Cellular immunotherapy compositions and uses thereof

Abstract

The present disclosure relates to cellular immunotherapy compositions comprising combinations of immune cells or subsets of cells modified with chimeric phagocytic receptors and chimeric antigen receptor / or T cell receptor binding proteins, and methods of using such cellular immunotherapy compositions.

Description

[0001] Declaration of sequence list

[0002] The sequence listings related to this application are provided in the form of a text file in lieu of a paper copy and are incorporated herein by reference. The text file containing the sequence listings is named 200265_407WO_SEQUENCE_LISTING.txt. This text file is 479KB in size, was created on March 26, 2019, and submitted electronically via EFS-Web. Background Technology

[0003] Genetically engineered receptor-modified immune cells (e.g., T cells) targeting cancer antigens have demonstrated clinical success in hematologic malignancies (e.g., CD19-specific chimeric antigen receptor therapy in leukemia). To name just a few, numerous clinical trials are underway using engineered receptors targeting CEA, GD2, mesothelin, IL13Rα, HER2, FAP, and L1CAM for adoptive cell immunotherapy of solid tumors. Engineered receptors include chimeric antigen receptors (CARs) and affinity-enhanced T-cell receptors (TCRs). However, treating solid tumors presents unique challenges, including: transport to the tumor site, physical barriers to reaching the tumor microenvironment, a stressful metabolic environment, and immunosuppressive mechanisms (e.g., expression of immune checkpoint molecules, production of inhibitory cytokines). Efforts are underway to increase the persistence and activity of immune cells in adoptive immunotherapy. Attached Figure Description

[0004] Figure 1 This is a schematic diagram illustrating different mechanisms of cell elimination achieved through the use of combinations of cell immunotherapy compositions disclosed herein. Cells expressing chimeric phagocytic receptors (CERs) (right) internalize and kill target cells within cell compartments using phagocytosis, while cells expressing CARs or TCRs (left) lyse target cells by releasing lysozyme molecules (such as granzymes and perforin) or inducing death ligands (e.g., Fas ligands) to “burst” the cells. Combinations of immunotherapy compositions containing both lysing and phagocytic cells can be used to enhance adoptive cell therapy (ACT).

[0005] Figure 2This is a schematic diagram illustrating an exemplary treatment method of adoptive cell immunotherapy, which utilizes a combination of modified immune cells that eliminate target cells through cell lysis and phagocytosis. After leukocyte removal, the graft is divided into defined cell populations. CD4+ T cells are transduced using a chimeric phagocytic receptor (CER), which specifically phagocytoses target cells using a phagocytic mechanism. CD8+ T cells are transduced using a chimeric antigen receptor (CAR) or T cell receptor (TCR) that promotes antigen-specific cell lysis. The cells are then expanded ex vivo and reintroduced in a defined proportion into the patient's body to target tumor cells expressing antigens. Autologous cell infusion utilizing both phagocytic and cytolytic modes of cell elimination can work in combination.

[0006] Figure 3 This is a schematic diagram of an exemplary in vitro co-culture experiment. CD8+ T cells were activated and transduced using a lentiviral cassette encoding a TCR specific to human papillomavirus 16 (HPV16) E7 protein, while CD4+ T cells from the same graft were activated and transduced using a lentiviral cassette encoding a CER. Both cell groups were expanded in vitro, combined at a 1:1 ratio, and co-cultured with HPV16 E7+ head and neck squamous cell carcinoma cells (SCC152).

[0007] Figure 4 This is a bar graph showing the number of caspase-positive SCC152 target cells in a co-culture assay using HPV16 E7 TCR-transduced CD8+ T cells and control or selected CER-transduced CD4+ T cells, as shown on the x-axis. Caspase intensity was measured by quantifying the red fluorescence intensity from a caspase 3 / 7 apoptosis reagent, which conjugates activated caspase 3 / 7 recognition motifs to a red reagent that fluoresces upon lysis. The caspase 3 / 7 apoptosis reagent was added to the co-culture assay after 6 hours, and fluorescence was detected using a BZ-X710 Keyence microscope and hybridization capture software. SCC152 target cells were similarly identified (using green fluorescent protein (GFP) transduction). The Y-axis represents the percentage of caspase-positive targets (caspase events # / GFP target cells #) * 100.

[0008] Figure 5This is a bar graph showing the intensity of caspase in SCC152 target cells quantified in a co-culture assay containing CD8+ T cells transduced using HPV16 E7 TCR and CD4+ T cells transduced using control or selected CER, as shown on the x-axis. Caspase intensity was measured by quantifying the red fluorescence intensity from a caspase 3 / 7 apoptosis reagent, which conjugates activated caspase 3 / 7 recognition motifs to a red reagent that fluoresces upon lysis. The caspase 3 / 7 apoptosis reagent was added to the co-culture assay after 6 hours, and fluorescence was detected using a BZ-X710 Keyence microscope and hybridization capture software. The Y-axis represents the intensity of the caspase reagent in arbitrary units (au).

[0009] Figure 6 These are fluorescence micrographs of a co-culture assay containing CD8+ T cells transduced with HPV16 E7 TCR at a 1:1 ratio, CD4+ T cells transduced with control (blue), and SCC152 cells (green). The red signals (representative red signals are indicated by white arrows) are fluorescence signals from the caspase reagent, which was added to the co-culture assay 6 hours later.

[0010] Figure 7 These are fluorescence micrographs of a co-culture assay containing CD8+ T cells transduced with HPV16 E7 TCR at a 1:1 ratio, CD4+ T cells transduced with CER5 (blue), and SCC152 cells (green). The red signals (representative red signals are indicated by white arrows) are fluorescence signals from the caspase reagent added to the co-culture assay 6 hours later.

[0011] Figure 8 These are fluorescence micrographs of a co-culture assay containing CD8+ T cells transduced with HPV16 E7 TCR at a 1:1 ratio, CD4+ T cells transduced with CER17 (blue), and SCC152 cells (green). The red signals (representative red signals are indicated by white arrows) are fluorescence signals from the caspase reagent, which was added to the co-culture assay 6 hours later.

[0012] Figure 9These are fluorescence micrographs of a co-culture assay containing CD8+ T cells transduced with HPV16 E7 TCR at a 1:1 ratio, CD4+ T cells transduced with CER19 (blue), and SCC152 cells (green). The red signals (representative red signals are indicated by white arrows) are fluorescence signals from the caspase reagent added to the co-culture assay 6 hours later.

[0013] Figure 10 These are fluorescence micrographs of a co-culture assay containing CD8+ T cells transduced with HPV16 E7 TCR at a 1:1 ratio, CD4+ T cells transduced with CER21 (blue), and SCC152 cells (green). The red signals (representative red signals are indicated by white arrows) are fluorescence signals from the caspase reagent added to the co-culture assay 6 hours later.

[0014] Figure 11 These are fluorescence micrographs of a co-culture assay containing CD8+ T cells transduced with HPV16 E7 TCR at a 1:1 ratio, CD4+ T cells transduced with CER23 (blue), and SCC152 cells (green). The red signals (representative red signals are indicated by white arrows) are fluorescence signals from the caspase reagent added to the co-culture assay 6 hours later.

[0015] Figure 12 These are fluorescence micrographs of a co-culture assay containing CD8+ T cells transduced with HPV16 E7 TCR at a 1:1 ratio, CD4+ T cells transduced with CER26 (blue), and SCC152 cells (green). The red signals (representative red signals are indicated by white arrows) are fluorescence signals from the caspase reagent added to the co-culture assay 6 hours later.

[0016] Figure 13 These are fluorescence micrographs of a co-culture assay containing CD8+ T cells transduced with HPV16 E7 TCR at a 1:1 ratio, CD4+ T cells transduced with CER27 (blue), and SCC152 cells (green). The red signals (representative red signals are indicated by white arrows) are fluorescence signals from the caspase reagent added to the co-culture assay 6 hours later.

[0017] Figure 14These are fluorescence micrographs of a co-culture assay containing CD8+ T cells transduced with HPV16 E7 TCR at a 1:1 ratio, CD4+ T cells transduced with CER103B (blue), and SCC152 cells (green). The red signals (representative red signals are indicated by white arrows) are fluorescence signals from the caspase reagent added to the co-culture assay 6 hours later.

[0018] Figure 15 These are fluorescence micrographs of a co-culture assay containing CD8+ T cells transduced with HPV16 E7 TCR at a 1:1 ratio, CD4+ T cells transduced with CER104 (blue), and SCC152 cells (green). The red signals (representative red signals are indicated by white arrows) are fluorescence signals from the caspase reagent, which was added to the co-culture assay 6 hours later.

[0019] Figure 16 These are fluorescence micrographs of a co-culture assay containing CD8+ T cells transduced with HPV16 E7 TCR at a 1:1 ratio, CD4+ T cells transduced with CER105 (blue), and SCC152 cells (green). The red signals (representative red signals are indicated by white arrows) are fluorescence signals from the caspase reagent added to the co-culture assay 6 hours later.

[0020] Figure 17 These are fluorescence micrographs of a co-culture assay containing CD8+ T cells transduced with HPV16 E7 TCR at a 1:1 ratio, CD4+ T cells transduced with CER106 (blue), and SCC152 cells (green). The red signals (representative red signals are indicated by white arrows) are fluorescence signals from the caspase reagent added to the co-culture assay 6 hours later.

[0021] Figure 18 These are fluorescence micrographs of a co-culture assay containing CD8+ T cells transduced with HPV16 E7 TCR at a 1:1 ratio, CD4+ T cells transduced with CER116 (blue), and SCC152 cells (green). The red signals (representative red signals are indicated by white arrows) are fluorescence signals from the caspase reagent added to the co-culture assay 6 hours later.

[0022] Figure 19This is a bar chart showing the lactate dehydrogenase (LDH) cytotoxicity assay. CD8+ T cells transduced with HPV16E7TCR and CD4+ T cells transduced with CER104 were co-cultured with SCC152 target cells at different target cell:effect cell ratios (0.5:1, 1:1, 1:2.5, 1:5, 1:10, 1:20) for 4 hours.

[0023] Figure 20 This is a bar chart showing the quantification of SCC152 HPV+ head and neck squamous cell carcinomas over time. SCC152 target cells were co-cultured at a 1:1:1 ratio with CD8+ T cells transduced using HPV E7 TCR + CD4+ T cells transduced using selected CER or control (CD8+ T cells transduced using HPV E7 TCR + CD4+ T cells transduced using control). The number of target cells was quantified using imaging software. 0 hours, 24 hours, and 48 hours are shown from left to right.

[0024] Figure 21 This is a time-lapse image from a 96-well plate co-culture assay, which involved co-culturing SCC152 target cells with CD8+ T cells transduced using HPV16E7TCR and CD4+ T cells transduced using CER5, CER17, or CER19 at a 1:1:1 ratio. SCC152 HPV+ target cells were quantified using automated cell counting software. Compared to the control (CD8+ T cells + HPV16E7TCR + CD4+ T cells control), the number of SCC152 cells showed a decrease over time in the co-culture assay with CD8+ T cells transduced using HPV16E7TCR and CD4+ T cells transduced using CER5, CER17, or CER19.

[0025] Figure 22 This is a time-lapse image from a 96-well plate co-culture assay, which involved co-culturing SCC152 target cells with CD8+ T cells transduced using HPV16E7TCR and CD4+ T cells transduced using CER21, CER23, or CER26 at a 1:1:1 ratio. SCC152 HPV+ target cells were quantified using automated cell counting software. Compared to the control (CD8 T cells + HPV16 E7TCR + CD4 T cells), the number of SCC152 cells showed a decrease over time in the co-culture assay with CD8+ T cells transduced using HPV16 E7TCR and CD4+ T cells transduced using CER21, CER23, or CER26.

[0026] Figure 23This is a time-lapse image from a 96-well plate co-culture assay, which involved co-culturing SCC152 target cells with CD8+ T cells transduced using HPV16E7TCR and CD4+ T cells transduced using CER103b, CER104, or CER105 at a 1:1:1 ratio. SCC152 HPV+ target cells were quantified using automated cell counting software. Compared to the control (CD8+ T cells + HPV16 E7 TCR + CD4+ T cells control), the number of SCC152 cells showed a decrease over time in the co-culture with CD8+ T cells transduced using HPV16 E7 TCR and CD4+ T cells transduced using CER103b, CER104, or CER105.

[0027] Figure 24 This is a time-lapse image from a 96-well plate co-culture assay, which involved co-culturing SCC152 target cells with CD8+ T cells transduced using HPV16E7 TCR and CD4+ T cells transduced using CER106, CER116, or CER27 at a 1:1:1 ratio. SCC152 HPV+ target cells were quantified using automated cell counting software. Compared to the control (CD8+ T cells + HPV16E7 TCR + CD4+ T cells control), the number of SCC152 cells showed a decrease over time in the co-culture assay with CD8+ T cells transduced using HPV16E7 TCR and CD4+ T cells transduced using CER106, CER116, or CER27.

[0028] Figure 25 This is a time-lapse image from a 96-well plate co-culture experiment. SCC152 target cells were co-incubated with CD4+ T cells (control) and quantified using automated cell counting software. The number of SCC152 cells co-cultured with control CD4+ T cells did not decrease over time.

[0029] Figure 26 This is a line graph showing caspase 3 / 7 induction over time in a co-culture assay. The graph shows the number of caspase-positive SCC52 target cells in the co-culture assay, which included CD8+ T cells transduced using HPV16 E7 TCR and CD4+ T cells transduced using control or selected CER. Caspase intensity was measured by quantifying the red fluorescence intensity from a caspase 3 / 7 apoptosis reagent that couples activated caspase 3 / 7 recognition motifs to a red reagent that fluoresces upon lysis. Measurements were taken at 2, 6, 8, and 10 hours of co-culture.

[0030] Figure 27 This is a 3D bar chart showing the profile of enhanced effector cytokines elicited after co-culturing SCC152 cells with CD8+ T cells transduced using HPV16 E7 TCR and CD4+ T cells transduced using selected CER. SCC152 target cells were co-treated with CD8+ T cells transduced using HPV16 E7 TCR and CD4+ T cells transduced using selected CER at a 1:1 ratio of effector cells to target cells. The secretion of antigen-specific cytokines was determined by measuring cytokine concentrations in the cell supernatant from each co-culture experiment using a medium-sized multiarray cytokine plate. The combination of CD8+ T cells / HPV16 E7 TCR + CD4+ T cells / CER enhanced IFN-γ, IL-2, TNFα, and IL-13 responses compared to either CD8+ T cells / HPV16 E7 TCR alone or with CD4+ T cells transduced using truncated EGFR. The following cytokines were measured in the assay: IFN-γ, IL-2, TNFα, IL-4, IL-6, IL-12b, IL-13, IL-1b and IL-10. The columns shown from front to back are: untransduced CD8+ T cells; CD8+ T cells transduced with HPV E7 TCR; CD8+ T cells transduced with HPV E7 TCR + CD4+ T cells transduced with EGFR; CD8+ T cells transduced with HPV E7 TCR + CD4+ T cells transduced with CER21; CD8+ T cells transduced with HPV E7 TCR + CD4+ T cells transduced with CER25; CD8+ T cells transduced with HPV E7 TCR + CD4+ T cells transduced with CER29; CD8+ T cells transduced with HPV E7 TCR + CD4+ T cells transduced with CER31; and CD8+ T cells transduced with HPV E7 TCR + CD4+ T cells transduced with CER112.

[0031] Figure 28This is a bar chart representing the quantification of CD4+ T cell-CER-mediated phagocytosis of SCC152 target cells. Results are calculated as ((number of phagocytic target events) / (total number of effectors))*100, from 3x3 40x images taken 4 hours after the start of co-culture assays. CD8+ T cells transduced with HPV16 E7 TCR at a 1:1:0.5 ratio and CD4+ T cells transduced with selected CERs (CER5, CER17, CER19, CER21, CER23, CER26, CER27, CER103b, CER104, CER105, CER106, or CER116) were co-cultured with SCC152 head and neck squamous cell carcinoma target cells for 4 hours and then imaged.

[0032] Figure 29 This is a bar chart representing the quantification of CD4+ T cell-CER-mediated phagocytosis of SCC152 target cells. Results are calculated as (median area of ​​target events in effector cells * phagocytosis %), from 3x3 40x images taken 4 hours after the start of co-culture assays. CD8+ T cells transduced with HPV16 E7 TCR at a 1:1:0.5 ratio and CD4+ T cells transduced with selected CERs (CER5, CER17, CER19, CER21, CER23, CER26, CER27, CER103b, CER104, CER105, CER106, or CER116) were co-cultured with SCC152 head and neck squamous cell carcinoma target cells for 4 hours and then imaged.

[0033] Figure 30 The images show fluorescence micrographs (40X, left panel) of CD8+ T cells transduced with HPV16 E7 TCR and truncated EGFR tags (unstained), CD4+ T cells transduced with controls (blue), and SCC152 target cells (red) in a 4-hour co-culture assay. Phagocytic events were identified by red target events labeled on blue effector cells. These events were quantified using hybridization capture software (Keyence BZ-X710) to provide the number of phagocytic target sites, the total number of effector cells, and the area occupied by phagocytosed target cells within effector cells (right panel).

[0034] Figure 31The images show fluorescence micrographs (40X, left panel) of CD8+ T cells (unstained) transduced with HPV16 E7 TCR and truncated EGFR tags, CD4+ T cells (blue) transduced with CER5, and SCC152 target cells (red) in a 4-hour co-culture assay. Engulfment events were identified by red target events labeled on blue effector cells. These events were quantified using hybridization capture software (Keyence BZ-X710) to provide the number of engulfed target sites, the total number of effector cells, and the area occupied by engulfed target cells within effector cells (right panel).

[0035] Figure 32 The images show fluorescence micrographs (40X, left panel) of CD8+ T cells transduced with HPV16 E7 TCR and truncated EGFR tags (unstained), CD4+ T cells transduced with CER17 (blue), and SCC152 target cells (red) in a 4-hour co-culture assay. Engulfment events were identified by red target events labeled on blue effector cells. These events were quantified using hybridization capture software (Keyence BZ-X710) to provide the number of engulfed target sites, the total number of effector cells, and the area occupied by engulfed target cells within effector cells (right panel).

[0036] Figure 33 The images show fluorescence micrographs (40X, left panel) of CD8+ T cells (unstained) transduced with HPV16 E7 TCR and truncated EGFR tags, CD4+ T cells (blue) transduced with CER19, and SCC152 target cells (red) in a 4-hour co-culture assay. Engulfment events were identified by red target events labeled on blue effector cells. These events were quantified using hybridization capture software (Keyence BZ-X710) to provide the number of engulfed target sites, the total number of effector cells, and the area occupied by engulfed target cells within effector cells (right panel).

[0037] Figure 34 The images show fluorescence micrographs (40X, left panel) of CD8+ T cells (unstained) transduced with HPV16 E7 TCR and truncated EGFR tags, CD4+ T cells (blue) transduced with CER21, and SCC152 target cells (red) in a 4-hour co-culture assay. Engulfment events were identified by red target events labeled on blue effector cells. These events were quantified using hybridization capture software (Keyence BZ-X710) to provide the number of engulfed target sites, the total number of effector cells, and the area occupied by engulfed target cells within effector cells (right panel).

[0038] Figure 35The images show fluorescence micrographs (40X, left panel) of CD8+ T cells (unstained) transduced with HPV16 E7 TCR and truncated EGFR tags, CD4+ T cells (blue) transduced with CER23, and SCC152 target cells (red) in a 4-hour co-culture assay. Engulfment events were identified by red target events labeled on blue effector cells. These events were quantified using hybridization capture software (Keyence BZ-X710) to provide the number of engulfed target sites, the total number of effector cells, and the area occupied by engulfed target cells within effector cells (right panel).

[0039] Figure 36 The images show fluorescence micrographs (40X, left panel) of CD8+ T cells (unstained) transduced with HPV16 E7 TCR and truncated EGFR tags, CD4+ T cells (blue) transduced with CER26, and SCC152 target cells (red) in a 4-hour co-culture assay. Engulfment events were identified by red target events labeled on blue effector cells. These events were quantified using hybridization capture software (Keyence BZ-X710) to provide the number of engulfed target sites, the total number of effector cells, and the area occupied by engulfed target cells within effector cells (right panel).

[0040] Figure 37 The images show fluorescence micrographs (40X, left panel) of CD8+ T cells (unstained) transduced with HPV16 E7 TCR and truncated EGFR tags, CD4+ T cells (blue) transduced with CER27, and SCC152 target cells (red) in a 4-hour co-culture assay. Engulfment events were identified by red target events labeled on blue effector cells. These events were quantified using hybridization capture software (Keyence BZ-X710) to provide the number of engulfed target sites, the total number of effector cells, and the area occupied by engulfed target cells within effector cells (right panel).

[0041] Figure 38 The images show fluorescence micrographs (40X, left panel) of CD8+ T cells (unstained) transduced with HPV16 E7 TCR and truncated EGFR tags, CD4+ T cells (blue) transduced with CER103b, and SCC152 target cells (red) in a 4-hour co-culture assay. Engulfment events were identified by red target events labeled on blue effector cells. These events were quantified using hybridization capture software (Keyence BZ-X710) to provide the number of engulfed target sites, the total number of effector cells, and the area occupied by engulfed target cells within effector cells (right panel).

[0042] Figure 39The images show fluorescence micrographs (40X, left panel) of CD8+ T cells (unstained) transduced with HPV16 E7 TCR and truncated EGFR tags, CD4+ T cells (blue) transduced with CER104, and SCC152 target cells (red) in a 4-hour co-culture assay. Engulfment events were identified by red target events labeled on blue effector cells. These events were quantified using hybridization capture software (Keyence BZ-X710) to provide the number of engulfed target sites, the total number of effector cells, and the area occupied by engulfed target cells within effector cells (right panel).

[0043] Figure 40 The images show fluorescence micrographs (40X, left panel) of CD8+ T cells (unstained) transduced with HPV16 E7 TCR and truncated EGFR tags, CD4+ T cells (blue) transduced with CER105, and SCC152 target cells (red) in a 4-hour co-culture assay. Engulfment events were identified by red target events labeled on blue effector cells. These events were quantified using hybridization capture software (Keyence BZ-X710) to provide the number of engulfed target sites, the total number of effector cells, and the area occupied by engulfed target cells within effector cells (right panel).

[0044] Figure 41 The images show fluorescence micrographs (40X, left panel) of CD8+ T cells transduced with HPV16 E7 TCR and truncated EGFR tags (unstained), CD4+ T cells transduced with CER106 (blue), and SCC152 target cells (red) in a 4-hour co-culture assay. Engulfment events were identified by red target events labeled on blue effector cells. These events were quantified using hybridization capture software (Keyence BZ-X710) to provide the number of engulfed target sites, the total number of effector cells, and the area occupied by engulfed target cells within effector cells (right panel).

[0045] Figure 42 The images show fluorescence micrographs (40X, left panel) of CD8+ T cells transduced with HPV16 E7 TCR and truncated EGFR tags (unstained), CD4+ T cells transduced with CER116 (blue), and SCC152 target cells (red) in a 4-hour co-culture assay. Engulfment events were identified by red target events labeled on blue effector cells. These events were quantified using hybridization capture software (Keyence BZ-X710) to provide the number of engulfed target sites, the total number of effector cells, and the area occupied by engulfed target cells within effector cells (right panel).

[0046] Figure 43This is a bar graph showing the enhanced cell lysis of SCC152 target cells in combination with CD4+ T cells transduced with various CERs containing the TRAF signaling domain, as measured by caspase induction, compared to administration of HPV16 E7 TCR alone. Human primary CD8+ cells transduced with HPV16 E7 TCR were co-cultured with SCC152 cells alone or with CD4+ T cells transduced with selected CERs (from left to right: control, CER29, CER30, CER110, CER112, CER113, CER114, CER115, CER116, or CER117) in a 1:1 ratio (CD4:CD8). In the co-culture assay, the number of caspase-positive SCC152 target cells was measured by quantifying the red fluorescence intensity from a caspase 3 / 7 apoptosis reagent, which conjugates activated caspase 3 / 7 recognition motifs to a red reagent that fluoresces upon lysis. The caspase 3 / 7 apoptosis reagent was added to the co-culture assay after 6 hours, and fluorescence was detected using a BZ-X710 Keyence microscope and hybridization capture software. SCC152 target cells were transduced using green fluorescent protein (GFP) for visualization. The Y-axis represents (caspase event # / GFP target cell #)*100.

[0047] Figure 44This is a bar graph showing the enhanced cell lysis of SCC152 target cells in combination with CD4+ T cells transduced with various CERs containing the TRAF signaling domain, as measured by caspase induction, compared to administration of HPV16 E7 TCR alone. Human primary CD8+ cells transduced with HPV16 E7 TCR were co-cultured with SCC152 cells alone or with CD4+ T cells transduced with selected CERs (from left to right: control, CER29, CER30, CER110, CER112, CER113, CER114, CER115, CER116, or CER117) in a 1:1 ratio (CD4:CD8). In the co-culture assay, the number of caspase-positive SCC152 target cells was measured by quantifying the red fluorescence intensity from the caspase 3 / 7 apoptosis reagent, which conjugates activated caspase 3 / 7 recognition motifs to the red reagent that fluoresces upon lysis. Six hours later, the caspase 3 / 7 apoptosis reagent was added to the co-culture assay, and fluorescence was detected using a BZ-X710 Keyence microscope and hybridization capture software. The Y-axis represents the intensity of the caspase reagent in arbitrary units (au).

[0048] Figure 45 These are fluorescence micrographs of a co-culture assay that included CD8+ T cells transduced with HPV16 E7 TCR at a 1:1 ratio (CD8:CD4) and control-transduced CD4+ T cells (blue), as well as head and neck squamous cell carcinoma SCC152 cells (green). The red signal is the fluorescence signal from the caspase reagent, which was added to the co-culture assay 6 hours later.

[0049] Figure 46 These are fluorescence micrographs of a co-culture assay that included CD8+ T cells transduced with HPV16 E7 TCR and CD4+ T cells transduced with CER29 at a 1:1 ratio (CD8:CD4) (blue), as well as head and neck squamous cell carcinoma SCC152 cells (green). The red signal is the fluorescence signal from the caspase reagent, which was added to the co-culture assay 6 hours later.

[0050] Figure 47These are fluorescence micrographs of a co-culture assay that included CD8+ T cells transduced with HPV16 E7 TCR and CD4+ T cells transduced with CER30 at a 1:1 ratio (CD8:CD4) (blue), as well as head and neck squamous cell carcinoma SCC152 cells (green). The red signal is the fluorescence signal from the caspase reagent, which was added to the co-culture assay 6 hours later.

[0051] Figure 48 These are fluorescence micrographs of a co-culture assay that included CD8+ T cells transduced with HPV16 E7 TCR and CD4+ T cells transduced with CER110 at a 1:1 ratio (CD8:CD4) (blue), as well as head and neck squamous cell carcinoma SCC152 cells (green). The red signal is the fluorescence signal from the caspase reagent, which was added to the co-culture assay 6 hours later.

[0052] Figure 49 These are fluorescence micrographs of a co-culture assay that included CD8+ T cells transduced with HPV16 E7 TCR and CD4+ T cells transduced with CER112 at a 1:1 ratio (CD8:CD4) (blue), as well as head and neck squamous cell carcinoma SCC152 cells (green). The red signal is the fluorescence signal from the caspase reagent, which was added to the co-culture assay 6 hours later.

[0053] Figure 50 These are fluorescence micrographs of a co-culture assay that included CD8+ T cells transduced with HPV16 E7 TCR and CD4+ T cells transduced with CER113 at a 1:1 ratio (CD8:CD4) (blue), as well as head and neck squamous cell carcinoma SCC152 cells (green). The red signal is the fluorescence signal from the caspase reagent, which was added to the co-culture assay 6 hours later.

[0054] Figure 51 These are fluorescence micrographs of a co-culture assay that included CD8+ T cells transduced with HPV16 E7 TCR and CD4+ T cells transduced with CER114 at a 1:1 ratio (CD8:CD4) (blue), and head and neck squamous cell carcinoma SCC152 cells (green). The red signal is the fluorescence signal from the caspase reagent, which was added to the co-culture assay 6 hours later.

[0055] Figure 52These are fluorescence micrographs of a co-culture assay that included CD8+ T cells transduced with HPV16 E7 TCR and CD4+ T cells transduced with CER115 at a 1:1 ratio (CD8:CD4) (blue), as well as head and neck squamous cell carcinoma SCC152 cells (green). The red signal is the fluorescence signal from the caspase reagent, which was added to the co-culture assay 6 hours later.

[0056] Figure 53 These are fluorescence micrographs of a co-culture assay that included CD8+ T cells transduced with HPV16 E7 TCR and CD4+ T cells transduced with CER116 at a 1:1 ratio (CD8:CD4) (blue), and head and neck squamous cell carcinoma SCC152 cells (green). The red signal is the fluorescence signal from the caspase reagent, which was added to the co-culture assay 6 hours later.

[0057] Figure 54 These are fluorescence micrographs of a co-culture assay that included CD8+ T cells transduced with HPV16 E7 TCR and CD4+ T cells transduced with CER117 at a 1:1 ratio (CD8:CD4) (blue), as well as head and neck squamous cell carcinoma SCC152 cells (green). The red signal is the fluorescence signal from the caspase reagent, which was added to the co-culture assay 6 hours later.

[0058] Figure 55 This is a bar chart representing the quantification of CD4+ T cell-CER-mediated phagocytosis of SCC152 target cells. The percentage of phagocytosis is calculated as ((number of target cell phagocytosis events) / (total number of effector cells)) * 100. These quantities were captured from 3x3 images using a Keyence BZ-X710 fluorescence microscope (40x resolution) 4 hours after the start of the co-culture assay. CD8+ T cells transduced with HPV16 E7 TCR and CD4+ T cells transduced with selected CERs (CER29, CER30, CER110, CER112, CER113, CER114, CER116, or CER117) were co-cultured with SCC152 target cells for 4 hours and imaged at a 1:1:0.5 ratio (CD8:CD4:target cells).

[0059] Figure 56This is a bar chart representing the quantification of CD4+ T cell-CER-mediated phagocytosis of SCC152 target cells. The adjusted phagocytosis index was calculated as (median area of ​​target events in effector cells * phagocytosis %). These quantities were calculated from 3x3 images using a Keyence BZ-X710 fluorescence microscope (40x resolution) 4 hours after the start of the co-culture assay. CD8+ T cells transduced with HPV16 E7 TCR and CD4+ T cells transduced with selected CERs (CER29, CER30, CER110, CER112, CER113, CER114, CER116, or CER117) were co-cultured with SCC152 target cells for 4 hours and imaged at a 1:1:0.5 ratio (CD8:CD4:target cells).

[0060] Figure 57 This is a bar graph showing the quantification of SCC152 HPV16+ target cell loss over time during the co-culture assay. SCC152 cells were transduced with green fluorescent protein (GFP) and co-cultured at a 1:1:1 ratio (CD8:CD4:target cells) with CD8+ T cells transduced with HPV16 E7 TCR and CD4+ T cells transduced with selected CERs (CER29, CER30, CER110, CER112, CER113, CER114, CER116, or CER117) or CD4+ T cell controls. The number of target cells was quantified at various time points (0 h, 12 h, 24 h, and 36 h) during the co-culture process using fluorescence microscopy and imaging software. Many CERs (CER30, CER112, CER113, CER114, CER116, and CER117) showed almost complete clearance of SCC152 target cells at 36 hours.

[0061] Figure 58 Is Figure 57 The fluorescence microscopy images of the co-culture assay described in the figure show the clearance process of SCC152 cells (pink) co-cultured with CD8+ T cells transduced using HPV16 E7 TCR and CD4+ T cells transduced using selected CERs (columns from left to right: control, CER29, CER30, CER110, or CER112) (rows from top to bottom: 0 h r, 12 h, 24 h, and 36 h).

[0062] Figure 59 Is Figure 58The fluorescence microscopy images of the co-culture assay described in the figure show the clearance process of SCC152 cells (pink) co-cultured with CD8+ T cells transduced using HPV16 E7 TCR and CD4+ T cells transduced using selected CERs (columns from left to right: control, CER113, CER114, CER116, or CER117) (rows from top to bottom: 0 hours, 12 hours, 24 hours, and 36 hours).

[0063] Figures 60A-60B The study demonstrated the phagocytic activity of HPV+SCC152 cells by analyzing CD4+ T cells expressing CER. Figure 60A The amplitude and breadth curves of CD4+ T cell phagocytosis, expressed as CER type, are shown. Figure 60B This image shows fluorescence micrographs of SCC152 target cells phagocytosed by CD4+ CER126-transduced T cells. The top image is a magnified view of the cells in the lower left image, showing SCC152 cells phagocytosed by CER126-transduced CD4+ T cells. The lower left image shows SCC152 cells phagocytosed by CE126R-transduced CD4+ T cells (stained with pHrodo red); the lower right image is the same micrograph showing CER126-transduced CD4+ T cells luminescent with CELLTRACE violet. White arrows indicate CD8+ T cells transduced using E7-specific TCRs, which are pHrodo red negative. Figure 60B (Lower left image). Software reproduction of swallowing ( Figure 60B (The image at the bottom right).

[0064] Figure 61 This study demonstrates cytokine secretion from co-cultured CD4+ T cells expressing CER with CD8+ T cells expressing E7-specific TCR. Adding CER-expressing CD4+ T cells to CD8+ T cells expressing E7-specific TCR enhances IFNγ secretion.

[0065] Figure 62 A schematic diagram of an exemplary antigen presentation assay is shown. In the phagocytosis assay step, CD4+ and CD8+ T cell lines expressing CER were co-cultured overnight with CD4+ and CD8+ T cells expressing HPV E7-specific TCRs and SCC152 (HPV+) cells. The following day, CER+ T cells were subsequently sorted by FACS. The FACS image depicts CT purple + CER. After FACS purification, antigen presentation of HPV oncoproteins was evaluated. Cells expressing CER were co-cultured with E6 and E7-specific TCR / NFAT reporter cell lines at a 1:2 ratio, and NFAT activation over time was measured using a microplate reader.

[0066] Figure 63 A line graph showing NFAT activation in E6 / E7 TCR-transduced T cells containing the NFAT reporter gene after co-culturing with CD4+ and CD8+ CER123-transduced T cells, which were co-cultured with HPV+ tumor cells and CD4+ / CD8+ E7 TCR-transduced T cells, as shown. Figure 62 As illustrated in the diagram, CD4+ and CD8+ T cell lines expressing CER were able to cross-present the E7 HPV oncoprotein to T cells expressing the E7 TCR / NFAT reporter protein after engulfing HPV+ tumor cells, as measured by NFAT activation.

[0067] Figures 64A-64B The viSNE atlas displays mass cytometry data of CER-transduced CD4+ T cells after antigen encounter. CER-transduced CD4+ T cells were co-cultured with E7-specific TCR-transduced CD8+ T cells and HPV+SCC152 target cells, and then questioned using CyTof mass cytometry. Intact CER-CD4+ T cells are shown in the figure displaying the tSNE1 and tSNE2 axes. Nine intracellular markers were used for viSNE analysis. Each point represents a single cell. Figure 64A The graph is colored using measured markers (GM-CSF, MIP1b, perforin, TNF, IL-17, granzyme B, IL-4, IL-2, and IFNγ) to show the phenotype across viSNE “islands.” For each marker, red indicates high expression and blue indicates low expression. Figure 64B CD4+ T cell populations were generated from all 32 markers using a clustering algorithm and overlaid on the viSNE map. Arrows indicate the enrichment of islands expressing the intracellular marker IFNγ in samples containing CER104, CER116, and CER117.

[0068] Figures 65A-65B This image shows a viSNE atlas of mass cytometry data of CER-expressing CD4+ T cells upon antigen encounter. CER-transduced CD4+ T cells were co-cultured with E7-specific TCR-transduced CD8+ T cells and HPV+SCC152 target cells, and then questioned using CyTof mass cytometry. Intact CER-CD4+ T cells are shown in the figure displaying the tSNE1 and tSNE2 axes. Eighteen cell surface markers were used for viSNE analysis. Each point represents a single cell. Figure 65ACD4+ T cell populations were generated from all 18 markers using a clustering algorithm and overlaid on a viSNE map. Arrows indicate the enrichment of islands expressing the T cell activation marker CD69 in samples containing CER104 and CER116. Figure 65B The color-coded plot shows the phenotype of viSNE "islands". For each marker, red indicates high expression and blue indicates low expression. The highlighted areas represent cells expressing the T cell activation marker C69.

[0069] Figure 66A-65B This image shows a viSNE atlas of mass cytometry data of CER-expressing CD4+ T cells upon antigen encounter. CER-transduced CD4+ T cells were co-cultured with E7-specific TCR-transduced CD8+ T cells and HPV+SCC152 target cells, and then questioned using CyTof mass cytometry. Intact CER-CD4+ T cells are shown in the figure displaying the tSNE1 and tSNE2 axes. Eighteen cell surface markers were used for viSNE analysis. Each point represents a single cell. Figure 66A CD4+ T cell populations were generated from all 18 markers using a clustering algorithm and overlaid on a viSNE atlas. Arrows indicate the loss of islands expressing the initial T cell marker CD45RA in the CCR7+ populations in CER104 and CER116 samples compared to the control. Figure 66B The color-coded plot shows the phenotype of viSNE "islands". For each marker, red indicates high expression and blue indicates low expression. The highlighted areas represent cells with the naïve T cell marker CD45RA. CD4+ T cells transduced by CER104 and CER116 are associated with the formation of memory after encountering antigens.

[0070] Figures 67A-67B The study showed that CD4+ and CD8+ T cell subtypes, and that CER-modified CD4+ T cells had most of the phagocytic activity. Figure 67A The FACS atlas of phagocytosis assay is shown, in which hCER104-modified CD4+ and CD8+ T cells were cultured overnight with pHrodo-labeled HCC827 NSCLC adenocarcinoma cells, and pHrodo positivity was evaluated by FACS (boxes indicate phagocytosis % - CD8+ T cells 9.81% and CD4+ T cells 42.0%). Figure 67B This is a bar chart comparing the phagocytic frequency of CER-modified CD4+ T cells and CER-modified CD8+ T cells.

[0071] Figure 68The viability of HCC827 NSCLC adenocarcinoma cells co-cultured with hCER104-modified T cells was demonstrated. Cell viability was measured at a 5:1 effector cell to target cell ratio, with or without the EGFR small molecule inhibitor (1 nM osimertinib). Co-cultures of hCER104-modified CD3+ T cells were compared with co-cultures of purified hCER104-modified CD4+ T cells. Cell viability was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

[0072] Figure 69 Phase-contrast microscopy images of hCER104-modified CD3+ T cells or purified hCER104-modified CD4+ T cells after co-culturing with HCC827 cells and 1 nM osimertinib for 48 hours are shown. Specific target-cell killing of HCC827 was observed in hCER104-modified T cells in the presence of osimertinib.

[0073] Figure 70 Tumor growth of SCC152 HPV+ squamous cell carcinoma of the head and neck is shown in NSG mice. One x 10^6 CD8+ T cells engineered with HPV E7-specific TCRs were injected with or without three x 10^6 CD4+ T cells engineered with CER104. Data represent the mean of tumor caliper measurements (n = 5 mice / group).

[0074] Figure 71 This study demonstrates HCC827 xenografting. The growth of HCC827 / luciferase+ cells in NSG mice was measured using bioluminescence imaging following adoptive transfer of purified hCER122-modified CD4+ T cells or hCER122-modified CD+ T cells with a concomitant targeting inhibitor. Data represent mean values ​​(n = 5 mice / treatment group).

[0075] Figure 72 Immunofluorescence staining from the HCC827 xenograft study is shown on day 16. Tumor staining shows PD1+CD4+ T cells (actively functioning) infiltrating the tumor stroma. Tumor specimens were stained with anti-EGFR (tumor antigen), anti-CD4, anti-PD1, and DAPI for contrast (left panel).

[0076] Figures 73A-73BThe study demonstrated that the phagolysosome V-ATPase inhibitor bafloxacin eliminated CER-induced phagocytosis. CER104-modified CD4+ T cells and simulated transduced CD4+ T cells (control) were co-cultured with HCC827 cells labeled with TAMRA-SE fluorescent dye and treated with osimertinib. Phagocytosis was quantified by FACS. Figure 73A The bar chart shows the phagocytosis of CER104-modified T cells on HCC827 NSCLC cells treated with TAMRA-SE and osimertinib, and how bafloxacin (20 nM) blocked the uptake of osimertinib-treated and TAMRA-SE-labeled HCC827 NSCLC cells by CER104-modified T cells. Figure 73B This is a FACS graph showing the in vitro phagocytosis assay of CER104-modified (left column) or simulated transduced T cells (control, right column) from untreated HCC827 NSCLC cells or HCC827 cells treated with osimertinib or osimertinib + bafloxacin.

[0077] Figures 74A-74B The addition of hCER104-modified CD4+ T cells to HPV E7 TCR-modified CD8+ T cells enhanced tumor-killing activity in vivo. HPV E7 TCR-modified CD8+ T cells + CD4+ T cells (mimicking transduction), HPV E7 TCR-modified CD8+ T cells + hCER104-modified CD4+ T cells were administered to NSG mice transplanted with HPV+SCC152, or the mice were left untreated. Tumor volume was measured by sequential bioluminescence imaging and displayed... Figure 74A middle. Figure 74B This is a graph showing the average intensity of bioluminescent signals obtained in the treatment group. Detailed Implementation

[0078] This disclosure provides combinations of cell immunotherapy compositions comprising combinations of immune cells or subsets of cells modified with different recombinant cell immunotherapy molecules. Embodiments of this disclosure include a first composition and a second composition, the first composition comprising immune cells containing a CER, and the second composition comprising immune cells containing a cell immunotherapy molecule (e.g., CER, CAR, or TCR-binding protein). Exemplary cell immunotherapy compositions may comprise: a first composition comprising CD4+ T cells containing a first chimeric phagocytic receptor (CER), and a second composition comprising CD8+ T cells containing a second CER; a first composition comprising CD4+ T cells containing a CER, and a second composition comprising CD8+ T cells containing a chimeric antigen receptor (CAR) or recombinant T cell receptor (TCR) binding protein; a first composition comprising CD4+ T cells containing a CER, and a second composition comprising CD4+ T cells containing a CAR or recombinant TCR binding protein; a first composition comprising CD8+ T cells containing a CER, and a second composition comprising CD8+ T cells containing a CAR or recombinant TCR binding protein; a first composition comprising CD8+ T cells containing a CER, and a second composition comprising CD4+ T cells containing a CAR or recombinant TCR binding protein; a first composition comprising B cells containing a CER, and a second composition comprising CD4+ T cells containing a CAR or recombinant TCR binding protein. T cells; a first composition comprising B cells containing a CER, and a second composition comprising CD8+ T cells containing a CAR or recombinant TCR-binding protein; a first composition comprising NK cells containing a CER, and a second composition comprising CD4+ T cells containing a CAR or recombinant TCR-binding protein; a first composition comprising NK cells containing a CER, and a second composition comprising CD8+ T cells containing a CAR or recombinant TCR-binding protein; a first composition comprising γδ T cells containing a CER, and a second composition comprising CD4+ T cells containing a CAR or recombinant TCR-binding protein; a first composition comprising γδ T cells containing a CER, and a second composition comprising CD8+ T cells containing a CAR or recombinant TCR-binding protein; a first composition comprising mucosa-associated invariant T (MAIT) cells containing a CER, and a second composition comprising CD4+ T cells containing a CAR or recombinant TCR-binding protein; a first composition comprising mucosa-associated invariant T (MAIT) cells containing a CER, and a second composition comprising CD8+ T cells containing a CAR or recombinant TCR-binding protein. T cells; a first composition comprising monocytes containing CER; and a second composition comprising CD4+ T cells containing CAR or recombinant TCR-binding protein;A first composition comprising monocytes containing a CER, and a second composition comprising CD8+ T cells containing a CAR or recombinant TCR-binding protein; a first composition comprising macrophages containing a CER, and a second composition comprising CD4+ T cells containing a CAR or recombinant TCR-binding protein; or a first composition comprising macrophages containing a CER, and a second composition comprising CD8+ T cells containing a CAR or recombinant TCR-binding protein. The cell immunotherapy compositions provided in this disclosure can be combined in the same pharmaceutical composition or in different pharmaceutical compositions for administration to a subject. Such cell immunotherapy compositions provided in this disclosure provide multiple, non-redundant target cell killing modes and enhanced effector function.

[0079] In addition, a method for delivering such cell immunotherapy compositions to subjects in need is provided.

[0080] Before elaborating on this disclosure in more detail, providing definitions for certain terms used herein may help in understanding this disclosure.

[0081] In this specification, unless otherwise stated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the range, and where appropriate, to include fractions (e.g., one-tenth and one-hundredth of an integer). Furthermore, unless otherwise stated, any numerical range relating to any physical characteristic (such as polymer monomer, size, or thickness) described herein should be understood to include any integer within the range. As used herein, unless otherwise stated, the term “about” means ±20% of the range, value, or structure shown. It should be understood that, as used herein, the term “a / some” refers to “one / some or more / some” of the listed components. The use of alternative words (e.g., “or”) should be understood to mean one, both, or any combination thereof. The terms “comprising,” “having,” and “including” as used herein are used synonymously, and these terms and variations thereof are intended to be construed as non-limiting.

[0082] Unless otherwise explicitly defined herein, those skilled in the art should understand the terminology as it is given in the art. The term "antibody" is used in the broadest sense and includes both polyclonal and monoclonal antibodies. "Antibody" can refer to a complete antibody comprising at least two heavy chains (H) and two light chains (L) linked together by disulfide bonds, and an antigen-binding moiety (or antigen-binding domain) that has or retains the ability to bind to a target molecule. Antibodies can be naturally occurring, recombinantly produced, genetically engineered, or modified forms of immunoglobulins, such as intracellular antibodies, peptide bodies, nanobodies, single-domain antibodies, SMIPs, and multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, tandem bi-scFVs, tandem tri-scFVs, ADAPTIR). Monoclonal antibodies or their antigen-binding moieties can be non-human, chimeric, humanized, or human, preferably humanized or human. The structure and function of immunoglobulins are reviewed in, for example, Chapter 14 of *Antibodies: A Laboratory Manual*, edited by Harlow et al. (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). The term "antigen-binding portion" or "antigen-binding domain" of a complete antibody is intended to include "antibody fragments" that represent a portion of the complete antibody and refer to the antigen-determining variable region or complementarity-determining region of the complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, Fab'-SH, F(ab')2, bifunctional antibodies, linear antibodies, scFv antibodies, VH, and multispecific antibodies formed from antibody fragments. A "Fab" (antigen-binding fragment) is a portion of an antibody that binds to an antigen and includes a variable region of a heavy chain linked to the light chain via interchain disulfide bonds and CH1. Antibodies can be of any class or subclass, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.

[0083] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the natural antibody heavy and light chains typically have similar structures, each containing four conserved frame regions (FRs) and three core regions (CDRs) (see, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated by screening libraries of complementary VL or VH domains, using the VH or VL domains of antibodies that bind to that antigen, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0084] The terms “complementarity-determining region” and “CDR”, known in the art as synonyms for “hypervariant region” or “HVR,” refer to discontinuous amino acid sequences within the antibody variable region that confer antigen specificity and / or binding affinity. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0085] As used herein, the terms “binding domain,” “binding region,” and “binding moiety” refer to molecules (such as peptides, oligopeptides, polypeptides, or proteins) that have the ability to specifically and non-covalently bind to, associate with, unite with, recognize, or combine with target molecules (e.g., tumor antigens). Binding domains include any naturally occurring, synthetic, semi-synthetic, or recombinant binding conjugate against a target biomolecule or other target. In some embodiments, the binding domain is an antigen-binding domain, such as an antibody or its functional binding domain or antigen-binding moiety. Exemplary binding domains include single-chain antibody variable regions (e.g., domain antibodies, sFv, scFv, Fab), receptor extracellular domains (e.g., TNF-α), ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for their specific binding ability to biomolecules.

[0086] A “T-cell receptor” (TCR) is a molecule present on the surface of T cells (also called T lymphocytes) that is typically responsible for recognizing antigens that bind to the major histocompatibility complex (MHC) molecule. In most T cells, the TCR typically consists of a heterodimer of highly variable α and β chains linked by disulfide bonds (also referred to as TCRα and TCRβ, respectively). In a small subset of T cells, the TCR consists of a heterodimer of γ and δ chains (also referred to as TCRγ and TCRδ, respectively). Each chain of the TCR is a member of the immunoglobulin superfamily and has an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus (see Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd ed., Current Biology Publications, p. 4:33, 1997). The TCRs of this disclosure can be derived from a variety of animal species, including humans, mice, rats, cats, dogs, goats, horses, or other mammals. TCRs can be cell-bound (i.e., possessing transmembrane regions or domains) or soluble. TCRs include recombinant-produced, genetically engineered, fusion-based, or modified forms, including, for example, scTCRs, soluble TCRs, and TCR fusion constructs (TRuCs). TM (See U.S. Patent Publication No. 2017 / 0166622).

[0087] The terms "variable regions" or "variable domains" (Vα or Vβ) of the TCR α-chain and β-chain, or Vγ and Vδ of the TCR, are associated with the binding of the TCR to the antigen. The Vγ of the natural TCR... α and V β They typically have similar structures, with each variable structural domain containing four conservative FRs and three CDRs. α The domain is encoded by two separate DNA segments: the variable gene segment (V gene) and the linker gene segment (J gene); V β The domain is encoded by three separate DNA segments: the variable gene segment (V gene), the diverse gene segment (D gene), and the connecting gene segment (J gene). A single V gene... α or V β The domain may be sufficient to confer antigen binding specificity.

[0088] Major histocompatibility complex (MHC) molecules are glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers composed of a transmembrane α-chain (containing three α-domains) and a non-covalently bound β2-microglobulin. MHC class II molecules consist of two transmembrane glycoproteins, α and β, both of which are transmembrane. Each chain has two domains. MHC class I molecules deliver peptides originating from the cytoplasm to the cell surface. The peptide:MHC complex is activated at the cell surface by CD8+. + T cell recognition. Class II MHC molecules deliver peptides originating from the vesicle system to the cell surface, where they are recognized by CD4+. + T-cell recognition. MHC molecules can originate from various animal species, including humans, mice, rats, or other mammals.

[0089] A chimeric antigen receptor (CAR) is a chimeric protein containing two or more distinct domains that can act as a receptor when expressed on the cell surface. A CAR typically consists of an extracellular domain (containing a binding domain that binds to the target antigen), optionally an extracellular spacer domain, a transmembrane domain, and an intracellular signaling domain (e.g., containing a T-cell activation motif based on an immune receptor tyrosine activation motif (ITAM), and optionally an intracellular co-stimulatory domain). In some embodiments, the intracellular signaling domain of the CAR has an ITAM-containing T-cell activation domain (e.g., CD3ζ) and an intracellular co-stimulatory domain (e.g., CD28). In some embodiments, the CAR is synthesized as a single polypeptide chain or encoded as a single-chain polypeptide by a nucleic acid molecule.

[0090] Various assays are known to be used to identify the binding domains of this disclosure that specifically bind to a particular target, and to determine the affinity of the binding domains, such as Western blotting, ELISA, analytical ultracentrifugation, spectroscopy, and surface plasmon resonance. Analysis and MHC tetramer analysis (see also, e.g., Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; and U.S. Patent Nos. 5,283,173, 5,468,614 or equivalents). As used herein, “specific binding” means binding the domain or its fusion protein at a concentration of 10 or greater. 5 M -1 Affinity or K a (i.e., the equilibrium association constant of a specific binding interaction, in units of 1 / M) associates or binds with the target molecule, but does not significantly associate or bind with any other molecule or component in the sample.

[0091] The terms “antigen” and “Ag” refer to molecules that elicit an immune response. The elicited immune response may involve antibody production, activation of specific immune cells, or both. Macromolecules (including proteins, glycoproteins, and glycolipids) can serve as antigens. Antigens can be derived from recombinant or genomic DNA. As considered herein, antigens do not need to be (i) encoded solely by the full-length nucleotide sequence of a gene or (ii) not encoded at all by a “gene.” Antigens can be produced or synthesized, or they can originate from biological samples. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or bodily fluids.

[0092] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant within an antigen that is specifically bound by a homologous immune-binding molecule, such as an antibody or fragment thereof (e.g., scFv), a T-cell receptor (TCR), a chimeric phagocytic receptor, or other binding molecules, domains, or proteins. Epitope determinants typically contain chemically active surface groups of the molecule, such as amino acid or sugar side chains, and may possess specific three-dimensional structural features and specific charge characteristics. Epitopes can be linear or conformational.

[0093] As used herein, an "effect domain" is the intracellular portion of a fusion protein or receptor that, upon receiving an appropriate signal, can directly or indirectly promote a biological or physiological response in cells expressing the effector domain. In some embodiments, the effector domain is part of a protein or protein complex that receives a signal upon binding, or its direct binding triggers a signal from the effector domain to a target molecule. For example, in response to the binding of a CER to a target molecule, the effector domain can transduce signals into the host cell to stimulate effector functions, such as phagocytosis, heterolysosomal maturation, or secretion of inflammatory and / or immunosuppressive cytokines. When the effector domain contains one or more signal transduction domains or motifs, it can directly promote a cellular response. In other embodiments, the effector domain will indirectly promote a cellular response by binding to one or more other proteins that directly promote a cellular response.

[0094] A "phagocytic signaling domain" refers to an intracellular effector domain that, upon binding to a target molecule (e.g., phosphatidylserine) targeted by the extracellular domain of the CER expressed by the host cell, activates one or more signaling pathways in the host cell, leading to phagocytosis (in certain embodiments, this includes cytoskeleton rearrangement of the host cell and internalization of target molecules or particles associated with the target antigen). In some embodiments, the phagocytic signaling domain activates one or more signaling pathways, resulting in the phagocytosis of target cells or particles. In further embodiments, the phagocytic signaling domain includes a primary phagocytic signaling domain and a secondary phagocytic signaling domain.

[0095] "Linking amino acid" or "linking amino acid residue" refers to one or more (e.g., about 2-20) amino acid residues located between two adjacent motifs, regions, or domains of a polypeptide. Linking amino acids can be generated as a result of designing the construct of a chimeric protein (e.g., amino acid residues generated using restriction enzyme sites during the construction of a nucleic acid molecule encoding a fusion protein).

[0096] "Disease" is a state of health in which an individual cannot maintain homeostasis, and in which, if the disease is not improved, the individual's health will continue to deteriorate. In contrast, an individual's "illness" or "adverse condition" is a state of health in which the individual can maintain homeostasis, but the individual's health is not as good as it would be without illness or adverse condition. Illness or adverse condition will not necessarily lead to a further decline in the individual's health without treatment.

[0097] "Nucleic acid molecules" and "polynucleotides" can be in the form of RNA or DNA, including cDNA, genomic DNA, and synthetic DNA. Nucleic acid molecules can consist of naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., α-enantiomers of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have modifications or substitutions of sugar, pyrimidine, or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester bonds include thiophosphates, dithiophosphates, selenite phosphates, diselenophosphates, aniline thiophosphates, aniline phosphates, aminophosphates, etc. Nucleic acid molecules can be double-stranded or single-stranded, and if single-stranded, can be a coding strand or a non-coding strand (antisense strand). Coding molecules can have the same coding sequence as those known in the art, or can have different coding sequences that encode the same polypeptide due to redundancy or degeneracy of the genetic code, or through splicing.

[0098] "Encoding" refers to the inherent property of a specific polynucleotide sequence (such as DNA, cDNA, and mRNA sequences) as a template for the synthesis of other polymers and macromolecules during bioprocessing, having a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to that polynucleotide produces a protein in a cell or other biological system, then that polynucleotide encodes a protein. Both the coding and non-coding strands can be referred to as encoding proteins or other products of that polynucleotide. Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences encoding degenerate versions of each other that encode the same amino acid sequence.

[0099] As used herein, the terms “endogenous” or “natural” refer to a gene, protein, compound, molecule, or activity that is normally present in the host or host cells, including naturally occurring variants of that gene, protein, compound, molecule, or activity.

[0100] As used herein, the term "homologous" or "homogeneous" refers to a molecule or activity derived from a host cell that is related to a second gene or activity by a common ancestor. The associated second gene or activity is, for example, derived from the same host cell, from different host cells, from different organisms, from different strains, or from different species. For example, heterologous or exogenous molecules or genes encoding said molecules may be homologous to natural host cell molecules or genes encoding said molecules, but they may have altered structures, sequences, expression levels, or combinations thereof.

[0101] As used herein, a "heterologous" nucleic acid molecule, construct, or sequence refers to a nucleic acid molecule or portion thereof that is not naturally occurring in the host cell but may be homologous to a nucleic acid molecule or portion thereof derived from the host cell. Heterologous nucleic acid molecules, constructs, or sequences may originate from different genera or species. In some embodiments, the heterologous nucleic acid molecule is not naturally occurring. In some embodiments, a heterologous nucleic acid molecule (i.e., not endogenous or natural) is added to a host cell or host genome via methods such as conjugation, transformation, transfection, transduction, electroporation, etc., wherein the added molecule may integrate into the host cell genome or exist as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and multiple copies may exist. Furthermore, "heterologous" refers to a non-natural enzyme, protein, or other activity encoded by a non-endogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes a homologous protein or activity.

[0102] As used herein, the terms “engineered,” “recombinant,” “modified,” or “non-natural” refer to organisms, microorganisms, cells, nucleic acid molecules, or vectors that have been modified by introducing exogenous nucleic acid molecules, or to cells or microorganisms that have been genetically engineered through human intervention, i.e., modified by introducing heterologous nucleic acid molecules, or to cells or microorganisms that have been altered to control, dysregulate, or constitutively modify the expression of endogenous nucleic acid molecules or genes, wherein such alterations or modifications can be introduced through genetic engineering. Human-derived genetic alterations may include, for example, modifications to introduce nucleic acid molecules encoding one or more proteins, chimeric receptors, or enzymes (which may include expression control elements such as promoters), or the addition, deletion, substitution, or functional disruption of other cellular genetic material. Exemplary modifications include modifications to the coding region or functional fragment of a heterologous or homologous polypeptide from a reference or parental molecule. Other exemplary modifications include, for example, modifications in non-coding regulatory regions, wherein said modifications alter the expression of a gene or operon.

[0103] As used herein, the term "transgenic" refers to a gene or polynucleotide encoding a target protein (e.g., CER, CAR, TCR) that requires expression in a host cell and has been transferred into the cell using genetic engineering techniques. Transgenics can encode proteins of therapeutic significance, or they can be reporter proteins, tags, biomarkers, suicide proteins, etc. Transgenics can originate from natural sources, modifications of natural genes, or recombinant or synthetic molecules. In some embodiments, the transgenic is a component of a vector.

[0104] The term "overexpressed" or "overexpressed" antigen refers to an abnormally high level of antigen expression in cells. Overexpressed antigens or antigen overexpression are often associated with disease states, such as in hematologic malignancies and in cells that form solid tumors within specific tissues or organs of a subject. Solid tumors or hematologic malignancies characterized by tumor antigen overexpression can be identified by standard assays known in the art.

[0105] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains and longer chains; short chains are generally referred to in the art, for example, as peptides, oligopeptides, and oligomers, while longer chains are generally referred to in the art, and there are many types of proteins. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0106] As used in this application, the terms "mature polypeptide" or "mature protein" refer to a protein or polypeptide that is secreted or located within the cell membrane or certain organelles (e.g., endoplasmic reticulum, Golgi apparatus, or introns) and does not contain an N-terminal signal peptide.

[0107] A signal peptide, also known as a signal sequence, leader sequence, or localization signal, is a short peptide (typically 15-30 amino acids long) located at the N-terminus of a newly synthesized protein and transported to the secretory pathway. A signal peptide typically consists of a short, positively charged hydrophilic amino acid at the N-terminus, a hydrophobic domain of 5-15 residues in the middle, and a C-terminal region containing a signal peptidase cleavage site. In eukaryotes, the signal peptide prompts the translocation of the newly synthesized protein to the endoplasmic reticulum (ER), where it is cleaved by a signal peptidase to produce the mature protein, which then proceeds to its appropriate destination.

[0108] The "percentage of identity" between two or more nucleic acid or amino acid sequences is a function of the number of common positions shared by the sequences (i.e., identity % = number of common positions / total number of positions x 100), taking into account the number of gaps that need to be introduced to optimize the alignment of the two or more sequences and the length of each gap. Mathematical algorithms, such as the BLAST and Gapped BLAST programs, can be used with their default parameters (e.g., Altschul et al., J.Mol.Biol.215:403,1990; see also BLASTN at www.ncbi.nlm.nih.gov / BLAST) to perform sequence alignment and determine the percentage of identity between two or more sequences.

[0109] In the art, a “conservative substitution” is considered to be an amino acid substitution for another amino acid with similar properties. Exemplary conservative substitutions are well known in the art (see, for example, WO 97 / 09433, p. 10, publication date: March 13, 1997; Lehninger, Biochemistry, 2nd edition; Worth Publishers, Inc. NY:NY (1975), pp. 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8).

[0110] The term "chimerism" refers to any non-endogenous nucleic acid molecule or protein that contains sequences that are joined or linked together (sequences that would not normally join or link together in nature). For example, a chimeric nucleic acid molecule may contain multiple domains encoding multiple different genes. In another instance, a chimeric nucleic acid molecule may contain regulatory and coding sequences from different sources, or regulatory and coding sequences from the same source but arranged in a manner different from that found in nature.

[0111] As used herein, the term “promoter” is defined as a DNA sequence that is recognized by the cell’s synthetic mechanisms or introduced synthetic mechanisms and is required to initiate specific transcription of a polynucleotide sequence.

[0112] As used herein, the term "promoter / regulatory sequence" refers to the nucleic acid sequence required to express a gene product operatively linked to that promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence; in others, it may contain enhancer sequences and other regulatory elements required for the expression of the gene product. A promoter / regulatory sequence may, for example, be a sequence that expresses the gene product in a tissue-specific manner.

[0113] A "constitutive" promoter is a nucleotide sequence that, when operatively linked to a polynucleotide sequence encoding or defining a gene product, results in the production of said gene product in the cell under most or all physiological conditions.

[0114] An "inducible" promoter is a nucleotide sequence that, when operatively linked to a polynucleotide sequence encoding or defining a gene product, results in the production of the gene product in large quantities in a cell only when an inducer corresponding to the promoter is present in the cell.

[0115] A tissue-specific promoter is a nucleotide sequence that, when operatively linked to a polynucleotide encoding or defined by a gene, results in the production of the gene product in large quantities in a cell only if the cell is a cell in a tissue type corresponding to the promoter.

[0116] As used herein, the phrase “under transcriptional control” or “operably linked” refers to the promoter being in the correct position and orientation relative to the polynucleotide to control the transcriptional initiation of RNA polymerase and the expression of that polynucleotide.

[0117] A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. Vectors can be, for example, plasmids, granules, viruses, or bacteriophages. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells. An "expression vector" is a vector that, when present in a suitable environment, directs the expression of a protein encoded by one or more genes carried by the vector.

[0118] In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, gamma retrovirus vectors, and lentiviral vectors. A "retrovirus" is a virus with an RNA genome. A "gamma retrovirus" refers to a genus within the family Retroviridae. Examples of gamma retroviruses include mouse stem cell virus, mouse leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendothelial cell proliferation virus. A "lentivirus" refers to a genus of retroviruses capable of infecting both dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0119] In other embodiments, the vector is a non-viral vector. Examples of non-viral vectors include lipid-based DNA vectors, modified mRNA (modRNA), self-amplifying mRNA, closed linear double-stranded (CELiD) DNA, and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty). When using a non-viral delivery system, the delivery carrier can be a liposome. Lipid formulations can be used to introduce nucleic acids into host cells in vitro, ex vivo, or in vivo. Nucleic acids can be encapsulated within liposomes, dispersed within the lipid bilayer of the liposome, attached to the liposome by linker molecules that bind the nucleic acid to the liposome, contained within or complexed with the liposome, or otherwise bound to the lipids.

[0120] "Particle" refers to cell debris or small objects with a diameter of at least 10 nm and at most 50 μm. Particles can originate from living cells or organisms, the environment, or be synthetic. Particles can be viral particles, prions, protein particles, synthetic particles, small mineral particles, or cell debris.

[0121] As used herein, the term “phagocytosis” refers to a receptor-mediated process in which endogenous or exogenous cells or particles larger than 10 nm in diameter are internalized by the phagocytes or host cells of this disclosure. Phagocytosis typically consists of several steps: (1) binding of the target cell or particle to a prophagocytic marker or antigen marker directly or indirectly (via a bridging molecule) to the target cell or particle; and (2) internalization or phagocytosis of the entire target cell or particle or a portion thereof. In some embodiments, internalization may occur by rearranging the cytoskeleton of the phagocytic cell or host cell to form a phagosome (containing a membrane-bound chamber containing the internalized target). Phagocytosis may also include the maturation of the phagosome, wherein the phagosome becomes more acidic and fuses with a lysosome (to form a heterolysosome), after which the phagosome is degraded (e.g., “phagocytosis”). Alternatively, phagosome-lysosome fusion may not be observed in phagocytosis. In yet another embodiment, the phagosome may reflux or expel its contents into the extracellular environment before complete degradation. In some embodiments, phagocytosis refers to phagocytosis. In some embodiments, the phagocytic cells that engulf host cells including those of this disclosure bind target cells or particles but do not internalize them. In some embodiments, the phagocytic cells that engulf host cells including those of this disclosure bind target cells or particles and partially internalize the target cells or particles.

[0122] As used herein, the term "phagocytosis" refers to the phagocytosis of cells or large particles (>0.5 μm) in which the binding of the target cell or particle, the phagocytosis of the target cell or particle, and the degradation of the internalized target cell or particle occur. In some embodiments, phagocytosis includes the formation of a phagosome surrounding the internalized target cell or particle, and the fusion of the phagosome with a lysosome to form a heterolysosome, wherein the contents are degraded. In some embodiments, a phagocytic synapse is formed after the CER expressed on the host cell of this disclosure binds to the target antigen expressed by the target cell or particle; an actin-rich phagocytic cup is generated at the phagocytic synapse; a phagocytic arm extends around the target cell or particle via cytoskeleton rearrangement; and finally, the target cell or particle is pulled into the phagocytic cell or host cell by forces generated by motor proteins. As used herein, "phagocytosis" includes the process of "cell burial," which specifically refers to the phagocytosis of apoptotic or necrotic cells in a non-inflammatory manner.

[0123] The term "immune system cells" or "immune cells" refers to any cell in the immune system that originates from hematopoietic stem cells in the bone marrow. Hematopoietic stem cells produce two main lineages: myeloid progenitor cells (myeloid cells that produce monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (lymphoid cells that produce T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4 cells. + T cells, CD8 + T cells, CD4 - CD8 - Double-negative T cells, γδ T cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells can also be called "antigen-presenting cells" or "APCs," which are specialized cells that can activate T cells when the major histocompatibility complex (MHC) receptor on the surface of APCs, which are complexed with peptides, interacts with the TCR on the surface of T cells.

[0124] The term "T cell" refers to cells of the T cell lineage. "Cells of the T cell lineage" are cells exhibiting at least one phenotypic characteristic of a T cell or its precursor or progenitor cell, distinct from other lymphocytes and erythrocyte or myeloid cell lineage cells. Such phenotypic characteristics may include the expression of one or more T cell-specific proteins (e.g., CD3). + CD4 + CD8 + ( ), or T cell-specific physiological, morphological, functional, or immunological characteristics. For example, cells in a T cell lineage can be progenitor cells or precursor cells directed towards a T cell lineage; CD25 + Immature and unactivated T cells; cells that have undergone CD4 or CD8 lineage orientation; CD4+CD8+ double-positive thymocyte progenitor cells; single-positive CD4+ T cells. + or CD8 + TCRαβ or TCRγδ; or mature and functional or activated T cells. The term "T cell" includes naïve T cells (CD45RA). + CCR7 + CD62L + CD27 + CD45RO - ), central memory T cells (CD45RO) + CD62L + CD8 + ), effector memory T cells (CD45RA) + CD45RO - CCR7 - CD62L - CD27- Mucosa-associated inert T (MAIT) cells, Tregs, natural killer T cells, and tissue-resident T cells.

[0125] The term "B cell" refers to cells of the B cell lineage. "Cells of the B cell lineage" are cells exhibiting at least one phenotypic characteristic of B cells or their precursors or progenitors, distinguishing them from other lymphocytes and erythrocyte or myeloid lineage cells. Such phenotypic characteristics may include the expression of one or more B cell-specific proteins (e.g., CD19). + CD72 + CD24 + CD20 + B cells can be defined by their specific physiological, morphological, functional, or immunological characteristics. For example, cells in a B cell lineage can be progenitor or precursor cells directed towards that lineage (e.g., pro-proto-B cells, pro-B cells, and pre-B cells); immature and unactivated B cells; or mature and functional or activated B cells. Therefore, "B cell" includes naïve B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytic-like cells, plasmablasts, and memory B cells (e.g., CD27 cells). + IgD - ).

[0126] For immune cells (e.g., NK cells or T cells) that express immune receptors (e.g., TCRs) on their surfaces, the term "cytotoxic activity," also known as "cytolytic activity," refers to the induction of apoptosis in target cells following antigen-specific signaling (e.g., via TCRs). In some embodiments, cytotoxic cells can induce apoptosis in target cells by releasing cytotoxins (such as perforin, granzymes, and granulosin) from granules. Perforin inserts into the target cell membrane and forms pores, allowing rapid entry of water and salts into the target cell. Granzymes are serine proteases that induce apoptosis in target cells. Granosin is also capable of forming pores on the target cell membrane and is a pro-inflammatory molecule. In some embodiments, cytotoxic cells can induce apoptosis in target cells through interactions with Fas ligands, which are upregulated on T cells following antigen-specific signaling as Fas molecules are expressed on target cells. Fas are apoptosis signaling receptor molecules on a variety of different cell surfaces. Cytotoxic activity on target cells can expose prophagocytic markers (e.g., phosphatidylserine) on the surface of the target cells.

[0127] A "disease" is a health condition in which an object cannot maintain homeostasis, and whereby if the disease is not improved, the object's health will continue to deteriorate. In contrast, an object's "illness" or "adverse condition" is a health condition in which the object can maintain homeostasis, but the object's health is not as good as it would be without the illness or adverse condition. An illness or adverse condition will not necessarily lead to a further decline in the object's health without treatment.

[0128] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. These abnormal cells can form solid tumors or constitute malignant hematologic disorders. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer.

[0129] The terms “object,” “patient,” and “individual” are used interchangeably in this document and are intended to include living organisms (e.g., mammals) in which an immune response can be elicited. Examples of objects include humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0130] "Adoptive cell immunotherapy" or "cell immunotherapy" refers to the administration of naturally occurring or genetically engineered disease antigen-specific immune cells (e.g., T cells). Adoptive cell immunotherapy can be autologous (immune cells come from the recipient), allogeneic (immune cells come from a donor of the same species), or genetically identical (immune cells come from a donor with the same genes as the recipient).

[0131] "Autologous" refers to a graft (e.g., organ, tissue, cell) that is subsequently reintroduced from the same object.

[0132] "Also-heterogeneous" refers to grafts from different objects of the same species.

[0133] The "therapeutic effective amount" or "effective amount" of the chimeric protein or cells expressing the chimeric protein (e.g., chimeric phagocytic receptor or cells expressing the chimeric phagocytic receptor) disclosed herein refers to the amount of protein or cells sufficient to improve one or more symptoms of the treated disease, condition, or adverse condition. When referring to the administration of a single active ingredient or cells expressing a single active ingredient alone, the therapeutic effective amount refers to the effective amount of the ingredient or cells expressing the single ingredient. When referring to combination therapy, the therapeutic effective amount refers to the combined amount of the active ingredient, or the amount of the adjuvant active ingredient, used in combination with cells expressing the active ingredient to produce a therapeutic effect, whether administered sequentially or simultaneously.

[0134] "Treatment" or "improvement" refers to the medical management of a disease, symptom, or adverse condition. Typically, it involves administering an appropriate dose or treatment regimen of host cells expressing the chimeric protein of this disclosure in an amount sufficient to elicit a therapeutic or preventative benefit. The therapeutic or preventative / avoidant benefits include improved clinical outcomes; reduction or alleviation of symptoms associated with the disease, symptom, or adverse condition; reduction of symptom occurrence; improved quality of life; prolongation of disease-free status; reduction of the severity of the disease, symptom, or adverse condition; stabilization of disease status; delay of disease progression; remission; survival; extended survival; or any combination thereof.

[0135] The term "anti-tumor effect" refers to biological effects that can manifest as shrinking tumor volume, reducing the number of tumor cells, reducing the number of metastases, prolonging life expectancy, or improving various physiological symptoms associated with cancer. The "anti-tumor effect" can also manifest as preventing the formation of malignant hematological diseases or tumors.

[0136] Other definitions are provided throughout this disclosure.

[0137] genetically modified

[0138] The cell immunotherapy compositions of this disclosure comprise combinations of immune cells modified to include transgenes encoding cell immunotherapy molecules, such as chimeric phagocytic receptors (CERs), chimeric antigen receptors, and T-cell receptor (TCR) binding proteins. The cell immunotherapy compositions of this disclosure comprise specific combinations of immune cell types or subtypes modified with specific cell immunotherapy molecules that exhibit unique mechanisms for eliminating target cells in the host (i.e., cell lysis and phagocytosis) (see...). Figure 1 ).

[0139] I. Chimeric phagocytic receptor (CER)

[0140] The compositions of this disclosure partially comprise immune cells containing transgenes encoding a chimeric phagocytic receptor (CER). A chimeric phagocytic receptor typically comprises: (a) an extracellular domain including a binding domain that binds to a target antigen; (b) a phagocytic signaling domain; and (c) a transmembrane domain located between and connecting the extracellular domain and the phagocytic signaling domain. In some embodiments, the extracellular domain of the chimeric phagocytic receptor described herein optionally includes an extracellular spacer domain located between and connecting the binding domain and the transmembrane domain.

[0141] The chimeric phagocytic receptors described herein can confer a phagocytic phenotype specific to a target antigen onto host cells that express the chimeric phagocytic receptor. In some embodiments, expression of the CER as described herein confers a phagocytic phenotype onto host cells that do not naturally possess such a phagocytic phenotype. In some embodiments, phagocytic activity activates phagocytes. The CER of this disclosure can be used to redirect phagocytic specificity to target cells expressing the target antigen.

[0142] extracellular domain

[0143] As described herein, the CER contains an extracellular domain specifically targeting a target antigen. In some embodiments, the extracellular domain contains a binding domain that specifically binds to the target antigen (e.g., phosphatidylserine). Binding of the binding domain can block the interaction between the target molecule (e.g., a receptor or ligand) and another molecule, and, for example, interfere with, attenuate, or eliminate certain functions of the target molecule (e.g., signal transduction). In some embodiments, binding of the target molecule can induce certain biological pathways or identify the target molecule or cells expressing the target molecule for elimination.

[0144] The binding domain suitable for use in the CER of this disclosure can be any polypeptide or peptide that specifically binds to a target molecule (e.g., phosphatidylserine). Sources of the binding domain include the extracellular domain of a receptor, ligands of cell surface receptors or molecules, and antibody or antigen-binding moieties, such as variable regions of antibodies from various species. For example, the binding domain can contain sFv, scFv, Fab, scFv-based grababody, VH domain, VL domain, single-domain camel antibody (VHH), or domain antibody. The binding domain can be derived from humans, primates, rodents, birds, or sheep. Other sources of the binding domain include variable regions of antibodies from other species, such as camelids (from camels, dromedaries, or llamas; Ghahroudi et al., FEBS Lett. 414:521, 1997; Vincke et al., J. Biol. Chem. 284:3273, 2009; Hamers-Casterman et al., Nature). 363:446,1993 and Nguyen et al., J.Mol.Biol.275:413,1998), nurse shark (Roux et al., Proc.Nat′l.Acad.Sci.(USA)95:11804,1998), spotted chinchilla (Nguyen et al., Immunogen.54:39,2002) or lamprey (Herrin et al., Proc.Nat′l.Acad.Sci.(USA)105:2040,2008 and Alder et al., Nat.Immunol.9:319,2008). These antibodies use only the variable region of the heavy chain to form the antigen-binding region; that is, these functional antibodies are simply homodimers of the heavy chain (referred to as "heavy chain antibodies") (Jespers et al., Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008). In some embodiments, the binding domain is mouse, chimeric, human, or humanized.

[0145] In some embodiments, the CER-binding domain comprises an antibody or an antigen-binding fragment thereof, such as a single-chain Fv fragment (scFv), which includes VH and VL regions specific to the target disease antigen. In some embodiments, the antibody or antigen-binding fragment is chimeric, human, or humanized. In a further embodiment, V H and V L The area is human or humanized.

[0146] Target molecules that specifically bind to the extracellular domain of the CER of this disclosure may be present on or bound to target cells (“target cells”). Exemplary target cells include cancer cells, cells associated with autoimmune diseases or conditions, neurodegenerative diseases, or inflammatory diseases or conditions, as well as infectious microorganisms (e.g., bacteria, viruses, or fungi) or infected cells (e.g., virus-infected cells). Cells of infectious organisms (such as mammalian parasites) are also considered target cells.

[0147] In some embodiments, the extracellular domain binds to a phagocytic marker. As used herein, a phagocytic marker is a portion (e.g., a protein, lipid, or polysaccharide) that apoptotic, necrotic, pyroptotic, or infected cells display on their surface that distinguishes them from non-apoptotic, non-necrotic, non-pyroptotic, oncolytic, or non-infected cells. A phagocytic marker can be an intracellular portion exposed on the surface of apoptotic or necrotic cells, a portion on apoptotic or necrotic cells with altered glycosylation or altered surface charge, or a serum portion bound to apoptotic, necrotic, pyroptotic, or oncolytic cells. Examples of phagocytic markers targeting apoptotic cells include phosphatidylserine (PtdSer), ICAM-3, oxidized low-density lipoprotein, calreticulin, annexin I, complement C1q, and platelet-reactive protein. Necrotic, oncolytic, and pyroptotic cells also expose the PtdSer phagocytic marker on their cell surface. Phagocytic receptors can use soluble bridging molecules as intermediates for binding to prophagocytic markers to directly or indirectly detect (or bind) prophagocytic markers on target cells (e.g., damaged, infected, apoptotic, necrotic, pyroptotic, or oncolytic cells). In some such embodiments, prophagocytic markers targeted by the extracellular domain are phosphatidylserine (PtdSer), ICAM-3, oxidized low-density lipoprotein, calreticulin, annexin I, complement C1q, or platelet-reactive protein. Embodiments of the binding domain for the CER of this disclosure include those derived from Tim1, Tim4, Tim3, stabilin-2, receptor for advanced glycation end products (RAGE), brain-specific angiogenesis inhibitor 1 (BAI1), milk fat globulin-EGF receptor 8 protein (MFG-E8) (e.g., FA58C2 mediating high affinity binding to PtdSer), growth arrest-specific 6 (GAS6), protein S, protein C, factor II, factor VII, factor IX, factor X, β The PtdSer binding domain of 2-glycoprotein I, α5β3 integrin and other integrins, CR3 complement receptor, CR4 complement receptor, CD14, CD93, annexin V, phosphatidylserine receptor (PSr), prothrombin, or scavenger receptors such as scavenger receptor B (SRB) (e.g., SRB1 (CD36)), scavenger receptor C (SRC) (e.g., LOX-1, SRCL), scavenger receptor D (SRD) (e.g., CD68, macrophage sialic acid protein), and PSOX. An exemplary human Tim4 binding domain comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with amino acids 25-314 of SEQ ID NO:90 or SEQ ID NO:90.In some embodiments, the Tim4 binding domain comprises or consists of the amino acid sequence shown in SEQ ID NO:90, SEQ ID NO:85, SEQ ID NO:90 amino acids 25-314 or SEQ ID NO:85 amino acids 23-279.

[0148] In some implementations, the extracellular domain binds to tumor antigens, viral antigens, bacterial antigens, fungal antigens, parasitic antigens, neurodegenerative disease antigens, or autoimmune disease antigens. Exemplary tumor antigens include CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, ROR1, MUC-16, L1CAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPV E7, MUC-1, HER2, folate receptor α, CD97, CD171, CD179a, CD44v6, WT1, VEGF-α, VEGFR1, IL-13Rα1, IL-13Rα2, IL-11Rα, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, liver glycoside A2, liver glycoside B2, Lewis A antigen, Lewis... Y antigen, MAGE, MAGE-A1, RAGE-1, folate receptor β, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucose GM1, GM3, o-acetyl GD2 and GD2.

[0149] In some implementations, the extracellular domain binds to viral antigens, bacterial antigens, fungal antigens, protozoan antigens, or parasitic antigens.

[0150] In some embodiments, the extracellular domain optionally includes an extracellular, non-signal transduction spacer region or a linker domain. When included, such spacer regions or linker domains can position the binding domain away from the host cell surface to further facilitate suitable cell / cell contact, binding, and activation. An extracellular spacer region is typically present between the extracellular binding domain and the transmembrane domain of the CER. The length of the extracellular spacer region can be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size, and affinity (see, for example, Guest et al., J. Immunother. 28:203-11, 2005; PCT Publication No. WO2014 / 031687). In some embodiments, the extracellular spacer region is an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgD). The hinge region can be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. The modified IgG4 hinge region is described in PCT Publication No. WO2014 / 031687, the entire contents of which are incorporated herein by reference. In one particular embodiment, the extracellular spacer domain comprises the modified IgG4 hinge region having the amino acid sequence ESKYGPPCPPCP (SEQ ID NO: 1).

[0151] Other examples of hinge regions that can be used in the CER described herein include hinge regions present in the extracellular regions of type 1 membrane proteins (e.g., CD8a, CD4, CD28, and CD7, which may be wild-type or variants thereof). In a further embodiment, the extracellular spacer region domain comprises all or part of the Fc domains of immunoglobulins selected from: CH1 domain, CH2 domain, CH3 domain, or combinations thereof (see, for example, PCT Publication WO2014 / 031687, the entire contents of which are spacers are incorporated herein by reference). In yet another embodiment, the extracellular spacer region domain may comprise the stem region of type II C-lectins (the extracellular domain located between the C-lectin domain and the transmembrane domain). Type II C-lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D. In yet another embodiment, the extracellular spacer region domain may be derived from the juxtamembrane domain of a toll-like receptor (TLR). The TLR juxtamembrane domain comprises an acidic amino acid located between a leucine-rich repeat sequence (LRR) and the transmembrane domain of the TLR. In some embodiments, the TLR juxtamembrane domain is a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 juxtamembrane domain. An exemplary TLR juxtamembrane domain is a TLR4 juxtamembrane domain comprising the amino acid sequence of SEQ ID NO:2.

[0152] The extracellular domain can be derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species. In some embodiments, the extracellular domain is mouse, chimeric, human, or humanized.

[0153] Swallowing signal transduction domain

[0154] The phagocytic signaling domain of the cytokinetic echogenic molecule (CER) is an intracellular effector domain and is capable of delivering functional signals to the cell in response to the binding of the CER's extracellular domain to a target molecule. The phagocytic signaling domain can be any portion of a phagocytic signaling molecule that retains sufficient signaling activity. In some embodiments, a full-length phagocytic signaling molecule or its full-length intracellular portion is used. In some embodiments, a phagocytic signaling molecule or a truncated portion of the intracellular portion of a phagocytic signaling molecule is used, provided that the truncated portion retains sufficient signaling activity. In a further embodiment, the phagocytic signaling domain is a variant of the complete or truncated portion of a phagocytic signaling molecule, provided that the variant retains sufficient signaling activity (i.e., a functional variant).

[0155] Exemplary signal transduction domains that can be used in CER include the MRC1 signal transduction domain, MERTK signal transduction domain, Tyro3 signal transduction domain, Axl signal transduction domain, ELMO signal transduction domain, Traf6 signal transduction domain, Syk signal transduction domain, MyD88 signal transduction domain, PI3K signal transduction domain, and FcR signal transduction domain (e.g., FcγR1, FcγR2A, FcγR2C, FcγR2B2, FcγR3A, FcγR2C, FcγR3A, FcεR1, or FcαR1 signal transduction domains). The signal transduction domains include: B cell activating factor receptor (BAFF-R) signal transduction domain, DAP12 (also known as TYRO protein tyrosine kinase binding protein (TYROBP)) signal transduction domain, NFAT activating protein with ITAM motif 1 (NFAM1) signal transduction domain, CD79b signal transduction domain, TLR signal transduction domains (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 signal transduction domains), Traf2 signal transduction domain, or Traf3 signal transduction domain.

[0156] In some embodiments, the phagocytic signal transduction domain comprises a sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with the following sequences: an MRC1 signal transduction domain containing the amino acid sequence shown in SEQ ID NO:3; a MERTK signal transduction domain containing the amino acid sequence shown in SEQ ID NO:4; a Tyro3 signal transduction domain containing the amino acid sequence shown in SEQ ID NO:6; an Axl signal transduction domain containing the amino acid sequence shown in SEQ ID NO:7; an ELMO signal transduction domain containing the amino acid sequence shown in SEQ ID NO:8; a Traf6 signal transduction domain containing the amino acid sequence shown in SEQ ID NO:9; a Syk signal transduction domain containing the amino acid sequence shown in SEQ ID NO:10; a MyD88 signal transduction domain containing the amino acid sequence shown in SEQ ID NO:11; and a sequence containing SEQ ID NO:3. The amino acid sequence shown in NO:13 contains the FcγR1 signal transduction domain, the amino acid sequence shown in SEQ ID NO:14 contains the FcγR2A signal transduction domain, the amino acid sequence shown in SEQ ID NO:15 contains the FcγR2C signal transduction domain, the amino acid sequence shown in SEQ ID NO:16 contains the FcγR3A signal transduction domain, the amino acid sequence shown in SEQ ID NO:17 contains the BAFF-R signal transduction domain, the amino acid sequence shown in SEQ ID NO:18 contains the DAP12 signal transduction domain, the amino acid sequence shown in SEQ ID NO:19 contains the NFAM1 signal transduction domain, the amino acid sequence shown in SEQ ID NO:20 contains the CD79b signal transduction domain, the amino acid sequence shown in SEQ ID NO:22 contains the TLR1 signal transduction domain, the amino acid sequence shown in SEQ ID NO:23 contains the TLR2 signal transduction domain, the amino acid sequence shown in SEQ ID NO:24 contains the TLR3 signal transduction domain, and the amino acid sequence shown in SEQ ID NO:25 contains the FcγR1 signal transduction domain, the amino acid sequence shown in SEQ ID NO:24 contains the TLR2 signal transduction domain, and the amino acid sequence shown in SEQ ID NO:25 contains the FcγR2A signal transduction domain, ... The amino acid sequence shown in NO:26 contains the TLR4 signal transduction domain, the amino acid sequence shown in SEQ ID NO:27 contains the TLR5 signal transduction domain, the amino acid sequence shown in SEQ ID NO:28 contains the TLR6 signal transduction domain, the amino acid sequence shown in SEQ ID NO:29 contains the TLR7 signal transduction domain, and the amino acid sequence shown in SEQ ID NO:30 contains the TLR8 signal transduction domain.The signal transduction domain contains the TLR9 signal transduction domain of the amino acid sequence shown in SEQ ID NO:31, the Traf2 signal transduction domain of the amino acid sequence shown in SEQ ID NO:32, or the Traf3 signal transduction domain of the amino acid sequence shown in SEQ ID NO:33.

[0157] In some embodiments, the phagocytic signal transduction domain is an MRC1 signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:3, a MERTK signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:4, a Tyro3 signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:6, an Axl signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:7, an ELMO signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:8, a Traf6 signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:9, a Syk signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:10, a MyD88 signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:11, an FcεRIγ signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:13, an FcγR1 signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:14, or a domain comprising or composed of the amino acid sequence shown in SEQ ID NO:3. The FcγR2A signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:15, the FcγR2C signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:16, the FcγR3A signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:17, the BAFF-R signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:18, the DAP-12 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:19, the NFAM1 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:20, the CD79b signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:22, the TLR1 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:23, the TLR2 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:24, the TLR3 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:25, the TLR4 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:26, and the FcγR2A signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:15. The TLR5 signal transduction domain, composed of the amino acid sequence shown in SEQ ID NO:27, includes or is composed of the TLR6 signal transduction domain, composed of the amino acid sequence shown in SEQ ID NO:28, and the TLR7 signal transduction domain, composed of or is composed of the amino acid sequence shown in SEQ ID NO:29.The signal transduction domain comprises or is composed of the amino acid sequence shown in SEQ ID NO:30, the signal transduction domain comprises or is composed of the amino acid sequence shown in SEQ ID NO:31, the signal transduction domain comprises or is composed of the amino acid sequence shown in SEQ ID NO:32, or the signal transduction domain comprises or is composed of the amino acid sequence shown in SEQ ID NO:33.

[0158] A truncated swallowing signal transduction domain can be truncated at its N-terminus, its C-terminus, or both its N-terminus and C-terminus. In some embodiments, the MRC1 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:3; the MERTK phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:4; the Tyro3 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:6; the Axl phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:7; the ELMO phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:8; the Traf6 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:9; and the Syk phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:9. The amino acid sequence shown in NO:10 has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the MyD88 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:11, which has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the FcεRIγ phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:13, which has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the FcγR1 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:14, which has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the FcγR2A phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:15, which has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the FcγR2C phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:16, which has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the FcγR3A ...5, which has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the FcγR3A phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:16, which has The amino acid sequence shown in NO:17 has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the BAFF-R phagocytic signal transduction domain has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids in the amino acid sequence corresponding to SEQ ID NO:18; the DAP-12 phagocytic signal transduction domain has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids in the amino acid sequence corresponding to SEQ ID NO:19.The NFAM1 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 20; the CD79b phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 22; the TLR1 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 23; the TLR2 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 24; the TLR3 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 25; the TLR4 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 26; and the TLR5 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 26. The amino acid sequence shown in NO:27 has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the TLR6 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:28, which has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the TLR7 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:29, which has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the TLR8 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:30, which has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the TLR9 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:31, which has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the Traf2 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:32, which has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; or the Traf3 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:28, which has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; or the Traf3 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:29, which has its N-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; or the Traf2 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:32, which has its N-terminus truncated by 1, 2, The amino acid sequence shown in NO:33 has its N-terminus truncated by 1, 2, 3, 4, 5, or more amino acids.

[0159] In some embodiments, the MRC1 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:3; the MERTK phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:4; the Tyro3 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:6; the Axl phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:7; the ELMO phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:8; the Traf6 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:9; and the Syk phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:9. The amino acid sequence shown in NO:10 has its C-terminus shortened by 1, 2, 3, 4, 5 or more amino acids; the MyD88 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:11, which has its C-terminus shortened by 1, 2, 3, 4, 5 or more amino acids; the FcεRIγ phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:13, which has its C-terminus shortened by 1, 2, 3, 4, 5 or more amino acids; the FcγR1 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:14, which has its C-terminus shortened by 1, 2, 3, 4, 5 or more amino acids; the FcγR2A phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:15, which has its C-terminus shortened by 1, 2, 3, 4, 5 or more amino acids; the FcγR2C phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:16, which has its C-terminus shortened by 1, 2, 3, 4, 5 or more amino acids; the FcγR3A ...5, which has its C-terminus shortened by 1, 2, 3, 4, 5 or more amino acids; the FcγR3A phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:16, which has its C-terminus shortened by 1, 2, The amino acid sequence shown in NO:17 has its C-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the BAFF-R phagocytic signal transduction domain has its C-terminus truncated by 1, 2, 3, 4, 5 or more amino acids corresponding to the amino acid sequence shown in SEQ ID NO:18; the DAP-12 phagocytic signal transduction domain has its C-terminus truncated by 1, 2, 3, 4, 5 or more amino acids corresponding to the amino acid sequence shown in SEQ ID NO:19.The NFAM1 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:20; the CD79b phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:22; the TLR1 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:23; the TLR2 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:24; the TLR3 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:25; the TLR4 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:26; and the TLR5 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:26. The amino acid sequence shown in NO:27 has its C-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the TLR6 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:28, which has its C-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the TLR7 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:29, which has its C-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the TLR8 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:30, which has its C-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the TLR9 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:31, which has its C-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; the Traf2 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:32, which has its C-terminus truncated by 1, 2, 3, 4, 5 or more amino acids; or the Traf3 phagocytic signal transduction domain is the amino acid sequence corresponding to SEQ ID NO:32, which has its C-terminus truncated by 1, 2, 3, 4, 5 or more amino acids;

[0160] The amino acid sequence shown in SEQ ID NO:33 has its C-terminus truncated by 1, 2, 3, 4, 5 or more amino acids.

[0161] In some embodiments, the truncated MyD88 phagocytic signaling domain includes a death domain but lacks the Toll / interleukin 1 receptor (TIR) ​​homologous domain. Examples of such truncated MyD88 phagocytic signaling domains include the amino acid sequence shown in SEQ ID NO:34. In some embodiments, the truncated MyD88 phagocytic signaling domain includes a TIR domain. Examples of truncated MyD88 phagocytic signaling domains include a TIR domain containing the amino acid sequence shown in SEQ ID NO:91. An exemplary truncated Traf6 signaling domain contains the amino acid sequence shown in SEQ ID NO:35. An exemplary truncated NFAM1 signaling domain contains the amino acid sequence shown in SEQ ID NO:36. An exemplary truncated CD79b signaling domain contains the amino acid sequence shown in SEQ ID NO:21.

[0162] In some implementations, the CER includes a first (or primary) and a second (or secondary) swallowing signal transduction domain selected from any of the swallowing signal transduction domains provided herein. An exemplary first swallowing signal transduction domain is selected from the MRC1, MERTK, Tyro3, ​​Axl, ELMO, Traf6, Syk, MyD88, PI3K, and FcR signal transduction domains (e.g., FcγR1, FcγR2A, FcγR2C, FcγR2B2, FcγR3A, FcγR2C, FcγR3A, FcεR1, or FcαR1 signal transduction domains). The signal transduction domains include: B cell activating factor receptor (BAFF-R) domain, DAP12 (also known as TYRO protein tyrosine kinase-binding protein (TYROBP)) domain, NFAT activating protein with ITAM motif 1 (NFAM1) domain, CD79b domain, TLR signal transduction domains (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domains), Traf2 domain, and Traf3 domain. The exemplary second swallowing signal transduction domain is selected from the MRC1 signal transduction domain, MERTK signal transduction domain, Tyro3 signal transduction domain, Axl signal transduction domain, ELMO signal transduction domain, Traf6 signal transduction domain, Syk signal transduction domain, MyD88 signal transduction domain, PI3K signal transduction domain, and FcR signal transduction domain (e.g., FcγR1, FcγR2A, FcγR2C, FcγR2B2, FcγR3A, FcγR2C, FcγR3A, FcεR1, or cαR1 signal transduction domain). The signal transduction domains include: B cell activating factor receptor (BAFF-R), DAP12 (also known as TYRO protein tyrosine kinase binding protein (TYROBP)) signal transduction domain, NFAT activating protein with ITAM motif 1 (NFAM1) signal transduction domain, CD79b signal transduction domain, TLR signal transduction domains (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 signal transduction domains), Traf2 signal transduction domain, and Traf3 signal transduction domain.

[0163] It should be understood that the positions of the first and second phagocytic signaling domains in the CER are interchangeable. Therefore, in one instance, the first phagocytic signaling domain in the CER may be located at the N-terminus of the second phagocytic signaling domain. In another instance, the first phagocytic signaling domain in the CER may be located at the C-terminus of the second phagocytic signaling domain. In some embodiments, the CER comprises the first and second phagocytic signaling domains from the same molecule. In other embodiments, the first and second phagocytic signaling domains originate from different molecules.

[0164] Phagocytic signal transduction domains can originate from mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0165] Transmembrane domain

[0166] The CER disclosed herein includes a transmembrane domain that connects and lies between the extracellular domain and the phagocytic signaling domain. The transmembrane domain is a hydrophobic α-helix that traverses the host cell membrane and anchors the CER within it. The transmembrane domain may be directly fused to a binding domain or an extracellular spacer domain (if present). In some embodiments, the transmembrane domain is derived from an intact membrane protein (e.g., receptors, differentiation cluster (CD) molecules, enzymes, transport proteins, cell adhesion molecules, etc.). The transmembrane domain may be selected from the same molecule as the extracellular domain or the phagocytic signaling domain (e.g., the CER contains a TLR4 phagocytic signaling domain and a TLR4 transmembrane domain, or the CER contains a Tim4 binding domain and a Tim4 transmembrane domain). In some embodiments, the transmembrane domain and the extracellular domain are selected from different molecules. In other embodiments, the transmembrane domain and the phagocytic signaling domain are selected from different molecules. In still other embodiments, the transmembrane domain, the extracellular domain, and the phagocytic signaling domain are selected from different molecules.

[0167] In some embodiments, the transmembrane domains include Tim1, Tim4, Tim3, FcR (e.g., FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1 or FcαR1), CD8a, CD28, MERTK, Axl, Tyro3, ​​CD4, DAP12, MRC1, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 or TLR9 transmembrane domains.

[0168] In some embodiments, the transmembrane domain comprises a sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with the following sequences: a Tim1 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:37; a Tim4 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:38 or 39; a Tim3 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:40; an FcγR1 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:41; an FcγR2A transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:42; an FcγR2B2 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:43; an FcγR2C transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:44; and a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:44. The FcγR3A transmembrane domain of the amino acid sequence shown in SEQ ID NO:45 includes the FcεR1 transmembrane domain of the amino acid sequence shown in SEQ ID NO:46, the FcαR1 transmembrane domain of the amino acid sequence shown in SEQ ID NO:47, the CD8a transmembrane domain of the amino acid sequence shown in SEQ ID NO:48, the CD28 transmembrane domain of the amino acid sequence shown in SEQ ID NO:49, the MERTK transmembrane domain of the amino acid sequence shown in SEQ ID NO:50, the Axl transmembrane domain of the amino acid sequence shown in SEQ ID NO:51, the Tyro3 transmembrane domain of the amino acid sequence shown in SEQ ID NO:52, the CD4 transmembrane domain of the amino acid sequence shown in SEQ ID NO:53, the DAP12 transmembrane domain of the amino acid sequence shown in SEQ ID NO:54, the RC1 transmembrane domain of the amino acid sequence shown in SEQ ID NO:55, and the TLR1 transmembrane domain of the amino acid sequence shown in SEQ ID NO:56. The TLR2 transmembrane domain of the amino acid sequence shown in NO:57 includes the TLR3 transmembrane domain of the amino acid sequence shown in SEQ ID NO:58, the TLR4 transmembrane domain of the amino acid sequence shown in SEQ ID NO:59, the TLR5 transmembrane domain of the amino acid sequence shown in SEQ ID NO:60, the TLR6 transmembrane domain of the amino acid sequence shown in SEQ ID NO:61, the TLR7 transmembrane domain of the amino acid sequence shown in SEQ ID NO:62, the TLR8 transmembrane domain of the amino acid sequence shown in SEQ ID NO:63, or the TLR9 transmembrane domain of the amino acid sequence shown in SEQ ID NO:64.

[0169] In some embodiments, the transmembrane domain is a Tim1 transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:37, a Tim4 transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:38 or 39, a Tim3 transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:40, an FcγR1 transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:41, an FcγR2A transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:42, an FcγR2B2 transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:43, an FcγR2C transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:44, an FcγR3A transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:45, an FcεR1 transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:46, an FcαR1 transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:47, and a transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:47. The following domains are included: the CD8a transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:48; the CD28 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:59; the MERTK transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:50; the Axl transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:51; the Tyro3 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:52; the CD4 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:53; the DAP12 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:54; the MRC1 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:55; the TLR1 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:56; the TLR2 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:57; the TLR3 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:58; and the domains containing or formed by the amino acid sequence shown in SEQ ID NO:48. The TLR4 transmembrane domain, composed of the amino acid sequence shown in SEQ ID NO:59, includes or is composed of the TLR5 transmembrane domain, composed of or is composed of the amino acid sequence shown in SEQ ID NO:60, the TLR6 transmembrane domain, composed of or is composed of the amino acid sequence shown in SEQ ID NO:61, the TLR7 transmembrane domain, composed of or is composed of the amino acid sequence shown in SEQ ID NO:62, and the TLR8 transmembrane domain, composed of or is composed of the amino acid sequence shown in SEQ ID NO:63.It may contain or consist of a TLR9 transmembrane domain composed of the amino acid sequence shown in SEQ ID NO:64.

[0170] Transmembrane domains can originate from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0171] It should be understood that the direct fusion of one domain of a CER to another described herein does not preclude the presence of an intermediate linking amino acid. The linking amino acid can be natural or non-natural (e.g., the result of chimeric protein construct design).

[0172] In some embodiments, the chimeric phagocytic receptor comprises a multinucleotide sequence from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species, or any combination thereof. In some embodiments, the chimeric phagocytic receptor is mouse, chimeric, human, or humanized.

[0173] Embodiments of the CER for use in the cell immunotherapy composition combinations of this disclosure are provided in the Examples, Table 1, and Sequence List, as well as in PCT applications PCT / US2017 / 053553, PCT / US / 2018 / 52297, and U.S. Provisional Applications 62 / 563,615 and 62 / 649,529 (the entire contents of which are incorporated herein by reference).

[0174] Table 1: Exemplary chimeric phagocytic receptors

[0175]

[0176]

[0177]

[0178]

[0179]

[0180] Other embodiments of the CER disclosed herein include an extracellular domain that binds to a prophagocytic marker or target antigen, optionally an extracellular spacer domain, a transmembrane domain, and a phagocytic signaling domain, wherein the phagocytic signaling domain includes a primary phagocytic signaling domain selected from TLR signaling domains and a secondary phagocytic signaling domain selected from TRAF2, TRAF3, or TRAF6 signaling domains. Other embodiments of the CER include an extracellular domain that binds to a prophagocytic marker or target antigen, optionally an extracellular spacer domain, a transmembrane domain, and a phagocytic signaling domain, wherein the phagocytic signaling domain includes a primary phagocytic signaling domain selected from TLR2 or TLR8 signaling domains and a secondary phagocytic signaling domain selected from TRAF2, TRAF6, or DAP12 signaling domains. In some embodiments, the extracellular domain includes scFv. In some embodiments, the extracellular domain includes a Tim4 binding domain. The exemplary Tim4 binding domain comprises the amino acid sequence shown in SEQ ID NO:90 or amino acids 25-314 shown in SEQ ID NO:90. In some embodiments, the transmembrane domain comprises the Tim4 transmembrane domain. The exemplary Tim4 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:38. Exemplary TLR2 and TLR8 signal transduction domains that can be used comprise amino acid sequences comprising SEQ ID NO:24 and SEQ ID NO:30, respectively. The exemplary TRAF2 signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:32. The exemplary DAP12 signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:19. The exemplary TRAF6 signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:9 or 35.

[0181] II. Chimeric antigen receptor

[0182] In some embodiments, the compositions of this disclosure partially comprise immune cells containing transgenes encoding a chimeric antigen receptor (CAR). A chimeric antigen receptor is a recombinant receptor that typically comprises: an extracellular domain including a binding domain for binding to a target antigen; an intracellular signaling domain; and a transmembrane domain located between and connecting the extracellular and intracellular signaling domains. Chimeric antigen receptors typically confer antigen-specific cytotoxic activity to host cells expressing them.

[0183] The binding domain of the CAR applicable to this disclosure includes any antigen-binding polypeptide. The binding domain may comprise an antibody or an antigen-binding fragment thereof, including, for example, a full-length heavy chain, a Fab fragment, Fab', F(ab'), sFv, a VH domain, a VL domain, dAb, VHH, CDR, and scFv. In some embodiments, the CAR binding domain is mouse, chimeric, human, or humanized.

[0184] In some embodiments, the extracellular domain of the CAR provided in this disclosure optionally includes an extracellular, non-signal transduction spacer region or a linker domain. When included, such spacer regions or linker domains can position the binding domain away from the host cell surface to further achieve suitable cell / cell contact, binding, and activation. There is typically an extracellular spacer region between the extracellular binding domain and the transmembrane domain of the CAR. The length of the extracellular spacer region can be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size, and affinity (see, for example, Guest et al., J. Immunother. 28:203-11, 2005; PCT Publication No. WO2014 / 031687). In some embodiments, the extracellular spacer region is an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgD). The hinge region can be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. The modified IgG4 hinge region is described in PCT Publication No. WO2014 / 031687, the entire contents of which are incorporated herein by reference. In one particular embodiment, the extracellular spacer domain comprises the modified IgG4 hinge region having the amino acid sequence ESKYGPPCPPCP (SEQ ID NO:1).

[0185] Other examples of hinge regions that can be used in the CAR described herein include hinge regions of extracellular regions of type 1 membrane proteins (such as CD8a, CD4, CD28, and CD7), which may be wild-type or variants thereof. In a further embodiment, the extracellular spacer region domain comprises all or part of the immunoglobulin Fc domains selected from: CH1 domain, CH2 domain, CH3 domain, or combinations thereof (see, for example, PCT Publication WO2014 / 031687, the entire contents of which are spacers are incorporated herein by reference). In yet another further embodiment, the extracellular spacer region domain may comprise the stem region of type II C-lectins (the extracellular domain located between the C-lectin domain and the transmembrane domain). Type II C-lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D.

[0186] The CAR disclosed herein includes a transmembrane domain that connects and lies between an extracellular domain and an intracellular signaling domain. The transmembrane domain is a hydrophobic α-helix that traverses the host cell membrane and anchors the CAR within it. The transmembrane domain may be directly fused to a binding domain or an extracellular spacer domain (if present). In some embodiments, the transmembrane domain is derived from an intact membrane protein (e.g., receptors, differentiation cluster (CD) molecules, enzymes, transport proteins, cell adhesion molecules, etc.). The transmembrane domain may be selected from molecules that are the same as the extracellular domain or the phagocytic signaling domain (e.g., the CAR includes a CD28 co-stimulatory signaling domain and a CD28 transmembrane domain). In some embodiments, the transmembrane domain and the extracellular domain are selected from different molecules. In other embodiments, the transmembrane domain and the intracellular signaling domain are selected from different molecules. In still other embodiments, the transmembrane domain, the extracellular domain, and the phagocytic signaling domain are selected from different molecules.

[0187] Exemplary transmembrane domains used in the CAR of this disclosure include CD28, CD2, CD3ε, CD3δ, CD3ζ, CD25, CD27, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), C The amino acid sequences listed are D274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, and Zap70. An exemplary CD28 transmembrane domain contains the amino acid sequence shown in SEQ ID NO:49.

[0188] The intracellular signaling domain of a CAR is an intracellular effector domain and is capable of delivering functional signals to the cell in response to the binding of the CAR's extracellular domain to a target molecule. The intracellular signaling domain can be any portion of an intracellular signaling molecule that retains sufficient signal transduction activity. In some embodiments, a full-length intracellular signaling molecule or a full-length intracellular component is used. In some embodiments, a truncated portion of an intracellular signaling molecule or an intracellular component of an intracellular signaling molecule is used, provided that the truncated portion retains sufficient signal transduction activity. In a further embodiment, the intracellular signaling domain is a variant of the entire or truncated portion of an intracellular signaling molecule, provided that the variant retains sufficient signal transduction activity (i.e., is a functional variant).

[0189] In some embodiments, the intracellular signal transduction domain of the CAR includes an immune receptor tyrosine activation motif (ITAM) containing the signal transduction domain. The ITAM containing the signal transduction domain typically comprises at least one (one, two, three, four, or more) ITAMs, referring to the conserved YXXL / IX. 6-8 -YXXL / I motif. ITAMs containing signal transduction domains can initiate T cell activation signaling upon antigen or ligand binding. ITAM signal transduction domains include, for example, intracellular signal transduction domains of CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD278 (ICOS), DAP10, DAP12, and CD66d. An exemplary CD3ζ signal transduction domain in a CAR that can be used in this disclosure comprises the amino acid sequence shown in SEQ ID NO: 158 or 159.

[0190] The intracellular signal transduction domain of the CAR optionally includes a co-stimulatory signal transduction domain that, when activated by binding to a primary or classical (e.g., ITAM-driven) activation signal, initiates or enhances T cell responses such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or a combination thereof. The co-stimulatory signal transduction domains used in CAR include, for example, CD27, CD28, CD40L, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, LFA-1, LIGHT, NKG2C, SLP76, TRIM, ZAP70, or any combination thereof. In one particular embodiment, the co-stimulatory signal transduction domain comprises an OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB (CD137) signal transduction domain. An exemplary CD28 co-stimulatory signal transduction domain that can be used in a CAR of this disclosure comprises the amino acid sequence shown in SEQ ID NO: 161 or 162. An exemplary 4-1BB co-stimulatory signal transduction domain comprises the amino acid sequence shown in SEQ ID NO: 160.

[0191] In some embodiments, the chimeric antigen receptor comprises a multinucleotide sequence from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, their transgenic species, or any combination thereof. In some embodiments, the chimeric antigen receptor is mouse, chimeric, human, or humanized.

[0192] In some implementations, the CAR is a first-generation CAR, a second-generation CAR, or a third-generation CAR. First-generation CARs typically have an intracellular signaling domain containing an intracellular signaling domain of CD3ζ, an FcγRI, or other ITAM-containing activation domain to provide T cell activation signals. Second-generation CARs also contain a co-stimulatory signaling domain (e.g., a co-stimulatory signaling domain derived from an endogenous T cell co-stimulatory receptor, such as CD28, 4-1BB, or ICOS). Third-generation CARs contain an ITAM-containing activation domain, a first co-stimulatory signaling domain, and a second co-stimulatory signaling domain.

[0193] In some implementations, the CAR is a chimeric antigen receptor based on the T-cell receptor (TCR-CAR). A TCR-CAR is a heterodimeric fusion protein that typically comprises a soluble TCR (a polypeptide chain containing Vα and Cα domains and a polypeptide chain containing Vβ and Cβ domains), wherein the VβCβ polypeptide chain is linked to a transmembrane domain and an intracellular signaling component (e.g., an activation domain containing ITAM and optionally a co-stimulatory signaling domain) (see, for example, Walseng et al., 2017 Scientific Reports 7:10713).

[0194] The CAR disclosed herein can target a variety of antigens, including viral antigens, bacterial antigens, fungal antigens, parasitic antigens, tumor antigens, neurodegenerative disease antigens, or autoimmune disease antigens. Exemplary tumor antigens that CARs can target include CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, ROR1, MUC-16, L1CAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPV E7, MUC-1, HER2, folate receptor α, CD97, CD171, CD179a, CD44v6, WT1, VEGF-α, VEGFR1, IL-13Rα1, IL-13Rα2, IL-11Rα, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, liver glycoside A2, liver glycoside B2, Lewis... A antigen, Lewis Y antigen, MAGE, MAGE-A1, RAGE-1, folate receptor β, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucose GM1, GM3, o-acetyl GD2 and GD2.

[0195] III. T cell receptor-binding protein

[0196] In some embodiments, the compositions of this disclosure partially comprise immune cells containing a transgene encoding a recombinant TCR-binding protein. The recombinant TCR-binding protein includes "conventional" TCRs composed of heterodimers of α-chain and β-chain polypeptides (αβTCR) or γ-chain and δ-chain polypeptides (γδTCR), their binding fragments, and fusion proteins, including, for example: single-chain TCRs, single-domain TCRs, soluble TCR fusion TCR proteins, and TCR fusion constructs (TRuC). TM In some implementations, the recombinant TCR is an affinity-enhanced TCR.

[0197] In some embodiments, the recombinant TCR-binding protein is a single-chain TCR (scTCR) containing Vα linked to Vβ via a flexible linker. In some embodiments, the scTCR contains a Vα-linker-Vβ polypeptide. In other embodiments, the scTCR contains a Vβ-linker-Vα polypeptide.

[0198] In some implementations, the recombinant TCR-binding protein is a single-domain TCR (e.g., Vβ).

[0199] In some embodiments, the recombinant TCR-binding protein is a single-chain TCR (scTCR) fusion protein. The scTCR fusion protein comprises a binding domain containing the scTCR (TCR Vα domain linked to the TCR Vβ domain), optionally an extracellular spacer region, a transmembrane domain, and an intracellular signal transduction domain containing the CD3ζITAM activation domain and optionally a co-stimulatory signal transduction domain (see Aggen et al., 2012, Gene Ther. 19:365-374; Stone et al., Cancer Immunol. Immunother. 2014, 63:1163-76).

[0200] In some implementations, the recombinant TCR-binding protein is a TCR fusion construct (TRuC). TM (See, U.S. Patent Publication No. 2017 / 0166622). TRuC TM The construct contains an antigen-specific binding domain (e.g., scFv) that fuses at least one component of the TCR complex (CD3γ, CD3ε, or CD3δ) to form a TCR complex component fusion protein. The human TCR complex contains CD3ε, CD3γ, CD3δ, CD3ζ, TCRα, and TCRβ chain peptides. The TCR complex component fusion protein can bind to other components of the TCR complex to form a functional, complete TCR fusion complex. Unlike TCR, TruC... TM The construct is able to bind to the target antigen in a manner independent of MHC.

[0201] In some embodiments, the TCR-binding protein comprises a multinucleotide sequence from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, their transgenic species, or any combination thereof. In some embodiments, the TCR-binding protein is mouse-derived, chimeric, human-derived, or humanized.

[0202] The TCR-binding protein of this disclosure can bind to a variety of antigens, including tumor antigens, viral antigens, bacterial antigens, fungal antigens, parasitic antigens, neurodegenerative disease antigens, and autoimmune disease antigens. Exemplary tumor antigens that the recombinant TCR-binding protein can target include WT-1, mesothelin, MART-1, NY-ESO-1, MAGE-A3, HPV E7, survivin, alpha-fetoprotein, and tumor-specific neoantigens. An exemplary HPV16 E7 protein-specific TCR that can be used in combination with the cell immunotherapy compositions of this disclosure is provided in PCT Publication No. WO2015 / 184228 (incorporated in its entirety by reference). In some embodiments, the HPV16 E7 TCR comprises the amino acid sequence shown in SEQ ID NO:84. The amino acid sequence of SEQ ID NO:84 contains a P2A self-cleaving peptide between the TCRβ chain sequence and the TCRα chain sequence, which will cleave in the host cell to form two polypeptide chains. Therefore, in some embodiments, the TCR represented by SEQ ID NO:84 comprises separate TCRβ and TCRα polypeptide chains capable of dimerizing to form αβTCR. In some embodiments, the HPV16 E7 TCR comprises Vβ, which contains the amino acid sequence shown in SEQ ID NO:86. In some embodiments, the HPV16 E7 TCR comprises Vα, which contains the amino acid sequence shown in SEQ ID NO:88. In a further embodiment, the HPV16 E7 TCR comprises Vβ containing the amino acid sequence shown in SEQ ID NO:86 and Vα containing the amino acid sequence shown in SEQ ID NO:88.

[0203] In some embodiments, the TCRCα domain, Cβ domain, or both contain cysteine ​​substitutions to form interchain disulfide bonds between cysteine ​​residues in the two constant domains, which are absent in the unmodified TCR. Such modified TCRs can form more stable heterodimers. In a particular embodiment, the Cα domain contains a Thr→Cys substitution at position 48 of the wild-type protein sequence, and the Cβ domain contains a Ser→Cys substitution at position 56 of the wild-type protein sequence (see PCT publication WO2015 / 184228). An exemplary cysteine-modified TCR Cβ constant region contains the amino acid sequence shown in SEQ ID NO:87.

[0204] In some embodiments, the TCR includes replacing one, two, or three amino acids in the transmembrane domain of one or both of the α and β chains with hydrophobic amino acids to increase the hydrophobicity of the transmembrane domain. In some embodiments, one, two, or three residues of the TCR α chain selected from Ser112, Met114, and Gly115 are substituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp. An exemplary cysteine-modified, “LVL”-substituted TCR Cα region comprises the amino acid sequence shown in SEQ ID NO: 89.

[0205] In some embodiments, the CER and CAR or CER and TCR-binding proteins in the combined cell immunotherapy composition target the same antigen. In other embodiments, the CER and CAR or CER and TCR-binding proteins in the combined cell immunotherapy composition target different antigens.

[0206] Polynucleotides, vectors and host cells

[0207] In some aspects, this disclosure provides nucleic acid molecules encoding any one or more cell immunotherapy molecules described herein (e.g., CER, CAR, and TCR-binding proteins). Nucleic acids can refer to single-stranded or double-stranded DNA, cDNA, or RNA, and can include positive and negative strands of nucleic acids complementary to each other, including antisense DNA, cDNA, and RNA. Nucleic acids can be naturally occurring or synthetically produced DNA or RNA. The nucleic acid sequence encoding the desired receptor can be obtained or generated using standard techniques and recombinant methods known in the art, such as by screening a library from cells expressing the desired sequence or a portion thereof, by obtaining the sequence from a vector known to contain the same sequence, or by directly isolating the sequence or a portion thereof from cells or tissues containing the same sequence, as described, for example, in Sambrook et al. (1989 and 2001; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY) and Ausubel et al. (Current Protocols in Molecular Biology, 2003). Alternatively, the target sequence can be produced synthetically rather than cloned.

[0208] The polynucleotide encoding the cell immunotherapy molecule provided herein can be derived from any animal, such as human, primate, cow, horse, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, or a combination thereof. In some embodiments, the polynucleotide encoding the cell immunotherapy molecule is derived from the same animal species as the host cell into which the polynucleotide is inserted.

[0209] In some embodiments, the polynucleotide encoding the cell immunotherapy molecule includes a 5' end sequence encoding a signal peptide (also referred to as a leader peptide or signal sequence) for targeting the precursor protein to the secretion pathway. Optionally, the signal peptide is cleaved from the N-terminus of the extracellular domain during cellular processing and receptor localization to the host cell membrane. A polypeptide with the signal peptide sequence cleaved or removed may also be referred to as a mature polypeptide. Examples of signal peptides that can be used in the receptors of this disclosure include signal peptides derived from endogenous secretory proteins, including, for example, GM-CSF (amino acid sequence shown in SEQ ID NO: 67) or Tim4 (amino acid sequence shown in SEQ ID NO: 68). As used herein, the polynucleotide or polypeptide sequences of the cell immunotherapy molecules (e.g., CER, CAR, or TCR-binding proteins) provided herein may or may not include a signal sequence. Those skilled in the art will understand that for sequences including a signal peptide sequence disclosed herein, another signal peptide capable of transporting the encoded protein to the extracellular membrane may be used instead of that signal peptide sequence.

[0210] In some embodiments, the polynucleotide encoding the cell immunotherapy molecule disclosed herein is codon-optimized for efficient expression in target host cells containing the polynucleotide (see, for example, Scholten et al., Clin. Immunol. 119:135-145 (2006)). As used herein, the “codon-optimized” polynucleotide comprises a heteropolynucleotide having a codon modified by a silenced mutation corresponding to the tRNA abundance in the target host cell.

[0211] The polynucleotide encoding the cell immunotherapy molecule disclosed herein can be operatively linked to expression regulatory sequences. Expression regulatory sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNA; sequences enhancing translation efficiency (i.e., Kozak concordant sequences); sequences enhancing protein stability; and possible sequences enhancing protein secretion.

[0212] In some embodiments, the polynucleotide encoding the cell immunotherapy molecule of this disclosure can be constructed to optimize spatial and temporal control. For example, the polynucleotide encoding the cell immunotherapy molecule may include promoter elements to optimize spatial and temporal control. In some embodiments, the polynucleotide encoding the cell immunotherapy molecule includes a tissue-specific promoter or enhancer that is capable of specifically delivering the polynucleotide encoding the cell immunotherapy molecule to an organ, cell type (e.g., immune cells), or pathological microenvironment, such as tumor or infected tissue. An "enhancer" is an additional promoter element that can act synergistically or independently to activate transcription. In some embodiments, the polynucleotide encoding the cell immunotherapy molecule includes a constitutive promoter. An exemplary constitutive promoter for expressing the polynucleotide of this disclosure is the EF-1α promoter. In some embodiments, the polynucleotide encoding the cell immunotherapy molecule includes an inducible promoter. In some embodiments, the polynucleotide encoding the cell immunotherapy molecule includes a tissue-specific promoter.

[0213] Polynucleotides encoding the cell immunotherapy molecules disclosed herein can be inserted into suitable vectors, such as viral vectors, non-viral plasmid vectors, and non-viral vectors like lipid-based DNA vectors, modified mRNA (modRNA), self-amplified mRNA, CELID, and transposon-mediated gene transfer (PiggyBac), to introduce them into target host cells (e.g., immune cells). Polynucleotides encoding the cell immunotherapy molecules disclosed herein can be cloned into any suitable vector, such as expression vectors, replication vectors, probe-generating vectors, or sequencing vectors. In some embodiments, polynucleotides encoding extracellular domains, transmembrane domains, and phagocytic signaling domains are linked together to form a single polynucleotide encoding a CER, which is then inserted into the vector. In other embodiments, polynucleotides encoding extracellular domains, transmembrane domains, and phagocytic signaling domains can be inserted into the vector separately, such that the expressed amino acid sequence produces a functional CER. Similarly, components encoding CAR or TCR-binding proteins can be assembled before insertion into the vector, or inserted into the vector separately and then assembled. In this paper, vectors encoding CER are referred to as "CER" vectors. Vectors encoding CAR are referred to as "CAR" vectors. Vectors encoding TCR-binding proteins are referred to as "TCR-binding protein vectors." All of these vectors are collectively referred to as "molecular vectors for cell immunotherapy" in this paper.

[0214] In some implementations, vectors are used that allow cellular immunotherapy molecular peptides to integrate long-term and proliferate into progeny cells. Examples include viral vectors such as adenovirus, adeno-associated virus, vaccinia virus, herpesvirus, cytomegalovirus, poxvirus, or retroviral vectors such as lentiviral vectors. Lentiviral-derived vectors can be used to achieve long-term gene transfer and offer several advantages over traditional vectors, including the ability to transduce non-proliferating cells (such as hepatocytes) and low immunogenicity.

[0215] In some embodiments, a free, non-integrating vector is used to encode a polynucleotide of the cell immunotherapy molecule disclosed herein. Examples of non-integrating viral vectors include adenovirus vectors and integrated viral vectors that have been mutated to be non-integrating, such as non-integrating lentiviral vectors and non-integrating foamy virus vectors.

[0216] In this document, the vector encoding the core virus is referred to as a "viral vector." A wide variety of viral vectors are suitable for the compositions of this disclosure, including those identified for human gene therapy applications (see, Pfeifer and Verma, Ann. Rev. Genomics Hum. Genet. 2:177, 2001). Suitable viral vectors include RNA virus-based vectors, such as those derived from retroviruses, for example, vectors derived from Moloney mouse leukemia virus (MLV), as well as vectors derived from more complex retroviruses, such as vectors derived from lentiviruses. Vectors derived from HIV-1 fall into this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (sheep lentivirus). The use of retroviral and lentiviral vectors and packaging cells for transducing mammalian host cells with viral particles containing chimeric receptor transgenes is well known in the art and has been previously described, for example, in U.S. Patent No. 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0217] In some embodiments, the viral vector is used to introduce a non-endogenous polynucleotide sequence encoding a cellular immunotherapy molecule into host cells. The viral vector may be a retroviral vector or a lentiviral vector. The viral vector may also contain a nucleic acid sequence encoding a marker for transduction. Transduction markers used with the viral vector are known in the art and include selectable markers that may confer drug resistance, or detectable markers, such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry. In certain embodiments, the viral vector also contains a genetic marker for transduction, which includes a fluorescent protein (e.g., green, yellow), the extracellular domain of human CD2, or a truncated human EGFR (EGFRt or tEGFR; see Wang et al., Blood 118:1255, 2011). An exemplary tEGFR sequence contains the amino acid sequence shown in SEQ ID NO:70.

[0218] Other viral vectors can also be used for polynucleotide delivery, including DNA viral vectors, such as adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex virus (HSV), including amplicon vectors, replication-defective HSV, and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

[0219] Other viral vectors recently developed for gene therapy applications can also be used in conjunction with the compositions and methods disclosed herein. Such vectors include those derived from baculoviruses and alpha viruses (Jolly, DJ. 1999. Emerging Viral Vectors. pp. 209-40, Friedmann T. ed., The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors).

[0220] Where time control is required, cell immunotherapy molecules can contain elements that induce depletion of transduced cells. For example, such vectors can contain an inducible suicide gene. The suicide gene can be an apoptosis gene or a gene that confers sensitivity to a reagent (e.g., a drug), such as chemically inducible caspase 9 (iCASP9) (US Patent Publication No. 2013 / 0071414), chemically inducible Fas, or herpes simplex virus thymidine kinase (HSV-TK) (conferring sensitivity to ganciclovir). In a further embodiment, cell immunotherapy molecule vectors can be designed to express known cell surface antigens that, upon infusion of an associated antibody, deplete transduced cells. Examples of cell surface antigens and associated antibodies that can be used to deplete transduced cells include CD20 and rituximab, RQR8 (a combination of CD34 and CD20 epitopes capable of CD34 selection and anti-CD20 deletion) and rituximab, and EGFR and cetuximab.

[0221] Inducible vector systems can also be used for the expression of inducible cell immunotherapy molecules, such as the tetracycline (Tet)-On vector system that activates transgenic expression with doxycycline (Heinz et al., Hum. Gene Ther. 2011, 22:166-76). Small molecule reactive transcription factors can also be used to regulate expression. The expression of inducible cell immunotherapy molecules can also be accomplished using a selective hook (RUSH) system, which is based on streptavidin anchored to the endoplasmic reticulum membrane and a streptavidin-binding protein of the introduced cell immunotherapy molecule structure, wherein the addition of biotin to the system causes the cell immunotherapy molecule to be released from the endoplasmic reticulum (Agaugue et al., 2015, Mol. Ther. 23(Suppl. 1):S88).

[0222] In some embodiments, CER-modified host cells may also be modified to co-express one or more small GTPases. Rho GTPases are small (~21 kDa) signaling G protein family members and subfamily members of the Ras superfamily, regulating actin cytoskeleton organization in various cell types during phagocytosis and promoting pseudopodia expansion and phagosome closure (see, for example, Castellano et al., 2000, J. Cell Sci. 113:2955-2961). Phagocytosis requires the recruitment of F-actin to tethered cells or granules, and F-actin rearrangement to allow membrane extension leading to cell or granule internalization. Rho GTPases include RhoA, Rac1, Rac2, RhoG, and CDC42. Other small GTPases (such as Rap1) are involved in the regulation of complement-mediated phagocytosis. Co-expression of small GTPases with CER can promote or enhance granule internalization and / or phagosome formation in target or host cells. In some embodiments, the recombinant nucleic acid molecule encoding the GTPase is encoded on a separate vector compared to a vector containing the CER. In other embodiments, the recombinant nucleic acid molecule encoding the GTPase and the CER are encoded on the same vector. The GTPase and CER can be expressed on the same vector under the regulation of different promoters (e.g., at different multiple cloning sites). Alternatively, the CER and GTPase can be expressed under the regulation of one promoter in a polycistronic vector.

[0223] Examples of GTPases that can be co-expressed with CER include Rac1, Rac2, Rab5 (also known as Rab5a), Rab7, Rap1, RhoA, RhoG, CDC42, or any combination thereof. In a particular embodiment, the GTPase comprises or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence of Rac1 shown in SEQ ID NO:71, the amino acid sequence of Rab5 shown in SEQ ID NO:72, the amino acid sequence of Rab7 shown in SEQ ID NO:73, the amino acid sequence of Rap1 shown in SEQ ID NO:74, the amino acid sequence of RhoA shown in SEQ ID NO:75, the amino acid sequence of CDC42 shown in SEQ ID NO:76, or any combination thereof. In some embodiments, GTPase expression is induced or regulated in host cells such that GTPase expression is activated only after a sufficiently long period of time has elapsed for the CER to bind to its target antigen. In a further embodiment, GTPase expression can be shut down after a sufficient period of time following phagocytosis of the target antigen by a CER-mediated cell.

[0224] In some embodiments, the polynucleotides or vectors of this disclosure may include an internal ribosome entry site (IRES), a furin cleavage site, or a viral 2A peptide positioned between multiple genes encoded therein to allow co-expression of multiple proteins from a single mRNA. For example, the IRES, furin cleavage site, or viral 2A peptide may be located between a polynucleotide encoding a TCRα chain polypeptide and a polynucleotide encoding a TCRβ chain polypeptide. In another example, the IRES, furin cleavage site, or viral 2A peptide may be located between a polynucleotide encoding a CER and a polynucleotide encoding a transduction marker (e.g., a truncated EGFR). In some embodiments, the viral 2A peptide is porcine swine cirrhosis virus-1 (P2A), *T. cirrhosis var. cirrhosis virus* (T2A), *E. cirrhosis var. cirrhosis virus* (E2A), foot-and-mouth disease virus (F2A), or a variant thereof. An exemplary T2A peptide comprises the amino acid sequence shown in any one of SEQ ID NO: 77, 78, 79, and 168. An exemplary P2A peptide comprises the amino acid sequence shown in SEQ ID NO: 80 or 81. The exemplary E2A peptide sequence comprises the amino acid sequence shown in SEQ ID NO:82. The exemplary F2A peptide sequence comprises the amino acid sequence shown in SEQ ID NO:83.

[0225] In some embodiments, by introducing a polynucleotide encoding a cell immunotherapy molecule as described herein, cells (e.g., immune cells) obtained from a subject can be genetically modified into non-natural or recombinant cells (e.g., non-natural or recombinant immune cells) to express cell immunotherapy molecules (e.g., CER, CAR, or TCR-binding proteins) localized to the cell surface. In some embodiments, the host cell is an immune cell, such as a myeloid progenitor cell or a lymphoid progenitor cell. Exemplary immune cells that can be modified to contain a cell immunotherapy molecule or a carrier containing a cell immunotherapy molecule include T cells, natural killer cells, B cells, lymphoid progenitor cells, antigen-presenting cells, dendritic cells, Langerhans cells, myeloid progenitor cells, mature myeloid cells, monocytes, or macrophages.

[0226] In some embodiments, B cells are genetically modified to express the CER of this disclosure. B cells possess certain properties that may be advantageous as host cells, including: transport to sites of inflammation, ability to internalize and present antigens, ability to co-stimulate T cells, high proliferation and self-renewal (lifelong). In some embodiments, CER-modified B cells are capable of digesting phagocytosed target cells or phagocytosed target particles into smaller peptides and presenting them to T cells via MHC molecules. Antigen presentation by CER-modified B cells may facilitate antigen diffusion in immune responses against untargeted antigens. B cells include progenitor cells or precursor cells associated with B cell lineages (e.g., pre-proto-B cells, pro-B cells, and pre-B cells); immature and inactive B cells; or mature and functional or activated B cells. In some embodiments, B cells may be naïve B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytic-like cells, plasmablasts, memory B cells, or any combination thereof. Memory B cells can be distinguished from naïve B cells based on the lack of CD27 expression on naïve B cells. In some embodiments, B cells may be primary cells or cell lines derived from humans, mice, rats, or other mammals. B cell lines are well known to those skilled in the art. If obtained from mammals, B cells can be obtained from many sources, including blood, bone marrow, spleen, lymph nodes, or other tissues or body fluids. B cell components may be enriched or purified.

[0227] In some embodiments, T cells are genetically modified to express the cell immunotherapy molecules of this disclosure (e.g., CER, CAR, and TCR-binding proteins). Exemplary T cells include CD4+. + Helper cells, CD8 + Effector (cytotoxic) cells, naïve (CD45 RA+, CCR7+, CD62L+, CD27+, CD45RO-) cells, central memory (CD45RO-) cells + CD62L + CD8 +The T cells may include effector memory cells (CD45RA+, CD45RO-, CCR7-, CD62L-, CD27-), T memory stem cells, regulatory cells, mucosa-associated invariance (MAIT) cells, γδ (gd) cells, tissue-resident T cells, natural killer T cells, or any combination thereof. In some embodiments, the T cells may be primary cells or cell lines derived from humans, mice, rats, or other mammals. If derived from mammals, the T cells may be obtained from many sources, including blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. The T cell composition may be enriched or purified. T cell lines are well known in the art, some of which are described in Sandberg et al., Leukemia 21:230, 2000. In some embodiments, the T cells lack the TCRα gene, the TCRβ gene, or endogenous expression of both. These T cells may naturally lack endogenous expression of TCRα and β chains, or may have been modified to prevent expression (e.g., T cells from transgenic mice that do not express TCRα and β chains or cells that have been manipulated to suppress TCRα and β chain expression), or by knocking out the TCRα chain, α TCRβ chain, or both genes.

[0228] In some embodiments, the host cell expressing the cell immunotherapy molecules of this disclosure is not a T cell or a cell of the T cell lineage, but a progenitor cell, stem cell, or a cell that has been modified to express anti-CD3 on its cell surface.

[0229] In some embodiments, gene editing methods are used to modify the host cell genome to include polynucleotides encoding the cell immunotherapy molecules disclosed herein. Gene editing, or genome editing, is a genetic engineering method in which genetically engineered endonucleases are used to insert, replace, or delete DNA from the host cell genome. The nucleases form specific double-strand breaks at target loci in the genome. The host cell's endogenous DNA repair pathways then repair one or more induced breaks, for example, through non-homologous end joining (NHEJ) and homologous recombination. Exemplary endonucleases used in gene editing include zinc finger nucleases (ZFNs), transcription activation-like effector (TALE) nucleases, clustered regularly spaced short palindromic repeat (CRISPR) / Cas nuclease systems (e.g., CRISPR-Cas9), large-scale nucleases, or combinations thereof. Methods for disrupting or knocking out genes or gene expression in immune cells, including B cells and T cells, using gene-editing endonucleases are known in the art and are described, for example, in PCT Publications WO 2015 / 066262; WO2013 / 074916; WO 2014 / 059173; Cheong et al., Nat. Comm. 2016 7:10934; Chu et al., Proc. Natl. Acad. Sci. USA 2016 113:12514-12519; the entire contents of the methods from each of these publications are incorporated herein by reference.

[0230] In some implementations, the expression of endogenous genes in host cells is suppressed, knocked down, or eliminated. Examples of endogenous genes that can be suppressed, knocked down, or eliminated in B cells include IGH, IGκ, IGλ, or any combination thereof. Examples of endogenous genes that can be suppressed, knocked down, or eliminated in T cells include TCR genes (TRA or TRB), HLA genes (class I or class II HLA genes), immune checkpoint molecules (PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244 / 2B4, CD160, TIGIT, LAIR-1, or PVRIG / CD112R) or any combination thereof. Endogenous gene expression can be suppressed, knocked down, or eliminated at the gene level, transcription level, translation level, or a combination thereof. For example, suppression, knockdown, or elimination of endogenous genes can be achieved using RNA interference agents (e.g., siRNA, shRNA, miRNA, etc.) or engineered endonucleases (e.g., CRISPR / Cas nuclease systems, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), macronucleases), or any combination thereof. In some embodiments, endogenous B-cell genes (e.g., IGH, IGκ, or IGλ) are knocked out by inserting a polynucleotide encoding CER of this disclosure into a locus of an endogenous B-cell gene, such as using an engineered endonuclease. In some embodiments, endogenous T-cell genes (e.g., TCR genes, HLA genes, or immune checkpoint genes) are knocked out by inserting a polynucleotide encoding CER, CAR, or TCR-binding protein of this disclosure into a locus of an endogenous T-cell gene, such as using an engineered endonuclease.

[0231] This disclosure also provides compositions comprising host cell populations modified with cell immunotherapy molecules. In some embodiments, the host cell population modified with cell immunotherapy molecules may be a population of B cells, T cells, natural killer cells, lymphocyte precursor cells, antigen-presenting cells, dendritic cells, Langerhans cells, bone marrow precursor cells, mature myeloid cells, or any combination thereof. Furthermore, the host cell population modified with cell immunotherapy molecules for a specific cell type may consist of one or more subtypes. For example, the B cell population may consist of CER-modified naive B cells, plasma cells, regulatory B cells, marginal zone B cells, lymphoplasmacytic-like cells, plasmablasts, memory B cells, or any combination thereof. In another example, the T cell population may consist of CAR-modified CD4+. + Helper T cells, CD8 +Effector (cytotoxic) T cells, naive (CD45RA+, CCR7+, CD62L+, CD27+, CD45RO-) T cells, central memory (CD45RO-) T cells, and other T cells. + CD62L + CD8 + T cells, effector memory (CD45RA+, CD45RO-, CCR7-, CD62L-, CD27-) T cells, T memory stem cells, regulatory T cells, mucosa-associated invariant T cells (MAIT), γδ (gd), tissue-resident T cells, natural killer T cells, or any combination thereof.

[0232] In some embodiments, when preparing host cells (e.g., B cells or T cells) modified with cell immunotherapy molecules, one or more growth factor cytokines that promote the proliferation of host cells (e.g., B cells or T cells) may be added to the cell culture medium. The cytokines may be human or non-human. Exemplary growth factor cytokines that can be used to promote T cell proliferation include IL-2, IL-15, etc. Exemplary growth factor cytokines that can be used to promote B cell proliferation include CD40L, IL-2, IL-4, IL-15, IL-21, BAFF, etc.

[0233] Before genetically modifying host cells with polynucleotides encoding cell immunotherapy molecules, the host cells (e.g., T cells, B cells, natural killer cells, etc.) are obtained from an object (e.g., whole blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue), from which host cells are isolated using methods known in the art. Specific subpopulations of host cells can be collected according to known techniques and enriched or depleted according to known techniques, such as antibody affinity binding, flow cytometry, and / or immunomagnetic selection. After the enrichment and / or depletion steps and the introduction of polynucleotides encoding cell immunotherapy molecules, the desired modified host cells can be expanded in vitro according to known techniques or variations of such techniques that will be obvious to those skilled in the art.

[0234] The expression of cell immunotherapy molecules on host cells can be functionally characterized using many methods recognized in the art for assessing host cell (e.g., T cell) activity, including determining T cell binding, activation, or induction, as well as determining antigen-specific T cell responses. Examples include determining T cell proliferation, T cytokine release, antigen-specific T cell stimulation, and CTL activity (e.g., by detecting preloaded target cells). 51The release of Cr or europium, the induction of caspase activity in target cells, the extracellular release of lactate dehydrogenase from target cells, changes in the expression of T cell phenotypic markers, and other indicators of T cell function. Procedures for performing these and similar assays can be found, for example, in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998). See also Current Protocols in Immunology; Weir, Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA (1986); Mishell and Shigii, eds., Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed, Science 281:1309 (1998) and the references cited therein. Cytokine levels can be determined using methods known in the art, including, for example, ELISA, ELISPOT, intracellular cytokine staining, flow cytometry, and any combination thereof (e.g., intracellular cytokine staining and flow cytometry). The proliferation and clonal expansion of immune cells caused by antigen-specific initiation or stimulation of an immune response can be determined by isolating lymphocytes (such as peripheral blood cells or circulating lymphocytes from cell samples from lymph nodes), stimulating the cells with antigens, and measuring cytokine production, cell proliferation, and / or cell viability (such as by incorporation of tritium-substituted thymine or by non-radioactive assays such as MTT assays).

[0235] In some embodiments, the CER-modified host cells have a phagocytic index of approximately 20 to approximately 1,500 for target cells. The “phagocytic index” is a measure of the phagocytic activity of the transduced host cells, determined by counting the number of target cells or particles ingested by each CER-modified host cell in a suspension of target cells or particles and CER-modified host cells incubated in a culture medium over a set time period. It can be expressed as a multiplier [total number of phagocytosed target cells / total number of counted CER-modified cells (e.g., phagocytic frequency)] x [per CER-modified host cell]. + [Average area of ​​target cell or particle staining in host cells x 100 (e.g., hybridization capture)] or [Total number of phagocytosed particles / Total number of CER-modified host cells counted] x [Number of CER-modified host cells containing phagocytosed particles / Number of CER cells counted] +The phagocytic index is calculated by multiplying the total number of cells by 100. In some embodiments, the tandem expression cassette modified cells have approximately 30 to approximately 1,500; approximately 40 to approximately 1,500; approximately 50 to approximately 1,500; approximately 75 to approximately 1,500; approximately 100 to approximately 1,500; approximately 200 to approximately 1,500; approximately 300 to approximately 1,500; approximately 400 to approximately 1,500; approximately 500 to approximately 1,500; approximately 20 to approximately 1,400; approximately 30 to approximately 1,400; approximately 40 to approximately 1,400; approximately 50 to approximately 1,400; approximately 100 to approximately 1,400; approximately 200 to approximately 1,400; approximately 300 to approximately 1, 400; about 400 to about 1,400; about 500 to about 1,400; about 20 to about 1,300; about 30 to about 1,300; about 40 to about 1,300; about 50 to about 1,300; about 100 to about 1,300; about 200 to about 1,300; about 300 to about 1,300; about 400 to about 1,300; about 500 to about 1,300; about 20 to about 1,200; about 30 to about 1,200; about 40 to about 1,200; about 50 to about 1,200; about 100 to about 1,200; about 200 to about 1,200; about 3 00 to about 1,200; about 400 to about 1,200; about 500 to about 1,200; about 20 to about 1,100; about 30 to about 1,100; about 40 to about 1,100; about 50 to about 1,100; about 100 to about 1,100; about 200 to about 1,100; about 300 to about 1,100; about 400 to about 1,100; or about 500 to about 1,100; about 20 to about 1,000; about 30 to about 1,000; about 40 to about 1,000; about 50 to about 1,000; about 100 to about 1,000; about 200 to about A phagocytic index of 1,000; about 300 to about 1,000; about 400 to about 1,000; or about 500 to about 1,000; about 20 to about 750; about 30 to about 750; about 40 to about 750; about 50 to about 750; about 100 to about 750; about 200 to about 750; about 300 to about 750; about 400 to about 750; or about 500 to about 750; about 20 to about 500; about 30 to about 500; about 40 to about 500; about 50 to about 500; about 100 to about 500; about 200 to about 500; or about 300 to about 500. In a further embodiment, the incubation time is about 2 hours to about 4 hours, for example about 2 hours, about 3 hours, or about 4 hours. In a further embodiment, the phagocytic index of CER-modified cells is statistically significantly higher than that of cells transduced using truncated EGFR control.The phagocytic index can be calculated using methods known in the art and further described in the examples and PCT application number PCT / US2017 / 053553 (which are incorporated herein by reference in their entirety), including quantification by flow cytometry or fluorescence microscopy.

[0236] The host cells can be derived from animals such as humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, or combinations thereof. In a preferred embodiment, the animal is a human. The host cells can be derived from a healthy subject or a subject suffering from a disease associated with antigen expression.

[0237] Cellular immunotherapy compositions

[0238] This disclosure provides combinations of cell immunotherapy compositions. The combinations of cell immunotherapy compositions comprise a first composition and a second composition, the first composition comprising immune cells containing a CER (also referred to as "Composition #1"), and the second composition comprising immune cells containing a cell immunotherapy molecule (e.g., CER, CAR, or TCR) (also referred to as "Composition #2"). The CER present in the immune cells of the first composition may be selected from any one or more CER-binding proteins described herein. The CER, CAR, or TCR present in the immune cells of the second composition may be selected from any one or more CER, CAR, or TCR-binding proteins described herein. In a particular embodiment, the immune cells containing a CER in the first composition are CER-modified host cells as described herein, and the immune cells containing the cell immunotherapy molecule in the second composition are host cells modified using CER, CAR, or TCR as described herein. Exemplary embodiments of the combination of host immune cells and cell immunotherapy molecules can be found in Tables 2 and 3.

[0239] In some embodiments, the CER of the first composition and the cell-mediated immunotherapy molecule of the second composition (e.g., CER, CAR, or TCR-binding protein) bind to a target antigen associated with the same disease or condition (e.g., cancer). The CER of the first composition and the cell-mediated immunotherapy molecule of the second composition (e.g., CER, CAR, or TCR-binding protein) may bind to the same target antigen or to different target antigens. In some embodiments, the CER of the first composition binds to a phagocytic marker (e.g., phosphatidylserine) and the cell-mediated immunotherapy molecule of the second composition (e.g., CER, CAR, or TCR-binding protein) binds to a target antigen associated with a disease (e.g., cancer). Other embodiments provide a CER of the first composition that binds to a first tumor antigen and a cell-mediated immunotherapy molecule of the second composition (e.g., CER, CAR, or TCR-binding protein) that binds to the first tumor antigen. Still other embodiments provide a CER of the first composition that binds to a first tumor antigen and a cell-mediated immunotherapy molecule (e.g., CER, CAR, or TCR-binding protein) that binds to a second tumor antigen.

[0240] In one embodiment of the compositions shown in Table 2, specific immune cells of the first composition containing CER target phagocytic markers, while the CER, CAR, or TCR of specific immune cells of the second composition targets tumor antigens. In another embodiment of the compositions shown in Table 2, specific immune cells of the first composition containing CER target phagocytic markers, while the CER, CAR, or TCR of specific immune cells of the second composition targets bacterial, viral, or parasitic antigens. In yet another embodiment of the compositions shown in Table 2, specific immune cells of the first composition containing CER target phagocytic markers, while the CER, CAR, or TCR of specific immune cells of the second composition targets autoimmune disease antigens. In yet another embodiment of the compositions shown in Table 2, specific immune cells of the first composition containing CER target tumor antigens, and the CER, CAR, or TCR of specific immune cells of the second composition targets tumor antigens.

[0241] Table 2: Combinations of Exemplary Cell Immunotherapy Compositions

[0242]

[0243]

[0244]

[0245] Table 3: Combinations of Specific Cell Immunotherapy Compositions

[0246]

[0247]

[0248] In some embodiments, the type of immune cells in the cell immunotherapy composition may include any one or more specific cell subtypes as provided herein. In one example, a cell immunotherapy composition comprising CD4+ T cells includes naive CD4+ T cells, effector memory CD4+ T cells, central memory CD4+ T cells, or any combination thereof. In another example, a cell immunotherapy composition comprising CD8+ T cells includes naive CD8+ T cells, effector memory CD8+ T cells, central memory CD8+ T cells, or any combination thereof. In yet another example, a cell immunotherapy composition comprising B cells includes naive B cells, memory B cells, or both.

[0249] In some embodiments, the combination of cell immunotherapy compositions further comprises a third composition containing immune cells, said immune cells containing cell immunotherapy molecules according to any of the embodiments provided herein, such as CER, CAR, or TCR-binding proteins.

[0250] The cell immunotherapy compositions provided in this disclosure can be formulated into a single pharmaceutical composition comprising a first composition and a second composition. Alternatively, the cell immunotherapy compositions provided in this disclosure can be formulated into different pharmaceutical compositions having a first composition formulated as the first pharmaceutical composition and a second composition formulated as a second pharmaceutical composition different from the first pharmaceutical composition. Embodiments of the cell immunotherapy compositions provided in this disclosure offer multiple non-redundant modes of target cell killing and enhanced effector function. Examples of enhanced effector function include: cytolytic activity against target cells; enhanced activation (e.g., enhanced cytokine production, IFNγ); enhanced cell proliferation; enhanced cell expansion; enhanced persistence; enhanced memory formation; antigen-presenting activity; induction of antigen-specific phagocytic signaling or enhanced antigen-specific phagocytic signaling; degradation of phagocytosed target cells; or any combination thereof. In some embodiments, such cell immunotherapy compositions have a synergistic effect on effector function.

[0251] The relative amounts of the first and second compositions used in the cell immunotherapy compositions according to this specification (whether contained in the same or different formulations) can be adjusted to achieve a specific cell ratio for the recipient. As used herein, the term "cell ratio" refers to the ratio of the number of immune cells contained in the first composition to the number of immune cells contained in the second composition. For example, when a combination of cell immunotherapy compositions according to this specification comprises a first composition having 100 immune cells containing CERs and a second composition having 100 immune cells containing CERs, CARs, or TCRs, the ratio of the first composition to the second composition will be 1:1. In another example, when a combination of cell immunotherapy compositions according to this specification comprises a first composition having 50 immune cells containing CERs and a second composition having 100 immune cells containing CERs, CARs, or TCRs, the ratio of the first composition to the second composition will be 1:2. In another example, when the cell immunotherapy composition according to this specification comprises a first composition having 100 immune cells containing CERs and a second composition having 50 immune cells containing CERs, CARs, or TCRs, the ratio of the first composition to the second composition will be 2:1. In some embodiments, the cell immunotherapy composition according to this specification comprises a first composition and a second composition selected from the following ratios: about 0.1:1, about 0.25:1, about 0.5:1, about 0.75:1, about 1:1, about 1.25:1, about 1.5:1, about 1.75:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 2... 0:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 1:1.1, about 1:1.25, about 1:1.5, about 1:1.75, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, 1:25, about 1:30, about 1:35, about 1:40, about 1:45, and about 1:50. In another embodiment, the cell immunotherapy composition according to this specification comprises the first composition and the second composition in a ratio range selected from the following: from about 1:1 to about 1:2, from about 1:1 to about 1:5, from about 1:1 to about 1:7.5, from about 1:1 to about 1:10, from about 1:1 to about 1:15, from about 1:1 to about 1:20, from about 1:1 to about 1:30, from about 1:1 to about 1:40, and from about 1:1 to about 1:50.In another embodiment, the cell immunotherapy composition according to this specification comprises a first composition and a second composition in a ratio range selected from the following: from about 50:1 to about 1:1, from about 40:1 to about 1:1, from about 30:1 to about 1:1, from about 20:1 to about 1:1, from about 15:1 to about 1:1, from about 10:1 to about 1:1, from about 7.5:1 to about 1:1, from about 5:1 to about 1:1, and from about 2:1 to about 1:1. In each such embodiment, the cell immunotherapy composition may comprise a first composition (i.e., composition #1) and a second composition (i.e., composition #2) according to any combination described herein.

[0252] Embodiments of the combination of specific cell immunotherapy molecules and defined immune cell populations used in the cell immunotherapy compositions disclosed herein provide multiple non-redundant modes of target cell killing and enhanced effector function. For example, the cell immunotherapy composition may comprise: a first composition comprising CD4+ T cells containing a CER, and a second composition comprising CD8+ T cells containing a CAR or TCR-binding protein. Upon binding to an expressed CAR or TCR antigen, the CAR or TCR-modified CD8+ T cells are able to induce target cell apoptosis (cell lysis) by releasing the contents of cytotoxic granules (e.g., granzymes, granzymes, perforin). The CER-modified CD4+ T cells are also able to induce target cell apoptosis upon antigen binding and also secrete Th1 cytokines (e.g., IFN-γ, IL-2) that support cytotoxic CD8+ T cell responses. Furthermore, the CER-modified CD4+ T cells are able to phagocytose target cells bound by the CER. In another example, the cell immunotherapy composition comprises: a first composition comprising B cells containing a CER, and a second composition comprising CD8+ T cells containing a CAR or TCR-binding protein. Upon binding to expressed CAR or TCR antigens, CAR / or TCR-modified CD8+ T cells can induce target cell apoptosis (cell lysis) by releasing the contents of cytotoxic granules (e.g., granzymes, granzymes, perforin). CER-modified B cells can phagocytose target cells bound by CERs. Furthermore, B cells can present internalized antigens to T cells and co-stimulate them. Therefore, the cell immunotherapy compositions provided herein possess unique specificity and functionality conferred by specific cell immunotherapy molecules expressed by host immune cells.

[0253] In some embodiments, the cytotoxic activity of a combination of cell immunotherapy compositions is increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more compared to a combination containing only a first or second cell immunotherapy composition. In further embodiments, a synergistic cytotoxic response is shown. In some embodiments, the host cells are T cells or NK cells. Methods for measuring the cytotoxic activity of host cells (particularly immune cells, such as T cells and NK cells) include chromium (… 51 Cr) release assays, β-gal or firefly luciferase release assays, and flow cytometry methods for measuring target cell death and effector cell activity (see, for example, Expert Rev. Vaccines, 2010, 9:601-616). In some embodiments, host cell cytotoxic activity, such as caspase 3 / 7 activity and lactate dehydrogenase release, can be measured by monitoring apoptosis of target cells after exposure to host cells.

[0254] How to use

[0255] In one aspect, a combination of cell immunotherapy compositions according to any of the embodiments provided herein may be used in a method of treating a subject suffering from a disease, condition, or adverse condition. Embodiments of these methods include administering to the subject a therapeutically effective amount of one or more pharmaceutical compositions comprising a combination of cell immunotherapy compositions according to this specification.

[0256] Diseases that can be treated with the combination of cell immunotherapy compositions provided in this disclosure include cancer, autoimmune diseases, neurodegenerative diseases, and infectious diseases (viral, bacterial, fungal, protozoan infections). Adoptive immunotherapy and gene therapy are promising therapies for various types of cancer (Morgan et al., Science 314:126, 2006; Schmitt et al., Hum. Gene Ther. 20:1240, 2009; June, J. Clin. Invest. 117:1466, 2007) and infectious diseases (Kitchen et al., PLoS One 4:38208, 2009; Rossi et al., Nat. Biotechnol. 25:1444, 2007; Zhang et al., PLoS Pathog. 6:e1001018, 2010; Luo et al., J. Mol. Med. 89:903, 2011).

[0257] A variety of cancers, including solid tumors and leukemias, are suitable for combination therapy using the cell immunotherapy compositions described herein. Exemplary cancer types that can be treated with the combination of the cell immunotherapy compositions described herein include breast, prostate, and colonic adenocarcinoma; all forms of bronchogenic carcinoma; myeloid leukemia; melanoma; liver cancer; neuroblastoma; papilloma; amine precursor uptake and decarboxylation cell tumors; vacuole tumors; branchial protozoa; malignant carcinoid syndromes; carcinoid heart disease; and carcinomas (e.g., Walker carcinoma, basal cell carcinoma, basal squamous cell carcinoma, Brown-Pearce carcinoma, ductal carcinoma, Ehrlich tumor, Krebs 2 carcinoma, Merkel cell carcinoma, mucinous carcinoma, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, scleroderma, bronchiolar carcinoma, bronchial carcinoma, squamous cell carcinoma, and transitional cell carcinoma). Other cancer types that can be treated using the receptors, modified host cells, and compositions described herein include histiocytosis; malignant histiocytosis; leukemia; Hodgkin's disease; immunoproliferative enteropathy; non-Hodgkin's lymphoma; plasmacytoma; multiple myeloma; plasmacytoma; reticuloendothelial proliferation; melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giant cell tumor; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma; dysgerminoma; hamartoma; stromal tumor; mesonephroma; sarcoma; ameloblastoma; cementum tumor; odontoma; teratoma; thymoma; and trophoblastic tumor. Furthermore, it is also considered suitable for treating the following types of cancer: adenoma; cholangioma; cholesteatoma; cylindrica; cystadenocarcinoma; cystadenoma; granulosa cell tumor; germinal cell tumor; liver cancer; hidradenoma; islet cell tumor; Ledich's cell tumor; papilloma; Sertoli cell tumor; theca cell tumor; leiomyoma; leiomyosarcoma; myoblastoma; myoma; sarcoma; rhabdomyosarcoma; rhabdomyosarcoma; ependymoma; ganglionoma; glioma; medulloblastoma; meningioma; schwannoma; neuroblastoma; neuroepithelial tumor; neurofibroma; neuroma; paraganglioma; paraganglioma; non-pheochromocytoma. Treatable cancer types also include angiokeratoma; angiolymphoid hyperplasia with eosinophilia; hemangioma sclerosis; hemangiomatosis; glomus tumor; hemangioendothelioma; hemangioma; hemangiopericytoma; angiosarcoma; lymphangioma; lymphangiomyoma; lymphangiosarcoma; pineal tumor; carcinosarcoma; chondrosarcoma; phyllodes sarcoma; fibrosarcoma; angiosarcoma; leiomyosarcoma; leukemic sarcoma; liposarcoma; lymphangiosarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; sarcoma; growths; neurofibromatosis; and cervical dysplasia.

[0258] Exemplary hyperproliferative disorders suitable for combination therapy using the cell immunotherapy compositions described herein are B-cell cancers, including B-cell lymphomas (such as various forms of Hodgkin's disease, non-Hodgkin's lymphoma (NHL), or central nervous system lymphoma), leukemias (such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, B-blast transformation of chronic myeloid leukemia), and myelomas (such as multiple myeloma). Other B-cell cancers that can be treated with the combination of the cell immunotherapy compositions described herein include small lymphocytic lymphoma, B-cell prelymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytic myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extra-joint marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), lymph node marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma / leukemia, B-cell proliferation with uncertain malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorders.

[0259] Infectious diseases include those associated with infectious agents and include any number of bacteria (e.g., pathogenic E. coli, S. typhimurium, P. aeruginosa, B. anthracis, C. botulinum, C. difficile, C. perfringens, H. pylori, V. cholerae, Listeria spp., Rickettsia spp., Chlamydiaspp., etc.), mycobacteria, and parasites (including any known parasitic member of protozoa). Infectious viruses include eukaryotic viruses such as adenoviruses, Bunyaviruses, herpesviruses, polymorphonuclear papillomaviruses, papillomaviruses (e.g., HPV), paramyxoviruses, picornaviruses, rhabdoviruses (e.g., rabies virus), orthomyxoviruses (e.g., influenza virus), poxviruses (e.g., vaccinia virus), reoviruses, retroviruses, lentiviruses (e.g., HIV), flaviviruses (e.g., HCV, HBV), etc. In some embodiments, a combination of cell immunotherapy compositions according to the present disclosure is used to treat infections caused by microorganisms that establish a persistent infection in a subject.

[0260] The method of treating a subject includes administering an effective amount of a combination of the cell immunotherapy compositions of this disclosure. The combination of cell immunotherapy compositions may be allogeneic, syngeneic, allogeneic, or autologous for the subject. Furthermore, each individual cell immunotherapy composition in the cell immunotherapy composition may independently be allogeneic, syngeneic, allogeneic, or autologous for the subject.

[0261] Pharmaceutical compositions comprising cell immunotherapy compositions can be administered in a manner suitable for the disease or condition being treated (or prevented) as determined by a person skilled in the medical field. The appropriate dose, duration, and frequency of administration of the composition will be determined by factors such as the patient's condition, physique, weight, body surface area, age, sex, type and severity of the disease, the specific therapy being administered, the specific form of the active ingredient, the time and method of administration, and any other medications administered concurrently. This disclosure provides pharmaceutical compositions comprising a cell immunotherapy composition and a pharmaceutically acceptable carrier, diluent, or excipient. Suitable excipients include water, saline, dextran, glycerol, and combinations thereof. Other suitable infusion media may be any isotonic media formulation, including saline, Normosol R (Abbott), Plasma-Lyte A (Baxter), 5% dextran aqueous solution, or Ringer's lactate. In some embodiments, the cell immunotherapy compositions in the combination are formulated together in the same pharmaceutical composition. In other embodiments, each cell immunotherapy composition in the combination is formulated as a separate pharmaceutical composition.

[0262] The therapeutically effective amount of cells in the pharmaceutical composition is at least one cell (e.g., one CER-modified T cell) or more generally more than 10. 2 Cells, for example, up to 10 6 Up to 10 7 Up to 10 8 Cells, up to 10 9 Cells, up to 10 10 One cell or up to 10 11 One or more cells. In some embodiments, cells are arranged from about 10 6 To about 10 10 cells / m 2 The range, preferably about 10 7 To about 10 9 Application is carried out at a rate of cells / m². In a particular embodiment, at least about 1 x 10⁻⁶ cells / m² is used. 6 1 cell, 2 x 10 6 1 cell, 3 x 10 6 1 cell, 4 x 10 6 5 x 10 cells 6 1 cell, 6 x 10 6 1 cell, 7x10 6 1 cell, 8 x 10 6 1 cell, 9 x 10 6 1 cell, 1 x 10 7 1 cell, 2 x 10 7 1 cell, 3 x 107 1 cell, 4x10 7 5 x 10 cells 7 1 cell, 6 x 10 7 1 cell, 7 x 10 7 1 cell, 8 x 10 7 1 cell, 9 x 10 7 1 cell, 1x10 8 1 cell, 2 x 10 8 1 cell, 3 x 10 8 1 cell, 4x10 8 5 x 10 cells 8 1 cell, 6 x 10 8 1 cell, 7x10 8 1 cell, 8 x 10 8 1 cell, 9 x 10 8 1 cell, 1 x 10 9 1 cell, 2 x 10 9 1 cell, 3 x 10 9 1 cell, 4x10 9 5 x 10 cells 9 1 cell, 6 x 10 9 1 cell, 7 x 10 9 1 cell, 8 x 10 9 1 cell, 9 x 10 9 1 cell, 1x10 10 1 cell, 2 x 10 10 1 cell, 3 x 10 10 1 cell, 4 x 10 10 5 x 10 cells 10 1 cell, 6 x 10 10 1 cell, 7 x 10 10 1 cell, 8 x 10 10 1 cell, 9 x 10 10 1 cell, 1 x 10 11 1 cell, 2 x 10 11 1 cell, 3 x 10 11 1 cell, 4 x 10 11 5 x 10 cells 11 1 cell, 6 x 10 11 1 cell, 7 x 10 11 1 cell, 8 x 10 11 1 cell or 9 x 10 11The amount of cells applied is CER-modified cells. The number of cells will depend on the intended end use of the composition and the cell types contained therein. For example, a composition containing CER-modified cells will contain a cell population containing about 5% to about 95% or more of such cells. In some embodiments, the composition containing CER-modified cells contains a cell population containing at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. For the purposes described herein, the cells are typically in volumes of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Therefore, the desired cell density is typically greater than 10. 4 cells / ml, and generally greater than 10 7 10 cells / ml, typically 10 8 Cells / ml or higher. Cells can be administered as a single infusion or multiple infusions over a period of time. If there is a recurrence of disease or disease activity, repeated infusions of molecularly modified cells for cell immunotherapy can be administered at intervals of days, weeks, months, or even years. Clinically relevant numbers of immune cells can be allocated across multiple infusions, accumulating to equal or greater than 10⁻⁶ cells / ml. 6 10 7 10 8 10 9 10 10 Or 10 11 One cell. The preferred dose for administering host cells containing the recombinant expression vector as described herein is about 10. 7 cells / m 2 Approximately 5x10 7 cells / m 2 Approximately 10 8 cells / m 2 Approximately 5 x 10 8 cells / m 2 Approximately 10 9 cells / m 2 Approximately 5 x 10 9 cells / m 2 Approximately 10 10 cells / m 2 Approximately 5 x 10 10 cells / m 2 Or about 10 11 cells / m 2 .

[0263] In some embodiments, the first composition, the second composition, or both are administered at a dose that may be considered a subtherapeutic treatment if administered as a monotherapy. The first composition comprises immune cells containing a CER, and the second composition comprises immune cells containing a cell immunotherapy molecule (e.g., CER, CAR, or TCR-binding protein). In such embodiments, a combination of the first and second compositions may provide an additive or synergistic effect, allowing the first composition, the second composition, or both to be administered at a lower dose.

[0264] The cell immunotherapy compositions described herein can be administered intravenously, intraperitoneally, intranasally, intratumorally, intramedullary, intramedullary, intralymphatic, and / or intracerebrospinal fluid.

[0265] When a cell immunotherapy composition in a combination therapy is formulated into a separate pharmaceutical composition, the treatment method includes administering a first composition containing immune cells expressing CER, CAR, or TCR-binding proteins as described herein, before (e.g., 1 to 7 days, 1 to 10 days, 1 to 14 days, 1 to 30 days, or more days before the second composition), simultaneously (on the same day) with the second composition, or after the second composition (e.g., 1 to 7 days, 1 to 10 days, 1 to 14 days, 1 to 30 days, or more days after the second composition). In some embodiments, the first composition containing immune cells expressing CER is administered after the administration of the second composition containing immune cells expressing CER, CAR, or TCR. In a further embodiment, the first composition is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the administration of the second composition. In yet another further embodiment, the first composition is administered within 4 weeks, 3 weeks, 2 weeks, or 1 week after the administration of the second composition. When the second composition involves multiple doses, the first composition may be administered after the initial dose of the second composition, after the final dose of the second composition, or between multiple doses of the second composition.

[0266] The cell immunotherapy composition can be combined with one or more other therapeutic agents and administered to the subject. Examples of therapeutic agents that can be administered in combination with the cell immunotherapy composition according to this specification include radiotherapy, antibody therapy, immune checkpoint molecule inhibitor therapy, UV phototherapy, electroporation therapy, high-intensity focused ultrasound therapy, oncolytic virus therapy, or drug therapy such as chemotherapy agents, therapeutic peptides, hormone therapy, aptamers, antibiotics, antiviral agents, antifungal agents, anti-inflammatory agents, and small molecule therapies.

[0267] Radiation therapy includes external beam radiation therapy (e.g., conventional external beam radiation therapy, stereotactic radiation therapy, three-dimensional conformal radiation therapy, intensity-modulated radiation therapy, volume-modulated arc therapy, particle therapy, proton therapy, and auger therapy), brachytherapy, whole-body radioisotope therapy, intraoperative radiation therapy, or any combination thereof. In some embodiments, the radiation therapy dose used in conjunction with CER therapy is lower than the typical dose. Low-dose radiation therapy may be sufficient to cause sublytic membrane damage to cells, but not necessarily cell lysis. Sublytic membrane disruption is sufficient to expose prophagocytic markers (e.g., phosphatidylserine) that can be targeted by CER therapy.

[0268] Exemplary antibodies used in combination with the cell immunotherapy compositions described herein include rituximab, pertuzumab, trastuzumab, alemtuzumab, teimomab, bentuximab, cetuximab, bevacizumab, abciximab, adalimumab, afasicept, baliximab, belimumab, bezoolomab, canatumab, cetuzumab, dacrolimus, denosumab, efalizumab, golimumab, olaratumab, pallizumab, panitumumab, and tocilizumab.

[0269] Inhibitors of exemplary immune checkpoint molecules that can be used in combination with the cell immunotherapy compositions described herein include checkpoint inhibitors targeting PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244 / 2B4, CD160, TIGIT, LAIR-1, PVRIG / CD112R, or any combination thereof. In some embodiments, the immune checkpoint inhibitor may be an antibody, peptide, RNAi agent, or small molecule. An antibody specific to CTLA-4 may be ipilimumab or tramemumab. An antibody specific to PD-1 may be pidilizumab, nivolumab, or pembrolizumab. Antibodies that specifically target PD-L1 can be durvalumab, atezolizumab, or avelumab.

[0270] Chemotherapy agents include nonspecific cytotoxic agents that inhibit mitosis or cell division, and molecularly targeted therapies that stop cancer cell growth and spread by targeting specific molecules (e.g., oncogenes) associated with tumor growth, progression, and metastasis. Exemplary nonspecific chemotherapeutic agents for use in combination with the cell immunotherapy compositions described herein include alkylating agents, platinum-based agents, cytotoxic agents, chromatin function inhibitors, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (such as folic acid antagonists, pyrimidine analogs, purine analogs, and glycomodification analogs), DNA synthesis inhibitors, DNA interactors (such as intercalating agents), and DNA repair inhibitors.

[0271] Exemplary molecularly targeted inhibitors for use in combination with the cell immunotherapy compositions described herein include inhibitors of target molecules involved in cancer cell growth and survival, including, for example, hormones, signal transduction inhibitors, gene expression inhibitors (e.g., translation inhibitors), apoptosis inducers, angiogenesis inhibitors (e.g., VEGF pathway inhibitors), GTPase inhibitors, receptor tyrosine kinase inhibitors, growth factor inhibitors, serine / threonine kinase inhibitors, transcription factor inhibitors, and tyrosine kinase inhibitors (e.g., EGF / EGFR pathway inhibitors). Other exemplary molecularly targeted inhibitors include B-Raf inhibitors, MEK inhibitors, mTOR inhibitors, adenosine pathway inhibitors, EGFR inhibitors, ALK inhibitors, VEGFR inhibitors, MET inhibitors, MYC inhibitors, ABS inhibitors, HER2 inhibitors, H-RAS inhibitors, K-RAS inhibitors, PDGFR inhibitors, PI3K inhibitors, BCR-ABL inhibitors, ALK / ROS1 inhibitors, and BTK inhibitors. In some embodiments, the use of molecularly targeted therapy includes administering a molecularly targeted therapy specific to the molecular target to a subject identified as having a tumor with a molecular target (e.g., a driver oncogene). In some embodiments, the molecular target has an activating mutation. In some embodiments, combining CER-modified cells with a molecularly targeted inhibitor can enhance the intensity, durability, or both of the antitumor response. In some embodiments, a sub-typical or sub-therapeutic dose of molecularly targeted therapy is combined with CER-modified cells.

[0272] Examples of chemotherapy agents considered for use in the combination therapies discussed in this article include vemurafenib, dabrafenib, trametinib, cobimetinib, and anastrozole. Bicalutamide Bleomycin sulfate Bai Xiaoan Busulfan Injection Capecitabine N4-pentoxycarbonyl-5-deoxy-5-fluorocytosine nucleoside, carboplatin Camustin Chlorinated nitrogen mustard Cisplatin Kratsubin Cyclophosphamide ( or ), cytarabine, cytarabine Cytarabine liposome injection Dacarbazine Actinomycin (Actinomycin D, Cosmegan), Daunorubicin Hydrochloride Daunorubicin Citrate Liposome Injection Dexamethasone, Docetaxel Doxorubicin Hydrochloride Etoposide Fludarabine phosphate 5-Fluorouracil Flutamide Tezatabin, gemcitabine (difluorodeoxycytidine), hydroxyurea Idabi Star Ifosfamide Irinotecan L-asparaginase Calcium formyltetrahydrofolate, melphalan 6-Mercaptopurine Methotrexate Mitothrone Gemtuzumab, Paclitaxel Phoenix (Yttrium 90 / MX-DTPA), Pentostatin, and polyphenylene 20 containing carmustine implants Tamoxifen Citrate teniposide 6-Thioguanine, thiotepa, telazamine Topotecan Hydrochloride for Injection Vincristine Changchun New Alkali Ibrutinib, Venetocin, Crizotinib, Alectinib, Brigatinib, Ceritinib, and Vinorelbine

[0273] Exemplary alkylating agents used in the combination therapies considered herein include nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazines: uramustine (Aminouracil) Uracilnitrogen nitrogen mustard Cyclophosphamide ( Revimmune TM), ifosfamide Meifalun Chlorinated nitrogen mustard piperobromine Triethylene melamine Triethylenethiophosphoramide, temozolomide Thiotepa Bai Xiaoan Camustin Lomustine streptozotocin and dacarbazine Other exemplary alkylating agents used in the combination therapies considered in this article include, but are not limited to, oxaliplatin. Temozolomide ( and Actinomycin (also known as actinomycin D), ); Mefarin (also known as L-PAM, L-oncolytic toxin, and phenylalanine mustard) ); Hexamethylmelamine (also known as hexamethylmelamine (HMM), ); Camustin Bendamustine Bai Xiaoan ( and Carboplatin Lomustine (also known as CCNU, ); Cisplatin (also known as CDDP, and -AQ); Chlorobutyric acid mustard Cyclophosphamide ( and ); Dacarbazine (also known as DTIC, DIC, and imidazole carboxamide, ); Hexamethylmelamine (also known as hexamethylmelamine (HMM), Ifosfamide Prednumustine; Methylbenzylhydrazine Dichloromethyldiethylamine (also known as nitrogen mustard, nitrogen mustard, and methylchloroethylamine hydrochloride) Streptozotocin Thiotepa (also known as thiophosphoramide, TESPA, and TSPA) ); Cyclophosphamide and bendamustine hydrochloride

[0274] Exemplary platinum-based agents used for the combination therapies considered herein include carboplatin, cisplatin, oxaliplatin, nedaplatin, pyridine, saxaplatin, phenanthreneplatin, and triplatintetranitrate.

[0275] Exemplary angiogenesis inhibitors for use in combination with the cell immunotherapy compositions described herein may include, but are not limited to, A6 (Angstrom Pharmaceuticals), ABT-510 (Abbott Laboratories), ABT-627 (Atrasentan) (Abbott Laboratories / Xinlay), ABT-869 (Abbott Laboratories), Actimid (CC4047, Pomalidomide) (Celgene Corporation), AdGVPEDF.11D (GenVec), ADH-1 (Exherin) (Adherex Technologies), AEE788 (Novartis), AG-013736 (Axitinib) (Pfizer), AG3340 (Promasitol) (Agouron Pharmaceuticals), AGX1053 (AngioGenex), AGX51 (AngioGenex), ALN-VSP (ALN-VSP O2) (Alnylam Pharmaceuticals), AMG 386 (Amgen), AMG706 (Amgen), Apatinib (YN968D1) (Jiangsu Hengrui Medicine), AP23573 (Desfotiam / MK8669) (Ariad Pharmaceuticals), AQ4N (Novavea), ARQ 197 (ArQule), ASA404 (Novartis / Antisoma), Atemod (Callisto Pharmaceuticals), ATN-161 (Attenuon), AV-412 (Aveo Pharmaceuticals), AV-951 (Aveo Pharmaceuticals), Avastin (Bevacizumab) (Genentech), AZD2171 (Sildenafil / Recentin) (AstraZeneca), BAY57-9352 (Tiratitinib) (Bayer), BEZ235 (Novartis), BIBF1120 (Boehringer Ingelheim) Pharmaceuticals), BIBW 2992 (Boehringer Ingelheim Pharmaceuticals), BMS-275291 (Bristol-Myers Squibb), BMS-582664 (brivanib) (Bristol-Myers Squibb),BMS-690514 (Bristol-Myers Squibb), Calcitriol, CCI-779 (Wyeth), CDP-791 (ImClone Systems), High-Crude Torreya (HHT) (ChemGenex Therapeutics), Celebrex (Celexicob) (Pfizer), CEP-7055 (Cephalon / Sanofi), CHIR-265 (Chiron Corporation), NGR-TNF, COL-3 (Metastat) (Collagenex Pharmaceuticals), Comparepine (Oxigene), CP-751, 871 (Figitumumab) (Pfizer), CP-547, 632 (Pfizer), CS-7017 (Daiichi Sankyo) Pharma), CT-322 (Angiocept) (Adnexus), Curcumin, Dalteparin (Fanamin) (Pfizer), Disulfiram (Antabuse), E7820 (Eisai Limited), E7080 (Eisai Limited), EMD 121974 (Silengitide) (EMD Pharmaceuticals), ENMD-1198 (EntreMed), ENMD-2076 (EntreMed), Endostar (Simcere), Erbitux (ImClone / Bristol-Myers Squibb), EZN-2208 (Enzon Pharmaceuticals), EZN-2968 (Enzon Pharmaceuticals) Pharmaceuticals), GC1008 (Genzyme), Genistein, GSK1363089 (Foretinib) (GlaxoSmithKline), GW786034 (Pazopanib) (GlaxoSmithKline), GT-111 (Vascular Biogenics Ltd.), IMC-1121B (Ramucumab) (ImClone Systems), IMC-18F1 (ImClone Systems), IMC-3G3 (ImClone LLC), INCB007839 (Incyte Corporation), INGN 241 (Introgen Therapeutics), Iressa (ZD1839 / Gefitinib), LBH589 (Faridak / Panobinostst) (Novartis), Lucentis (Ramucumab) (Genentech / Novartis),LY317615 (Enzastaurin) (Eli Lilly and Company), Macugen (Pegatani) (Pfizer), MEDI522 (Abegrin) (MedImmune), MLN518 (Tandutinib) (Millennium), Neovastat (AE941 / Benifen) (Aeterna Zentaris), Nexavar (Bayer / Onyx), NM-3 (Genzyme Corporation), Noscapine (Cougar Biotechnology), NPI-2358 (Nereus Pharmaceuticals), OSI-930 (OSI), Palomid 529 (Paloma Pharmaceuticals, Inc.), Panzem Capsules (2ME2) ​​(EntreMed), Panzem NCD (2ME2) ​​(EntreMed), PF-02341066 (Pfizer), PF-04554878 (Pfizer), PI-88 (Progen) Industries / Medigen Biotechnology), PKC412 (Novartis), Tea Polyphenol E (Green Tea Extract) (Polypheno E International, Inc.), PPI-2458 (Praecis Pharmaceuticals), PTC299 (PTC Therapeutics), PTK787 (Valtarani) (Novartis), PXD101 (Belisitar) (CuraGen Corporation), RAD001 (Everolimus) (Novartis), RAF265 (Novartis), Regorafenib (BAY73-4506) (Bayer), Revlimid (Celgene), Anacostat (Alcon) Research), SN38 (liposomes) (Neopharm), SNS-032 (BMS-387032) (Sunesis), SOM230 (paretide) (Novartis), squalamine (Genaera), suramin, sunitin (Pfizer), taraxer (Genentech), TB-403 (Thrombogenics), tempostatin (Collard Biopharmaceuticals), tetrathiomolybdate (Sigma-Aldrich), TG100801 (TargeGen), thalidomide (Celgene Corporation), tinzaparin sodium, TKI258 (Novartis),TRC093 (Tracon Pharmaceuticals Inc.), VEGF Trap (Aflibercept) (Regeneron Pharmaceuticals), VEGF Trap-Eye (Regeneron Pharmaceuticals), Veglin (VasGene Therapeutics), Bortezomib (Millennium), XL184 (Exelixis), XL647 (Exelixis), XL784 (Exelixis), XL820 (Exelixis), XL999 (Exelixis), ZD6474 (AstraZeneca), Vorinostat (Merck), and ZSTK474.

[0276] Exemplary vascular endothelial growth factor (VEGF) receptor inhibitors that can be used in combination with the cell immunotherapy compositions described herein include, but are not limited to, bevacizumab. Axitinib Alanine brinib (BMS-582664, (S)-((R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-yloxy)propyl-2-yl)2-aminopropionate); sorafenib Pazopanib Sunitinib malate Sildenafil (AZD2171, CAS 288383-20-1); Nintedanib (BIBF1120, CAS 928326-83-4); Foretinib (GSK1363089); Tilatinib (BAY57-9352, CAS332012-40-5); Apatinib (YN968D1, CAS 811803-05-1); Imatinib Panatinib (AP24534, CAS 943319-70-8); Tivozanib (AV951, CAS475108-18-0); Regorafenib (BAY73-4506, CAS 755037-03-7); Vatalanib dihydrochloride (PTK787, CAS 212141-51-0); Brinib (BMS-540215, CAS 649735-46-6); Vandetanib ( Or AZD6474); Moteseni diphosphate (AMG706, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridylmethyl)amino]-3-pyridinecarboxamide, as described in PCT Publication No. WO 02 / 066470); Dovirtinib dilactate (TKI258, CAS852433-84-2); Linfanib (ABT869, CAS 796967-16-3); Cabozantinib (XL184, CAS 849217-68-1); Lettatinib (CAS 111358-88-4); N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS38703, CAS) 345627-80-7); (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopentan[c]pyrrolo-5-yl]methoxy]-4-quinazolinamine (XL647, CAS781613-23-8); 4-methyl-3-[[1-methyl-6-(3-pyridyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]amino]-N-[3-(trifluoromethyl)phenyl]-benzamide (BHG712, CAS 940310-85-0); and Abercet

[0277] Exemplary EGF pathway inhibitors that can be used in combination with the cell immunotherapy compositions described herein include, but are not limited to, tyrosine phosphorylation inhibitors 46, EKB-569, and erlotinib. Gefitinib Cerbitux, Nimotuzumab, Lapatinib Cetuximab (anti-EGFR mAb) 188 Re-labeled nimotuzumab (anti-EGFR mAb), and those compounds generally and specifically disclosed in WO 97 / 02266, EP 0 564 409, WO 99 / 03854, EP0 520 722, EP 0 566 226, EP 0 787 722, EP 0 837 063, U.S. Patent Nos. 5,747,498, WO 98 / 10767, WO 97 / 30034, WO 97 / 49688, WO 97 / 38983, and WO 96 / 33980. Exemplary EGFR antibodies include, but are not limited to, cetuximab. Panitumumab Martuzumab (EMD-72000); Trastuzumab Nimotuzumab (hR3); Zatumab; TheraCIM h-R3; MDX0447 (CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1). Exemplary epidermal growth factor receptor (EGFR) inhibitors include, but are not limited to, osimertinib. Erlotinib Hydrochloride Osimertinib, Erlotinib, Brigatinib N-[4-[(3-chloro-4-fluorobenzene)amino]-7-[[(3″S″)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide, ); Van der Tani Lapatinib (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); cannabinib dihydrochloride (CI-1033); 6-[4-[(4-ethyl-1-piperazinyl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine (AEE788, CAS) 497839-62-0); Muritinib (TAK165); Peritinib (EKB569); Afatinib (BIBW2992); Lenatinib (HKI-272); N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]carbamic acid, (3S)-3-morpholinomethyl ester (BMS599626); N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopentan[c]pyrrolo-5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8); and 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]phenol (PKI166, CAS 187724-61-4).

[0278] Exemplary mTOR inhibitors that can be used in combination with the cell immunotherapy compositions described herein include, but are not limited to, rapamycin. and its analogues and derivatives; SDZ-RAD; tesimolimus ( Also known as CCI-779); deferolimus (formerly known as deferolimus, dimethylphosphonic acid (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentoxy-11,36-dioxa-4-azatricyclo[30.3.1.0]). 4,9 [36-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl ester, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03 / 064383); everolimus ( Or RAD001); Rapamycin (AY22989, ); Simapimod (CAS 164301-51-3); (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N 2 -[1,4-dioxo-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholin-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-asparticyl-L-serine-inner salt (SF1126, CAS 936487-67-1).

[0279] In some embodiments, the tyrosine kinase inhibitor that can be used in combination with the cell immunotherapy compositions described herein is an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK inhibitors include crizotinib, ceritinib, alectinib, brigatinib, dalantercept, entrectinib, and lorlatinib.

[0280] Exemplary phosphoinositol 3-kinase (PI3K) inhibitors that can be used in combination with the cell immunotherapy compositions described herein include, but are not limited to, 4-[2-(1H-indazol-4-yl)-6-[[4-(methanesulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as GDC0941 and described in PCT publications WO 09 / 036082 and WO09 / 055730); 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile (also known as BEZ 235 or NVP-BEZ 235 and described in PCT publication WO 09 / 036082 and WO09 / 055730). 06 / 122806); 4-(trifluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridine-2-amine (also known as BKM120 or NVP-BKM120, and described in PCT Publication No. WO2007 / 084786); Tóuzhärtyl (VX680 or MK-0457, CAS) 639089-54-6); (5Z)-5-[[4-(4-pyridyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione (GSK1059615, CAS958852-01-2); (1E,4S,4aR,5R,6aS,9aR)-5-(acetoxy)-1-[(di-2-propenylamino)methylene]-4,4a,5,6,6a,8,9,9a-octahydro-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-cyclopentan[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione (PX866, CAS 502632-66-8); and 8-phenyl-2-(morpholin-4-yl)-chromen-4-one (LY294002, CAS 154447-36-6).Exemplary protein kinase B (PKB) or AKT inhibitors include, but are not limited to, 8-[4-(1-aminocyclobutyl)benzene]-9-phenyl-1,2,4-thiazo[3,4-f][1,6]naphthyl-3(2H)-one (MK-2206, CAS1032349-93-1); perifoxine (KRX0401); 4-dodecyl-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (PHT-427, CAS 1191951-57-1); 4-[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-7-[(3S)-3-piperidinylmethoxy]-1H-imidazo[4,5-c]pyridin-4-yl]-2-methyl-3-butyn-2-ol (GSK690693, CAS 937174-76-0); 8-(1-hydroxyethyl)-2-methoxy-3-[(4-methoxyphenyl)methoxy]-6H-dibenzo[b,d]pyran-6-one (palomid 529, P529 or SG-00529); Tricirbine (6-amino-4-methyl-8-(β-D-furanribosyl)-4H,8H-pyrrolo[4,3,2-de]pyrimidino[4,5-c]pyridazine); (αS)-α-[[[5-(3-methyl-1H-indazol-5-yl)-3-pyridyl]oxy]methyl]phenethylamine (A674563, CAS) 552325-73-2); 4-[(4-chlorophenyl)methyl]-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidineamine (CCT128930, CAS 885499-61-6); 4-(4-chlorophenyl)-4-[4-(1H-pyrazol-4-yl)phenyl]-piperidine (AT7867, CAS 857531-00-1); and Archexin (RX-0201, CAS663232-27-7).

[0281] In some embodiments where a combination of cell immunotherapy compositions is administered in combination with one or more other therapies, the one or more other therapies may be administered at a dose that would be considered subtherapeutic when administered as a single therapy. In such embodiments, the combination of cell immunotherapy compositions may provide an additive or synergistic effect, allowing one or more other therapies to be administered at a lower dose. Combination therapies include administering the combination of cell immunotherapy compositions described herein before (e.g., 1 to 30 days or longer before other therapies), concurrently with (on the same day) with other therapies, or after (e.g., 1 to 30 days or longer after other therapies). In some embodiments, the combination of cell immunotherapy compositions is administered after administration of one or more other therapies. In further embodiments, cells modified with cell immunotherapy molecules are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after administration of one or more other therapies. In a further embodiment, the combination of cell immunotherapy compositions is administered within 4 weeks, 3 weeks, 2 weeks, or 1 week after administration of one or more other therapies. When one or more other therapies involve multiple doses, the combination of cell immunotherapy compositions may be administered after the initial dose of one or more other therapies, after the final dose of one or more other therapies, or between multiple doses of one or more other therapies.

[0282] In some embodiments, the methods of this disclosure include an exhaustion step. An exhaustion step can be performed after a sufficiently long period of therapeutic benefit to remove cells modified with the cell immunotherapy molecule from the subject, thereby mitigating toxicity to the subject. In such embodiments, the vector containing the cell immunotherapy molecule (e.g., CER, CAR, or TCR-binding protein) may contain an inducible suicide gene, such as iCASP9, inducible Fas, or HSV-TK. Similarly, the vector can be engineered to express a known cell surface antigen, such as CD20 or a truncated EGFR (SEQ ID NO:70), which promotes exhaustion of transduced cells by infusion of an associated monoclonal antibody (mAb). For example, rituximab targeting CD20 or cetuximab targeting EGFR. Alemtuzumab, which targets CD52 present on the surface of mature lymphocytes, can also be used to exhaust transduced B cells, T cells, or natural killer cells.

[0283] Subjects that can be treated with the compositions and methods disclosed herein include animals such as humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, or pigs. Subjects can be male or female, and can be of any suitable age, including infants, adolescents, teenagers, adults, and the elderly.

[0284] Example

[0285] Example 1: Construction of CER, TCR and modified T cells

[0286] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domain of TLR4 to form a chimeric phagocytic receptor “CER5” encoding the amino acid sequence shown in SEQ ID NO:94.

[0287] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domain of TLR3 to form a chimeric phagocytic receptor “CER17” encoding the amino acid sequence shown in SEQ ID NO:112.

[0288] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domain of TLR5 to form a chimeric phagocytic receptor “CER19” encoding the amino acid sequence shown in SEQ ID NO:95.

[0289] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domain of TLR8 to form a chimeric phagocytic receptor “CER21” encoding the amino acid sequence shown in SEQ ID NO:96.

[0290] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domain of TLR9 to form a chimeric phagocytic receptor “CER23” encoding the amino acid sequence shown in SEQ ID NO:116.

[0291] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domain of TLR1 to form a chimeric phagocytic receptor “CER26” encoding the amino acid sequence shown in SEQ ID NO:118.

[0292] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domain of TLR2 to form a chimeric phagocytic receptor “CER27” encoding the amino acid sequence shown in SEQ ID NO:98.

[0293] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domain of TRAF6 to form a chimeric phagocytic receptor “CER29” encoding the amino acid sequence shown in SEQ ID NO:99.

[0294] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domain of TRAF2 to form a chimeric phagocytic receptor “CER30” encoding the amino acid sequence shown in SEQ ID NO:120.

[0295] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domain of TLR8 and a truncated phagocytic signaling domain of CD79b to form a chimeric phagocytic receptor “CER103B” encoding the amino acid sequence shown in SEQ ID NO: 138.

[0296] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domains of TLR8 and DAP12 to form a chimeric phagocytic receptor “CER104” encoding the amino acid sequence shown in SEQ ID NO:139.

[0297] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domains of TLR8 and BAFF-R to form a chimeric phagocytic receptor “CER105” encoding the amino acid sequence shown in SEQ ID NO:140.

[0298] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domains of NFAM1 and TLR8 to form a chimeric phagocytic receptor “CER106” encoding the amino acid sequence shown in SEQ ID NO:141.

[0299] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domains of TRAF6 and DAP12 to form a chimeric phagocytic receptor “CER110” encoding the amino acid sequence shown in SEQ ID NO:145.

[0300] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domains of TRAF6 and NFAM1 to form a chimeric phagocytic receptor “CER112” encoding the amino acid sequence shown in SEQ ID NO:148.

[0301] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domains of TRAF6 and BAFF-R to form a chimeric phagocytic receptor “CER113” encoding the amino acid sequence shown in SEQ ID NO:149.

[0302] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domains of TRAF6 and MERTK to form a chimeric phagocytic receptor “CER114” encoding the amino acid sequence shown in SEQ ID NO:150.

[0303] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domains of MERTK and TRAF6 to form a chimeric phagocytic receptor “CER115” encoding the amino acid sequence shown in SEQ ID NO:151.

[0304] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domains of Traf6 and TLR8 to form a chimeric phagocytic receptor “CER116” encoding the amino acid sequence shown in SEQ ID NO:152.

[0305] A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim 4 was fused with the phagocytic signaling domains of TLR8 and TRAF6 to form a chimeric phagocytic receptor “CER117” encoding the amino acid sequence shown in SEQ ID NO:153.

[0306] A polynucleotide encoding the TCRβ chain and a polynucleotide encoding the TCRα chain of the HPV16 E7-specific TCR (see PCT Publication No. WO2015 / 184228) are fused together with a sequence encoding a P2A self-cleaving peptide. The TCR Vα domain contains the amino acid sequence shown in SEQ ID NO:88, and the TCR Vβ region contains the amino acid sequence shown in SEQ ID NO:86. The Cα domain contains cysteine ​​and LVL substitutions at positions 12, 14, and 15, and contains the amino acid sequence shown in SEQ ID NO:89. The Cβ domain also contains cysteine ​​substitutions and contains the amino acid sequence shown in SEQ ID NO:87. The encoded HPV16 E7-specific TCR contains the amino acid sequence shown in SEQ ID NO:84.

[0307] Selected CER polynucleotides and HPV16 E7 TCR polynucleotides were each inserted into pLenti lentiviral vectors. Peripheral blood was collected from human donors via venipuncture, and human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using lymphocyte separation media. CD8+ or CD4+ T cells were enriched from PBMCs using a commercially available isolation kit and activated with anti-CD3 and anti-CD28 in complete cell growth medium. 50 μl of the viral vector expressing CER-HPV16 E7 TCR was diluted in 0.5 ml of complete cell growth medium and added to CD8+ T cells. 50 μl of the viral vector expressing the selected CER was diluted in 0.5 ml of complete cell growth medium and added to CD4+ T cells. The transduced T cells were then centrifuged at 270 x g rpm for 1 h in a preheated centrifuge at 32 °C. The T cells were incubated at 37 °C for 24 h. T cells were further expanded in complete cell growth medium for 72 hours, the beads were removed, and the cells were allowed to expand for 5 days before being used for functional assays. Transduced CD4+ and CD8+ T cells were combined in a 1:1 ratio for functional assays.

[0308] Example 2: The combination of CD8+ T cells-TCR+CD4+ T cells-CER showed enhanced antigen-specific cells. Dissolution and phagocytic activity

[0309] Dual elimination of SCC152 target cells mediated by HPV16 E7 TCR and CER was detected using cytotoxicity and phagocytosis assays (see [link to assay]). Figure 3 SCC152 cells are HPV16+ cells derived from hypopharyngeal squamous cell carcinoma. Apoptosis was achieved using caspase 3 / 7 apoptosis reagent. The cytotoxic activity of HPV16E7-specific TCR-transduced CD8+ T cells was assessed using a reagent that conjugates an activated caspase 3 / 7 recognition motif to a red reagent that fluoresces upon lysis. Fluorescence signals were measured using fluorescence microscopy. CD8+ T cells transduced with HPV16E7 TCR and selected CER-transduced CD4+ T cells were co-cultured 1:1 with HPV16E7+ head and neck squamous cell carcinoma cells (SCC152), and the caspase 3 / 7 apoptosis reagent was added to the co-culture. Cytotoxic activity over time was measured by fluorescence. A control sample consisted of CD8+ T cells transduced with HPV16E7 TCR alone. Figure 4 , 5 And the diagram in 26, and Figure 6-18 As shown in the fluorescence micrographs, adding CD4+ T cells transduced with most of the tested CERs to CD8+ T cells transduced with HPV16 E7 TCRs enhances cell lysis activity compared to single treatment with CD8+ T cells transduced with HPV16 E7 TCRs.

[0310] When measured using the lactate dehydrogenase (LDH) cytotoxicity assay, enhanced cytolytic activity was observed in CD4+ T cells transduced with CER104 and CD8+ T cells transduced with HPV16 E7 TCR (see [link to assay]). Figure 19 LDH is a cytoplasmic enzyme that cells release into the cell culture medium when the plasma membrane is damaged. Therefore, the presence of LDH in the culture medium is a marker of cell death. LDH assays can detect low-level damage to the cell membrane that other methods cannot. LDH can be detected using colorimetric or fluorescence methods.

[0311] Elimination of SCC152 target cells was also detected by quantifying the expression of green fluorescent protein in SCC152 cells over time (0 h, 24 h, 48 h) during co-incubation with CD8+ T cells transduced with HPV16 E7-specific TCR and CD4 T cells transduced with selected CER (see [link to original text]). Figure 20 By 48 hours, all co-cultures of CD4+ T cells / CER+CD8+ T cells / HPV16 E7 TCR combinations showed enhanced elimination of SCC152 cells compared to the control. Time-lapse imaging of the co-culture experiments similarly showed that the co-cultures of CD4+ T cells / CER+CD8+ T cells / HPV16 E7 TCR combinations enhanced the elimination of SCC152 cells compared to the control (see [link to co-culture experiments]). Figure 21-25 ).

[0312] Cytokine responses in co-culture experiments were measured by sampling cell supernatant using a medium-sized multi-array cytokine plate. The following cytokines were measured: IFNγ, IL-2, TNFα, IL-4, IL-6, IL-12b, IL-13, IL-1b, and IL-10. Compared to control, co-cultures with CD4+ T cells / CER+CD8+ T cells / HPV16 E7 TCRs elicited enhanced production of cytokines indicating activation signatures (e.g., IFNγ, IL-2) (see [link to relevant documentation]). Figure 27 ).

[0313] The phagocytic activity of CD4+ T cells / CER+CD8+ T cells / HPV16 E7 TCR combination co-cultured with SCC152 cells was visualized and quantified using a KEYENCE BZ-X710 fluorescence microscope (20x objective) and hybridization capture software. Figure 28-42 The study showed that various CER-transduced CD4+ T cells used in co-culture with CD8+ T cells / HPVE7 TCRs exhibited enhanced phagocytosis of SCC152 target cells compared to co-culture with control CD8+ T cells / HPV16 E7 TCRs alone.

[0314] The cell lysis and phagocytic activity of the composition, comprising CD8+ T cells transduced using HPV16 E7 TCR and CD4+ T cells transduced using selected CERs containing the TRAF signaling domain, were also tested. HPV16 E7 TCR-transduced CD8+ T cells and selected CERs (CER29, CER30, CER110, CER112, CER113, CER114, CER115, CER116, or CER117)-transduced CD4+ T cells were mixed 1:1 and co-cultured 1:1 with HPV16 E7+ head and neck squamous cell carcinoma cells (SCC152). Caspase 3 / 7 apoptosis reagent was added to the co-culture after 6 hours. Cytotoxic activity over time was measured by fluorescence. The control sample consisted of CD8+ T cells transduced using HPV16 E7 TCR + CD4+ T cells transduced using control. Figure 43 and 44 The bar chart in the middle and Figures 45-54 As shown in the fluorescence microscopy images, adding CER-transduced CD4+ T cells to CD8+ T cells transduced with HPV16 E7 TCR enhanced cell lysis activity.

[0315] The phagocytic activity of CD4+ T cells / CER+CD8+ T cells / HPV16 E7 TCR co-cultured with SCC152 cells was visualized and quantified using a KEYENCE BZ-X710 fluorescence microscope (20x objective) and hybridization capture software. CD4+ T cells / control + CD8+ T cells / HPV16 E7 TCR served as a control. Figure 55 and Figure 56 The bar charts show that, compared with the control, using CD4+ T cells transduced with all detected CERs (CER29, CER30, CER110, CER112, CER113, CER114, CER115, CER116, or CER117) to co-culture with CD8 T cells / HPV E7 TCRs enhanced the phagocytosis of SCC152 target cells.

[0316] Elimination of SCC152 target cells was also detected by quantifying the expression of green fluorescent protein in SCC152 cells over time (0 h, 12 h, 24 h, 36 h) during co-incubation with HPV16 E7-specific TCR-transduced CD8+ T cells and CD4 T cells transduced with selected CERs (CER29, CER30, CER110, CER112, CER113, CER114, CER115, CER116, or CER117). (See [link to relevant documentation]). Figure 57 By 36 hours, co-cultures treated with a composition containing CD4+ T cells transduced with CER30, CER112, CER113, CER114, CER116, or CER117 showed near-complete elimination of SCC152 cells compared to controls (see [link to study]). Figure 57 Time-lag imaging of co-culture experiments similarly showed enhanced elimination of SCC152 cells in co-cultures treated with CD4+ T cells transduced with CER30, CER112, CER113, CER114, CER116, or CER117 compared to controls. Figure 58 and Figure 59 ).

[0317] Example 3: Characterization of CER-modified CD4 T cells

[0318] The breadth of response of various CER-modified CD4+ T cells was also evaluated to determine whether a particular CER conferred a low-intensity, broad phagocytic response in host cells (e.g., 10% phagocytosis in 90% of cells) or a low-frequency but strong phagocytic response (e.g., 90% phagocytosis in 10% of cells). As described in Example 1, CD8+ T cells were transduced using an HPV16 E7-specific TCR. CD4+ T cells were transduced using a lentiviral vector containing CER21, CER27, CER104, CER116, or CER117 nucleic acids. Simulated transduced (vector only) CD4+ T cells served as a control. CD4+ / CER+ and CD8+ / E7 TCR+ T cells were stained with CELLTRACE violet. HPV16 E7+ head and neck squamous cell carcinoma cells (SCC152) were stained with pHrodo red. HPV16 E7TCR-transduced CD8+ T cells and selected CER-transduced CD4+ T cells were mixed at a 1:1 ratio and co-cultured with SCC152 cells at a 1:1 ratio for 8 hours. The phagocytic effect of CER-transduced CD4+ T cells on SCC152 target cells was analyzed by fluorescence microscopy. Figure 60A The amplitude and extent of phagocytosis for CER types are shown in the curves. The horizontal axis represents the percentage of CER-transduced CD4+ T cells phagocytosed or the percentage of CER-transduced CD4+ T cells taken up by SCC152 target cells. This metric rarely exceeds 40% across all tested CER types. The vertical axis represents the proportion of phagocytogenic CER-transduced CD4+ T cells. For CER104, approximately 20% of CER104-transduced CD4+ T cells exhibited phagocytosis exceeding 10%. For CER117-transduced CD4+ T cells, less than 10% exhibited phagocytosis exceeding 10%. Figure 60B Fluorescence micrographs of SCC152 target cells phagocytosed by CER126-transduced CD4+ T cells are shown.

[0319] CD4+ T cells were transduced using a lentiviral vector containing CER21, CER27, CER102, CER103A, CER103B, CER104, CER106, CER116, or CER117 nucleic acids. Simulated transduced (vector only) CD4+ T cells served as a control. CD8+ T cells were transduced using an HPV16E7-specific TCR. CD8+ T cells transduced using the HPV16E7 TCR and selected CER-transduced CD4+ T cells were mixed 1:1 and co-cultured 1:1 with SCC152 cells for 10 hours. The supernatant was then collected, and the secretion of bulk cytokines was analyzed. Figure 61As shown, adding CD4+ T cells expressing CER to CD8+ T cells transduced with E7 TCR enhanced the level of IFNγ secretion.

[0320] Example 4: Antigen presentation by CER-modified T cells

[0321] One strategy for enhancing tumor-killing effects through cytotoxic CD8+ T cells (CTLs) is the use of antigen-presenting cells (APCs), which possess a unique ability to “cross-present” exogenous antigens on MHC I molecules. Expanding tumor-specific CTL responses has the potential to induce effective immune responses against tumors. In this example, viral HPV E6 and E7 oncoproteins are used as model antigens to characterize the antigen processing and presentation capabilities of cells expressing chimeric phagocytic receptors (CERs).

[0322] T cell lines expressing CD4+ and CD8+ CER were established from human PBMCs. After activation with CD3 and CD28 microbeads, purified T cells were channeled using a lentivirus encoding CER123 (SEQ ID NO:164) and a truncated EGFR (transduction marker), and then expanded for 5 days in medium containing IL-7, IL-15, and IL-2. The percentage of tEGFR+ T cells ranged from 40% to 60%.

[0323] Jurkat cell lines with a stably integrated NFAT-inducible luciferase reporter gene construct were used to study T cell responses. Human E6 and E7-specific engineered TCRs were transduced into Jurkat NFAT reporter cell lines to identify NFAT activation after co-culturing with engineered CERs.

[0324] To assess MHC-I cross-presentation, SCC152HPV+ cells were co-cultured overnight with CER123-expressing CD4+ and CD8+ T cells or vector-mimicked T cells (vector only) in the presence of T cells expressing E7-specific TCRs. After overnight co-culture, CER123-expressing T cells or vector-mimicked T cells were purified using FACS, washed, and then cultured at a 1:1 ratio with E6 / E7-specific human TCR / NFAT reporter cell lines. NFAT activation was assessed in the cell culture supernatant by measuring luciferase activity at a series of time points (0, 6, 12, 24, and 72 hours). A schematic diagram of this assay is shown below. Figure 62As shown in the figure. Cells were cultured in RPMI / 10% FCS in 96-well round-bottom culture plates. After engulfing HPV+ tumors, CD4+ and CD8+ T cell lines expressing CER123 were co-cultured overnight with Jurkat T cells expressing E711-19-specific TCR and NFAT reporter proteins. At the time points shown, the induction of E711-19-specific Jurkat T cells was quantified by the luminescence of NFAT signaling and compared with T cells transduced by mimic (vector only). Figure 63 After engulfing HPV+ tumor cells, T cells expressing CER123 showed enhanced cross-presentation of HPV E7 oncoprotein.

[0325] Example 5: Marker analysis of CER-modified CD4 T cells

[0326] CD4+ T cells were transduced using a lentiviral vector containing CER10, CER116, or CER117 nucleic acid. CER104 (SEQ ID NO: 139) contains a Tim4 binding domain, a Tim4 transmembrane domain, and a phagocytic signaling domain, wherein the phagocytic signaling domain includes a major phagocytic signaling domain containing a TLR8 signaling domain and a minor phagocytic signaling domain containing a DAP12 signaling domain. CER116 (SEQ ID NO: 152) contains a Tim4 binding domain, a Tim4 transmembrane domain, and a phagocytic signaling domain, wherein the phagocytic signaling domain includes a major phagocytic signaling domain containing a TRAF6 signaling domain and a minor phagocytic signaling domain containing a TLR8 signaling domain. CER117 (SEQ ID NO:153) comprises a Tim4 binding domain, a Tim4 transmembrane domain, and a phagocytic signaling domain, wherein the phagocytic signaling domain comprises a major phagocytic signaling domain containing a TLR8 signaling domain and a minor phagocytic signaling domain containing a TRAF6 signaling domain. CER-transduced CD4+ T cells were co-cultured with E7 TCR-transduced CD8+ T cells and HPV+ SCC152 target cells, and questioned using CyTOF mass cytometry with viSNE for visualization of high-dimensional single-cell data (Figures 64-66). Complete CER-induced CD4+ T cells are shown in the figure displaying the tSNE1 (horizontal axis) and tSNE2 (vertical axis) axes. Twenty-seven intracellular markers were used for viSNE analysis. Each point represents a single cell. The graph was colored using several measured markers (GM-CSF, MIP1b, perforin, TNF, IL-17, granzyme B, IL-4, IL-2, and IFNγ) to show the phenotype across viSNE “islands”. Figure 64AFor each marker, red represents high expression and blue represents low expression. CD4+ T cell populations were generated from all 27 markers using a clustering algorithm and overlaid on a viSNE plot. Arrows indicate the enrichment of islands expressing the intracellular marker IFNγ in samples containing CER104, CER116, and CER117. Figure 64B CD4+ T cell populations were generated from all 18 markers using a clustering algorithm and overlaid on the viSNE map. Figure 65A The arrows indicate the enrichment of islands expressing the T cell activation marker CD69 in samples containing CD4+ T cells transduced with CER104 and CER116. A color plot using 18 intracellular markers (CD28, CCR7, CD45RA, PD1, CD127, perforin, CD49d, CD85j, CD38, CD27, granzyme B, CD57, CD25, CD69, CD154, CD56, HLA-DR, and TCRγδ) shows the phenotype of the transviSNE “islands”. Figure 65B For each marker, red represents high expression and blue represents low expression. Highlighted areas with arrows indicate cells expressing the T cell activation marker CD69. CD4+ T cell populations were generated from 18 intracellular markers (CD28, CCR7, CD45RA, PD1, CD127, perforin, CD49d, CD85j, CD38, CD27, granzyme B, CD57, CD25, CD69, CD154, CD56, HLA-DR, and TCRγδ) using a clustering algorithm and overlaid on a viSNE atlas. Arrows indicate the loss of islands expressing the initial T cell marker CD45RA in the CCR7+ population in CER104 and CER116 samples compared to the control. Figure 66A Color images of the viSNE “islands” were displayed using 18 intracellular markers. Figure 66B For each marker, red represents high expression and blue represents low expression. The highlighted areas with arrows indicate cells expressing the naïve T cell marker CD45RA. Therefore, this data suggests that CD4+ T cells transduced by CER104 and CER116 are associated with the formation of post-antigen exposure memory.

[0327] Example 6: Most CD4+CER-modified T cells exhibit phagocytic activity and enhance tumor-killing effects in vivo.

[0328] CD4+ or CD3+ T cells were purified from PBMCs, activated, and transduced using a lentiviral vector containing hCER104 nucleic acid. hCER104 is a human CER104 construct containing a Tim4 binding domain, a Tim4 transmembrane domain, a TLR8 phagocytic signaling domain, and a Dap12 phagocytic signaling domain, and contains the amino acid sequence shown in SEQ ID NO:176. HCC827 cells carried an EGFR mutation, and treatment with the EGFR inhibitor osimertinib induced the exposure of phosphatidylserine on the cell surface. Transduced T cells were expanded. CER-modified T cells were co-cultured overnight with pHrodo red-labeled HCC827 NSCLC adenocarcinoma cells, and the phagocytosis of HCC827 target cells was evaluated by FACS for pHrodo positive signal (see [link to FACS]). Figure 67A (The boxes in the FACS figure represent phagocytosis %). T cell subsets were analyzed for their phagocytic capacity using antibody staining. CD4+CER-modified T cells exhibited a significantly higher frequency of phagocytosis in vitro than CD8+CER-modified T cells (see [link to FACS figure]). Figure 67B ).

[0329] hCER104-modified CD3+ T cells and hCER104-modified CD4+ T cells were co-cultured with HCC827 NSCLC adenocarcinoma cells for 48 hours, with or without 1 nM osimertinib, at an effector cell to target cell ratio of 5:1. Simulated transduced T cells were used as a control. The viability of HCC827 cells after co-culture was measured by MTT assay, and... Figure 68 As shown in the figure. Compared with hCER104-modified CD4+ T cells, HCC827 cells exhibited lower cell viability when co-cultured with hCER104-modified CD4+ T cells, which represented a mixture of CD4+ and CD8+ T cell subsets. Phase-contrast microscopy after 48 hours of co-culture showed that, in the presence of osimertinib, hCER104-modified CD4+ T cells and CD3+ T cells specifically killed HCC827 cells (…). Figure 69 ).

[0330] CD8+ T cells containing HPV E7-specific TCR modification (1 x 10^6 cells) 6 (as described in Example 1) and hCER104-modified CD4+ T cells (3 x 10) 6The combination of cell immunotherapy compositions was injected into a mouse model of head and neck squamous cell carcinoma induced by injecting HPV+ SCC152 cells into NSG mice, and compared with SCC152 xenograft mice treated only with HPV E7-specific TCR-modified CD8+ T cells (n = 5 mice / treatment group). Tumor volume was measured over time using calipers, and as shown in the figure. Figure 70 As shown in the figure, adding CER104-modified CD4+ T cells to E7-specific TCR-modified CD8+ T cells enhanced tumor killing in vivo.

[0331] CD3+ or CD4+ T cells were purified from PBMCs, activated, and transduced using a lentiviral vector containing hCER122 nucleic acid. hCER122 is a human CER122 construct containing a Tim4 binding domain, a Tim4 transmembrane domain, a TLR2 phagocytic signaling domain, and a Dap12 phagocytic signaling domain, and contains the amino acid sequence shown in SEQ ID NO:179. The transduced T cells were expanded. hCER122-modified CD3+ T cells (2.5 x 10⁻⁶) were... 6 Or 5 x 10 6 ) or hCER122-modified CD4+ T cells (2.5 x 10⁻⁶) 6 Or 5 x 10 6 The drug was injected into NSG mice transplanted with HCC827 adenocarcinoma cells (2 million cells / mouse). Following phagocytosis, the HCC827 xenograft mice also received 1 mg / kg osimertinib. Compared with osimertinib alone, hCER122-modified CD3+ and CD4+ T cells showed enhanced in vivo antitumor responses. Figure 71 hCER122-modified CD4+ T cells eliminated tumor xenograft models. Figure 71 Microscopic images of tumor cells from the HCC827 xenograft model, collected on day 16 and stained with immunofluorescence. Tumor specimens were stained using anti-EGFR (tumor antigen), anti-CD4, anti-PD1, and DAPI counterstains. Tumor staining showed PD1+CD4+ T cells (functionally active) infiltrating into the tumor stroma. Figure 72 (Image on the left). Figure 72 (The image on the right) shows T cells stained with anti-PD1.

[0332] Example 7: Inhibition of heterolysosomal V-ATPase eliminated CER-induced phagocytosis.

[0333] As described in Example 6, T cells were transduced using a lentiviral vector containing hCER104 nucleic acid. hCER104-modified T cells were co-cultured with HCC827 adenocarcinoma cells with or without osimertinib. Bafloxacin, an inhibitor of V-ATPase, disrupts the acidification of phagosomes. hCER104-modified T cells exhibited phagocytosis of HCC827 cells labeled with TAMRA-SE fluorescent dye. Figure 73A Adding bafloxacin (20 nM) to the co-culture prevented hCER104-modified T cells from taking up the labeled HCC827 cells. Figure 73A FACS plots of in vitro phagocytosis assays from hCER104-modified T cells or control T cells with simulated transduction are shown in [images]. Figure 73B middle.

[0334] Example 8: In vivo enhancement of E7-specific TCR-modified CD8+ T cells by HCE R104-modified CD4+ T cells Tumor killing effect

[0335] HPV+SCC152 / luciferase+ cells were transplanted into NSG mice. Once tumors formed, mice were treated with either HPV E7-specific TCR (SEQ ID NO:84)-modified CD8+ T cells + mimicked transduced CD4+ T cells, HPV E7-specific TCR-modified CD8+ T cells + hCER104 (SEQ ID NO:176)-modified CD4+ T cells, or no treatment (n=5 per treatment group). HPV E7 CD8+ T cells and hCER104 CD4+ T cells were administered at a 1:1 ratio. Tumor volume over time was measured by bioluminescence imaging (see [link to relevant documentation]). Figures 74A-74B In xenograft models, HPV E7 CD8+ T cells exhibited an anti-tumor response compared to untreated controls. Adding hCER104 CD4+ T cells to HPV E7 CD8+ T cells enhanced tumor-killing activity in vivo.

[0336] The various embodiments described above can be combined to provide other embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in and / or listed in the Application Data Sheet, including but not limited to U.S. Provisional Patent Application No. 62 / 649,541, filed March 28, 2018; U.S. Provisional Patent Application No. 62 / 652,838, filed April 4, 2018; and U.S. Provisional Patent Application No. 62 / 734,863, filed September 21, 2018, are incorporated herein by reference in their entirety. If necessary, aspects of the embodiments can be modified using the concepts of the various patents, applications, and publications described above to provide other embodiments.

[0337] These and other changes can be made to the embodiments based on the detailed description above. Generally, the terminology used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted to include all possible embodiments and the full scope of equivalents enjoyed by these claims. Therefore, the claims are not limited to the disclosure. sequence list <110> Shinro Therapy Company D.M. Corey <120> Cellular Immunotherapy Compositions and Their Uses <130> 200265.407WO <140> PCT <141> 2019-03-27 <150> US 62 / 649,541 <151> 2018-03-28 <150> US 62 / 652,838 <151> 2018-04-04 <150> US 62 / 734,863 <151> 2018-09-21 <160> 181 <170> FastSEQ, version 4.0 for Windows <210> 1 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Modified IgG4 hinge <400> 1 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 2 <211> 13 <212> PRT <213> Homo sapiens <220> <223> TLR4 near-membrane domain <400> 2 Pro Val Leu Ser Leu Asn Ile Thr Cys Gln Met Asn Lys 1 5 10 <210> 3 <211> 46 <212> PRT <213> Homo sapiens <220> <223> MRC1 signal conduction domain <400> 3 Tyr Lys Lys Arg Arg Val His Leu Pro Gln Glu Gly Ala Phe Glu Asn 1 5 10 15 Thr Leu Tyr Phe Asn Ser Gln Ser Ser Pro Gly Thr Ser Asp Met Lys 20 25 30 Asp Leu Val Gly Asn Ile Glu Gln Asn Glu His Ser Val Ile 35 40 45 <210> 4 <211> 473 <212> PRT <213> Homo sapiens <220> <223> MERTK signal conduction domain <400> 4 Lys Arg Val Gln Glu Thr Lys Phe Gly Asn Ala Phe Thr Glu Glu Asp 1 5 10 15 Ser Glu Leu Val Val Asn Tyr Ile Ala Lys Lys Ser Phe Cys Arg Arg 20 25 30 Ala Ile Glu Leu Thr Leu His Ser Leu Gly Val Ser Glu Glu Leu Gln 35 40 45 Asn Lys Leu Glu Asp Val Val Ile Asp Arg Asn Leu Leu Ile Leu Gly 50 55 60 Lys Ile Leu Gly Glu Gly Glu Phe Gly Ser Val Met Glu Gly Asn Leu 65 70 75 80 Lys Gln Glu Asp Gly Thr Ser Leu Lys Val Ala Val Lys Thr Met Lys 85 90 95 Leu Asp Asn Ser Ser Gln Arg Glu Ile Glu Glu Phe Leu Ser Glu Ala 100 105 110 Ala Cys Met Lys Asp Phe Ser His Pro Asn Val Ile Arg Leu Leu Gly 115 120 125 Val Cys Ile Glu Met Ser Ser Gln Gly Ile Pro Lys Pro Met Val Ile 130 135 140 Leu Pro Phe Met Lys Tyr Gly Asp Leu His Thr Tyr Leu Leu Tyr Ser 145 150 155 160 Arg Leu Glu Thr Gly Pro Lys His Ile Pro Leu Gln Thr Leu Leu Lys 165 170 175 Phe Met Val Asp Ile Ala Leu Gly Met Glu Tyr Leu Ser Asn Arg Asn 180 185 190 Phe Leu His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Arg Asp Asp 195 200 205 Met Thr Val Cys Val Ala Asp Phe Gly Leu Ser Lys Lys Ile Tyr Ser 210 215 220 Gly Asp Tyr Tyr Arg Gln Gly Arg Ile Ala Lys Met Pro Val Lys Trp 225 230 235 240 Ile Ala Ile Glu Ser Leu Ala Asp Arg Val Tyr Thr Ser Lys Ser Asp 245 250 255 Val Trp Ala Phe Gly Val Thr Met Trp Glu Ile Ala Thr Arg Gly Met 260 265 270 Thr Pro Tyr Pro Gly Val Gln Asn His Glu Met Tyr Asp Tyr Leu Leu 275 280 285 His Gly His Arg Leu Lys Gln Pro Glu Asp Cys Leu Asp Glu Leu Tyr 290 295 300 Glu Ile Met Tyr Ser Cys Trp Arg Thr Asp Pro Leu Asp Arg Pro Thr 305 310 315 320 Phe Ser Val Leu Arg Leu Gln Leu Glu Lys Leu Leu Glu Ser Leu Pro 325 330 335 Asp Val Arg Asn Gln Ala Asp Val Ile Tyr Val Asn Thr Gln Leu Leu 340 345 350 Glu Ser Ser Glu Gly Leu Ala Gln Gly Ser Thr Leu Ala Pro Leu Asp​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<223> MERTK signaling domain <400> 5 Ala Leu Arg Arg Arg Val Gln Glu Thr Lys Phe Gly Gly Ala Phe Ser 1 5 10 15 Glu Glu Asp Ser Gln Leu Val Val Asn Tyr Arg Ala Lys Lys Ser Phe 20 25 30 Cys Arg Arg Ala Ile Glu Leu Thr Leu Gln Ser Leu Gly Val Ser Glu 35 40 45 Glu Leu Gln Asn Lys Leu Glu Asp Val Val Ile Asp Arg Asn Leu Leu 50 55 60 Val Leu Gly Lys Val Leu Gly Glu Gly Glu Phe Gly Ser Val Met Glu 65 70 75 80 Gly Asn Leu Lys Gln Glu Asp Gly Thr Ser Gln Lys Val Ala Val Lys 85 90 95 Thr Met Lys Leu Asp Asn Phe Ser Gln Arg Glu Ile Glu Glu Phe Leu 100 105 110 Ser Glu Ala Ala Cys Met Lys Asp Phe Asn His Pro Asn Val Ile Arg 115 120 125 Leu Leu Gly Val Cys Ile Glu Leu Ser Ser Gln Gly Ile Pro Lys Pro 130 135 140 Met Val Ile Leu Pro Phe Met Lys Tyr Gly Asp Leu His Thr Phe Leu 145 150 155 160 Leu Tyr Ser Arg Leu Asn Thr Gly Pro Lys Tyr Ile His Leu Gln Thr 165 170 175 Leu Leu Lys Phe Met Met Asp Ile Ala Gln Gly Met Glu Tyr Leu Ser 180 185 190 Asn Arg Asn Phe Leu His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu 195 200 205 Arg Asp Asp Met Thr Val Cys Val Ala Asp Phe Gly Leu Ser Lys Lys 210 215 220 Ile Tyr Ser Gly Asp Tyr Tyr Arg Gln Gly Arg Ile Ala Lys Met Pro 225 230 235 240 Val Lys Trp Ile Ala Ile Glu Ser Leu Ala Asp Arg Val Tyr Thr Ser 245 250 255 Lys Ser Asp Val Trp Ala Phe Gly Val Thr Met Trp Glu Ile Thr Thr 260 265 270 Arg Gly Met Thr Pro Tyr Pro Gly Val Gln Asn His Glu Met Tyr Asp 275 280 285 Tyr Leu Leu His Gly His Arg Leu Lys Gln Pro Glu Asp Cys Leu Asp 290 295 300 Glu Leu Tyr Asp Ile Met Tyr Ser Cys Trp Ser Ala Asp Pro Leu Asp 305 310 315 320 Arg Pro Thr Phe Ser Val Leu Arg Leu Gln Leu Glu Lys Leu Ser Glu 325 330 335 Ser Leu Pro Asp Ala Gln Asp Lys Glu Ser Ile Ile Tyr Ile Asn Thr 340 345 350 Gln Leu Leu Glu Ser Cys Glu Gly Ile Ala Asn Gly Pro Ser Leu Thr 355 360 365 Gly Leu Asp Met Asn Ile Asp Pro Asp Ser Ile Ile Ala Ser Cys Thr 370 375 380 Pro Gly Ala Ala Val Ser Val Val Thr Ala Glu Val His Glu Asn Asn 385 390 395 400 Leu Arg Glu Glu Arg Tyr Ile Leu Asn Gly Gly Asn Glu Glu Trp Glu 405 410 415 Asp Val Ser Ser Thr Pro Phe Ala Ala Val Thr Pro Glu Lys Asp Gly 420 425 430 Val Leu Pro Glu Asp Arg Leu Thr Lys Asn Gly Val Ser Trp Ser His 435 440 445 His Ser Thr Leu Pro Leu Gly Ser Pro Ser Pro Asp Glu Leu Leu Phe 450 455 460 Val Asp Asp Ser Leu Glu Asp Ser Glu Val Leu Met 465 470 475 <210> 6 <211> 440 <212> PRT <213> Homo sapiens <220> <223> Tyro3 signaling domain <400> 6 Leu Arg Lys Arg Arg Lys Glu Thr Arg Phe Gly Gln Ala Phe Asp Ser 1 5 10 15 Val Met Ala Arg Gly Glu Pro Ala Val His Phe Arg Ala Ala Arg Ser 20 25 30 Phe Asn Arg Glu Arg Pro Glu Arg Ile Glu Ala Thr Leu Asp Ser Leu 35 40 45 Gly Ile Ser Asp Glu Leu Lys Glu Lys Leu Glu Asp Val Leu Ile Pro 50 55 60 Glu Gln Gln Phe Thr Leu Gly Arg Met Leu Gly Lys Gly Glu Phe Gly 65 70 75 80 Ser Val Arg Glu Ala Gln Leu Lys Gln Glu Asp Gly Ser Phe Val Lys 85 90 95 Val Ala Val Lys Met Leu Lys Ala Asp Ile Ile Ala Ser Ser Asp Ile 100 105 110 Glu Glu Phe Leu Arg Glu Ala Ala Cys Met Lys Glu Phe Asp His Pro 115 120 125 His Val Ala Lys Leu Val Gly Val Ser Leu Arg Ser Arg Ala Lys Gly 130 135 140 Arg Leu Pro Ile Pro Met Val Ile Leu Pro Phe Met Lys His Gly Asp 145 150 155 160 Leu His Ala Phe Leu Leu Ala Ser Arg Ile Gly Glu Asn Pro Phe Asn 165 170 175 Leu Pro Leu Gln Thr Leu Ile Arg Phe Met Val Asp Ile Ala Cys Gly 180 185 190 Met Glu Tyr Leu Ser Ser Arg Asn Phe Ile His Arg Asp Leu Ala Ala 195 200 205 Arg Asn Cys Met Leu Ala Glu Asp Met Thr Val Cys Val Ala Asp Phe 210 215 220 Gly Leu Ser Arg Lys Ile Tyr Ser Gly Asp Tyr Tyr Arg Gln Gly Cys 225 230 235 240 Ala Ser Lys Leu Pro Val Lys Trp Leu Ala Leu Glu Ser Leu Ala Asp 245 250 255 Asn Leu Tyr Thr Val Gln Ser Asp Val Trp Ala Phe Gly Val Thr Met 260 265 270 Trp Glu Ile Met Thr Arg Gly Gln Thr Pro Tyr Ala Gly Ile Glu Asn 275 280 285 Ala Glu Ile Tyr Asn Tyr Leu Ile Gly Gly Asn Arg Leu Lys Gln Pro 290 295 300 Pro Glu Cys Met Glu Asp Val Tyr Asp Leu Met Tyr Gln Cys Trp Ser 305 310 315 320 Ala Asp Pro Lys Gln Arg Pro Ser Phe Thr Cys Leu Arg Met Glu Leu 325 330 335 Glu Asn Ile Leu Gly Gln Leu Ser Val Leu Ser Ala Ser Gln Asp Pro 340 345 350 Leu Tyr Ile Asn Ile Glu Arg Ala Glu Glu Pro Thr Ala Gly Gly Ser 355 360 365 Leu Glu Leu Pro Gly Arg Asp Gln Pro Tyr Ser Gly Ala Gly Asp Gly 370 375 380 Ser Gly Met Gly Ala Val Gly Gly Thr Pro Ser Asp Cys Arg Tyr Ile 385 390 395 400 Leu Thr Pro Gly Gly Leu Ala Glu Gln Pro Gly Gln Ala Glu His Gln 405 410 415 Pro Glu Ser Pro Leu Asn Glu Thr Gln Arg Leu Leu Leu Leu Gln Gln 420 425 430 Gly Leu Leu Pro His Ser Ser Cys 435 440 <210> 7 <211> 422 <212> PRT <213> Homo sapiens <220> <223> Axl signaling domain <400> 7 His Arg Arg Lys Lys Glu Thr Arg Tyr Gly Glu Val Phe Glu Pro Thr 1 5 10 15 Val Glu Arg Gly Glu Leu Val Val Arg Tyr Arg Val Arg Lys Ser Tyr 20 25 30 Ser Arg Arg Thr Thr Glu Ala Thr Leu Asn Ser Leu Gly Ile Ser Glu 35 40 45 Glu Leu Lys Glu Lys Leu Arg Asp Val Met Val Asp Arg His Lys Val<00​​​​​​​​​​​​​​​​​ Ile Gly Val Cys Phe Gln Gly Ser Glu Arg Glu Ser Phe Pro Ala Pro 130 135 140 Val Val Ile Leu Pro Phe Met Lys His Gly Asp Leu His Ser Phe Leu 145 150 155 160 Leu Tyr Ser Arg Leu Gly Asp Gln Pro Val Tyr Leu Pro Thr Gln Met 165 170 175 Leu Val Lys Phe Met Ala Asp Ile Ala Ser Gly Met Glu Tyr Leu Ser 180 185 190 Thr Lys Arg Phe Ile His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu 195 200 205 Asn Glu Asn Met Ser Val Cys Val Ala Asp Phe Gly Leu Ser Lys Lys 210 215 220 Ile Tyr Asn Gly Asp Tyr Tyr Arg Gln Gly Arg Ile Ala Lys Met Pro 225 230 235 240 Val Lys Trp Ile Ala Ile Glu Ser Leu Ala Asp Arg Val Tyr Thr Ser 245 250 255 Lys Ser Asp Val Trp Ser Phe Gly Val Thr Met Trp Glu Ile Ala Thr 260 265 270 Arg Gly Gln Thr Pro Tyr Pro Gly Val Glu Asn Ser Glu Ile Tyr Asp 275 280 285 Tyr Leu Arg Gln Gly Asn Arg Leu Lys Gln Pro Ala Asp Cys Leu Asp 290 295 300 Gly Leu Tyr Ala Leu Met Ser Arg Cys Trp Glu Leu Asn Pro Gln Asp 305 310 315 320 Arg Pro Ser Phe Thr Glu Leu Arg Glu Asp Leu Glu Asn Thr Leu Lys 325 330 335 Ala Leu Pro Pro Ala Gln Glu Pro Asp Glu Ile Leu Tyr Val Asn Met 340 345 350 Asp Glu Gly Gly Gly Tyr Pro Glu Pro Pro Gly Ala Ala Gly Gly Ala 355 360 365 Asp Pro Pro Thr Gln Pro Asp Pro Lys Asp Ser Cys Ser Cys Leu Thr 370 375 380 Ala Ala Glu Val His Pro Ala Gly Arg Tyr Val Leu Cys Pro Ser Thr 385 390 395 400 Thr Pro Ser Pro Ala Gln Pro Ala Asp Arg Gly Ser Pro Ala Ala Pro 405 410 415 Gly Gln Glu Asp Gly Ala 420 <210> 8 <211> 727 <212> PRT <213> Homo sapiens <220> <223> ELMO signal transmission structure <400> 8 Met Pro Pro Pro Ala Asp Ile Val Lys Val Ala Ile Glu Trp Pro Gly 1 5 10 15 Ala Tyr Pro Lys Leu Met Glu Ile Asp Gln Lys Lys Pro Leu Ser Ala 20 25 30 Ile Ile Lys Glu Val Cys Asp Gly Trp Ser Leu Ala Asn His Glu Tyr 35 40 45 Phe Ala Leu Gln His Ala Asp Ser Ser Asn Phe Tyr Ile Thr Glu Lys 50 55 60 Asn Arg Asn Glu Ile Lys Asn Gly Thr Ile Leu Arg Leu Thr Thr Ser 65 70 75 80 Pro Ala Gln Asn Ala Gln Gln Leu His Glu Arg Ile Gln Ser Ser Ser 85 90 95 Met Asp Ala Lys Leu Glu Ala Leu Lys Asp Leu Ala Ser Leu Ser Arg 100 105 110 Asp Val Thr Phe Ala Gln Glu Phe Ile Asn Leu Asp Gly Ile Ser Leu 115 120 125 Leu Thr Gln Met Val Glu Ser Gly Thr Glu Arg Tyr Gln Lys Leu Gln 130 135 140 Lys Ile Met Lys Pro Cys Phe Gly Asp Met Leu Ser Phe Thr Leu Thr 145 150 155 160 Ala Phe Val Glu Leu Met Asp His Gly Ile Val Ser Trp Asp Thr Phe 165 170 175 Ser Val Ala Phe Ile Lys Lys Ile Ala Ser Phe Val Asn Lys Ser Ala 180 185 190 Ile Asp Ile Ser Ile Leu Gln Arg Ser Leu Ala Ile Leu Glu Ser Met 195 200 205 Val Leu Asn Ser His Asp Leu Tyr Gln Lys Val Ala Gln Glu Ile Thr 210 215 220 Ile Gly Gln Leu Ile Pro His Leu Gln Gly Ser Asp Gln Glu Ile Gln 225 230 235 240 Thr Tyr Thr Ile Ala Val Ile Asn Ala Leu Phe Leu Lys Ala Pro Asp 245 250 255 Glu Arg Arg Gln Glu Met Ala Asn Ile Leu Ala Gln Lys Gln Leu Arg 260 265 270 Ser Ile Ile Leu Thr His Val Ile Arg Ala Gln Arg Ala Ile Asn Asn 275 280 285 Glu Met Ala His Gln Leu Tyr Val Leu Gln Val Leu Thr Phe Asn Leu 290 295 300 Leu Glu Asp Arg Met Met Thr Lys Met Asp Pro Gln Asp Gln Ala Gln 305 310 315 320 Arg Asp Ile Ile Phe Glu Leu Arg Arg Ile Ala Phe Asp Ala Glu Ser 325 330 335 Glu Pro Asn Asn Ser Ser Gly Ser Met Glu Lys Arg Lys Ser Met Tyr 340 345 350 Thr Arg Asp Tyr Lys Lys Leu Gly Phe Ile Asn His Val Asn Pro Ala 355 360 365 Met Asp Phe Thr Gln Thr Pro Pro Gly Met Leu Ala Leu Asp Asn Met 370 375 380 Leu Tyr Phe Ala Lys His His Gln Asp Ala Tyr Ile Arg Ile Val Leu 385 390 395 400 Glu Asn Ser Ser Arg Glu Asp Lys His Glu Cys Pro Phe Gly Arg Ser 405 410 415 Ser Ile Glu Leu Thr Lys Met Leu Cys Glu Ile Leu Lys Val Gly Glu 420 425 430 Leu Pro Ser Glu Thr Cys Asn Asp Phe His Pro Met Phe Phe Thr His 435 440 445 Asp Arg Ser Phe Glu Glu Phe Phe Cys Ile Cys Ile Gln Leu Leu Asn 450 455 460 Lys Thr Trp Lys Glu Met Arg Ala Thr Ser Glu Asp Phe Asn Lys Val 465 470 475 480 Met Gln Val Val Lys Glu Gln Val Met Arg Ala Leu Thr Thr Lys Pro 485 490 495 Ser Ser Leu Asp Gln Phe Lys Ser Lys Leu Gln Asn Leu Ser Tyr Thr 500 505 510 Glu Ile Leu Lys Ile Arg Gln Ser Glu Arg Met Asn Gln Glu Asp Phe 515 520 525 Gln Ser Arg Pro Ile Leu Glu Leu Lys Glu Lys Ile Gln Pro Glu Ile 530 535 540 Leu Glu Leu Ile Lys Gln Gln Arg Leu Asn Arg Leu Val Glu Gly Thr 545 550 555 560 Cys Phe Arg Lys Leu Asn Ala Arg Arg Arg Gln Asp Lys Phe Trp Tyr 565 570 575 Cys Arg Leu Ser Pro Asn His Lys Val Leu His Tyr Gly Asp Leu Glu 580 585 590 Glu Ser Pro Gln Gly Glu Val Pro His Asp Ser Leu Gln Asp Lys Leu 595 600 605 Pro Val Ala Asp Ile Lys Ala Val Val Thr Gly Lys Asp Cys Pro His 610 615 620 Met Lys Glu Lys Gly Ala Leu Lys Gln Asn Lys Glu Val Leu Glu Leu 625 630 635 640 Ala Phe Ser Ile Leu Tyr Asp Ser Asn Cys Gln Leu Asn Phe Ile Ala 645 650 655 Pro Asp Lys His Glu Tyr Cys Ile Trp Thr Asp Gly Leu Asn Ala Leu 660 665 670 Leu Gly Lys Asp Met Met Ser Asp Leu Thr Arg Asn Asp Leu Asp Thr 675 680 685 Leu Leu Ser Met Glu Ile Lys Leu Arg Leu Leu Asp Leu Glu Asn Ile 690 695 700 Gln Ile Pro Asp Ala Pro Pro Pro Ile Pro Lys Glu Pro Ser Asn Tyr 705 710 715 720 Asp Phe Val Tyr Asp Cys Asn 725 <210> 9 <211> 522 <212> PRT <213> Homo sapiens <220> <223> Traf6 signaling domain - full length <400> 9 Met Ser Leu Leu Asn Cys Glu Asn Ser Cys Gly Ser Ser Gln Ser Glu 1 5 10 15 Ser Asp Cys Cys Val Ala Met Ala Ser Ser Cys Ser Ala Val Thr Lys 20 25 30 Asp Asp Ser Val Gly Gly Thr Ala Ser Thr Gly Asn Leu Ser Ser Ser 35 40 45 Phe Met Glu Glu Ile Gln Gly Tyr Asp Val Glu Phe Asp Pro Pro Leu 50 55 60 Glu Ser Lys Tyr Glu Cys Pro Ile Cys Leu Met Ala Leu Arg Glu Ala 65 70 75 80 Val Gln Thr Pro Cys Gly His Arg Phe Cys Lys Ala Cys Ile Ile Lys 85 90 95 Ser Ile Arg Asp Ala Gly His Lys Cys Pro Val Asp Asn Glu Ile Leu 100 105 110 Leu Glu Asn Gln Leu Phe Pro Asp Asn Phe Ala Lys Arg Glu Ile Leu 115 120 125 Ser Leu Met Val Lys Cys Pro Asn Glu Gly Cys Leu His Lys Met Glu 130 135 140 Leu Arg His Leu Glu Asp His Gln Ala His Cys Glu Phe Ala Leu Met 145 150 155 160 Asp Cys Pro Gln Cys Gln Arg Pro Phe Gln Lys Phe His Ile Asn Ile 165 170 175 His Ile Leu Lys Asp Cys Pro Arg Arg Gln Val Ser Cys Asp Asn Cys 180 185 190 Ala Ala Ser Met Ala Phe Glu Asp Lys Glu Ile His Asp Gln Asn Cys 195 200 205 Pro Leu Ala Asn Val Ile Cys Glu Tyr Cys Asn Thr Ile Leu Ile Arg 210 215 220 Glu Gln Met Pro Asn His Tyr Asp Leu Asp Cys Pro Thr Ala Pro Ile 225 230 235 240 Pro Cys Thr Phe Ser Thr Phe Gly Cys His Glu Lys Met Gln Arg Asn 245 250 255 His Leu Ala Arg His Leu Gln Glu Asn Thr Gln Ser His Met Arg Met 260 265 270 Leu Ala Gln Ala Val His Ser Leu Ser Val Ile Pro Asp Ser Gly Tyr 275 280 285 Ile Ser Glu Val Arg Asn Phe Gln Glu Thr Ile His Gln Leu Glu Gly 290 295 300 Arg Leu Val Arg Gln Asp His Gln Ile Arg Glu Leu Thr Ala Lys Met 305 310 315 320 Glu Thr Gln Ser Met Tyr Val Ser Glu Leu Lys Arg Thr Ile Arg Thr 325 330 335 Leu Glu Asp Lys Val Ala Glu Ile Glu Ala Gln Gln Cys Asn Gly Ile 340 345 350 Tyr Ile Trp Lys Ile Gly Asn Phe Gly Met His Leu Lys Cys Gln Glu 355 360 365 Glu Glu Lys Pro Val Val Ile His Ser Pro Gly Phe Tyr Thr Gly Lys 370 375 380 Pro Gly Tyr Lys Leu Cys Met Arg Leu His Leu Gln Leu Pro Thr Ala 385 390 395 400 Gln Arg Cys Ala Asn Tyr Ile Ser Leu Phe Val His Thr Met Gln Gly 405 410 415 Glu Tyr Asp Ser His Leu Pro Trp Pro Phe Gln Gly Thr Ile Arg Leu 420 425 430 Thr Ile Leu Asp Gln Ser Glu Ala Pro Val Arg Gln Asn His Glu Glu 435 440 445 Ile Met Asp Ala Lys Pro Glu Leu Leu Ala Phe Gln Arg Pro Thr Ile 450 455 460 Pro Arg Asn Pro Lys Gly Phe Gly Tyr Val Thr Phe Met His Leu Glu 465 470 475 480 Ala Leu Arg Gln Arg Thr Phe Ile Lys Asp Asp Thr Leu Leu Val Arg 485 490 495 Cys Glu Val Ser Thr Arg Phe Asp Met Gly Ser Leu Arg Arg Glu Gly 500 505 510 Phe Gln Pro Arg Ser Thr Asp Ala Gly Val 515 520 <210> 10 <211> 261 <212> PRT <213> Homo sapiens <220> <223> Syk signaling domain <400> 10 Thr Leu Glu Asp Lys Glu Leu Gly Ser Gly Asn Phe Gly Thr Val Lys 1 5 10 15 Lys Gly Tyr Tyr Gln Met Lys Lys Val Val Lys Thr Val Ala Val Lys 20 25 30 Ile Leu Lys Asn Glu Ala Asn Asp Pro Ala Leu Lys Asp Glu Leu Leu 35 40 45 Ala Glu Ala Asn Val Met Gln Gln Leu Asp Asn Pro Tyr Ile Val Arg 50 55 60 Met Ile Gly Ile Cys Glu Ala Glu Ser Trp Met Leu Val Met Glu Met 65 70 75 80 Ala Glu Leu Gly Pro Leu Asn Lys Tyr Leu Gln Gln Asn Arg His Val 85 90 95 Lys Asp Lys Asn Ile Ile Glu Leu Val His Gln Val Ser Met Gly Met 100 105 110 Lys Tyr Leu Glu Glu Ser Asn Phe Val His Arg Asp Leu Ala Ala Arg 115 120 125 Asn Val Leu Leu Val Thr Gln His Tyr Ala Lys Ile Ser Asp Phe Gly 130 135 140 Leu Ser Lys Ala Leu Arg Ala Asp Glu Asn Tyr Tyr Lys Ala Gln Thr 145 150 155 160 His Gly Lys Trp Pro Val Lys Trp Tyr Ala Pro Glu Cys Ile Asn Tyr 165 170 175 Tyr Lys Phe Ser Ser Lys Ser Asp Val Trp Ser Phe Gly Val Leu Met 180 185 190 Trp Glu Ala Phe Ser Tyr Gly Gln Lys Pro Tyr Arg Gly Met Lys Gly 195 200 205 Ser Glu Val Thr Ala Met Leu Glu Lys Gly Glu Arg Met Gly Cys Pro 210 215 220 Ala Gly Cys Pro Arg Glu Met Tyr Asp Leu Met Asn Leu Cys Trp Thr 225 230 235 240 Tyr Asp Val Glu Asn Arg Pro Gly Phe Ala Ala Val Glu Leu Arg Leu 245 250 255 Arg Asn Tyr Tyr Tyr 260 <210> 11 <211> 296 <212> PRT <213> Homo sapiens <220> <223> MyD88 signaling domain <400> 11 Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser 1 5 10 15 Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg 20 25 30 Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr 35 40 45 Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu ​​​​​​​​​​​​​​​​​​​ 130 135 140 Ala Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly His Met Pro Glu Arg 145 150 155 160 Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile Gln Phe Val Gln 165 170 175 Glu Met Ile Arg Gln Leu Glu Gln Thr Asn Tyr Arg Leu Lys Leu Cys 180 185 190 Val Ser Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp Ser Ile Ala 195 200 205 Ser Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val Val Val Ser 210 215 220 Asp Asp Tyr Leu Gln Ser Lys Glu Cys Asp Phe Gln Thr Lys Phe Ala 225 230 235 240 Leu Ser Leu Ser Pro Gly Ala His Gln Lys Arg Leu Ile Pro Ile Lys 245 250 255 Tyr Lys Ala Met Lys Lys Glu Phe Pro Ser Ile Leu Arg Phe Ile Thr 260 265 270 Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Ser Trp Phe Trp Thr Arg 275 280 285 Leu Ala Lys Ala Leu Ser Leu Pro 290 295 <210> 12 <400> 12 000 <210> 13 <211> 42 <212> PRT <213> Homo sapiens <220> <223> FcεR1γ signal conduction domain <400> 13 Arg Leu Lys Ile Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys 1 5 10 15 Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr 20 25 30 Glu Thr Leu Lys His Glu Lys Pro Pro Gln 35 40 <210> 14 <211> 61 <212> PRT <213> Homo sapiens <220> <223> FcγR1 signal conduction domain <400> 14 Arg Lys Glu Leu Lys Arg Lys Lys Lys Trp Asp Leu Glu Ile Ser Leu 1 5 10 15 Asp Ser Gly His Glu Lys Lys Val Ile Ser Ser Leu Gln Glu Asp Arg 20 25 30 His Leu Glu Glu Glu Leu Lys Cys Gln Glu Gln Lys Glu Glu Gln Leu 35 40 45 Gln Glu Gly Val His Arg Lys Glu Pro Gln Gly Ala Thr 50 55 60 <210> 15 <211> 77 <212> PRT <213> Homo sapiens <220> <223> FcγR2A signal conduction domain <400> 15 Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala 1 5 10 15 Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg 20 25 30 Gln Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr 35 40 45 Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr 50 55 60 Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn 65 70 75 <210> 16 <211> 77 <212> PRT <213> Homo sapiens <220> <223> FcγR2c signal transduction domain <400> 16 Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala 1 5 10 15 Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg 20 25 30 Gln Pro Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr 35 40 45 Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr 50 55 60 Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn 65 70 75 <210> 17 <211> 25 <212> PRT <213> Homo sapiens <220> <223> FcγR3A signal transduction domain <400> 17 Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe 1 5 10 15 Lys Trp Arg Lys Asp Pro Gln Asp Lys 20 25 <210> 18 <211> 85 <212> PRT <213> Homo sapiens <220> <223> BAFF-R signal conduction domain <400> 18 Ser Trp Arg Arg Arg Gln Arg Arg Leu Arg Gly Ala Ser Ser Ala Glu 1 5 10 15 Ala Pro Asp Gly Asp Lys Asp Ala Pro Glu Pro Leu Asp Lys Val Ile 20 25 30 Ile Leu Ser Pro Gly Ile Ser Asp Ala Thr Ala Pro Ala Trp Pro Pro 35 40 45 Pro Gly Glu Asp Pro Gly Thr Thr Pro Pro Gly His Ser Val Pro Val 50 55 60 Pro Ala Thr Glu Leu Gly Ser Thr Glu Leu Val Thr Thr Lys Thr Ala 65 70 75 80 Gly Pro Glu Gln Gln 85 <210> 19 <211> 52 <212> PRT <213> Homo sapiens <220> <223> DAP12 signal conduction domain <400> 19 Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala 1 5 10 15 Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu 20 25 30 Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg 35 40 45 Pro Tyr Tyr Lys 50 <210> 20 <211> 86 <212> PRT <213> Homo sapiens <220> <223> NFAM1 signal conduction domain <400> 20 Leu Trp Asn Lys Lys Arg Met Arg Gly Pro Gly Lys Asp Pro Thr Arg 1 5 10 15 Lys Cys Pro Asp Pro Arg Ser Ala Ser Ser Pro Lys Gln His Pro Ser 20 25 30 Glu Ser Val Tyr Thr Ala Leu Gln Arg Arg Glu Thr Glu Val Tyr Ala 35 40 45 Cys Ile Glu Asn Glu Asp Gly Ser Ser Pro Thr Ala Lys Gln Ser Pro 50 55 60 Leu Ser Gln Glu Arg Pro His Arg Phe Glu Asp Asp Gly Glu Leu Asn 65 70 75 80 Leu Val Tyr Glu Asn Leu 85 <210> 21 <211> 29 <212> PRT <213> Homo sapiens <220> <223> CD79b signaling domain 185 - 213 <400> 21 Asp Ser Lys Ala Gly Met Glu Glu Asp His Thr Tyr Glu Gly Leu Asp 1 5 10 15<00​​​​​​​​​​​​​​<223> CD79b signal transduction domain 185-229 <400> twenty two Asp Ser Lys Ala Gly Met Glu Glu Asp His Thr Tyr Glu Gly Leu Asp 1 5 10 15 Ile Asp Gln Thr Ala Thr Tyr Glu Asp Ile Val Thr Leu Arg Thr Gly 20 25 30 Glu Val Lys Trp Ser Val Gly Glu His Pro Gly Gln Glu 35 40 45 <210> twenty three <211> 185 <212> PRT <213> Homo sapiens <220> <223> TLR1 signal conduction domain <400> twenty three Ser Tyr Leu Asp Leu Pro Trp Tyr Leu Arg Met Val Cys Gln Trp Thr 1 5 10 15 Gln Thr Arg Arg Arg Ala Arg Asn Ile Pro Leu Glu Glu Leu Gln Arg 20 25 30 Asn Leu Gln Phe His Ala Phe Ile Ser Tyr Ser Gly His Asp Ser Phe 35 40 45 Trp Val Lys Asn Glu Leu Leu Pro Asn Leu Glu Lys Glu Gly Met Gln 50 55 60 Ile Cys Leu His Glu Arg Asn Phe Val Pro Gly Lys Ser Ile Val Glu 65 70 75 80 Asn Ile Ile Thr Cys Ile Glu Lys Ser Tyr Lys Ser Ile Phe Val Leu 85 90 95 Ser Pro Asn Phe Val Gln Ser Glu Trp Cys His Tyr Glu Leu Tyr Phe 100 105 110 Ala His His Asn Leu Phe His Glu Gly Ser Asn Ser Leu Ile Leu Ile 115 120 125 Leu Leu Glu Pro Ile Pro Gln Tyr Ser Ile Pro Ser Ser Tyr His Lys 130 135 140 Leu Lys Ser Leu Met Ala Arg Arg Thr Tyr Leu Glu Trp Pro Lys Glu 145 150 155 160 Lys Ser Lys Arg Gly Leu Phe Trp Ala Asn Leu Arg Ala Ala Ile Asn 165 170 175 Ile Lys Leu Thr Glu Gln Ala Lys Lys 180 185 <210> 24 <211> 175 <212> PRT <213> Homo sapiens <220> <223> TLR2 signaling domain <400> 24 His Arg Phe His Gly Leu Trp Tyr Met Lys Met Met Trp Ala Trp Leu 1 5 10 15 Gln Ala Lys Arg Lys Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr 20 25 30 Asp Ala Phe Val Ser Tyr Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn 35 40 45 Leu Met Val Gln Glu Leu Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys 50 55 60 Leu His Lys Arg Asp Phe Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile 65 70 75 80 Ile Asp Ser Ile Glu Lys Ser His Lys Thr Val Phe Val Leu Ser Glu 85 90 95 Asn Phe Val Lys Ser Glu Trp Cys Lys Tyr Glu Leu Asp Phe Ser His 100 105 110 Phe Arg Leu Phe Asp Glu Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu 115 120 125 Glu Pro Ile Glu Lys Lys Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg 130 135 140 Lys Ile Met Asn Thr Lys Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala 145 150 155 160 Gln Arg Glu Gly Phe Trp Val Asn Leu Arg Ala Ala Ile Lys Ser 165 170 175 <210> 25 <211> 179 <212> PRT <213> Homo sapiens <220> <223> TLR3 doesn't work either. <400> 25 Glu Gly Trp Arg Ile Ser Phe Tyr Trp Asn Val Ser Val His Arg Val 1 5 10 15 Leu Gly Phe Lys Glu Ile Asp Arg Gln Thr Glu Gln Phe Glu Tyr Ala 20 25 30 Only Tyr Ile Ile His Only Tyr Lys Asp Lys Asp Trp Val Trp Glu His 35 40 45 Phe Ser Ser Met Glu Lys Glu Asp Gln Ser Leu Lys Phe Cys Leu Glu 50 55 60 Glu Arg Asp Phe Glu Ala Gly Val Phe Glu Leu Glu Ala Ile Val Asn 65 70 75 80 Ser Ile Lys Arg Ser Arg Lys Ile Ile Phe Val Ile Thr His His Leu 85 90 95 Leu Lys Asp Pro Leu Cys Lys Arg Phe Lys Val His Ala Val Gln 100 105 110 Gln Ala With Glu Gln Asn With Asp Ser With With Val Phe With Glu 115 120 125 Glu Ile Pro Asp Tyr Lys Leu Asn His Ala Cys Leu Arg Arg Gly 130 135 140 Met Phe Lys Ser His Cys Ile Leu Asn Trp Pro Val Gln Lys Glu Arg 145 150 155 160 Ile Gly Ala Phe Arg His Lys Leu Gln Val Ala Leu Gly Ser Lys Asn 165 170 175 Ser Val His <210> 26 <211> 187 <212> PRT <213> Homo sapiens <220> <223> TLR4 signaling domain <400> 26 Lys Phe Tyr Phe His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly<## [[ID=2##6]]1 5 10 15 Arg Gly Glu Asn Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp 20 25 30 Glu Asp Trp Val Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val 35 40 45 [[ID=3##6]]Pro Pro Phe Gln Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val 50 55 60 Ala Ile Ala Ala Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys 65 7##0 75 80[[ID=##3]] Val Ile Val Val Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile 85 90 95 Phe Glu Tyr Glu Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala 100 105 110 Gly Ile Ile Phe Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg 115 120 125 Gln Gln Val Glu Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu 130 135 140 Trp Glu Asp Ser Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg 145 150 155 160 Lys Ala Leu Leu Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly 165 170 175​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 35 40 45 Asn Ala Leu Leu Lys His Leu Asp Thr Gln Tyr Ser Asp Gln Asn Arg 50 55 60 Phe Asn Leu Cys Phe Glu Glu Arg Asp Phe Val Pro Gly Glu Asn Arg 65 70 75 80 Ile Ala Asn Ile Gln Asp Ala Ile Trp Asn Ser Arg Lys Ile Val Cys 85 90 95 Leu Val Ser Arg His Phe Leu Arg Asp Gly Trp Cys Leu Glu Ala Phe 100 105 110 Ser Tyr Ala Gln Gly Arg Cys Leu Ser Asp Leu Asn Ser Ala Leu Ile 115 120 125 Met Val Val Val Gly Ser Leu Ser Gln Tyr Gln Leu Met Lys His Gln 130 135 140 Ser Ile Arg Gly Phe Val Gln Lys Gln Gln Tyr Leu Arg Trp Pro Glu 145 150 155 160 Asp Phe Gln Asp Val Gly Trp Phe Leu His Lys Leu Ser Gln Gln Ile 165 170 175 Leu Lys Lys Glu Lys Glu Lys Lys Lys Asp Asn Asn Ile Pro Leu Gln 180 185 190 Thr Val Ala Thr Ile Ser 195 <210> 28 <211> 189 <212> PRT <213> Homo sapiens <220> <223> TLR6 signaling domain <400> 28 Tyr Leu Asp Leu Pro Trp Tyr Leu Arg Met Val Cys Gln Trp Thr Gln 1 5 10 15 Thr Arg Arg Arg Ala Arg Asn Ile Pro Leu Glu Glu Leu Gln Arg Asn 20 25 30 Leu Gln Phe His Ala Phe Ile Ser Tyr Ser Glu His Asp Ser Ala Trp 35 40 45 Val Lys Ser Glu Leu Val Pro Tyr Leu Glu Lys Glu Asp Ile Gln Ile 50 55 60 Cys Leu His Glu Arg Asn Phe Val Pro Gly Lys Ser Ile Val Glu Asn 65 70 75 80 Ile Ile Asn Cys Ile Glu Lys Ser Tyr Lys Ser Ile Phe Val Leu Ser 85 90 95 Pro Asn Phe Val Gln Ser Glu Trp Cys His Tyr Glu Leu Tyr Phe Ala 100 105 110 His His Asn Leu Phe His Glu Gly Ser Asn Asn Leu Ile Leu Ile Leu 115 120 125 Leu Glu Pro Ile Pro Gln Asn Ser Ile Pro Asn Lys Tyr His Lys Leu 130 135 140 Lys Ala Leu Met Thr Gln Arg Thr Tyr Leu Gln Trp Pro Lys Glu Lys 145 150 155 160 Ser Lys Arg Gly Leu Phe Trp Ala Asn Ile Arg Ala Ala Phe Asn Met 165 170 175 Lys Leu Thr Leu Val Thr Glu Asn Asn Asp Val Lys Ser 180 185 <210> 29 <211> 189 <212> PRT <213> Homo sapiens <220> <223> TLR7 signaling domain <400> 29 His Leu Tyr Phe Trp Asp Val Trp Tyr Ile Tyr His Phe Cys Lys Ala 1 5 10 15 Lys Ile Lys Gly Tyr Gln Arg Leu Ile Ser Pro Asp Cys Cys Tyr Asp 20 25 30 Ala Phe Ile Val Tyr Asp Thr Lys Asp Pro Ala Val Thr Glu Trp Val 35 40 45 Leu Ala Glu Leu Val Ala Lys Leu Glu Asp Pro Arg Glu Lys His Phe 50 55 60 Asn Leu Cys Leu Glu Glu Arg Asp Trp Leu Pro Gly Gln Pro Val Leu 65 70 75 80 Glu Asn Leu Ser Gln Ser Ile Gln Leu Ser Lys Lys Thr Val Phe Val 85 90 95 Met Thr Asp Lys Tyr Ala Lys Thr Glu Asn Phe Lys Ile Ala Phe Tyr 100 105 110 Leu Ser His Gln Arg Leu Met Asp Glu Lys Val Asp Val Ile Ile Leu 115 120 125 Ile Phe Leu Glu Lys Pro Phe Gln Lys Ser Lys Phe Leu Gln Leu Arg 130 135 140 Lys Arg Leu Cys Gly Ser Ser Val Leu Glu Trp Pro Thr Asn Pro Gln 145 150 155 160 Ala His Pro Tyr Phe Trp Gln Cys Leu Lys Asn Ala Leu Ala Thr Asp 165 170 175 Asn His Val Ala Tyr Ser Gln Val Phe Lys Glu Thr Val[[ID=2​​​​​​​​​​​​​​​​​​​​​ 20 25 30 Asp Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp 35 40 45 Val Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn 50 55 60 Val Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile 65 70 75 80 Ile Asp Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe 85 90 95 Val Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe 100 105 110 Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile 115 120 125 Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu 130 135 140 Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro 145 150 155 160 Lys Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu Thr 165 170 175 Glu Asn Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln 180 185 190 Tyr <210> 31 <211> 193 <212> PRT <213> Homo sapiens <220> <223> TLR9 signaling domain <400> 31 Gly Trp Asp Leu Trp Tyr Cys Phe His Leu Cys Leu Ala Trp Leu Pro 1 5 10 15 Trp Arg Gly Arg Gln Ser Gly Arg Asp Glu Asp Ala Leu Pro Tyr Asp 20 25 30 Ala Phe Val Val Phe Asp Lys Thr Gln Ser Ala Val Ala Asp Trp Val 35 40 45 Tyr Asn Glu Leu Arg Gly Gln Leu Glu Glu Cys Arg Gly Arg Trp Ala 50 55 60 Leu Arg Leu Cys Leu Glu Glu Arg Asp Trp Leu Pro Gly Lys Thr Leu 65 70 75 80 Phe Glu Asn Leu Trp Ala Ser Val Tyr Gly Ser Arg Lys Thr Leu Phe 85 9​​​​​​​​​Leu Val Ile Leu Ser Pro Asp Gly Arg Arg Ser Arg Tyr Val Arg Leu 130 135 140 Arg Gln Arg Leu Cys Arg Gln Ser Val Leu Leu Trp Pro His Gln Pro 145 150 155 160 Ser Gly Gln Arg Ser Phe Trp Ala Gln Leu Gly Met Ala Leu Thr Arg 165 170 175 Asp Asn His His Phe Tyr Asn Arg Asn Phe Cys Gln Gly Pro Thr Ala 180 185 190 Glu <210> 32 <211> 303 <212> PRT <213> Homo sapiens <220> <223> TRAF2 signaling domain <400> 32 Met Ala Ala Ala Ser Val Thr Pro Pro Gly Ser Leu Glu Leu Leu Gln 1 5 10 15 Pro Gly Phe Ser Lys Thr Leu Leu Gly Thr Lys Leu Glu Ala Lys Tyr 20 25 30 Leu Cys Ser Ala Cys Arg Asn Val Leu Arg Arg Pro Phe Gln Ala Gln 35 40 45 Cys Gly His Arg Tyr Cys Ser Phe Cys Leu Ala Ser Ile Leu Ser Ser 50 55 60 Gly Pro Gln Asn Cys Ala Ala Cys Val His Glu Gly Ile Tyr Glu Glu 65 70 75 80 Gly Ile Ser Ile Leu Glu Ser Ser Ser Ala Phe Pro Asp Asn Ala Ala 85 90 95 Arg Arg Glu Val Glu Ser Leu Pro Ala Val Cys Pro Ser Asp Gly Cys 100 105 110 Thr Trp Lys Gly Thr Leu Lys Glu Tyr Glu Ser Cys His Glu Gly Arg 115 120 125 Cys Pro Leu Met Leu Thr Glu Cys Pro Ala Cys Lys Gly Leu Val Arg 130 135 140 Leu Gly Glu Lys Glu Arg His Leu Glu His Glu Cys Pro Glu Arg Ser 145 150 155 160 Leu Ser Cys Arg His Cys Arg Ala Pro Cys Cys Gly Ala Asp Val Lys 165 170 175 Ala His His Glu Val Cys Pro Lys Phe Pro Leu Thr Cys Asp Gly Cys 180 185 190 Gly Lys Lys Lys Ile Pro Arg Glu Lys Phe Gln Asp His Val Lys Thr 195 200 205 Cys Gly Lys Cys Arg Val Pro Cys Arg Phe His Ala Ile Gly Cys Leu 210 215 220 Glu Thr Val Glu Gly Glu Lys Gln Gln Glu His Glu Val Gln Trp Leu 225 230 235 240 Arg Glu His Leu Ala Met Leu Leu Ser Ser Val Leu Glu Ala Lys Pro 245 250 255 Leu Leu Gly Asp Gln Ser His Ala Gly Ser Glu Leu Leu Gln Arg Cys 260 265 270 Glu Ser Leu Glu Lys Lys Thr Ala Thr Phe Glu Asn Ile Val Cys Val 275 280 285 Leu Asn Arg Glu Val Glu Arg Val Ala Met Thr Ala Glu Ala Cys 290 295 300 <210> 33 <21l> 274 <212> PRT <213> Homo sapiens [[ID=??]]<220> <223> TRAF3 signaling domain <400> 33 Met Glu Ser Ser Lys Lys Met Asp Ser Pro Gly Ala Leu Gln Thr Asn 1 5 10 15 Pro Pro Leu Lys Leu His Thr Asp Arg Ser Ala Gly Thr Pro Val Phe 20 25 30 Val Pro Glu Gln Gly Gly Tyr Lys Glu Lys Phe Val Lys Thr Val Glu 35 40 45 It should be noted that there seems to be an error in the original text where "21l" in should probably be "211". The above translation is based on the corrected understanding.Asp Lys Tyr Lys Glu Lys Cys His Leu Val Leu Cys Ser Pro Lys 50 55 60 Gln Thr Glu Cys Gly His Arg Phe Cys Glu Ser Cys Met Ala Ala Leu 65 70 75 80 Leu Ser Ser Ser Ser Pro Lys Cys Thr Ala Cys Gln Glu Ser Ile Val 85 90 95 Lys Asp Lys Val Phe Lys Asp Asn Cys Cys Lys Arg Glu Ile Leu Ala 100 105 110 Leu Gln With Tyr Cys Arg Asn Glu Ser Arg Gly Cys Ala Glu Gln Leu 115 120 125 Met Leu Gly His Leu Leu Val His Leu Lys Asn Asp Cys His Phe Glu 130 135 140 Glu Leu Pro Cys Val Arg Pro Asp Cys Lys Glu Lys Val Leu Arg Lys 145 150 155 160 Asp Leu Arg Asp His Val Glu Lys Ala Cys Lys Tyr Arg Glu Ala Thr 165 170 175 Cys Ser His Cys Lys Ser Gln Val Pro Met Ile Ala Leu Gln Lys His 180 185 190 Glu Asp Thr Asp Cys Pro Cys Val Val Val Ser Cys Pro His Lys Cys 195 200 205 Ser Val Gln Thr Leu Leu Arg Ser Glu Leu Ser Ala His Leu Ser Glu 210 215 220 Cys Val Asn Ala Pro Ser Thr Cys Ser Phe Lys Arg Tyr Gly Cys Val 225 230 235 240 Phe Gln Gly Thr Asn Gln Gln Ile Lys Ala His Glu Ala Ser Ser Ala 245 250 255 Val Gln His Val Asn Leu Leu Lys Glu Trp Ser Asn Ser Leu Glu Lys 260 265 270 Lys Val <210> 34 <211> 155 <212> PRT <213> Homo sapiens <220> <223> The MyD88 signal conduction domain is truncated, and there is no TIR domain. <400> 34 Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser 1 5 10 15 Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg 20 25 30 Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr 35 40 45 Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu 50 55 60 Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly 65 70 75 80 Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu 85 90 95 Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Glu Glu Asp 100 105 110 Cys Gln Lys Tyr Ile Leu Lys Gln Gln Gln Glu Glu Ala Glu Lys Pro 115 120 125 Leu Gln Val Ala Ala Val Asp Ser Ser Val Pro Arg Thr Ala Glu Leu 130 135 140 Ala Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly ​​​​​​​​​​​​​​​​​​​​​​​​​ 35 40 45 Phe Met Glu Glu Ile Gln Gly Tyr Asp Val Glu Phe Asp Pro Pro Leu 50 55 60 Glu Ser Lys Tyr Glu Cys Pro Ile Cys Leu Met Ala Leu Arg Glu Ala 65 70 75 80 Val Gln Thr Pro Cys Gly His Arg Phe Cys Lys Ala Cys Ile Ile Lys 85 90 95 Ser Ile Arg Asp Ala Gly His Lys Cys Pro Val Asp Asn Glu Ile Leu 100 105 110 Leu Glu Asn Gln Leu Phe Pro Asp Asn Phe Ala Lys Arg Glu Ile Leu 115 120 125 Ser Leu Met Val Lys Cys Pro Asn Glu Gly Cys Leu His Lys Met Glu 130 135 140 Leu Arg His Leu Glu Asp His Gln Ala His Cys Glu Phe Ala Leu Met 145 150 155 160 Asp Cys Pro Gln Cys Gln Arg Pro Phe Gln Lys Phe His Ile Asn Ile 165 170 175 His Ile Leu Lys Asp Cys Pro Arg Arg Gln Val Ser Cys Asp Asn Cys 180 185 190 Ala Ala Ser Met Ala Phe Glu Asp Lys Glu Ile His Asp Gln Asn Cys 195 200 205 Pro Leu Ala Asn Val Ile Cys Glu Tyr Cys Asn Thr Ile Leu Ile Arg 210 215 220 Glu Gln Met Pro Asn His Tyr Asp Leu Asp Cys Pro Thr Ala Pro Ile 225 230 235 240 Pro Cys Thr Phe Ser Thr Phe Gly Cys His Glu Lys Met Gln Arg Asn 245 250 255 His Leu Ala Arg His Leu Gln Glu Asn Thr Gln Ser His Met Arg Met 260 265 270 Leu Ala <210> 36 <211> 29 <212> PRT <213> Homo sapiens <220> <223> Truncated NFAM1 signal conduct...

Claims

1. A combination cell immunotherapy composition, said composition comprising: (a) A first composition comprising CD4+ T cells containing a first chimeric phagocytic receptor (CER), the first chimeric phagocytic receptor (CER) comprising: The extracellular domain includes a Tim4-binding domain that binds phosphatidylserine. The signal transduction structure domain is a swallowed signal transduction structure domain, which includes a TLR2 signal transduction structure domain, and A transmembrane domain, located between and connecting the extracellular domain and the phagocytic signaling domain; and (b) A second composition comprising CD8+ T cells containing a recombinant T cell receptor (TCR) that binds to a target antigen.

2. The combined cell immunotherapy composition according to claim 1, wherein the recombinant TCR is an αβTCR, γδTCR, affinity-enhanced TCR, soluble TCR, single-chain TCR, or single variable domain TCR.

3. The combined cell immunotherapy composition according to any one of claims 1 or 2, wherein the extracellular domain of the CER further comprises a spacer region structural domain.

4. The combined cell immunotherapy composition according to claim 1, wherein the CER transmembrane domain comprises a Tim1, Tim4, Tim3, FcR, CD8, CD28, MERTK, Axl, Tyro3, ​​BAI1, CD4, DAP12, MRC1, FcR, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain.

5. The combined cell immunotherapy composition according to claim 1, wherein the CER phagocytic signaling domain comprises a primary phagocytic signaling domain and a secondary phagocytic signaling domain.

6. The composition according to claim 5, wherein the primary and secondary phagocytic signal transduction domains of the CER are different.

7. The combined cell immunotherapy composition according to claim 5, wherein the secondary phagocytic signaling domain is selected from the MERTK, BAFF-R, DAP12, NFAM1, CD79b, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, Traf6, Traf2, and Traf3 signaling domains.

8. The combined cell immunotherapy composition according to claim 6, wherein the secondary phagocytic signaling domain is selected from the MERTK, BAFF-R, DAP12, NFAM1, CD79b, TLR1, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, Traf6, Traf2, and Traf3 signaling domains.

9. The combined cell immunotherapy composition according to claim 1, wherein the CER is composed of the amino acid sequence shown in SEQ ID NO:

98.

10. The combined cell immunotherapy composition according to claim 1, wherein the target antigen of the TCR is WT-1, mesothelin, MART-1, NY-ESO-1, MAGE, HPV E6, HPV E7, survivin, alpha-fetoprotein, or a neotumor antigen.

11. The combined cell immunotherapy composition according to claim 1, wherein the recombinant TCR comprises Vα and Vβ, wherein Vα is composed of the amino acid sequence shown in SEQ ID NO: 88, and wherein Vβ is composed of the amino acid sequence shown in SEQ ID NO:

86.

12. The combined cell immunotherapy composition of claim 1, wherein the CD4+ T cells are naive CD4+ T cells, effector memory CD4+ T cells, or central memory CD4+ T cells.

13. The combined cell immunotherapy composition according to claim 1, wherein the CD8+ T cells are naive CD8+ T cells, effector memory CD8+ T cells, or central memory CD8+ T cells.

14. The combined cell immunotherapy composition according to claim 1, wherein the CD4+ T cells, CD8+ T cells, or both are human cells.

15. The combined cell immunotherapy composition according to claim 1, wherein the ratio of CD4+ T cells to CD8+ T cells in the composition is 1:1, 1:2, 1:4, 1:8, 1:10 or 1:

20.

16. The combination cell immunotherapy composition of claim 1, wherein the first composition and the second composition each further comprise a pharmaceutically acceptable carrier.

17. The combined cell immunotherapy composition according to claim 1, wherein the first composition and the second composition are in the same formulation or in different formulations.

18. Use of the combined cell immunotherapy composition according to any one of claims 1-9 and 11-17 in the preparation of a medicament for treating hypopharyngeal squamous cell carcinoma or cervical cancer in a subject, wherein, The target antigen of the TCR is HPV E7.

19. The use according to claim 18, wherein the CD4+ T cells are autologous or allogeneic to the subject, the CD8+ T cells are autologous or allogeneic to the subject, or any combination thereof.

20. The use according to claim 18 or 19, wherein the medicament further comprises an additional therapeutic agent.

21. The use according to claim 20, wherein the additional therapeutic agent is a small molecule therapy.

22. The use according to claim 20, wherein the additional therapeutic agent is an antibody, radiotherapy, chemotherapy agent, immune checkpoint molecule inhibitor therapy, cellular immunotherapy, oncolytic virus, electroporation therapy, UV phototherapy, high intensity focused ultrasound therapy, oncolytic virus, peptide, hormone, aptamer, anti-inflammatory agent, antibiotic, antifungal agent or antiviral agent.

23. The use according to claim 18, wherein the first composition and the second composition are applied to the object simultaneously or sequentially.

24. The use according to claim 23, wherein the first composition is applied 1 to 7 days after the second composition.

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