Compositions and methods related to populations of cell-derived nanoparticles (CDNPS)

Abstract

Provided herein, at least in part, are compositions comprising distinct populations of cell-derived nanoparticles (CDNPs) and related methods. Such compositions can be used in the expansion of effector cells, such as T cells or NK cells. Related methods comprise methods of expanding effector cells, administering the expanded effector cells for therapeutic purposes as well as methods of producing the compositions comprising CDNP populations.

Description

[0001] COMPOSITIONS AND METHODS RELATED TO POPULATIONS OF CELL- DERIVED NANOPARTICLES (CDNPs)

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 704,001, entitled “COMPOSITIONS AND METHODS RELATED TO POPULATIONS OF CELL-DERIVED NANOPARTICLES (CDNPs)”, filed on October 6, 2024, the entire contents of which are incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] Provided herein, at least in part, are compositions comprising distinct populations of cell-derived nanoparticles (CDNPs) and related methods. Such compositions can be used in the expansion of effector cells, such as T cells or NK cells. Related methods comprise methods of expanding effector cells, administering the expanded effector cells for therapeutic purposes as well as methods of producing the compositions comprising CDNP populations.

[0006] SUMMARY OF THE INVENTION

[0007] In an aspect, a composition comprising a first population of cell-derived nanoparticles CDNPs, and a second population of CDNPs; wherein the CDNPs of the first population and the second population express a cell surface signal, or set of cell surface signals, and wherein the cell surface signal, or set of cell surface signals, of the first population and the second population are distinct, is provided.

[0008] In an aspect, a composition comprising a first population of cell-derived nanoparticles CDNPs, and a second population of CDNPs; wherein the CDNPs of the first population and the second population are produced from cells that were engineered to express a cell surface signal, or set of cell surface signals, and wherein the cell surface signal, or set of cell surface signals, of the first population and the second population are distinct, is provided.

[0009] In an embodiment, any one of the compositions provided herein can further comprise a third population of CDNPs, wherein the CDNPs of the third population are produced from cells that were engineered to express a cell surface signal, or set of cell surface signals, such as IL-7, IL-15, IL-21 or 4-1BBL, and wherein the cell surface signal, or set of cell surface signals, of the first population, the second population, and the third population are distinct.

[0010] #14471824vl In an aspect, a composition comprising a first population of first cell line population cell-derived nanoparticles CDNPs, and a second population of second cell line population CDNPs; wherein the first cell line population and the second cell line population are engineered to express a cell surface signal, or set of cell surface signals, and wherein the first cell line and second cell line each comprise a cell surface signal, or set of cell surface signals, that are distinct, is provided.

[0011] In an embodiment, any one of the compositions provided herein can further comprise a third population of third cell line population CDNPs, wherein the third cell line population is engineered to express a cell surface signal, or set of cell surface signals, such as IL-7, IL- 15, IL-21 or 4-1BBL and wherein the first cell line, second cell line and third cell line comprise cell surface signals, or sets of cell surface signals, that are distinct.

[0012] In an aspect, a method comprising placing a first population of CDNPs and a second population of CDNPs in contact with effector cells; wherein the CDNPs of the first population and the second population each express a cell surface signal, or set of cell surface signals, and wherein the cell surface signal, or set of cell surface signals, of the first population and the second population are distinct, is provided.

[0013] In an embodiment, any one of the methods provided herein can further comprise placing a third population of CDNPs in contact with the effector cells, wherein the CDNPs of the third population express a cell surface signal, or set of cell surface signals, such as IL-7, IL-15, IL-21 or 4-1BBL, and wherein the cell surface signal, or set of cell surface signals, of the first population, the second population and third population are distinct.

[0014] In an embodiment of any one of the compositions or methods provided herein, the cell surface signal, or set of cell surface signals, is / comprises stimulatory molecule(s) and / or ligand(s), costimulatory molecule(s) and / or ligand(s) and / or cytokine(s). In an embodiment of any one of the compositions or methods provided herein, the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) comprise CD3L (e.g., an anti-CD3 antibody), CD86, IL-7, IL-15 and 4-1BBL. In an embodiment of any one of the compositions or methods provided herein, the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) is IL-7, IL- 15, IL-21 or 4-1BBL.

[0015] In an embodiment of any one of the compositions or methods provided herein, the CDNPs of the first population comprise CD3L (e.g., an anti-CD3 antibody), CD86, IL-7, IL- 15 and 4-1BBL. In an embodiment of any one of the compositions or methods provided herein, the CDNPs of the second population comprise IL-7, IL-15, IL-21 or 4-1BBL. In an

[0016] #14471824vl embodiment of any one of the compositions or methods provided herein, the CDNPs of the third population comprise IL-7, IL-15, IL-21 or 4-1BBL. In an embodiment of any one of the compositions or methods provided herein, the CDNPs of the second or third population comprise IL-7 and / or IL- 15. In an embodiment of any one of the compositions or methods provided herein, the CDNPs of the second or third population comprise IL-21. In an embodiment of any one of the compositions or methods provided herein, the CDNPs of the second or third population comprise 4-1BBL.

[0017] In an embodiment of any one of the compositions or methods provided herein, the CDNPs of the first population comprise IL-7, IL-15, IL-21 or 4-1BBL, such as IL-7, and the CDNPs of the second population comprise IL-7, IL- 15, IL-21 or 4-1BBL, such as IL- 15, with the cell surface signal, or set of cell surface signals, of the first and second population being distinct.

[0018] In an embodiment of any one of the compositions or methods provided herein, the CDNPs of the third population comprise CD3L (e.g., an anti-CD3 antibody), CD86, IL-7, IL- 15 and 4-1BBL.

[0019] In an embodiment of any one of the compositions or methods provided herein, the IL- 15 is in combination with IL-15Ra.

[0020] In an embodiment of any one of the methods provided herein, the placing in contact of the second and / or third populations occurs at the same time as the first population of CDNPs. In an embodiment of any one of the methods provided herein, the placing in contact of the second and / or third populations does not occur at the same time as the first population of CDNPs.

[0021] In an embodiment of any one of the methods provided herein, the placing in contact of the second and / or third populations occurs, or also occurs, at least three days after the first population of CDNPs. In an embodiment of any one of the methods provided herein, the placing in contact of the second and / or third populations occurs, or also occurs, at least six days after the first population of CDNPs. In an embodiment of any one of the methods provided herein, the placing in contact of the second and / or third populations occurs, or also occurs, at least seven days after the first population of CDNPs. In an embodiment of any one of the methods provided herein, the placing in contact of the second and / or third populations occurs, or also occurs, at least nine days after the first population of CDNPs.

[0022] In an embodiment of any one of the methods provided herein, the placing in contact of the second and / or third populations occurs, or also occurs, at least three days after and at

[0023] #14471824vl least six days after the first population of CDNPs. In an embodiment of any one of the methods provided herein, the placing in contact of the second and / or third populations occurs, or also occurs, at least three days after and at least seven days after the first population of CDNPs. In an embodiment of any one of the methods provided herein, the placing in contact of the second and / or third populations occurs, or also occurs, at least three days after and at least nine days after the first population of CDNPs.

[0024] In an embodiment of any one of the methods provided herein, the second population of CDNPs is placed in contact with the effector cells at the same time as the first population of CDNPs. In an embodiment of such a method, the second population of CDNPs comprise IL-15, IL-21 or 4-1BBL. In an embodiment of any one of the methods provided herein, the second population of CDNPs is not placed in contact with the effector cells at the same time as the first population of CDNPs. In an embodiment of such a method, the second population of CDNPs comprise IL-7.

[0025] In an embodiment of any one of the methods provided herein, the second population of CDNPs is placed in contact with the effector cells at least three days after the first population of CDNPs. In an embodiment of such a method, the second population of CDNPs comprise IL-7 and / or IL- 15. In an embodiment of any one of the methods provided herein, the second population of CDNPs is placed in contact with the effector cells at least six or seven days after the first population of CDNPs. In an embodiment of such a method, the second population of CDNPs comprise IL-21 or 4-1BBL or comprise IL-7 and / or IL- 15. In an embodiment of any one of the methods provided herein, the second population of CDNPs is placed in contact with the effector cells at least three days and at least seven days after the first population of CDNPs. In an embodiment of such a method, the second population of CDNPs comprise IL-7 and / or IL- 15. In an embodiment of any one of the methods provided herein, the second population of CDNPs is placed in contact with the effector cells at the same time as the first population and at least three days and at least seven days after the first population of CDNPs. In an embodiment of such a method, the second population of CDNPs comprise IL- 15. In an embodiment of any one of the methods provided herein, the second population of CDNPs is not placed in contact with the effector cells at the same time as the first population but in contact with the effector cells at least three days and at least seven days after the first population of CDNPs. In an embodiment of such a method, the second population of CDNPs comprise IL-7.

[0026] #14471824vl In an embodiment of any one of the methods provided herein, the second population of CDNPs is placed in contact with the effector cells at the same time as the first population and at least six days after the first population of CDNPs. In an embodiment of such a method, the second population of CDNPs comprise IL-21 or 4-1BBL. In an embodiment of any one of such methods, the CDNPs of the first population comprise CD3L (e.g., an anti-CD3 antibody), CD86, IL-7, IL-15 and 4-1BBL.

[0027] In an embodiment of any one of the methods provided herein, a third population of CDNPs is placed in contact with the effector cells at the same time as the second population of CDNPs. In an embodiment of any one of such methods, the third population of CDNPs and second population of CDNPs are placed in contact with the effector cells at least three days after the first population of CDNPs. In an embodiment of any one of such methods, the third population of CDNPs and second population of CDNPs are placed in contact with the effector cells at least seven days after the first population of CDNPs. In an embodiment of any one of such methods, the third population of CDNPs and second population of CDNPs are placed in contact with the effector cells at least three days after and at least seven days after the first population of CDNPs. In an embodiment of any one of such methods, the second population of CDNPs comprise IL-7 and the third population of CDNPs comprise IL- 15. In an embodiment of any one of such methods, the second population of CDNPs is not placed in contact with the effector cells at the same time as the first population of CDNPs, but, optionally, the third population of CDNPs are placed in contact with the effector cells at the same time as the first population of CDNPs. In an embodiment of any one of such methods, the CDNPs of the first population comprise CD3L (e.g., an anti-CD3 antibody), CD86, IL-7, IL-15 and 4-1BBL.

[0028] In an embodiment of any one of the methods provided herein, the first population of CDNPs comprise IL-7, IL-15, IL-21 or 4-1BBL, such as IL-7, and the second population of CDNPs comprise IL-7, IL-15, IL-21 or 4-1BBL, such as IL-15. In an embodiment of any one of such methods, the first population of CDNPs and the second population of CDNPs are placed in contact with the effector cells at the same time. In an embodiment of any one of such methods, the first population of CDNPs and the second population of CDNPs are placed in contact with the effector cells at least six or seven days after a third population of CDNPs had been placed in contact with the effector cells. In an embodiment of any one of such methods, the first population of CDNPs and the second population of CDNPs are placed in contact with the effector cells at least three days and / or at least seven days after a third

[0029] #14471824vl population of CDNPs had been placed in contact with the effector cells. In an embodiment of any one of such methods, the CDNPs of the third population comprise CD3L (e.g., an anti- CD3 antibody), CD86, IL-7, IL- 15 and 4-1BBL. In an embodiment of any one of such methods, the method further comprises a step of placing the CDNPs of the third population in contact with the effector cells.

[0030] In an embodiment of any one of the methods provided herein, the contacting is according to any one of the methods provided herein, including as shown in the Examples.

[0031] In an embodiment, any one of the methods provided herein can be used for producing, obtaining, increasing or enriching the number of effector cells with a desirable phenotype. In one embodiment of any one of such methods, the cells with a desirable phenotype are less or non-fully diffentiated cells. In one embodiment of any one of such methods, the cells with a desirable phenotype are central memory cells. In one embodiment of any one of such methods, the desirable phenotype is CD62L+. In one embodiment of any one of such methods, the cells with a desirable phenotype are TSCM cells. In one embodiment of any one of such methods, the cells with a desirable phenotype are Tcm cells.

[0032] In an embodiment of any one of the compositions or methods provided herein, the composition further comprises effector cells. In an embodiment of any one of the compositions or methods provided herein, the effector cells are T cells. In an embodiment of any one of the compositions or methods provided herein, the effector cells are NK cells.

[0033] In an embodiment of any one of the methods provided herein, the placing in contact occurs in vitro or ex vivo.

[0034] In an embodiment of any one of the methods provided herein, the method further comprises obtaining the expanded effector cells.

[0035] In an embodiment of any one of the methods provided herein, the method further comprises purifying the expanded effector cells.

[0036] In an embodiment of any one of the methods provided herein, further comprising administering the expanded effector cells to a subject.

[0037] In an aspect, a method for producing population(s) of CDNPs comprising processing two or more cell lines to form population(s) of CDNPs, wherein each cell line is engineered to express a cell surface signal, or set of cell surface signals, and wherein the cell surface signal, or set of cell surface signals, of the cell lines are distinct; and obtaining the population(s) of CDNPs, is provided. In an embodiment of any one of such methods, the cell lines are processed separately or together and / or the populations of CDNPs that results are

[0038] #14471824vl separate in different compositions or together, when the cell lines are mixed during the process or when the CDNPs from the separately processed cell lines are obtained and subsequently mixed.

[0039] In an embodiment of any one of the methods provided herein, the two or more cell lines are maintained in the same cell culture.

[0040] In an embodiment of any one of the methods provided herein, the two or more cell lines are maintained in separate cell cultures.

[0041] In an embodiment of any one of the methods provided herein, one population of CDNPs is obtained with the population comprising CDNPs formed from each of the two or more cell lines. In an embodiment of any one of the methods provided herein, two or more populations of CDNPs are obtained with each of the two or more populations of CDNPs being formed from one of the two or more cell lines, respectively. In an embodiment of such a method, each of the two or more populations are comprised in a composition separate from the other population(s) or each of the two or more populations are mixed to be comprised in a single composition.

[0042] In an embodiment of any one of the methods provided herein, the method further comprises engineering each cell line of the two or more cell lines to express the cell surface signal, or set of cell surface signals.

[0043] In an embodiment of any one of the compositions or methods provided herein, the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) and / or cytokine(s) of each of the at least two cell lines are distinct from the other. In an embodiment of any one of the compositions or methods provided herein, the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) and / or cytokine(s) of one of the at least two cell lines comprise CD3L (e.g., an anti-CD3 antibody), CD86, 4-1BBL, IL-7 and IL- 15. In an embodiment of any one of the compositions or methods provided herein, the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) and / or cytokine(s) of one of the at least two cell lines comprise IL-7, IL-15, IL-21 or 4-1BBL, such as IL-7 and / or IL-15. In an embodiment of any one of the compositions or methods provided herein, the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) and / or cytokine(s) of one of the at least two cell lines comprise IL-21. In an embodiment of any one of the compositions or methods provided herein, the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) and / or cytokine(s) of one of the at least two cell lines comprise 4-1BBL.

[0044] #14471824vl In an embodiment of any one of the compositions or methods provided herein, the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) and / or cytokine(s) of one of the at least two cell lines comprise IL-7, IL- 15, IL-21 or 4- 1BBL, such as IL-7, and the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) and / or cytokine(s) of another one of the at least two cell lines comprise IL-7, IL-15, IL-21 or 4-1BBL, such as IL-15, with the cell surface signal, or set of cell surface signals, of the cell lines being distinct.

[0045] In an embodiment of any one of the compositions or methods provided herein, the at least two cell lines are three cell lines, and the third cell line is engineered to express a cell surface signal, or set of cell surface signals, such as CD3L (e.g., an anti-CD3 antibody), CD86, IL-7, IL-15 and 4-1BBL, with the cell surface signal, or set of cell surface signals, of the cell lines being distinct. In an embodiment of any one of the compositions or methods provided herein, the at least two cell lines are three cell lines, and the third cell line is engineered to express a cell surface signal, or set of cell surface signals, such as IL-7, IL-15, IL-21 or 4-1BBL, with the cell surface signal, or set of cell surface signals, of the cell lines being distinct.

[0046] In an embodiment of any one of the compositions or methods provided herein, the at least two cell lines are three cell lines, wherein the third cell line is engineered to express a cell surface signal, or set of cell surface signals, such as IL-7 or IL- 15, with the cell surface signal, or set of cell surface signals, of the cell lines being distinct.

[0047] In an embodiment of any one of the compositions or methods provided herein, the IL- 15 is in combination with IL-15Ra.

[0048] In an embodiment of any one of the methods provided herein, the processing comprises disruption of the cells of the two or more cell lines. In an embodiment of any one of the methods provided herein, the disruption is mechanical disruption. In an embodiment of any one of the methods provided herein, the mechanical disruption is cavitation, homogenization, or extrusion. In an embodiment of any one of the methods provided herein, the disruption is chemical disruption.

[0049] In an embodiment of any one of the methods provided herein, when the two or more cell lines are maintained in separate cell cultures, the processing comprises mixing the cells of the two or more cell lines to form a mixed cell culture. In an embodiment of any one of the methods provided herein, when the processing comprises disruption, the mixing occurs prior to the disruption of the cells of the mixed cell culture.

[0050] #14471824vl In an embodiment of any one of the methods provided herein, when the two or more cell lines are maintained in separate cell cultures and the processing comprises disruption, the processing further comprises collecting and mixing CDNPs from each cell culture subsequent to disruption of the cells of each cell culture.

[0051] In an embodiment of any one of the methods provided herein, the obtaining comprises extracting filtering and / or purifying.

[0052] In an aspect, a composition comprising any one or any one combination of population(s) of CDNPs provided herein, such as produced by any one of the methods provided herein, is provided.

[0053] In an aspect, a method comprising administering the expanded effector cells produced by any one of the methods provided herein, to a subject in need thereof, is provided.

[0054] In an aspect, a cell line or set of cell lines that are each engineered to express a cell surface signal or set of cell surface signals, which cell surface signal(s) being distinct for each cell line or set of cell lines, is provided. In an embodiment, the cell surface signal or set of cell surface signals is / are those of any one or any one combination of cell surface signal(s) provided herein.

[0055] In an aspect, a composition comprising any one or any one combination of cell lines provided herein, such as produced by any one of the methods provided herein, is provided. Further provided by this disclosure are kits comprising any one of the compositions as described herein or any one of the compositions as produced by or used in any one of the methods described herein. In an embodiment, the kit comprising any one or any one combination of CDNPs provided herein, such as produced by any one of the methods provided herein, is provided. In an embodiment, the kit comprising any one or any one combination of the cell lines provided herein, such as produced by any one of the methods provided herein, is provided. In an embodiment of any one of the kits provided herein, the populations of CDNPs or cell lines are contained within separate containers in the kit. In an embodiment of any one of the kits provided herein, the container is a vial or an ampoule. In an embodiment of any one of the kits provided herein, other component(s) are contained within a solution separate from the container(s), such that the other component(s) may be added to the container(s) at a subsequent time. In an embodiment of any one of the kits provided herein, the kit further comprises instructions for reconstitution, mixing, administration, extraction or distribution, etc. In an embodiment of any one of the kits provided herein, the instructions include a description of any one of the methods described

[0056] #14471824vl herein. In an embodiment of any one of the kits provided herein, the kit further comprises one or more syringes or other means for administering, extracting or distributing the CDNPs or cells or any other components.

[0057] BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG. 1 shows results from the addition of CDNP-IL-7 / CDNP-IL-15, which addition pronouncedly boosted cell division. T cells from healthy donors were initially stimulated on day 0 under various conditions as shown. DL1 is dose level one (equivalent to soluble IL-7 and IL- 15 concentrations of 5 ng / mL and 10 ng / mL, respectively). DL1 / 2 is dose level 0.5 x DLL On day 6 post-stimulation, activated T cells were reseeded and restimulated under the conditions shown. On day 9, cell counts were determined using NC-200, and the population doubling levels (PDLs) and fold expansion were calculated.

[0059] FIG. 2 shows a summary of the fold expansion (FIG. 2A) and population doubling levels (PDLs) (FIG. 2B) of T cells by initial stimulation / restimulation on day 9 (as indicated). T cells were from healthy donors. N=3 healthy donors.

[0060] FIG. 3 also demonstrated that CDNPs comprising cell surface signals, such as cytokines, can boost cell division. T cells were from healthy donors. N=3 healthy donors. Fold expansion (FIG. 3A) and PDL (FIG. 3B) results are shown for stimulation / restimulation as indicated.

[0061] FIG. 4 demonstrates that boost in T cell division can occur in a dose-dependent manner with cell surface signals. T cells from healthy donors were stimulated / restimulated (on day 6) as indicated. DL1 is dose level one (equivalent to soluble IL-7 and IL- 15 concentrations of 5 ng / mL and 10 ng / mL, respectively). DL1 / 2 is dose level 0.5 x DLL DL1 / 4 is dose level 0.25 x DLL DL1 / 8 is 1 / 8 x DL1, and DL1 / 16 is 1 / 16 x DLL On day 9, cell counts were determined using NC-200, and the fold expansion was calculated. N=2 healthy donors.

[0062] FIG. 5 shows the addition of CDNP-IL-21 boosted T cell proliferation (FIG. 5A), including CD8+ T cell proliferation (FIG. 5B). T cells from healthy donors were stimulated on day 0 under various conditions as indicated. On day 7, cell counts from all conditions were determined using NC-200, and the fold expansion was calculated. On day 9, T cell populations were analyzed by a flow cytometry assay.

[0063] FIG. 6 demonstrates that the inclusion of CDNP-4-1BBL enhances T cell expansion. T cells from healthy donors were stimulated on day 0 under various conditions as indicated.

[0064] #14471824vl The indicated dose levels were as defined above. On day 7, cell counts were determined using NC-200, and the fold expansion was calculated.

[0065] FIG. 7 shows that CDNP-IL-21 supplementation improves NK cell expansion. PBMCs from a leukemia patient were stimulated on day 0 under various conditions as indicated. NK cells were defined as CD3- / CD56+ population by flow cytometry assay.

[0066] FIG. 8 shows that CDNP-IL-21 (FIG. 8A) and CDNP-4-1BBL (FIG. 8B) supplementation improves NK cell expansion. PBMCs from a leukemia patient were stimulated on day 0 under various conditions as indicated. NK cells were defined as CD3- / CD56+ population by flow cytometry assay.

[0067] FIG. 9 demonstrates that T cell expansion (calculated as a ratio of total cells at indicated timepoints to initial number of calls plated on day 0) measured at 7 and 9 days (D7 or D9 within each bar) after treatment with 35K of CDNPs alone or combined with soluble or cell-derived IL-7 / IL-15. Doses and timing of cytokine administration are indicated on the X- axis. Averages ± SD are shown for each group at both time points. Fold expansion at both time-points over CDNPs used alone indicated for select groups (rounded to the second digit after the decimal).

[0068] FIG. 10 shows the total number of CD4+CD8- and CD4-CD8+ T cells (calculated as % of each population (as assessed by flow cytometry) multiplied by a total T cell number) measured at 7 (FIG. 10A) and 9 (FIG. 10B) days after treatment with 35K of CDNPs alone or combined with soluble or cell-derived IL-7 / IL-15. Doses and timing of cytokine administration are indicated on the X-axis. Averages ± SD are shown for each population within each group.

[0069] FIG. 11 shows the total number of TN, TSCM, TCM, TEM-like (CD62L+), TEFF-like (CD62L+), TEM-like (CCR7+), TEM and TEFF cells (calculated as % of each population as assessed by flow cytometry multiplied by a total T cell number) measured at 7 (FIG. 11A) and 9 (FIG. 11B) days after treatment with 35K of CDNPs alone or combined with soluble or cell-derived IL-7 / IL-15. Doses and timing of cytokine administration are indicated on the X- axis. Averages ± SD are shown for each population within each group.

[0070] FIG. 12 shows the total number of CD62L+ cells (calculated as % of each population as assessed by flow cytometry multiplied by a total T cell number) measured at 3, 7 and 9 days after treatment with 35K of CDNPs alone or combined with soluble or cell-derived IL- 7 / IL-15. Doses and timing of cytokine administration are indicated on the X-axis. Averages ± SD are shown for each population within each group at all timepoints.

[0071] #14471824vl DETAILED DESCRIPTION OF THE INVENTION

[0072] Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified materials or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting of the use of alternative terminology to describe the present invention.

[0073] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety for all purposes.

[0074] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a CDNP" includes a mixture of two or more such CDNPs, a plurality of such CDNPs, or a population of such CDNPs, and the like.

[0075] As used herein, the term “comprise” or variations thereof such as “comprises” or “comprising” are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, elements, characteristics, properties, method / process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein, the term “comprising” is inclusive and does not exclude additional, unrecited integers or method / process steps.

[0076] In embodiments of any one of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of’. The phrase “consisting essentially of’ is used herein to require the specified integer(s) or steps as well as those which do not materially affect the character or function of the claimed invention. As used herein, the term “consisting of’ is used to indicate the presence of the recited integer (e.g. a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, elements, characteristics, properties, method / process steps or limitations) alone.

[0077] Although this specification, including any examples, fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto.

[0078] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if

[0079] #14471824vl it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0080] Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0081] Introduction

[0082] Currently, cell-based therapeutics, such as adoptive transfer therapies involving the use of effector cells, such as T cells, hold the promise for the treatment of many diseases, such as cancer and infectious disease. Such therapies generally involve the infusion of cells that have been stimulated, cloned, and expanded for many weeks in vitro, for example, using autologous dendritic cells (DC), virally infected B cells, and / or allogeneic feeder cells. However, a major challenge for such therapies is the need for large numbers of therapeutic cells. Adoptive T cell immunotherapy clinical trials routinely require billions of cells per patient. For patients with low initial cell numbers, efficient ex vivo expansion can be crucial. For allogeneic therapies, efficient and robust cell production is beneficial. Commonly, to produce these quantities of cells, the cells must be subjected to manyfold expansion in vitro.

[0083] Current methods for expanding cells, however, have proven to have drawbacks. In particular, obtaining a sufficient number of useful therapeutic cells, especially effector cells, has been particularly difficult. Additionally, the various techniques available for expanding such cells have relied primarily on the use of accessory cells. The requirement for accessory cells presents a significant problem for long-term culture systems because these cells are

[0084] #14471824vl relatively short-lived. A need exists to provide robust methods to expand therapeutic cells, such as effector cells, for example T cells, and to do so in a controllable, efficient and cost- effective manner. The compositions and methods provided herein address such needs. The compositions and methods provided herein can not only provide for robust effector cell expansion but can do so in a way that is controllable, with cell surface signals of interest, and cost effective. Moreover, the compositions and methods provided herein, surprisingly and unexpectedly, can provide beneficial features in effector cell expansion.

[0085] Cell-derived Nanoparticles (CDNPs) and Compositions Thereof

[0086] It has been surprisingly and unexpectedly found that CDNPs that express cell surface signals can be produced and used to boost the expansion of effector cells, such as T cells and NK cells, in a way that can be more robust and / or controllable than current methods. Additionally, more robust expansion has been seen with cell surface signals contained in CDNPs as compared to the same signals but in soluble form. As described in the Examples, combinations of different CDNP populations after initial stimulation of effector cells were found to significantly increase effector cell expansion as compared to soluble signals, such as cytokines, with the same signals. In other words, the addition of cell surface signals to boost effector cell expansion of effector cell cultures is more robust when such signals are contained in CDNPs as provided herein rather than in soluble form. For example, as described in more detail in the Examples, the addition of CDNPs carrying IL-21 as well as CDNPs carrying 4-1BBL significantly improved activated T cell proliferation and expansion. Consistent with these results was the additional expansion of NK cells stimulated by CDNPs carrying IL-21 as well as CDNPs carrying 4-1BBL when activated by CDNP4. Further, again as described in more detail in the Examples, CDNPs carrying IL-7 and / or IL- 15 boosted the expansion of T cells that had been activated by CDNPs carrying a set of costimulatory molecules more robustly with the combination of CDNP populations comprising IL-7 and IL- 15 (in two populations of CDNPs in the Examples showing superior results).

[0087] Accordingly, these findings demonstrate that the use of populations of CDNPs with different signals can result in improved effector cell expansion. Also importantly, such improved expansion does not require the creation of a new cell line or the use of all initial stimulation signals. Instead, it has been shown that the subsequent addition of specific signals provided in CDNPs can allow for effector cell expansion improvements. Thus, the compositions and methods provided herein represent an efficient and cost-effective way to

[0088] #14471824vl “mix and match” cell signals for the optimization and improvement of effector cell expansion. Such mixing and matching of cell signals also allows for more rapid exploration of the optimization and improvement of effector cell therapies for patients in need thereof. Overall, the compositions and methods provided herein allow for flexibility in the expansion of effector cells similar to the flexibility afforded by the use of soluble signals. Such flexibility can be helpful in the optimization of processes for effector cell expansion in culture. Moreover, the compositions and methods provided herein have been demonstrated to be able to provide superior results in effector cell expansion as compared to soluble signals, such as cytokines. Such superior performance is shown in the Examples with IL-7, IL- 15, IL-21 and 4-1BBL, and it will be readily apparent to one of ordinary skill in the art that the results from these examples can be extended to other cell surface signals as provided herein.

[0089] Moreover, in addition to the aforementioned benefits, the compositions and methods provided herein were also suprisingly found to provide not only increased numbers of effector cells with desirable phenotypes but also an enrichment of effector cells with such phenotypes within a population of cells that are undergoing expansion. The effector cells with such phenotypes include those that are less or non-fully diffentiated, such as TSCM cells. Such cells are desirable for their ability to differentiate, grow and / or migrate within the body. Such cells are also less exhausted than others in cell culture and have the opportunity for renewal. This is also shown in the Examples. For example, it was found that at Day 9, the levels of effector cells carrying CD62L, such as TSCM cells, were not only increased in number but also increased preferentially as compared to the other cell phenotypes, such as those not carrying CD62L. Any one of the methods or compositions provided herein can be used for producing or enriching effector cells with any one of such desirable phenotypes.

[0090] As used herein, “cell-derived nanoparticles” or “CDNPs” refer to cell membranecontaining particles or vesicles derived from cells engineered to express a specific cell surface signal or specific set of cell surface signals. Cell surface signals of interest as provided herein are contained in these particles or vesicles accordingly are also referred to herein as being cell-derived. The CDNPs provided herein are not those produced naturally (z.e., not naturally produced extracellular vesicles) but rather are derived and obtained from cells engineered to express a desired cell surface signal or set of cell surface signals. The CDNPs provided herein are thus designed to comprise specific cell surface signal(s) and can be used in combination with other CDNPs also designed to comprise specific cell surface signal(s), which cell surface signal(s) are desirable in the expansion of effector cells.

[0091] #14471824vl Provided herein are CDNPs as well as compositions comprising CDNPs that comprise a cell surface signal or set of cell surface signals, which CDNPs and compositions thereof can be used to expand effector cells. Preferably, the compositions comprising CDNPs as provided herein comprise populations of CDNPs that comprise a specific cell surface signal or specific set of cell surface signals where each population of CDNPs is derived from cells engineered to express the specific cell surface signal or specific set of cell surface signals. Compositions comprising any one, two or more of the populations of CDNPs are provided herein.

[0092] As used herein, a “population of CDNPs” refers to a number of CDNPs of like composition and cell surface signal(s) of interest. Preferably, in some embodiments, a population of CDNPs are derived from substantially similar, preferably the same, cell lines and have at least substantially similar, preferably the same, cell surface signal or set of cell surface signals of interest. A population of CDNPs may be different or distinct from another population of CDNPs in that the cell surface signal or set of cell surface signals of interest of the CDNPs are not identical, meaning that at least one cell surface signal is not shared between the two populations although one or more other cell surface signals may be found on both populations of CDNPs. In other words, a population of CDNPs is “distinct” from another population of CDNPs where the cells from which the CDNPs of the population are derived are those of a different cell line and / or are engineered to express at least one cell surface signal that the cells from which the other population of CDNPs are derived are not engineered to express, or vice versa. For example, a population of CDNPs that comprise IL-7 is considered distinct from a population comprising IL-7 and IL- 15, when such cell surface signal(s) are of interest for the compositions and methods as provided herein for effector cell expansion. In such an embodiment, the two populations of CDNPs are distinct. It is noted, however, that the use of “first” or “second” (or “third”, in some embodiments) with respect to CDNP populations provided herein are intended to refer to two (or more) populations that are different from each other but are not intended to denote order in time. It is also noted, that the foregoing use of “first” or “second” (or “third”, in some embodiments) may refer to the same distinct type of CDNP in a population that can be used more than once, such as at a different point in time, in some embodiments. For example, a particular type of CDNP may be referred to as a “first” population but may be used and placed in contact with effector cells at Day 0 and at Day 6, or at Day 3 and at Day 7, etc. In such embodiments, the “first” and “second” (and “third”, in some embodiments) denote distinctness.

[0093] #14471824vl A population of CDNPs as provided herein can be from a cell line engineered to express cell surface signal(s) of interest. As used herein, “cell line population CDNPs” refers to the CDNPs obtained from such a cell line. The CDNPs, when more than one, form a population. As above, the population of CDNPs can be, in some instances, referred to as a “first” or “second” (or “third”, in some embodiments) population of CDNPs. Also as above, the CDNPs of the populations may be distinct but are not necessarily so. They may be two (or three, in some embodiments) populations of CDNPs that are both derived from the same cell line. When “first” or “second” (or “third”, in some embodiments) is used to refer to the cell lines from which the populations of CDNPs are derived, the terms are intended to refer to two (or three) cell lines that are in some embodiments distinct whereby the cells of the cell lines are of a different type of cell, different composition and / or are engineered to express cell surface signals(s) that are distinct.

[0094] The term “expand” or the like as used herein refers to increasing in number, as in an increase in the number of effector cells, which increase may be due to an increase in proliferation of the effector cells. The effector cells that are expanded may increase in number relative to the number originally present in the culture, in an embodiment. In another embodiment, the effector cells that are expanded increase in number relative to other cell types in the culture, such as those carrying CD62L or those that are less differentiated cells, such as TSCM cells, over more differentiated cells. The expansion of the effector cells may occur ex vivo. The term "ex vivo " as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor). It is noted that the term “expand” is intended to include “causing to expand”. “Causing to expand” means causing, urging, encouraging, aiding, inducing or directing, directly or indirectly, another party to takes steps, such as described herein, that can result in an increase in the number of effector cells, such as by taking one or more or all of the steps of any one of the methods provided herein.

[0095] As used herein, “effector cells” are any immune cell that can be activated or differentiated to carry out immune response functions, which can in some embodiments result in pathogenic cell killing or the modulation of other immune cells. Examples of immune effector cells include plasma cells, T cells (CD4+ and CD8+ T cells), natural killer (NK) cells, macrophages, etc. Examples of effector cells also include modified or engineered effector cells and, thus, include CAR-effector cells. The CAR-effector cell may be a modified or engineered version of any naturally occurring effector cell, such as a modified or

[0096] #14471824vl engineered version of any one of the foregoing types of effector cells. Accordingly, CAR- effector cells include, but are not limited to CAR-T cells, CAR-NK cells, CAR-macrophages, etc. CAR-effector cells are known in the art, descriptions of which can be found, for example, in Patel et al., Mol Then 2025 May 7;33(5):2123-2140. doi: 10.1016 / j.ymthe.2025.03.005, Jprgcnscn, L.V., Christensen, E.B., Barnkob, M.B. et al. The clinical landscape of CAR NK cells. Exp Hematol Oncol 14, 46 (2025). doi.org / 10.1186 / s40164-025-00633-8, and Klichinsky et al. Nat Biotechnol. 2020 Aug;38(8):947-953. doi: 10.1038 / s41587-020-0462-y. Epub 2020 Mar 23. The effector cells may also be tumor infiltrating lymphocytes (TILs).

[0097] In one embodiment of any one of the methods or compositions provided herein, a cell line is engineered to express a cell surface signal or set of cell surface signals. Similar to above, the cell surface signal or set of cell surface signals the cells of the cell line are engineered to express may be distinct from the cell surface signal or set of cell surface signals other cells are engineered to express. The other cells may be of the same cell line or another cell line. The “cell surface signal” or “set of cell surface signals” refer to molecule(s) that can be expressed by the cells from which CDNPs are derived and are on or in the membrane of such cells. Such signal(s) are preferably those that can result in the expansion of effector cells. As the cells comprise the cell surface signal(s) on or in their cell membrane, when the CDNPs are produced from such cells, the CDNPs, which comprise portions of the cell membrane of the cells, can thus include cell surface signal(s) on or in those portions of the cell membrane.

[0098] Expansion of the effector cells can be the result of stimulation by a cell surface signal or set of cell surface signals. By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and / or reorganization of cytoskeletal structures, and the like. In some embodiments, the populations of CDNPs are used or placed in contact with effector cells that have already been initially stimulated. “Initially stimulated” refers to any prior stimulation of the cells to expand them in culture. This initial stimulation may have occurred with another population of CDNPs or with another method of stimulation. Thus, in some embodiments, the populations of CDNPs are used to boost these effector cells that have been exposed to a prior stimulation in culture. In some embodiment, however, the CDNPs are used or placed in contact with effector cells at the start of an initial stimulation of the effector

[0099] #14471824vl cells and / or may include a population of CDNPs that is used for initial stimulation. In some embodiments, the population of CDNPs that is used for initial stimulation comprise CD3L (e.g., anti-CD3 antibody), CD86, 4-1BBL, IL-7 and IL- 15.

[0100] A “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand. A “stimulatory ligand,” as used herein, means a ligand can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, a CD3 ligand (CD3L), such as an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody. As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells, such as in response to the presentation of antigen. The TCR is generally responsible for recognizing antigens bound to major histocompatibility complex molecules. Cell surface signals include stimulatory molecules and stimulatory ligands.

[0101] Cell surface signals also include costimulatory ligands and costimulatory molecules. “Costimulatory ligands”, as used herein, refers to a molecule that specifically binds a cognate costimulatory molecule on an effector cell, such as a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR / CD3 complex with an MHC molecule loaded with peptide, mediates an effector cell response, such as a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A co-stimulatory ligand can include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A “co stimulatory molecule” refers to the cognate binding partner on an effector cell, such as a T cell, that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the effector cell, such as a T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to CD86, 4-1BBL, BTLA and a Toll ligand receptor.

[0102] Cell surface signals include cytokines. As used herein “cytokines” refer to proteinbased cell signaling molecules that aid cell-to-cell communication in immune responses. In

[0103] #14471824vl some embodiments, the cytokine is an interleukin, such as IL-7, IL-15, or IL-21. IL-7, IL-15 and IL-21 are members of the common y-chain cytokine family.

[0104] In embodiments of any one of the compositions or methods provided herein the CDNPs of a population of CDNPs comprise CD3L (e.g., an anti-CD3 antibody), CD86, 4- 1BBL, IL-7 and IL-15. In another embodiment, the CDNPs of the population comprise at least one of IL-7, IL-15, IL-21 and 4-1BBL. In one embodiment, the CDNPs of the population comprise IL-7 or IL- 15. In one embodiment, the CDNPs of the population comprise IL-7 and IL- 15. In one embodiment, the CDNPs of the population comprise IL-21. In one embodiment, the CDNPs of the population comprise 4-1BBL.

[0105] When the CDNPs comprise IL- 15 as the cell surface signal, or as one of the sets of cell surface signals, as provided herein, the IL-15 signal can be provided or expressed as a chimeric protein encoding IL-15Ra (a-chain of IL- 15 receptor) and IL- 15. In an embodiment, this chimeric protein comprises a Kozak sequence, followed by huIL-15Ra (798bp), followed by T2A translation skipping sequence (60bp), in turn followed by huIL-15 (489bp).

[0106] CDNP Production

[0107] In embodiments of any one of the methods provided herein, CDNPs are produced by processing cells of a cell line, such as of the cells of two or more cell lines, each engineered to express a cell surface signal or set of cell surface signals. In an embodiment, the cell surface signal or set of cell surface signals may be distinct as between the two or more cell lines. In an embodiment, the processing comprises disruption of the cells, such as of the two or more cell lines. In embodiments, the disruption is mechanical disruption, such as cavitation, homogenization, or extrusion. In some embodiments, the disruption is chemical disruption. The CDNPs can be generated as described in Oyer, J.L., et al., Biology of blood and marrow transplantation: Journal of the American Society for Blood and Marrow Transplantation vol. 21, 4 (2015). The CDNPs can also be generated by the disruption of cells as described herein. A number of methods of cell disruption are known in the art including, but not limited to, mechanical homogenization such as with a handheld or motorized device, ultrasonic homogenization, pressure homogenization, temperature treatments comprising repeated cycles of freezing and thawing, osmotic lysis in hypotonic solutions, chemical lysis including the use of various detergents or solvents, nitrogen cavitation, among others. The skilled artisan would recognize which method of disruption would be most applicable to specific applications of generating CDNPs. In some embodiments, the method of disruption

[0108] #14471824vl is nitrogen cavitation. This method involves placing a culture of cells in a pressure vessel, often called a “bomb”, and equilibrating the cells at high pressure under a nitrogen-rich atmosphere, thereby resulting in increased nitrogen and other gasses dissolved in the cytoplasm of the cells. The sudden exposure of the cells to atmospheric pressure results in nitrogen bubbles forming in the cytoplasm of the cells, which results in cell lysis. Pressure and release time can be varied to result in differing degrees of cell lysis and preservation of cellular, membrane, protein, and organelle structures. For example, relatively low pressures will disrupt the plasma membrane and endoplasmic reticulum only, while high pressures will result in the disruption of the nucleus and other organelles including lysosomes and mitochondria. The pressure vessel can consist of a thick stainless steel or similar metal casing that is capable of withstanding high pressure, with an inlet for delivery of nitrogen gas from a tank and an outlet port with an adjustable discharge valve. The skilled artisan would recognize the conditions of nitrogen cavitation sufficient to produce the CDNPs provided herein.

[0109] In one embodiment of any one of the methods provided herein, the method comprises obtaining, such as collecting, extracting, isolating, separating, filtering and / or purifying, the population of CDNPs from cell culture. The CDNPs may be so obtained by methods known in the art such as ultracentrifugation (Pisitkun et al (2004) PNAS 101:13368-13373) or ultrafiltration (Cheruvanky et al (2007) Am. J. Physiol. Renal Physiol. 292:F1657-F1661). Methods are also known involving continuous flow electrophoresis and chromatography procedures (Taylor and Gercel- Taylor (2005) Br J Cancer 92:305-311). In some embodiments, the obtaining comprises extracting, filtering and / or purifying the CDNPs from cell culture. “Obtaining” as used herein means an act of acquiring a material(s) by any means. This term is intended to include “causing to obtain”. “Causing to obtain” means causing, urging, encouraging, aiding, inducing or directing or acting in coordination with an entity for the entity to obtain a material(s) as provided herein. In some embodiments of any one of the methods provided herein, the method may comprise or further comprise any one of the steps of obtaining as described herein.

[0110] In an embodiment of any one of the compositions or methods provided herein the populations of CDNPs can be produced with a construct as described herein. The cells from which the CDNPs are derived can be engineered to express the cell surface signal or set of cell surface signals. Thus, the cells of any one of the methods provided herein may be transfected, transformed or transduced with one or more constructs encoding the cell surface

[0111] #14471824vl signal or set of cell surface signals provided herein. The term “transfected” or “transformed” or “transduced”, as used herein, refers to a process by which exogenous nucleic acid is transferred or introduced into a host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with an exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0112] The construct may comprise a signal sequence. In some embodiments, the signal sequence is a human GM-CSF signal sequence. In some embodiments, the signal sequence is a signal sequence as provided in Huse, M., et al. Nature immunology vol. 7, 3 (2006): 247- 55; Conradt, H. S., et al., Carbohydrate research vol. 149, 2 (1986): 443-50, e.g., an IL-7 signal sequence. The construct may comprise a linker. In some embodiments, the linker comprises a modified IgG4 hinge region, an IgG4 CH2 Domain, or an IgG4 CH3 domain. In some embodiments, the linker may comprise a GS linker.

[0113] Since the cell surface signals when expressed in the cells as provided herein are on or in or part of the cell membranes, constructs that encode the cell surface signal(s) can be one that results in the cell surface signal(s) being inserted into a membrane or otherwise anchored in or to the cell membrane. Thus, the construct may be appended to a membrane domain or comprise a membrane-insertion moiety. In some embodiments, the membrane insertion moiety comprises a transmembrane domain, such as a human transmembrane domain, a CD4 transmembrane domain, such as a human CD4 transmembrane domain, a CD8 transmembrane domain, or a CD28 transmembrane domain. In some embodiments, the transmembrane domain is derived either from a natural or from a synthetic source. In some embodiments, where the source is natural, the domain is derived from any membrane-bound or transmembrane protein, e.g., a Type I transmembrane protein. In some embodiments, where the source is synthetic, the transmembrane domain is any artificial sequence that facilitates insertion into a cell membrane, e.g., an artificial hydrophobic sequence. Examples of the transmembrane regions of particular use in this disclosure include, without limitation, transmembrane domains derived from (e.g., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD3 epsilon, CD86, CD137 (4-1BB), etc. In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some instances, a triplet of phenylalanine, tryptophan and valine is found at each end of a synthetic transmembrane domain.

[0114] #14471824vl In some embodiments, a construct as described herein is encoded by a polynucleotide. The polynucleotide may comprise recombinant or isolated or synthetic DNA or RNA. As used herein, the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides or a defined sequence of amino acids. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0115] The term “polynucleotide” as used herein is defined as a chain of nucleotides. As nucleic acids are polymers of nucleotides, “nucleic acid” and “polynucleotide” as used herein are interchangeable. As used herein, polynucleotides include, but are not limited to, nucleic acid sequences (i.e., “nucleotide sequences”) which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0116] In some embodiments, a vector encodes the construct. The vector may be an expression vector. As used herein, the term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter. As used herein, the term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. The term "recombinant" as used herein is defined produced by joining pieces of material from different sources.

[0117] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides,

[0118] #14471824vl polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like. As used herein, the term “host cell” includes an individual cell or cell culture that can be or has been a recipient of exogenous polynucleotide(s). Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. An exemplary host cell for use as provided herein are K562 cells.

[0119] In some embodiments, the polynucleotide encoding the construct is operably linked to a transcriptional control element, e.g., a promoter, and enhancer, and so forth. Suitable promoter and enhancer elements are known to those of skill in the art. In some embodiments, the polynucleotide encoding the construct is operably linked to a promoter. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, light and / or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, and so forth, is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), and so forth), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, and so forth), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, and so forth), metal regulated

[0120] #14471824vl promoters (e.g., metallothionein promoter systems, and so forth), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, and so forth), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, and so forth), light regulated promoters, synthetic inducible promoters, and the like.

[0121] Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Other constitutive promoter sequences may also be used, including, but not limited to a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, the EF-1 alpha promoter, as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0122] In some embodiments, the polynucleotide or vector containing the suitable promoter is irreversibly switched through the induction of an inducible system. Suitable systems for induction of an irreversible switch are well known in the art, e.g., induction of an irreversible switch may make use of a Cre-lox-mediated recombination (see, e.g., Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, and so forth known to the art may be used in generating an irreversibly switchable promoter. Methods, mechanisms, and requirements for performing site-specific recombination, described elsewhere herein, find use in generating irreversibly switched promoters and are well known in the art, see, e.g., Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.)

[0123] #14471824vl A polynucleotide encoding the construct of the present disclosure can be present within an expression vector and / or a cloning vector. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example and should not be construed in anyway as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pST-Ki, pST-KiT, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0124] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0125] #14471824vl Additional expression vectors suitable for use are, e.g., without limitation, a lentivirus vector, a gamma retrovirus vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a pox virus vector, a herpes virus vector, an engineered hybrid virus vector, a transposon mediated vector, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.

[0126] In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0127] In some embodiments, an expression vector can be used to introduce the construct into a cell. Accordingly, an expression vector may comprise a nucleic acid encoding for the construct. In some embodiments, the expression vector will comprise additional elements that will aid in the functional expression of the construct encoded therein. In some embodiments, an expression vector comprising a nucleic acid encoding for the construct further comprises a mammalian promoter. In one embodiment, the vector further comprises an elongation-factor- 1-alpha promoter (EF- la promoter). Use of an EF-la promoter may increase the efficiency in expression of downstream transgenes. Physiologic promoters (e.g., an EF-la promoter) may be less likely to induce integration mediated genotoxicity and may abrogate the ability of the retroviral vector to transform stem cells. Other physiological promoters suitable for use in a vector are known to those of skill in the art and may be incorporated into a vector of the present invention. In some embodiments, the vector further comprises a non-requisite cis acting sequence that may improve titers and gene expression. One non-limiting example of a non-requisite cis acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS) which is important for efficient reverse transcription and nuclear import. Other non-requisite cis acting sequences are known to those of skill in the art and may be incorporated into a vector of the present invention. In some embodiments, the vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Accordingly, in some embodiments, a vector for the present invention further comprises a WPRE sequence. Various

[0128] #14471824vl posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector of the present invention. A vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, packaging sequences, and 5’ and 3’ long terminal repeats (LTRs). The term “long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which comprise U3, R and U5 regions. LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication. In one aspect, a vector of the present invention includes a 3’ U3 deleted LTR. Accordingly, a vector of the present invention may comprise any combination of the elements described herein to enhance the efficiency of functional expression of transgenes.

[0129] In some embodiments, a polynucleotide encoding the construct is RNA, e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those of skill in the art; any known method can be used to synthesize RNA comprising a sequence encoding the construct of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding the construct of the present disclosure into a host cell can be carried out in vitro, ex vivo or in vivo. For example, a host cell can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding a construct of the present disclosure.

[0130] In order to assess the expression of a polypeptide or portions thereof, the expression vector to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, without limitation, antibiotic-resistance genes.

[0131] Methods of Expanding Effector Cells and Related Compositions

[0132] Provided in the present disclosure are methods for expanding, or producing expanded, effector cells, such as an T cells, using the compositions comprising the population(s) of CDNPs as provided herein. “Producing”, as used herein, refers to any action that results in a

[0133] #14471824vl material being made. An act of producing includes preparing the material or processing it in some manner. In some embodiments, an act of producing includes any act that makes that material available for use by another. This term is intended to include “causing to produce”. “Causing to produce” means causing, urging, encouraging, aiding, inducing or directing or acting in coordination with an entity for the entity to make a material(s) as provided herein.

[0134] In one aspect, the present disclosure generally relates to a method for expanding or producing effector cells, comprising contacting the effector cells with a composition comprising the population(s) of CDNPs as provided herein. When effector cells are in the same composition as one or more populations of CDNPs, such as when added to a composition comprising one or more populations of CDNPs (or vice versa), the effector cells and population(s) of CDNPs are “placed in contact” or the like. Such contact occurs in vitro or ex vivo, such as in cell culture. “Contacting” or “placing in contact” includes anything that allows for a physical and / or chemical interaction. Such terms are intended to also include “causing to contact” and “causing to be placed in contact”, which means causing, urging, encouraging, aiding, inducing or directing, directly or indirectly, another party to takes steps, such as described herein, that can result in contact to occur, such as by taking one or more or all of the steps of any one of the methods provided herein.

[0135] The populations of CDNPs may be placed in contact with the effector cells at the same time or a different point in time. “At the same time” refers to placing two or more populations of CDNPs in contact with the effector cells at the very same point in time or within a short period of time of each other, but no more than within the same day, and / or that is considered by the ordinarily skilled artisan to be negligible, such as no more than within a few second, minutes or hours of each other. Such contact may be simultaneous, which can be by means of placing the effector cells and the populations of CDNPs in contact with each other at the very same time or by placing the effector cells in contact with composition(s) comprising the populations of CDNPs, such as simultaneously or substantially simultaneously.

[0136] Populations of CDNPs may be placed in contact with effector cells at different points in time. Such different points in time generally are at a certain number of days after an initial stimulation. In some embodiments, populations of CDNPs as provided herein are placed in contact with the effector cells at least three days after an initial stimulation (e.g., at Day 0). In some embodiments, populations of CDNPs as provided herein are placed in contact with the effector cells at least six days after an initial stimulation (e.g., at Day 0). In some

[0137] #14471824vl embodiments, populations of CDNPs as provided herein are placed in contact with the effector cells at least seven days after an initial stimulation (e.g., at Day 0). In some embodiments, populations of CDNPs as provided herein are placed in contact with the effector cells at least nine days after an initial stimulation (e.g., at Day 0). In some embodiments, populations of CDNPs as provided herein are placed in contact with the effector cells at least three days and at least six days after an initial stimulation (e.g., at Day 0). In some embodiments, populations of CDNPs as provided herein are placed in contact with the effector cells at least three days and at least seven days after an initial stimulation (e.g., at Day 0). In some embodiments, populations of CDNPs as provided herein are placed in contact with the effector cells at least three days and at least nine days after an initial stimulation (e.g., at Day 0).

[0138] In some embodiments, these populations of CDNPs comprise IL-7, IL- 15, IL-21 or 4- 1BBL as provided herein, which have been found to result in robust effector cell expansion after an initial stimulation, such as with CDNPs that comprise CD3L (e.g., an anti-CD3 antibody), CD86, 4-1BBL, IL-7 and IL-15. In some embodiments, these populations of CDNPs may be also placed in contact with the effector cells at the time of initial stimulation; however, in some embodiments, these populations of CDNPs are not placed in contact with the effector cells at the time of initial stimulation. For example, in some embodiments, it may be advantageous to not place effector cells in contact with CDNPs comprising IL-7 at the time of initial stimulation.

[0139] Accordingly, methods for placing one or more populations of CDNPs in contact with effector cells (or vice versa), such as above, are also provided herein. Such methods are useful for expanding the effector cells. In some embodiments of any one of the methods provided herein, the method may comprise or further comprise any one or more or all of the steps of expanding or producing as described herein. Also provided are compositions comprising the population(s) of CDNPs in contact with the effector cells, the expansion of which is desired and / or that have been expanded by such contact. The effector cells may be T cells, such as CD4+and / or CD8+cells, and natural killer (NK cells). In some embodiments of any one of the methods or compositions provided herein, the effector cells may be autologous effector cells.

[0140] In an embodiment, the methods provided herein may comprise or further comprise a step of obtaining the resulting expanded effector cells. In an embodiment, the obtaining includes collecting, extracting, isolating, separating, filtering and / or purifying, the expanded

[0141] #14471824vl effector cells, such as from cell culture. Methods of cell culture as well as obtaining, such as separating, purifying, isolating, etc., the cells are known in the art. These methods include, but are not limited to, fluorescence activated cell sorting (FACS), immunomagnetic cell separation, density gradient centrifugation, immunodensity cell isolation, and microfluidic cell sorting. In some embodiments, the obtaining comprises extracting, separating, isolating and / or purifying the expanded effector cells from the cell culture in which they were in contact with one or more populations of CDNPs as provided herein.

[0142] In an aspect, the expanded effector cells obtained using any one of the methods provided herein may be administered to a subject in whom the expanded effector cells may have a clinical benefit. Any one of the methods provided herein may comprise or further comprise a step of administering the expanded effector cells provided herein. As used herein, “administering” or “administration” or “administer” means giving or dispensing a material to a subject in a manner that is pharmacologically useful. The term is intended to include “causing to be administered”. “Causing to be administered” means causing, urging, encouraging, aiding, inducing or directing, directly or indirectly, another party to administer the material. Any one of the methods provided herein may comprise or further comprise a step of administering the expanded effector cells provided herein.

[0143] In one embodiment of any one of the compositions or methods provided herein, the CDNPs or populations thereof are in an amount effective to result in effector cell expansion. In one embodiment of any one of the compositions or methods provided herein, the expanded effector cells are in an amount effective to result in a therapeutic effect in a subject. An “amount effective” in the context of effector cell expansion as provided herein refers to an amount of a composition comprising population(s) of CDNPs used in vitro or ex vivo that can result or is expected to result in the expansion of effector cells.

[0144] “Amount effective” in the context of a composition for administration to a subject as provided herein refers to an amount of the composition that produces one or more desired results in the subject. Such an amount can be one that a clinician would believe may have a clinical benefit for a subject. An amount effective can also be an amount that results in a desired therapeutic endpoint or a desired therapeutic result. In some embodiments, amounts effective can depend, of course, on the particular subject being treated; the severity of a condition, disease or disorder; the individual patient parameters including age, physical condition, size and weight; the duration of the treatment; the nature of concurrent therapy (if any); the specific route of administration and like factors within the knowledge and expertise

[0145] #14471824vl of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. As used herein, “subject” means animals, including warm blooded mammals such as humans and primates; avians; domestic household or farm animals such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals such as mice, rats and guinea pigs; fish; reptiles; zoo and wild animals; and the like. As used herein, a subject may be one in need of any one of the methods or compositions provided herein. In preferred embodiments, the subject is human. The achievement of any of the foregoing can be monitored by routine methods. In any one of the methods provided herein, the composition(s) may be in an amount effective as provided herein.

[0146] Compositions provided herein may further comprise a “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier”, which terms refer to a pharmacologically inactive material used together with a pharmacologically active material to formulate the compositions. Pharmaceutically acceptable excipients comprise a variety of materials known in the art, including but not limited to saccharides (such as glucose, lactose, and the like), preservatives such as antimicrobial agents, reconstitution aids, colorants, saline (such as phosphate buffered saline), and buffers.

[0147] In some embodiments, the present disclosure encompasses the preparation and use of pharmaceutical compositions comprising expanded effector cells as described herein as an active ingredient. Such pharmaceutical compositions may consist of the active ingredient alone, as a combination of at least one active ingredient in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional (active and / or inactive) ingredients, or some combination of these. The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0148] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of

[0149] #14471824vl pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation.

[0150] A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or for use in the expansion of effector cells from a subject (or treatment therewith) or a convenient fraction of such a dosage such as, for example, one -half or one-third of such a dosage.

[0151] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0152] In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents. Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[0153] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intravenous injection, and infusion techniques.

[0154] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable

[0155] #14471824vl formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions, and implantable sustained- release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.

[0156] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a nontoxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides.

[0157] In some embodiments, when the expanded effector cells are administered, the amount of cells administered can range from about 1 million cells to about 300 billion. In some embodiments, the expanded effector cells can be administered in an amount ranging from about 100,000 to about ten billion cells wherein the cells are infused into the animal, preferably, a human patient in need thereof. In some embodiments, the precise dosage administered varies depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration.

[0158] In some embodiments, the expanded effector cells are co-administered with various other compounds (cytokines, chemotherapeutic and / or antiviral drugs, among many others). The frequency and administration regimen will be readily apparent to the skilled artisan and will depend upon any number of factors such as, but not limited to, the type and severity of the disease being treated, the age and health status of the animal, the identity of the compound or compounds being administered, the route of administration of the various compounds, and the like.

[0159] Also provided are kits comprising any one of the compositions provided herein, such as any one or any one combination of the populations of CDNPs or cell lines provided herein. A “kit” comprises any one or any one combination of the populations of CDNPs or cell lines

[0160] #14471824vl provided herein in one or more containers, such as vials or ampoules, which can be used by a practicing end user in a laboratory or clinical setting. In an embodiment of any one of the kits provided, the kit comprises any one of the compositions provided herein in combination with a pharmaceutically acceptable carrier. The composition of any one of the kits provided may be in solution. In an embodiment of any one of the kits provided herein, the populations of CDNPs or cell lines are contained within separate containers in the kit. In an embodiment of any one of the kits provided herein, other component(s) are contained within a solution separate from the container(s), such that the other component(s) may be added to the container(s) at a subsequent time. In an embodiment of any one of the kits provided herein, the kit further comprises one or more syringes or other means for administering, extracting or distributing the CDNPs or cells or any other components. In an embodiment of any one of the kits provided herein, the kit further comprises instructions for reconstitution, mixing, administration, extraction or distribution, etc. In an embodiment of any one of the kits provided herein, the instructions include a description of any one of the methods described herein. A kit provided herein may be used to perform any one of the methods provided herein. In one embodiment of any one of the kits provided, the kit further comprises indicia comprising at least one of: instructions for performing any one of the methods provided herein, for preparing any one of the compositions provided herein, and / or for using any one of the compositions as provided herein in a method of use, such as any one of the methods of use provided herein.

[0161] EXAMPLES

[0162] Example 1: CDNP Production and Purification

[0163] CDNPs were produced by culturing and expanding transduced K562 cells, expressing relevant transgenes, e.g., IL-7 transgene for CDNP-IL-7. CDNP4s were derived from K562 cells transduced to express CD3, CD86, m-IL-7, IL-15Ra / IL-15 and 4-1BBL. The transduced cell lines were generated using previously published general methods, such as described in U.S. published patent application US 2023 / 0000916 Al to Riley et al, the methods of which are herein incorporated by reference.

[0164] The transduced cells were thawed as necessary, and transferred into culture flasks, such as T75 and T175 flasks. The cells were then expanded to the required numbers and maintained at log phase of growth by passaging the cells every 2-3 days. Once the required

[0165] #14471824vl cell numbers were obtained, the cells were transferred to a bigger culture flask such as G-Rex 500M (Wilson Wolf Manufacturing) for 5-6 days to expand to ~4-5xl09cells total using a media system such as CTS OpTmizer® (Gibco). During this expansion period, the cells were monitored for glucose, lactate, CO2, and 02 levels, and the cells harvested once the glucose level was << 200 mg / dL and the lactate level reached 12-15 mg / dL.

[0166] Harvested cells were washed by removing the culture medium and then resuspending in an isotonic buffer for cell disruption. The cells were disrupted by cavitation using nitrogen gas pumped into a sealed chamber (aka “bomb” cavitator) that houses the cells and allowed to diffuse into the cells for 2x 30 minutes at a target cavitator head pressure ranging from 300 - 500 psi using a pressure vessel such as one manufactured by Parr Instrument Company, Vessel Number 4636. This was followed by a quick release of the nitrogen pressure. Without committing to a particular mechanism, the assumption was that the nitrogen pressure release abruptly increased cell size and subsequently caused an implosion in the cells that disrupted the cells. Hydrophobic parts of the cells were subsequently allowed to reconstitute into a composition comprising nanoparticulate-containing structures termed cell-derived nanoparticles, i.e., CDNPs. The hydrophobic parts include lipid bilayer of the cell membrane which are expected to hold the transgene cell surface signal(s). They are thought to reconstitute into spherical liposome-like nano particle structures.

[0167] The CDNPs were then subjected to a clarification and purification process, and concentration methods, using such instruments such as the Lovo® automated cell processing system (Fresenius Kabi) and the tangential flow filtration (TFF) TFF KrosFlo ® KR2i System (Repligen) and / or other chemical reactions and physical handling (such as sedimentation) to obtain purified CDNPs, and were concentrated for use. Samples of CDNPs were further subjected to QC tests such as ELISA, particle counting, and potency assessment.

[0168] CDNPs were stored at -80°C in an aliquot for single use after the first thawing on ice. Repeated freezing and thawing cycles were avoided. CDNPs were resuspended by pipetting over 10 times before aliquoting or taking for T cell activation.

[0169] Example 2: The Addition of Cytokine-containing CDNPs (CDNP-IL-7 and CDNP-IL- 15) at Different Timepoints Optimize Expansion

[0170] Protocol for T Cell Activation for Healthy Donor T Cells (starting with 5x105T cells)

[0171] #14471824vl Table 1 : Experiment Conditions with Healthy Donor 1 T Cells

[0172] Table 2: Experiment Conditions with Healthy Donors 2 and 3 T Cells

[0173] #14471824vl

[0174]

[0175] Procedure

[0176] Culture medium was prepared following the recipe in Table 3. The cOpt medium was stored wrapped in foil at 4°C for up to 2 weeks. The medium was warmed at 37 °C for 30 min prior to use.

[0177] Table 3. Culture Media Preparation

[0178] Frozen total human T cells obtained from healthy donors were stored in the liquid nitrogen storage until use for the experiments. Cells were thawed prior to use, washed by centrifugation, counted, and resuspended at the targeted concentration per 1 mL.

[0179] CDNP4 at the indicated dose levels was then added to the T cells. For the costimulation conditions the pre-defined levels of supplemental CDNPs, such as CDNP-IL-7, CDNP-IL-15, CDNP-IL-21, CDNP-4-1BBL, WT-CDNP, etc. were added to CDPN4- activated cultures either at DL1 levels or at 2x dilutions of DL1 level, such as DL1 / 2, DL1 / 4,

[0180] #14471824vl DL1 / 8, DL1 / 16, etc. Alternatively, soluble cytokines IL-7 and IL-15 were added to the CDNP4-activated T cells.

[0181] The cells were then expanded in culture at 37 °C and 5% CO2 until day 6. On day 6 for the restimulated conditions, the cells were collected, washed by centrifugation, and resuspended at targeted concentrations per 1 mL as on day 0. For the restimulation conditions, the pre-defined levels of supplemental CDNPs, such as CDNP-IL-7, CDNP-IL- 15, WT-CDNP, were added to CDPN4-activated cultures either at DL1 levels or at 2x serial dilutions of DL1 level, such as DL1 / 2, DL1 / 4, DL1 / 8, DL1 / 16, etc. Alternatively, soluble cytokines IL-7 and IL- 15 were added to the activated T cells. The DL1 level of CDNP-IL-7 and CDNP-IL-15 was equivalent to the dose of soluble IL-7 and IL- 15 used in this study.

[0182] The cells, stimulated on day 0 or stimulated on day 0 and restimulated on day 6, were maintained in culture until day 9. On day 9, the cells were collected and counted to define the total cell number per culture condition. Where needed, cells were also characterized by flow cytometry to determine the phenotypes by staining for desired markers.

[0183] Results

[0184] The data for these experiments revealed that expansion of activated T cells slows down after initial stimulation. Surprisingly and significantly, the addition of CDNP-IL-7 and CDNP-IL-15 elevated T cell expansion after the initial stimulation. CDNP4 was used as a T cell activator, and soluble IL-7 5ng / mL and IL-15 lOng / mL were used as control. CDNP-IL-7 and CDNP-IL-15 were added to T cells either at the start of the stimulation with CDNP4 or on day 6 post-stimulation. The dose of CDNP-IL-7 and CDNP-IL-15 were based on ELISA assay, with two dose levels: one equivalent to the dose of soluble IL-7 and IL-15, and the other at half the dose. It was found that neither soluble IL-7 and IL- 15 nor CDNP-IL-7 and CDNP-IL-15 impacted T cell expansion when added at the beginning of stimulation with CDNP4. However, the addition of either soluble IL-7 and IL- 15 or CDNP-IL-7 and CDNP- IL-15 significantly increased T cell expansion compared to CDNP4 control without signal, such as cytokine, restimulation. Notably, CDNP-IL-7 and CDNP-IL-15 induced a significantly greater T cell expansion compared to soluble IL-7 and IL- 15, even at half the dose. Data can be found in FIGS. 1-3.

[0185] A dose-response analysis for stimulation with CDNP-IL-7 and CDNP-IL-15 on day 6 was then performed. As shown in FIG. 4, CDNP-IL-7 and CDNP-IL-15 increased T cell expansion in a dose dependent manner, with half the dose of CDNP-IL-7 and CDNP-IL-15

[0186] #14471824vl reaching to the plateaus of cell expansion. Unexpectedly, 1 / 16 dose of CDNP-IL-7 and CDNP-IL-15, the lowest dose tested, led to 50% more T cell expansion compared to the full dose of soluble IL-7 and IL- 15. This shows that cell surface signals, such as cytokines, carried by CDNPs provide pronouncedly more potent stimulation than soluble signals, such as cytokines.

[0187] Example 3: Addition of CDNP-IL-21 Enhances CD8 T Cell Proliferation

[0188] To evaluate the additive effect of the combination of CDNP4 with CDNP-IL-21, a CDNP4 dose DL1 / 2 was used in conjunction with different doses of CDNP-IL-21 (DL1, DL1 / 2, and DL1 / 4). When CDNP-IL-21 was combined with CDNP4, T cell proliferation, including CD8+ T cells, was markedly increased with all tested doses (FIG. 5). Overall, these findings demonstrated that addition of IL-21 into ex vivo T cell expansion can improve cell expansion and that the use of different populations of CDNPs with different signals can result in improved effector cell expansion. Importantly, such improved expansion did not require the creation of a new cell line or the use of all initial stimulation signals with the added signal.

[0189] Example 4: Addition of CDNP-4-1BBL Improves Ex vivo T Cell Expansion

[0190] The addition of CDNP-4-1BBL to activated T cells was also assessed. As shown in FIG. 6, CDNP-4-1BBL moderately increased expansion of CDNP4-activated T cells across all tested doses while also pronouncedly increased the expansion of T cells activated with Dynabeads. These findings show that addition of CDNP-4-1BBL to T cell manufacturing with surface-bound CD3 / CD28 stimulators can enhance T cell expansion. Accordingly, this experiment also demonstrates that the use of populations of CDNPs with different signals can result in improved effector cell expansion. Further, the CDNPs provided herein can also allow for improvements to older technologies, such as bead-mounted cell signals (e.g., Dynabeads (thermofisher.com / order / catalog / product / 11131D)) in the expansion of effector cells.

[0191] Example 5: CDNP4 Supplementation Boosts Expansion NK Cells Stimulated by CDNP- IL-21 and CDNP-4-1BBL

[0192] NK cells typically die in ex vivo culture when cytokines are absent. In this experiment, peripheral blood mononuclear cells (PBMCs) were stimulated under various

[0193] #14471824vl conditions. CDNP4 and CDNP-IL-21 and with CDNP-4-1BBL were effective in maintaining NK cell survival and increasing NK cells (FIGS. 7 and 8). Interestingly, combinations of CDNPs exhibited increased NK cell expansion where in other circumstances the NK cells died or barely survived. However, with CDNP supplementation NK cells could be significantly expanded. Here, again, it is demonstrated that CDNPs as provided herein can allow for the mixing and matching of cell surface signals such that effector cell expansion improvements can result. The results also demonstrate that such an approach can effectively and efficiently enable the discovery of cell surface signals that can result in positive effects on effector cell expansion (here as an example on NK cell expansion).

[0194] Example 6: Addition of CDNP-IL-7 and / or CDNP-IL-15 to other CDNPs Elevates T Cell Expansion to Higher Extent than Soluble IL-7 and / or IL- 15 and at Lower Cytokine Doses

[0195] CDNPs were produced as provided above in Example 1. Purified T cells (250,000 in total 500 pL volume) from four different normal human donors were activated by CDNPs, with scFvOKT3, CD86, 4-1BBL, IL-7 and fused IL-15 / IL-15Ra signals, alone or in combination with soluble or CDNP-IL-7 and / or CDNP-IL-15. CDNP-IL-7 is essentially a CDNP from cells engineered to carry only an IL-7 signal. CDNP-IL-15 is essentially a CDNP from cells engineered to carry only IL- 15 signal, which IL- 15 signal in this example is a chimeric protein encoding IL-15Ra (a-chain of IL- 15 receptor) and IL- 15. Soluble or CDNP- IL-7 was administered on days 3 and 7, soluble or CDNP-IL-15 was administered on days 0, 3, and 7. The dose of CDNP-IL-7 was two times lower than of sIL-7, the dose of CDNP-IL- 15 was either eight (day 0), four (day 3) or two (day 7) times less than that of sIL-15.

[0196] Cells were transferred to G-Rex24 plates on day 3 and cultures topped with media (OpTmizer™) to 7 mL. Cells were assessed for viability and counted using NC-200 cell counter (Chemometec, Denmark) and analyzed by flow (NovoCyte Quanteon; Agilent Technologies, CA, USA) on days 3, 7 and 9 using the following fluorescently-labeled antibodies to T cell surface markers: CD3-FITC, CD4-BV421, CD8-BV510, CD45RA-PE, CD62L-BV605, CD95-BV650, and CCR7-PE-Cy7 (all from BioLegend, CA, USA) with LIVE / DEAD™ Fixable Near-IR Dead Cell stain kit (Invitrogen MA, USA) used to gate on living cells.

[0197] While soluble IL-7 and IL- 15 used singly did not result in any elevation of T cell expansion, when used in combination, led to 24-25% increase of total T cells at day 7 and 9

[0198] #14471824vl (FIG. 9). The CDNP carrying cytokines were significantly more efficient when added to the CDNPs carrying scFvOKT3, CD86, 4-1BBL, IL-7 and fused IL-15 / IL-15Ra signals. Specifically, CDNP-IL-15 used alone led to a 70-73% increase in T cell expansion and CDNP-IL-7 led to more than a 2-fold increase in T cell expansion compared to the CDNPs, carrying scFvOKT3, CD86, 4-1BBL, IL-7 and fused IL-15 / IL-15Ra signals, used alone. Moreover, this effect was synergistic with the combination of CDNP-IL-7 and CDNP-IL-15 used with the CDNPs carrying scFvOKT3, CD86, 4-1BBL, IL-7 and fused IL-15 / IL-15Ra signals, resulting in nearly 3-fold increase in T cell expansion by day 7 and in more than 3- fold elevation of T cell expansion by day 9 (FIG. 9). This increase of T cell expansion seen after combination treatment with CDNPs carrying scFvOKT3, CD86, 4-1BBL, IL-7 and fused IL-15 / IL-15Ra signals, CDNP-IL-7 and CDNP-IL-15 was statistically significant (p<0.05; Mann- Whitney test).

[0199] Example 7. Addition of CDNP-IL-7 and / or CDNP-IL-15 to other CDNPs has Proportionally Elevated both CD4+and CD8+Cell Populations

[0200] In the same series of studies from Example 6, the fractions and the total number of CD4+CD8 and CD4 CD8+cells were assessed by flow cytometry. For these studies, there was no noticeable change in CD4+CD8 ZCD4 CD8+cell ratio at any measured timepoint resulting from addition of soluble or CDNP-IL-7 and / or or CDNP-IL-15 to CDNPs carrying scFvOKT3, CD86, 4-1BBL, IL-7 and fused IL-15 / IL-15Ra signals. As a consequence, the total number of both CD4+CD8 and CD4 CD8+cells were proportionally elevated with their expansion (the greatest expansion seen on days 7 and 9) after the treatment with CDNPs carrying scFvOKT3, CD86, 4-1BBL, IL-7 and fused IL-15 / IL-15Ra signals and combined with both CDNP-IL-7 and CDNP-IL-15 (FIG. 10). Only a minor and statistically insignificant elevation of CD4+CD8 and CD4 CD8+cell populations was seen if both soluble IL-7 and IL- 15 were used in combination with the CDNPs carrying scFvOKT3, CD86, 4-1BBL, IL-7 and fused IL-15 / IL-15Ra signals. These results demonstrate that like the soluble signals, such as cytokines, the cell-derived signals, such as cytokines, resulted in comparable proportions of CD4+and CD8+cells in the exansion; however, importantly, the expansion of these cells was much greated with the use of cell-derived IL-7 and IL- 15.

[0201] Example 8. Addition of CDNP-IL-7 and / or CDNP-IL-15 to other CDNPs has Initially Resulted in Proportional Elevation on Low-differentiated T cells with TSCM, TCM, TEM-

[0202] #14471824vl like and TEFF-like Phenotypes, Followed by Preferential Elevation of Undifferentiated TSCM cells by Day 9

[0203] In the same series of studies from Example 6, the fractions and the total number of T cells exhibiting surface markers of different T memory cell phenotypes were assessed by flow cytometry. The key populations were the following: T stem cell-like memory cells (TSCM, defined as CD45RA+CD62L+CD95+CCR7+), T central memory cells (TCM, defined as CD45R A CD62L+CD95+CCR7+), T effector memory-like cells (T| \i-likc, similar to typical TEM population, but carrying CD62L, defined as CD45R A CD62L+CD95+CCR7 ), and T effector-like cells (TEFF-like, similar to typical TEFF population, but carrying CD62L, defined as CD45RA+CD62L+CD95+CCR7 ). Additional populations assessed were naive T cells (TN, defined as CD45R A+CD62L+CD95 CCR7+), another T| \i-likc population, similar to typical TEM cells, but carrying CCR7 (defined as CD45RA CD62L CD95+CCR7+), T effector memory cells (TEM, defined as CD45RA CD62L CD95+CCR7 ) and T effector cells (TEFF, defined as CD45RA+CD62L CD95+CCR7 ).

[0204] At day 7, the expansion of key T cell memory populations was generally proportional to total T cell expansion (2.2-2.8-fold additional elevation in groups treated with CDNP-IL-7 or CDNP-IL-7 / IL-15, FIG. 9) with the elevation of TSCM by CDNP-IL-7 or CDNP-IL- 7 / CDNP-IL-15 combination at 2.2-2.3-fold, TCM at 1.9-2.5-fold, TEM-like cells at 2.6-4.2- fold, and TEFF-like cells at 1.9-2.4-fold (FIG. 11A). This has dramatically shifted by day 9 with TSCM being more profoundly elevated than other populations by either CDNP-IL-7 or especially, CDNP-IL-7 / CDNP-IL-15 combination (FIG. 11B). Specifically, TSCM elevated

[0205] 3.5-fold if CDNP-IL-7 was used, compared to 2.0-fold total T cell expansion (FIG. 9) and

[0206] 1.5-, 0.9- and 1.3-fold elevation in TCM, TEM-like and TEFF-like cells, respectively (FIG. 11B). The same effect was seen if CDNP-IL-7 / CDNP-IL-15 was used, with 4.2-fold TSCM elevation vs. 3.2-fold increase in total T cell expansion (FIG. 9), and 2.9-, 2.0 and 2.1-fold additional expansion of TCM, TEM-like and TEFF-like cells, respectively (FIG. 11B).

[0207] The results show that not only can cell-derived signals result in increased numbers of effector cells but the expansion of the effector cells can result in preferential expansion of desirable phenotypes within effector cells in a cell culture. Thus, an enrichment of effector cells with desirable phenotypes can result, such as CD62L+effector cells, including TSCM cells, which are less or non-fully diffentiated. Such cells are desirable for their ability to differentiate, renew or grow, and / or migrate within the body. Such features are important in the expansion of effector cells for cell therapies. For effector T cells, the presence of the

[0208] #14471824vl CD62L+ marker indicates a central memory T cell (Tcm) phenotype, not a terminally differentiated effector cell. The Tcm cells have superior proliferative potential, patrol lymph nodes, and differentiate into potent effector cells upon restimulation, which gives them high therapeutic value in cancer immunotherapy.

[0209] Example 9. Addition of CDNP-IL-7 and / or CDNP-IL-15 other CDNPs does not affect Elevation of CD62L Expression and Results in Total Number of CD62L-positive Cells that is Proportional to Overall T Cell Expansion

[0210] In the same series of studies from Example 6, the surface expression of selectin molecule CD62L, known to be strongly elevated by the CDNPs carrying scFvOKT3, CD86, 4-1BBL, IL-7 and fused IL-15 / IL-15Ra signals, was assessed by flow cytometry. There was no significant effect of cell-derived cytokines on CD62L expression with the total number of CD62L-positive cells generally reflecting the overall T cell expansion. Therefore, the highest number of cells expressing surface CD62L was seen after treatment with CDNPs carrying scFvOKT3, CD86, 4-1BBL, IL-7 and fused IL-15 / IL-15Ra signals complemented with CDNP-IL-7 and CDNP-IL-15 at all timepoints measured (FIG. 12). Like above, such results show greatly increased numbers of effector cells with desirable phenotypes (CD62L+in this example). This is evidenced by the superior expansion in such cells that results from cell- derived cell surface signals, such as cytokines.

[0211] #14471824vl

Claims

CLAIMSWhat is claimed is:

1. A composition comprising: a first population of cell-derived nanoparticles (CDNPs), and a second population of CDNPs; wherein the CDNPs of the first population and the second population are produced from cells that were engineered to express a cell surface signal, or set of cell surface signals, and wherein the cell surface signal, or set of cell surface signals, of the first population and the second population are distinct.

2. The composition of claim 1, wherein the cell surface signal, or set of cell surface signals, is / comprises stimulatory molecule(s) and / or ligand(s), costimulatory molecule(s) and / or ligand(s) and / or cytokine(s).

3. The composition of claim 2, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) comprise CD3L, CD86, IL-7, IL- 15 and 4- 1BBL.

4. The composition of claim 2, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) is IL-7, IL-15, IL-21 or 4-1BBL.

5. The composition of claim 2, wherein the CDNPs of the first population comprise CD3L, CD86, IL-7, IL- 15 and 4-1BBL.

6. The composition of claim 2 or 5, wherein the CDNPs of the second population comprise IL-7, IL- 15, IL-21 or 4-1BBL.

7. The composition of claim 6, wherein the CDNPs of the second population comprise IL-7 and / or IL- 15.#14471824vl8. The composition of any one of the preceding claims, further comprising a third population of CDNPs, wherein the CDNPs of the third population are produced from cells that were engineered to express IL-7 or IL-15, and wherein the cell surface signal, or set of cell surface signals, of the first population, the second population, and the third population are distinct.

9. The composition of any one of claims 3-8, wherein the IL- 15 is in combination with IL-15Ra.

10. The composition of any one of the preceding claims, further comprising effector cells.

11. The composition of claim 10, wherein the effector cells are T cells.

12. The composition of claim 10, wherein the effector cells are NK cells.

13. A composition comprising: a first population of first cell line population cell-derived nanoparticles (CDNPs), and a second population of second cell line population CDNPs; wherein the first cell line population and the second cell line population are engineered to express a cell surface signal, or set of cell surface signals, and wherein the first cell line and second cell line comprise cell surface signals, or sets of cell surface signals, that are distinct.

14. The composition of claim 13, wherein the cell surface signal, or set of cell surface signals, is / comprises stimulatory molecule(s) and / or ligand(s), costimulatory molecule(s) and / or ligand(s) and / or cytokine(s).

15. The composition of claim 14, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) comprise CD3L, CD86, IL-7, IL- 15 and 4- 1BBL.

16. The composition of claim 14, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) is IL-7, IL-15, IL-21 or 4-1BBL.#14471824vl17. The composition of claim 14, wherein the CDNPs of the first population comprise CD3L, CD86, IL-7, IL- 15 and 4-1BBL.

18. The composition of claim 14 or 17, wherein the CDNPs of the second population comprise IL-7, IL- 15, IL-21 or 4-1BBL.

19. The composition of claim 18, wherein the CDNPs of the second population comprise IL-7 and / or IL- 15.

20. The composition of any one of 13-19, further comprising a third population of third cell line population CDNPs, wherein the third cell line population is engineered to express IL-7 or IL- 15, and wherein the first cell line, second cell line and third cell line comprise cell surface signals, or sets of cell surface signals, that are distinct.

21. The composition of any one of claims 15-20, wherein the IL- 15 is in combination with IL-15Ra.

22. The composition of any one of claims 13-21, further comprising effector cells.

23. The composition of claim 22, wherein the effector cells are T cells.

24. The composition of claim 22, wherein the effector cells are NK cells.

25. A method comprising: placing a first population of CDNPs and a second population of CDNPs in contact with effector cells; wherein the CDNPs of the first population and the second population express a cell surface signal, or set of cell surface signals, and wherein the cell surface signal, or set of cell surface signals, of the first population and the second population are distinct.#14471824vl26. The method of claim 25, wherein the cell surface signal, or set of cell surface signals, is / comprises stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) and / or cytokine(s).

27. The method of claim 26, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) comprise CD3L, CD86 IL-7, IL-15 and 4-1BBL.

28. The method of claim 26, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) is IL-7, IL- 15, IL-21 or 4-1BBL.

29. The method of claim 26, wherein the CDNPs of the first population comprise CD3L, CD86, 4-1BBL, IL-7 and IL- 15.

30. The method of claim 26 or 29, wherein the CDNPs of the second population comprise IL-7, IL-15, IL-21 or 4-1BBL.

31. The method of claim 30, wherein the CDNPs of the second population comprise IL-7 and / or IL- 15.

32. The method of any one of claims 25-31, wherein the method further comprises placing a third population of CDNPs in contact with the effector cells, wherein the CDNPs of the third population express IL-7 or IL-15, and wherein the cell surface signal, or set of cell surface signals, of the first population, the second population and third population are distinct.

33. The method of any one of claims 27-32, wherein the IL- 15 is in combination with IL- 15Ra.

34. The method of any one of claims 25-33, wherein the effector cells are T cells.

35. The method of any one of claims 25-33, wherein the effector cells are NK cells.#14471824vl36. The method of any one of claims 25-35, wherein the placing in contact occurs in vitro or ex vivo.

37. The method of any one of claims 25-36, wherein the placing in contact of the second and / or third populations occurs at the same time as the first population of CDNPs.

38. The method of any one of claims 25-36, wherein the placing in contact of the second and / or third populations does not occur at the same time as the first population of CDNPs.

39. The method of any one of claims 25-38, wherein the placing in contact of the second and / or third populations occurs, or also occurs, at least three days after the first population of CDNPs.

40. The method of any one of claims 25-39, wherein the placing in contact of the second and / or third populations occurs, or also occurs, at least six days after the first population of CDNPs.

41. The method of any one of claims 25-39, wherein the placing in contact of the second and / or third populations occurs, or also occurs, at least seven days after the first population of CDNPs.

42. The method of any one of claims 25-41, wherein the placing in contact of the second and / or third populations occurs, or also occurs, at least nine days after the first population of CDNPs.

43. The method of any one of claims 25-42, wherein the method further comprises obtaining the expanded effector cells.

44. The method of claim 43, wherein the method further comprises purifying the expanded effector cells.

45. The method of claim 43 or 44, further comprising administering the expanded effector cells to a subject.#14471824vl46. A method for producing a population of CDNPs comprising: processing two or more cell lines to form a population of CDNPs, wherein each cell line is engineered to express a cell surface signal, or set of cell surface signals, and wherein the cell surface signal, or set of cell surface signals, of the cell lines are distinct; and obtaining the population of CDNPs.

47. The method of claim 46, wherein the two or more cell lines are maintained in the same cell culture.

48. The method of claim 46, wherein the two or more cell lines are maintained in separate cell cultures.

49. The method of any one of claims 46-48, further comprising engineering each cell line of the two or more cell lines to express the cell surface signal, or set of cell surface signals.

50. The method of any one of claims 46-49, wherein the cell surface signal, or set of cell surface signals, is / comprises stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) and / or cytokine(s).

51. The method of claim 50, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) comprise CD3L, CD86, IL-7, IL-15 and 4-1BBL.

52. The method of claim 50, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) is IL-7, IL- 15, IL-21 or 4-1BBL.

53. The method of any one of claims 46-52, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) and / or cytokine(s) of each of the at least two cell lines are distinct from the other.

54. The method of claim 53, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) and / or cytokine(s) of one of the at least two cell lines comprise CD3L, CD86, 4-1BBL, IL-7 and IL-15.#14471824vl55. The method of claim 53 or 54, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) and / or cytokine(s) of one of the at least two cell lines comprise IL-7, IL- 15, IL-21 or 4-1BBL.

56. The method of claim 55, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) and / or cytokine(s) of one of the at least two cell lines comprise IL-7 and / or IL15.

57. The method of any one of claims 46-56, wherein the two or more cell lines are three cell lines, and wherein the third cell line is engineered to express IL-7 or IL- 15.

58. The method of any one of claims 46-57, wherein the processing comprises disruption of the cells of the two or more cell lines.

59. The method of claim 58, wherein the disruption is mechanical disruption.

60. The method of claim 59, wherein the mechanical disruption is cavitation, homogenization, or extrusion.

61. The method of claim 58, wherein the disruption is chemical disruption.

62. The method of any one of claims 46-61, wherein when the two or more cell lines are maintained in separate cell cultures, the processing comprises mixing the cells of the two or more cell lines to form a mixed cell culture.

63. The method of claim 62, wherein when the processing comprises disruption, the mixing occurs prior to the disruption of the cells of the mixed cell culture.

64. The method of any one of claims 48-61, wherein when the two or more cell lines are maintained in separate cell cultures and the processing comprises disruption, the processing further comprises collecting and mixing CDNPs from each cell culture subsequent to disruption of the cells of each cell culture.#14471824vl65. The method of any one of claims 46-64, wherein the obtaining comprises extracting filtering and / or purifying.

66. A composition comprising a population of CDNPs produced by the method of any one of claims 46-65.

67. The composition of claim 66, wherein the population of CDNPs comprise a first population of CDNPs and a second population of CDNPs, and optionally a third population of CDNPs, wherein each of the first, second and optional third populations of CDNPs comprise cell surface signals, or sets of cell surface signals, that are distinct from the other populations.

68. The composition of claim 66 or 67, wherein the cell surface signal, or set of cell surface signals, is / comprises stimulatory molecule(s) and / or ligand(s), costimulatory molecule(s) and / or ligand(s) and / or cytokine(s).

69. The composition of claim 68, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) comprise CD3L, CD86, IL-7, IL- 15 and 4- 1BBL.

70. The composition of claim 68, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) is IL-7, IL-15, IL-21 or 4-1BBL.

71. The composition of claim 68, wherein the CDNPs of the first population comprise CD3L, CD86, IL-7, IL- 15 and 4-1BBL.

72. The composition of claim 68 or 71, wherein the CDNPs of the second population comprise IL-7, IL- 15, IL-21 or 4-1BBL.

73. The composition of claim 72, wherein the CDNPs of the second population comprise IL-7 and / or IL- 15.#14471824vl74. The composition of any one of claims 68-73, wherein the CDNPs of the third population comprise IL-7 or IL- 15.

75. The composition of any one of claims 69-74, wherein the IL- 15 is in combination with IL-15Ra.

76. A method comprising administering the expanded effector cells produced by the method of any one of claims 25-45, to a subject in need thereof.

77. A cell line or set of cell lines that are each engineered to express a cell surface signal or set of cell surface signals, which cell surface signal(s) being distinct for each cell line or set of cell lines.

78. The cell line or set of cell lines of claim 77, wherein the cell surface signal or set of cell surface signals is / are those of any one or any one combination of cell surface signal(s) provided herein.

79. The cell line or set of cell lines of claim 77 or 78, comprising a first cell line and a second cell line, and optionally a third cell line.

80. The cell line or set of cell lines of any one of claims 77-79, wherein the cell surface signal, or set of cell surface signals, is / comprises stimulatory molecule(s) and / or ligand(s), costimulatory molecule(s) and / or ligand(s) and / or cytokine(s).

81. The cell line or set of cell lines of claim 80, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) comprise CD3L, CD86, IL-7, IL- 15 and 4-1BBL.

82. The cell line or set of cell lines of claim 80, wherein the stimulatory molecule(s) and / or ligand(s) and / or costimulatory molecule(s) and / or ligand(s) is IL-7, IL-15, IL-21 or 4- 1BBL.

83. The cell line or set of cell lines of any one of claims 80, wherein the first cell line expresses CD3L, CD86, IL-7, IL-15 and 4-1BBL.#14471824vl84. The cell line or set of cell lines of any one of claims 80 or 83, wherein the second cell line expresses IL-7, IL- 15, IL-21 or 4-1BBL.

85. The cell line or set of cell lines of any one of claims 80 or 83, wherein the second cell line expresses IL-7 and / or IL- 15.

86. The cell line or set of cell lines of any one of claims 79-85, wherein the third cell line expresses IL-7 or IL-15.

87. The cell line or set of cell lines of any one of claims 81-86, wherein the IL- 15 is in combination with IL-15Ra.#14471824vl

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