Use of membrane anchoring protein motif as a means of lytic granule repositioning to the cellular cortex to increase bystander killing as an adjuvant for solid tumor cell therapy

Abstract

Methods to purposefully target lytic granules to the cell membrane following NK cell activation for the benefit of bystander killing in a solid tumor setting as novel cell therapy approach. These methods can be used alone or in combination with dispersion related strategies. Methods of using truncated proteins from the dynein-dynactin complex to block dynein function to induce lytic granule dispersion. The methods and compositions enhance the lethality of degranulation events. The methods and compositions can boost cytotoxicity in a solid tumor directly or be combined with other technologies, such as immunotherapies to improve their outcomes.

Description

Attorney Docket 44010.215WO-PCT / / CU24341USE OF MEMBRANE ANCHORING PROTEIN MOTIF AS A MEANS OF LYTIC GRANULE REPOSITIONING TO THE CELLULAR CORTEX TO INCREASE BYSTANDER KILLING AS AN ADJUVANT FOR SOLID TUMOR CELL THERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 742,294, filed January 6, 2025, to The Trustees of Columbia University, titled “USE OF MEMBRANE ANCHORING PROTEIN MOTIF AS A MEANS OF LYTIC GRANULE REPOSITIONING TO THE CELLULAR CORTEX TO INCREASE BYSTANDER KILLING AS AN ADJUVANT FOR SOLID TUMOR CELL THERAPY,” the entirety of the disclosure of which is hereby incorporated by this reference. The entire contents of the above-identified applications are hereby fully incorporated herein by reference.SEQUENCE LISTING

[0002] In accordance with 37 C.F.R. § 1.831, the present specification makes reference to a Sequence Listing submitted electronically in the form of an XML file (entitled “215WO-PCT.xml”, created on January 6, 2026, (64,133 bytes in size). The entire contents of the Sequence Listing are herein incorporated by reference in their entirety, with the intention that, upon publication (including issuance), this incorporated Sequence Listing will be inserted in the published document immediately before the claims.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under AI067946 and AH20989 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0004] The subject matter disclosed herein is generally directed to methods, compositions, and systems to actively redirect lytic granules to the plasma membrane of cytotoxic lymphocytes.Attorney Docket 44010.215WO-PCT / / CU24341BACKGROUND

[0005] Natural killer (NK) cells are innate immune cells specialized for the surveillance of malignancy and defense against viral infection and utilize cell-mediated cytotoxicity.1During recognition of diseased cells, NK cells form a lytic immunological synapse (IS) to organize their cytotoxic machinery, precisely eliminating the triggering cell. The lytic IS enables the directed release of perforin and granzymes contained within a specialized secretory lysosomal organelle known as a lytic granule (LG), specifically onto the diseased cell.2The release of only one LG is capable of eradicating a targeted cell, although an individual NK cell can contain over a hundred, necessitating precise control of LG positioning and release.3

[0006] While LGs are scattered throughout the cytoplasm in resting cells, prior to release onto a target, they converge to the microtubule-organizing center (MTOC) and then polarize to the IS.4LG convergence requires dynein motors and utilizes minus-end movement along microtubules.5Once the converged LGs are polarized toward the IS, they are precisely directed onto the target cell. This tightly regulated process promotes efficiency in eradicating the triggering cell and prevents the killing of neighboring healthy cells, known as bystander killing.2,5This “honing activity” is highly desirable for anNK cell’s surveillance functions in which a diseased cell may be amid many that are not targeted for elimination.

[0007] Much of the precision in lytic functions of cytotoxic cells after their activation by a diseased cell derives from the control and specific release of the LG. As an organelle with such truly destructive potential, its precise harnessing is critical to cell survival while allowing access to critical host defense. The cytotoxic cell protects itself from the contents of released LGs via membrane lipid ordering, which is reinforced locally by the hyper-ordered LG membrane itself.6

[0008] The process of LG convergence, however, is critical in ensuring control of these destructive payloads. Many proteins that interact with the LG surface are required for their motility, docking, and ultimately fusing with the synaptic membrane. Some proteins also allow LGs to navigate the dense cortical F-actin meshwork that defines the IS, which can further serve to govern their release. Maintaining a converged LG state, however, would seem to be an efficient stance for an armed cytotoxic cell to take. This process, however, is LG-associated dynein driven, which seems inefficient when considering that the polarization of the MTOC with converged LGs to the IS also requires dynein function.7Importantly, LG convergence can be uncoupled from polarization and does not represent a commitment to killing.2,7LGAttorney Docket 44010.215WO-PCT / / CU24341convergence occurs rapidly and is sustained throughout cytotoxic cell activation. How this occurs, however, and any internal LG “dock” are unclear.

[0009] The Golgi complex represents a known source of vesicle biogenesis and sorting. It also exists in proximity to the MTOC under certain circumstances and in immune cells at the IS under others?10Understanding of these processes may allow for lytic granule redirection in cytotoxic cells to promote improved cellular therapies.

[0010] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.SUMMARY

[0011] In certain example embodiments, the present invention provides compositions and methods for targeting lytic granules (LGs) to the surface of cytotoxic lymphocyte cell plasma membrane to increase cell cytotoxicity and / or increase bystander killing and / or anti-tumor effectiveness.

[0012] In one aspect, the present invention provides for a fusion protein for targeting LGs to the plasma membrane of a cytotoxic lymphocyte, said fusion protein comprising: a plasma membrane directing peptide; and a LG associated peptide comprising a structural component of LGs or a transient adaptor peptide linked to LGs, wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell.

[0013] In certain embodiments, the plasma membrane directing peptide comprises hydrophobic amino acids and / or is capable of being post translationally modified with hydrophobic-related moieties. In certain embodiments, the hydrophobic-related moieties comprise hydrophobic carbon chains. In certain embodiments, the hydrophobic carbon chains comprise fatty acids.

[0014] In certain embodiments, the plasma membrane directing peptide comprises a CAAX motif. In certain embodiments, the CAAX motif is derived from the C terminal region of KRAS.

[0015] In certain embodiments, the plasma membrane directing peptide comprises a C-terminal peptide from a Type IV transmembrane domain. In certain embodiments, the plasma membrane directing peptide comprises a C-terminal transmembrane domain from a syntaxin family member. In certain embodiments, the syntaxin family member is syntaxin 1 A (STX1).Attorney Docket 44010.215WO-PCT / / CU24341

[0016] In certain embodiments, the plasma membrane directing peptide comprises a signal-anchor sequence from a Type II transmembrane (TII) protein. In certain embodiments, the plasma membrane directing peptide comprises an N-terminal peptide from a TII protein.

[0017] In certain embodiments, the plasma membrane directing peptide comprises an N-terminal domain of a Src Family Kinase (SFK). In certain embodiments, the SFK is selected from the group consisting of Src, Yes, Fyn, Fgr, Lek, Hck, Blk, Lyn, and Frk.

[0018] In certain embodiments, the plasma membrane directing peptide comprises a transmembrane and / or juxtamembrane region of LAT (Linker for Activation of T cells).

[0019] In certain embodiments, the structural component of the LG comprises a transmembrane domain from an LG expressed protein. In certain embodiments, the transmembrane domain comprises the transmembrane domain from LAMP1, LAMP2, LAMP3, CD63, CD68, NKG7, or functional fragments or variants thereof. In certain embodiments, the structural component of the LG comprises a transmembrane domain comprising a YXX motif. In certain embodiments, the YXX motif is either N- or C-terminal of the transmembrane domain.

[0020] In certain embodiments, the transient adaptor peptide linked to LGs is derived from a protein associated with LG tethering. In certain embodiments, the protein associated with LG tethering is GCC2, Golgin-97 (G-97), a coiled-coil domain that binds an LG associated coiled-coil domain, or a truncated GCC2 lacking the C-terminal GRIP domain. In certain embodiments, the coiled-coil domain that binds an LG associated coiled-coil domain is the coiled-coil domain 2 (CC2) of GCC2.

[0021] In certain embodiments, the fusion protein further comprises a C-terminal signal peptide that sorts the fusion protein to the LG and is derived from a protein specifically expressed on LGs. In certain embodiments, the signal peptide is derived from an LG protein selected from the group consisting of perforin, a granzyme, and NKG7.

[0022] In certain embodiments, the plasma membrane directing peptide is a CAAX motif and the LG associated peptide is GCC2 or Golgin-97. In certain embodiments, the plasma membrane directing peptide is a CAAX motif and the LG associated peptide is a LAMP1 transmembrane domain. In certain embodiments, the plasma membrane directing peptide is a STX1 motif and the LG associated peptide is a LAMP1 transmembrane domain. In certain embodiments, the plasma membrane directing peptide is an N-terminal domain of a SFK, the LG associated peptide is the N-terminal domain of NKG7, and the C-terminal signal peptide that sorts the fusion protein to the LG is derived from perforin, NKG7, or a granzyme. In certainAttorney Docket 44010.215WO-PCT / / CU24341embodiments, the plasma membrane directing peptide is an N-terminal domain of Lek, the LG associated peptide is the N-terminal domain of NKG7, and the C-terminal signal peptide that sorts the fusion protein to the LG is derived from perforin (SEQ ID NO: 48). In certain embodiments, the plasma membrane directing peptide is a transmembrane and / or juxtamembrane region of LAT, and the LG associated peptide is the N-terminal domain of NKG7 (SEQ ID NO: 49). In certain embodiments, the plasma membrane directing peptide is a transmembrane and / or juxtamembrane region of LAT, the LG associated peptide is the N-terminal domain of NKG7, and the C-terminal signal peptide that sorts the fusion protein to the LG is derived from perforin (SEQ ID NO: 50). In certain embodiments, the plasma membrane directing peptide is a is a CAAX motif and the LG associated peptide is a coiled-coil domain that binds an LG associated coiled-coil domain. In certain embodiments, the coiled-coil domain that binds an LG associated coiled-coil domain is the coiled-coil domain 2 (CC2) of GCC2 (SEQ ID NO: 51).

[0023] In another aspect, the present invention provides for a fusion protein for targeting LGs comprising: a LG associated peptide comprising a structural component of LGs; a protein of interest; and a signal peptide that sorts the fusion protein to the LG. In certain embodiments, the structural component of LGs is derived from LAMP1, NKG7, or any transmembrane domain flanked by a YXX motif. In certain embodiments, the protein of interest is a detectable marker, therapeutic protein, or toxic protein. In certain embodiments, the detectable marker is a fluorescent protein. In certain embodiments, the signal peptide is derived from an LG protein selected from the group consisting of perforin, a granzyme, and NKG7. In certain embodiments, the fusion protein further comprises a plasma membrane directing peptide. In certain embodiments, the fusion protein comprises the N-terminal domain of NKG7, the protein of interest, and a C-terminal domain from perforin, NKG7, or a granzyme.

[0024] In another aspect, the present invention provides for one or more vectors encoding the fusion protein according to any embodiment herein.

[0025] In another aspect, the present invention provides for a system for targeting LGs to the plasma membrane of a cytotoxic lymphocyte comprising: a first fusion protein comprising an LG associated coiled-coil peptide and a signal peptide that sorts the fusion protein to the LG and is derived from a protein specifically expressed on LGs, optionally, coiled-coil domain 2 (CC2) of GCC2; and a second fusion protein comprising a transmembrane domain specific to the plasma membrane and a coiled-coil containing protein capable of binding the first fusionAttorney Docket 44010.215WO-PCT / / CU24341protein, wherein the first and second fusion proteins are encoded for by one or more vectors, wherein the cytotoxic lymphocyte is a NK cell, CD8+CTL, or NKT cell.

[0026] In another aspect, the present invention provides for an isolated cytotoxic lymphocyte modified to express the fusion protein of any embodiment herein, wherein the cytotoxic lymphocyte is aNK cell, CD8+CTL, or NKT cell, optionally, wherein expression of the fusion protein is inducible.

[0027] In another aspect, the present invention provides for an isolated cytotoxic lymphocyte genetically modified to delete GCC2, wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell.

[0028] In another aspect, the present invention provides for an isolated cytotoxic lymphocyte genetically modified to overexpress one or more exophilin proteins selected from the group consisting of SYTL-1, SYTL-2, SYTL-2, SYTL-3, SYTL4, exophilin-5, and melanophilin (MLPH), wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell.

[0029] In another aspect, the present invention provides for an isolated cytotoxic lymphocyte modified to express the system of any embodiment herein, wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell.

[0030] In another aspect, the present invention provides for a method of targeting lytic granules (LGs) to the plasma membrane of a cytotoxic lymphocyte to increase cytotoxicity, increase bystander killing, and / or enhance anti-tumor activity, the method comprising anchoring LGs to the plasma membrane using plasma membrane-directing peptides that bind directly or indirectly to the LG, wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell. In certain embodiments, the method comprises expressing the fusion protein according to any embodiment herein in the cytotoxic lymphocyte, thereby anchoring LGs to the plasma membrane.

[0031] In another aspect, the present invention provides for a method of targeting lytic granules (LGs) to the plasma membrane of a cytotoxic lymphocyte to increase cytotoxicity, increase bystander killing, and / or enhance anti-tumor activity, the method comprising anchoring LGs to the plasma membrane by overexpressing one or more exophilin proteins, wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell. In certain embodiments, the one or more exophilin proteins are selected from the group consisting of SYTL-1, SYTL-2, SYTL-3, SYTL4, exophilin-5, and melanophilin (MLPH).Attorney Docket 44010.215WO-PCT / / CU24341

[0032] In another aspect, the present invention provides for a method of treating cancer in a subject in need thereof comprising administering the isolated cytotoxic lymphocyte according to any embodiment herein to the tumor of the subject. In certain embodiments, the isolated cytotoxic lymphocyte expresses an exogenous T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0033] In another aspect, the present invention provides for a method of treating cancer in a subject in need thereof comprising administering lipid nanoparticles carrying modified mRNA targeting cytotoxic lymphocytes in vivo and encoding the fusion protein or system of any embodiment herein.

[0034] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0036] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0037] FIG. 1A-1G - The Golgi associates with lytic granules after NK cell activation. (FIG. 1A-C) YTS cells stably transduced with GALT-mCherry allow Golgi visualization (teal) imaged in combination with F-actin (via phalloidin, orange), the MTOC (via a-tubulin foci, purple), and LGs (via perforin, yellow) in cells adhered with non-activating antiCD 18(IB4) (FIG. 1A), cells activated with anti-CD18(IB4) / anti-CD28 (FIG. IB), or in conjugation with 721.221 target cells (FIG. 1C). Left: original images; right: reconstructions using Imaris software (target cells, gray; scale bar, 10 pm). Left images in each pair show x, z; right images, x,y projections. (FIG. ID and E) Average distance of LGs to the MTOC (FIG. ID) and the Golgi (FIG. IE) for 20 to 25 cells per condition: each point represents average distance in one cell derivative from 3 independently repeated experiments (”” = p < 0.0001 Mann-Whitney U tests). (FIG. IF) Visualization of interaction of proteins identified on the LG surface by mass spectrometry after their surface biotinylation and streptavidin-based isolation.Attorney Docket 44010.215WO-PCT / / CU24341Inset denotes known protein associations with Golgi via STRING. (FIG. 1G) Western blot analysis confirmation of / ra / .s-Golgins within proteins extracted from purified LGs.

[0038] FIG.2A-2G - GCC2 is required for activation-induced LG convergence. (FIG.2A and B) GCC2 expression was confirmed by (FIG. 2A) western blot analysis of whole-cell lysate and (FIG. 2B) confocal microscopy: F-actin (phalloidin, orange), Golgi (GALT-mCherry, teal), and GCC2 (anti-GCC2, yellow); scale bar, 10 pm. (FIG.2C) Representative 4 h51Cr-release assay cytotoxicity using YTS GCC2 KO or parental cells against 721.221 target cells (*** =p < 0.001 chi-squared tests). (FIG.2D) Degranulation in YTS GCC2 KO or parental cells after 60 min of incubation with 721.221 target cells measured by mean fluorescence intensity (MFI) of CD 107a. Data represented as mean ± SD of 3 independent repeats with individual values depicted (* = p < 0.05, *** = p < 0.001 Mann- Whitney U tests). (FIG. 2E) Representative rendered images from YTS parental or GCC2 KO cells stimulated with antiCD 18(IB4) / anti-CD28-coated glass or 721.221 target cells for 60 min: LGs (perforin, yellow), F-actin (phalloidin, orange), Golgi (GALT-mCherry, teal), and MTOC (a-tubulin purple); scale bar, 10 pm. The left image in each pair shows an x, z and the right image an x,y projection.(FIG. 2F and G) Mean distance of LGs to MTOC (FIG. 2F) and Golgi (FIG. 2G) measured from 20 to 25 cells per condition across 3 independent experiments in YTS parental (left) and YTS GCC2 KO (right, shaded) cells, (noted comparisons were different **** = p < 0.0001, *** = p < 0.001, * =p < 0.05, Mann-Whitney U tests).

[0039] FIG. 3A-3G - GCC2 prevents bystander killing in multicellular microenvironments. (FIG. 3A) TheCOS stacks were digested, and cells were stained using LIVE / DEAD Near-IR and anti-CD56, -CD45, and -CD19 to allow for identification of live and dead effector, target, and bystander cells via gating. (FIG. 3B) Representative results of resistant K562 target cell “bystander” killing in TheCOS via flow cytometry by parental YTS (red) or YTS GCC2 KO (blue) with increasing percentages of susceptible 721.221 cells from 0 to 80% of the target cells in the TheCOS stack. (FIG. 3C) TheCOS stacks were cut and placed upon glass and imaged via confocal microscopy through the z axis. Three-dimensional reconstruction of images were generated to show vital dye-labeled parental YTS (teal), 721.221 triggering target cells (red), and resistant K562 “bystander” cells (yellow) with staining for perforin to visualize LGs (white); scale bar, 10 pm. (FIG. 3D-G) TheCOS experiments using the osteosarcoma cell lines LM7 (FIG. 3D and E) or 143B (FIG. 3F and G) substituted for the K562 “bystander” cells. After digestion of TheCOS stacks, (FIG.3D) cells were evaluated by flow cytometry to measure LM7 osteosarcoma cell “bystander” killing by parental YTSAttorney Docket 44010.215WO-PCT / / CU24341(red) or YTS GCC2 KO (blue) cells with increasing percentages of susceptible 721.221 cells ranging from 0 to 80%. (FIG. 3E) Three-dimensional reconstruction of a TheCOS stack: vital dye-labeled parental YTS (teal), 721.221 triggering target cells (red), resistant LM7 osteosarcoma “bystander” cells (yellow), and perforin for LGs (white); scale bar, 10 pm. (FIG.3F) After digestion of a TheCOS stack, cells were evaluated by flow cytometry to measure the 143B osteosarcoma cell “bystander” killing mediated by parental YTS (red) or YTS GCC2 KO (blue) cells with increasing percentages of susceptible 721.221 cells ranging from 0 to 80%.(FIG. 3G) Three-dimensional reconstruction of a TheCOS stack: vital dye-labeled parental YTS (teal), 721.221 triggering target cells (red), resistant 143B osteosarcoma “bystander” cells (yellow), and perforin for LGs (white); scale bar, 10 pm.

[0040] FIG. 4A-4G - Ectopic expression of GCC2 at the cell membrane blocks LG convergence and enhances bystander killing. (FIG. 4A) Construct used for targeting GCC2 to the cell membrane (SEQ ID NO: 41). (FIG. 4B) Western blot analysis for GCC2 of wholecell lysates confirming GCC2-KRAS expression (178 kDa) vs. endogenous GCC2 (185 kDa).(FIG. 4C) Representative reconstructed YTS cell images from parental and GCC2 KO, or GCC2 KO YTS reconstituted with the GCC2-KRAS, after stimulation with antiCD 18(IB4) / anti-CD28-coated glass for 60 min. LGs (perforin, yellow), F-actin (phalloidin, orange), Golgi (GALT-mCherry, teal), and the MTOC (a-tubulin purple); scale bar, 10 pm, x, z (left) and x, y (right) projections. (FIG. 4D and E) Mean distance of LGs to MTOC (FIG.4D) and to the F-actin cortex (FIG. 4E) measured from 20 to 25 cells per condition across 2 independent experiments in YTS parental and GCC2 KO (left) and when stably transduced with GCC2-KRAS cells (right, shadowed), (noted comparisons were different **** =p < 0.0001, *** = p < 0.001, ** =p < 0.01, * =p < 0.05, Mann-Whitney U tests). (FIG. 4F) Cytotoxicity of YTS or GCC2 KO YTS cells with or without the GCC2-KRAS construct against 721.221 target cells measured by51Cr-release assay. Individual points show technical triplicate means and represent 3 independent repeats (*** = p < 0.001 chi-squared test). (FIG. 4G) Representative flow cytometry viability assays after 4 h killing in TheCOS. Resistant K562 target cell “bystander” killing by parental YTS (brown), YTS GCC2 KO (blue), or GCC2 KO YTS+GCC2 KRAS (purple) with increasing percentages of susceptible 721.221 cells from 0 to 80% of the target cells in the TheCOS stack.

[0041] FIG. 5A-5E - Golgi maintains LG convergence after activation. (FIG. 5A) Representative rendered images of YTS cells untreated or treated with 1 pM Brefeldin A and stimulated with anti-CD18(IB4) / anti-CD28-coated glass or by 721.221 target cells for 60 min.Attorney Docket 44010.215WO-PCT / / CU24341LGs (perforin, yellow), F-actin (phalloidin, orange), Golgi (GALT-mCherry, teal), and the MTOC (a-tubulin purple), and target cells (gray), scale bar, 10 pm, x, z (left) and x, y (right) projections. (FIG.5B) Mean distance of LGs to the MTOC in 20-25 cells per condition across 3 independent experiments in YTS that were unstimulated (anti-CD18 [ZB4]) or stimulated (anti-CD18 [IB4] / anti-CD28) on coated glass or by conjugation with 721.221 target cells for 60 min; pretreated with (right, shadowed) or without (left) 1 pM Brefeldin A (noted comparisons were different **** =p < 0.0001, * = p < 0.05, Mann-Whitney U tests). (FIG. 5C) Representative 4 h51Cr release killing assay against 721.221 target cells using YTS cells untreated (red) or pretreated (blue) with 1 pM Brefeldin A (means of technical triplicates, representative assay of n = 3, *p < 0.05 chi-squared tests). (FIG. 5D) Rendered images of YTS cells untreated (top) or treated with 1 pM Brefeldin A (bottom); unstimulated (left), stimulated with 100 units / mL IL-2 for 30 min (center), or stimulated for 30 min followed by IL-2 washout and 1 h incubation at 37°C (right). LGs (perforin, yellow), F-actin (phalloidin, orange), Golgi (GALT-mCherry, teal), and MTOC (a-tubulin, purple), x, z (left) and the right image x,y(right) projections. (FIG. 5E) Mean distance of LGs to the MTOC from 20 to 25 cells per condition (2 independent experiments) in untreated (left) or Brefeldin-treated (right, shadowed) cells after IL-2 stimulation, and 1 or 3 h after washing the stimulus, (noted comparisons were different *** =p < 0.0001, ** =p < 0.01, * =p < 0.05 Mann-Whitney U tests).

[0042] FIG. 6A-6J - Biallelic mutations in GCC2 lead to an NK cell deficiency with impaired LG convergence. (FIG. 6A) Patients pedigree with biallelic GCC2 variants. (FIG.6B) Flow cytometric analysis of total NK cells among PBMC. (FIG. 6C) Clinical and variant summary. (FIG. 6D) Schematic GCC2 with interspaced CC-D and GRIP domain regions (green) and disordered regions (gray) showing variant localization. (FIG. 6E) Western blot analysis of GCC2 expression in PBMC whole-cell lysates (HD relative expression shown at the bottom). (FIG. 6F) 4 h51Cr-release assay of PBMC (patients, blue; HD purple) against K562 targets with (dashed line) or without (solid line) 1,000 U / mL added IL-2 (points = means of technical triplicates; representative assay of n = 2 is shown; **** = p < 0.0001 chi-squared test). (FIG.6G) Confocal microscopy (x, y plane) of isolated NK cells from patient 2 incubated 60 min with 721.221 targets showing F-actin (phalloidin, orange), LGs (perforin, yellow), and MTOC (a-tubulin, purple); scale bar, 10 pm. (FIG. 6H) Mean distance of LGs to the MTOC after adherence to non-activating (anti-CD18 [IB4]) glass surface or conjugated to 721.221 target cells; minimum 20 cells per condition (noted comparisons were different **** = p < 0.0001, *** = p < 0.001, or not significant [ns] Mann-Whitney U tests). (FIG. 61) ConfocalAttorney Docket 44010.215WO-PCT / / CU24341microscopy (x, y plane) of isolated NK cells from patient 2 incubated 60 min on glass coated with anti-CD18(IB4) and anti-NKp30 showing F-actin (phalloidin, orange), LGs (perforin, yellow), and MTOC (a-tubulin, purple); scale bar, 10 pm. (FIG. 6 J) Mean distance of LGs to the MTOC from a minimum of 20 cells per condition (noted comparisons were different; **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, or not significant (ns), Mann- Whitney U tests).

[0043] FIG. 7A-7D - The common NKD GCC2 variant impairs directed killing and LG convergence. (FIG. 7A) GCC2 reconstitution confirmed by western blot analysis of wholecell lysates. (FIG. 7B) 4 h51Cr-release assay against 721.221 targets by YTS GCC2 KO cells reconstituted with either WT GCC2 or GCC2 E1608G. Individual points show technical triplicate means, showing a representative assay of n = 3 (noted comparisons were different **** =p < 0.0001, *** =p < 0.001, chi-squared test). (FIG. 7C) Mean distance of LGs to the MTOC in YTS GCC2 KO cells reconstituted with GCC2 WT, or GCC2 E1608G (after 40 min of stimulation on IB4 / anti-CD28-coated glass; 20-25 cells per condition from 2 independent experiments; noted comparisons were different, *** =p < 0.001, * =p < 0.05). (FIG.7D) Timelapse live-cell confocal microscopy of conjugates between 721.221 targets (red) and YTS parental, YTS GCC2 KO, and YTS GCC2 KO reconstituted with WT GCC2 or GCC2 E1608G pre-loaded with Lysotracker Deep Red, LGs (yellow) (scale bar, 20 pm). Imaging began after 30 min of conjugation (time = 0), and images collected every 5 s (total = 100 frames, images from every ~2 min shown).

[0044] FIG. 8A-8E - Control experiments for NK cell activation, LG isolation, bystander killing and degranulation. (FIG. 8A) LG convergence to the MTOC is driven only by activating anti-CD18 monoclonal antibodies. LG convergence to MTOC mediated by different clones of anti-CD18 or anti-CD28 fixed to glass surfaces. Glass bottom 18-well plates were coated with different monoclonal antibodies, and parental YTS cells were added and incubated at 37°C for Ih. Cells were then fixed, permeabilized, and stained for F-actin (phalloidin-Alexa Fluor 405), LG (anti-perforinAlexa Fluor 488), and the MTOC (a-tubulin-Alexa Fluor 592). Mean distance of LG to the MTOC was measured from 20 to 25 cells per condition obtained from 3 independent experiments. The non-activating anti-CD18 mAb clone IB4 was used as basis for cell attachment without LG convergence (non-activation control). Anti-CD28 was used as a positive activation control for LG convergence. Noted comparisons were statistically different (*=p<0.05, Mann-Whitney U test). (FIG. 8B) Quality control for Golgi contamination during LG isolation. Protein extracts from isolated LG using the same procedure as that utilized for LG proteomic analysis were blotted for Giantin (a cis-GolgiAttorney Docket 44010.215WO-PCT / / CU24341protein) (top) and CD107A as a marker for LG (bottom). (FIG. 8C) “Bystander cells” are resistant to direct killing by YTS cells. Representative 4h51Cr release killing assay of parental YTS cells against susceptible 721.221 target (green), and resistant target cells K562, 143B and SaoS-LM7. (FIG. 8D) Degranulation assay using LAMPl-Phluorin reporter.YTS parental cells or YTS GCC2 KO cells transduced an mApple-LAMPl-pHluorin reporter were stimulated on a glass surface coated with anti-CD18(IB4) and anti-CD28 for 1 hour and imaged every 2 minutes. The number of pHluorin transitions on the glass surface were manually counted and plotted for YTS parental (red) and YTS GCC2 KO (blue). Noted comparisons were statistically different using a non-parametric T test with Welsh correction (*=p<0.05 MannWhitney U tests). (FIG.8E) Bystander Killing by Brefeldin A treated cells.YTS cells were pretreated with 1 pM of Brefeldin for 1 hour and then TheCOS stacks were generated, incubated and digested. Isolated cells were stained for flow cytometric analysis using LIVE / DEAD Near IR and directly conjugated antibodies anti-CD56, -CD45, and -CD 19 to allow for identification of live and dead effector, target, and bystander cells using the gating strategy utilized in Fig. 3A. Representative results of resistant K562 target cell “bystander” killing in TheCOS via flow cytometry by untreated YTS (green) or Brefeldin-A treated YTS cells (blue) with increasing percentages of susceptible 721.221 cells (0 to 80%) of the target cells in the TheCOS stack.

[0045] FIG. 9A-9D - Specificity of Golgi and LG interaction. (FIG. 9A) GCC2 requirement for LG proximity to Golgi fragments. YTS Parental or YTS GCC2 KO cells stably expressing GALT-mCherry, were pre-incubated for 1 hour with lug / ml of Brefeldin A, washed twice and stimulated on a glass surface coated with anti-CD18(IB4) and anti-CD28 for 1 hour. Cells were fixed and stained with Phalloidin (orange), and anti-Perforin (yellow), and then imaged using spinning disk confocal microscopy and Golgi visualized via mCherry (teal). Untreated cells (left) compared to those that were Brefeldin A-treated allow for the visualization of Golgi fragmentation. Magnified insets from the Brefeldin A-treated cells show approximation of Golgi fragments to LG primarily when GCC2 was present (top) and not in GCC2 KO cells (bottom) (scale bar = 10pm). (FIG. 9B) quantitative analysis LG and Golgi fragment approximation with or without GCC2. Brefeldin A-treated YTS parental or GCC2 KO cells shown in the images were analyzed and the LG distance to Golgi fragments were quantified using Imaris Software. At least 40 cells per condition for YTS parental (yellow) and GCC2 KO (green) were measured and the mean distance in each cell plotted as an individual point. Noted comparisons were statistically different (*=p<0.05 Mann-Whitney U tests). (FIG.Attorney Docket 44010.215WO-PCT / / CU243419C) Golgi-dependent convergence is specific to LG. YTS Parental cells were stained with Mitospy (teal), pretreated with Brefeldin A and stimulated on a glass surface coated with antiCD 18(IB4) and anti-CD28 for 1 hour. Cells were fixed, permeabilized and stained with Phalloidin (orange) and anti-Perforin (yellow) and imaged using confocal microscopy. In each case, activation induced the convergence of LG while the positioning of mitochondria was diffuse and unaffected by NK cell activation or Brefeldin A treatment, (scale bar = 10pm).(FIG. 9D) Quantitative analysis for the specificity of LG convergence and Brefeldin A effect. Cells from the images and at least 20 others were evaluated for the distance of the LG distance to MTOC (left) and Mitochondria distance to MTOC (right) using Imaris software. The mean distance in individual cells of each was plotted in both untreated and Brefeldin A-treated cells in the presence of only non-activating adhesion anti-CD18(IB4) or anti-CD28-induced activation. Noted comparisons were significant (****=p<0.0001,**=p<0.01, *=p<0.05), or not significant (ns) as determined using Mann-Whitney U tests.

[0046] FIG. 10 A- 10C - In-silico analysis of patient-derived mutations. (FIG. 10 A) In-silico protein modeling of patient mutations. GCC2 protein sequence (Uniprot Q8IWJ2) was used to generate predicted homodimer structures using Alphafold2, which were visualized using Molstar. Each residue (WT left column and mutants right column) was modeled within 50 amino acids in each direction with enlargements to show details of relevant residues. (FIG.10B) Protein alignment of mutated residues. GCC2 protein sequence within 10 amino acids of each of the mutated residues were aligned against the predicted sequence from pig, mouse, Chicken and frog using Clustal default coloring (SEQ ID NOS: 1-15). (FIG. 10C) In silico scoring of deleteriousness for patient-derived mutations. In silico prediction of the deleteriousness of each of the patient derived mutations was obtained from CADD and polyPhen-2, and the evolutionary conservation was retrieved from PhyloP.

[0047] FIG. 11A-11D - Endogenous expression of patient-derived mutations and reconstitution analyses in GCC2 KO YTS cells. (FIG. 11 A) GCC2 expression in patient and healthy donor (HD) ex vivo cells. Whole cell lysate of PBMC from patients 1 and 2 as well as 5 different HD were evaluated for GCC2 (top) and GAPDH (bottom) protein levels by Western blot analysis. The relative expression of GCC2 in each sample (corrected for GAPDH loading) relative to HD2 was measured and is shown. (FIG. 11B) GCC2 expression in patient cells. Purified ex vivo NK cells from a healthy control or patient 2 were fixed, permeabilized and staining with fluorophore-conjugated Phalloidin (red) and anti-GCC2 (detected using secondary anti rabbitAF647, teal). Cells were then imaged using confocal microscopy, (scaleAttorney Docket 44010.215WO-PCT / / CU24341bar = 10pm). (FIG. 11C) Killing efficiency of YTS GCC2 KO cells reconstituted with WT GCC2 or patient-derived GCC2 variants. Representative 4h51Cr release killing assay against susceptible 721.221 target using either parental YTS cells or YTS cells stably reconstituted with full length GCC2 WT or each of the patient derived GCC2 variants. Noted comparisons were significantly different (****=p<0.0001, ***=p<0.001, **=p<0.01 or *=p<0.05 Chi-squared tests). (FIG. 11D) Presence of variant GCC2 protein on LG in reconstituted YTS cells. Whole cell lysates and purified LG (left and right in each panel, respectively) from parental YTS cells (P) or GCC2 KO YTS cells expressing either the GCC2-KRAS, GCC2-WT, GCC2 K77I, GCC2 Q815P, or GCC2 E1608G constructs. Evaluation of GCC2 (top) and CD 107a (bottom) levels by Western blot analysis were performed and in each the enrichment of CD 107a signifies the effective purification of the LG relative to the whole cell lysate.

[0048] FIG. 12A-12F - Extended NK cell phenotyping in GCC2 NKD patients. Flow cytometric analysis of PBMC from GCC2 NKD patients and a healthy donor evaluating NK cell developmental subsets. (FIG. 12A) Uniform manifold approximation and projection (UMAP) analysis of NK cell developmental subsets (Lineage: CD3 CD14 CD19") with expression data overlaid from 1 healthy donor and patients 1 and 2. (FIG. 12B) Frequency of each NK cell developmental subset (Stages 4-6) gated on CD45+LimLiveCD94+with bars showing the mean of 3 distinct healthy donors ±SD and 2 GCC2 patients ±range with points showing the individual values (red=healthy donors, black=GCC2 patients). Frequency of NK cell developmental subsets positive for (FIG. 12C) KIRs [CD158a / b / el / g / h / j], (FIG. 12D) Granzyme B, (FIG. 12E) TBET, and (FIG. 12F) EOMES. Bars display the mean of 3 distinct healthy donors ±SD and 2 GCC2 patients ±range with points showing the individual values (red=healthy donors, black=GCC2 patients).

[0049] FIG. 13A-13C - Effect of Rab6A deletion in YTS cells. (FIG. 13A) Validation of Rab6A deletion in YTS cells KO cells. Whole cell lysate from YTS parental or Rab6A CRISPR CAS9 KO cells were evaluated using anti-Rab6A antibody (top) and antiGAPDH (bottom) via Western blot analysis. (FIG. 13B) Localization of LG and Golgi in Rab6A KO cells. YTS parental or Rab6A KO cells were attached on anti-CD18(IB4), or stimulated using anti-CD18(IB4) / anti-CD28-coated glass surfaces for 40 minutes and then fixed, permeabilized and stained using Phalloidin, anti-Perforin, anti-a-Tubulin and anti-Giantin. Representative images using confocal microscopy and showing the Golgi (teal), LG (yellow) and filamentous actin (red) are shown after activation in a parental (left) and Rab6AKO cell, (scale bar = 10pm).Attorney Docket 44010.215WO-PCT / / CU24341(FIG. 13C) LG distance to MTOC in Rab6A KO cells. Cells from all of the conditions in B (adhered or activated, in parental or Rab6AKO cells) were evaluated for the positioning of the LG relative to the MTOC after activation using Imaris software. Noted comparisons were either significant (****=p<0.0001) or not significant, (ns) via Mann-Whitney U tests.

[0050] FIG. 14 - Graphs showing cytotoxicity of patient 1, 200 healthy donors, and a control set without or with added IL-2 stimulation in vitro.

[0051] FIG. 15 - Graphs showing cytotoxicity of patient 2, 200 healthy donors, and a control set without or with added IL-2 stimulation in vitro.

[0052] FIG. 16 - Graphical abstract showing that the absence of GCC2 leads to LG dispersion, non-directed degranulation, and bystander killing.

[0053] FIG. 17 - shows three genetic strategies to target lytic granules to the surface of the NK cell plasma membrane for the purpose of increased NK cell cytotoxicity and / or increased bystander killing.

[0054] FIG. 18 - Alignment of C terminal sequences of Syntaxin family proteins, showing in blue hydrophobic amino acids, important for the membrane insertion of the protein (SEQ ID NOS: 16-27).

[0055] FIG. 19 - Alignment of example CAAX containing proteins (SEQ ID NOS: 28-40) and illustration of prenylation of a protein of interest containing a CAAX motif (SEQ ID NO: 41).

[0056] FIG. 20 - Golgin-97 KRAS construct. Western blot analysis for G-97 of whole cell lysates confirming expression of the G97-KRAS construct in transduced YTS G-97KO cells (theoretical MW=97kDa) compared to endogenous G-97 (theoretical MW=81 kDa) in YTS parental cells (top), with GAPDH used as a loading control (bottom).

[0057] FIG. 21 - G-97 KRAS construct showing increased lytic approximation to actin cortex. Representative confocal images of parental and G-97 KO, or G-97-KO YTS reconstituted with the G-97-KRAS construct, all after stimulation with anti-CD18(IB4) / anti-CD28-coated glass for 60 min. LG (perforin, yellow), F-actin (phalloidin, red), and the MTOC (a-tubulin cyan). The left image in each pair shows an x,z and the right image an x,y projection.

[0058] FIG. 22A-22B - Increased degranulation and killing capacity of G97. (FIG. 22A) degranulation assay for indicated cell lines. Cells were stimulated for 60 min with Target cells 721.221 or unstimulated (UNS), stained for CD107a (i.e., LAMP1) and analyzed by FACS (FIG. 22B) Cytotoxicity of YTS or G-97 KO YTS cells with or without the G-97-KRASAttorney Docket 44010.215WO-PCT / / CU24341construct against 721.221 target cells measured by 51Cr-release assay. Individual points represent means of technical triplicates and a representative assay.

[0059] FIG. 23 - G-97 THECOS. Representative flow cytometry viability assay results of cells isolated after 4h killing in TheCOS. Points represent resistant K562 target cell “bystander” killing by parental YTS (blue), YTS Parental + G-97 KRAS (green) or YTS G-97 KO + G97-KRAS (red) with increasing percentages of susceptible 721.221 cells ranging from 0 to 80% of the target cells in the TheCOS stack.

[0060] FIG.24 - LAMP1-KRAS and LAMP1-STX1 transduced cells show increased lytic granule approximation toward to actin cortex. Representative confocal images of YTS parental and YTS transduced with KRAS LAMP1, or STX1 LAMP1, all after stimulation with anti-CD18(IB4) / anti-CD28-coated glass for 60 min. LG (perforin, yellow), F-actin (phalloidin, red), and the MTOC (a-tubulin cyan). The left image in each pair shows an x,z and the right image an x,y projection.

[0061] FIG.25A-25B - LAMP1-KRAS and LAMP1-STX1 transduced cells have reduced LG distance to the actin cortex. Mean distance of LG to MTOC (FIG. 25A) measured from 20 cells per condition in YTS parental and YTS parental cells transduced with LAMP1 KRAS and LAMP1-STX1 on anti-CD18 surface with (+) or without (-) anti-CD28 activating surface.(FIG. 25B) Mean distance to the F-actin cortex of cells on anti-CD18 and Anti-CD28 surface.

[0062] FIG. 26A-26B - Increased degranulation and killing capacity of LAMP1-KRAS and LAMP1-STX1 transduced cells. (FIG.26A) degranulation assay for indicated cell lines. Cells were stimulated for 60 min with Target cells 721.221 or unstimulated (UNS), stained for CD107a (i.e LAMP1) and analyzed by FACS (FIG. 26B) Cytotoxicity of YTS parental, KRAS_LAMP1 or STX1_LAMP1 against 721.221 target cells measured by 51Cr-release assay. Individual points represent means of technical triplicates and a representative assay.

[0063] FIG. 27 - Increased bystander killing with LAMP1-KRAS and LAMP1-STX1 transduced cells. Representative flow cytometry viability assay results of cells isolated after 4h killing in TheCOS. Points represent resistant K562 target cell “bystander” killing by parental YTS parental (blue), YTS KRAS-LAMP1 (red), or YTS STX- LAMPl(green) with increasing percentages of susceptible 721.221 cells ranging from 0 to 80% of the target cells in the TheCOS stack.

[0064] FIG. 28 - Membrane targeting of lytic granules specifically. Construct includes the N-terminal domain of a Src Family Kinases (SFKs) followed by the N-terminal region of theAttorney Docket 44010.215WO-PCT / / CU24341family of Type II transmembrane (TII) proteins or by the N-terminal domain of NKG7 and the C-terminal domains of Perforin or NKG7 or GZMB / A, linked by a flexible linker.

[0065] FIG. 29 - Specific Degranulation tool to interrogate lytic granules. Construct that includes an N-terminal domain of NKG7, followed by a protein of interest and by C-terminal Perf / NKG7 / GZMB / A.

[0066] FIG. 30 - Membrane targeting of LG specifically with increase cytotoxic payload for more efficient bystander killing. Construct that includes an N-terminal domain of a Src Family Kinase (SFK), an N-terminal domain of NKG7, followed by a protein of interest followed by C-terminal domain of C-terminal Perf / NKG7 / GZMB / A.

[0067] FIG.31 - Membrane targeting of LG using calmodulin (CaM). Illustration showing CaM anchored to LG binding to transmembrane anchored Ml 3 upon cell activation and increased release of cytoplasmic Ca2+.

[0068] FIG. 32 - C-terminal alignment of lysosomal proteins. An alignment of the C-terminus of LAMP1 (SEQ ID NO: 52), LAMP2 (SEQ ID NO: 53), LAMP3 (SEQ ID NO: 54), CD68 (SEQ ID NO: 55), LAMP5 (SEQ ID NO: 56), CD63 (SEQ ID NO: 57), and NKG7 (SEQ ID NO: 58). Each alignment includes a YXXcj) motif.

[0069] FIG. 33 - C-terminal alignment of human syntaxins. An alignment of the C-terminus of STX10 (SEQ ID NO: 59), STX6 (SEQ ID NO: 60), STX12 (SEQ ID NO: 61), STX7 (SEQ ID NO: 62), STX16 (SEQ ID NO: 63), STX1A (SEQ ID NO: 64), STX1B (SEQ ID NO: 65), STX2 (SEQ ID NO: 66), STX3 (SEQ ID NO: 67), STX4 (SEQ ID NO: 68), STX8 (SEQ ID NO: 69), STX5 (SEQ ID NO: 70), and STX18 (SEQ ID NO: 71).

[0070] FIG.34A-34B - Membrane targeting of LG using LCK-NKG7-PERFORIN (SEQ ID NO: 48). (FIG. 34A) Confocal images showing parental cells stained for actin, tubulin, perforin. (FIG. 34B) Confocal images showing LCK NKG7 PERF modified cells stained for actin, tubulin, perforin. Confocal: 100X Oil, NA 1.4.

[0071] FIG. 35A-35B - Membrane targeting of LG using LAT-NKG7 (SEQ ID NO: 49). (FIG. 35A) Confocal images showing parental cells stained for actin, tubulin, perforin. (FIG.35B) Confocal images showing LAT NKG7 modified cells stained for actin, tubulin, perforin. Confocal :100X Oil, NA 1.4.

[0072] FIG. 36 - Convergence using LCK-NKG7-PERFORIN and LAT-NKG7. Graph showing distance to the MTOC in control, LCK-NKG7 -PERFORIN, and LAT-NKG7 cells. NK cells were incubated for 45 mins with 721.221 target cells, and the degree of convergence was measured.Attorney Docket 44010.215WO-PCT / / CU24341

[0073] FIG.37A-37B - Bystander killing using using LCK-NKG7-PERFORIN and LAT-NKG7-PERFORIN (SEQ ID NO: 50). Representative flow cytometry viability assay results of cells isolated after 4h killing in two TheCOS experiments. Points represent resistant K562 target cell “bystander” killing by parental YTS parental (blue), positive control LAMP1-STX1 (green), LAT-NKG7 -Perforin (purple) (FIG. 37A), and LCK-NKG7 -Perforin (black) (FIG.37B) with increasing percentages of susceptible 721.221 cells from 0 to 80% of the target cells in the TheCOS stack.

[0074] FIG. 38A-38B - Membrane targeting of LG using CC2-KRAS (SEQ ID NO: 51). (FIG. 38A) Confocal images showing parental, CC2-KRAS, and CC3-KRAS cells stained for actin, tubulin, perforin. (FIG. 38B) Graph showing distance to the MTOC in control, CC2-KRAS, and CC3-KRAS cells. NK cells were incubated for 45 mins on functionalized surfaces with anti-CD18 antibody and in the presence or absence of activation signal from anti-CD28 (+ / - CD28), and the degree of convergence was measured.

[0075] FIG. 39A-39C - Membrane targeting of LG using exophilin family overexpression. (FIG.39 A) Chromium release assay using control parental cells and parental cells overexpressing melanophilin. (FIG. 39B) Degranulation assay using control parental cells, parental cells overexpressing melanophilin, and STX1-LAMP1 cells. (FIG. 39C) Representative flow cytometry viability assay results of cells isolated after 4h killing TheCOS experiment. Points represent resistant K562 target cell “bystander” killing by parental YTS parental (blue), positive control LAMP1-STX1 (green) and Melanophilin overexpressing (purple) with increasing percentages of susceptible 721.221 cells from 0 to 80% of the target cells in the TheCOS stack.

[0076] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTSGeneral Definitions

[0077] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); theAttorney Docket 44010.215WO-PCT / / CU24341series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011).

[0078] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0079] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0080] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0081] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / - 10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0082] As used herein, a “biological sample” may contain whole cells and / or live cells and / or cell debris. The biological sample may contain (or be derived from) a “bodily fluid”. The present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum,Attorney Docket 44010.215WO-PCT / / CU24341synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures.

[0083] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0084] All gene name symbols refer to the gene as commonly known in the art. Any reference to the gene symbol is a reference made to the entire gene or variants of the gene. Any reference to the gene symbol is also a reference made to the gene product (e.g., protein). For ease of discussion, when discussing gene expression, any of gene or genes, or protein or proteins may be substituted. The term “expressed” refers to the production of a polypeptide by a cell, including transcription of a corresponding nucleic acid and translation into protein. As used herein “expressed” encompasses not only the synthesis of the protein but also its proper folding, post-translational modification, and localization within the cell. In example embodiments, “expressed” includes the presence of the protein at, or targeted to, a specific intracellular compartment or organelle, such as the endoplasmic reticulum, Golgi apparatus, plasma membrane, secretory vesicle, or a lytic granule. Thus, a protein is considered “expressed” where it is detectably present in such organelle or compartment. Gene names are to be understood to encompass human genes, as well as genes in any other organism (e.g., homologous, orthologous genes). The term, homolog, may apply to the relationship between genes separated by the event of speciation (e.g., ortholog). Orthologs are genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Gene symbols may be those referred to by the HUGO Gene Nomenclature Committee (HGNC) or National Center for Biotechnology Information (NCBI).

[0085] As used herein, the terms “functional fragments,” “derivatives,” “analogs,” and “variants” of a protein (including, without limitation, GCC2, Golgin-97, STX1A, Src-family kinases, and any membrane-anchoring, lytic-granule-associated, or signaling polypeptides described herein) refer to polypeptides that retain at least one biological activity of the corresponding full-length protein. Functional fragments include polypeptides comprisingAttorney Docket 44010.215WO-PCT / / CU24341truncations, deletions, internal deletions, N-terminal or C-terminal truncations, or combinations thereof, provided that the resulting fragment maintains the ability to (i) localize to the plasma membrane, Golgi, or lytic granule; (ii) bind its natural partner (e.g., binding of Src-family N-terminal domains to membranes; binding of Golgin domains to vesicular cargo; or STX1 A transmembrane anchoring); or (iii) perform the intended function in the engineered fusion proteins disclosed herein. Variants include polypeptides comprising amino acid substitutions, additions, or deletions that do not materially alter functional activity, including conservative substitutions (e.g., replacement of one hydrophobic residue with another, one acidic residue with another, or one basic residue with another). In certain embodiments, variants share at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the reference amino-acid sequence while retaining the functional characteristics described herein. Variants may further include post-translational modifications, codon-optimized sequences, species homologs, synthetic sequences, or engineered stability or trafficking motifs, provided that they preserve the capacity to direct, tether, anchor, or reposition lytic granules, or otherwise support the membrane-targeting and cytotoxic functions of the disclosed constructs. Functional fragments and variants explicitly include any sequence that (i) preserves membrane-targeting (e.g., CAAX motifs, N-terminal SH4 domains, STX1A transmembrane helices), (ii) preserves lytic-granule association (e.g., LAMP1 / NKG7 transmembrane domains, GCC2 / Gol gin-97 tethering motifs), or (iii) preserves inducible interaction domains (e.g., calmodulin or M13-derived peptides), whether occurring naturally or engineered.

[0086] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in otherAttorney Docket 44010.215WO-PCT / / CU24341embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0087] Reference is made to Pedroza LA, van den Haak F, Frumovitz A, Hernandez E, Hegewisch-Solloa E, Orange TK, Sheehan KB, Prockop S, Bodansky A, Chinn IK, Lupski JR, Posey JE, Mace EM, Li Y, Orange JS. The Golgi complex governs natural killer cell lytic granule positioning to promote directionality in cytotoxicity. Cell Rep. 2025 Jan 28;44(1): 115156. doi: 10.1016 / j.celrep.2024.115156. Epub 2025 Jan 14. During NK cell activation, lytic granules (LGs) converge to the microtubule-organizing center (MTOC), enabling precision in cytotoxicity. Pedroza et al. demonstrate that GCC2 maintains convergence via tethering LGs to the Golgi. Absence of GCC2 leads to LG dispersion, nondirected degranulation, and bystander killing, and biallelic missense variants result in human NK cell deficiency.

[0088] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.Overview

[0089] Embodiments disclosed herein provide methods, compositions, and systems to actively redirect lytic granules to the plasma membrane of cytotoxic lymphocytes, by fusing plasma membrane anchoring protein sequences to LG-expressing proteins. Embodiments disclosed herein relate to immunotherapy and cellular engineering, specifically to fusion proteins and systems for directing lytic granules to the plasma membrane of cytotoxic lymphocytes to enhance cytotoxicity, bystander killing, and anti-tumor activity. As used herein, “cytotoxic lymphocyte” refers to any lymphocyte capable of killing target cells and encompasses natural killer (NK) cells, CD8+cytotoxic T cells (CTLs), and natural killer T (NKT) cells. As used herein, “natural killer (NK) cells” refers to a type of innate immune lymphocytes specialized for the surveillance of malignancy and defense against viral infection, functioning through cell-mediated cytotoxicity. NK cells form a lytic immunological synapse with diseased cells and mediate directed release of LGs containing perforin and granzymes to eradicate the target cell. NK cells maintain multiple LGs and rely on precise intracellular positioning via convergence to the MTOC and polarization toward the immune synapse to ensure efficient,Attorney Docket 44010.215WO-PCT / / CU24341targeted cytotoxicity and to prevent bystander killing. As used herein, “CD8+cytotoxic T cells (CTLs)” refers to a subset of cytotoxic lymphocytes within the adaptive immune system that express the CD8 co-receptor and possess the capacity to kill target cells, including virally infected or malignant cells. Like NK cells, CTLs contain lytic granules loaded with perforin and granzymes and eliminate target cells through directed degranulation at the immune synapse. As used herein “natural killer T cell” refers to a lymphocyte that co-expresses receptors characteristic of both NK cells and conventional T lymphocytes, and that recognizes lipid antigens presented by CD Id molecules, thereby functioning as a hybrid innate-like and adaptive immune effector capable of rapid cytokine secretion and cytotoxic activity (see, e.g., Nair S, Dhodapkar MV. Natural Killer T Cells in Cancer Immunotherapy. Front Immunol.2017;8:l 178).

[0090] As used herein “lytic granules” refer to specialized cytotoxic organelles found in NK cells, CD8+T cells, and NKT cells. Lytic granules contain effector proteins such as perforin, granzymes, and other cytotoxic mediators. LGs undergo regulated trafficking and fusion with the plasma membrane to mediate target-cell killing.

[0091] LG positioning is critical for malignant cell targeting, as it allows for concentration of the lytic machinery towards a single (often malignant or infected) cell, (Hsu HT, Mace EM, Carisey AF, et al. NK cells converge lytic granules to promote cytotoxicity and prevent bystander killing. J Cell Biol. 2016;215(6):875-889; Ham H, MedlynM, BilladeauDD. Locked and Loaded: Mechanisms Regulating Natural Killer Cell Lytic Granule Biogenesis and Release. Front Immunol. 2022; 13:871106) while sparing the surrounding healthy tissue. This is in part mediated by the process of LG convergence, where these specialized organelles concentrate around the MTOC and Golgi apparatus and the entire machinery is subsequently positioned near the interface of the NK cell and a single target cell, this interface is the immune synapse (IS). This process enables precise delivery of LG cargo (perforin, and granzymes) towards a single cell at a single time point. Convergence is mediated by the mobilization and transient interactions of LG with microtubules and Golgi (Hsu HT, Mace EM, Carisey AF, et al. NK cells converge lytic granules to promote cytotoxicity and prevent bystander killing. J Cell Biol. 2016;215(6):875-889; and Pedroza LA, van den Haak F, Frumovitz A, et al. The Golgi complex governs natural killer cell lytic granule positioning to promote directionality in cytotoxicity. Cell Rep. 2025;44(l):l 15156). Once the lytic machinery is at the IS, SNARE proteins on the LG and cell membrane interact; permitting fusion of the LG with the cell membrane, ensuing the release of LG content on the delimited space of the IS (one NK cell:Attorney Docket 44010.215WO-PCT / / CU24341one target cell) (Lesteberg K, Orange J, Makedonas G. Recycling endosomes in human cytotoxic T lymphocytes constitute an auxiliary intracellular trafficking pathway for newly synthesized perforin. Immunol Res. 2017;65(5): 1031-1045; Gwalani LA, Orange JS. Single Degranulations in NK Cells Can Mediate Target Cell Killing. J Immunol. 2018;200(9):3231-3243).

[0092] In summary, LG positioning in NK cells is critical for targeting malignant cells. It results in focused and directed release toward a single (often malignant or infected) cell, while sparing the healthy surrounding tissue. This process is in part mediated by LG convergence, the concentration of lytic granules around the MTOC and Golgi apparatus, in activated NK cells. In addition, polarization occurs, where the cytolytic machinery (LG, MTOC, and Golgi) move along microtubules to the interface of the NK cell and target cell, also referred to as the immune synapse. At the immune synapse, the LG interact with SNARE molecules that allow the LG membrane to fuse with the plasma membrane, thereby releasing its content onto the target cell.

[0093] Applicants demonstrate that blocking convergence during NK cell activation results in LG to be dispersed in the cytoplasm. These dispersed LG are released outside the context of the IS and consequently induce bystander killing (See, Example 1; and Pedroza, et al. 2025). In a reductionist context, an activated NK cell only requires LG to be positioned close to the cell membrane to initiate the degranulation process, either mediated via the IS resulting in specific target killing or outside the IS, resulting in bystander killing. The process of LG repositioning could be random by blocking LG convergence or could be actively initiated by direct repositioning the LG to the cell membrane.

[0094] Here Applicants describe a method to actively redirect the LG to the membrane, by fusing cell-membrane anchoring protein sequences to LG-expressing proteins. Described are two strategies that rely on modification of anchoring proteins. The first approach includes utilization of highly hydrophobic amino acid peptide sequences that naturally insert into the cell membrane. The second approach harnesses post translational modification events where non-proteinic hydrophobic sequences are added (fatty acid addition) that enables association with the cell membrane. In both cases these modifications can occur at the N-terminal or C-terminal regions of the LG-expressing proteins. These cell membrane-targeting peptides can be fused to either a transient adapter protein linked to the LG or to a structural component of the LG; allowing for either a transient or more permanent re-localization of the LG on the cell membrane.Attorney Docket 44010.215WO-PCT / / CU24341

[0095] To test both approaches, the C-terminal peptides from any Type IV transmembrane domain can be utilized. Here, as example, Syntaxin 1 A, is used as a representative C-terminal signal peptide that can be inserted into the cell membrane based on its high non-polar amino acid sequence. Also, demonstrated is the use of a representative CAAX peptide, which contains hydrophobic molecules with post-translational modifications (such as prenylation) that can associate with the cell membrane.

[0096] Using the aforementioned cell membrane anchoring peptides, a fusion protein was created containing the sequence for GCC2 (previously demonstrated protein with transient interactions with LG), which resulted in transient association of LG with the cell membrane. Additionally, cell membrane anchoring peptides were used to generate a fusion protein containing the transmembrane domain of LAMP 1 or NKG7. These transmembrane regions have previously been demonstrated to be the minimum requirement for insertion into the LG membrane, therefore creating stable expression and stable association of membrane anchoring proteins to the LG membrane. This is predicted to be a more enduring re-redirection of LG to the cell membrane.

[0097] A third additional approach to target LG related proteins to the cell membrane requires modifications that rely on N-terminal membrane targeting domains which could include the N-terminal domain of Src Family Kinases (Src, Yes, Fyn, Fgr, Lek, Hck, Blk, Lyn, and Frk). These N-terminal sequences are part of the larger family of type II transmembrane N-terminal domains. SFKs N-terminal domains are myristoylated, and this post translational modification helps with insertion into the plasma membrane. Here as example, the N-terminal region of a SFK is fused to the transmembrane containing N-terminal domain of NKG7. To ensure N-terminal SFK peptides are specifically inserted into LG membranes (and not other lysosome related organelles), they are also be linked to the C terminal region of any protein known to be specifically expressed on LG, such as perforin, granzymes or NKG7.

[0098] An additional approach to target LG to the membrane is to use specific peptide interactions, that can be modulated upon cell activation. As an example, calmodulin, which is a highly conserved protein among all species, is known to interact with other proteins when Ca2+is present. Ca2+is released from the ER and works as a second messenger during cell activation, this similarly occurs following NK cell engagement with a target cell. Phage display approaches have identified specific peptides that interact with calmodulin or its domains with a high affinity and specificity only in the presence of calcium. One example is the Ml 3 peptide from skeletal muscle myosin light chain kinase (Bayley PM, Findlay WA, Martin SR. TargetAttorney Docket 44010.215WO-PCT / / CU24341recognition by calmodulin: dissecting the kinetics and affinity of interact! on using short peptide sequences. Protein Sci. 1996;5(7): 1215-1228).

[0099] With this in mind, an approach was generated where the M13 peptide (or any peptide with high affinity or specificity for calmodulin) is used as cell membrane anchor. In combination, the sequence for calmodulin will be inserted on the LG through a type II transmembrane domain and will be linked to the C-terminal region of any protein know to be expressed on LG, such as perforin, granzymes or NKG7. This approach ensures that LG are specifically redirected to the cell-membrane for bystander killing following NK cell engagement with a target cell.

[0100] Here, Applicants present multiple strategies to purposefully target lytic granules to the cell membrane following NK cell activation for the benefit of bystander killing in a solid tumor setting as novel cell therapy approach. As such, the use of the fusion proteins described herein for targeting lytic granules to the cell membrane or for the treatment of cancer (for example, a solid tumor cancer) are also disclosed. These strategies can be used alone or in combination with dispersion related strategies.METHODS OF LYTIC GRANULE REDIRECTION TO THE PLASMA MEMBRANE IN CYTOTOXIC CELLS TO PROMOTE BYSTANDER KILLING IN CELLULAR THERAPIESFusion proteins

[0101] In example embodiments, a fusion protein targets LGs to the plasma membrane of cytotoxic cells. In example embodiments, the fusion protein includes a plasma membrane directing peptide and a protein associated with LGs. In example embodiments, the fusion protein includes an LG-sorting signal peptide.Plasma membrane-directing peptides

[0102] As used herein “plasma membrane-directing peptide” refers to any peptide or protein domain that, when fused to another protein, is sufficient to drive, enhance, or bias localization of the fusion protein toward the inner leaflet of the plasma membrane, the cortical actin interface, or other membrane-proximal cytoplasmic regions. In example embodiments, a plasma membrane-directing peptide can be targeted to the plasma membrane of a cell. In example embodiments, a plasma membrane directing peptide is any peptide sequence that drives or enhances localization of a protein to the inner plasma membrane. Such peptides mayAttorney Docket 44010.215WO-PCT / / CU24341(i) contain hydrophobic residues, (ii) undergo post-translational lipid modifications (e.g., prenylation of CAAX motifs, fatty-acid addition), or (iii) include transmembrane segments or membrane-anchoring domains. Exemplary sources include CAAX motifs (e.g., from KRAS), type II or type IV transmembrane domains, a C-terminal transmembrane domain of syntaxin 1A (STX1) or syntaxin family member, N-terminal domains of SFKs, and calmodulin via a calmodulin binding protein engineered to be targeted to the plasma membrane. Plasma membrane-directing peptides include, but are not limited to: (i) hydrophobic or lipid-modified motifs, including CAAX motifs (e.g., KRAS C-terminal CAAX) and sequences capable of undergoing farnesylation, geranylgeranylation, myristoylation, or palmitoylation; (ii) C-terminal tail-anchored transmembrane segments, including those derived from Type IV membrane proteins (e.g., syntaxin family members); (iii) signal-anchor sequences from Type II transmembrane proteins, including N-terminal membrane-targeting helices; (iv) N-terminal SH4 domains of Src-family kinases (SFKs) known to target proteins to the plasma membrane through lipidation; and (v) adaptor proteins or domains, such as the transmembrane and juxtamembrane regions of LAT (Linker for Activation of T cells) that naturally localize to the plasma membrane and have been demonstrated herein to redirect lytic granules toward the cell cortex when incorporated into fusion constructs.

[0103] Non-limiting hydrophobic amino acid residues that would insert into a plasma membrane include leucine, isoleucine, and valine, as well as phenylalanine, due to their nonpolar side chains. Other residues like alanine, methionine, and cysteine can also contribute to membrane insertion, particularly in transmembrane alpha-helices.

[0104] As used herein a “CAAX motif’ refers to a C-terminal lipidation sequence consisting of a cysteine (C), two aliphatic amino acids (AA), and a terminal residue (X). CAAX motifs undergo prenylation and mediate strong plasma membrane association.

[0105] As used herein, a “Type II transmembrane domain” refers to a single-pass transmembrane segment that spans the lipid bilayer once and adopts an N-cytosolic / C-luminal (or extracellular) orientation. In such proteins, the N-terminus remains on the cytoplasmic side of the membrane, while a hydrophobic a-helical transmembrane segment anchors the protein within the membrane and positions the C-terminal region within the luminal or extracellular space. Type II transmembrane domains are characteristic of single-pass membrane proteins whose topology is determined co-translationally, typically through an internal signal-anchor sequence that functions both as a membrane insertion signal and as the transmembrane helix. This signal-anchor sequence directs insertion into the endoplasmic reticulum membrane in anAttorney Docket 44010.215WO-PCT / / CU24341orientation in which the positively charged cytosolic N-terminus is retained on the cytoplasmic side in accordance with the “positive-inside” rule. In the context of the present disclosure, Type II transmembrane domains may be used as membrane-targeting elements to direct fusion proteins to the plasma membrane and to establish the desired N-cytosolic orientation of engineered protein constructs.

[0106] As used herein a “Type IV transmembrane domain” refers to a class of integral membrane proteins distinguished by having its single transmembrane segment near the C-terminus, making it a “tail-anchored” protein. Type IV proteins typically pass through the cell Type IV transmembrane proteins are defined as those with their N-terminus in the cytosol and the transmembrane domain (TMD) located near the C-terminus, which acts as the membrane anchor.

[0107] As used herein a “Src Family Kinase (SFK)” refers to a group of non-receptor tyrosine kinases that regulate crucial cellular functions like cell proliferation, differentiation, migration, and survival by phosphorylating tyrosine residues on other proteins. The SFK N-terminal domain contains myristoylation and / or palmitoylation sites that drive strong association with the plasma membrane. SFKs have an N-terminal SH4 domain that acts as a membranetargeting sequence, enabling their localization to the plasma membrane and other cellular membranes through lipid modifications like myristoylation and palmitoylation. These fatty acid attachments are crucial for anchoring SFKs to the inner leaflet of the cell membrane, which is essential for their signaling functions. Examples include domains from Src, Lek, Fyn, Blk, Lyn, Yes, Fgr, Hck, and Frk (see, e.g., Src: NP_005408, Lek: NP_005347, Fyn: NP_002026, Blk: NP_001714, Lyn: NP_002343, Yes: NP_005424, Fgr: NP_005247, Hck: NP_002097, Frk: NP_002024).

[0108] As used herein “syntaxin family members” refers to a class of membrane-anchored SNARE proteins typically characterized by a C-terminal Type IV (tail-anchored) transmembrane domain. Syntaxin proteins possess a predominantly cytosolic N-terminal region containing regulatory and SNARE-interacting domains, followed by a single C-terminal hydrophobic helix that anchors the protein into the plasma membrane or intracellular membranes. The C-terminal transmembrane segment of syntaxins functions as a membrane-directing peptide capable of inserting into lipid bilayers to position the cytosolic SNARE-binding region for vesicle docking and fusion. Syntaxin family members are generally considered type II or type IV transmembrane proteins, depending on the specific classification system used, but both classifications describe the same structural orientation. They are typicallyAttorney Docket 44010.215WO-PCT / / CU24341tail-anchored proteins with a single pass through the membrane and their bulk (amino-terminal) facing the cytoplasm.

[0109] As used herein “syntaxin 1A (STX1, STX1A)” refers to a protein essential for neurotransmitter release at synapses, acting as a key component of the SNARE complex that mediates synaptic vesicle docking and fusion with the presynaptic membrane (see, e.g., NP 004594). It also has independent roles in neuronal survival and function, including regulation of ion channels. Mutations in the STX1A gene are associated with neurodevelopmental disorders, such as autism spectrum disorder and epilepsy. STX1A has a C-terminal transmembrane domain. Syntaxin l isa type II transmembrane protein, also referred to as a type IV membrane protein in some classification systems. The classification “type II” refers to its specific orientation within the membrane (N-terminus in the cytoplasm / P-side, C-terminus extracellular / E-side or in the lumen of organelles), while “type IV” is sometimes used for tail-anchored proteins in general because they are inserted into the membrane via a C-terminal tail anchor, a category that includes all syntaxins except syntaxin 11.

[0110] As used herein, “LAT” refers to the Linker for Activation of T cells, a transmembrane adaptor protein involved in T-cell and natural killer (NK) cell signal transduction. LAT is encoded by the human LAT gene (NCBI Gene ID: 27040) and is represented in the NCBI RefSeq database by multiple transcript variants, including the canonical mRNA transcript NM_014387 and corresponding protein NP_055202. Alternative RefSeq isoforms include NM_001014987, NM_001014988, and NM_001014989, which encode proteins NP_001014987, NP_001014988, and NP_001014989, respectively. LAT functions as a plasma-membrane-resident adaptor that becomes phosphorylated upon lymphocyte activation and serves as a scaffold for SH2-domain containing signaling proteins.LG associated peptides[OHl] As used herein “LG associated peptides” are any peptides that associate with the LG. An LG associated peptide is any peptide sequence that either (i) forms a structural part of an LG (e.g., a transmembrane domain of an LG-resident protein such as LAMP1 or NKG7), or (ii) transiently associates with LGs, such as adaptor proteins or tethering proteins (e.g., GCC2 or Golgin-97). The term includes full-length proteins, fragments, truncations, motifs, and engineered variants that retain LG association. For example, LG associated peptides can be inserted into the LG membrane or be tethered either directly or indirectly to LGs. LG associated peptides may not exclusively interact with LGs. For example, LG associated peptides mayAttorney Docket 44010.215WO-PCT / / CU24341interact with LGs but may also interact with other lysosome related vesicles. In example embodiments, the LG associated peptides have the highest affinity for LGs. In example embodiments, the LG associated peptides interact specifically with LGs. In example embodiments, the modification of LG associated peptides with plasma membrane-directing polypeptides can occur at the N-terminal or C-terminal regions of the LG associated proteins.Transient adapter proteins linked to the LG

[0112] In example embodiments, the adaptor proteins or tethering proteins are transient. As used herein “transient adaptor peptide” refers to a peptide derived from a protein that interacts with or tethers to LGs but is not a stable structural component. Representative examples include GCC2, truncated GCC2 variants, coiled-coil domain of GCC2, and Golgin-97.

[0113] As used herein “GRIP and coiled-coil domain containing 2 (GCC2)” encodes a protein that is important for vesicle trafficking and maintaining the structure of the Golgi apparatus (see, e.g., NP_852118). This protein plays a role in the movement of vesicles between the Golgi and endosomes, such as recycling the mannose 6-phosphate receptor. GCC2 is a large (~l,684-aa) coiled-coil-rich tethering protein composed of five interspersed coiled-coil domains (CC-Ds) separated by intrinsically disordered regions, followed by a highly conserved C-terminal GRIP (Golgin-97 / RanBP2a-interacting protein) domain. The coiled-coil domains comprise extended a-helical heptad-repeat motifs that mediate long-range interactions with vesicular Rab GTPases, including Rab9 (interaction region approximately residues 805-889) and Rab6A (interaction region approximately residues 1609-1659). These CC-Ds provide the structural platform by which GCC2 engages transport vesicles and contributes to vesicle tethering and positioning within the trans-Golgi network. The C-terminal GRIP domain, beginning just after residue E1608, encodes the conserved targeting module responsible for Golgi localization through ARL1 -dependent binding. This GRIP region directs GCC2 to the trans-Golgi and enables stable tethering of incoming vesicles once they have been captured through the upstream coiled-coil regions. Variants located at the CC-3 region (e.g., K777I, Q851P) or at the start of the GRIP domain (e.g., E1608G) disrupt the structure or function of these motifs, further confirming the functional importance of these sequences.

[0114] As used herein “golgin 97,” also known as GOLGA1, encodes a protein that acts as a tethering factor in the trans-Golgi network (TGN) (see, e.g., NP_002068). This protein is crucial for vesicular trafficking, maintaining cell polarity, and is involved in the transport of specific proteins like E-cadherin from the TGN to other cellular destinations.Attorney Docket 44010.215WO-PCT / / CU24341Structural component of the LG

[0115] A structural component of LGs is a peptide or protein domain that is physically present in LG membranes or LG-resident proteins. Non-limiting examples include transmembrane regions of LAMP 1, NKG7, LAMP2, LAMP3, CD63, CD68, or any LG-expressed protein.

[0116] As used herein “lysosomal-associated membrane protein 1 (LAMP-1),” also known as lysosome-associated membrane glycoprotein 1 and CD 107a (Cluster of Differentiation 107a), is a protein that in humans is encoded by the LAMP1 gene. LAMP1 encodes lysosomal-associated membrane protein 1, with the primary human RefSeq mRNA accession NM_00556L

[0117] As used herein “natural killer cell granule protein 7 (NKG7 protein)” is a protein crucial for immune cell function, particularly in natural killer (NK) cells and CD8+ T cells. It is a protein coding gene involved in the regulation of cytotoxic granule exocytosis, which is essential for killing target cells and fighting cancer. NKG7 encodes natural-killer-cell granule protein 7, with the primary human RefSeq mRNA accession NM_005601.

[0118] As used herein “lysosomal-associated membrane protein 2 (LAMP2),” also known as CD 107b and lysosome-associated membrane glycoprotein 2, is a heavily glycosylated integral membrane protein that localizes predominantly to the lysosomal membrane and is encoded by the A4A P2 gene. LAMP2 encodes lysosomal-associated membrane protein 2, with the primary human RefSeq mRNA accession NM_013995.

[0119] As used herein “lysosomal-associated membrane protein 3 (LAMP3),” also referred to as DC-LAMP or CD208, is a member of the LAMP family expressed predominantly in activated human dendritic cells and encoded by the LAMP3 gene. LAMP3 participates in lysosomal and endosomal processes, including phagocytosis, autophagy, and immune activation. LAMP3 encodes lysosomal-associated membrane protein 3, with the primary human RefSeq mRNA accession NM_014398.

[0120] As used herein “CD63,” also known as a lysosomal membrane-associated glycoprotein and a member of the tetraspanin (transmembrane-4 superfamily), is a multi-pass transmembrane protein encoded by the CD63 gene. CD63 is primarily localized to late endosomes, multivesicular bodies, and lysosomes, and is translocated to the cell surface upon cellular activation. It functions in vesicular trafficking, signal transduction, and is widely used as a marker of exosomes and cellular degranulation. CD63 encodes the tetraspanin lysosomal membrane protein CD63, with the primary human RefSeq mRNA accession NM_001780.Attorney Docket 44010.215WO-PCT / / CU24341

[0121] As used herein “CD68,” also known as macrosialin, gpllO, or LAMP4, is a heavily glycosylated lysosomal membrane-associated glycoprotein structurally related to the LAMP family and encoded by the CD68 gene. CD68 contains a mucin-like luminal domain, a proline-rich hinge, a LAMP -like domain, and a single transmembrane region, and is predominantly expressed in macrophages and other phagocytic immune cells. Lysosomal / endosomal-associated membrane glycoprotein highly expressed in macrophages, with the primary human RefSeq mRNA accession NM_001251.

[0122] In example embodiments, the structural component of LGs includes a YXX<b sequence (i.e., transmembrane domain). In preferred embodiments, the YXX sequence is either N- or C- terminal of the transmembrane domain. The sequence YXX (Tyrosine-Any amino acid-Any amino acid -Bulky Hydrophobic amino acid (e.g., I / L / M / F / V)) is a critical tyrosine-based sorting motif in cell biology, directing proteins for clathrin-dependent endocytosis (internalizing from the cell surface) and intracellular trafficking, interacting with adaptor proteins like AP-2, and playing roles in viral entry (like HCV, SARS-CoV) and immune signaling.LG-sorting signal peptide

[0123] In example embodiments, the fusion protein further comprises a C-terminal signal peptide that sorts the fusion protein to the LG and is derived from a protein specifically expressed on LGs. As used herein, a “sorting signal peptide” is any peptide sequence, typically C-terminal in the described constructs, that drives trafficking to lytic granules. In example embodiments, sorting peptides are used to enhance LG localization and retention. Such peptides may be derived from perforin, granzymes, NKG7, or similar LG-specific proteins. In example embodiments, the signal peptide is derived from perforin, a granzyme, or NKG7. In example embodiments, LG associated transmembrane domains, or adaptor proteins or tethering proteins are further linked to the signal sequences of proteins specifically expressed on LGs, such as NKG7, perforin, or a granzyme. In example embodiments, LG specific transmembrane proteins are modified to retain their signal peptides for correct sorting into the LG.Linkers

[0124] In example embodiments, the fusion protein includes one or more linkers. As used herein, “linker” refers to a short peptide sequence incorporated between two functional domains of a fusion protein to provide flexibility, reduce steric hindrance, and maintainAttorney Docket 44010.215WO-PCT / / CU24341independent folding and activity of each domain. In example embodiments, linkers comprise small, neutral amino acids that impart flexibility and minimize structural interference. Nonlimiting examples include glycine-serine (G / S) linkers, which are widely used due to their high conformational flexibility and low immunogenicity. G / S linkers typically consist of repeating units of glycine and serine (e.g., (G4S)n, where n = 1-3), providing rotational freedom and reducing steric clashes between fused domains. Additional linker designs may include flexible linkers (rich in glycine and serine), rigid linkers (e.g., proline-rich sequences), or cleavable linkers for controlled release applications (see, e.g., US patent application US20200392512A1). Linker length and composition may be tailored to the needs of the particular fusion construct, including but not limited to fusion proteins comprising plasma membrane directing peptides, LG associated peptides, calmodulin based interaction modules, or LG sorting signal peptides described herein.Calmodulin to target LGs to the membrane

[0125] In example embodiments, specific peptide interactions are used to target LGs to the plasma membrane. In example embodiments, calmodulin or a domain is tethered to LGs and a calmodulin binding protein is tethered to the plasma membrane. In example embodiments, specific peptides interact with calmodulin or its domains with a high affinity and specificity only in the presence of calcium. In example embodiments, when the NK cell or cytotoxic T cell is activated, calcium is released thus allowing binding. As used herein “Calmodulin (CaM)” refers to a calcium-dependent regulatory protein that binds calmodulin-binding peptides (e.g., M13). In this invention, CaM is used as a membrane-anchoring or interactionmediating component for LG targeting systems. In example embodiments, any calmodulin binding protein or any peptide with high affinity or specificity for calmodulin can be used. As used herein “Ml 3” refers to a synthetic peptide having a sequence that is the same as the calmodulin-binding domain of skeletal muscle myosin light chain kinase (skMLCK) (residues 577-602). In example embodiments, calmodulin binding protein or any peptide is inserted on the plasma membrane through any of the plasma membrane-directing peptides described herein. In example embodiments, calmodulin is inserted on the LG through any of the LG associated peptides described herein. In example embodiments, calmodulin is also linked to the C-terminal region of any protein known to be expressed on LG. In example embodiments, the sequence for calmodulin is inserted on the LG through a type II transmembrane domainAttorney Docket 44010.215WO-PCT / / CU24341and is linked to the C-terminal region of any protein known to be expressed on LGs, such as perforin, a granzyme, or NKG7.Genetically Modifying Cytotoxic Lymphocytes

[0126] In example embodiments, a genetically modified cytotoxic lymphocyte targets lytic granules (LGs) to the plasma membrane. In example embodiments, GCC2 expression is reduced or eliminated in a cytotoxic lymphocyte. In example embodiments, GCC2 expression is reduced or eliminated using a CRISPR system, RNAi, antisense oligonucleotides, or any other method of genetically modifying a cytotoxic lymphocyte. As discussed further herein, GCC2 normally tethers LGs to the Golgi for convergence and directional killing. Loss of GCC2 disperses LGs, causing non directional release and bystander killing.

[0127] In example embodiments, one or more exophilin proteins (Rab27 effector proteins) are overexpressed in a cytotoxic lymphocyte (e.g., SYTL-1, SYTL-2, SYTL-3, SYTL4, exophilin-5, or melanophilin (MLPH)). Exophilins (also known as synaptotagmin-like proteins or SYTLs, plus melanophilin and others) are Rab27 effector proteins (see, Table A) (see, e.g., Izumi T. In vivo Roles of Rab27 and Its Effectors in Exocytosis. Cell Struct Funct.2021;46(2):79-94). Exophilins bind RAB27a, a protein necessary for docking of LG on the membrane, prior to membrane fusion occurring via the SNARE complex. Rab27 and exophilins position, tether, dock, and traffic secretory vesicles toward the cell membrane. Exophilins bind Rab27 on LGs and simultaneously interact with membrane-associated scaffolds. Overexpression of exophilin proteins increases the docking efficiency of RAB27a positive granules on the plasma membrane. This suggest increased degranulation and could be beneficial for increased killing. In example embodiments, exophilin overexpression increases the number of LGs that accumulate at the cell membrane, instead of waiting for natural activation cues, and primes the cell for release.Table A.Attorney Docket 44010.215WO-PCT / / CU24341Fusion proteins for targeting LGs

[0128] In example embodiments, a fusion protein targeting LGs is used to target a protein of interest to LGs. In example embodiments, the fusion protein comprises an LG associated peptide comprising a structural component of LGs, a protein of interest, and a signal peptide that sorts the fusion protein to the LG. A protein of interest refers to any polypeptide to be delivered to LGs, including but not limited to fluorescent markers, therapeutic proteins, immune-modifying proteins, toxins, or engineered effector proteins.Systems for Targeting LGs to the Plasma Membrane

[0129] In example embodiments, the fusion proteins described herein are used as part of a system for targeting LGs to the plasma membrane. The systems include vectors and reagents for modifying NK cells or cytotoxic lymphocytes. In example embodiments, the fusion proteins or exophilin protein for targeting LGs to the plasma membrane are encoded for on one or more vectors. As used herein a “vector” is any nucleic-acid construct that encodes one or more fusion proteins described herein. Non-limiting vectors include plasmids, viral vectors (e.g., lentiviral, retroviral, AAV), and RNA vectors. In example embodiments, the vectors include promoters that are constitutive, inducible, NK-specific, T-cell specific, or combinations thereof. In example embodiments, the vectors include regulatory elements, such as 573' UTRs, poly A sequences, codon optimization.

[0130] In example embodiments, a system comprises two or more fusion proteins whose coordinated interaction directs LGs to the plasma membrane. For example, one component is LG-localized (e.g., CaM-containing LG-associated fusion), and the second is plasmamembrane anchored and comprises a CaM-binding peptide such as M13. In example embodiments, a first fusion protein comprises an LG associated coiled-coil peptide and a signal peptide that sorts the fusion protein to the LG and is derived from a protein specifically expressed on LGs; and a second fusion protein comprises a transmembrane domain specific to the plasma membrane and a coiled-coil containing protein capable of binding the first fusion protein, wherein the first and second fusion proteins are encoded for by one or more vectors.

[0131] As used herein, an “LG-associated coiled-coil peptide” refers to any polypeptide comprising a coiled-coil structural motif that interacts directly or indirectly with a lytic granule (LG) and is capable of mediating, stabilizing, or enhancing the association of the peptide, or any fusion protein comprising the peptide, with the LG membrane (e.g., CC2). In certain embodiments, the LG-associated coiled-coil peptide is derived from a naturally occurringAttorney Docket 44010.215WO-PCT / / CU24341tethering or scaffold protein that associates transiently or constitutively with lytic granules, including but not limited to members of the Golgin family, such as GCC2 or Golgin-97, whose coiled-coil domains participate in vesicle tethering and intracellular positioning of LGs. In exemplary embodiments, the LG-associated coiled-coil peptide comprises one or more heptad-repeat regions capable of adopting a parallel or antiparallel a-helical coiled-coil conformation sufficient to maintain its LG-binding function when incorporated into a fusion protein. Functional fragments of coiled-coil domains that retain LG-targeting activity are additionally encompassed, including fragments that preserve Rab-interaction regions, Golgi / LG docking interfaces, or other sequence determinants enabling transient or stable association with LGs. Variants having conservative amino-acid substitutions, engineered stabilizing residues, or modifications enhancing helical propensity are included, provided that the variant peptide maintains LG-association capacity. In example embodiments, the LG-associated coiled-coil peptide is linked to a plasma-membrane directing peptide, a sorting signal peptide derived from an LG-resident protein, or both, to generate a fusion protein capable of repositioning LGs within cytotoxic lymphocytes. In other embodiments, coiled-coil peptides are used as engineered LG-tethering modules irrespective of their native biological origin, provided that they confer the ability to recruit, anchor, or localize a construct to lytic granules following expression in a natural killer (NK) cell, cytotoxic T lymphocyte, or other cytotoxic immune effector cell.METHODS OF TREATING CANCER

[0132] In example embodiments, the invention comprises genetically modified NK cells or cytotoxic lymphocytes for use in adoptive cell transfer to a subject in need thereof. In example embodiments, NK cells or cytotoxic lymphocytes are modified ex vivo or in vitro to express any of the fusion proteins described herein. In example embodiments, NK cells or cytotoxic lymphocytes are genetically modified ex vivo or in vitro to promote bystander killing by blocking convergence without interfering with degranulation (e.g., by altering or deleting expression of GCC2). In example embodiments, fusion proteins are expressed in NK cells or cytotoxic lymphocytes genetically modified ex vivo or in vitro by altering or deleting expression of GCC2.

[0133] In example embodiments, the NK cells or cytotoxic lymphocytes can be administered to a subject to treat a tumor. In example embodiments, the cells have increased cytotoxicityAttorney Docket 44010.215WO-PCT / / CU24341and bystander killing (e.g., enhanced immediate release of perforin / granzymes upon target contact). In example embodiments, the cells are administered intratum orally.

[0134] The cancer may include, without limitation, liquid tumors such as leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin’s disease, nonHodgkin’s disease), Waldenstrom’s macroglobulinemia, heavy chain disease, or multiple myeloma.

[0135] The cancer may include, without limitation, solid tumors such as sarcomas and carcinomas. Examples of solid tumors include, but are not limited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, epithelial carcinoma, bronchogenic carcinoma, hepatoma, colorectal cancer (e.g., colon cancer, rectal cancer), anal cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors), breast cancer (e.g., ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma), ovarian carcinoma (e.g., ovarian epithelial carcinoma or surface epithelial-stromal tumour including serous tumour, endometrioid tumor and mucinous cystadenocarcinoma, sex-cord-stromal tumor), prostate cancer, liver and bile duct carcinoma (e.g., hepatocelluar carcinoma, cholangiocarcinoma, hemangioma), choriocarcinoma, seminoma, embryonal carcinoma, kidney cancer (e.g., renal cell carcinoma, clear cell carcinoma, Wilm's tumor, nephroblastoma), cervical cancer, uterine cancer (e.g., endometrial adenocarcinoma, uterine papillary serous carcinoma, uterine clear-cell carcinoma, uterine sarcomas and leiomyosarcomas, mixed mullerian tumors), testicular cancer, germ cell tumor, lung cancer (e.g., lung adenocarcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, non-small-cell carcinoma, small cell carcinoma, mesothelioma), bladder carcinoma, signet ring cell carcinoma, cancer of the head and neck (e.g., squamous cell carcinomas), esophageal carcinoma (e.g., esophageal adenocarcinoma), tumors of the brain (e.g., glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,Attorney Docket 44010.215WO-PCT / / CU24341hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma), neuroblastoma, retinoblastoma, neuroendocrine tumor, melanoma, cancer of the stomach (e.g., stomach adenocarcinoma, gastrointestinal stromal tumor), or carcinoids. Lymphoproliferative disorders are also considered to be proliferative diseases.

[0136] As used herein, “ACT”, “adoptive cell therapy” and “adoptive cell transfer” may be used interchangeably. In certain embodiments, Adoptive cell therapy (ACT) can refer to the transfer of cells to a patient with the goal of transferring the functionality and characteristics into the new host by engraftment of the cells (see, e.g., Mettananda et al., Editing an a-globin enhancer in primary human hematopoietic stem cells as a treatment for P-thalassemia, Nat Commun. 2017 Sep 4;8(1):424). As used herein, the term “engraft” or “engraftment” refers to the process of cell incorporation into a tissue of interest in vivo through contact with existing cells of the tissue. Adoptive cell therapy (ACT) can refer to the transfer of cells, most commonly immune-derived cells (e.g., T cells or NK cells), back into the same patient or into a new recipient host with the goal of transferring the immunologic functionality and characteristics into the new host. If possible, use of autologous cells helps the recipient by minimizing GVHD issues. The adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) (Zacharakis et al., (2018) Nat Med. 2018 Jun;24(6):724-730; Besser et al., (2010) Clin. Cancer Res 16 (9) 2646-55; Dudley et al., (2002) Science 298 (5594): 850-4; and Dudley et al., (2005) Journal of Clinical Oncology 23 (10): 2346-57.) or genetically re-directed peripheral blood mononuclear cells (Johnson et al., (2009) Blood 114 (3): 535-46; and Morgan et al., (2006) Science 314(5796) 126-9) has been used to successfully treat patients with advanced solid tumors, including melanoma, metastatic breast cancer and colorectal carcinoma, as well as patients with CD19-expressing hematologic malignancies (Kalos et al., (2011) Science Translational Medicine 3 (95): 95ra73). In certain embodiments, allogenic cells immune cells are transferred (see, e.g., Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266). As described further herein, allogenic cells can be edited to reduce alloreactivity and prevent graft-versus-host disease. Thus, use of allogenic cells allows for cells to be obtained from healthy donors and prepared for use in patients as opposed to preparing autologous cells from a patient after diagnosis.

[0137] Aspects of the invention involve the adoptive transfer of immune system cells, such as T cells or NK cells, specific for selected antigens, such as tumor associated antigens or tumor specific neoantigens (see, e.g., Maus et al., 2014, Adoptive Immunotherapy for Cancer or Viruses, Annual Review of Immunology, Vol. 32: 189-225; Rosenberg and Restifo, 2015,Attorney Docket 44010.215WO-PCT / / CU24341Adoptive cell transfer as personalized immunotherapy for human cancer, Science Vol. 348 no.6230 pp. 62-68; Restifo et al., 2015, Adoptive immunotherapy for cancer: harnessing the T cell response. Nat. Rev. Immunol. 12(4): 269-281; and Jenson and Riddell, 2014, Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells. Immunol Rev. 257(1): 127-144; and Rajasagi et al., 2014, Systematic identification of personal tumorspecific neoantigens in chronic lymphocytic leukemia. Blood. 2014 Jul 17;124(3):453-62).

[0138] In certain embodiments, an antigen (such as a tumor antigen or self antigen) to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) may be selected from a group consisting of: MR1 (see, e.g., Crowther, et al., 2020, Genome-wide CRISPR-Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1, Nature Immunology volume 21, pagesl78-185), B cell maturation antigen (BCMA) (see, e.g., Friedman et al., Effective Targeting of Multiple BCMA-Expressing Hematological Malignancies by Anti-BCMA CAR T Cells, Hum Gene Ther. 2018 Mar 8; Berdeja JG, et al. Durable clinical responses in heavily pretreated patients with relapsed / refractory multiple myeloma: updated results from a multicenter study of bb2121 anti-Bcma CAR T cell therapy. Blood.2017; 130:740; and Mouhieddine and Ghobrial, Immunotherapy in Multiple Myeloma: The Era of CAR T Cell Therapy, Hematologist, May-June 2018, Volume 15, issue 3); PSA (prostatespecific antigen); prostate-specific membrane antigen (PSMA); PSCA (Prostate stem cell antigen); Tyrosine-protein kinase transmembrane receptor ROR1; fibroblast activation protein (FAP); Tumor-associated glycoprotein 72 (TAG72); Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); Mesothelin; Human Epidermal growth factor Receptor 2 (ERBB2 (Her2 / neu)); Prostase; Prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); Insulin-like growth factor 1 receptor (IGF-1R); gplOO; BCR-ABL (breakpoint cluster region-Abelson); tyrosinase; New York esophageal squamous cell carcinoma 1 (NY-ESO-1); K-light chain, LAGE (L antigen); MAGE (melanoma antigen); Melanoma-associated antigen 1 (MAGE-A1); MAGE A3; MAGE A6; legumain; Human papillomavirus (HPV) E6; HPV E7; prostein; survivin; PCTA1 (Galectin 8); Melan-A / MART-1; Ras mutant; TRP-1 (tyrosinase related protein 1, or gp75); Tyrosinase-related Protein 2 (TRP2); TRP-2 / INT2 (TRP-2 / intron 2); RAGE (renal antigen); receptor for advanced glycation end products 1 (RAGE1); Renal ubiquitous 1, 2 (RU1, RU2); intestinal carboxyl esterase (iCE); Heat shock protein 70-2 (HSP70-2) mutant; thyroid stimulating hormone receptor (TSHR); CD 123; CD171; CD 19; CD20; CD22; CD26; CD30; CD33; CD44v7 / 8 (cluster ofAttorney Docket 44010.215WO-PCT / / CU24341differentiation 44, exons 7 / 8); CD53; CD92; CD100; CD148; CD150; CD200; CD261; CD262; CD362; CS-1 (CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); Tn antigen (Tn Ag); Fms-Like Tyrosine Kinase 3 (FLT3); CD38; CD138; CD44v6; B7H3 (CD276); KIT (CD117); Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2); Interleukin 11 receptor alpha (IL-1 IRa); prostate stem cell antigen (PSCA); Protease Serine 21 (PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); Mucin 1, cell surface associated (MUC1); mucin 16 (MUC16); epidermal growth factor receptor (EGFR); epidermal growth factor receptor variant III (EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); ephrin type-A receptor 2 (EphA2); Ephrin B2; Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); TGS5; high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor alpha; Folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Poly sialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1 A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); CT (cancer / testis (antigen)); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53; p53 mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl -transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl; Cyclin DI; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Cytochrome P450 1B1Attorney Docket 44010.215WO-PCT / / CU24341(CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS); Squamous Cell Carcinoma Antigen Recognized By T Cells- 1 or 3 (SART1, SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint- 1, -2, -3 or -4 (SSX1, SSX2, SSX3, SSX4); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); mouse double minute 2 homolog (MDM2); livin; alphafetoprotein (AFP); transmembrane activator and CAML Interactor (TACI); B-cell activating factor receptor (BAFF-R); V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS); immunoglobulin lambda-like polypeptide 1 (IGLL1); 707-AP (707 alanine proline); ART-4 (adenocarcinoma antigen recognized by T4 cells); BAGE (B antigen; b-catenin / m, b-catenin / mutated); CAMEL (CTL-recognized antigen on melanoma); CAP1 (carcinoembryonic antigen peptide 1); C ASP-8 (caspase-8); CDC27m (cell-division cycle 27 mutated); CDK4 / m (cycline-dependent kinase 4 mutated); Cyp-B (cyclophilin B); DAM (differentiation antigen melanoma); EGP-2 (epithelial glycoprotein 2); EGP-40 (epithelial glycoprotein 40); Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4); FBP (folate binding protein); , fAchR (Fetal acetylcholine receptor); G250 (glycoprotein 250); GAGE (G antigen); GnT-V (N-acetylglucosaminyltransferase V); HAGE (helicose antigen); ULA-A (human leukocyte antigen- A); HST2 (human signet ring tumor 2); KIAA0205; KDR (kinase insert domain receptor); LDLR / FUT (low density lipid receptor / GDP L-fucose: b-D-galactosidase 2-a-L fucosyltransferase); LI CAM (LI cell adhesion molecule); MC1R (melanocortin 1 receptor); Myosin / m (myosin mutated); MUM-1, -2, -3 (melanoma ubiquitous mutated 1, 2, 3); NA88-A (NA cDNA clone of patient M88); KG2D (natural killer group 2, member D) ligands; oncofetal antigen (h5T4); pl 90 minor bcr-abl (protein of 190KD bcr-abl); Pml / RARa (promyelocytic leukaemia / retinoic acid receptor a); PRAME (preferentially expressed antigen of melanoma); SAGE (sarcoma antigen); TEL / AML 1 (translocation Ets-family leukemia / acute myeloid leukemia 1); TPI / m (triosephosphate isomerase mutated); CD70; and any combination thereof.Attorney Docket 44010.215WO-PCT / / CU24341

[0139] In certain embodiments, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) is a tumor-specific antigen (TSA).

[0140] In certain embodiments, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) is a neoantigen.

[0141] In certain embodiments, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) is a tumor-associated antigen (TAA).

[0142] In certain embodiments, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) is a universal tumor antigen. In certain preferred embodiments, the universal tumor antigen is selected from the group consisting of: a human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 IB 1 (CYP1B), HER2 / neu, Wilms' tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, cyclin (DI), and any combinations thereof.

[0143] In certain embodiments, an antigen (such as a tumor antigen) to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) may be selected from a group consisting of: CD19, BCMA, CD70, CLL-1 , MAGE A3 , MAGE A6, HP V E6, HP V E7, WT 1 , CD22, CD 171 , ROR1 , MUC 16, and S SX2. In certain preferred embodiments, the antigen may be CD19. For example, CD19 may be targeted in hematologic malignancies, such as in lymphomas, more particularly in B-cell lymphomas, such as without limitation in diffuse large B-cell lymphoma, primary mediastinal b-cell lymphoma, transformed follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia including adult and pediatric ALL, non-Hodgkin lymphoma, indolent non-Hodgkin lymphoma, or chronic lymphocytic leukemia. For example, BCMA may be targeted in multiple myeloma or plasma cell leukemia (see, e.g., 2018 American Association for Cancer Research (AACR) Annual meeting Poster: Allogeneic Chimeric Antigen Receptor T Cells Targeting B Cell Maturation Antigen). For example, CLL1 may be targeted in acute myeloid leukemia. For example, MAGE A3, MAGE A6, SSX2, and / or KRAS may be targeted in solid tumors. For example, HPV E6 and / or HPV E7 may be targeted in cervical cancer or head and neck cancer. For example, WT1 may be targeted in acute myeloidAttorney Docket 44010.215WO-PCT / / CU24341leukemia (AML), myelodysplastic syndromes (MDS), chronic myeloid leukemia (CML), nonsmall cell lung cancer, breast, pancreatic, ovarian or colorectal cancers, or mesothelioma. For example, CD22 may be targeted in B cell malignancies, including non-Hodgkin lymphoma, diffuse large B-cell lymphoma, or acute lymphoblastic leukemia. For example, CD171 may be targeted in neuroblastoma, glioblastoma, or lung, pancreatic, or ovarian cancers. For example, ROR1 may be targeted in ROR1+ malignancies, including non-small cell lung cancer, triple negative breast cancer, pancreatic cancer, prostate cancer, ALL, chronic lymphocytic leukemia, or mantle cell lymphoma. For example, MUC16 may be targeted in MUC16ecto+ epithelial ovarian, fallopian tube or primary peritoneal cancer. For example, CD70 may be targeted in both hematologic malignancies as well as in solid cancers such as renal cell carcinoma (RCC), gliomas (e.g., GBM), and head and neck cancers (HNSCC). CD70 is expressed in both hematologic malignancies as well as in solid cancers, while its expression in normal tissues is restricted to a subset of lymphoid cell types (see, e.g., 2018 American Association for Cancer Research (AACR) Annual meeting Poster: Allogeneic CRISPR Engineered Anti-CD70 CAR-T Cells Demonstrate Potent Preclinical Activity Against Both Solid and Hematological Cancer Cells).

[0144] In example embodiments, Yescarta is an FDA-approved CAR-T cell therapy that is particularly used to treat relapsed or refractory large B-cell lymphoma. Yescarta has shown relatively high efficacy in terms of patient response and preventing cancer progression or the need for additional cancer treatment. In example embodiments, Abecma is an FDA-approved CAR T-cell therapy used to treat adult patients with relapsed or refractory multiple myeloma following other therapeutic approaches. In example embodiments, Tecartus is an FDA-approved CAR T-cell therapy to treat relapsed or refractory mantle cell lymphoma as well as B-cell precursor acute lymphoblastic leukemia. It is used following previous therapies and has previously been reported to show relatively high complete and overall response rate in treating lymphoma. In example embodiments, Kymriah is an FDA-approved CAR T-cell therapy to treat diffuse large B-cell lymphoma and relapsed or refractory acute lymphoblastic leukemia. Novartis has relatively long-term data showing durable remission and long-term survival in children and young adults with this form of leukemia. Furthermore, Kymriah has a relatively good safety profile according to currently reported results. In example embodiments, Carvykti is an FDA-approved CAR T-cell therapy to treat relapsed or refractory multiple myeloma following four lines of treatment. In prior studies involving a follow-up of approximately 18Attorney Docket 44010.215WO-PCT / / CU24341months, patients showed a relatively high overall response rate as well as in previous pilot studies. It was also shown to reduce risk of progression and relapse

[0145] Various strategies may for example be employed to genetically modify T cells by altering the specificity of the T cell receptor (TCR) for example by introducing new TCR a and P chains with selected peptide specificity (see U.S. Patent No. 8,697,854; PCT Patent Publications: W02003020763, W02004033685, W02004044004, W02005114215, W02006000830, W02008038002, W02008039818, W02004074322, W02005113595, WO2006125962, WO2013166321, WO2013039889, WO2014018863, WO2014083173; U.S. Patent No. 8,088,379).

[0146] As an alternative to, or addition to, TCR modifications, chimeric antigen receptors (CARs) may be used in order to generate immunoresponsive cells, such as T cells or natural killer cells (NK), specific for selected targets, such as malignant cells, with a wide variety of receptor chimera constructs having been described (see U.S. PatentNos. 5,843,728; 5,851,828; 5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014; 6,753,162; 8,211,422; and, PCT Publication WO9215322).

[0147] In general, CARs are comprised of an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antigen-binding domain that is specific for a predetermined target (see, e.g., Gong Y, Klein Wolterink RGJ, Wang J, Bos GMJ, Germeraad WTV. Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. J Hematol Oncol. 2021;14(l):73; Guedan S, Calderon H, Posey AD Jr, Maus MV. Engineering and Design of Chimeric Antigen Receptors. Mol Ther Methods Clin Dev. 2018;12:145-156; Petersen CT, Krenciute G. Next Generation CAR T Cells for the Immunotherapy of High-Grade Glioma. Front Oncol. 2019;9:69; Lu H, Zhao X, Li Z, Hu Y, Wang H. From CAR-T Cells to CAR-NK Cells: A Developing Immunotherapy Method for Hematological Malignancies. Front Oncol. 2021; and Miliotou AN, Papadopoulou LC. CAR T-cell Therapy: A New Era in Cancer Immunotherapy. Curr Pharm Biotechnol. 2018; 19(1): 5-18). While the antigen-binding domain of a CAR is often an antibody or antibody fragment (e.g., a single chain variable fragment, scFv), the binding domain is not particularly limited so long as it results in specific recognition of a target. For example, in some embodiments, the antigen-binding domain may comprise a receptor, such that the CAR is capable of binding to the ligand of the receptor. Alternatively, the antigenbinding domain may comprise a ligand, such that the CAR is capable of binding the endogenous receptor of that ligand.Attorney Docket 44010.215WO-PCT / / CU24341

[0148] The antigen-binding domain of a CAR is generally separated from the transmembrane domain by a hinge or spacer. The spacer is also not particularly limited, and it is designed to provide the CAR with flexibility. For example, a spacer domain may comprise a portion of a human Fc domain, including a portion of the CH3 domain, or the hinge region of any immunoglobulin, such as IgA, IgD, IgE, IgG, or IgM, or variants thereof. Furthermore, the hinge region may be modified so as to prevent off-target binding by FcRs or other potential interfering objects. For example, the hinge may comprise an IgG4 Fc domain with or without a S228P, L235E, and / or N297Q mutation (according to Kabat numbering) in order to decrease binding to FcRs. Additional spacers / hinges include, but are not limited to, CD4, CD8, and CD28 hinge regions.

[0149] The transmembrane domain of a CAR may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane bound or transmembrane protein. Transmembrane regions of particular use in this disclosure may be derived from CD8, CD28, CD3, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154, TCR. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0150] Alternative CAR constructs may be characterized as belonging to successive generations. First-generation CARs typically consist of a single-chain variable fragment of an antibody specific for an antigen, for example comprising a VL linked to a VH of a specific antibody, linked by a flexible linker, for example by a CD8a hinge domain and a CD8a transmembrane domain, to the transmembrane and intracellular signaling domains of either CD3(^ or FcRy (scFv-CD3(^ or scFv-FcRy; see U.S. Patent No. 7,741,465; U.S. Patent No.5,912,172; U.S. Patent No. 5,906,936). Second-generation CARs incorporate the intracellular domains of one or more costimulatory molecules, such as CD28, 0X40 (CD134), or 4-1BB (CD137) within the endodomain (for example scFv-CD28 / OX40 / 4-lBB-CD3^; see U.S. Patent Nos. 8,911,993; 8,916,381; 8,975,071; 9,101,584; 9,102,760; 9,102,761). Third-generation CARs include a combination of costimulatory endodomains, such a CD3^-chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, 0X40, 4-1BB, CD2, CD7, LIGHT, LFA-1,Attorney Docket 44010.215WO-PCT / / CU24341NKG2C, B7-H3, CD30, CD40, PD-1, or CD28 signaling domains (for example scFv-CD28-4-lBB-CD3(^ or scFv-CD28-OX40-CD3i ; see U.S. Patent No. 8,906,682; U.S. Patent No.8,399,645; U.S. Pat. No. 5,686,281; PCT Publication No. WO2014134165; PCT Publication No. WO2012079000).

[0151] In certain embodiments, the primary signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCERIG), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fc gamma Rlla, DAP10, and DAP12. In certain preferred embodiments, the primary signaling domain comprises a functional signaling domain of CD3(^ or FcRy. In certain embodiments, the one or more costimulatory signaling domains comprise a functional signaling domain of a protein selected, each independently, from the group consisting of: CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD lid, ITGAE, CD 103, ITGAL, CD 11 a, LFA-1, ITGAM, CD 11b, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD 18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, and NKG2D. In certain embodiments, the one or more costimulatory signaling domains comprise a functional signaling domain of a protein selected, each independently, from the group consisting of: 4-1BB, CD27, and CD28. In certain embodiments, a chimeric antigen receptor may have the design as described in U.S. Patent No. 7,446,190, comprising an intracellular domain of CD3(^ chain (such as amino acid residues 52-163 of the human CD3 zeta chain, as shown in SEQ ID NO: 14 of US 7,446,190), a signaling region from CD28 and an antigenbinding element (or portion or domain; such as scFv). The CD28 portion, when between the zeta chain portion and the antigen-binding element, may suitably include the transmembrane and signaling domains of CD28 (such as amino acid residues 114-220 of SEQ ID NO: 10, full sequence shown in SEQ ID NO: 6 of US 7,446,190; these can include the following portion of CD28 as set forth in Genbank identifier NM_006139 (sequence version 1, 2 or 3). Alternatively, when the zeta sequence lies between the CD28 sequence and the antigen-bindingAttorney Docket 44010.215WO-PCT / / CU24341element, intracellular domain of CD28 can be used alone (such as amino sequence set forth in SEQ ID NO: 9 of US 7,446,190). Hence, certain embodiments employ a CAR comprising (a) a zeta chain portion comprising the intracellular domain of human CD3(^ chain, (b) a costimulatory signaling region, and (c) an antigen-binding element (or portion or domain), wherein the costimulatory signaling region comprises the amino acid sequence encoded by SEQ ID NO: 6 of US 7,446,190.

[0152] Alternatively, costimulation may be orchestrated by expressing CARs in antigenspecific T cells, chosen so as to be activated and expanded following engagement of their native aPTCR, for example by antigen on professional antigen-presenting cells, with attendant costimulation. In addition, additional engineered receptors may be provided on the immunoresponsive cells, for example to improve targeting of a T-cell attack and / or minimize side effects

[0153] By means of an example and without limitation, Kochenderfer et al., (2009) J Immunother. 32 (7): 689-702 described anti-CD19 chimeric antigen receptors (CAR). FMC63-28Z CAR contained a single chain variable region moiety (scFv) recognizing CD 19 derived from the FMC63 mouse hybridoma (described in Nicholson et al., (1997) Molecular Immunology 34: 1157-1165), a portion of the human CD28 molecule, and the intracellular component of the human TCR-(^ molecule. FMC63-CD828BBZ CAR contained the FMC63 scFv, the hinge and transmembrane regions of the CD8 molecule, the cytoplasmic portions of CD28 and 4- IBB, and the cytoplasmic component of the TCR-(^ molecule. The exact sequence of the CD28 molecule included in the FMC63-28Z CAR corresponded to Genbank identifier NM 006139; the sequence included all amino acids starting with the amino acid sequence IEVMYPPPY (SEQ. I.D. No. 2) and continuing all the way to the carboxy-terminus of the protein. To encode the anti-CD19 scFv component of the vector, the authors designed a DNA sequence which was based on a portion of a previously published CAR (Cooper et al., (2003) Blood 101: 1637-1644). This sequence encoded the following components in frame from the 5’ end to the 3’ end: an Xhol site, the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor a-chain signal sequence, the FMC63 light chain variable region (as in Nicholson et al., supra), a linker peptide (as in Cooper et al., supra), the FMC63 heavy chain variable region (as in Nicholson et al., supra), and a Notl site. A plasmid encoding this sequence was digested with Xhol and Noth To form the MSGV-FMC63-28Z retroviral vector, the Xhol and Notl-digested fragment encoding the FMC63 scFv was ligated into a second Xhol and Notl-digested fragment that encoded the MSGV retroviral backbone (as in Hughes et al.,Attorney Docket 44010.215WO-PCT / / CU24341(2005) Human Gene Therapy 16: 457-472) as well as part of the extracellular portion of human CD28, the entire transmembrane and cytoplasmic portion of human CD28, and the cytoplasmic portion of the human TCR-(^ molecule (as in Maher et al., 2002) Nature Biotechnology 20: 70-75). The FMC63-28Z CAR is included in the KTE-C19 (axicabtagene ciloleucel) anti-CD19 CAR-T therapy product in development by Kite Pharma, Inc. for the treatment of inter alia patients with relap sed / refractory aggressive B-cell non-Hodgkin lymphoma (NHL).

[0154] Accordingly, in certain embodiments, cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may express the FMC63-28Z CAR as described by Kochenderfer et al. (supra). Hence, in certain embodiments, cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may comprise a CAR comprising an extracellular antigen-binding element (or portion or domain; such as scFv) that specifically binds to an antigen, an intracellular signaling domain comprising an intracellular domain of a CD3(^ chain, and a costimulatory signaling region comprising a signaling domain of CD28. Preferably, the CD28 amino acid sequence is as set forth in Genbank identifier NM_006139 (sequence version 1, 2 or 3). Preferably, the antigen is CD19, more preferably the antigen-binding element is an anti-CD19 scFv, even more preferably the anti-CD19 scFv as described by Kochenderfer et al. (supra).

[0155] Additional anti-CD19 CARs are further described in WO2015187528. More particularly Example 1 and Table 1 of WO2015187528, incorporated by reference herein, demonstrate the generation of anti-CD19 CARs based on a fully human anti-CD19 monoclonal antibody (47G4, as described in US20100104509) and murine anti-CD19 monoclonal antibody (as described in Nicholson et al. and explained above). Various combinations of a signal sequence (human CD8-alpha or GM-CSF receptor), extracellular and transmembrane regions (human CD8-alpha) and intracellular T-cell signaling domains (CD28-CD3(^; 4-lBB-CD3(^; CD27-CD3 CD28-CD27-CD3^, 4-lBB-CD27-CD3i ; CD27-4-lBB-CD3i ; CD28-CD27-FcsRI gamma chain; or CD28-FcsRI gamma chain) were disclosed. Hence, in certain embodiments, cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may comprise a CAR comprising an extracellular antigen-binding element that specifically binds to an antigen, an extracellular and transmembrane region as set forth in Table 1 of WO2015187528 and an intracellular T-cell signaling domain as set forth in Table 1 of WO2015187528. Preferably, the antigen is CD19, more preferably the antigen-binding element is an anti-CD19 scFv, even more preferably the mouse or human anti-CD19 scFv as described in Example 1 of WO2015187528. In certain embodiments, the CAR comprises,Attorney Docket 44010.215WO-PCT / / CU24341consists essentially of or consists of an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 as set forth in Table 1 of WO2015187528.

[0156] By means of an example and without limitation, chimeric antigen receptor that recognizes the CD70 antigen is described in W02012058460A2 (see also, Park et al., CD70 as a target for chimeric antigen receptor T cells in head and neck squamous cell carcinoma, Oral Oncol. 2018 Mar;78: 145-150; and Jin et al., CD70, a novel target of CAR T-cell therapy for gliomas, Neuro Oncol. 2018 Jan 10;20(l):55-65). CD70 is expressed by diffuse large B-cell and follicular lymphoma and also by the malignant cells of Hodgkins lymphoma, Waldenstrom's macroglobulinemia and multiple myeloma, and by HTLV-1- and EBV-associated malignancies. (Agathanggelou et al. Am.J.Pathol. 1995;147: 1152-1160; Hunter et al., Blood 2004; 104:4881. 26; Lens et al., J Immunol. 2005;174:6212-6219; Baba et al., J Virol. 2008;82:3843-3852.) In addition, CD70 is expressed by non-hematological malignancies such as renal cell carcinoma and glioblastoma. (Junker et al., J Urol.2005;173:2150-2153; Chahlavi et al., Cancer Res 2005;65:5428-5438) Physiologically, CD70 expression is transient and restricted to a subset of highly activated T, B, and dendritic cells.

[0157] By means of an example and without limitation, chimeric antigen receptor that recognizes BCMA has been described (see, e.g., US20160046724A1; WO2016014789A2; W02017211900A1; WO2015158671A1; US20180085444A1; WO2018028647A1; US20170283504A1 ; and WO2013154760A1).

[0158] Additional CAR T cell therapies with enhanced efficacy are further described in WO / 2017 / 049166.

[0159] In certain embodiments, the immune cell may, in addition to a CAR or exogenous TCR as described herein, further comprise a chimeric inhibitory receptor (inhibitory CAR) that specifically binds to a second target antigen and is capable of inducing an inhibitory or immunosuppressive or repressive signal to the cell upon recognition of the second target antigen. In certain embodiments, the chimeric inhibitory receptor comprises an extracellular antigen-binding element (or portion or domain) configured to specifically bind to a target antigen, a transmembrane domain, and an intracellular immunosuppressive or repressive signaling domain. In certain embodiments, the second target antigen is an antigen that is not expressed on the surface of a cancer cell or infected cell or the expression of which is downregulated on a cancer cell or an infected cell. In certain embodiments, the second targetAttorney Docket 44010.215WO-PCT / / CU24341antigen is an MHC-class I molecule. In certain embodiments, the intracellular signaling domain comprises a functional signaling portion of an immune checkpoint molecule, such as for example PD-1 or CTLA4. Advantageously, the inclusion of such inhibitory CAR reduces the chance of the engineered immune cells attacking non-target (e.g., non-cancer) tissues.

[0160] Alternatively, T-cells expressing CARs may be further modified to reduce or eliminate expression of endogenous TCRs in order to reduce off-target effects. Reduction or elimination of endogenous TCRs can reduce off-target effects and increase the effectiveness of the T cells (U.S. 9,181,527). T cells stably lacking expression of afunctional TCRmay be produced using a variety of approaches. T cells internalize, sort, and degrade the entire T cell receptor as a complex, with a half-life of about 10 hours in resting T cells and 3 hours in stimulated T cells (von Essen, M. et al. 2004. J. Immunol. 173:384-393). Proper functioning of the TCR complex requires the proper stoichiometric ratio of the proteins that compose the TCR complex. TCR function also requires two functioning TCR zeta proteins with IT AM motifs. The activation of the TCR upon engagement of its MHC-peptide ligand requires the engagement of several TCRs on the same T cell, which all must signal properly. Thus, if a TCR complex is destabilized with proteins that do not associate properly or cannot signal optimally, the T cell will not become activated sufficiently to begin a cellular response.

[0161] Accordingly, in some embodiments, TCR expression may eliminated using RNA interference (e.g., shRNA, siRNA, miRNA, etc.), CRISPR, or other methods that target the nucleic acids encoding specific TCRs (e.g., TCR-a and TCR-P) and / or CD3 chains in primary T cells. By blocking expression of one or more of these proteins, the T cell will no longer produce one or more of the key components of the TCR complex, thereby destabilizing the TCR complex and preventing cell surface expression of a functional TCR.

[0162] In some instances, CAR may also comprise a switch mechanism for controlling expression and / or activation of the CAR. For example, a CAR may comprise an extracellular, transmembrane, and intracellular domain, in which the extracellular domain comprises a targetspecific binding element that comprises a label, binding domain, or tag that is specific for a molecule other than the target antigen that is expressed on or by a target cell. In such embodiments, the specificity of the CAR is provided by a second construct that comprises a target antigen binding domain (e.g., an scFv or a bispecific antibody that is specific for both the target antigen and the label or tag on the CAR) and a domain that is recognized by or binds to the label, binding domain, or tag on the CAR. See, e.g., WO 2013 / 044225, WO 2016 / 000304, WO 2015 / 057834, WO 2015 / 057852, WO 2016 / 070061, US 9,233,125, USAttorney Docket 44010.215WO-PCT / / CU243412016 / 0129109. In this way, a T-cell that expresses the CAR can be administered to a subject, but the CAR cannot bind its target antigen until the second composition comprising an antigenspecific binding domain is administered.

[0163] Alternative switch mechanisms include CARs that require multimerization in order to activate their signaling function (see, e.g., US 2015 / 0368342, US 2016 / 0175359, US 2015 / 0368360) and / or an exogenous signal, such as a small molecule drug (US 2016 / 0166613, Yung et al., Science, 2015), in order to elicit a T-cell response. Some CARs may also comprise a “suicide switch” to induce cell death of the CAR T-cells following treatment (Buddee et al., PLoS One, 2013) or to downregulate expression of the CAR following binding to the target antigen (WO 2016 / 011210).

[0164] Alternative techniques may be used to transform target immunoresponsive cells, such as protoplast fusion, lipofection, transfection or electroporation. A wide variety of vectors may be used, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, plasmids or transposons, such as a Sleeping Beauty transposon (see U.S. Patent Nos.6,489,458; 7,148,203; 7,160,682; 7,985,739; 8,227,432), may be used to introduce CARs, for example using 2nd generation antigen-specific CARs signaling through CD3(^ and either CD28 or CD137. Viral vectors may for example include vectors based on HIV, SV40, EBV, HSV or BPV. In certain embodiments, inducible gene switches are used to regulate expression of a CAR or TCR (see, e.g., Chakravarti, Deboki et al. “Inducible Gene Switches with Memory in Human T Cells for Cellular Immunotherapy.” ACS synthetic biology vol. 8,8 (2019): 1744-1754).

[0165] Cells that are targeted for transformation may for example include T cells, natural Killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, human embryonic stem cells, tumor-infiltrating lymphocytes (TIL) or a pluripotent stem cell from which lymphoid cells may be differentiated. T cells expressing a desired CAR may for example be selected through coculture with y-irradiated activating and propagating cells (AaPC), which co-express the cancer antigen and co-stimulatory molecules. The engineered CAR T-cells may be expanded, for example by co-culture on AaPC in presence of soluble factors, such as IL-2 and IL-21. This expansion may for example be carried out so as to provide memory CAR+ T cells (which may for example be assayed by non-enzymatic digital array and / or multi-panel flow cytometry). In this way, CAR T cells may be provided that have specific cytotoxic activity against antigenbearing tumors (optionally in conjunction with production of desired chemokines such asAttorney Docket 44010.215WO-PCT / / CU24341interferon-y). CAR T cells of this kind may for example be used in animal models, for example to treat tumor xenografts.

[0166] In certain embodiments, ACT includes co-transferring CD4+ Thl cells and CD8+ CTLs to induce a synergistic antitumor response (see, e.g., Li et al., Adoptive cell therapy with CD4+ T helper 1 cells and CD8+ cytotoxic T cells enhances complete rejection of an established tumor, leading to generation of endogenous memory responses to non-targeted tumor epitopes. Clin Transl Immunology. 2017 Oct; 6(10): el60).

[0167] In certain embodiments, ACT may include autologous iPSC-based vaccines, such as irradiated iPSCs in autologous anti-tumor vaccines (see e.g., Kooreman, Nigel G. et al., Autologous iPSC-Based Vaccines Elicit Anti-tumor Responses In Vivo, Cell Stem Cell 22, 1-13, 2018, doi.org / 10.1016 / j. stem.2018.01.016).

[0168] Unlike T-cell receptors (TCRs) that are MHC restricted, CARs can potentially bind any cell surface-expressed antigen and can thus be more universally used to treat patients (see Irving et al., Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don’t Forget the Fuel, Front. Immunol., 03 April 2017, doi.org / 10.3389 / fimmu.2017.00267). In certain embodiments, in the absence of endogenous T-cell infiltrate (e.g., due to aberrant antigen processing and presentation), which precludes the use of TIL therapy and immune checkpoint blockade, the transfer of CAR T-cells may be used to treat patients (see, e.g., Hinrichs CS, Rosenberg SA. Exploiting the curative potential of adoptive T-cell therapy for cancer. Immunol Rev (2014) 257(1):56- 71. doi: 10.1111 / imr.12132).

[0169] Approaches such as the foregoing may be adapted to provide methods of treating and / or increasing survival of a subject having a disease, such as a neoplasia, for example by administering an effective amount of an immunoresponsive cell comprising an antigen recognizing receptor that binds a selected antigen, wherein the binding activates the immunoresponsive cell, thereby treating or preventing the disease (such as a neoplasia, a pathogen infection, an autoimmune disorder, or an allogeneic transplant reaction).

[0170] In certain embodiments, the treatment can be administered after lymphodepleting pretreatment in the form of chemotherapy (typically a combination of cyclophosphamide and fludarabine) or radiation therapy. Initial studies in ACT had short lived responses and the transferred cells did not persist in vivo for very long (Houot et al., T-cell-based immunotherapy: adoptive cell transfer and checkpoint inhibition. Cancer Immunol Res (2015) 3(10): 1115-22; and Kamta et al., Advancing Cancer Therapy with Present and Emerging Immuno-Oncology Approaches. Front. Oncol. (2017) 7:64). Immune suppressor cells likeAttorney Docket 44010.215WO-PCT / / CU24341Tregs and MDSCs may attenuate the activity of transferred cells by outcompeting them for the necessary cytokines. Not being bound by a theory lymphodepleting pretreatment may eliminate the suppressor cells allowing the TILs to persist.

[0171] In one embodiment, the treatment can be administrated into patients undergoing an immunosuppressive treatment (e.g., glucocorticoid treatment). The cells or population of cells may be made resistant to at least one immunosuppressive agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent. In certain embodiments, the immunosuppressive treatment provides for the selection and expansion of the immunoresponsive T cells within the patient.

[0172] In certain embodiments, the treatment can be administered before primary treatment (e.g., surgery or radiation therapy) to shrink a tumor before the primary treatment. In another embodiment, the treatment can be administered after primary treatment to remove any remaining cancer cells.

[0173] In certain embodiments, immunometabolic barriers can be targeted therapeutically prior to and / or during ACT to enhance responses to ACT or CAR T-cell therapy and to support endogenous immunity (see, e.g., Irving et al., Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don’t Forget the Fuel, Front. Immunol., 03 April 2017, doi.org / 10.3389 / fimmu.2017.00267).

[0174] The administration of cells or population of cells, such as immune system cells or cell populations, such as more particularly immunoresponsive cells or cell populations, as disclosed herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The cells or population of cells may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intrathecally, by intravenous or intralymphatic injection, or intraperitoneally. In some embodiments, the disclosed CARs may be delivered or administered into a cavity formed by the resection of tumor tissue (i.e. intracavity delivery) or directly into a tumor prior to resection (i.e. intratumoral delivery). In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection.

[0175] The administration of the cells or population of cells can consist of the administration of 104- 109cells per kg body weight, preferably 105to 106cells / kg body weight including all integer values of cell numbers within those ranges. Dosing in CAR T cell therapies may for example involve administration of from 106to 109cells / kg, with or without a course of lymphodepletion, for example with cyclophosphamide. The cells or population of cells can beAttorney Docket 44010.215WO-PCT / / CU24341administrated in one or more doses. In another embodiment, the effective amount of cells are administrated as a single dose. In another embodiment, the effective amount of cells are administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions are within the skill of one in the art. An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.

[0176] In another embodiment, the effective amount of cells or composition comprising those cells are administrated parenterally. The administration can be an intravenous administration. The administration can be directly done by injection within a tumor.

[0177] To guard against possible adverse reactions, engineered immunoresponsive cells may be equipped with a transgenic safety switch, in the form of a transgene that renders the cells vulnerable to exposure to a specific signal. For example, the herpes simplex viral thymidine kinase (TK) gene may be used in this way, for example by introduction into allogeneic T lymphocytes used as donor lymphocyte infusions following stem cell transplantation (Greco, et al., Improving the safety of cell therapy with the TK-suicide gene. Front. Pharmacol. 2015; 6: 95). In such cells, administration of a nucleoside prodrug such as ganciclovir or acyclovir causes cell death. Alternative safety switch constructs include inducible caspase 9, for example triggered by administration of a small-molecule dimerizer that brings together two nonfunctional icasp9 molecules to form the active enzyme. A wide variety of alternative approaches to implementing cellular proliferation controls have been described (see U.S. Patent Publication No. 20130071414; PCT Patent Publication WO2011146862; PCT Patent Publication W02014011987; PCT Patent Publication W02013040371; Zhou et al. BLOOD, 2014, 123 / 25:3895 - 3905; Di Stasi et al., The New England Journal of Medicine 2011; 365:1673-1683; Sadelain M, The New England Journal of Medicine 2011; 365:1735-173; Ramos et al., Stem Cells 28(6): 1107-15 (2010)).

[0178] In a further refinement of adoptive therapies, genome editing may be used to tailor immunoresponsive cells to alternative implementations, for example providing edited CAR T cells (see Poirot et al., 2015, Multiplex genome edited T-cell manufacturing platform for “off-the-shelf’ adoptive T-cell immunotherapies, Cancer Res 75 (18): 3853; Ren et al., 2017,Attorney Docket 44010.215WO-PCT / / CU24341Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition, Clin Cancer Res. 2017 May l;23(9):2255-2266. doi: 10.1158 / 1078-0432.CCR-16-1300. Epub 2016 Nov 4; Qasim et al., 2017, Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells, Sci Transl Med. 2017 Jan 25;9(374); Legut, et al., 2018, CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells. Blood, 131(3), 311-322; Georgiadis et al., Long Terminal Repeat CRISPR-CAR-Coupled “Universal” T Cells Mediate Potent Anti-leukemic Effects, Molecular Therapy, In Press, Corrected Proof, Available online 6 March 2018; Roth, T.L. Editing of Endogenous Genes in Cellular Immunotherapies. Curr Hematol Malig Rep 15, 235-240 (2020); and Webber BR, Lonetree CL, Kluesner MG, et al. Highly efficient multiplex human T cell engineering without doublestrand breaks using Cas9 base editors [published correction appears in Nat Commun. 2019 Dec 6;10(l):5659. doi: 10.1038 / s41467-019-13778-y], Nat Commun. 2019; 10(l):5222). Cells may be edited using any CRISPR system and method of use thereof as described herein. CRISPR systems may be delivered to an immune cell by any method described herein.

[0179] In preferred embodiments, cells are edited ex vivo and transferred to a subject in need thereof. Immunoresponsive cells, CAR T cells or any cells used for adoptive cell transfer may be edited. Editing may be performed for example to insert or knock-in an exogenous gene, such as an exogenous gene encoding a CAR or a TCR, at a preselected locus in a cell (e.g. TRAC locus); to eliminate potential alloreactive T-cell receptors (TCR) or to prevent inappropriate pairing between endogenous and exogenous TCR chains, such as to knock-out or knock-down expression of an endogenous TCR in a cell; to disrupt the target of a chemotherapeutic agent in a cell; to block an immune checkpoint, such as to knock-out or knock-down expression of an immune checkpoint protein or receptor in a cell; to knock-out or knock-down expression of other gene or genes in a cell, the reduced expression or lack of expression of which can enhance the efficacy of adoptive therapies using the cell; to knock-out or knock-down expression of an endogenous gene in a cell, said endogenous gene encoding an antigen targeted by an exogenous CAR or TCR; to knock-out or knock-down expression of one or more MHC constituent proteins in a cell; to activate a T cell; to modulate cells such that the cells are resistant to exhaustion or dysfunction; and / or increase the differentiation and / or proliferation of functionally exhausted or dysfunctional CD8+ T-cells (see PCT Patent Publications: WO2013176915, WO2014059173, WO2014172606, WO2014184744, and WO2014191128).

[0180] In certain embodiments, editing may result in inactivation of a gene. By inactivating a gene, it is intended that the gene of interest is not expressed in a functional protein form. In aAttorney Docket 44010.215WO-PCT / / CU24341particular embodiment, the CRISPR system specifically catalyzes cleavage in one targeted gene thereby inactivating said targeted gene. The nucleic acid strand breaks caused are commonly repaired through the distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ). However, NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the site of the cleavage. Repair via non-homologous end joining (NHEJ) often results in small insertions or deletions (Indel) and can be used for the creation of specific gene knockouts. Cells in which a cleavage induced mutagenesis event has occurred can be identified and / or selected by well-known methods in the art. In certain embodiments, homology directed repair (HDR) is used to concurrently inactivate a gene (e.g., TRAC) and insert an endogenous TCR or CAR into the inactivated locus.

[0181] Hence, in certain embodiments, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to insert or knock-in an exogenous gene, such as an exogenous gene encoding a CAR or a TCR, at a preselected locus in a cell. Conventionally, nucleic acid molecules encoding CARs or TCRs are transfected or transduced to cells using randomly integrating vectors, which, depending on the site of integration, may lead to clonal expansion, oncogenic transformation, variegated transgene expression and / or transcriptional silencing of the transgene. Directing of transgene(s) to a specific locus in a cell can minimize or avoid such risks and advantageously provide for uniform expression of the transgene(s) by the cells. Without limitation, suitable ‘safe harbor’ loci for directed transgene integration include CCR5 or AAVS1. Homology-directed repair (HDR) strategies are known and described elsewhere in this specification allowing to insert transgenes into desired loci (e.g., TRAC locus).

[0182] Further suitable loci for insertion of transgenes, in particular CAR or exogenous TCR transgenes, include without limitation loci comprising genes coding for constituents of endogenous T-cell receptor, such as T-cell receptor alpha locus (TRA) or T-cell receptor beta locus (TRB), for example T-cell receptor alpha constant (TRAC) locus, T-cell receptor beta constant 1 (TRBC1) locus or T-cell receptor beta constant 2 (TRBC1) locus. Advantageously, insertion of a transgene into such locus can simultaneously achieve expression of the transgene, potentially controlled by the endogenous promoter, and knock-out expression of the endogenous TCR. This approach has been exemplified in Eyquem et al., (2017) Nature 543: 113-117, wherein the authors used CRISPR / Cas9 gene editing to knock-in a DNA molecule encoding a CD19-specific CAR into the TRAC locus downstream of the endogenous promoter;Attorney Docket 44010.215WO-PCT / / CU24341the CAR-T cells obtained by CRISPR were significantly superior in terms of reduced tonic CAR signaling and exhaustion.

[0183] T cell receptors (TCR) are cell surface receptors that participate in the activation of T cells in response to the presentation of antigen. The TCR is generally made from two chains, a and P, which assemble to form a heterodimer and associates with the CD3 -transducing subunits to form the T cell receptor complex present on the cell surface. Each a and P chain of the TCR consists of an immunoglobulin-like N-terminal variable (V) and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region. As for immunoglobulin molecules, the variable region of the a and P chains are generated by V(D)J recombination, creating a large diversity of antigen specificities within the population of T cells. However, in contrast to immunoglobulins that recognize intact antigen, T cells are activated by processed peptide fragments in association with an MHC molecule, introducing an extra dimension to antigen recognition by T cells, known as MHC restriction. Recognition of MHC disparities between the donor and recipient through the T cell receptor leads to T cell proliferation and the potential development of graft versus host disease (GVHD). The inactivation of TCRa or TCRP can result in the elimination of the TCR from the surface of T cells preventing recognition of alloantigen and thus GVHD. However, TCR disruption generally results in the elimination of the CD3 signaling component and alters the means of further T cell expansion.

[0184] Hence, in certain embodiments, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to knock-out or knock-down expression of an endogenous TCR in a cell. For example, NHEJ-based or HDR-based gene editing approaches can be employed to disrupt the endogenous TCR alpha and / or beta chain genes. For example, gene editing system or systems, such as CRISPR / Cas system or systems, can be designed to target a sequence found within the TCR beta chain conserved between the beta 1 and beta 2 constant region genes (TRBC1 and TRBC2) and / or to target the constant region of the TCR alpha chain (TRAC) gene.

[0185] Allogeneic cells are rapidly rejected by the host immune system. It has been demonstrated that, allogeneic leukocytes present in non-irradiated blood products will persist for no more than 5 to 6 days (Boni, Muranski et al. 2008 Blood 1;112(12):4746-54). Thus, to prevent rejection of allogeneic cells, the host's immune system usually has to be suppressed to some extent. However, in the case of adoptive cell transfer the use of immunosuppressive drugs also have a detrimental effect on the introduced therapeutic T cells. Therefore, to effectivelyAttorney Docket 44010.215WO-PCT / / CU24341use an adoptive immunotherapy approach in these conditions, the introduced cells would need to be resistant to the immunosuppressive treatment. Thus, in a particular embodiment, the present invention further comprises a step of modifying T cells to make them resistant to an immunosuppressive agent, preferably by inactivating at least one gene encoding a target for an immunosuppressive agent. An immunosuppressive agent is an agent that suppresses immune function by one of several mechanisms of action. An immunosuppressive agent can be, but is not limited to a calcineurin inhibitor, a target of rapamycin, an interleukin-2 receptor a-chain blocker, an inhibitor of inosine monophosphate dehydrogenase, an inhibitor of dihydrofolic acid reductase, a corticosteroid or an immunosuppressive antimetabolite. The present invention allows conferring immunosuppressive resistance to T cells for immunotherapy by inactivating the target of the immunosuppressive agent in T cells. As non-limiting examples, targets for an immunosuppressive agent can be a receptor for an immunosuppressive agent such as: CD52, glucocorticoid receptor (GR), a FKBP family gene member and a cyclophilin family gene member.

[0186] In certain embodiments, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to block an immune checkpoint, such as to knock-out or knock-down expression of an immune checkpoint protein or receptor in a cell. Immune checkpoints are inhibitory pathways that slow down or stop immune reactions and prevent excessive tissue damage from uncontrolled activity of immune cells. In certain embodiments, the immune checkpoint targeted is the programmed death-1 (PD-1 or CD279) gene (PDCD1) (see, e.g., Rupp LJ, Schumann K, Roybal KT, et al. CRISPR / Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci Rep. 2017;7(l):737). In other embodiments, the immune checkpoint targeted is cytotoxic T-lymphocyte-associated antigen (CTLA-4). In additional embodiments, the immune checkpoint targeted is another member of the CD28 and CTLA4 Ig superfamily such as BTLA, LAG3, ICOS, PDL1 or KIR. In further additional embodiments, the immune checkpoint targeted is a member of the TNFR superfamily such as CD40, 0X40, CD 137, GITR, CD27 or TIM-3.

[0187] Additional immune checkpoints include Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) (Watson HA, et al., SHP-1: the next checkpoint target for cancer immunotherapy? Biochem Soc Trans. 2016 Apr 15;44(2):356-62). SHP-1 is a widely expressed inhibitory protein tyrosine phosphatase (PTP). In T-cells, it is a negative regulator of antigen-dependent activation and proliferation. It is a cytosolic protein, and therefore notAttorney Docket 44010.215WO-PCT / / CU24341amenable to antibody-mediated therapies, but its role in activation and proliferation makes it an attractive target for genetic manipulation in adoptive transfer strategies, such as chimeric antigen receptor (CAR) T cells. Immune checkpoints may also include T cell immunoreceptor with Ig and ITIM domains (TIGIT / Vstm3 / WUCAM / VSIG9) and VISTA (Le Mercier I, et al., (2015) Beyond CTLA-4 and PD-1, the generation Z of negative checkpoint regulators. Front. Immunol. 6:418).

[0188] WO2014172606 relates to the use of MT1 and / or MT2 inhibitors to increase proliferation and / or activity of exhausted CD8+ T-cells and to decrease CD8+ T-cell exhaustion (e.g., decrease functionally exhausted or unresponsive CD8+ immune cells). In certain embodiments, metallothioneins are targeted by gene editing in adoptively transferred T cells.

[0189] In certain embodiments, targets of gene editing may be at least one targeted locus involved in the expression of an immune checkpoint protein. Such targets may include, but are not limited to CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, ICOS (CD278), PDL1, KIR, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244 (2B4), TNFRSF10B, TNFRSF10A, CASP8, C ASP 10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HM0X2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, VISTA, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, MT1, MT2, CD40, 0X40, CD 137, GITR, CD27, SHP-1, TIM-3, CEACAM-1, CE AC AM-3, or CEACAM-5. In preferred embodiments, the gene locus involved in the expression of PD-1 or CTLA-4 genes is targeted. In other preferred embodiments, combinations of genes are targeted, such as but not limited to PD-1 and TIGIT.

[0190] By means of an example and without limitation, WO2016196388 concerns an engineered T cell comprising (a) a genetically engineered antigen receptor that specifically binds to an antigen, which receptor may be a CAR; and (b) a disrupted gene encoding a PD-Ll, an agent for disruption of a gene encoding a PD- LI, and / or disruption of a gene encoding PD-L1, wherein the disruption of the gene may be mediated by a gene editing nuclease, a zinc finger nuclease (ZFN), CRISPR / Cas9 and / or TALEN. WO2015142675 relates to immune effector cells comprising a CAR in combination with an agent (such as CRISPR, TALEN or ZFN) that increases the efficacy of the immune effector cells in the treatment of cancer, wherein the agent may inhibit an immune inhibitory molecule, such as PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFR beta, CEACAM-1, CEACAM-Attorney Docket 44010.215WO-PCT / / CU243413, or CEACAM-5. Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266 performed lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, P-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.

[0191] In certain embodiments, cells may be engineered to express a CAR, wherein expression and / or function of methylcytosine dioxygenase genes (TET1, TET2 and / or TET3) in the cells has been reduced or eliminated, such as by CRISPR, ZNF or TALEN (for example, as described in WO201704916).

[0192] In certain embodiments, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to knock-out or knock-down expression of an endogenous gene in a cell, said endogenous gene encoding an antigen targeted by an exogenous CAR or TCR, thereby reducing the likelihood of targeting of the engineered cells. In certain embodiments, the targeted antigen may be one or more antigen selected from the group consisting of CD38, CD138, CS-1, CD33, CD26, CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, CD362, human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2 / neu, Wilms’ tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, cyclin (DI), B cell maturation antigen (BCMA), transmembrane activator and CAML Interactor (TACI), and B-cell activating factor receptor (BAFF-R) (for example, as described in W02016011210 and WO2017011804).

[0193] In certain embodiments, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to knock-out or knock-down expression of one or more MHC constituent proteins, such as one or more HLA proteins and / or beta-2 microglobulin (B2M), in a cell, whereby rejection of non-autologous (e.g., allogeneic) cells by the recipient’s immune system can be reduced or avoided. In preferred embodiments, one or more HLA class I proteins, such as HLA-A, B and / or C, and / or B2M may be knocked-out or knocked-down. Preferably, B2M may be knocked-out or knocked-down. By means of an example, Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266 performed lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, P-2 microglobulin (B2M) and PD1Attorney Docket 44010.215WO-PCT / / CU24341simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.

[0194] In other embodiments, at least two genes are edited. Pairs of genes may include, but are not limited to PD1 and TCRa, PD1 and TCRp, CTLA-4 and TCRa, CTLA-4 and TCRp, LAG3 and TCRa, LAG3 and TCRP, Tim3 and TCRa, Tim3 and TCRP, BTLA and TCRa, BTLA and TCRP, BY55 and TCRa, BY55 and TCRp, TIGIT and TCRa, TIGIT and TCRp, B7H5 and TCRa, B7H5 and TCRp, LAIR1 and TCRa, LAIR1 and TCRp, SIGLEC10 and TCRa, SIGLEC10 and TCRp, 2B4 and TCRa, 2B4 and TCRp, B2M and TCRa, B2M and TCRp.

[0195] In certain embodiments, a cell may be multiply edited (multiplex genome editing) as taught herein to (1) knock-out or knock-down expression of an endogenous TCR (for example, TRBC1, TRBC2 and / or TRAC), (2) knock-out or knock-down expression of an immune checkpoint protein or receptor (for example PD1, PD-L1 and / or CTLA4); and (3) knock-out or knock-down expression of one or more MHC constituent proteins (for example, HLA-A, B and / or C, and / or B2M, preferably B2M).

[0196] Whether prior to or after genetic modification of the T cells, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631. T cells can be expanded in vitro or in vivo.

[0197] Immune cells may be obtained using any method known in the art. In one embodiment, allogenic T cells may be obtained from healthy subjects. In one embodiment T cells that have infiltrated a tumor are isolated. T cells may be removed during surgery. T cells may be isolated after removal of tumor tissue by biopsy. T cells may be isolated by any means known in the art. In one embodiment, T cells are obtained by apheresis. In one embodiment, the method may comprise obtaining a bulk population of T cells from a tumor sample by any suitable method known in the art. For example, a bulk population of T cells can be obtained from a tumor sample by dissociating the tumor sample into a cell suspension from which specific cell populations can be selected. Suitable methods of obtaining a bulk population of T cells may include, but are not limited to, any one or more of mechanically dissociating (e.g., mincing) the tumor, enzymatically dissociating (e.g., digesting) the tumor, and aspiration (e.g., as with a needle).Attorney Docket 44010.215WO-PCT / / CU24341

[0198] The bulk population of T cells obtained from a tumor sample may comprise any suitable type of T cell. Preferably, the bulk population of T cells obtained from a tumor sample comprises tumor infiltrating lymphocytes (TILs).

[0199] The tumor sample may be obtained from any mammal. Unless stated otherwise, as used herein, the term “mammal” refers to any mammal including, but not limited to, mammals of the order Logomorpha, such as rabbits; the order Carnivora, including Felines (cats) and Canines (dogs); the order Artiodactyla, including Bovines (cows) and Swines (pigs); or of the order Perssodactyla, including Equines (horses). The mammals may be non-human primates, e.g., of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In some embodiments, the mammal may be a mammal of the order Rodentia, such as mice and hamsters. Preferably, the mammal is a non-human primate or a human. An especially preferred mammal is the human.

[0200] T cells and NK cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, spleen tissue, and tumors. In certain embodiments of the present invention, T cells and NK cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll separation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS).

[0201] In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.Attorney Docket 44010.215WO-PCT / / CU24341

[0202] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. A specific subpopulation of T cells, such as CD28+, CD4+, CDC, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one preferred embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3*28)-conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, or XCYTE DYNABEADS™ for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In one preferred embodiment, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells.

[0203] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CDllb, CD16, HLA-DR, and CD8.

[0204] Further, monocyte populations (i.e., CD14+ cells) may be depleted from blood preparations by a variety of methodologies, including anti-CD14 coated beads or columns, or utilization of the phagocytotic activity of these cells to facilitate removal. Accordingly, in one embodiment, the invention uses paramagnetic particles of a size sufficient to be engulfed by phagocytotic monocytes. In certain embodiments, the paramagnetic particles are commercially available beads, for example, those produced by Life Technologies under the trade name Dynabeads™. In one embodiment, other non-specific cells are removed by coating the paramagnetic particles with “irrelevant” proteins (e.g., serum proteins or antibodies). Irrelevant proteins and antibodies include those proteins and antibodies or fragments thereof that do notAttorney Docket 44010.215WO-PCT / / CU24341specifically target the T cells to be isolated. In certain embodiments, the irrelevant beads include beads coated with sheep anti-mouse antibodies, goat anti-mouse antibodies, and human serum albumin.

[0205] In brief, such depletion of monocytes is performed by preincubating T cells isolated from whole blood, apheresed peripheral blood, or tumors with one or more varieties of irrelevant or non-antibody coupled paramagnetic particles at any amount that allows for removal of monocytes (approximately a 20:1 bead:cell ratio) for about 30 minutes to 2 hours at 22 to 37 degrees C., followed by magnetic removal of cells which have attached to or engulfed the paramagnetic particles. Such separation can be performed using standard methods available in the art. For example, any magnetic separation methodology may be used including a variety of which are commercially available, (e.g., DYNAL® Magnetic Particle Concentrator (DYNAL MPC®)). Assurance of requisite depletion can be monitored by a variety of methodologies known to those of ordinary skill in the art, including flow cytometric analysis of CD14 positive cells, before and after depletion.

[0206] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[0207] In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads),Attorney Docket 44010.215WO-PCT / / CU24341interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one embodiment, the concentration of cells used is 5><106 / ml. In other embodiments, the concentration used can be from about 1 x 105 / ml to 1 x 106 / ml, and any integer value in between.

[0208] T cells can also be frozen. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media, the cells then are frozen to -80° C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C. or in liquid nitrogen.

[0209] T cells for use in the present invention may also be antigen-specific T cells. For example, tumor-specific T cells can be used. In certain embodiments, antigen-specific T cells can be isolated from a patient of interest, such as a patient afflicted with a cancer or an infectious disease. In one embodiment, neoepitopes are determined for a subject and T cells specific to these antigens are isolated. Antigen-specific cells for use in expansion may also be generated in vitro using any number of methods known in the art, for example, as described in U.S. Patent Publication No. US 20040224402 entitled, Generation and Isolation of Antigen-Specific T Cells, or in U.S. Pat. Nos. 6,040,177. Antigen-specific cells for use in the present invention may also be generated using any number of methods known in the art, for example, as described in Current Protocols in Immunology, or Current Protocols in Cell Biology, both published by John Wiley & Sons, Inc., Boston, Mass.

[0210] In a related embodiment, it may be desirable to sort or otherwise positively select (e.g. via magnetic selection) the antigen specific cells prior to or following one or two rounds of expansion. Sorting or positively selecting antigen-specific cells can be carried out using peptide-MHC tetramers (Altman, et al., Science. 1996 Oct. 4; 274(5284): 94-6). In another embodiment, the adaptable tetramer technology approach is used (Andersen et al., 2012 Nat Protoc. 7:891-902). Tetramers are limited by the need to utilize predicted binding peptides based on prior hypotheses, and the restriction to specific HLAs. Peptide-MHC tetramers canAttorney Docket 44010.215WO-PCT / / CU24341be generated using techniques known in the art and can be made with any MHC molecule of interest and any antigen of interest as described herein. Specific epitopes to be used in this context can be identified using numerous assays known in the art. For example, the ability of a polypeptide to bind to MHC class I may be evaluated indirectly by monitoring the ability to promote incorporation of125I labeled P2-microglobulin (P2m) into MHC class I / p2m / peptide heterotrimeric complexes (see Parker et al., J. Immunol. 152:163, 1994).

[0211] In one embodiment cells are directly labeled with an epitope-specific reagent for isolation by flow cytometry followed by characterization of phenotype and TCRs. In one embodiment, T cells are isolated by contacting with T cell specific antibodies. Sorting of antigen-specific T cells, or generally any cells of the present invention, can be carried out using any of a variety of commercially available cell sorters, including, but not limited to, MoFlo sorter (DakoCytomation, Fort Collins, Colo.), FACSAria™, FACSArray™, FACSVantage™, BD™ LSR II, and FACSCalibur™ (BD Biosciences, San Jose, Calif.).

[0212] In a preferred embodiment, the method comprises selecting cells that also express CD3.The method may comprise specifically selecting the cells in any suitable manner. Preferably, the selecting is carried out using flow cytometry. The flow cytometry may be carried out using any suitable method known in the art. The flow cytometry may employ any suitable antibodies and stains. Preferably, the antibody is chosen such that it specifically recognizes and binds to the particular biomarker being selected. For example, the specific selection of CD3, CD8, TIM-3, LAG-3, 4-1BB, or PD-1 may be carried out using anti-CD3, anti-CD8, anti-TIM-3, anti-LAG-3, anti-4-lBB, or anti-PD-1 antibodies, respectively. The antibody or antibodies may be conjugated to a bead (e.g., a magnetic bead) or to a fluorochrome. Preferably, the flow cytometry is fluorescence-activated cell sorting (FACS). TCRs expressed on T cells can be selected based on reactivity to autologous tumors. Additionally, T cells that are reactive to tumors can be selected for based on markers using the methods described in patent publication Nos. WO2014133567 and WO2014133568, herein incorporated by reference in their entirety. Additionally, activated T cells can be selected for based on surface expression of CD 107a.

[0213] In one embodiment of the invention, the method further comprises expanding the numbers of T cells in the enriched cell population. Such methods are described in U.S. Patent No. 8,637,307 and is herein incorporated by reference in its entirety. The numbers of T cells may be increased at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold), more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold), more preferably at least about 100-fold, more preferably at least about 1,000 fold, or most preferably at least about 100,000-Attorney Docket 44010.215WO-PCT / / CU24341fold. The numbers of T cells may be expanded using any suitable method known in the art. Exemplary methods of expanding the numbers of cells are described in patent publication No. WO 2003057171, U.S. Patent No. 8,034,334, and U.S. Patent Application Publication No.2012 / 0244133, each of which is incorporated herein by reference.

[0214] In one embodiment, ex vivo T cell expansion can be performed by isolation of T cells and subsequent stimulation or activation followed by further expansion. In one embodiment of the invention, the T cells may be stimulated or activated by a single agent. In another embodiment, T cells are stimulated or activated with two agents, one that induces a primary signal and a second that is a co-stimulatory signal. Ligands useful for stimulating a single signal or stimulating a primary signal and an accessory molecule that stimulates a second signal may be used in soluble form. Ligands may be attached to the surface of a cell, to an Engineered Multivalent Signaling Platform (EMSP), or immobilized on a surface. In a preferred embodiment both primary and secondary agents are co-immobilized on a surface, for example a bead or a cell. In one embodiment, the molecule providing the primary activation signal may be a CD3 ligand, and the co-stimulatory molecule may be a CD28 ligand or 4- IBB ligand.

[0215] In certain embodiments, T cells comprising a CAR or an exogenous TCR, may be manufactured as described in W02015120096, by a method comprising: enriching a population of lymphocytes obtained from a donor subject; stimulating the population of lymphocytes with one or more T-cell stimulating agents to produce a population of activated T cells, wherein the stimulation is performed in a closed system using serum-free culture medium; transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule which encodes the CAR or TCR, using a single cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; and expanding the population of transduced T cells for a predetermined time to produce a population of engineered T cells, wherein the expansion is performed in a closed system using serum-free culture medium. In certain embodiments, T cells comprising a CAR or an exogenous TCR, may be manufactured as described in W02015120096, by a method comprising: obtaining a population of lymphocytes; stimulating the population of lymphocytes with one or more stimulating agents to produce a population of activated T cells, wherein the stimulation is performed in a closed system using serum-free culture medium; transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule which encodes the CAR or TCR, using at least one cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closedAttorney Docket 44010.215WO-PCT / / CU24341system using serum-free culture medium; and expanding the population of transduced T cells to produce a population of engineered T cells, wherein the expansion is performed in a closed system using serum-free culture medium.

[0216] The predetermined time for expanding the population of transduced T cells may be 3 days. The time from enriching the population of lymphocytes to producing the engineered T cells may be 6 days. The closed system may be a closed bag system. Further provided is population of T cells comprising a CAR or an exogenous TCR obtainable or obtained by said method, and a pharmaceutical composition comprising such cells.

[0217] In certain embodiments, T cell maturation or differentiation in vitro may be delayed or inhibited by the method as described in W02017070395, comprising contacting one or more T cells from a subject in need of a T cell therapy with an AKT inhibitor (such as, e.g., one or a combination of two or more AKT inhibitors disclosed in claim 8 of W02017070395) and at least one of exogenous Interleukin-7 (IL-7) and exogenous Interleukin- 15 (IL- 15), wherein the resulting T cells exhibit delayed maturation or differentiation, and / or wherein the resulting T cells exhibit improved T cell function (such as, e.g., increased T cell proliferation; increased cytokine production; and / or increased cytolytic activity) relative to a T cell function of a T cell cultured in the absence of an AKT inhibitor.

[0218] In certain embodiments, a patient in need of a T cell therapy may be conditioned by a method as described in WO2016191756 comprising administering to the patient a dose of cyclophosphamide between 200 mg / m2 / day and 2000 mg / m2 / day and a dose of fludarabine between 20 mg / m2 / day and 900 mg / m2 / day.

[0219] In certain embodiments, a patient in need of adoptive cell transfer may be administered a TLR agonist to enhance anti-tumor immunity (see, e.g., Urban-Wojciuk, et al., The Role of TLRs in Anti-cancer Immunity and Tumor Rejection, Front Immunol. 2019; 10: 2388; and Kaczanowska et al., TLR agonists: our best frenemy in cancer immunotherapy, J Leukoc Biol.2013 Jun; 93(6): 847-863). In certain embodiments, TLR agonists are delivered in a nanoparticle system (see, e.g., Buss and Bhatia, Nanoparticle delivery of immunostimulatory oligonucleotides enhances response to checkpoint inhibitor therapeutics, Proc Natl Acad Sci USA. 2020 Jun 3;202001569). In certain embodiments, the agonist is a TLR9 agonist. Id.

[0220] The invention is further described by the following numbered paragraphs:1. A fusion protein for targeting lytic granules (LGs) to the plasma membrane of a cytotoxic lymphocyte, said fusion protein comprising: a plasma membrane directing peptide;Attorney Docket 44010.215WO-PCT / / CU24341and a LG associated peptide comprising a structural component of LGs or a transient adaptor peptide linked to LGs, wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell.2. The fusion protein of paragraph 1, wherein the plasma membrane directing peptide comprises hydrophobic amino acids and / or is capable of being post translationally modified with hydrophobic-related moieties.3. The fusion protein of paragraph 2, wherein the hydrophobic-related moieties comprise hydrophobic carbon chains.4. The fusion protein of paragraph 3, wherein the hydrophobic carbon chains comprise fatty acids.5. The fusion protein of any of paragraphs 2-4, wherein the plasma membrane directing peptide comprises a CAAX motif.6. The fusion protein of paragraph 5, wherein the CAAX motif is derived from the C terminal region of KRAS.7. The fusion protein of paragraph 1, wherein the plasma membrane directing peptide comprises a C-terminal peptide from a Type IV transmembrane domain.8. The fusion protein of paragraph 1, wherein the plasma membrane directing peptide comprises a C-terminal transmembrane domain from a syntaxin family member.9. The fusion protein of paragraph 8, wherein syntaxin family member is syntaxin 1A (STX1).10. The fusion protein of paragraph 1, wherein the plasma membrane directing peptide comprises a signal-anchor sequence from a Type II transmembrane (TII) protein.11. The fusion protein of paragraph 1, wherein the plasma membrane directing peptide comprises an N-terminal peptide from a Type II transmembrane (TII) protein.12. The fusion protein of paragraph 1, wherein the plasma membrane directing peptide comprises an N-terminal domain of a Src Family Kinase (SFK).13. The fusion protein of paragraph 12, wherein the SFK is selected from the group consisting of Src, Yes, Fyn, Fgr, Lek, Hck, Blk, Lyn, and Frk.14. The fusion protein of paragraph 1, wherein the plasma membrane directing peptide comprises a transmembrane and / or juxtamembrane region of LAT (Linker for Activation of T cells).15. The fusion protein of any of paragraphs 1-14, wherein the structural component of the LG comprises a transmembrane domain from an LG expressed protein.Attorney Docket 44010.215WO-PCT / / CU2434116. The fusion protein of paragraph 15, wherein the transmembrane domain comprises the transmembrane domain from LAMP1, LAMP2, LAMP3, CD63, CD68, NKG7, or functional fragments or variants thereof.17. The fusion protein of any one of paragraphs 1-16, wherein the structural component of the LG comprises a transmembrane domain comprising a YXX motif.18. The fusion protein of paragraph 17, wherein the YXX motif is either N- or C- terminal of the transmembrane domain.19. The fusion protein of any one of paragraphs 1-14, wherein the transient adaptor peptide linked to LGs is derived from a protein associated with LG tethering.20. The fusion protein of paragraph 19, wherein the protein associated with LG tethering is GCC2, Golgin-97 (G-97), a coiled-coil domain that binds an LG associated coiled-coil domain, or a truncated GCC2 lacking the C-terminal GRIP domain.21. The fusion protein of paragraph 20, wherein the coiled-coil domain that binds an LG associated coiled-coil domain is the coiled-coil domain 2 (CC2) of GCC2.22. The fusion protein of any one of paragraphs 1-21, wherein the fusion protein further comprises a C-terminal signal peptide that sorts the fusion protein to the LG and is derived from a protein specifically expressed on LGs.23. The fusion protein of paragraph 22, wherein the signal peptide is derived from an LG protein selected from the group consisting of perforin, a granzyme, and NKG7.23. The fusion protein of any one of paragraphs 1-23, wherein the plasma membrane directing peptide is a CAAX motif and the LG associated peptide is GCC2 or Golgin-97. 24. The fusion protein of any one of paragraphs 1-23, wherein the plasma membrane directing peptide is a CAAX motif and the LG associated peptide is a LAMP1 transmembrane domain.25. The fusion protein of any one of paragraphs 1-23, wherein the plasma membrane directing peptide is a STX1 motif and the LG associated peptide is a LAMP1 transmembrane domain.26. The fusion protein of any one of paragraphs 1-23, wherein the plasma membrane directing peptide is an N-terminal domain of a Src Family Kinase (SFK), the LG associated peptide is the N-terminal domain of NKG7, and the C-terminal signal peptide that sorts the fusion protein to the LG is derived from perforin, NKG7, or a granzyme.27. The fusion protein of any one of paragraphs 1-23, wherein the plasma membrane directing peptide is an N-terminal domain of Lek, the LG associated peptide is the N-terminalAttorney Docket 44010.215WO-PCT / / CU24341domain of NKG7, and the C-terminal signal peptide that sorts the fusion protein to the LG is derived from perforin (SEQ ID NO: 48).28. The fusion protein of any one of paragraphs 1-23, wherein the plasma membrane directing peptide is a transmembrane and / or juxtamembrane region of LAT, and the LG associated peptide is the N-terminal domain of NKG7 (SEQ ID NO: 49).29. The fusion protein of any one of paragraphs 1-23, wherein the plasma membrane directing peptide is a transmembrane and / or juxtamembrane region of LAT, the LG associated peptide is the N-terminal domain of NKG7, and the C-terminal signal peptide that sorts the fusion protein to the LG is derived from perforin (SEQ ID NO: 50).30. The fusion protein of any one of paragraphs 1-23, wherein the plasma membrane directing peptide is a is a CAAX motif and the LG associated peptide is a coiled-coil domain that binds an LG associated coiled-coil domain.31. The fusion protein of paragraph 30, wherein the coiled-coil domain that binds an LG associated coiled-coil domain is the coiled-coil domain 2 (CC2) of GCC2 (SEQ ID NO: 51).32. A fusion protein for targeting lytic granules (LGs) comprising:a) a LG associated peptide comprising a structural component of LGs;b) a protein of interest; andc) a signal peptide that sorts the fusion protein to the LG.33. The fusion protein of paragraph 32, wherein the structural component of LGs is derived from LAMP1, NKG7, or any transmembrane domain flanked by a YXX<b motif.34. The fusion protein of paragraph 32 or 33, wherein the protein of interest is a detectable marker, therapeutic protein, or toxic protein.35. The fusion protein of paragraph 34, wherein the detectable marker is a fluorescent protein.36. The fusion protein of any of paragraphs 32-35, wherein the signal peptide is derived from an LG protein selected from the group consisting of perforin, a granzyme, and NKG7.37. The fusion protein of any of paragraphs 32-36, wherein the fusion protein further comprises a plasma membrane directing peptide.38. The fusion protein of any of paragraphs 32-37, wherein the fusion protein comprises the N-terminal domain of NKG7, the protein of interest, and a C-terminal domain from perforin, NKG7, or a granzyme.39. One or more vectors encoding the fusion protein according to any one of paragraphs 1- 38.Attorney Docket 44010.215WO-PCT / / CU2434140. A system for targeting lytic granules (LGs) to the plasma membrane of a cytotoxic lymphocyte comprising: a first fusion protein comprising an LG associated coiled-coil peptide and a signal peptide that sorts the fusion protein to the LG and is derived from a protein specifically expressed on LGs, optionally, coiled-coil domain 2 (CC2) of GCC2; and a second fusion protein comprising a transmembrane domain specific to the plasma membrane and a coiled-coil containing protein capable of binding the first fusion protein, wherein the first and second fusion proteins are encoded for by one or more vectors, wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell.41. An isolated cytotoxic lymphocyte modified to express the fusion protein of any of paragraphs 1-38, wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell, optionally, wherein expression of the fusion protein is inducible.42. An isolated cytotoxic lymphocyte genetically modified to delete GCC2, wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell.43. An isolated cytotoxic lymphocyte genetically modified to overexpress one or more exophilin proteins selected from the group consisting of SYTL-1, SYTL-2, SYTL-2, SYTL-3, SYTL4, exophilin-5, and melanophilin (MLPH), wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell.44. An isolated cytotoxic lymphocyte modified to express the system of paragraph 40, wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell.45. A method of targeting lytic granules (LGs) to the plasma membrane of a cytotoxic lymphocyte to increase cytotoxicity, increase bystander killing, and / or enhance anti-tumor activity, the method comprising anchoring LGs to the plasma membrane using plasma membrane-directing peptides that bind directly or indirectly to the LG, wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell.46. The method of paragraph 45, comprising expressing the fusion protein according to any one of paragraphs 1-38 in the cytotoxic lymphocyte, thereby anchoring LGs to the plasma membrane.Attorney Docket 44010.215WO-PCT / / CU2434147. A method of targeting lytic granules (LGs) to the plasma membrane of a cytotoxic lymphocyte to increase cytotoxicity, increase bystander killing, and / or enhance anti-tumor activity, the method comprising anchoring LGs to the plasma membrane by overexpressing one or more exophilin proteins, wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell.48. The method of paragraph 47, wherein the one or more exophilin proteins are selected from the group consisting of SYTL-1, SYTL-2, SYTL-3, SYTL4, exophilin-5, and melanophilin (MLPH).49. A method of treating cancer in a subject in need thereof comprising administering the isolated cytotoxic lymphocyte according to any of paragraphs 41-44 to the tumor of the subject.50. The method of paragraph 49, wherein the isolated cytotoxic lymphocyte expresses an exogenous T cell receptor (TCR) or a chimeric antigen receptor (CAR).51. A method of treating cancer in a subject in need thereof comprising administering lipid nanoparticles carrying modified mRNA targeting cytotoxic lymphocytes in vivo and encoding the fusion protein or system of any one of paragraphs 1-40.

[0221] Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the invention.EXAMPLES EXAMPLE 1 - THE GOLGI COMPLEX GOVERNS NATURAL KILLER CELL LYTIC GRANULE POSITIONING TO PROMOTE DIRECTIONALITY IN CYTOTOXICITY

[0222] This example shows that the Golgi serves as a regulator of lytic granule convergence in natural killer cells, GCC2 is present on LGs and helps them tether to the Golgi to sustain convergence, KO of GCC2 results in LG dispersion, non-directional degranulation, and bystander killing, and humans with biallelic GCC2 missense variants have NK deficiency. Thus, blocking convergence during NK cell activation results in LG to be dispersed in the cytoplasm. These dispersed LG are released outside the context of the IS and consequently induce bystander killing.

[0223] Cytotoxic immune cells mediate precise attacks against diseased cells to maintain organismal health. Their operational unit of killing and host defense is lytic granules (LGs), which are specialized lysosomal-related organelles. Precision in cytotoxicity is achieved by converging the many LGs to the microtubule-organizing center (MTOC) and polarizing theseAttorney Docket 44010.215WO-PCT / / CU24341to the diseased cell for secretion. Applicants identify unappreciated intimate relationships between the Golgi, MTOC, and LGs after cytotoxic cell activation, as well as the trans-Golgin protein GCC2 on the LG surface. GCC2 serves to tether LGs to the Golgi following convergence, and both GCC2 and the Golgi are required for the persistence of convergence. GCC2 allows LGs to utilize the Golgi as a docking station preventing LG dispersion and innocent bystander killing in complex three-dimensional environments. Applicants also identify GCC2 variants causing human natural killer cell deficiency, further emphasizing the importance of LG convergence and Golgi linkage in precision targeting for human immunity.

[0224] Specifically, Applicants augmented this observation in NK cells after activation and identified the Golgin family member GCC2 (also known as GCC185) on the surface of the LG as a critical component of the Golgi -LG interaction. Applicants found that GCC2 is required for NK cell LG convergence and promotes their tethering to the Golgi after activation. The loss of GCC2 leads to the dispersion of LGs and non-directional bystander killing. Applicants also identified GCC2 as essential to physiological NK cell function and host defense through its definition as a human NK cell deficiency (NKD) gene. Human biallelic GCC2 variants block LG convergence and reduce directed killing efficiency, thus linking LG convergence to human host defense.ResultsGolgi is associated with LG convergence to the MTOC

[0225] Early electron microscopy studies have demonstrated that the Golgi approximates the IS in cytotoxic cells11Given the robust coordination of the lytic machinery involved in cytotoxic host defense function, Applicants considered the potential for the Golgi as a physical regulator of LG positioning and release. To evaluate an interaction between LGs and the Golgi, the YTS human NK cell line was stably transduced with bl,4-galactosyltransferase, fused with the fluorescent protein mCherry (mCherry-GalT).12In resting cells (adhered using nonactivating anti-CD18 [IB4]) monoclonal antibody-coated glass) (FIG. 8A), LGs were scattered in the cytoplasm having no clear association with the Golgi or the MTOC despite the Golgi and MTOC being in close proximity to each other (FIG. 1A). However, after stimulation with an antiCD18(IB4) / anti-CD28-coated surface (FIG. IB), or 721.221 target cells (FIG.1C), the LG converged to the MTOC (FIG. ID) and occupied the same space as the Golgi (FIG. IE), raising the possibility of a role for the Golgi in LG function.Attorney Docket 44010.215WO-PCT / / CU24341

[0226] To investigate the possibility of a physical interaction between LGs and the Golgi, LGs were purified from YTS cells, and their surface proteins were biotinylated and isolated using streptavidin-coated magnetic beads as previously described.13This isolated set of LG surface proteins was then sequenced using mass spectrometry (FIG. IF; Table 1), and Applicants focused on those known to associate with the Golgi (FIG. IF inset). These included GCC2, known to be involved in retrograde transport of vesicles,14 16which is part of a family of trans-Golgi proteins that includes GCC1, GOLGA1 (Golgin-97), and GOLGA4 (Golgin-245).17GCC2 has no known cargoes.18Table 1. LG surface proteins identified via mass spectrometry, related to FIG. IFAttorney Docket 44010.215WO-PCT / / CU24341

[0227] Although GCC2 is a known / ra / .s-Golgi protein, its interaction with Rab6A and ARL1 leads to its Golgi association being indirect and transient, allowing it to switch between different compartments.14A proposed model is that cytosolic GCC2 interacts with vesicles on microtubules that subsequently tether to the Golgi.14To confirm the presence of GCC2 and other Golgins on LGs independently of the Golgi, western blot analysis was performed using lysates from purified LGs compared to whole YTS cell lysates. The presence of CD107a was used as a control and was enriched in LGs as expected. The four / ra / r.s-Golgin proteins GCC2, GCC1, Golgin-97, and Golgin-245 were evaluated, and while all were found in NK cells, only GCC2 and Golgin-97 were identified with LGs (FIG. 1G). To ensure the lack of Golgi contamination in the isolated LG fraction, western blot analysis for giantin, a c / .s-Golgi protein, was performed and was absent (FIG.8B). Collectively, these data demonstrate the presence of select / ra / r.s-Golgi resident proteins with LGs and raise the question of whether they possess any functional relevance.Attorney Docket 44010.215WO-PCT / / CU24341GCC2 tethers LGs during activation-induced convergence

[0228] A GCC2-deficient cell line was generated in YTS cells using CRISPR-Cas9 editing (YTS GCC2 knockout [KO]) in order to determine the role of GCC2 in NK cells, and the deletion of GCC2 was confirmed by western blot (FIG. 2A) and confocal microscopy (FIG.2B). YTS cells are clonal and have a large number of LGs that are measurably converged after activation and prior to polarization to the IS.7,19This parallels ex vivo NK cell biology and makes YTS cells suitable for the study of LG dynamics. The deletion of GCC2 in YTS cells did not affect the gross morphology of the Golgi (FIG.2B). Their function in directed cytotoxicity against a transformed target cell was overall maintained albeit slightly reduced as measured using a51Crrelease assay (FIG. 2C). To evaluate for a potential explanation for this reduction, the ability of YTS GCC2 KO cells to degranulate after stimulation was measured by flow cytometry using CD107a as a measure of granule release. Interestingly, however, the GCC2-deficient cells demonstrated increased and not decreased degranulation after activation (FIG.2D) This was confirmed using cells expressing a fluorescence-based degranulation indicator (mApple-LAMPl-pHluorin) and measuring the number of degranulation events (pHluorin fluorescence transitions)3on an activating anti-CD18(IB4) / anti-CD28-coated surface (FIG.8D) Thus, directed cytotoxicity in the absence of GCC2 was decreased, but with increased degranulation.

[0229] To explain this unusual combination of behaviors, Applicants considered a decreased efficiency of directed secretion for cytotoxicity that might allow for more degranulation with less targeted killing. Although increased degranulation could suggest a direct role of GCC2 in LG exocytosis, its primary expression on the Golgi suggested a role in earlier events when LGs approach the Golgi, specifically convergence. Given that convergence is a major driver of LG positioning and efficiency in directed synaptic degranulation, Applicants measured convergence to the MTOC in GCC2 KO and parental YTS stimulated on an antiCD 18(IB4) / anti-CD28-activating surface or with 721.221 target cells. Following stimulation, LG convergence was incomplete in YTS GCC2 KO cells, with many remaining dispersed relative to the MTOC (FIGS. 2E and 2F) or the Golgi (FIGS. 2E and 2G). Thus, GCC2 was required for obtaining activation-induced LG convergence and approximation to both the MTOC and the Golgi.

[0230] One function of LG convergence is to prevent non-directional LG release and bystander cell killing. Thus, Applicants next wanted to determine how the dispersed LGs in GCC2 KO cells function in complex three-dimensional environments containing both targeted and non-Attorney Docket 44010.215WO-PCT / / CU24341targeted cells. Applicants utilized a thermal collapse of strata (TheCOS) system to approximate LCdifferent types of cells in specified ratios, quickly upon demand via utilizing cells embedded in layers of temperature-sensitive hydrogels (manuscript in submission). Using TheCOS, YTS parental or GCC2 KO cells embedded in layers could be approximated to triggering 721.221 target cells and non-targeted K562 cells at the same time after the alteration of ambient temperature. To assess the function of the YTS cells against the different 721.221 and K562 cells, a TheCOS stack was digested after 4 h, and individual cells were isolated, stained, and fixed for flow cytometric analysis (FIG.3A). This allows for the identification of the different types of cells after mixing via TheCOS as well as their viability using nuclear dye. As expected, the parental YTS cells were unable to kill the resistant K562 “bystander cells” even in the presence of a majority of 721.221 -triggering target cells (FIG.3B, red). When GCC2 KO cells were evaluated, however, killing of K562 cells was apparent, and increased with increasing percentages of triggering 721.221 cells (FIG. 3B, blue). In parallel and from the same experiment, a slice of an undigested TheCOS gel stack was evaluated using confocal microscopy and demonstrated the intimate contact of the YTS cells with both the 721.221 and K562 cells (FIG. 3C). Furthermore, the LGs, as denoted by Lysotracker staining, were converged in the parental YTS cells but dispersed in the YTS GCC2 KO cells, demonstrating that the convergence and dispersion of LGs were maintained in the NK cells even when in a complex three-dimensional environment. Importantly, the YTS GCC2 KO cells with dispersed LGs were able to mediate off-target killing in a three-dimensional environment and promote the destruction of a bystander cell.

[0231] To evaluate the impact of GCC2-directed LG convergence in a setting more relevant to a human solid tumor, Applicants performed similar experiments using human osteosarcoma cell lines, including the highly resistant LM7 and the relatively resistant 143B tumor cells. Neither YTS nor GCC2 KO cells demonstrated measurable killing of either 143B or LM7 cells in the absence of a triggering target using TheCOS, and thus both were resistant (FIGS. 3D, 3F, and 8E for51Cr killing assay). When triggering 721.221 target cells were added, however, the killing of osteosarcoma cells was detectable, but only by the GCC2 KO cells. This “bystander” killing effect against the 143B and LM7 cells increased with increasing percentages of triggering 721.221 target cells present in TheCOS. As an example of the complex cell arrangement between YTS, 721.221, and osteosarcoma cells, undigested TheCOS stack slices from the highly resistant LM7 and 143B cell experiments were visualized using confocal microscopy (FIGS. 3E and 3F). In experiments using either the YTS parental orAttorney Docket 44010.215WO-PCT / / CU24341GCC2 KO cells, these cells could be seen in close approximation with a 721.221 target cell and osteosarcoma cells. The LGs in the parental YTS cell were converged, but those in the GCC2 KO were dispersed. Thus, LG dispersion owing to the absence of GCC2 was associated with increased bystander cell killing after triggering even when these bystander cells were otherwise resistant human osteosarcoma cells. This suggests that GCC2 regulates LG convergence to promote efficiency against a triggering target cell and prevents killing of neighboring cells in a simulated three-dimensional tumor microenvironment setting.Ectopic expression of GCC2 at the cell membrane redirects LGs to the cell surface after activation

[0232] Since endogenous GCC2 appeared to link LGs to the Golgi during convergence, Applicants wanted to determine if altering the cellular localization of the protein could redirect LG positioning. GCC2 localization is directed by its GRIP domain that is responsible for its Golgi anchoring, and replacing this with a monoamine oxidase A sequence has been shown to divert it to mitochondria.20Since under some circumstances LGs can interact with mitochondria,21Applicants developed a different localization strategy and replaced the C terminus of the GCC2 GRIP domain with the last 14 residues of the KRAS protein22(FIG.4A).This residue is known to be farnesylated, which should promote cytoplasmic membrane interaction. When YTS GCC2 KO cells were stably transduced with the GCC2 1532-C14KRAS construct (GCC2-KRAS), the fusion protein deleted of the C-terminal 104 residues could be identified by western blot analysis of total lysates (FIG. 4B). Applicants next stimulated YTS parental and GCC2 KO GCC2-KRAS cells to evaluate if the localization of LGs was altered relative to the MTOC and cell surface in the presence of the GCC2 fusion protein. Applicants used the F-actin cortex labeled via phalloidin as a surrogate for the cell membrane owing to its proximity to it and its robustness in fixed cells. As expected, activation via CD28 induced granule convergence to the MTOC in parental cells, but not GCC2 KO cells, irrespective of the presence of the GCC2-KRAS construct (FIGS. 4C and 4D). When the GCC2-KRAS construct was present, however, the LGs were found closer to the cell cortex after CD28 activation, with increased significance in the absence of endogenous GCC2 (FIGS.3C and 3E). Thus, ectopic localization of GCC2 promoted mislocalization of LGs toward targeted sites of GCC2 expression.

[0233] To evaluate the effect of forced LG localization, directed cytotoxic activity was measured in the presence of the GCC2-KRAS construct. The expression of GCC2-KRAS inAttorney Docket 44010.215WO-PCT / / CU24341parental YTS cells reduced their killing efficiency against 721.221 target cells to the level of GCC2 KO cells (FIG.4F). Directed killing efficiency was reduced further still in GCC2 KO cells containing GCC2KRAS. This construct, however, did not impair the ability of GCC2 to interact with LGs as isolated LGs from these cells could still be found to contain the GCC2-KRAS construct (FIG. 11D). Given that the LGs in the GCC2-KRAS-expressing cells were dispersed and more peripherally localized after activation, Applicants wanted to evaluate if there was an impact upon bystander killing of neighboring non-targeted cells after NK cell triggering. Thus, Applicants measured the killing of non-targeted K562 cells using TheCOS when increasing percentages of triggering 721.221 cells were present. As triggering target cells were added, there was increased measurable specific killing of K562 bystanders in the absence of GCC2, but not in parental YTS cells (FIG.4G). This specific bystander killing in the GCC2 KO cells was increased further still by the addition of GCC2-KRAS. While this may relate to the level of GCC2-KRAS expression, it suggests that convergence to the MTOC and Golgi dominates in preventing bystander killing. Conversely, redirection of LGs to the cell membrane results in reduced IS-mediated killing and increased non-IS-mediated killing.The Golgi complex modulates LG positioning during convergence

[0234] Since the Golgi-related protein GCC2 appears essential for at least part of LG convergence after NK cell activation, Applicants wanted to determine any direct role of the Golgi in LG positioning. To eliminate the Golgi as an MTOC proximal presence, the Golgi was fragmented using Brefeldin A prior to and during NK cell stimulation. In Brefel din-treated cells, the Golgi as labeled by mCherry-GALT were no longer aggregated around the MTOC in either resting or activated cells (FIG. 5A). Surprisingly, the fragmentation of the Golgi did not grossly impact NK cell LG convergence after their activation on antiCD18(IB4) / anti-CD28-coated glass or when in conjugation with 721.221 target cells. Detailed measurements of convergence after activation confirmed no significant alteration of LG positioning relative to the MTOC (FIG. 5B). When fragmented, however, the proximity of Golgi fragments to LGs was still maintained, but only when GCC2 was present (FIGS. 9A-9B). The specificity of LG positioning was further considered in this context by

[0235] contrasting their localization to that of mitochondria, which were unaffected by either NK cell activation or Golgi fragmentation (FIGS. 9C-9D). Finally, killing efficiency as determined by51Cr-release assay was only minimally affected by Brefeldin treatment andAttorney Docket 44010.215WO-PCT / / CU24341Golgi fragmentation (FIGS. 5A-5C). Thus, the positioning of the Golgi appears to be dispensable for LG convergence to the MTOC and resulting directed cytotoxicity.

[0236] Given that GCC2 was required for full LG convergence to the MTOC after activation and LGs to Golgi approximation, Applicants instead considered a role for the Golgi in sustaining LG convergence. To test this, Applicants induced LG convergence using soluble interleukin-2 (IL-2) that promotes LG movement to the MTOC dependent upon non-canonical signaling via Src family kinase-mediated convergence.4IL-2 added to media induced significant LG convergence to the MTOC in YTS cells (FIGS. 5D and 5E), albeit at levels lower than those measured after target cell engagement. The disaggregation of the Golgi using Brefeldin A treatment did not affect the IL-2-induced convergence of LG, consistent with the observation in glass surface or target cell-activated NK cells. Applicants next removed the IL-2 convergence signal after IL-2-induced NK cell activation by washing the cells and placing them into media without IL-2. Despite the removal of IL-2, there was sustained convergence of LGs in otherwise untreated NK cells after 3 h (FIGS. 5D and 5E). Importantly, however, in the presence of Brefeldin A, the removal of IL-2 resulted in a dispersion of LGs after 1 h. Thus, when the Golgi were fragmented and not MTOC localized, LG convergence was not sustained. This suggests that the LGs use tethering to the Golgi as a means for controlling their positioning and maintaining a converged state after having experienced a convergence signal. This, in turn, promotes maximal efficiency in directed cytotoxicity and prevents bystander killing.Biallelic GCC2 mutations cause human NK cell deficiency

[0237] Since directed cytotoxicity as enabled by LG convergence and polarization is a central theme in killing efficiency, Applicants suspected that interference with these functions could impair the ability of NK cells to serve typical human host defense. Since removal of GCC2 reduced the convergence function in NK cells without preventing their ability to divide and proliferate, Applicants considered GCC2 as a candidate gene for causing human NKD. NKD is defined as a stable deficiency of NK cell number or function that represents an individual’s major clinical immunodeficiency, mostly leading to susceptibility to viral infections and cancers.23,24Applicants have been evaluating individuals suspected of having NKD since 2006 and have performed exome sequencing on many of them. Thus, Applicants queried the unsolved cases for variants in GCC2 or related genes and identified two unrelated individuals with compound heterozygous biallelic variants in GCC2 (FIG.6A). Both patient percentages of total NK cells in the peripheral blood were within normal limits (FIG. 6B). The two individualsAttorney Docket 44010.215WO-PCT / / CU24341presented in childhood (age 1 and age 16) had a history of recurrent viral infection and lacked known immunodeficiency gene variation (FIG. 6C and Clinical histories and presentations for patients Pl and P2).

[0238] Interestingly, both individuals shared a single GCC2 variant E1608G in combination with a second Q851P in patient 1 and K777I in patient 2 (FIG. 6D). The maximal estimated combinatorial incidence of these variants would be 2 in 10 million according to GnomAD.25GCC2 has 1,684 residues and contains 5 coiled-coil domains (CC-Ds) and a C-terminal GRIP domain all separated by disordered regions (FIG. 6D). The shared E1608G variant is at the beginning of the GRIP domain, and the other two are in or near the third CC-D, and in silico modeling using AlphaFold326,27predicts that Q851P, but not K777I, and E1608G are within a CC-D region (FIGS. 10A-10C). Interestingly, all three variants lie within proximity of known Rab-interacting regions, Rab9 (residues 805-889) and Rab6a (residues 1,609-1, 659).28To determine if there was expression of GCC2 protein in the setting of these biallelic variants, peripheral blood mononuclear cells (PBMCs) from patients 1 and 2 were used for western blot analysis (FIG. 6E), and GCC2 was detectable in both, albeit at somewhat lower levels than those found in a set of healthy control donors (FIGS. 6E and 11 A). Finally, to confirm the expression pattern of the patient GCC2 variants, microscopy was performed using patient NK cells. GCC2 demonstrated a Golgi pattern of expression in both patient and control cells suggesting that the variant GCC2 protein expressed in patient NK cells was not impaired in localization (FIG. 11B).

[0239] To determine the potential effects of these GCC2 variants in NK cells from patients, patient samples were evaluated directly, via51Cr-release assay against K562 target cells, and demonstrated reduced killing in both patients (FIG.6F). A substantive portion of killing could be restored by short-term stimulation with IL-2, but not to the levels of stimulated healthy control donor cells. Although the percentage of NK cells in the patients was normal, Applicants evaluated the maturity and developmental trajectory of their peripheral NK cells in detail by high-parameter flow cytometry. The relative developmental subset frequency and phenotype found in the patient NK cells was generally indistinguishable from healthy control donor cells (FIG. 12). This suggests that the defect associated with biallelic GCC2 variants was functional rather than developmental (unlike most known NKD). Therefore, Applicants next evaluated LG positioning in purified ex vivo patient NK cells conjugated with 721.221 target cells or an activating surface with anti-NKp30. After 1 h, both healthy donor and patient NK cells formed lytic immune synapses with LGs converged to the MTOC (FIG.6G). LG convergence measuredAttorney Docket 44010.215WO-PCT / / CU24341across multiple synapses, however, was significantly reduced in patient NEC cells (FIG.6H). Thus, the impact of biallelic GCC2 variants in NEC cells from patients with NECD appeared similar to that of GCC2 KO YTS cells with reduced directed cytotoxicity and LG convergence.

[0240] To identify the impact of the common GCC2 variant in patients with NKD on NK cell biology, it was evaluated directly in YTS cells (thus, independently of any patient genetic background). YTS GCC2 KO cells were reconstituted with the shared E1608G variant or wildtype (WT) GCC2. Levels of GCC2 protein reconstituted in stably transduced YTS GCC2 KO cells measured by western blot analysis were less than in parental cells but similar between the WT and E1608G variant (FIG. 7A). Expression of the WT GCC2 restored the cytotoxicity of GCC2 KO cells to near parental cell levels (FIG. 7B). Expression of the El 608G variant, however, failed to increase cytotoxicity relative to those lacking GCC2 altogether. This same trend was also observed in YTS cells transduced with either of the other two patient-derived GCC2 variants (FIG. 11C). In order to determine if the interaction of GCC2 variants with LGs was preserved, LGs from the reconstituted cells were isolated and evaluated for the presence of GCC2 by western blot analysis. The common E1608G and Q851P variants retained the ability to interact with LGs, while the K777I had reduced interaction (FIG. 11D).

[0241] Finally, Applicants evaluated the effect of the common patient variant on LG convergence when used to reconstitute GCC2 KO cells in direct imaging of lytic synapses. The expression of E1608G failed to restore LG convergence after lytic synapse formation (FIG.7C). Time-lapse imaging demonstrated that even the typical convergence found after activation was greatly reduced in the absence of GCC2 and largely eliminated in the presence of the common patient variant (FIG. 7D). Thus, GCC2 E1608G was unable to rescue NK cell function and resulted in dispersed LGs after activation. Variant GCC2 functions as an NKD-causing gene by eradicating LG convergence and underscoring that an LG Golgi linkage is needed for optimal LG convergence, directed killing, and human host defense.DISCUSSION

[0242] One of the main functions of NK cells is to kill virus-infected or malignant cells in a complex context where those cells are usually surrounded by healthy cells. As such, they are hypothesized to be important for immunological surveillance against cancer and other nascent threats.29,30This selectivity is dictated by a complex system of activating and inhibitory receptors engaged in the context of the IS. This in turn triggers intracellular pathways to ensure that LGs containing perforin and granzymes are only released at the activating IS with theAttorney Docket 44010.215WO-PCT / / CU24341targeted cell rather than toward bystander cells.2,31This directionality and specificity is dependent upon LG convergence and subsequent polarization.2While the LG convergence process is mediated by dynein / kinesin-dependent trafficking on microtubules,2,7how this convergence is sustained after activation and throughout polarization until LGs access the F-actin cortex during degranulation is unknown. Given that dynein motors use substantive energy, it would seem logical that there is another mechanism for maintaining convergence after the initial process has occurred. In the context of NK cells, this could represent long periods of time as might be required by an NK cell that has extravasated via a convergenceinducing integrin signal and is in the midst of mediating cytotoxic function within a tissue. It would essentially allow them to persist in an “armed” state.

[0243] In the context of restricted intracellular space including numerous organelles having distinct functions, interactions between these organelles are expected and have been studied, such as the interaction of LGs with the centriole5,7or mitochondria21in NK cells. While the Golgi complex is known to be relevant to LG biogenesis,32only incidental data in cytotoxic cells using electron microscopy have observed proximity of the Golgi to LGs and any association of the Golgi with the IS.8 11Primarily known for its role in the maturation and secretion of proteins and vesicles, the Golgi is also critical to endosomal recycling mediated by Golgi membrane proteins that tether and promote fusion.12,17,28,33A first tethering step is mediated in part by the / ra / .s-Golgins, a family of structurally conserved CC-D proteins that can interact with different vesicles and cargoes.17Generally, / ra / .s-Golgins have an N-terminal region that interacts with the cargo, a long CC-D region with several Rab interacting domains,14and a highly conserved C-terminal region (GRIP domain) important for Golgi interaction. Of the four / ra / .s-Golgins, cargo has been described for three, but not GCC2.18,20

[0244] Since Applicants identified the LGs as converged around the MTOC in the vicinity of the Golgi after NK cell activation, Applicants looked for proteins at the LG surface that could potentially facilitate interaction. Applicants expected to find Rab proteins, which are known to enable Golgi interaction in recycling,34but were surprised to find actual Golgi proteins. Although GCC2 is clearly a Golgi protein owing to its structure, its interaction with the Golgi through the GRIP domain could be considered dynamic due to its dependence on other proteins such as Rab6 and Aril.14,15,28While GCC2 could be acquired from LG biogenesis, it has been suggested that GCC2 is also cytoplasmic and can interact with vesicles to drive them to the Golgi.14Similar behavior has been suggested for another CC domain protein, HkRP3, in NK cells.35Interestingly, the preliminary unpublished proteomic data suggest an interaction ofAttorney Docket 44010.215WO-PCT / / CU24341GCC2 and HkRP3, and thus a model where HkRP3 promotes LG convergence via microtubules, while GCC2 enables tethering at the Golgi, is plausible. Alternatively, GCC2 has been reported as a microtubule-nucleating factor on the Golgi, via CLASP 1 interaction,36and could help generate noncentrosomal microtubule fibers that could facilitate the recruitment of LGs to the Golgi. Along these lines, cytotoxic T cells lacking centrioles have reduced, but not absent, MTOC function, suggesting that other nucleating centers such as those formed on the Golgi can function as rudimentary MTOCs.37

[0245] How GCC2 interacts with LG remains elusive. GCC2 has several Rab-interacting domains,28and Rabs are enriched on the LG surface.34Applicants have attempted to knock down the expression of the 2 main Rab proteins known to interact with GCC2: Rab9A and Rab6A, but found no clear phenotype (not shown). Interestingly, reduction of Rab6A decreased GCC2 presence on the Golgi and increased the LG convergence, similar to the results obtained in Brefeldin-treated cells (FIG. 13). While not conclusive, the lack of a clear phenotype with reducing expression of individual Rab proteins suggests a functional role for multiple Rab-GCC2 interactions and is a focus of future work.

[0246] GCC2 is involved with linking LGs to the Golgi after NK cell activation, presumably without affecting the association of LGs with microtubules. This is in part supported by the experiments with Brefeldin induced Golgi dispersion. In the absence of intact Golgi, LG convergence was more efficient. Applicants propose that this improved convergence may be due to the absence of a physical barrier caused by the Golgi, allowing LGs to more tightly pack into the MTOC. That said, when the activating stimulus was removed from the cells with fragmented Golgi, the LGs were easily dispersed, while in the cells with intact Golgi, LGs remain converged (for at least 3 h after withdrawal of activating stimuli). This further emphasizes a role for the Golgi as an anchor for converged LGs that were approximated to the Golgi via dynein-mediated activation-induced LG convergence.

[0247] The utility of converged LGs has been shown in reductionist single and multi-cell systems to promote killing efficiency and prevent non-directional degranulation and bystander cell killing. Using a recently developed three-dimensional complex cell arrangement system, TheCOS, Applicants have extended this into simulated multicellular environments. Here, Applicants found that, in a target / bystander or a more complex resistant tumor cell three-dimensional system, NK cells with converged LGs are focused upon killing their triggering target cell. When LG convergence was reduced via GCC2 absence, the dispersion of the LG was maintained even when in the midst of complex cell arrangements. Furthermore, these cellsAttorney Docket 44010.215WO-PCT / / CU24341with dispersed LGs were able to kill the triggering target cell, but also uncovered bystander killing of otherwise resistant tumor cells. Thus, the efficiency of convergence and nondirectionality of LG dispersion could be seen in complex multilayer cell environments, with efficient convergence associated with more efficient target killing, and the lack of convergence (dispersion) making the NK cells prone to degranulate outside of the lytic synapse increasing bystander killing. Although rapid and excessive synaptic degranulation could theoretically result in bystander killing, as could the release of predocked LGs, the bystander killing of cells that do not promote NK cell activation and are resistant, such as osteosarcoma cells used in the present study (FIG. 9A), suggests a direct role of LG dispersion (as prevented by GCC2) in bystander killing. This is reinforced by the observation that when Golgi are fragmented by Brefeldin A, convergence is more efficient and there is no bystander killing (FIG. 8E) as well as the previously demonstrated concept that the contents of a single granule can be enough to induce target cell death.3

[0248] The impact of LG convergence and dispersion in complex three-dimensional environments questions the in vivo relevance of these cellular processes. Specifically, does the decreased on target killing efficiency associated with dispersion create any host defense deficit? Having collected an 18-year cohort of individuals with NKD where a static deficit in NK cell activity results in clinically relevant immunodeficiency, this was a relevant place to search. This cohort has previously given rise to monogenic explanations for NKD and is characterized by susceptibility to viral infections, most notably those caused by herpesviruses and wart-causing viruses,3 42and it has been complemented by NKD-related discoveries from other programs.43 46Approximately one-quarter of the NKD cohort has been attached to a genetic explanation and many remain unsolved. Having identified two unrelated individuals in the NKD cohort with biallelic GCC2 variants led Applicants to explore potential causality, but it certainly raised the possibility of the relevance of GCC2 and LG positioning in NK cell-mediated human host defense.

[0249] The two patients with biallelic GCC2 shared a common mutation, E1608G, which is located close to the GRIP domain. Hypothetically, this proximity could destabilize the CC-D structure (similar to predictions for the other private variants) and potentially alter interaction with key downstream effectors. When modeled in GCC2-deficient YTS cells, GCC2 E1608G failed to restore GCC2 function in enabling LG convergence. This was similar to what was found in ex vivo patient NK cells that demonstrated reduced killing efficiency and a failure to converge LGs in lytic synapses. The only other listed association for GCC2 is for a homozygous missenseAttorney Docket 44010.215WO-PCT / / CU24341variant, Argl669His, present in a patient with a severe neurological disease, although it was not clear if there was any immunological phenotype in that individual.47Irrespective, a partial loss of GCC2 function is associated with a defect of LG movement and clinical presentation with NKD, suggesting an in vivo human relevance to killing efficiency and LG convergence.

[0250] The Golgi as a cellular center for production and recycling is well established but takes a new form and function via a role for the / ra / / .s-Golgin GCC2. Specifically, it serves as “docking station’ ’ for diffusely localized cellular organelles that need to be compartmentalized in order to enable a specialized cell function. That said, it is unlikely that NK cell LGs represent GCC2’s only cargo, and exploration of similar paradigms for other lysosomal-related organelles where strict control of positioning and release is relevant remains to be investigated. Similarly, using the Golgi as a docking station for other diffusely localized cellular organelles after cell activation is potentially an appealing means to preserve cellular energy as opposed to relying upon constant motor protein activity to dictate organelle positioning. A role for the Golgi as a docking station for lysosome-related organelles in NK cells to facilitate their cytolytic function, however, governs the persistence of convergence after microtubule-directed LG delivery. In this context and through extension to a new human genetic disease, Golgi-GCC2 linkages demonstrate a mechanism for preventing bystander killing while promoting killing efficiency that is required for human host defense. Conversely, perhaps hardwired mechanisms to promote bystander killing by blocking convergence without interfering with degranulation could prove to be useful in improving the therapeutic use of cytotoxic cells against otherwise difficult-to-treat solid tumors where the killing of neighboring cells would be of great value.Limitations of the study

[0251] The major limitation of the study is that Applicants have not established the molecular partner of GCC2 that enables its linkage to the LG governing its tethering function. Also, although Applicants were able to link biallelic GCC2 variants to human NKD in two unrelated patients, additional cases would be instructive. Finally, although the work identifies a role for GCC2 in NK cell function, Applicants do not know its specificity to NK cells or if it affects other cells that converge lysosome-related organelles such as cytotoxic CD8 T cells, mast cells, or melanocytes.Attorney Docket 44010.215WO-PCT / / CU24341EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILSHuman PBMC

[0252] Human PBMC were obtained from whole blood from research volunteers after informed consent and enrollment in a research protocol approved by the institutional review board for the protection of human subjects of either Columbia University Irving Medical Center (AAAR7377) or Baylor College of Medicine (H-30487) and isolated by density centrifugation using Ficoll-Paque medium (Cytiva) as previously described.40ex vivo NK cells were isolated from whole blood using RosetteSep Human NK Cell Enrichment Cocktail (Stemcell) according to manufacturer’s recommendations. For expansion and where indicated, primary NK cells were cultured in NK MACS Medium (Miltenyi) supplemented with 5% human AB serum (GeminiBio) and 500U / mL of IL-2 (Proleukin) for 2 weeks or until confluence.Human subjects and exome sequencing

[0253] Individuals suspected of having NKD were enrolled in research protocols approved by the institutional review board for the protection of human subjects Columbia University Irving Medical Center (AAAR7377) and Baylor College of Medicine (H-30487) and clinical data evaluated and blood samples obtained for research NK cell studies and exome sequencing. Sequencing was performed and variants analyzed as described at Baylor College of Medicine.48Cells and culture

[0254] The human NK cell line YTS and the MHC class-I-deficient 721.221 EBV-transformed B cell line were as originally described49and maintained in RPMI 1640 (Thermo) complete media (R10) supplemented with 10% heat inactivated fetal bovine serum (Thermo), lOmM HEPES, ImM sodium pyruvate, 2mM L-glutamine,l% non-essential amino acid, and lOU / mL penicillin / streptomycin (all from Thermo). The K562 erythroleukemia cell line was obtained from ATCC, and the LM7 and 143B osteosarcoma cells were the kind gift of Dr. Stephen Gottschalk (St. Jude, Memphis, TN) with the permission of Dr. Eugenie Kleinerman (MD Anderson, Houston, TX). Lenti-X 293T cells were obtained from Takara and cultured in DMEM Complete media (Thermo) (D10) supplemented with 10% heat inactivated fetal bovine serum, lOmM HEPES, ImM sodium pyruvate, 2mM L-glutamine,l% non-essential amino acid, and lOU / mL penicillin / streptomycin. Cultured cells were maintained at 37°C and 5% CO2 at an average concentration of 5xl05cells / mL and adherent cells or confluence of 80%.Attorney Docket 44010.215WO-PCT / / CU24341METHOD DETAILSGene editing and transduction

[0255] CRISPR / CAS9 gene editing was performed using 2xl06cells that were nucleofected with 5mM of sgRNA guide specific for GCC2 (Horizon; for the sequence, see Table 4) and 5mg of Cas9-GFP mRNA (Horizon) in 100 mL of nucleofection Kit-R supplemented (Lonza) solution using the R-024 program of an Amaxa Nucleofector II (Lonza). After 24h, cells were sorted for GFP positivity, and single clones generated in 96-well plates were then selected based upon GCC2 expression evaluated by Western Blot.

[0256] Gene transduction was performed using lentiviral particles produced in HEK293T LENTLX cells according to manufacturer’s instructions (Takara) using FuGene transfection reagent (Promega) in serum free DMEM (Thermo). Supernatants were collected and concentrated using PEG-it Virus Precipitation Solution (System Bioscience) and were used to transduce YTS cells via spinoculation. Lentiviral particles contained pCDH-CMV-MCS-EFla-puro plasmids with GCC2 NM_181453.4 mRNA WT (kind gift from Dr. Suzanne Pfeffer from the Department of Biochemistry, Stanford University School of Medicine), the El 608G variant, or truncated GCC2 (amino acids 1-1532) fused with the GGGGSGGGGSGGGGS-GI<I<I<I<I<I<SI<TI<CVIM (SEQ ID NO: 42) peptide. YTS cells were also transduced with a GALTmCherry fusion protein containing the amino acids 1-81 from b 1 ,4-galactosyltransferase (pmCherry-Nl-GalT was a gift from Lei Lu, Addgene plasmid # 87327)12or a fusion protein of pHluorin-LAMPl-m Apple as previously reported.3All constructs were generated or subcloned by Epoch Life Science custom cloning services (Sugarland, TX). After 72h, transduced cells were selected using progressively increasing concentrations of puromycin ending with 5 mg / ml.Flow cytometric analyses

[0257] Cells were stained for flow cytometric analyses as described,19and -IxlO6cells were analyzed using a NovoCyte Penteon flow cytometer (at the Columbia Stem Cell Initiative Flow Cytometry Core Facility); this data were analyzed using FlowJo software. Flow cytometric data were plotted and statistically analyzed using Prism 10 (GraphPad). Degranulation assay was performed with 5xl04YTS and 721.221 target cells mixed for 90 min at 37°C after which cells were fixed and stained as described previously, using anti-CD56 to identify NK cells and antiCD 107a as a marker for degranulation.19Detailed NK cell development was assessed using cryopreserved PBMC stained with surface antibodies as specified (Table 4), followed byAttorney Docket 44010.215WO-PCT / / CU24341permeabilization using FoxP3 fixative buffer (Thermo) for 30 min on ice. Cells were then washed and incubated with antibodies diluted in FoxP3 permeabilization buffer for intracellular staining as specified (antibodies utilized are listed in Table 4).Cytotoxicity assay

[0258] 4h51Cr-release assay using either K562 or 721.221 target cells was performed as described,19using IxlO4target cells per well of a 96-well U bottom plate. Effector cells were used in serial dilution starting at an effector to target cell ratio of 50: 1 for PBMC or 10: 1 for YTS cells. The activity of PBMC was measured using unstimulated cells or with lOOOU / mL of IL-2 (Proleukin) added during the assay. After incubation, supernatants were transferred into Lumaplates (Rewity) and measured using a TopCountXL (PerkinElmer). Specific lysis (%) was calculated as: (experimental cpm - spontaneously released cpm) / (total cpm - spontaneously released cpm) xlOO.Confocal microscopy

[0259] For single cell or single cell conjugate confocal microscopy, 18-well chamber slides (Cellvis) were precoated with 5 mg / mL of mouse anti -human CD 18 (IB4) (to adhere NK cells), a mix of mouse anti-human CD 18 (IB4) and CD28 clone CD28.2 (Biolegend) (to adhere and activate NK cells), or mouse anti-human CD19 clone EHB19 (Biolegend) (to attach 721.221 targets). Either 2xl04cells YTS cells or 4xl04equally mixed YTS and 721.221 cells in lOOmL of R10 media were added to each well and incubated for either Ih or 30 min at 37°C, respectively. The mixed YTS and 721.221 cells were preincubated in suspension for 30 min at 37°C prior to being added to the slide. After incubation, media was aspirated and cells were fixed with 4% paraformaldehyde in PBS, washed, and permeabilized with 0.25% of Triton X-100. Cells were then stained with fluorophore conjugated antibodies and Phalloidin (antibodies utilized are listed in Table 4). For live cell imaging, YTS cells were labeled with Lysotracker Deep Red (Thermo) and 721 :221 target cells with Cell Tracker Blue. 721.221 cells were added to chamber slides first and allowed to adhere for 30min, after which YTS cells were then added and images recorded every 5s to capture 100 frames (~8min). For all experiments, cells were visualized and images were acquired on a Zeiss Axioplan Observer Z1 with Yokigawa CSU-W 1 T2 50mm spinning disk and 63X 1.49 NA objective. Excitation was by 405, 488, 505, 561, and 637 nm lasers and light captured via a Prime 95B metal oxide camera. Data were acquired with Slidebook software (Intelligent Imaging Systems) and analyzed using Imaris (OxfordAttorney Docket 44010.215WO-PCT / / CU24341instruments) or ImageJ (Fiji) software. LG distance to MTOC reconstructions were generated using Imaris software, and the tool for spots measurement was utilized to identify LG and render the signal from Perforin and MTOC (using maximum a-Tubulin signal which has been validated as alternative to pericentrin50). For the Golgi rendering the tool for surface reconstruction was applied to the signal from mCherry-Galt. Upon 3D model reconstruction, the software was used to generate the shortest distance between the centroid of each spot (in the case of LG to MTOC distance) or the shortest distance between the LG centroid and the surface of Golgi. The average of those distances were computed using at least 20 cells per measurement.Lytic granules isolation and analysis

[0260] LG were isolated from YTS cells using a Lysosome Enrichment Kit for Tissues and Cultured Cells (Thermo) according to manufacturer’s recommendations. In brief, 3xl08cells were washed and sonicated, and mitochondria / cell debris were removed via centrifugation at 10000 x g. Supernatant was precipitated at 20000 x g, washed twice, resuspended, and layered on a percoll density gradient followed by ultracentrifugation at 340000 x g. LG were harvested from the top layer and washed. For Western blot analysis of LG contents, they were lysed with NP40 lysis buffer (lOmM HEPES pH8, lOmM KC1, ImM EDTA pH 8, 0.4% NP40, IX Halt proteinase phosphatase), separated using gradient 4-12% gel, and transferred to a nitrocellulose membrane. Membranes were blocked with skim milk, incubated with primary antibodies overnight (antibodies utilized are listed on Table 4), secondary antibodies for Ih, and imaged using an Odyssey CLx System (Licor). For surface biotinylation, LG were incubated with EZ-Link Sulfo-NHS-SS-Biotin (Thermo) in PBS pH 8.0 at RT for 30min following manufacturer’ s instructions, lysed, precleared, and then precipitated using streptavidin-agarose beads (Millipore) as Applicants had previously described.13Mass spectrometry was performed at the protein core facility of the Children’s Hospital of Philadelphia as described, and comparison of surface biotinylated to non-biotinylated LG was performed.13Only proteins identified in the lysates from surface biotinylated LG in both of two independently repeated experiments with R3 identified peptide sequences were considered present.Thermal collapse of strata (TheCOS)

[0261] The overarching strategy of TheCOS was to be able to bring into approximation specified ratios of cells on demand without having them be in contact a priori, while at the sameAttorney Docket 44010.215WO-PCT / / CU24341time enabling direct visualization via microscopy as well as cell isolation at the end of an experiment. Full details and protocols are reported separately (manuscript in submission) and are presented in brief and as relevant to present results. YTS cells, triggering 721.221 target cells and non-triggering target cells (K562 erythroleukemia, or LM7 or 143B osteosarcoma cells) were resuspended in 1% PolyN-isopropylacrylamide (PNIPAM) (Millipore Sigma) in Culture media at 4°C to enable 1:10 effector to target cell ratios with specified percentages of the different target cells. The individual cell suspensions were added (below the phase transition temperature of PNIPAM at 32°C) over a premade layer of 1% agarose hydrogel in culture media (lower layer) that had been previously poured into in a 3D-printed micromold assembly and allowed to cool and solidify. After adding a cell suspension in PNIPAM a 3D-printed piston was carefully inserted into the mircomold to create a flattened layer. The piston was then removed, exposing the newly created layer. This process was repeated for each subsequent layer and finally, a 1% agarose hydrogel at 45 °C was added to fill the micromold and seal the cell strata. The entire sealed assembly was then placed on an isothermal plate (37-40°C) for 5 min to allow for the gelation of the cell suspension and collapse of the PNIPAM layers brining the layered cells into contact with each other within the agarose encasement. To assess the bystander killing capacity of NK cells, a three-layer cell strata of triggering target cell / YTS cell / non-triggering target cell was assembled. Following the collapse of the PNIPAM hydrogel, the aggregated cells encased in agarose were incubated for 4 h. Subsequently, the assembly was cut open through an incision guide printed into the micromold, and either imaged by placing the exposed matrix onto a coverslip or individual cells isolated by soaking the agarose matrix on PBS and filtering through a 40-mm cell strainer, after which flow cytometric analysis was performed using fluorescently-conjugated antibodies and live / dead staining reagents to measure the viability of specific cells.Clinical histories and presentations for patients Pl and P2, related to FIG. 6.Patient 1

[0262] Patient 1 is a male second child of healthy parents, bom after an uneventful pregnancy and without perinatal complications. He had rhinovirus at 2mos of age with Imo of persistent symptoms, and respiratory syncytial virus at 5mos of age requiring hospitalization. At age 10 and 12mos he was hospitalized for respiratory compromise and presumed respiratory viral infection. Owing to the severity and recurrence of illness with fevers he had several immunological assessments performed that suggested accentuated / prolonged physiologic IgGAttorney Docket 44010.215WO-PCT / / CU24341nadir vs hypogammaglobulinemia. Evaluations for primary antibody deficiency were normal and his IgG levels ultimately normalized by 17 months with documentation of intact vaccinespecific IgG. Lymphocyte subset analyses were normal, but NK cell function was persistently low.

[0263] Respiratory symptoms persisted over his first 3 years. He also had skin rashes starting at age 1 which ultimately resolved at age 4 when he was diagnosed with molluscum contagiosum. Initial research NK cell studies performed at age 1 documented low cytotoxicity without or with added IL-2 stimulation in vitro compared to the range of over 200 healthy donors (FIG. 14) as well as NK cell percentages within normal ranges. This pattern continued through last evaluation at age 7.Table 2. Lymphocyte panel and NK cell cytotoxicity assay# / %: absolute number and percentage of lymphocytes.NK LU: Lytic units(L): below the normal levels for age.Patient 2

[0264] Patient 2 is a male first child of healthy unrelated parents. He experienced early life upper respiratory infections and otitis media and had tonsillectomy / adenoidectomy at age 15 for recurrent pharyngitis. At age 16 he presented with prolonged fever, fatigue and meningismus and was diagnosed with Lyme meningitis, receiving Imo of ceftriaxone treatment. Later that year he developed myalgias and diffuse arthralgias and was diagnosed with acute EBV infection followed by several months of persistent symptoms. A clinical immunological evaluation was performed and demonstrated normogammagi obulinemia and no protective titers against 23 pneumococcal polysaccharides. After receiving a 23-serotypeAttorney Docket 44010.215WO-PCT / / CU24341pneumococcal polysaccharide vaccine, only 10 serotypes had titers >1.3 pg / ml and in light of his history he was started on immunoglobulin replacement therapy. Lymphocyte subsets demonstrated a skewed CD4 / CD8 T cell ratio, but otherwise normal cell counts, and NK cell cytotoxic function was low (see table). He was referred for research evaluation at age 19 and initial studies demonstrated low NK cell cytotoxicity without or with added IL-2 stimulation in vitro compared to the range of over 200 healthy donors (FIG. 15), as well as NK cell percentages within normal ranges.Table 3. Lymphocyte panel and NK cell cytotoxicity assay# / %: absolute number and percentage of lymphocytes.NK LU: Lytic units(L): below the normal levels for age.QUANTIFICATION AND STATISTICAL ANALYSIS

[0265] Numerical data were analyzed and displayed using GraphPad Prism 10.0 (GraphPad Software). D’Agostino and Pearson tests were used to assess normality, two-tailed student’s T test for significance with normal distributed data, and Mann-Whitney U tests for significance with non-normal data. Statistical significance of the difference between arbitrary curves such as those generated on51Cr or in TheCOS were assessed using a modified Chi-squared method as previously reported.51Values considered to be statistically significant were noted as follows: *p % 0.05, **p % 0.01, ***p % 0.001, ****p % 0.0001 and non-significant diferences are not shown.Table 4. Key ResourcesAttorney Docket 44010.215WO-PCT / / CU24341Attorney Docket 44010.215WO-PCT / / CU24341Attorney Docket 44010.215WO-PCT / / CU24341Attorney Docket 44010.215WO-PCT / / CU24341Attorney Docket 44010.215WO-PCT / / CU24341EXAMPLE 2 - METHODS OF LYTIC GRANULE REDIRECTION TO THE PLASMA MEMBRANE IN CYTOTOXIC CELLS TO PROMOTE BYSTANDER KILLING IN CELLULAR THERAPIES

[0266] An activated NK cell only requires LG to be positioned close to the cell membrane to initiate the degranulation process, either mediated via the immune synapse (IS) resulting in specific target killing or outside the IS, resulting in bystander killing.

[0267] FIG. 17 shows three genetic strategies to target lytic granules to the surface of the NK cell plasma membrane for the purpose of increased NK cell cytotoxicity and / or increased bystander killing. Method 1: CAAX motif binds accessory molecules associated to LG tethering such as GCC2 or G-97. Method 2: CAAX motif fused with LAMP1 transmembrane domain. Method 3: transmembrane domain from STX1 motif fused with LAMP1 transmembrane domain. Under normal conditions, LG are not attached to cell membrane and upon NK cell activation they are moved to the MTOC and polarized to the IS ensuring a specific target cell killing (no bystander Killing). After cell modification with peptides allowing anchoring of LG to the membrane, LG can fuse with the membrane outside of the IS and thus increasing Bystander killing.

[0268] Applicants use STX1 and KRAS modifications to target lytic granules to the membrane. LG are anchored to the membrane by expression of highly hydrophobic amino acids or by post translational modification events where hydrophobic sequences are added to LG associated proteins. These membrane directing peptides can be fused to either a transient adaptor protein linked to LG or to a structural component of the LG, providing the flexibility for either more transient or more permanent re-localization of the LG to the membrane.

[0269] Syntaxin 1A (STX1): using the c-terminal peptide from STX1 (example amino acid seq: IMIIICCVILGIVIASTVGGIFA) (SEQ ID NO: 45) that can be inserted into plasmaAttorney Docket 44010.215WO-PCT / / CU24341membrane, based on the highly non-polar amino acid sequence. Other sequences of Syntaxin family proteins are shown in FIG. 18.

[0270] CAAX peptide (KRAS): a representative CAAX motif (example amino acid seq: KKKKKKSKTKCVIM (SEQ ID NO: 46) derived from KRAS C terminal region) that allows for post translational modification via prenylation of the motif with hydrophobic residues, resulting in association with the plasma membrane. Other CAAX containing proteins are shown in FIG. 19. CAAX motifs are post translationally modified via prenylation on the conserved cysteine residue (FIG. 19).

[0271] FIG. 32 and FIG. 33 show C-terminal alignment of lysosomal proteins and human syntaxins. Sequence alignment of lysosomal proteins identifies the high degree of sequence alignment between the selected proteins with emphasis on the contribution of polar and nonpolar (transmembrane region) amino acids for their function. Also, note the recurring motif YXXcj). Syntaxins have greater than 60% non-polar residues making them hydrophobic.Examples of Lytic granule membrane targeting approaches:

[0272] Genetic modification of “KRAS” or “STX1” domains to lytic granule associated proteins result in re-direction to the plasma membrane.1. CAAX (KRAS) approach, supporting data includes:1. GCC2 KRAS2. G97 KRAS3. LAMP1 KRAS2. STX1 approach, supporting data includes:1. LAMP1 STX1

[0273] A C-terminal modification of GCC2 with KRAS motif can target lytic granules to the membrane (FIGS. 4A-4G).

[0274] MTOC: LG from YTS GCC2 KO cells transduced with GCC2 KRAS, show reduced convergence. Parental cells stably expressing GCC2 KRAS show a trend to have decreased LG Distance to the actin cortex (i.e., LG closer to the actin cortex) than parental only cells. GCC2 KO cells stably expressing GCC2 KRAS significantly show a decreased LG distance (i.e., closer to the actin cortex) than GCC2 KO cells. (FIGS. 4D and 4E).

[0275] Chromium release assay: A decrease in cytotoxic capacity for YTS GCC2 KO with GCC2 KRAS.Attorney Docket 44010.215WO-PCT / / CU24341

[0276] TheCOs: GCC2 KO cells stably expressing GCC2 KRAS show increased bystander Killing towards resistant bystander K562 cells compared to YTS Parental and GCC2 KO only cells. (FIGS. 4F and 4G).

[0277] A fusion protein including a C-terminal modification of G-97 with a KRAS motif is expressed (FIG. 20). The G-97 KRAS construct shows increased lytic approximation to actin cortex (FIG. 21). Degranulation: G-97 KO cells transduced with G-97 KRAS show increased levels of degranulation compared to G-97 only or parental cells (FIGS. 22A). Chromium release assay: An increase in cytotoxic capacity for G-97 KO cells transduced with G-97 KRAS compared to compared to G-97 KO only or parental cells (FIG. 22B). No difference is found in the ability to kill resistant bystander cells (K562) cells between parental, Parental + G97 KRAS or G97-KO + G97 KRAS (FIG. 23).

[0278] LAMP 1 -KRAS and LAMP1-STX1 transduced cells show increased lytic granule approximation toward to actin cortex (FIG. 24). MTOC: LG from YTS cells transduced with either LAMP1 constructs, show reduced convergence (FIG. 25). Actin: KRAS LAMP1 and STX1-LAMP1 transduced cells show decreased distance to the cortex compared to parental cells (FIG.25). This suggests that LMAP1-KRAS and LMAP1-STX1 have more lytic granules approximating with the actin cortex under activating conditions. Degranulation: KRAS LAMP1 and STX1-LAMP1 transduced cells show increased levels of degranulation compared to parental cells (FIG. 26). Chromium release assay: An increase in cytotoxic capacity KRAS LAMP1 and STX1-LAMP1 transduced cells compared to parental cells (FIG.26). TheCOs: LAMP1 KRAS and LAMP1 STX1 transduced cells show increased bystander killing towards resistant bystander K562 cells compared to parental cells (FIG. 27).Examples of additional lytic granule membrane targeting approaches:

[0279] GCC2-KRAS, G97-KRAS, LAMP1-KRAS and LAMP1-STX1 are modifications that move lysosome related vesicles, including lytic granules, to the plasma membrane for the purpose of increased bystander killing following NK cell activation. These are all modifications of proteins that include C-terminal membrane targeting domains (i.e., KRAS, STX1).

[0280] To improve this approach, specific lytic granule related targets were pinpointed for modification to only direct lytic granules to the membrane and not other lysosome related organelles. These Lytic granule related proteins contain modifications that rely on N-terminal membrane targeting domains which could include:Attorney Docket 44010.215WO-PCT / / CU24341• The N-terminal domain of Src Family Kinases (Src, Yes, Fyn, Fgr, Lek, Hck, Blk, Lyn, and Frk). This N-terminal domain is myristoyl ated, and this post translational modification helps with insertion into the plasma membrane.• The N-terminal region of the family of Type II transmembrane (TII) proteins.• The C terminal Region of Perforin Granzymes or NKG7.

[0281] A construct for membrane targeting of lytic granules specifically is described (FIG.28). The construct includes the N-terminal domain of a Src Family Kinases (SFKs) followed by the N-terminal region of the family of Type II transmembrane (TII) proteins or by the N-terminal domain of NKG7 and the C-terminal domains of Perforin or NKG7 or GZMB / A, linked by a flexible linker. Membrane targeting is achieved by utilization of SFKs N-terminal domain that can be myristolated and inserted into the plasma membrane or utilization of the N-terminal region of the family of Type II transmembrane (TII) proteins. These domains will be linked to the sequences of specific LG proteins such as NKG7, Perforin, or Granzyme. These LG specific transmembrane proteins are modified to retain their signal peptides for correct sorting into the LG. This should ensure that lytic granules are moved to the membrane following activation for degranulation.

[0282] FIG.34 shows representative confocal images showing LCK-NKG7 -Perforin modified cells with more dispersed lytic granules and a portion of lytic granule in approximation with the cell cortex.

[0283] LCK NKG7 PERFORIN sequence:MGCVCSSNPELPVATMELCRSLALLGGSLGLMFCLIALSTDFWFEAVDYKDDDDKD YKDDDDKDYKDDDDKGGGTCLDYVPQMLLGEPPGNRSGAVW* (SEQ ID NO: 48)

[0284] FIG. 35 shows representative confocal images showing LAT NKG7 PERF modified cells with more dispersed lytic granules and a portion of lytic granule in approximation with the cell cortex.

[0285] LAT NKG7 Sequence:MEEAILVPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVA PWGGGSGGGGSGGGGSGELCRSLALLGGSLGLMFCLIALSTDFWFEAVDYKDDDD KDYKDDDDKDYKDDDDK* (SEQ ID NO: 49)

[0286] LAT-NKG7-PERF:Attorney Docket 44010.215WO-PCT / / CU24341MEEAILVPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVA PWGGGSGGGGSGGGGSGELCRSLALLGGSLGLMFCLIALSTDFWFEAVDYKDDDD KDYKDDDDKDYKDDDDKGGGTCLDYVPQMLLGEPPGNRSGAVW* (SEQ ID NO: 50)

[0287] LCK-NKG7-Perf and LAT-NKG7 show a trend for an increased distance to the MTOC, indicating more dispersed lytic granules (FIG.36). LAMP1-STX1, LAT-NKG7 -Perforin, and LCK-NKG7 -Perforin show increased bystander killing compared to parental cells (FIG.37A- B)

[0288] FIG. 38A shows representative confocal images showing CC2-KRAS modified cells with more dispersed lytic granules and a portion of lytic granule in approximation with the cell cortex. YTS parentals and CC3-KRAS are controls for convergence.

[0289] CC2 KRAS sequence:MENLRKATSNANQDNQICSILLQENTFVEQVVNEKVKHLEDTLKELESQHSILKDEV TYMNNLKLKLEMDAQHIKDEFFHEREDLEFKINELLLAKEEQGCVIEKLKSELAGLN KQFCYTVEQHNREVQSLKEQHQKEISELNETFLSDSEKEKLTLMFEIQGLKEQCENL QQEKQEAILNYESLREIMEILQTELGESAGKISQEFESMKQQQASDVHELQQKLRTAF TEKDALLETVNRLQGENEKLLSQQELVPELENTIKNLQEKNGVYLLSLSQRDTMLKE LEGKINSLTEEKDDFINKLKNSHEEMDNFHKKCEREERLILELGKKVEQTIQYNSELE QKVNELTGGLEETLKEKDQNDQKLEKLMVQMKVLSEDKEVLSAEVKSLYEENNKL SSEGGGGSGGGGSGGGGSDYKDHDGDYKDHDIDYKDDDDKLAIGGGGSGKKKKK KSKTKCVIM* (SEQ ID NO: 51)Over expression of exophilins to increase degranulation and bystander killing

[0290] YTS cells overexpressing melanophilin have increased killing (FIG. 39A). YTS cells overexpressing melanophilin have increased degranulation (FIG. 39B). YTS cells overexpressing melanophilin have increased bystander killing (FIG.39C).Constructs for targeting Lytic Granules

[0291] A specific degranulation tool to interrogate lytic granules is described (FIG. 29). The construct includes an N-terminal domain of NKG7, followed by a protein of interest, and byAttorney Docket 44010.215WO-PCT / / CU24341the C-terminal Perforin / NKG7 / GZMB / A. The protein of interest could be a fluorescent protein and could be used as a tool to:• Visualize lytic granules inside the cell (i.e., live cell imaging).• Visualize a degranulation event of a lytic granule via increased fluorescent protein accumulation on the membrane following NK cell activation.

[0292] Fluorescent proteins could include:• a pH- stable fluorescent molecule such as m-Cherry, mTurquise2, or Gamillus; or • Phluorin or LIME as fluorescent proteins. These are sensitive to pH changes that could be used as degranulation indicator, where the fluorescence changes from the acidic environment of the lytic granule to the more neutral environment on the cell surface following degranulation.

[0293] Applicants note that the lytic granule construct described herein has different targeting moieties and different transmembrane domains as compared to the construct in WO2013 / 025598 Al, which is a degranulation indicator for the lysosome compartment and not specifically for lytic granules. The construct of WO2013 / 025598 also includes an endoplasmic reticulum targeting signal sequence.

[0294] A lytic granule tool for increased delivery of cytotoxic proteins is described (FIG.29).The construct includes an N-terminal domain of NKG7, followed by a protein of interest, followed by C-terminal domain of C-terminal Perforin / NKG7 / GZMB / A.

[0295] The protein of interest could be a peptide that:• Could be toxic (e.g., a toxin) to target cells upon degranulation;• Could be a peptide that has the ability to puncture the membrane of target cells; or • Could be a therapeutic protein (e.g., a defensin).

[0296] The fusion protein is distinguished from any prior constructs, (e.g., US20170182096A1) by including both a structural component of LGs and a signal peptide that sorts the fusion protein to the LG.

[0297] Membrane targeting of LG specifically with an increased cytotoxic payload for more efficient bystander killing is described (FIG.30). The construct allows for membrane targeting of lytic granule specific proteins that contains a protein of interest.

[0298] This protein of interest could be a fluorescent protein and could be used as a tool to:• Visualize lytic granules inside the cell (i.e., live cell imaging); or• Visualize a degranulation event of a lytic granule via increased fluorescent protein accumulation on the membrane following NK cell activation.Attorney Docket 44010.215WO-PCT / / CU24341

[0299] Fluorescent proteins could include:• a pH- stable fluorescent molecule such as m-Cherry, mTurquise2, or Gamillus; or • Phluorin or LIME as fluorescent proteins. These are sensitive to pH changes that could be used as degranulation indicator, where the fluorescence changes from the acidic environment of the lytic granule to the more neutral environment on the cell surface following degranulation.

[0300] The protein of interest could be a peptide that:• Could be toxic (e.g., a toxin) to target cells upon degranulation;• Could be a peptide that has the ability to puncture the membrane of target cells; or • Could be a therapeutic protein (e.g., a defensin).Use of calmodulin for targeting Lytic Granules

[0301] A calmodulin system for membrane targeting is described (FIG. 31). Calmodulin (CaM) presents high affinity for the peptide Ml 3 (KRRWKKNFIAVSAANRFKKISSSGAL) (SEQ ID NO: 47) from skeletal muscle myosin light chain kinase. They interact upon Calcium binding. In this example, the M13 is anchored to the plasma membrane and the calmodulin to the LG. Upon activation of the NK cell and increased release of cytoplasmic Ca2+, it will result in the LG approximating to the plasma membrane through the interaction of CaM and Ml 3 (FIG. 31). Calmodulin can be anchored to the LG through an LG associated peptide comprising a structural component of LGs or a transient adaptor peptide linked to LGs. The fusion protein can further include a C-terminal signal peptide that sorts the fusion protein to the LG and is derived from a protein specifically expressed on LGs (e.g., perforin, a granzyme, or NKG7). The Ml 3 can be anchored to the plasma membrane by fusing it to any plasma membrane directing peptide described herein.EXAMPLE 3 - BIALLELIC GCC2 MUTATION AS A NOVEL CAUSE OF NATURAL KILLER CELL DEFICIENCY

[0302] Natural -Killer cell deficiencies (NKD) are inborn errors of immunity (IEI) in which the major clinically relevant immunodeficiency is that of NK cells leading to increased susceptibility to Herpesviruses, infections and malignancy. NKD can be subclassified as developmental or functional depending upon whether or notNK cell differentiation / maturation is impaired leading to decreased or absent NK cell activity.Attorney Docket 44010.215WO-PCT / / CU24341

[0303] Methods: Applicants describe 2 patients with recurrent EBV and Herpesvirus infections. Exome sequencing (ES) identified compound heterozygous variants in GCC2, a gene encoding Golgin protein essential for tethering cytoplasmic vesicles to Golgi. Applicants studied GCC2 in normal NK cells and modeled GCC2 variant alleles in order to identify roles for the protein in NK cell biology. Patient PBMC and ex-vivo NK cells were used to evaluate NK cell phenotype, cytotoxicity, and lytic immunological synapse formation including lytic granule (LG) positioning. Additionally using the YTS human NK cell line, deletion of GCC2 and introduction of patient derived mutation and its impact upon NK cell biology was investigated.

[0304] Results: GCC2 loss of function (LoF) variant alleles result in defective tethering of LG around the microtubule-organizing center (MTOC) and Golgi, reducing the effectiveness of LG convergence that normally promotes efficiency in cytotoxicity. As a result, dispersed release of LG occurs, reducing killing capacity and leading to a functional NKD.

[0305] Conclusions: Loss of function of GCC2 leads to defective tethering of LG around the MTOC and Golgi, resulting in a reduced killing capacity. To Applicants knowledge this novel immunodeficiency is the first NKD associated with impaired LG convergence.REFERENCES1. Cooper, M.A., Fehniger, T.A., and Caligiuri, M.A. (2001). The biology of human natural killer-cell subsets. Trends Immunol. 22, 633-640.2. Hsu, H.T., Mace, E.M., Carisey, A.F., Viswanath, D.I., Christakou, A.E., Wiklund, M., O nfelt, B., and Orange, J.S. (2016). NK cells converge lytic granules to promote cytotoxicity and prevent bystander killing. J. Cell Biol. 275, 875-889.3. Gwalani, L.A., and Orange, J.S. (2018). Single Degranulations in NK Cells Can Mediate Target Cell Killing. J. Immunol. 200, 3231-3243.4. James, A.M., Hsu, H.T., Dongre, P., Uzel, G., Mace, E.M., Baneijee, P.P., and Orange, J.S. (2013). 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Mace, E.M., Paust, S., Conte, M.I., Baxley, R.M., Schmit, M.M., Patil, S.L., Guilz, N.C., Mukherjee, M., Pezzi, A.E., Chmielowiec, J., et al. (2020). Human NK cell deficiency as a result of biallelic mutations in MCM10. J. Clin. Invest. 130, 5272-5286. doi.org / 10.1172 / JCH34966.Attorney Docket 44010.215WO-PCT / / CU2434141. Conte, ML, Poli, M.C., Taglialatela, A., Leuzzi, G., Chinn, I.K., Salinas, S.A., Rey-Jurado, E., Olivares, N., Veramendi-Espinoza, L., Ciccia, A., et al. (2022). Partial loss-of-function mutations in GINS4 lead to NK cell deficiency with neutropenia. JCI Insight 7, el54948.42. Salinas, S.A., Mace, E.M., Conte, M.I., Park, C.S., Li, Y., Rosario-Sepulveda, J.I., Mahapatra, S., Moore, E.K., Hernandez, E.R., Chinn, I.K., et al. (2022). An ELF4 hypomorphic variant results in NK cell deficiency. JCI Insight 7, el 55481.43. Hughes, C.R., Guasti, L., Meimaridou, E., Chuang, C.H., Schimenti, J.C., King, P.J., Costigan, C., Clark, A.J.L., and Metherell, L.A. (2012). 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Mitani, T., Isikay, S., Gezdirici, A., Gulec, E.Y., Punetha, J., Fatih, J.M., Herman, L, Akay, G., Du, H., Calame, D.G., et al. (2021). High prevalence of multilocus pathogenic variation in neurodevelopmental disorders in the Turkish population. Am. J. Hum. Genet. 108, 1981-2005.48. Stray-Pedersen, A., Sorte, H.S., Samarakoon, P., Gambin, T., Chinn, I.K., Coban Akdemir, Z.H., Erichsen, H.C., Forbes, L.R., Gu, S., Yuan, B., et al. (2017). Primary immunodeficiency diseases: Genomic approaches delineate heterogeneous Mendelian disorders. J. Allergy Clin. Immunol. 139, 232-245.49. Cohen, G.B., Gandhi, R.T., Davis, D.M., Mandelboim, O., Chen, B.K., Strominger, J.L., and Baltimore, D. (1999). The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIVinfected cells from NK cells. Immunity 10, 661-671.Attorney Docket 44010.215WO-PCT / / CU2434150. Hsu, H.T., Carisey, A.F., and Orange, J.S. (2017). Measurement of Lytic Granule Convergence After Formation of an NK Cell Immunological Synapse. Methods Mol. Biol.1584, 497-515.51. Hristova, K., and Wimley, W.C. (2023). Determining the statistical significance of the difference between arbitrary curves: A spreadsheet method. PLoS One 18, e0289619.***

[0306] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.

Claims

Attorney Docket 44010.215WO-PCT / / CU24341CLAIMSWhat is claimed is:

1. A fusion protein for targeting lytic granules (LGs) to the plasma membrane of a cytotoxic lymphocyte, said fusion protein comprising:a plasma membrane directing peptide; anda LG associated peptide comprising a structural component of LGs or a transient adaptor peptide linked to LGs,wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell.

2. The fusion protein of claim 1, wherein:the plasma membrane directing peptide comprises hydrophobic amino acids and / or is capable of being post translationally modified with hydrophobic-related moi eties, optionally, wherein the hydrophobic-related moieties comprise hydrophobic carbon chains, optionally, wherein the hydrophobic carbon chains comprise fatty acids; orthe plasma membrane directing peptide comprises a CAAX motif, optionally, wherein the CAAX motif is derived from the C terminal region of KRAS; orthe plasma membrane directing peptide comprises a C-terminal peptide from a Type IV transmembrane domain; orthe plasma membrane directing peptide comprises a C-terminal transmembrane domain from a syntaxin family member, optionally, syntaxin 1 A (STX1); orthe plasma membrane directing peptide comprises a signal-anchor sequence from a Type II transmembrane (TII) protein, optionally, an N-terminal peptide from a Type II transmembrane (TII) protein; orthe plasma membrane directing peptide comprises an N-terminal domain of a Src Family Kinase (SFK), optionally, wherein the SFK is selected from the group consisting of Src, Yes, Fyn, Fgr, Lek, Hck, Blk, Lyn, and Frk; orthe plasma membrane directing peptide comprises a transmembrane and / or juxtamembrane region of LAT (Linker for Activation of T cells).

3. The fusion protein of claim 1 or 2, wherein the structural component of the LG comprises a transmembrane domain from an LG expressed protein, optionally, wherein theAttorney Docket 44010.215WO-PCT / / CU24341transmembrane domain comprises the transmembrane domain from LAMP1, LAMP2, LAMP3, CD63, CD68, NKG7, or functional fragments or variants thereof.

4. The fusion protein of any one of claims 1-3, wherein the structural component of the LG comprises a transmembrane domain comprising a YXX motif, optionally, wherein the YXX<b motif is either N- or C- terminal of the transmembrane domain.

5. The fusion protein of any one of claims 1 -4, wherein the transient adaptor peptide linked to LGs is derived from a protein associated with LG tethering, optionally, wherein the protein associated with LG tethering is GCC2, Golgin-97 (G-97), a coiled-coil domain that binds an LG associated coiled-coil domain, optionally, coiled-coil domain 2 (CC2) of GCC2, or a truncated GCC2 lacking the C-terminal GRIP domain.

6. The fusion protein of any one of claims 1-5, wherein the fusion protein further comprises a C-terminal signal peptide that sorts the fusion protein to the LG and is derived from a protein specifically expressed on LGs, optionally, wherein the signal peptide is derived from an LG protein selected from the group consisting of perforin, a granzyme, and NKG7.

7. The fusion protein of any one of claims 1-6, wherein the plasma membrane directing peptide is a CAAX motif and the LG associated peptide is GCC2 or Golgin-97; or wherein the plasma membrane directing peptide is a CAAX motif and the LG associated peptide is a LAMP1 transmembrane domain; orwherein the plasma membrane directing peptide is a STX1 motif and the LG associated peptide is a LAMP 1 transmembrane domain; orwherein the plasma membrane directing peptide is an N-terminal domain of a Src Family Kinase (SFK), the LG associated peptide is the N-terminal domain of NKG7, and the C-terminal signal peptide that sorts the fusion protein to the LG is derived from perforin, NKG7, or a granzyme; orwherein the plasma membrane directing peptide is an N-terminal domain of Lek, the LG associated peptide is the N-terminal domain of NKG7, and the C-terminal signal peptide that sorts the fusion protein to the LG is derived from perforin; orwherein the plasma membrane directing peptide is a transmembrane and / or juxtamembrane region of LAT, and the LG associated peptide is the N-terminal domain of NKG7; orAttorney Docket 44010.215WO-PCT / / CU24341wherein the plasma membrane directing peptide is a transmembrane and / or juxtamembrane region of LAT, the LG associated peptide is the N-terminal domain of NKG7, and the C-terminal signal peptide that sorts the fusion protein to the LG is derived from perforin; orwherein the plasma membrane directing peptide is a is a CAAX motif and the LG associated peptide is a coiled-coil domain 2 (CC2) of GCC2.

8. A fusion protein for targeting lytic granules (LGs) comprising:a) a LG associated peptide comprising a structural component of LGs;b) a protein of interest; andc) a signal peptide that sorts the fusion protein to the LG.

9. The fusion protein of claim 8, wherein the structural component of LGs is derived from LAMP1, NKG7, or any transmembrane domain flanked by a YXX motif; and / or wherein the protein of interest is a detectable marker, therapeutic protein, or toxic protein, optionally, wherein the detectable marker is a fluorescent protein; and / or wherein the signal peptide is derived from an LG protein selected from the group consisting of perforin, a granzyme, and NKG7.

10. The fusion protein of claim 8 or 9, wherein the fusion protein further comprises a plasma membrane directing peptide.

11. The fusion protein of claim 9, wherein the fusion protein comprises the N-terminal domain of NKG7, the protein of interest, and a C-terminal domain from perforin, NKG7, or a granzyme.

12. One or more vectors encoding the fusion protein according to any one of claims 1-11.

13. A system for targeting lytic granules (LGs) to the plasma membrane of a cytotoxic lymphocyte comprising:a first fusion protein comprising an LG associated coiled-coil peptide and a signal peptide that sorts the fusion protein to the LG and is derived from a protein specifically expressed on LGs, optionally, coiled-coil domain 2 (CC2) of GCC2; anda second fusion protein comprising a transmembrane domain specific to the plasma membrane and a coiled-coil containing protein capable of binding the first fusion protein, wherein the first and second fusion proteins are encoded for by one or more vectors,Attorney Docket 44010.215WO-PCT / / CU24341wherein the cytotoxic lymphocyte is a natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or natural killer T (NKT) cell.

14. An isolated cytotoxic lymphocyte:modified to express the fusion protein of any of claims 1-11, optionally, wherein expression of the fusion protein is inducible; orgenetically modified to delete GCC2; orgenetically modified to overexpress one or more exophilin proteins selected from the group consisting of SYTL-1, SYTL-2, SYTL-2, SYTL-3, SYTL4, exophilin-5, and melanophilin (MLPH),wherein the cytotoxic lymphocyte is a Natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or Natural killer T (NKT) cell.

15. An isolated cytotoxic lymphocyte modified to express the system of claim 13, wherein the cytotoxic lymphocyte is a Natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or Natural killer T (NKT) cell.

16. A method of targeting lytic granules (LGs) to the plasma membrane of a cytotoxic lymphocyte to increase cytotoxicity, increase bystander killing, and / or enhance anti-tumor activity, the method comprising anchoring LGs to the plasma membrane:using plasma membrane-directing peptides that bind directly or indirectly to the LG; or by overexpressing one or more exophilin proteins,wherein the cytotoxic lymphocyte is a Natural killer (NK) cell, CD8+cytotoxic T cell (CTL), or Natural killer T (NKT) cell.

17. The method of claim 16, comprising expressing the fusion protein according to any one of claims 1-11 in the cytotoxic lymphocyte, thereby anchoring LGs to the plasma membrane.

18. The method of claim 16, wherein the one or more exophilin proteins are selected from the group consisting of SYTL-1, SYTL-2, SYTL-3, SYTL4, exophilin-5, and melanophilin (MLPH).

19. A method of treating cancer in a subject in need thereof comprising administering the isolated cytotoxic lymphocyte according to claim 14 or 15 to the tumor of the subject; or administering lipid nanoparticles carrying modified mRNA targeting cytotoxic lymphocytes in vivo and encoding the fusion protein or system of any one of claims 1-13.Attorney Docket 44010.215WO-PCT / / CU2434120. The method of claim 19, wherein the isolated cytotoxic lymphocyte expresses an exogenous T cell receptor (TCR) or a chimeric antigen receptor (CAR).

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