for expressing the polynucleotides and polypeptides of the invention in mammalian (e.g., human) cells include pThioHis A, B, and C, pcDNA3.1 / His, pEBVHis A, B, and C (Invitrogen, San Diego, California), MPS V vectors, and many other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and vectors based on SV40, papillomavirus, HBP Epstein-Barr virus, vaccinia virus, and Semliki Forest virus (SFV). See Brent et al., supra; Smith, 1995, Annu. Rev. Microbiol. 49:807; and Rosenfeld et al., 1992, Cell 68:143. Specifically, retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors are generally used to express in T cells. Examples of such vectors include the SFG retroviral expression vector (see Riviere et al., 1995, Proc. Natl. Acad. Sci. (USA) 92:6733-6737).

[0219] Preferably, the genetic modification includes delivery of a viral vector encoding an NKG2D polypeptide (and optionally other polypeptides disclosed) into the cell. More preferably, the viral vector is a lentiviral vector or a retroviral vector. The lentiviral vector can include a self-inactivating lentiviral vector (i.e., a SIN vector). The retroviral vector can include the SFG retroviral expression vector.

[0220] Vectors may contain expression control sequences such as origins of replication, promoters and enhancers (see, e.g., Queen, et al., 1986, Immunol. Rev. 89:49-68), as well as necessary processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. These vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific and / or regulatable or controllable. Useful promoters include, but are not limited to, metallothionein promoter, constitutive adenovirus major late promoter, dexamethasone-inducible MMTV promoter, SV40 promoter, MRP polIII promoter, constitutive MPS V promoter, tetracycline-inducible CMV promoter (e.g., human immediate early CMV promoter), constitutive CMV promoter, EF1α promoter, phosphoglycerate kinase (PGK) promoter, and promoter-enhancer combinations known in the art.

[0221] In some embodiments, the vector contains a polynucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% sequence identity with any of the polynucleotides encoding SEQ ID NOs: 60 to 69, 87, 90 and 102. In some embodiments, the vector contains a polynucleotide sequence encoding one or more of SEQ ID NOs: 60 to 69, 87, 90 and 102. In some embodiments, the vector contains a polynucleotide sequence as shown in any of SEQ ID NOs: 70 to 79, 88, 91 and 103.

[0222] Immune cell

[0223] Those skilled in the art should understand that immune cells refer to cells of the immune system. In other words, immune cells should be understood as cells capable of responding to infectious organisms. Any such immune cells are suitable for the present invention.

[0224] Exemplary immune cells may include, but are not necessarily limited to, neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells and lymphocytes.

[0225] Those skilled in the art should understand that lymphocytes include B cells and T cells.

[0226] In some embodiments, the immune cell is a neutrophil, macrophage, dendritic cell, natural killer cell or lymphocyte.

[0227] In some embodiments, the immune cells are B cells, T cells or natural killer cells. Preferably, the immune cells are T cells or natural killer (NK) cells.

[0228] More preferably, the immune cells are T cells. T cells include CD4 T cells, CD8 T cells and NKT cells.

[0229] In some embodiments, the T cells are CD4 + or CD8 + T cells. In some embodiments, the immune cells are CD4 + T cells. In other embodiments, the immune cells are CD8 + T cells.

[0230] In some embodiments, the immune cells are αβ or γδ T cells. In some embodiments, the immune cells are αβ T cells. In other embodiments, the immune cells are γδ T cells.

[0231] In some embodiments, the immune cells are peripheral blood mononuclear cells (PBMCs). As described above, after activation and genetic modification, the PBMCs can differentiate into another immune cell as described above. For example, after activation and genetic modification, the PBMCs can differentiate into T cells.

[0232] Exemplary method of the present invention

[0233] Preferably, the method is a method for preparing immune-responsive T cells or NK cells, comprising:

[0234] (i) activating PBMCs with anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix; and

[0235] (ii) genetically modifying the PBMCs to express NKG2D polypeptide, DAP10 / DAP12 fusion polypeptide and CXCR2 polypeptide, wherein the genetic modification comprises transducing the PBMCs with a retroviral vector encoding NKG2D polypeptide, DAP10 / DAP12 fusion polypeptide and CXCR2 polypeptide.

[0236] In some embodiments, the method is a method for preparing immune-responsive T cells or NK cells, comprising:

[0237] (i) activating PBMCs with anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix such that the PBMCs differentiate into T cells or NK cells; and

[0238] (ii) Genetically modify the T cells or NK cells to express NKG2D polypeptide, DAP10 / DAP12 fusion polypeptide and CXCR2 polypeptide, wherein the genetic modification includes transducing the T cells or NK cells with a retroviral vector encoding NKG2D polypeptide, DAP10 / DAP12 fusion polypeptide and CXCR2 polypeptide.

[0239] In some embodiments, the method is a method for preparing immune-responsive T cells or NK cells, comprising:

[0240] (i) Activate PBMCs with anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix, such that the PBMCs differentiate into T cells or NK cells; wherein the nanomatrix comprises iron oxide crystals embedded in a biocompatible polysaccharide matrix and has a diameter of about 100 nm; and

[0241] (ii) Genetically modify the T cells or NK cells to express NKG2D polypeptide, DAP10 / DAP12 fusion polypeptide and CXCR2 polypeptide, wherein the genetic modification includes transducing the T cells or NK cells with a retroviral vector encoding NKG2D polypeptide, DAP10 / DAP12 fusion polypeptide and CXCR2 polypeptide.

[0242] In some embodiments, the method is a method for preparing immune-responsive T cells or NK cells, comprising:

[0243] (i) Activate PBMCs with anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix, such that the PBMCs differentiate into T cells or NK cells; wherein the nanomatrix is in colloidal form and comprises iron oxide crystals embedded in a biocompatible polysaccharide matrix and has a diameter of about 100 nm; and

[0244] (ii) Genetically modify the T cells or NK cells to express NKG2D polypeptide, DAP10 / DAP12 fusion polypeptide and CXCR2 polypeptide, wherein the genetic modification includes transducing the T cells or NK cells with a retroviral vector encoding NKG2D polypeptide, DAP10 / DAP12 fusion polypeptide and CXCR2 polypeptide.

[0245] According to another aspect, there is provided a method for preparing immune-responsive cells, wherein the method comprises:

[0246] (a) Activate immune cells, wherein the activation does not include phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA) or magnetic beads; and

[0247] (b) Genetically modify the immune cells to express the NKG2D polypeptide.

[0248] The order of activation and genetic modification can be defined as above. Similarly, the genetic modification and immune cells can be defined as above. For example, the genetic modification can include transduction. The immune cells can be PBMCs. In some embodiments, the immune cells are T cells or neutrophils.

[0249] The genetic modification can include genetically modifying immune cells to express an NKG2D polypeptide and a DAP10 / DAP12 fusion polypeptide. In some embodiments, the genetic modification includes genetically modifying immune cells to express an NKG2D polypeptide and a CXCR2 polypeptide. In some embodiments, the genetic modification includes genetically modifying immune cells to express an NKG2D polypeptide, a DAP10 / DAP12 fusion polypeptide, and a CXCR2 polypeptide.

[0250] Immune response cell

[0251] The present invention also provides an immune response cell obtainable by any method of the present invention.

[0252] According to another aspect, there is also provided an immune response cell genetically modified to express an NKG2D polypeptide; wherein the immune response cell has been activated by anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix.

[0253] In some embodiments, the immune response cell is genetically modified to express an NKG2D polypeptide and a fusion polypeptide comprising: (i) a DNAX activation protein 10 (DAP10) polypeptide or a functional variant thereof, and (ii) a DNAX activation protein 12 (DAP12) polypeptide or a functional variant thereof. The NKG2D polypeptide can be defined as above. The fusion polypeptide can be defined as above.

[0254] In some embodiments, the DAP10 polypeptide is a functional variant of DAP10 that comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the DAP10 polypeptide shown in SEQ ID NO:1. In some embodiments, the DAP10 polypeptide comprises or consists of an amino acid sequence having the sequence shown in SEQ ID NO:1.

[0255] In some embodiments, the DAP10 polypeptide is a truncated form of DAP10 that comprises or consists of amino acids 19 to 93, 19 to 69, 1 to 71, 19 to 71, 19 to 48, 49 to 69, 49 to 93, or 70 to 93 of SEQ ID NO:1.

[0256] In some embodiments, the DAP10 polypeptide comprises or consists of any one of the sequences of SEQ ID NOs: 1 to 8.

[0257] In some embodiments, the DAP12 polypeptide is a functional variant of DAP12, which comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity with the DAP12 polypeptide having the sequence shown in SEQ ID NO: 9. In some embodiments, the DAP12 polypeptide comprises or consists of the amino acid sequence having the sequence shown in SEQ ID NO: 9.

[0258] In some embodiments, the DAP12 polypeptide is a truncated form of DAP12, which comprises or consists of the amino acids at positions 22 to 113, 62 to 113, 22 to 61 or 41 to 61 of SEQ ID NO: 9.

[0259] In some embodiments, the DAP12 polypeptide comprises or consists of any one of the sequences of SEQ ID NOs: 9 to 13.

[0260] In some embodiments, the NKG2D polypeptide is a human polypeptide. In some embodiments, the NKG2D polypeptide is a functional variant of NKG2D. The functional variant of NKG2D may comprise an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity with the NKG2D polypeptide having the sequence shown in SEQ ID NO: 14. In some embodiments, the NKG2D polypeptide comprises or consists of SEQ ID NO: 14.

[0261] The functional variant of NKG2D may comprise or consist of a truncated form of NKG2D. In some embodiments, the NKG2D polypeptide may be a truncated form of NKG2D, which only comprises the amino acids at positions 73 to 216 of SEQ ID NO: 14 (i.e., the extracellular domain). This sequence is referred to as SEQ ID NO: 15 herein. Other truncated forms may comprise the amino acids at positions 82 to 216 of SEQ ID NO: 14, which sequence comprises a partial extracellular domain of NKG2D and is referred to as SEQ ID NO: 16 herein. Another truncated form comprises the amino acids at positions 52 to 216 of SEQ ID NO: 14 (i.e., the transmembrane domain and the extracellular domain), which is referred to as SEQ ID NO: 17 herein.

[0262] In some embodiments, the functional variant of the NKG2D polypeptide is a chimeric NKG2D polypeptide. The chimeric NKG2D polypeptide can be a human-mouse chimeric polypeptide.

[0263] In some embodiments, the chimeric NKG2D polypeptide comprises, in the direction from the N-terminus to the C-terminus, a murine NKG2D transmembrane domain or a variant thereof, and a human NKG2D extracellular domain or a variant thereof.

[0264] In some embodiments, immune cells are genetically modified to further express a CXCR2 polypeptide. Thus, in some embodiments, the immune response cells are genetically modified to express an NKG2D polypeptide and a CXCR2 polypeptide. In some embodiments, the immune response cells are genetically modified to express an NKG2D polypeptide, a CXCR2 polypeptide, and a DAP10 / DAP12 fusion polypeptide as defined above.

[0265] The CXCR2 polypeptide can be as defined above. For example, the CXCR2 polypeptide can be a mammalian polypeptide. The CXCR2 polypeptide can be a human polypeptide.

[0266] In some embodiments, the CXCR2 polypeptide comprises a functional variant of the wild-type CXCR2 polypeptide. Thus, the CXCR2 polypeptide can be a functional variant of the CXCR2 polypeptide of SEQ ID NO:87. The functional variant of the CXCR2 polypeptide can comprise an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the CXCR2 polypeptide having the sequence shown in SEQ ID NO:87. In some embodiments, the CXCR2 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:87.

[0267] The immune response cells are immune cells. The immune response cells can include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, or lymphocytes. In some embodiments, the immune cells are neutrophils, macrophages, dendritic cells, natural killer cells, or lymphocytes.

[0268] Preferably, the immune response cells are lymphocytes or natural killer (NK) cells. Thus, the immune response cells can be T cells, B cells, or NK cells. Preferably, the immune response cells are T cells or natural killer (NK) cells. In some embodiments, the immune response cells are T cells. In some embodiments, the T cells are CD4 + or CD8 + T cells. In some embodiments, the immune response cells are CD4+ T cells. In other embodiments, the immune response cells are CD8 + T cells.

[0269] In some embodiments, the immune response cells are αβ or γδ T cells. In some embodiments, the immune response cells are αβ T cells. In other embodiments, the immune response cells are γδ T cells.

[0270] In some embodiments, the immune response cells are peripheral blood mononuclear cells (PBMCs). Alternatively, the immune response cells can be derived from PBMCs. In the context of the present invention, "derived from PBMCs" should be understood as cells that have differentiated from PBMCs.

[0271] Preferably, the immune response cells comprise a population of immune response cells. More preferably, the immune response cells comprise a population of T cells or a population of NK cells. Most preferably, the immune response cells comprise a population of T cells. The population of T cells can comprise CD4 + T cells. The population of T cells can comprise CD8 + T cells. In some embodiments, the population of T cells comprises CD4 + T cells and CD8 + T cells.

[0272] The inventors unexpectedly found that, compared with immune response cells activated with other activation stimulants, immune response cells activated with anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to the nanomatrix of the present invention have a higher amplification multiple and cell population number.

[0273] Thus, in some embodiments, the cell population comprises at least about 1×10 5 immune response cells, at least about 5×10 5 immune response cells, at least about 1×10 6 immune response cells, at least about 5×10 6 immune response cells, at least about 1×10 7 immune response cells, at least about 5×10 7 immune response cells, at least about 1×10 8 immune response cells, at least about 5×10 8 immune response cells, at least about 1×10 9 immune response cells, at least about 5×10 9 immune response cells, at least about 1×10 10 immune response cells, at least about 5×10 10 immune response cells or at least about 1×10 11 immune response cells.

[0274] In some embodiments, the cell population comprises at least about 1×10 6 immune-responsive cells.

[0275] In some embodiments, the immune-responsive cell population has an expansion rate of at least about 5-fold every ten days. In some embodiments, the immune-responsive cell population has an expansion rate of at least about 10-fold every ten days. In some embodiments, the immune-responsive cell population has an expansion rate of at least about 20-fold every ten days. In some embodiments, the immune-responsive cell population has an expansion rate of at least about 30-fold every ten days. In some embodiments, the immune-responsive cell population has an expansion rate of at least about 40-fold every ten days. In some embodiments, the immune-responsive cell population has an expansion rate of at least about 50-fold every ten days. In some embodiments, the immune-responsive cell population has an expansion rate of at least about 60-fold every ten days. In some embodiments, the immune-responsive cell population has an expansion rate of at least about 70-fold every ten days. In some embodiments, the immune-responsive cell population has an expansion rate of at least about 80-fold every ten days. In some embodiments, the immune-responsive cell population has an expansion rate of at least about 90-fold every ten days. In some embodiments, the immune-responsive cell population has an expansion rate of at least about 100-fold every ten days.

[0276] Preferably, the immune-responsive cells comprise a population of T cells that has an expansion rate of at least about 5-fold every ten days. More preferably, the immune-responsive cells comprise a population of T cells that has an expansion rate of at least about 10-fold every ten days. Most preferably, the immune-responsive cells comprise a population of T cells that has an expansion rate of at least about 20-fold every ten days.

[0277] The inventors also found that activating T cells with anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to the nanomatrix of the present invention results in an unexpected increase in CD4 + T cells compared to T cells activated with other stimulants. Thus, in some embodiments, the CD4 to CD8 ratio of the T cell population is at least about 1:1. In some embodiments, the CD4 to CD8 ratio of the T cell population is at least about 2:1. Alternatively, the CD4 to CD8 ratio of the T cell population can be at least about 3:1, optionally about 4:1.

[0278] Pharmaceutical composition

[0279] The present invention also provides a pharmaceutical composition comprising immune-responsive cells obtainable by any of the methods disclosed herein or immune-responsive cells of the above aspects, and a pharmaceutically or physiologically acceptable diluent and / or carrier.

[0280] As used herein, the term "pharmaceutical composition" refers to a combination of an active ingredient and an inert or active carrier such that the composition is particularly suitable for in vitro, in vivo or ex vivo therapeutic or diagnostic use.

[0281] As used herein, the term "pharmaceutically acceptable" or "pharmacologically acceptable" refers to a composition that, when administered to a subject, does not substantially produce adverse reactions such as toxic, allergic or immunological reactions.

[0282] The carrier is usually selected to be suitable for the intended mode of administration and may include agents for modifying, maintaining or preserving the composition, such as pH, osmotic pressure, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorptivity or permeability. Generally, these carriers include aqueous solutions or alcohol / aqueous solutions, emulsions or suspensions, including physiological saline and / or buffer media.

[0283] Agents suitable for inclusion in pharmaceutical compositions include, but are not limited to: amino acids (such as glycine, glutamine, asparagine, arginine or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite), buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates or other organic acids), fillers (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides and other carbohydrates (such as glucose, mannose or dextrin), proteins (such as free serum albumin, gelatin or immunoglobulins), coloring agents, flavoring agents and diluents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl paraben, propyl paraben, chlorhexidine, sorbic acid or hydrogen peroxide), solvents (such as glycerol, propylene glycol or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as poloxamer; PEG; sorbitan esters; polysorbates, such as polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapol), stability enhancers (such as sucrose or sorbitol), osmotic pressure enhancers (such as alkali metal halides, such as sodium chloride or potassium chloride, or mannitol sorbitol), delivery carriers, diluents, excipients and / or pharmaceutical adjuvants.

[0284] Parenteral carriers include sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, and lactated Ringer's solution. Suitable physiologically acceptable thickening agents may be included, such as carboxymethyl cellulose, polyvinylpyrrolidone, gelatin, and alginates. Intravenous injection carriers include fluid and nutritional supplements as well as electrolyte supplements, such as Ringer's dextrose-based supplements. In certain cases, agents for adjusting the osmotic pressure of the composition may be added to the pharmaceutical composition, such as sugars, polyols like mannitol and sorbitol, or sodium chloride. For example, in many cases, an essentially isotonic composition is desired. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases. The exact formulation depends on the route of administration. Other relevant principles, methods, and components of pharmaceutical formulations are well known (see, for example, Allen, Loyd V. Ed., (2012) Remington’s Pharmaceutical Sciences, 22nd Edition).

[0285] The pharmaceutical compositions of the present invention can be administered by one or more of the various methods known in the art via one or more routes of administration. Those skilled in the art will understand that the route of administration and / or mode will vary depending on the desired results. Routes of administration of the pharmaceutical compositions of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. As used herein, the term "parenteral administration" refers to a route of administration other than enteral and topical administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. In one embodiment, the pharmaceutical composition is administered by intratumoral injection. When parenteral administration is contemplated, the pharmaceutical composition is generally in the form of a sterile, pyrogen-free, and parenterally acceptable composition. A particularly suitable carrier for parenteral injection is a properly preserved sterile isotonic solution. The pharmaceutical composition may be in the form of a lyophilizate, such as a lyophilized cake.

[0286] Alternatively, the pharmaceutical compositions described herein can be administered by non-parenteral routes, such as topical, epidermal, or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical administration.

[0287] In some embodiments, the pharmaceutical composition is for subcutaneous administration. Suitable formulation ingredients and methods for polypeptide therapeutic agents (such as antibodies, fusion polypeptides, etc.) for subcutaneous administration are known in the art, see, for example, US2011 / 0044977, US8465739, and US8476239. Generally, pharmaceutical compositions for subcutaneous administration contain suitable stabilizers (such as amino acids like methionine, and / or sugars like sucrose), buffers, and tonicity agents.

[0288] Preferably, in cell therapy, the pharmaceutical composition comprising immune response cells is administered to a subject by intravenous infusion.

[0289] The pharmaceutically useful compositions of the present invention are preferably administered in a "therapeutically effective amount" or "prophylactically effective amount", which is sufficient to confer a benefit to an individual. The actual dosage administered, the dosing rate, and the dosing time depend on the nature and severity of the protein aggregation disease being treated. The prescription of a treatment regimen, such as the determination of dosage, etc., is the responsibility of general practitioners and other physicians and usually takes into account the disease being treated, the condition of the individual patient, the site of administration, the method of administration, and other factors known to the physician. Examples of such techniques and regimens can be found in Remington’s Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0290] The pharmaceutical composition can be administered alone or in combination with other treatment methods, either simultaneously or sequentially, depending on the condition being treated.

[0291] As used herein, the terms "administer" and "administration" refer to the act of giving a drug, prodrug, or other agent or treatment method (such as a peptide) to a subject or to cells, tissues, and organs in vivo, in vitro, or ex vivo. Exemplary routes of administration into the human body can be through the subarachnoid space of the brain or spinal cord (intrathecal), the eye (intraocular), the mouth (oral), the skin (topical or transdermal), the nose (nasal), the lung (inhalation), the oral mucosa (buccal or sublingual), the ear, the rectum, the vagina, by injection (such as intravenous, subcutaneous, intratumoral, intraperitoneal, etc.).

[0292] Kit

[0293] The present invention also provides a kit, which comprises immune response cells obtainable by the methods disclosed herein, the immune response cells of the present invention, or the pharmaceutical composition of the present invention.

[0294] The kit may further comprise instructions for use. In some embodiments, the immune response cells and / or the pharmaceutical composition in the kit are provided in an aqueous solution, optionally as a buffered solution and / or at a temperature not lower than -20 °C.

[0295] The system components or embodiments described herein may be provided in the form of a kit. For example, any vector, mammalian cells, associated buffers, media, triggers, or other components related to cell culture and virion production may be provided, where the optional components are frozen and packaged into a kit, which may be provided alone or together with separate containers of any other agent and optional instructions for use. In some embodiments, the kit may include culture vessels, vials, test tubes, etc.

[0296] Therapeutic method

[0297] Also provided is a method of treating or preventing cancer in a subject, the method comprising administering to the subject an immune response cell or pharmaceutical composition of the present invention.

[0298] As used herein, the term "treatment" refers to a method of obtaining a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes may include alleviating symptoms, reducing the severity of the disease, inhibiting the underlying cause of the disease or disorder, stabilizing the disease in a non-advanced state, delaying the progression of the disease and / or improving or alleviating the disease condition.

[0299] The method generally comprises administering a therapeutically effective amount or a prophylactically effective amount of the immune response cell or pharmaceutical composition of the present invention. A therapeutically effective amount is an amount that can improve one or more symptoms of the disease (e.g., all symptoms) and / or eliminate one or more symptoms of the disease (e.g., all symptoms). A therapeutically effective amount preferably can cure the disease. A prophylactically effective amount is an amount that can prevent the onset of the disease and / or prevent the appearance of one or more symptoms of the disease (e.g., all symptoms). A prophylactically effective amount preferably prevents the subject from suffering from the disease. Suitable amounts will be discussed in more detail below.

[0300] The immune response cell or pharmaceutical composition of the present invention may be administered to a subject exhibiting symptoms of the disease. The immune response cell or pharmaceutical composition of the present invention may be administered to an asymptomatic subject, i.e., a subject not exhibiting symptoms of the disease. The immune response cell or pharmaceutical composition of the present invention may be administered when the disease state of the subject is unknown or when the patient is expected to be disease-free. The immune response cell or pharmaceutical composition of the present invention may be administered to a subject susceptible to the disease, such as a subject genetically susceptible to the disease.

[0301] As used herein, the term "subject" generally refers to any animal, including but not limited to humans and non-human animals (e.g., dogs, cats, cows, horses, sheep, pigs, poultry, fish, crustaceans, etc.).

[0302] The subject may be a mammal. Optionally, the subject is a human, a horse, a dog or a cat. In some embodiments, the subject is a human. Alternatively, the subject may be a horse.

[0303] The cancer may include but is not necessarily limited to solid tumors, soft tissue tumors, metastatic lesions, and blood cancers. For example, the cancer may be liver cancer, lung cancer, breast cancer, prostate cancer, lymphoma, colon cancer, kidney cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal canal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), pediatric solid tumors, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma, myelodysplastic syndrome (MDS), chronic phase of chronic myeloid leukemia (CMLCP), diffuse large B cell lymphoma (DLBCL), cutaneous T cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), hepatocellular carcinoma (HCC), gastrointestinal stromal tumor (GIST), non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), environmentally induced cancers (including asbestos-induced cancers), and combinations of the above cancers. Specifically, the cancer may be breast cancer, such as estrogen receptor positive (ER pos) breast cancer and / or metastatic breast cancer.

[0304] The cancer may be a solid tumor.

[0305] In some embodiments, the cancer is selected from the group consisting of: head and neck cancer, ovarian cancer, malignant mesothelioma, breast cancer, pancreatic cancer, colorectal cancer, lung cancer, gastric cancer, bladder cancer, prostate cancer, esophageal cancer, endometrial cancer, hepatobiliary cancer, chronic or acute leukemia (including acute myeloid leukemia), duodenal cancer, thyroid cancer, central nervous system cancer, or renal cell cancer.

[0306] In some embodiments, the cancer is selected from ovarian cancer, breast cancer (optionally triple-negative breast cancer), pancreatic cancer, chronic or acute leukemia (including acute myeloid leukemia), malignant mesothelioma, and combinations of the above cancers.

[0307] In some embodiments, the cancer is breast cancer. Breast cancer includes but is not necessarily limited to estrogen receptor positive (ER pos) breast cancer, estrogen receptor negative (ER neg) breast cancer, progesterone receptor positive (PR pos) breast cancer, progesterone receptor negative (PR neg) breast cancer, HER2 positive (HER2 pos) breast cancer, HER2 negative (HER2 neg) breast cancer, and triple negative breast cancer (TNBC). Triple negative breast cancer refers to breast cancer in which cancer cells test negative (i.e., do not express or express negligibly) for estrogen receptor, progesterone receptor, and HER2. In some embodiments, the breast cancer includes ERpos, ER neg, PR pos, PR neg, HER2 pos, HER2 neg, and / or combinations thereof.

[0308] In some embodiments, the breast cancer includes triple negative breast cancer (TNBC).

[0309] In some embodiments, the treatment of cancer involves targeting non-tumor cells, such as tumor-associated stromal cells. Example types of such tumor-associated stromal cells include pancreatic stromal cells. Other types of non-tumor cells that can be targeted include macrophages, regulatory T cells, and myeloid-derived suppressor cells.

[0310] The subject may have been previously treated with chemotherapeutic drugs.

[0311] Compared to untreated tumors, administration of the immune response cells or pharmaceutical compositions of the present invention to a subject can reduce the tumor volume by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%.

[0312] In embodiments where immune response cells are administered, the number of cells administered to the subject should take into account the route of administration, the disease being treated, the body weight of the subject, and / or the age of the subject. Generally, about 1×10 6 to about 1×10 11 immune response cells can be administered to the subject. In some embodiments, about 1×10 7 to about 1×10 10 immune response cells, or about 1×10 8 to about 1×10 9 immune response cells are administered to the subject.

[0313] The present invention also provides the immune response cells or pharmaceutical compositions of the present invention for any one of the above-mentioned treatment methods. Accordingly, there is also provided the immune response cells or pharmaceutical compositions of the present invention for treating or preventing diseases. Preferably, the disease is cancer. This can also be referred to as for treatment. More preferably, the present invention provides the immune response cells or pharmaceutical compositions of the present invention for treating or preventing breast cancer. Most preferably, the present invention provides the immune response cells or pharmaceutical compositions of the present invention for treating or preventing triple-negative breast cancer.

[0314] There is also provided the use of the immune response cells or pharmaceutical compositions of the present invention in the preparation of a medicament for treating or preventing a disease. Preferably, the disease is cancer as described above. There is also provided the use of the immune response cells or pharmaceutical compositions of the present invention in (i) therapy or (ii) cancer treatment. In some embodiments, the cancer is breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer.

[0315] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0316] As used herein, the terms "comprise" and "contain" and their variants, such as "comprising" and "comprises", mean "including but not limited to", and do not exclude other ingredients, integers, or steps. Furthermore, unless the context otherwise requires, the singular includes the plural: in particular, when using the indefinite article, unless the context otherwise requires, this specification should be understood to include both the plural and the singular.

[0317] Examples

[0318] The following are specific examples for practicing the present invention. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Every effort has been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but of course some experimental error and deviation are to be allowed.

[0319] Unless otherwise specified, the practice of the present invention will employ conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the skill of the art. These techniques are well explained in the literature.

[0320] Materials and methods

[0321] T cell isolation and retroviral transduction

[0322] Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of healthy volunteers by density gradient centrifugation. T cells were activated as follows. 1×10 6An activated PBMC was seeded onto a RetroNectin-coated plate pre-treated with 3 mL of retroviral supernatant. Each well was then treated with 3 mL of fresh viral supernatant and 100 IU / mL of IL-2. Retroviral transduction was performed using viral particles produced by stable packaging cells of the stable gibbon leukemia virus (GALV) pseudotype 293TVec. Thereafter, T cells were cultured at a concentration of 100 IU / mL in RPMI-1640 medium + 5% normal human AB serum, and fresh medium and IL-2 (100 IU / mL) were supplemented three times a week.

[0323] T cell activation

[0324] T cells were activated using paramagnetic beads coated with anti-human CD3 and anti-human CD28 antibodies (cell-to-bead ratio of 1:2), phytohemagglutinin (PHA) at a concentration of 5 μg / mL, or 10 μL of TransAct reagent added per 1 × 10 6 peripheral blood mononuclear cells.

[0325] Flow cytometry

[0326] T cell transduction and 293T cell transfection were evaluated by flow cytometry and compared with appropriate isotype controls under specified conditions. To evaluate the expression of NKG2D-based constructs, cells were stained with mouse anti-human CD4-FITC, mouse anti-human NKG2D-PE, and mouse anti-human CD8-APC, and appropriate compensation was performed. Since the endogenous NKG2D expression level in CD8 + T cells is relatively high, its transduction efficiency was compared with the NKG2D expression level in untransduced CD4 + T cells. Before use, the transduction efficiency of different constructs was normalized by adding the necessary proportion of untransduced T cells. This ensured that the total number of CAR + T cells and the total T cell concentration were the same under all conditions.

[0327] To evaluate T cell differentiation after multiple rounds of stimulation, T cells were removed from the tumor cell co-culture and stained with FITC-conjugated anti-human CD45RO, allophycocyanin (APC)-conjugated anti-human CD62L, PE-conjugated anti-human CCR7, FITC-conjugated anti-human CD4, APC Cy7-conjugated anti-human CD8α, and phycoerythrin-cyanine 7 (PECy7)-conjugated anti-human CD27. After washing with 2 mL of ice-cold PBS, the cells were resuspended in 0.5 mL of ice-cold PBS and evaluated by flow cytometry. The staining efficiency was evaluated using T cells stained with the corresponding isotype control and fluorescence minus one (FMO) control.

[0328] Dose-response assay

[0329] 1×10 4 Tumor cells were seeded per well (100 μL) in a 96-well plate and incubated overnight at 37°C and 5% CO2. After 24 hours, the cells were plated at a log saturation ratio of 1:1 to 1:64. 2 T cells were added in the ratio of CAR T cells to tumor cells. After 72 hours, T cells were removed and 100 μL of MTT solution (500 μg / mL) was added, and the culture plate was then incubated at 37°C and 5% CO2 for about 1 hour. After removing the MTT solution, the resulting formazan crystals were dissolved in DMSO (100 μL / well) and the absorbance was measured at 560 nm. Tumor cell viability was calculated as follows: (absorbance of the monolayer containing T cells / absorbance of the monolayer without T cells)*100.

[0330] Re-stimulation assay

[0331] 1×10 5 Tumor cells were seeded in triplicate wells of a 24-well plate and incubated at 37°C and 5% CO2 for 24 hours. After 24 hours, 1 mL of 1×10 5 CAR + T cells. After 72 hours, gently remove the T cells and wash the wells with 1 mL of PBS. After removing the PBS, add 1 mL of MTT (final concentration of 500 μg / mL) to each well, and then incubate the culture plate at 37°C and 5% CO2 for about 1 hour. Measure the absorbance of the corresponding wells at 560 nm and calculate the tumor cell viability as detailed in the "Dose Response" section. If the measured tumor cell viability is less than 50%, the restimulation is considered successful.

[0332] The T cells removed from the culture plate were centrifuged at 400 × g for 5 minutes, and the supernatant was discarded. The pellet was resuspended in 3.2 mL of R5 medium, and 1 mL of fresh tumor monolayer cells was added to each well (1 × 10 per well of a 24-well plate). 5 A small portion of the remaining 200 μL sample was subjected to trypan blue exclusion to assess the total number of T cells.

[0333] In vivo studies

[0334] Bioluminescence imaging

[0335] 1×10 5 Firefly luciferase (ffLUC)-labeled BxPC3 cells were injected into the peritoneal cavity of NSG mice. Twelve days after tumor inoculation, each group of mice (n=5) were intraperitoneally injected with PBS, 4×10 6 (N1012 +(N1012(lo)) or 1×10 7 (N1012(hi) or NKG2D) CARs + T cells. Untransduced T cells were used as controls.

[0336] Tumor growth was monitored by BLI, and all data were expressed as total flux (photons / second) per treatment or average total flux (photons / second). Mice were closely monitored and weighed three times a week to detect signs of poor health.

[0337] Tumor volume

[0338] 1×10 5 CFPac-1 or BxPC3 cells were subcutaneously injected into the left flank of NSG mice together with 50 μL of Matrigel (mixed with PBS at a 1:1 ratio). Twenty-nine days after inoculation with CFPac-1 cells or 14 days after inoculation with BxPC3 cells, the mice were intravenously injected with PBS or 1×10 7 CAR T cells (N1012_CXCR2, N1012, NKG2D, CYAD-01 replicons, untransduced (UT)).

[0339] Tumor growth was measured weekly by caliper and expressed as tumor volume (mm 3 ).

[0340] Example 1: Evaluation of in vivo anti-tumor efficacy of N1012_CXCR2 T cells in a murine pancreatic cancer model

[0341] T cells expressing CAR constructs (e.g., N1012, N1012_CXCR2, CYAD-01_10) were generated by isolating peripheral blood mononuclear cells (PBMCs), activating T cells, and transducing T cells with a virus containing a polynucleotide encoding the CAR ( Figure 1 ).

[0342] Briefly, to generate the virus, 1.65×10 6 HEK293T cells were seeded in a 10 cm 2In a tissue culture dish, it was placed in 10 mL of IMDM medium (I10 medium) containing 10% FBS and 2 mM L-glutamine and incubated at 37 °C and 5% CO2 for 24 hours. The next day, transfection mixtures were generated for each CAR construct according to the protocol in Table 3. HEK293T cells were transfected with the following plasmids respectively: N1012 (encoding the DAP10 / 12 fusion protein and the human NKG2D receptor (SEQ ID NO: 74)), N1012_CXCR2 (encoding the DAP10 / 12 fusion protein, the human NKG2D receptor and CXCR2 (SEQ ID NO: 91)), CYAD-01 replicon (encoding the NKG2D receptor fused with CD3ζ (SEQ ID NO: 93)), and CYAD-01_10 (encoding the CYAD-01 replicon and DAP10 (SEQ ID NO: 94)).

[0343] Table 3: Transfection protocol for CAR constructs (per 10 cm 2 culture dish volume)

[0344]

[0345] After 15 minutes of incubation, mixture B was added dropwise to the HEK293T cells and gently stirred. Then the cells were returned to the incubator. The supernatant was collected 48 hours after transfection, placed in a pre-chilled 50 mL Falcon tube, and stored at 4 °C.

[0346] 10 mL of fresh I10 medium was added to the HEK293T cells and then returned to the incubator. After another 24 hours, the supernatant was collected from the HEK293T cells for the second time and combined with the supernatant collected 48 hours after transfection. The combined supernatant was aliquoted into pre-labeled tubes, quickly frozen, and stored at -80 °C.

[0347] PBMC were isolated by standard Ficoll Paque density gradient centrifugation. T cells were resuspended at a concentration of 1×10 6 cells / mL in RPMI + 5% normal human AB serum and 2 mM L-glutamine ("R5" medium) to activate the T cells. After 48 hours of activation, 1×10 6 T cells were seeded onto a non-tissue culture plate coated with RetroNectin and mixed with 3 mL of virus supernatant collected from transiently transfected HEK293T cells. The T cells were cultured in RPMI-1640 medium + 5% normal human AB serum with 100 IU / mL IL-2, and fresh medium and IL-2 (100 IU / mL) were supplemented three times a week.

[0348] To evaluate the efficacy of high-dose and low-dose CAR T cells against pancreatic tumors in vivo, CAR T cells were injected into mice bearing CFPac-1 tumor cells. Briefly, 1×10 5 CFPac-1 cells were injected into the peritoneal cavity of NSG mice. Twenty-eight days after tumor inoculation, mice were treated with PBS [n = 5], high-dose 10×10 6 CAR T cells (N1012 [n = 7], N1012_CXCR2 [n = 7]) or low-dose 4×10 6 CAR T cells (N1012 [n = 6], N1012_CXCR2 [n = 7]). Figure 2 The results shown in A of Figure 2 indicate that the efficacy of low-dose N1012_CXCR2 T cells is comparable to that of high-dose N1012 T cells. Figure 2 B of Figure 2 shows that mice treated with N1012_CXCR2, especially those treated with high-dose N1012_CXCR2 cells, had the highest survival rate.

[0349] Example 2: Anti-tumor efficacy of N1012_CXCR2 T cells against breast cancer cells

[0350] To verify the efficacy of N1012_CXCR2 T cells against another cancer model, the restimulation and cytotoxicity capabilities of N1012_CXCR2 T cells against triple-negative breast cancer cell lines MDA-MB-468 and MDA-MB-231 were compared with those of N1012 T cells.

[0351] Briefly, N1012 or N1012_CXCR2 T cells were co-cultured with fresh monolayer cells twice a week until no monolayer cell disruption was observed. For this purpose, 1×10 5 MDA-MB-468 or MDA-MB-231 cells were seeded in triplicate wells of a 24-well plate and incubated at 37 °C and 5% CO2 for 24 hours. NKG2D + T cells were used as a control. After 24 hours, 1×10 5 CAR + T cells were added to each well at a final concentration of 1×10 5 CAR + / mL. After 72 hours, the T cells were gently removed, centrifuged at 400×g for 5 minutes, and then the supernatant was discarded. The pellet was resuspended in 3.2 mL of R5 medium, 1 mL of fresh tumor monolayer cells was added to each well, and three replicates were set up. A small portion of the remaining 200 μL sample was subjected to trypan blue exclusion to evaluate the total number of T cells.

[0352] Compared with N1012 and NKG2D T cells, N1012_CXCR2 T cells underwent significantly more rounds of restimulation against MDA-MB-468 and MDA-MB-231 cells( Figure 3 ).

[0353] Example 3: Anti-tumor efficacy of N1012_CXCR2 T cells activated with different activation stimulants

[0354] Subsequently, the in vivo anti-tumor efficacy of CAR T cells activated with different activation stimulants was evaluated. CAR T cells were activated in vitro by PHA, the clinically compliant activation stimulant TransAct (Miltenyi Biotec GmbH), or clinically compliant immobilized anti-CD3 and anti-CD28 antibodies. N1012 was compared with untransduced (UT) T cells (also activated with three different stimulants).

[0355] Briefly, 1×10 5 firefly luciferase-labeled BxPC3 cells (BxPC3_LT) were injected intraperitoneally into NSG mice. Twelve days after tumor inoculation, 4×10 6 T cells (activated as described above) or PBS were administered to the mice. Figure 4 and Figure 5 showed that CAR T cells activated with TransAct, especially N1012_CXCR2 CAR T cells activated with TransAct, had the best efficacy. As shown by the second vertical dashed line in Figure 6 , an additional rechallenge test was performed on the mice that achieved complete remission on day 64, and this figure shows the data of each mouse in Figure 4 and Figure 5 .

[0356] Example 4: TransAct-activated N1012_CXCR2 T cells are biased towards CD4 + Cell characteristics

[0357] Subsequently, the structural characteristics of N1012_CXCR2 T cells activated with different activation stimulants were evaluated. As shown in Example 3, CAR T cells were activated in vitro by PHA or the clinically compliant activation stimulant TransAct. N1012, CYAD-01 replicating cells, and untransduced (UT) T cells (also activated with two different stimulants) were used as references. The CYAD-01 CAR is a humanized version of a murine CAR initially described by Sentman et al. (Zhang et al., 2005, Blood 106:1544-1551) and consists of the fusion of NKG2D and CD3ζ. Although nominally a first-generation CAR, it binds to endogenous DAP10 in T cells, meaning that both signal 1 and signal 2 are provided simultaneously. The CYAD-01 CAR is currently in clinical development by Celyad Oncology, and a replicant of this CAR is provided in these examples for comparison purposes only. In CYAD-01_10, the CYAD-01 replicant is co-expressed with additional DAP10.

[0358] Figure 7 showed that at the end of the in vitro culture period (days 10 to 12), the CD4 to CD8 ratio of N1012_CXCR2 T cells activated with TransAct was higher than that of N1012_CXCR2 T cells activated with PHA. There were more CD8 cells than CD4 cells in N1012_CXCR2 T cells activated with PHA, while there were more CD4 cells than CD8 cells in N1012_CXCR2 T cells activated with TransAct. This result was unexpected. However, the transduction efficiency and median fluorescence intensity (MFI) of N1012_CXCR2 T cells activated with TransAct were comparable to those of N1012_CXCR2 T cells activated with PHA ( Figure 8 ).

[0359] Then, the fold expansion of T cells activated with 5 μg / mL phytohemagglutinin-L (PHA-L) or 10 μL of TransAct reagent added per 1 × 10 6 peripheral blood mononuclear cells was evaluated. This value was calculated by dividing the number of T cells present at the end of the ex vivo culture period (10 to 12 days) by the number of initially transduced T cells.

[0360] In addition to the different CD4 to CD8 ratios, the fold expansion of cells activated with TransAct was greater than that of cells activated with PHA ( Figure 9 ).

[0361] Example 5: Expression of CAR in γδ T cells

[0362] Primary human γδ T cells were activated by immobilized pan-γδ TCR antibody (clone 11F2). After 48 hours of activation, the T cells were engineered by retroviral transduction to express N1012 or N1012_CXCR2. Surface expression of NKG2D was evaluated by flow cytometry, and co-staining for TCR and CD3 expression was performed. The median fluorescence intensity (MFI, Figure 10 of A) and percentage of expression ( Figure 10 of B) of NKG2D were compared with untransduced T cells. As shown, both N1012 and N1012_CXCR2 constructs enabled high-level and reproducible expression on the surface of primary human γδ T cells.

[0363] Example 6: Evaluation of N1012 and N1012_CXCR2 γδ T cell expansion and anti-tumor cytotoxicity

[0364] Subsequently, the in vitro functions of γδ T cells expressing N1012 or N1012_CXCR2 were evaluated.

[0365] To evaluate the fold expansion, γδ T cells expressing N1012 or γδ T cells expressing N1012_CXCR2 were activated as described above. As Figure 11 shown in A, the fold expansion was calculated based on the percentage of pan-γδ TCR + CD3 + cells on day 0, day 7, day 14, and day 21 of culture. Figure 11 Shown in B are the percentages of pan-γδ TCR + CD3 + cells on day 0, day 7, day 14, and day 21 (n = 11). As Figure 11 shown, the cell percentages and fold expansion were similar among untransduced, N1012-expressing, and N1012_CXCR2-expressing γδ T cells.

[0366] Then, the in vitro anti-tumor functions of γδ T cells expressing N1012 and N1012_CXCR2 were evaluated. Figure 12 Shown in A are the results of cytotoxicity dose-response assays of untransduced (UT) and CAR + (N1012 or N1012_CXCR2) γδ T cells against the triple-negative breast cancer cell line MDA-MB-468 at different effector-to-target ratios. Figure 12 Shown in B are the results of untransduced (UT) and CAR +(N1012 or N1012_CXCR2) γδ T cell cytotoxicity dose-response assay results against pancreatic cancer cell line BxPC-3 at different effector-to-target ratios (n = 3). Both N1012 and N1012_CXCR2 γδ T cells showed significant cytotoxicity against the cancer cell line. This cytotoxicity was maintained after multiple T cell restimulations, including against the acute myeloid leukemia AML cell line THP-1-LT (n = 2)( Figure 13 ). Figure 14 A of Figure 14 and + B of Figure 14 show the purity of CAR Figure 14 γδ T cells after restimulation on MDA-MB-468 cells or BxPC-3 cells. As shown in C of

[0367] and Example 7: In vivo anti-tumor efficacy of N1012 and N1012_CXCR2 γδ T cells D of

[0369]

[0368] 5 7 + 7 γδ T cells. Untransduced γδ T cells and PBS were used as controls. The results are shown in Figure 15 . Compared with mice injected with control PBS or untransduced γδ T cells, mice injected with N1012 or N1012_CXCR2 (N1012 and N1012_CXCR2, respectively) γδ T cells had reduced tumor burden ( Figure 15 ) and extended survival ([[]] Figure 16 ).

[0370] Example 8: N1012_CXCR2 T cells exhibit continuously enhanced anti-tumor efficacy

[0371] Summarizes the survival curves of various in vivo experiments. Specifically, for those receiving PBS or 1×10 7Survival curves of mice bearing CFPac1 (pancreatic cancer, two models), BxPC3 (pancreatic cancer, two models), Kuramochi (high-grade serous ovarian cancer, one model), Ovsaho (epithelial ovarian cancer, one model), mesothelioma (patient-derived xenograft, one model), triple-negative breast cancer (patient-derived xenograft, one model), or SKOV3 (epithelial ovarian cancer, one model) tumors treated with CAR T cells (N1012, N1012_CXCR2, or untransduced) were pooled.

[0372] These survival curves are shown in Figure 17 As described above, CAR T cells (N1012_CXCR2, CYAD-01, or PBS buffer as a control) were injected into mice bearing CFPac-1 tumor cells or BxPC3 tumor cells.

[0373] As shown in Figure 17 Compared to other cells, the survival rate of mice injected with N1012_CXCR2 cells was significantly increased. This result was further demonstrated in Table 4, which shows the median survival of pooled data for each cell group.

[0374] Table 4: Pooled median survival

[0375] Construct Median survival (days) Survival rate N1012_CXCR2 Not reached 83％ CYAD-01 analogue 90 10％ Untransduced (UT) 38 0％ PBS 48 2％

[0376] Example 9: Efficient transduction and amplification of N1012_CXCR2 can be achieved within a certain range of TransAct TM concentration, Including cases far below the manufacturer's recommended concentration

[0377] Primary human peripheral blood mononuclear cells (at a concentration of 1 × 10 6 cells / mL) were activated by TransAct TM at concentrations ranging from 0.1 μL / mL to 10 μL / mL recommended by the manufacturer. After 72 hours of activation, the cells were counted by trypan blue exclusion, and 5 × 10 5 cells were divided into an untransduced group and an N1012_CXCR2 retrovirally transduced group. Transduction efficiency was measured by the expression levels of CXCR2 and / or NKG2D in CD4 + T cells and was evaluated by flow cytometry three days and ten days after transduction. Detection was performed using fluorescein isothiocyanate (FITC)-conjugated anti-human CD4, allophycocyanin cyanine 7 (APC-Cy7)-conjugated anti-human CD8, phycoerythrin (PE)-conjugated anti-human NKG2D, and AlexaFluor647-conjugated anti-human CXCR2 antibodies, as shown in Figure 18

[0378] ​Three and ten days after transduction, the expansion of untransduced (UT) and N1012_CXCR2 T cells was evaluated by trypan blue exclusion. The exact cell number was obtained by multiplying the cell concentration by the total culture volume.

[0379] The results showed that good transduction efficiency could be achieved even when only 10% of the manufacturer-recommended concentration of TransAct was used. TM was used.

[0380] Example 10: Anti-tumor efficacy of N1012_CXCR2 T cells in a metastatic colorectal cancer xenograft model

[0381] 1×10 6 LS180 or SW620 tumor cells were subcutaneously injected into the left flank of NSG mice. Tumor growth was measured weekly by caliper. Twelve days after transplantation, the mice were treated with 1×10 7 CAR + T cells or 1×10 7 untransduced T cells. LS180 tumor-bearing mice were intravenously injected with N1012_CXCR2 (n = 6), N1012 (n = 7), CYAD-01 (n = 6), or untransduced (UT) T cells (n = 5). SW620 tumor-bearing mice were injected with N1012_CXCR2 (n = 5), N1012 (n = 4), CYAD-01 (n = 5), or untransduced T cells (n = 4). Figure 19 The results shown indicated that N1012_CXCR2 exhibited potent anti-tumor efficacy in both models.

[0382] Sequence

[0383] SEQ ID NO:1 (Full-length human DAP10 sequence)

[0384] MIHLGHILFL LLLPVAAAQT TPGERSSLPA FYPGTSGSCS GCGSLSLPLL AGLVAADAVA

[0385] SLLIVGAVFL CARPRRSPAQ EDGKVYINMP GRG

[0386] SEQ ID NO:2 (Amino acids 19 to 93 of DAP10 - without the leader sequence)

[0387] QTTPGERSSL PAFYPGTSGS CSGCGSLSLP LLAGLVAADA VASLLIVGAV FLCARPRRSP

[0388] AQEDGKVYIN MPGRG

[0389] SEQ ID NO:3 (Amino acids 19 to 69 of DAP10 - extracellular / transmembrane domain)

[0390] QTTPGERSSL PAFYPGTSGS CSGCGSLSLP LLAGLVAADA VASLLIVGAV F

[0391] SEQ ID NO:4 (Amino acids 1 to 71 of DAP10)

[0392] MIHLGHILFL LLLPVAAAQT TPGERSSLPA FYPGTSGSCS GCGSLSLPLL AGLVAADAVA

[0393] SLLIVGAVFL C

[0394] SEQ ID NO:5 (Amino acids 19 to 71 of DAP10)

[0395] QTTPGERSSL PAFYPGTSGS CSGCGSLSLP LLAGLVAADA VASLLIVGAV FLC

[0396] SEQ ID NO:6 (Amino acids 70 to 93 of DAP10 - intracellular domain)

[0397] LCARPRRSPA QEDGKVYINM PGRG

[0398] SEQ ID NO:7 (Amino acids 49 to 93 of DAP10 - transmembrane and intracellular domains)

[0399] LLAGLVAADA VASLLIVGAV FLCARPRRSP AQEDGKVYIN MPGRG

[0400] SEQ ID NO:8 (Amino acids 49 to 69 of DAP10 - transmembrane domain)

[0401] LLAGLVAADA VASLLIVGAV F

[0402] SEQ ID NO:9 (Full - length human DAP12 sequence)

[0403] MGGLEPCSRL LLLPLLLAVS GLRPVQAQAQ SDCSCSTVSP GVLAGIVMGD LVLTVLIALA

[0404] VYFLGRLVPR GRGAAEAATR KQRITETESP YQELQGQRSD VYSDLNTQRP YYK

[0405] SEQ ID NO:10 (Amino acids 22 to 113 of DAP12 - leader sequence deleted)

[0406] LRPVQAQAQSDCSCSTVSPG VLAGIVMGDL VLTVLIALAV YFLGRLVPRG RGAAEAATRK

[0407] QRITETESPY QELQGQRSDV YSDLNTQRPY YK

[0408] SEQ ID NO:11 (Amino acids 62 to 113 of DAP12 - cytoplasmic / intracellular domain)

[0409] YFLGRLVPRG RGAAEAATRK QRITETESPY QELQGQRSDV YSDLNTQRPY YK

[0410] SEQ ID NO:12 (Amino acids 41 to 61 of DAP12 - transmembrane domain)

[0411] GVLAGIVMGD LVLTVLIALA V

[0412] SEQ ID NO:13 (Amino acids 22 to 61 of DAP12 - extracellular and transmembrane domains)

[0413] LRPVQAQAQSDCSCSTVSPG VLAGIVMGDL VLTVLIALAV

[0414] SEQ ID NO:14 (Full - length sequence of human NKG2D)

[0415] MGWIRGRRSR HSWEMSEFHN YNLDLKKSDF STRWQKQRCP VVKSKCRENA SPFFFCCFIA

[0416] VAMGIRFIIM VAIWSAVFLN SLFNQEVQIP LTESYCGPCP KNWICYKNNC YQFFDESKNW

[0417] YESQASCMSQ NASLLKVYSK EDQDLLKLVK SYHWMGLVHIPTNGSWQWED GSILSPNLLT

[0418] IIEMQKGDCA LYASSFKGYI ENCSTPNTYI CMQRTV

[0419] SEQ ID NO:15 (Amino acids 73 to 216 of human NKG2D - extracellular domain)

[0420] IWSAVFLNSL FNQEVQIPLT ESYCGPCPKN WICYKNNCYQ FFDESKNWYE SQASCMSQNA

[0421] SLLKVYSKED QDLLKLVKSY HWMGLVHIPT NGSWQWEDGS ILSPNLLTII EMQKGDCALY

[0422] ASSFKGYIEN CSTPNTYICM QRTV

[0423] SEQ ID NO:16 (Amino acids 82 to 216 of human NKG2D - extracellular domain)

[0424] LFNQEVQIPL TESYCGPCPK NWICYKNNCY QFFDESKNWY ESQASCMSQN ASLLKVYSKE

[0425] DQDLLKLVKS YHWMGLVHIP TNGSWQWEDG SILSPNLLTI IEMQKGDCAL YASSFKGYIE

[0426] NCSTPNTYIC MQRTV

[0427] SEQ ID NO:17 (Amino acids 52 to 216 of human NKG2D - transmembrane and extracellular domains)

[0428] PFFFCCFIAV AMGIRFIIMV AIWSAVFLNS LFNQEVQIPL TESYCGPCPK NWICYKNNCY

[0429] QFFDESKNWY ESQASCMSQN ASLLKVYSKE DQDLLKLVKS YHWMGLVHIP TNGSWQWEDG

[0430] SILSPNLLTI IEMQKGDCAL YASSFKGYIE NCSTPNTYIC MQRTV

[0431] SEQ ID NO:18 (Linker)

[0432] GSG

[0433] SEQ ID NO:19 (Linker)

[0434] GSGGG

[0435] SEQ ID NO:20 (Linker)

[0436] GSGG

[0437] SEQ ID NO:21 (Linker)

[0438] SGGG

[0439] SEQ ID NO:22 (Linker)

[0440] GGGGS

[0441] SEQ ID NO:23 (Linker)

[0442] GGGGSGGGGSGGGGSGGGGS

[0443] SEQ ID NO:24 (Linker)

[0444] GGGGSGGGGSGGGGS

[0445] SEQ ID NO:25 (Linker)

[0446] GPPGS

[0447] SEQ ID NO:26 (Linker)

[0448] GGGS

[0449] SEQ ID NO:27 (Linker)

[0450] GGGGS

[0451] SEQ ID NO:28 (Linker)

[0452] GYS

[0453] SEQ ID NO:29 (Linker)

[0454] GS

[0455] SEQ ID NO:30 (Linker)

[0456] SGGGG

[0457] SEQ ID NO:31 (Linker)

[0458] SGGG

[0459] SEQ ID NO:32 (Linker)

[0460] SGG

[0461] SEQ ID NO:33 (Linker)

[0462] SGSG

[0463] SEQ ID NO:34 (Linker)

[0464] SG

[0465] SEQ ID NO:35 (Linker)

[0466] GGGGA

[0467] SEQ ID NO:36 (Linker)

[0468] GGGA

[0469] SEQ ID NO:37 (Linker)

[0470] EAAAK

[0471] SEQ ID NO:38 (Furin cleavage site)

[0472] RRKR

[0473] SEQ ID NO:39 (P2A skipping peptide)

[0474] ATNFSLLKQAGDVEENPGP

[0475] SEQ ID NO:40 (T2A skipping peptide)

[0476] EGRGSLLTCGDVEENPGP

[0477] SEQ ID NO:41 (SGSG + P2A)

[0478] SGSGATNFSLLKQAGDVEENPGP

[0479] SEQ ID NO:42 (SGSG + T2A)

[0480] SGSGEGRGSLLTCGDVEENPGP

[0481] SEQ ID NO:43 (Furin + SGSG + P2A)

[0482] RRKRSGSGATNFSLLKQAGDVEENPGP

[0483] SEQ ID NO:44(Furin + SGSG + T2A)

[0484] RRKRSGSGEGRGSLLTCGDVEENPGP

[0485] SEQ ID NO:45(F2A Skipping Peptide)

[0486] VKQTLNFDLLKLAGDVESNPGP

[0487] SEQ ID NO:46(E2A Skipping Peptide)

[0488] QCTNYALLKLAGDVESNPGP

[0489] SEQ ID NO:47(His Tag)

[0490] HHHHHH

[0491] SEQ ID NO:48(FLAG Tag)

[0492] DYKDDDDK

[0493] SEQ ID NO:49(Avi Tag)

[0494] GLNDIFEAQKIEWHE

[0495] SEQ ID NO:50(V5 Tag)

[0496] GKPIPNPLLGLDST

[0497] SEQ ID NO:51(V5 Tag)

[0498] IPNPLLGLD

[0499] SEQ ID NO:52(Myc Tag)

[0500] EQKLISEEDL

[0501] SEQ ID NO:53(AHF Tag)

[0502] GLNDIFEAQKIEWHEGGHHHHHHDYKDDDDK

[0503] SEQ ID NO:54(FHA Tag)

[0504] DYKDDDDKHHHHHHGGGLNDIFEAQKIEWHE

[0505] SEQ ID NO:55 (CD8α leader sequence)

[0506] MALPVTALLL PLALLLHAAR P

[0507] SEQ ID NO:56 (4-1BB intracellular domain)

[0508] KRGRKKLLYI FKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EL

[0509] SEQ ID NO:57 (CD27 intracellular domain)

[0510] QRRKYRSNKG ESPVEPAEPC HYSCPREEEG STIPIQEDYR KPEPACSP

[0511] SEQ ID NO:58 (human IgG1 hinge region - amino acids 218 to 229 of UniProt accession number P0DOX5)

[0512] EPKSCDKTHT CP

[0513] SEQ ID NO:59 (truncated CD8α hinge region)

[0514] TTTPAPRPPT PAPTIASQPL SLRPEACRPA AGGAVHTRGL DFACD

[0515] SEQ ID NO:60

[0516] MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0517] SEQ ID NO:61

[0518] MALPVTALLLPLALLLHAARPDYKDDDDKQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0519] SEQ ID NO:62

[0520] MALPVTALLLPLALLLHAARPDYKDDDDKEPKSCDKTHTCPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0521] SEQ ID NO:63

[0522] MALPVTALLLPLALLLHAARPDYKDDDDKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0523] SEQ ID NO:64 (Construct 1 / N1012)

[0524] MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0525] SEQ ID NO:65 (Construct 3)

[0526] MALPVTALLLPLALLLHAARPDYKDDDDKQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0527] SEQ ID NO:66 (Construct 8)

[0528] MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0529] SEQ ID NO:67 (Construct 9)

[0530] MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPDYKDDDDKQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0531] SEQ ID NO:68 (Construct 10)

[0532] MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

[0533] SEQ ID NO:69 (Construct 11)

[0534] MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPDYKDDDDKQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

[0535] SEQ ID NO:70 (encoding the polypeptide of SEQ ID NO:60)

[0536] ATGATCCACCTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAGACCACCCCTGGCGAGCGGAGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTGGCCGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGGCGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAG

[0537] SEQ ID NO:71 (encoding the polypeptide of SEQ ID NO:61)

[0538] ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGATTATAAGGACGACGACGACAAGCAGACCACCCCTGGCGAGCGGAGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTGGCtGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCTACTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAG

[0539] SEQ ID NO:72 (encoding the polypeptide of SEQ ID NO:62)

[0540] ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGATTATAAGGACGACGACGACAAGGAGCCCAAGAGCTGCGACAAGACACACACATGCCCTCTTctggccggCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGGCGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAG

[0541] SEQ ID NO:73 (encoding the polypeptide of SEQ ID NO:63)

[0542] ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGATTATAAGGACGACGACGACAAGACCACAACACCTGCTCCTAGACCTCCCACCCCTGCTCCCACCATCGCCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGCAGACCCGCTGCTGGCGGCGCTGTGCATACCAGAGGCCTGGATTTCGCCTGCGACCTTctggccggCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGGCGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAG

[0543] SEQ ID NO:74 (encoding the polypeptide of SEQ ID NO:64 / Construct 1 / N1012)

[0544]

[0545] SEQ ID NO:75 (polypeptide / construct 3 encoding SEQ ID NO:65)

[0546]

[0547] SEQ ID NO:76 (polypeptide / construct 8 encoding SEQ ID NO:66)

[0548]

[0549] SEQ ID NO:77 (polypeptide / construct 9 encoding SEQ ID NO:67)

[0550]

[0551] SEQ ID NO:78 (polypeptide encoding SEQ ID NO:68 / construct 10)

[0552]

[0553] SEQ ID NO:79 (polypeptide encoding SEQ ID NO:69 / construct 11)

[0554]

[0555] SEQ ID NO:80 (A20 FMDV2 peptide)

[0556] NAVPNLRGDLQVLAQKVART

[0557] SEQ ID NO:81 (CD124 signal peptide)

[0558] MGWLCSGLLFPVSCLVLLQVASSGN

[0559] SEQ ID NO:82 (Amino acids 114 to 220 of CD28)

[0560] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0561] SEQ ID NO:83 (Amino acids 52 to 164 of CD247)

[0562] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0563] SEQ ID NO:84 (SEQ ID NO:1 of WO2019 / 182425)

[0564] MGWSCIILFLVATATGVHSQIQLVQSGPELKKPGETVKISCKTSGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEPTYTDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARTAVYWGQGTTLTVSSGSTSGSGKPGSGEGSDIQMTQSPSSLSASLGERVSLTCRASQEISGSLSWLQQKPDGTIKRLIYAASTLNSGVPKRFSGRRSGSDYSLTISSLESEDFVDYYCLQYSSYPWSFGGGTKLEIKEPKSPDKTHTCPPCPSHTQPLGVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGKFWVLVVVGGVLACYSLLVTVAFIIFWVARPRRSPAQEDGKVYINMPGRGGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0565] SEQ ID NO:85 (SEQ ID NO:9 of WO2019 / 182425)

[0566] ARPRRSPAQEDGKVYINMPGRG

[0567] SEQ ID NO:86 (SEQ ID NO:11 of WO2019 / 182425)

[0568] GRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0569] SEQ ID NO:87 (Human CXCR2 polypeptide sequence expressed in N1012_CXCR2)

[0570] MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRTVYSSNVSPACYEDMGNNTANWRMLLRILPQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQKHRAMRVIFAVVLIFLLCWLPYNLVLLADTLMRTQVIQETCERRNHIDRALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKDSRPSFVGSSSGHTSTTL

[0571] SEQ ID NO:88 (Nucleic acid sequence encoding the polypeptide of SEQ ID NO:87)

[0572]

[0573] SEQ ID NO:89 (Nucleic acid sequence of SFG N1012_CXCR2)

[0574]

[0575] SEQ ID NO:90 (Protein encoded by SEQ ID NO:89, comprising: (i) a fused full-length intracellular domain of DAP10 / DAP12; (ii) a furin cleavage site (RRKR); (iii) an SGSG linker; (iv) a P2A skip sequence; (v) NKG2D; (vi) a furin cleavage site (RRKR); (vii) an SGSG linker; (viii) a T2A skip sequence; (ix) CXCR2) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRTVYSSNVSPACYEDMGNNTANWRMLLRILPQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQKHRAMRVIFAVVLIFLLCWLPYNLVLLADTLMRTQVIQETCERRNHIDRALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKDSRPSFVGSSSGHTSTTL

[0576] SEQ ID NO:91 (polypeptide encoding SEQ ID NO:90)

[0577]

[0578] SEQ ID NO:92 (Amino acid sequence of the replicon of CYAD-01CAR (fusion of NKG2D and the intracellular domain of CD3ζ)) MRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV SEQ ID NO:93 (Nucleic acid sequence encoding the polypeptide of SEQ ID NO:92)

[0579] ATGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCcGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGATGCATTGCACATGCAGGCCCTGCCCCCTCGCATGGGCTGGATCCGCGGCCGCAGGAGCCGGCACAGCTGGGAGATGAGCGAGTTCCACAACTACAACCTGGACCTGAAGAAGAGCGACTTCAGCACCCGGTGGCAGAAGCAGCGGTGCCCCGTGGTGAAGAGCAAGTGCCGGGAGAACGCCAGCCCCTTCTTCTTCTGCTGCTTCATCGCCGTGGCtATGGGCATCCGGTTTATAATCATGGTGGCCATCTGGAGCGCCGTGTTCCTGAACAGCCTGTTCAACCAGGAGGTGCAGATCCCCCTGACCGAGAGCTACTGCGGCCCCTGCCCCAAGAACTGGATCTGCTACAAGAACAACTGCTACCAGTTCTTCGACGAGAGCAAGAACTGGTACGAGAGCCAGGCCAGCTGCATGAGCCAGAACGCCAGCCTGCTGAAGGTGTACAGCAAGGAGGACCAGGACCTGCTGAAGCTGGTGAAGAGCTACCACTGGATGGGCCTGGTGCACATCCCCACCAACGGCAGCTGGCAGTGGGAGGACGGCAGCATCCTGAGCCCCAACCTGCTGACCATCATCGAGATGCAGAAGGGCGACTGCGCCCTGTACGCCAGCAGCTTCAAGGGCTACATCGAGAACTGCAGCACCCCCAACACCTACATCTGCATGCAGCGGACCGTGtaa

[0580]

[0581] SEQ ID NO:95 (Human NKG2D extracellular domain)

[0582] LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0583] SEQ ID NO:96 (Human NKG2D extracellular domain)

[0584] IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0585] SEQ ID NO:97 (SEQ ID NO:95 with the 8 most N-terminal amino acids removed)

[0586] PLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0587] SEQ ID NO:98 (Mouse NKG2D TM domain; UniProt accession number: 054709)

[0588] VVRVLAIALAIRFTLNTLMWLAI

[0589] SEQ ID NO:99 (Mouse NKG2D TM domain)

[0590] KISPMFVVRVLAIALAIRFTLNTLMWLAIFKETFQPV

[0591] SEQ ID NO:100 (Rat NKG2D)

[0592] MSKCHNYDLKPAKWDTSQEHQKQRSALPTSRPGENGIIRRRSSIEELKISPLFVVRVLVAAMTIRFTVITLTWLAVFITLLCNKEVSVSSREGYCGPCPNDWICHRNNCYQFFNENKAWNQSQASCLSQNSSLLKIYSKEEQDFLKLVKSYHWMGLVQSPANGSWQWEDGSSLSPNELTLVKTPSGTCAVYGSSFKAYTEDCSNPNTYICMKRAV

[0593] SEQ ID NO:101 (Rat NKG2D TM domain; amino acids 52 to 74 of UniProt accession number 070215)

[0594] LFVVRVLVAAMTIRFTVITLTWL

[0595] SEQ ID NO:102 (N5 polypeptide)

[0596] MALPVTALLLPLALLLHAARPDYKDDDDKLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGRRKRSGSGATNFSLLKQAGDVEENPGPMKISPMFVVRVLAIALAIRFTLNTLMWLAIFKETFQPVLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0597] SEQ ID NO:103 (Nucleic acid encoding the polypeptide of SEQ ID NO:102)

[0598]

[0599] SEQ ID NO:104 (Human IgG1 hinge region - amino acids 218 to 232 of UniProt accession number P0DOX5)

[0600] EPK SCDKTHTCPP CP

[0601] SEQ ID NO:105 (Amino acid sequence of CYAD - 01_10 (NKG2D - CD3ζ + ribosomal skipping peptide + DAP10))

[0602] MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGRRKRSGSGATNFSLLKQAGDVEENPGPMRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

Claims

1. A method for preparing immune response cells, wherein, The method includes: (a) Genetically modifying immune cells to express an NKG2D polypeptide; wherein the immune cells have been activated by anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix; or (b) Activating immune cells, wherein the immune cells have been genetically modified to express an NKG2D polypeptide, and wherein activation is carried out by anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix.

2. The method according to claim 1, wherein The method includes: (i) Activating the immune cells with anti-CD3 and anti-CD28 antibodies or fragments thereof conjugated to a nanomatrix; and (ii) Genetically modifying the immune cells to express the NKG2D polypeptide.

3. The method according to claim 1 or claim 2, wherein The immune cells are genetically modified to further express a DAP10 / DAP12 fusion polypeptide.

4. The method according to claim 3, wherein, The fusion polypeptide has the following structural formula from the N-terminus to the C-terminus: A-B-C-D-E, wherein A is an optional N-terminal sequence; B is a DAP10 polypeptide; C is an optional linker sequence; D is a DAP12 polypeptide; and E is an optional C-terminal sequence.

5. The method according to claim 3 or 4, wherein The DAP10 polypeptide is a functional variant of DAP10 comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity with the DAP10 polypeptide shown in SEQ ID NO:

1.

6. The method according to any one of claims 3 to 5, wherein The DAP10 polypeptide is a functional variant of the DAP10 polypeptide, and the DAP10 polypeptide is a truncated form of the polypeptide having the amino acid sequence shown in SEQ ID NO:

1.

7. The method according to claim 6, wherein, The truncated form of DAP10 comprises or consists of amino acids at positions 19 to 93, 19 to 69, 1 to 71, 19 to 71, 19 to 48, 49 to 69, 49 to 93 or 70 to 93 of SEQ ID NO:

1.

8. The method according to any one of claims 3 to 7, wherein, The DAP10 polypeptide comprises or consists of any one of the sequences of SEQ ID NO:1 to 8.

9. The method according to any one of claims 3 to 8, wherein The DAP12 polypeptide is a functional variant of DAP12 comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity with the DAP12 polypeptide shown in SEQ ID NO:

9.

10. The method according to any one of claims 3 to 9, wherein The DAP12 polypeptide is a functional variant of the DAP12 polypeptide, and the DAP12 polypeptide is a truncated form of the polypeptide having the amino acid sequence shown in SEQ ID NO:

9.

11. The method according to claim 10, wherein, The truncated form of DAP12 comprises or consists of amino acids at positions 22 to 113, 62 to 113, 22 to 61 or 41 to 61 of SEQ ID NO:

9.

12. The method according to any one of claims 3 to 11, wherein, The DAP12 polypeptide comprises or consists of any one of the sequences of SEQ ID NO:9 to 13.

13. The method according to any one of claims 3 to 12, wherein, The DAP10 polypeptide and the DAP12 polypeptide are linked by a linker.

14. The method according to claim 13, wherein, The linker comprises an amino acid sequence shown in any one of SEQ ID NO:18 to 46.

15. The method according to any one of claims 3 to 14, wherein The fusion polypeptide comprises a sequence shown in any one of SEQ ID NO:60 to 63.

16. The method according to any one of the preceding claims, wherein, The NKG2D polypeptide is a mammalian polypeptide, optionally a human polypeptide.

17. The method according to any one of the preceding claims, wherein, The amino acid sequence of the NKG2D polypeptide has at least about 85% sequence identity with the sequence shown in SEQ ID NO:

14.

18. The method according to any one of the preceding claims, wherein, The NKG2D polypeptide is a functional variant of the polypeptide having the amino acid sequence shown in SEQ ID NO:

14.

19. The method according to claim 18, wherein, The functional variant is a chimeric NKG2D polypeptide.

20. The method according to any one of the preceding claims, wherein, The immune cells are genetically modified to express a CXCR2 polypeptide.

21. The method according to claim 20, wherein, The CXCR2 polypeptide is a mammalian polypeptide, optionally a human polypeptide.

22. The method according to claim 20 or claim 21, wherein, The amino acid sequence of the CXCR2 polypeptide has at least about 85% sequence identity with the sequence shown in SEQ ID NO:

87.

23. The immune response cell according to claim 22, wherein The CXCR2 polypeptide has the amino acid sequence shown in SEQ ID NO:

87.

24. The method according to any one of the preceding claims, wherein, The nano - matrix comprises a mobile polymer - chain matrix conjugated with anti - CD3 and anti - CD28 antibodies or fragments thereof.

25. The method according to claim 24, wherein, The polymer chain comprises a polysaccharide.

26. The method according to any one of the preceding claims, wherein The diameter of the nano - matrix is from about 1 nm to about 500 nm.

27. The method according to any one of the preceding claims, wherein, The diameter of the nano - matrix is from about 50 nm to about 200 nm.

28. The method according to any one of the preceding claims, wherein The method does not include phorbol 12 - myristate 13 - acetate (PMA), phytohemagglutinin (PHA), or magnetic beads.

29. The method according to any one of the preceding claims, wherein, The immune cells are T cells or natural killer (NK) cells.

30. The method according to any one of the preceding claims, wherein, The immune cells are αβ or γδ T cells.

31. The method according to any one of the preceding claims, wherein, The immune cell is CD4 + T cell.

32. A method for preparing immune response cells, wherein, The method comprises: (a) activating the immune cells, wherein the activation does not include phorbol 12 - myristate 13 - acetate (PMA), phytohemagglutinin (PHA), or magnetic beads; and (b) genetically modifying the immune cells to express an NKG2D polypeptide.

33. An immune - responsive cell obtainable by the method according to any one of claims 1 to 32.

34. An immune response cell genetically modified to express an NKG2D polypeptide; wherein, The immune - responsive cell has been activated by anti - CD3 and anti - CD28 antibodies or fragments thereof conjugated with a nano - matrix.

35. The immune response cell according to claim 34, wherein, The immune - responsive cell is genetically modified to express an NKG2D polypeptide and a fusion polypeptide, the fusion polypeptide comprising: (i) a DNAX - activating protein 10 (DAP10) polypeptide or a functional variant thereof, and (ii) a DNAX - activating protein 12 (DAP12) polypeptide or a functional variant thereof.

36. The immune response cell according to claim 34 or claim 35, wherein The immune - responsive cell is genetically modified to further express a CXCR2 polypeptide.

37. The immune response cell according to any one of claims 34 to 36, wherein, The immune - responsive cell is a T cell or a natural killer (NK) cell.

38. The immune response cell according to claim 37, wherein, The immune - responsive cell comprises a population of T cells.

39. The immune response cell according to claim 38, wherein, The population of T cells has a CD4:CD8 ratio of at least about 1:

1.

40. The immune response cell according to claim 38 or 39, wherein, The population of T cells has a CD4:CD8 ratio of at least about 2:

1.

41. The immune response cell according to any one of claims 37 to 40, wherein, The amplification rate of the population of T cells is at least about 5 - fold every ten days.

42. A pharmaceutical composition comprising the immune - responsive cell according to any one of claims 33 to 41 and a pharmaceutically or physiologically acceptable diluent and / or carrier.

43. A kit comprising the immune - responsive cell according to any one of claims 33 to 41 or the pharmaceutical composition according to claim 42.

44. An immune response cell according to any one of claims 33 to 41 or a pharmaceutical composition according to claim 42, for treating or preventing a disease, optionally wherein the disease is cancer.

45. Use of an immune response cell according to any one of claims 33 to 41 or a pharmaceutical composition according to claim 42 in (i) therapy or (ii) the treatment of cancer.

46. A method for treating or preventing cancer in a subject, wherein, The method comprises administering to the subject an immune response cell according to any one of claims 33 to 41 or a pharmaceutical composition according to claim 42.

47. The immune response cells or pharmaceutical composition for the use according to claim 44, the use according to claim 45 or the method according to claim 46, wherein, The cancer is breast cancer.

48. An immune response cell or pharmaceutical composition, use or method according to the use of claim 47, wherein the breast cancer is triple-negative breast cancer.

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