Receptors that provide targeted costimulation for adoptive cell therapy

Abstract

The present invention relates to cells containing a chimeric costimulatory antigen receptor (CoStAR) useful in adoptive cell therapy (ACT). The present invention relates to cells containing a chimeric costimulatory antigen receptor (CoStAR), which is useful for adoptive cell therapy (ACT). CoStAR can act as a modulator of cellular activity to enhance responses to defined antigens. The present invention also provides the CoStAR protein, nucleic acids encoding CoStAR, and their therapeutic uses.

Description

[Technical Field]

[0001] Related Applications and Incorporation by Reference Reference is made to UK Patent Application No. 1900858.0, filed January 22, 2019, and US Patent Application No. 62 / 951,770, filed December 20, 2019. The above-referenced application, and all documents cited therein or during its prosecution ("documents cited in the application"), and all documents cited or referenced in the documents cited in the application, and all documents cited or referenced herein ("documents cited herein"), and all documents cited or referenced in the documents cited herein, together with manufacturer's instructions, manuals, product specifications, and product sheets for any products described herein and in any documents incorporated by reference herein, are hereby incorporated by reference and may be employed in the practice of this invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0002] Array Statements The present application contains a Sequence Listing which has been submitted electronically and is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION The present invention relates to cells containing a chimeric costimulatory antigen receptor (CoStAR), which is useful for adoptive cell therapy (ACT). CoStAR can act as a modulator of cellular activity, enhancing responses to defined antigens. The present invention also provides CoStAR proteins, nucleic acids encoding CoStAR, and their therapeutic uses. [Background technology]

[0003] Adoptive cell therapy (ACT), which uses autologous T cells to mediate cancer regression, has shown great promise in early clinical trials. Several common approaches have been tried, including the use of ex vivo-expanded tumor-reactive or tumor-infiltrating naturally occurring lymphocytes (TILs). Furthermore, T cells can be genetically engineered to retarget themselves toward defined tumor antigens. This can be achieved through the genetic transfer of peptide (p)-major histocompatibility complex (MHC)-specific T cell receptors (TCRs) or through synthetic fusions between tumor-specific single-chain antibody fragments (scFvs) and T cell signaling domains (e.g., CD3ζ), the latter termed chimeric antigen receptors (CARs). TIL and TCR transfer have proven particularly successful in targeting melanoma (Rosenberg et al. 2011, Morgan 2006), while CAR therapy has shown great promise in treating certain B-cell malignancies (Grupp et al. 2013).

[0004] The success of CAR therapy in leukemia has been attributed, in part, to the incorporation into CAR constructs of costimulatory domains (e.g., CD28 or CD137), whose signals synergize with those provided by CD3ζ to enhance antitumor activity. The basis for this view is related to the classical signal 1 / signal 2 paradigm of T cell activation, in which signal 1 provided by the TCR complex synergizes with signal 2 provided by costimulatory receptors such as CD28, CD137, or CD134, enabling cells to clonally expand, produce IL2, and survive long-term without the activation-induced cell death (AICD) associated with signal 1 alone. Furthermore, engagement of signal 2 enhances the signal generated through signal 1, enabling cells to better respond to low-avidity interactions, such as those encountered during antitumor responses. Targeted costimulation would have beneficial effects on non-CAR T cell therapies. For example, incorporating costimulatory domains into chimeric TCRs has been shown to enhance T cell responses to pMHC (Govers 2014). Tumor-infiltrating lymphocytes (TILs) utilize their endogenous TCRs to mediate tumor recognition, but it has not been possible to manipulate endogenous TCRs. Because tumor cells express very few costimulatory ligands, TILs are subject to significant limitations. The ability to induce targeted costimulation in TILs, or indeed any other adoptive T cell therapy, would be beneficial. Summary of the Invention

[0005] The novel CoStAR and cells containing or expressing CoStAR are beneficial for CAR-based and non-CAR-based T cell therapies alike. The present invention uses cells that express novel chimeric costimulatory receptors and provide costimulatory signals to T cells upon engagement with a defined disease-associated antigen, such as a tumor-associated antigen. Several reports have used signal 1 and signal 2 individually to drive antigen-specific responses in engineered T cells (Alvarez-Vallina & Hawkins 1996). However, none have utilized the full-length CD28 molecule. Using a full-length receptor, such as CD28, over a truncated form offers certain advantages. Full-length CD28 is a dimer, allowing the receptor to function in its native form. Chimeric antigen receptors cannot function optimally when expressed as a monomer (Bridgeman et al. 2010). The present invention also provides targeted costimulatory receptors that trigger signal 2 upon engagement with a defined antigen, such as a disease-associated or tumor-associated antigen. The full-length CD28 molecule contains motifs important for its natural function in binding members of the B7 family of receptors. This is a potentially dangerous property from the perspective of CARs with tandem CD28 and CD3ζ receptors, where ligation of the CAR with B7 can induce T cell activation, but an advantageous property for receptors with only the signal 2 receptor.

[0006] The present inventors have shown that T lymphocytes (T cells) can be engineered to express CoStAR (costimulatory antigen receptor) to enhance T cell activation upon engagement with defined tumor-associated antigens. The inventors have shown that when engineered cells are allowed to receive signal 2 via CoStAR upon engagement with the tumor-associated antigen CEA, T cell activation by signal 1 mitogen (OKT3) is enhanced as measured by secretion of IFNγ and IL-2. Thus, in a first aspect, the present invention provides (i) a tumor-associated antigen-specific domain, such as a single-chain antibody fragment; (ii) full-length costimulatory receptor signaling chain or domain; The present invention provides a recombinant costimulatory antigen receptor (CoStAR) comprising: The present invention also provides (i) a tumor-associated antigen-specific domain, such as a single-chain antibody fragment; (ii) full-length costimulatory receptor signaling chain or domain; The present invention provides cells, such as lymphocytes, including T cells or NK cells, that contain a recombinant costimulatory antigen receptor comprising the co-stimulatory antigen receptor.

[0007] The CoStAR described herein is designed so that binding of CoStAR to its designated tumor antigen results in receptor activation, providing a costimulatory signal to enhance the concurrent signal 1-mediated signal provided by recombinant TCR, chimeric antigen receptor (CAR), or endogenous TCR. The tumor antigen may be a tumor-associated antigen, such as one from the carcinoembryonic or carcinoembryonic antigen family of molecules, such as CEA or 5T4, and may be targeted using a single-chain antibody fragment. Alternatively, the tumor antigen may be an antigen presented as a peptide via MHC and may be targeted using a pMHC-specific single-chain antibody fragment or a single-chain TCR. In certain embodiments, the signaling domain or signaling chain comprises a full-length (entire protein sequence without signal peptide) costimulatory receptor, including, but not limited to, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6. The costimulatory domain may consist of a single receptor domain or multiple domains in tandem (i.e., a fusion receptor), linked directly or via flexible linkers up to 50 amino acids in length. In certain embodiments, the signaling domain comprises a fragment of a full-length costimulatory receptor, wherein the fragment is effective for dimerization. There may be a linker connecting the single-chain antibody fragment to the signaling domain. If present, the linker may be 1 to 50 amino acids in length. The lymphocyte may be a T cell, including a tumor-infiltrating lymphocyte (TIL), a T regulatory cell (Treg), or a primary T cell, or an NK cell. In addition to CoStAR, the lymphocyte, T cell, or NK cell may contain a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0008] Those with sufficient experience will recognize that it is difficult to accurately determine the exact point at which a signal peptide is cleaved from the mature protein. Although prediction software can be applied, artificial splicing of sequences upstream of the mature protein creates new sites for peptidase activity of the signal peptide. Therefore, the term "full-length" herein refers to the entire mature protein, including deletions or mutations of up to five amino acids at its N-terminal extreme, a process important for optimal splicing of the costimulatory domain into the chimeric receptor. This clearly differs from previous published sequences, such as those used in Lanitis et al. (2013), in which only the CD28 intracellular domain was used, and the CD8 transmembrane domain, and in Krause et al. (1998), in which a truncated CD28 receptor was used, shortened to the motif IEV.

[0009] In a second aspect, the present invention provides a nucleic acid sequence encoding CoStAR as described above and herein. In a third aspect, the present invention provides a vector comprising a nucleic acid sequence according to the second aspect and, if present, a TCR and / or CAR nucleic acid sequence. In a fourth aspect, the present invention provides a method for producing a lymphocyte, or a T cell or NK cell, according to the first aspect of the invention, comprising the step of introducing a nucleic acid or vector encoding CoStAR into the lymphocyte. In another aspect, the present invention provides a pharmaceutical composition comprising a vector according to the third aspect or a lymphocyte (including a T cell or an NK cell) according to the first aspect and a pharmaceutically acceptable carrier, diluent or excipient, and therapeutic uses thereof. In a fifth aspect, the present invention provides a cell, such as a lymphocyte, including a T cell or NK cell according to the first aspect, or a vector according to the third aspect, for use in adoptive cell therapy.

[0010] In a sixth aspect, the present invention provides a lymphocyte, including a T cell or an NK cell, according to the first aspect, or a vector according to the third aspect, including for use in a method of treating cancer. In a seventh aspect, the present invention provides the use of a lymphocyte according to the first aspect or a vector according to the third aspect in the manufacture of a medicament for treating cancer. In an eighth aspect, the present invention provides a lymphocyte according to the first aspect, a vector according to the third aspect, or a pharmaceutical composition as disclosed herein for use in the treatment of cancer. In one embodiment, the CoStAR receptor comprises a full-length CD28 receptor as defined herein. In one embodiment, the CoStAR receptor comprises a fusion signaling domain comprising or consisting of a full-length human CD28, such as that set forth in SEQ ID NO: 2 or a variant thereof having at least 80% sequence identity at the protein level, and an intracellular domain from human CD137, such as that set forth in SEQ ID NO: 4 or a variant thereof having at least 80% or 90% sequence identity at the protein level, an intracellular domain from human CD134, such as that set forth in SEQ ID NO: 5 or a variant thereof having at least 80% or 90% sequence identity at the protein level, an intracellular domain from human CD2, such as that set forth in SEQ ID NO: 6 or a variant thereof having at least 80% or 90% sequence identity at the protein level, an intracellular domain from human CD29, such as that set forth in SEQ ID NO: 7 or a variant thereof having at least 80% or 90% sequence identity at the protein level, an intracellular domain from human CD29, such as that set forth in SEQ ID NO: 8 or a variant thereof having at least 80% or 90% sequence identity at the protein level, an intracellular domain from human CD29, such as that set forth in SEQ ID NO: 9 or a variant thereof having at least 80% or 90% sequence identity at the protein level, an intracellular domain from human CD29, such as that set forth in SEQ ID NO: 10 or a variant thereof having at least 80% or 90% sequence identity at the protein level, an intracellular domain from human CD29, such as that set forth in SEQ ID NO: 11 or a variant thereof having at least 80% or 90% sequence identity at the protein level, an intracellular domain from human CD29, such as that set forth in SEQ ID NO: 12 or a variant thereof having at least 80% or 90% sequence identity at the protein level, an intracellular domain from human CD29, or a variant thereof having at least 80% or 90% sequence identity at the protein level, an intracellular domain from human IL2Rγ such as that set forth in SEQ ID NO:9 or a variant thereof having at least 80% or 90% sequence identity at the protein level, an intracellular domain from human CD40 such as that set forth in SEQ ID NO:10 or a variant thereof having at least 80% or 90% sequence identity at the protein level, an intracellular domain from human CD150 such as that set forth in SEQ ID NO:11 or a variant thereof having at least 80% or 90% sequence identity at the protein level, or an intracellular domain from human CD2 and human CD40 such as that set forth in SEQ ID NO:12 or a variant thereof having at least 80% or 90% sequence identity at the protein level. In one embodiment, a variant of one of the above-mentioned moieties lacks 1, 2, 3, 4, 5, or up to 10 N-terminal amino acid residues with respect to the fusion sequences listed above.

[0011] The following detailed description, given by way of example and not by way of limitation to the particular embodiments, can be best understood in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows the structural organization of single costimulatory and fused costimulatory domain receptors. A schematic diagram of the claimed CoStAR receptors is shown. First, a CoStAR based on a single costimulatory receptor, and second, a fused CoStAR consisting of a full-length costimulatory receptor signaling domain fused to a second costimulatory domain. [Figure 2]

[0023] Figure 1 shows the genomic organization of potential CoStAR constructs. As shown in the figure and as claimed, CoStAR consists of an antigen-binding domain, an optional spacer domain, and a costimulatory domain. CoStAR can be expressed: A) alone from a promoter, with CoStAR consisting of a single (Ai) or fusion (Aii) costimulatory receptor; B) with an N- or C-terminal epitope tag (e.g., His tag, DYKDDDDK (SEQ ID NO: 14), etc.) to enable direct staining of CoStAR; C) together with a marker gene separated using a 2A cleavage sequence or an internal ribosomal entry site (IRES); D) together with a marker gene expressed from a second promoter; E) together with a protein of interest, such as a chimeric antigen receptor or T cell receptor, separated using a 2A cleavage sequence or an internal ribosomal entry site (IRES); or F) together with a protein of interest, such as a chimeric antigen receptor or T cell receptor, expressed from a second promoter. It will be apparent to one skilled in the art that CoStAR and the marker gene / chimeric antigen receptor / T cell receptor / other protein of interest can be expressed in either orientation or 3' (3 prime) or 5' (5 prime) to each other. [Figure 3]Figure 1 shows functional activity of CoStAR in T cells in response to LS174T and LoVo tumor-presented antigens. Normal donor T cell populations from donor 1 (A and D), donor 2 (B), and donor 3 (C and E) were lentivirally engineered to express CoStAR targeting carcinoembryonic antigen (CEA) and magnetically sorted using CD34 magnetic selection to enrich for the transgene. T cells were mixed with wild-type, unengineered CEA+ tumor cells (non-activated tumors) or CEA+ tumor cells engineered to express a cell surface-anchored anti-CD3 single-chain antibody fragment (activated tumors) at the indicated effector-to-target ratios, and IL-2 was measured in the supernatants by ELISA. Data were obtained using LS174T cells (A, B, and C) and LoVo cells (D and E). [Figure 4] Figure 1. Effect of CoStAR on T cell proliferation. 5 x 10 transduced and untransduced T cells were mixed with 6.25 x 10 wild-type LoVo or LoVo-OKT3 cells in the presence (A) or absence (B) of IL-2, and cell numbers were counted after 3 days. In a separate assay at the same cell ratio, T cells from two donors were loaded with a proliferation dye, and the number of proliferation cycles the cells had undergone was determined by dye dilution after 6 days using flow cytometry. [Figure 5] Figure 1 shows the IL-2 activity of CoStAR fusion receptors in primary human T cells. Normal donor CD8+ T cells from seven donors (except for control CoStAR in three donors) were lentivirally transduced with the indicated CEA-targeted CoStAR and examined for IL-2 production after overnight stimulation in the presence of LoVo-OKT3 cells. The percentage of IL-2-positive cells was determined using intracellular flow staining in the CD34-negative (CoStAR-untransduced) and CD34-positive (CoStAR-transduced) populations. Asterisks indicate significant differences between transduced and untransduced populations using a paired Wilcoxon signed-rank test with *p<0.05. [Figure 6-1]Figure 6 shows multiparameter analysis of CoStAR activity in primary human T cells. Normal donor CD8+ T cells were lentivirally transduced with the indicated CEA-targeted CoStAR and IL-2 production was investigated after overnight stimulation in the presence of LoVo-OKT3 cells. The percentages of IL-2 (seven donors) (Figure 6A), IFNγ (seven donors) (Figure 6B), bcl-xL (five donors) (Figure 6C), and CD107a (six donors) (Figure 6D) positive cells were determined using intracellular flow staining in CD34-negative (CoStAR-untransduced) and CD34+ (CoStAR-transduced) populations. The control was nonspecific CA125-targeted CoStAR, from three donors in all cases. Heatmaps are averages across all donors, and color intensity relates to the percentage of cells positive for a particular readout under the defined conditions. [Figure 6-2] Figure 6 shows multiparameter analysis of CoStAR activity in primary human T cells. Normal donor CD8+ T cells were lentivirally transduced with the indicated CEA-targeted CoStAR and IL-2 production was investigated after overnight stimulation in the presence of LoVo-OKT3 cells. The percentages of IL-2 (seven donors) (Figure 6A), IFNγ (seven donors) (Figure 6B), bcl-xL (five donors) (Figure 6C), and CD107a (six donors) (Figure 6D) positive cells were determined using intracellular flow staining in CD34-negative (CoStAR-untransduced) and CD34+ (CoStAR-transduced) populations. The control was nonspecific CA125-targeted CoStAR, from three donors in all cases. Heatmaps are averages across all donors, and color intensity relates to the percentage of cells positive for a particular readout under the defined conditions. [Figure 7]Figure 1 shows that CD40 enhances IL-2 production from CD28-based CoStAR. Primary human T cells from three healthy donors were either untransduced or transduced with CD28.CD40-based CoStAR containing extracellular domain-truncated CD28 (TrCD28), full-length CD28 (FLCD28), or a CEA-specific scFv (MFE23). Transduced cells were selected using the CD34 marker gene and expanded prior to analysis. T cells were mixed with OKT3-expressing CEA+ LoVo cells at an effector-to-target ratio of 8:1 for 20 hours, after which IL-2 production was analyzed by ELISA. [Figure 8] Figure 1 shows the influence of signaling domain and target antigen on CoStAR-mediated T cell expansion. T cells were transduced with DYKDDDDK (SEQ ID NO: 14) epitope-tagged CD28 or CD28.CD40-based CoStAR containing CA125-, FolR-, or CEA-specific scFv or a FolR-specific binding peptide (C7). T cells were mixed with the OKT3-expressing CA125+ / FolR+ / CEA cell line OVCAR3. The number of transduced cells was counted every 7 days up to every 21 days, and fresh OVCAR3 cells were added after each count. [Figure 9] (A-I) Diagram showing the effect of signaling domain and target antigen on CoStAR-mediated T cell expansion. T cells were transduced with DYKDDDDK (SEQ ID NO: 14) epitope-tagged CD28 or CD28.CD40-based CoStAR containing CA125-, FolR-, or CEA-specific scFv or the FolR-specific binding peptide (C7). T cells were mixed with the OKT3-expressing CA125+ / FolR+ / CEA cell line OVCAR3. The number of transduced cells was counted every 7 days up to every 21 days, and fresh OVCAR3 cells were added after each count. [Figure 10](A and B) Diagram showing that CD40-based CoStAR enhances T cell costimulation in a TCR transfer model. Primary human T cells from three healthy donors were transduced with a CEA-specific TCR and DYKDDDK-labeled CD28 or CD28.CD40-based CoStAR containing MFE (open or closed circles) or CA125 (open square)-specific scFvs. T cells were mixed with CEA / CA125-H508 cells at a 1:1 effector-to-target ratio, and intracellular cytokine staining was performed to determine the number of responding CD4 or CD8 T cells within the TCR / CoStAR, TCR / CoStAR, TCR / CoStAR, and TCR / CoStAR populations. Two-way ANOVA (Tukey's test) was performed to determine significant differences in activity: *p>0.05, **p>0.01, ***p>0.001, ****p>0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0014] Costimulatory antigen receptor (CoStAR) Provided herein are recombinant costimulatory antigen receptors (CoStARs) comprising: (i) a disease- or tumor-associated antigen-binding domain; and (ii) an extracellular ligand-binding segment of a stimulatory receptor protein; and (iii) a first intracellular segment comprising an intracellular signaling domain of a receptor protein; and (iv) optionally, a second intracellular segment comprising an intracellular signaling domain of a second receptor protein. In certain embodiments, the extracellular segment and the first intracellular segment comprise segments of the same receptor protein. In certain embodiments, the extracellular segment and the first intracellular segment comprise segments of different receptor proteins. In embodiments of the invention, there is an intervening transmembrane domain between the extracellular segment and the first intracellular segment. Embodiments comprising an optional second intracellular segment and / or an optional third intracellular segment can further comprise a spacer between the intracellular segments. In a simple form, the CoStARs of the invention comprise a single-chain antibody fragment (scFv) fused to full-length CD28 that delivers a costimulatory signal dependent on engagement of the scFv to its cognate antigen and / or engagement of CD28 to its cognate ligand. As used herein, "full-length protein" or "full-length receptor" refers to a receptor protein, e.g., a CD28 receptor protein. The term "full-length" encompasses receptor proteins lacking up to about 5 or up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, at the N-terminus of the mature receptor protein after its signal peptide has been cleaved. For example, the specific cleavage site of the receptor N-terminal signal peptide can be defined, although variation in the exact cleavage point has been observed. The term "full-length" does not imply the presence or absence of amino acids in the receptor N-terminal signal peptide. In one embodiment, the term "full-length" (e.g., full-length CD28 according to certain aspects of the invention) encompasses mature receptor proteins (e.g., CD28 according to certain aspects of the invention) lacking an N-terminal signal peptide that lacks up to about 5, e.g., 1, 2, 3, 4, 5, or up to 10 amino acids at the N-terminus of the mature receptor protein after its signal peptide has been cleaved.SEQ ID NO: 2 shows the mature CD28 protein, including the signal peptide. As noted above, a "full-length" CD28 receptor according to various embodiments of the invention does not include the signal peptide and may lack up to about 5, e.g., 1, 2, 3, 4, 5, or up to 10 amino acids at the N-terminus of the mature receptor protein (e.g., N-terminal residues N, K, I, L, and / or V). This is shown in exemplary fusions, e.g., SEQ ID NOs: 4-12 (note that, as shown in the boxed regions, these may lack up to about 5, e.g., 1, 2, 3, 4, 5, or up to 10 amino acids at the N-terminus of the mature receptor protein). CoStAR has a modular format and can be constructed to contain extracellular, transmembrane, and intracellular domains from one or more proteins, together with scFvs derived from antibodies that bind to disease-associated antigens, e.g., tumor-associated antigens.

[0015] According to the present invention, in one embodiment, CoStAR comprises a disease-associated, e.g., tumor-associated, antigen receptor, such as, but not limited to, a tumor-associated antigen-specific scFv, and a primary costimulatory receptor protein capable of binding to its cognate ligand and providing an intracellular signal. In certain embodiments, the primary costimulatory receptor may be a protein less than full-length, but sufficient to bind to its cognate ligand and transmit a signal. In certain embodiments, the primary costimulatory receptor domain is full-length, such as, but not limited to, full-length CD28. Thus, the antigen-specific binding domain and the ligand-specific receptor are capable of binding to their cognate antigen and ligand, respectively. Figure 1 depicts two embodiments of CoStAR constructs. Both CoStARs comprise a full-length costimulatory receptor protein comprising an antigen-binding domain, an optional spacer, an extracellular ligand-binding segment, and an intracellular signaling domain. In another embodiment, CoStAR comprises an antigen-binding domain, an optional spacer, the extracellular ligand-binding portion of a costimulatory receptor protein, a transmembrane domain, and the intracellular signaling domain of a second, different costimulatory receptor protein. In certain embodiments, the extracellular ligand-binding portion comprises a CD28 truncation, e.g., a C-terminal CD28 truncation after amino acid IEV, as in SEQ ID NO: 13, followed by an intracellular signaling domain. In certain embodiments, the intracellular signaling domain is from CD40. The transmembrane domain separating the extracellular ligand-binding and intracellular signaling domains may be, but is not limited to, from CD28 or CD40. Compared to the construct on the left in FIG. 1, the construct on the right in FIG. 1 further comprises an additional second intracellular receptor signaling domain and an optional spacer. In further embodiments, CoStAR can comprise an additional costimulatory domain, e.g., a third intracellular costimulatory signaling domain, and in this respect may resemble certain chimeric antigen receptors (CARs), which are classified as first (CD3ζ only), second (one costimulatory domain + CD3ζ), or third generation (more than one costimulatory domain + CD3ζ).

[0016] Costimulatory receptor proteins useful in the CoStAR of the present invention include, but are not limited to, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6, which in their native form comprise an extracellular ligand-binding domain and an intracellular signaling domain. For example, CD2 is characterized as a cell adhesion molecule found on the surface of T cells and is capable of initiating intracellular signals necessary for T cell activation. CD27 is characterized as a type II transmembrane glycoprotein belonging to the TNF superfamily (TNFSF), whose expression on B cells is induced by antigen receptor activation in B cells. CD28 is one of the proteins on T cells and is a receptor for CD80 (B7.1) and CD86 (B7.2) ligands on antigen-presenting cells. CD137 (4-1BB) ligand is found on most leukocytes and some non-immune cells. OX4 ligand is expressed on many antigen-presenting cells, such as DC2 (dendritic cells), macrophages and B lymphocytes. In one embodiment, the costimulatory receptor protein is full-length CD28 as defined herein.

[0017] In embodiments of the present invention, CoStAR can be expressed alone under the control of a promoter in a therapeutic population of therapeutically active cells, e.g., tumor-infiltrating lymphocytes (TILs). Alternatively, CoStAR can be expressed together with a therapeutic transgene, such as a chimeric antigen receptor (CAR) and / or T cell receptor (TCR), as set forth in SEQ ID NOS: 3-12 (note that up to about five, e.g., 1, 2, 3, 4, 5, or up to 10 amino acids at the N-terminus of the mature receptor protein may be missing). Thus, in one aspect, the present invention also relates to a CoStar construct having a sequence set forth in any of SEQ ID NOS: 3-12, including one of these sequences lacking up to about five, e.g., 1, 2, 3, 4, 5, or up to 10 amino acids at the N-terminus of the mature receptor protein. Suitable TCRs and CARs, such as HLA-A*02-NYESO-1-specific TCRs (Rapoport et al. Nat Med 2015) or anti-CD19scFv.CD3ζ fusion CARs (Kochenderfer et al. J Clin Oncol 2015), are well known in the literature and have been successfully used to treat myeloma or B-cell malignancies, respectively. The CoStAR described herein can be expressed with any known CAR or TCR, thus providing cells with a tunable proliferation switch that allows in vitro or in vivo cell expansion, and a conventional activation mechanism in the form of a TCR or CAR for anti-cancer activity. Thus, the present invention provides cells for use in adoptive cell therapy that contain the CoStAR described herein and a TCR and / or CAR that specifically binds to a tumor-associated antigen. An exemplary CoStAR, including CD28, contains an extracellular antigen-binding domain and an extracellular transmembrane and intracellular signaling domain that includes the amino acid sequence set forth in SEQ ID NO:2.

[0018] As used herein, the term "antigen-binding domain" refers to an antibody fragment, including, but not limited to, a diabody, Fab, Fab', F(ab'), Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv), bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody comprising one or more CDRs, camelized single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise the complete antibody structure. An antigen-binding domain is capable of binding to the same antigen as bound by the parent antibody or parent antibody fragment (e.g., the parent scFv). In some embodiments, the antigen-binding fragment can comprise one or more complementarity-determining regions (CDRs) from a particular human antibody grafted onto framework regions (FRs) from one or more different human antibodies.

[0019] The antigen-binding domain can be specific for any disease-associated antigen, including, but not limited to, tumor-associated antigens (TAA) and infectious disease-associated antigens. In certain embodiments, the ligand-binding domain is bispecific. Antigens have been identified in most human cancers, including Burkitt's lymphoma, neuroblastoma, melanoma, osteosarcoma, renal cell carcinoma, breast cancer, prostate cancer, lung cancer, and colon cancer. TAAs include, but are not limited to, CD19, CD20, CD22, CD24, CD33, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα), mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, and HER-2. TAAs also include neoantigens, peptide / MHC complexes, and HSP / peptide complexes.

[0020] In certain embodiments, the antigen-binding domain comprises a T cell receptor or a binding fragment thereof that binds to a defined tumor-specific peptide-MHC complex. The term "T cell receptor" or "TCR" refers to a heterodimeric receptor composed of paired αβ or γδ chains on the surface of T cells. Each α, β, γ, and δ chain is composed of two Ig-like domains: a variable domain (V) that confers antigen recognition through complementarity-determining regions (CDRs), followed by a constant domain (C) that is anchored to the cell membrane by a connecting peptide and a transmembrane (TM) region. The TM region binds to the invariant subunit of the CD3 signaling apparatus. Each V domain has three CDRs. These CDRs interact with the complex between antigenic peptides bound to proteins encoded by the major histocompatibility complex (pMHC) (Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544; Murphy (2012), xix, 868 p.).

[0021] In certain embodiments, the antigen-binding domain comprises a natural ligand of a tumor-expressed protein or a tumor-binding fragment thereof. For example, transferrin receptor 1 (TfR1), also known as CD71, is a homodimeric protein that is a key regulator of cellular iron homeostasis and proliferation. TfR1 is expressed at low levels in a variety of cells, but it is expressed at higher levels in rapidly proliferating cells, including malignant cells, where overexpression is associated with a poor prognosis. In one embodiment of the present invention, the antigen-binding domain comprises transferrin or a transferrin receptor-binding fragment thereof. In certain embodiments, the antigen-binding domain is specific for a defined tumor-associated antigen, such as, but not limited to, FRα, CEA, 5T4, CA125, SM5-1, or CD71. In certain embodiments, the tumor-associated antigen may be a tumor-specific peptide-MHC complex. In certain such embodiments, the peptide is a neoantigen. In other embodiments, the tumor-associated antigen is a peptide-heat shock protein complex.

[0022] In an embodiment of the invention, the extracellular, transmembrane and intracellular signaling domains of CoStAR have the sequence shown in SEQ ID NO:1. As used herein, the terms "specifically bind" or "specific for" refer to a measurable and reproducible interaction, e.g., binding between a target and an antibody or antibody portion, that is determinative of the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody portion that specifically binds to a target (which may be an epitope) is an antibody portion that binds to the target with greater affinity, avidity, more readily, and / or with a longer duration than its binding to other targets. In some embodiments, an antibody portion that specifically binds to an antigen reacts with one or more antigenic determinants of the antigen (e.g., a cell surface antigen or peptide / MHC protein complex) with a binding affinity that is at least about 10 times greater than its binding affinity to other targets.

[0023] spacer domain The CoStAR of the present invention optionally comprises a spacer region between the antigen-binding domain and the costimulatory receptor. As used herein, the term "spacer" refers to the extracellular structural region of CoStAR that separates the antigen-binding domain from the external ligand-binding domain of the costimulatory protein. The spacer provides flexibility in accessing target antigens and receptor ligands. In certain embodiments, a long spacer is used, for example, to target membrane-proximal epitopes or glycosylated antigens (see Guest RD et al. The role of extracellular spacer regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens. J. Immunother. 2005;28:203-211; Wilkie S. et al., Retargeting of human T cells to tumor-associated MUC1: the evolution of a chimeric antigen receptor. J. Immunol. 2008;180:4901-4909). In other embodiments, CoStAR has a short spacer, e.g., to target membrane-distal epitopes (see Hudecek M. et al., Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells. Clin. Cancer Res. 2013;19:3153-3164; Hudecek M. et al., The nonsignalling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol. Res. 2015;3:125-135).In certain embodiments, the spacer comprises or is derived from all or a portion of an IgG hinge, including, but not limited to, IgG1, IgG2, or IgG4. "Derived from an Ig hinge" refers to a spacer that contains an insertion, deletion, or mutation in an IgG hinge. In certain embodiments, the spacer can comprise all or a portion of one or more antibody constant domains, such as the CH2 and / or CH3 domains. In certain embodiments, in spacers that contain all or a portion of the CH2 domain, the CH2 domain is modified so that it does not bind to Fc receptors. For example, Fc receptor binding in myeloid cells has been found to impair CAR T cell function. In certain embodiments, the spacer comprises all or a portion of an Ig-like hinge from CD28, CD8, or other proteins that contain a hinge region. In certain embodiments of the present invention that include a spacer, the spacer is 1 to 50 amino acids in length.

[0024] Signaling domains As mentioned above, in certain embodiments, CoStAR comprises a full-length primary costimulatory receptor, which can comprise, but is not limited to, the extracellular ligand binding and intracellular signaling domains of CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR) or EphB6. In other embodiments, the costimulatory receptor comprises a chimeric protein, for example, comprising one extracellular ligand binding domain of the protein and another intracellular signaling domain of the protein. In certain embodiments, the signaling portion of CoStAR comprises a single signaling domain. In other embodiments, the signal transduction portion of CoStAR comprises a second intracellular signal transduction domain, for example, but not limited to, CD2, CD27, CD28, CD40, CD134 (OX40), CD137 (4-1BB), CD150 (SLAM). In certain embodiments, the first and second intracellular signal transduction domains are the same. In other embodiments, the first and second intracellular signal transduction domains are different. In certain embodiments, the costimulatory receptor is capable of dimerization. Without being bound by theory, it is believed that CoStAR dimerizes or binds to other accessory molecules to initiate signals. In certain embodiments, CoStAR dimerizes or binds to accessory molecules through transmembrane domain interaction. In certain embodiments, dimerization or binding by accessory molecules is supported by costimulatory receptor interaction in the intracellular and / or extracellular portions of the costimulatory receptor.

[0025] Transmembrane domain Although the primary function of the transmembrane is to anchor CoStAR to the T cell membrane, in certain embodiments, the transmembrane domain influences CoStAR function. In certain embodiments, the transmembrane domain is included in the full-length primary costimulatory receptor domain. In embodiments of the present invention in which a CoStAR construct comprises the extracellular domain of one receptor and the intracellular signaling domain of a second receptor, the transmembrane domain may be that of the extracellular domain or the intracellular domain. In certain embodiments, the transmembrane domain is from CD4, CD8α, CD28, or ICO. Gueden et al. associated the use of the ICOS transmembrane domain with increased CAR T cell persistence and overall anti-tumor efficacy (Guedan S. et al., Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. JCI Insight. 2018;3:96976). In one embodiment, the transmembrane domain comprises a hydrophobic α-helix spanning the cell membrane. Examples of CoStAR signaling domains according to the present invention are provided in SEQ ID NOs: 3-12.

[0026] Mutants In some embodiments, amino acid sequence variants of the antibody moieties or other moieties provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody moiety. Amino acid sequence variants of the antibody moiety can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody moiety or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues in the amino acid sequence of the antibody moiety. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, e.g., antigen binding.

[0027] In some embodiments, antibody binding domain portions containing one or more amino acid substitutions, deletions, or insertions are provided. Targeted sites for mutation include the heavy and light chain variable regions (VRs) and framework regions (FRs) of the antibody binding domain. Amino acid substitutions can be introduced into the desired binding domain, and the products can be screened for desired activity, such as retained / improved antigen binding or reduced immunogenicity. In certain embodiments, amino acid substitutions can be introduced into one or more of the primary costimulatory receptor domain (extracellular or intracellular), the secondary costimulatory receptor domain, or the extracellular co-receptor domain. Thus, the present invention encompasses the CoStAR proteins and component portions specifically disclosed herein, as well as variants thereof, i.e., CoStAR proteins and component portions having at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequences specifically disclosed herein. The terms "percent similarity," "percent identity," and "percent homology," when referring to particular sequences, are used as set forth in the University of Wisconsin GCG software program BestFit. Other algorithms, such as BLAST, psiBLAST, or TBLASTN (using the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (using the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), can be used.

[0028] Particular amino acid sequence variants may differ from a reference sequence by the insertion, addition, substitution, or deletion of 1, 2, 3, 4, 5-10, 10-20, or 20-30 amino acids. In some embodiments, a variant sequence can comprise a reference sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more residues have been inserted, deleted, or substituted. For example, 5, 10, 15, up to 20, up to 30, or up to 40 residues may have been inserted, deleted, or substituted. In some preferred embodiments, a variant may differ from a reference sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more conservative substitutions. Conservative substitutions involve the replacement of an amino acid with a different amino acid having similar properties. For example, an aliphatic residue can be replaced with another aliphatic residue, an apolar residue can be replaced with another apolar residue, an acidic residue can be replaced with another acidic residue, a basic residue can be replaced with another basic residue, a polar residue can be replaced with another polar residue, or an aromatic residue can be replaced with another aromatic residue. Conservative substitutions can occur, for example, between amino acids within the following groups:

[0029] Conservative substitutions are shown in the table below. [Table 1]

[0030] Amino acids can be divided into different classes according to shared side chain properties: a. hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. acidic: Asp, Glu; d. basic: His, Lys, Arg; e. residues that affect chain orientation: Gly, Pro; aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another.

[0031] cell The cells used in the present invention can be any lymphocyte that is beneficial in adoptive cell therapy, such as T cells or natural killer (NK) cells, NKT cells, gamma / delta T cells, or T regulatory cells. The cells can be allogeneic or autologous to the patient. T cells or T lymphocytes are a type of lymphocyte that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of T cell receptors (TCRs) on their cell surface. There are various types of T cells, as summarized below. Cytotoxic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells and have also been implicated in transplant rejection. CTLs express CD8 molecules on their surface.

[0032] These cells recognize their targets by binding to antigens associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases such as experimental autoimmune encephalomyelitis. Memory T cells are a subset of antigen-specific T cells that persist long after an infection has resolved. They rapidly expand into large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with a "memory" of past infections. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO. Regulatory T cells (Treg cells), formerly known as suppressor T cells, are important for maintaining immune tolerance. Their primary role is to shut down T cell-mediated immunity toward the end of an immune response and to suppress autoreactive T cells that have escaped the negative selection process in the thymus.

[0033] Two major classes of CD4+ Treg cells have been described: naturally occurring Treg cells and adaptive Treg cells. + CD25 + FoxP3 + Treg cells (also known as Treg cells) arise in the thymus and interact with developing T cells and myeloid (CD11c) cells activated by TSLP. + ) and plasmacytoid (CD123 + ) are associated with interactions between dendritic cells. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Adaptive Treg cells (also known as Tr1 or Th3 cells) can arise during a normal immune response. Natural killer cells (or NK cells) are a type of cytolytic cell that forms part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner. NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGLs), and constitute a third type of cell distinct from the common lymphocyte precursor that generates B and T lymphocytes.

[0034] In certain embodiments, therapeutic cells of the present invention comprise autologous cells that are engineered to express CoStAR.In certain embodiments, therapeutic cells of the present invention comprise allogeneic cells that are engineered to express CoStAR.Autologous cells that express CoStAR may be advantageous in avoiding graft-versus-host disease (GVDH) due to TCR-mediated recognition of recipient alloantigens.In addition, the immune system of CoStAR recipients may attack injected CoStAR cells, causing rejection.In certain embodiments, to prevent GVHD and reduce rejection, endogenous TcR is removed from allogeneic CoStAR cells by genome editing.

[0035] nucleic acid An embodiment of the present invention provides nucleic acid sequences of the present invention encoding any of the CoStAR, polypeptides, or proteins (including functional portions and functional variants thereof) described herein. As used herein, the terms "polynucleotide," "nucleotide," and "nucleic acid" are synonymous with each other. As a result of the degeneracy of the genetic code, those skilled in the art will understand that numerous different polynucleotides and nucleic acids can encode the same polypeptide. Furthermore, those skilled in the art will understand that, using routine techniques, nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein can be made, e.g., for codon optimization, to reflect the codon usage of any particular host organism in which the polypeptide will be expressed. Nucleic acids according to the present invention can comprise DNA or RNA. They can be single-stranded or double-stranded. They can also be polynucleotides that contain synthetic or modified nucleotides within them. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For purposes of the present invention, it should be understood that polynucleotides can be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.

[0036] The terms "variant," "homologue," or "derivative" in reference to a nucleotide sequence include any substitution, alteration, modification, replacement, deletion, or addition of one or more nucleic acids from or to that sequence. The nucleic acid sequence can encode the protein sequence shown in SEQ ID NO:2 or a variant thereof. The nucleotide sequence can comprise the codon-optimized human CD28 nucleic acid sequence shown in SEQ ID NO:1 or a variant thereof (depicted in Figure 1). The present invention also provides a nucleic acid sequence comprising a nucleic acid sequence encoding CoStAR and an additional nucleic acid sequence encoding a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR). The nucleic acid sequence can be linked to a sequence that allows for the simultaneous expression of two or more nucleic acid sequences. For example, the construct can include an internal promoter, an internal ribosome entry site (IRES) sequence, or a sequence encoding a cleavage site. The cleavage site can be self-cleaving so that when the polypeptide is produced, it is immediately cleaved into individual proteins without the need for any external cleavage activity. Various self-cleavage sites are known, including the foot-and-mouth disease virus (FMDV) and 2A self-cleaving peptides. The co-expression sequence can be an internal ribosome entry site sequence (IRES). The co-expression sequence can be an internal promoter.

[0037] vector In one aspect, the invention provides a vector comprising a nucleic acid sequence or nucleic acid construct of the invention. Such vectors can be used to introduce nucleic acid sequences or constructs into host cells so that they express one or more CoStARs according to the first aspect of the invention, and optionally one or more other proteins of interest (POIs), such as TCRs or CARs. The vectors can be, for example, plasmids or viral vectors, such as retroviral or lentiviral vectors, or transposon-based vectors or synthetic mRNAs. The nucleic acid of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocol.Gene delivery method is known in the art.For example, see U.S. Patent No. 5,399,346, 5,580,859, 5,589,466, which are fully incorporated herein by reference.

[0038] Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, because they allow the long-term stable integration of a transgene or multiple transgenes and their propagation in daughter cells.The vector may be capable of transfecting or transducing lymphocytes, including T cells or NK cells.The present invention also provides a vector into which the nucleic acid of the present invention is inserted.The expression of natural or synthetic nucleic acids encoding CoStAR and optionally TCR or CAR is generally achieved by operably linking the nucleic acid encoding CoStAR and TCR / CAR polypeptides or a portion thereof to one or more promoters, and incorporating this construct into an expression vector.

[0039] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start site, although it has recently been shown that some promoters also contain functional elements downstream of the start site. Because the spacing between promoter elements is often flexible, promoter function is preserved even when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of promoting high-level expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation growth factor 1 alpha (EF-1 alpha). However, other constitutive promoter sequences can also be used, including the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0040] The vector may be suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), as well as other virology and molecular biology manuals, see also WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193. In some embodiments, the construct is as shown in SEQ ID NOS: 3-12 (schematically represented in FIG. 2). In some embodiments, the nucleic acid is a polycistronic construct that allows expression of multiple transgenes (e.g., CoStAR and TCR and / or CAR) under the control of a single promoter. In some embodiments, the transgenes (e.g., CoStAR and TCR and / or CAR) are separated by a self-cleaving 2A peptide. Examples of 2A peptides useful in the nucleic acid construct of the present invention include F2A, P2A, T2A, and E2A. In another embodiment of the present invention, the nucleic acid construct of the present invention is a polycistronic construct containing two promoters; one promoter drives the expression of CoStAR, and the other promoter drives the expression of TCR or CAR. In some embodiments, the dual promoter construct of the present invention is unidirectional. In other embodiments, the dual promoter construct of the present invention is bidirectional. To investigate the expression of a CoStAR polypeptide or a portion thereof, the expression vector introduced into cells can also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells that are sought to be transfected or transduced via a viral vector.

[0041] Cell Source Prior to expansion and genetic modification, a source of cells (e.g., immune effector cells, such as T cells or NK cells) is obtained from a subject. The term "subject" includes organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and removing monocytes, for example, by centrifugation through a PERCOLL™ gradient or counterflow centrifugal elutriation.

[0042] Specific subpopulations of T cells, e.g., CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 conjugated beads, e.g., DYNABEADS® M-450CD3 / CD28T, for a time sufficient to positively select the desired T cells. In one embodiment, this time is approximately 30 minutes. In a further embodiment, this time is 30 minutes to 36 hours or longer, including all integers therebetween. In a further embodiment, this time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, this time is 10 to 24 hours. In one embodiment, the incubation time is 24 hours. In any situation where there are few T cells compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals, longer incubation times can be used to isolate T cells. Furthermore, using longer incubation times can increase the efficiency of CD8+ T cell capture. Thus, by simply shortening or extending the time that T cells are bound to CD3 / CD28 beads and / or by increasing or decreasing the bead-to-T cell ratio (as further described herein), subpopulations of T cells can be preferentially positively or negatively selected at the beginning of culture or at other times during the process. Furthermore, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, subpopulations of T cells can be preferentially positively or negatively selected at the beginning of culture or at other desired times. Those skilled in the art will recognize that multiple rounds of selection can also be used in the context of the present invention. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in the activation and expansion process. The "unselected" cells can also be subjected to additional rounds of selection.

[0043] Enrichment of T cell populations by negative selection can be achieved with a combination of antibodies directed against surface markers specific to the cells being negatively selected. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on the cells being negatively selected. For example, to enrich CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich or positively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, CD137, PD1, TIM3, LAG-3, CD150, and FoxP3+. Alternatively, in certain embodiments, regulatory T cells are removed by anti-CD25-conjugated beads or other similar selection methods. The methods described herein can include, for example, selecting a specific subpopulation of immune effector cells, e.g., T cells, that are regulatory T cell-depleted cells, e.g., CD25+ depleted cells, e.g., using negative selection techniques described herein. Preferably, the population of regulatory T cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells.

[0044] Specific subpopulations of CoStAR effector cells that specifically bind to a target antigen can be enriched by positive selection techniques. For example, in some embodiments, effector cells are enriched by incubation with target antigen-conjugated beads for a time sufficient to positively select the desired abTCR effector cells. In some embodiments, this time is approximately 30 minutes. In some embodiments, this time is 30 minutes to 36 hours or longer (including all ranges between these values). In some embodiments, this time is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, this time is 10 to 24 hours. In some embodiments, the incubation time is 24 hours. For the isolation of effector cells present at low levels in a heterogeneous cell population, the use of longer incubation times, such as 24 hours, can increase cell yield. In any situation where there are few effector cells compared to other cell types, longer incubation times can be used to isolate effector cells. Those skilled in the art will recognize that multiple rounds of selection can also be used in the context of the present invention.

[0045] T cells for stimulation may be frozen after a washing step. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and useful in this regard, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or a medium containing 10% dextran 40 and 5% glucose, 20% human serum albumin, and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% glucose 5%, 0.45% NaCl, 10% dextran 40 and 5% glucose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A. The cells are then frozen to -80°C at a rate of 1 degree per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as immediate, uncontrolled freezing at -20°C or liquid nitrogen, can also be used.

[0046] Allogeneic CoStAR In embodiments described herein, the immune effector cell can be an allogeneic immune effector cell, e.g., a T cell or an NK cell. For example, the cell can be an allogeneic T cell, e.g., an allogeneic T cell that lacks expression of an endogenous T cell receptor (TCR) and / or human leukocyte antigen (HLA), e.g., HLA class I and / or HLA class II. A T cell that lacks a functional endogenous TCR can, for example, be engineered so that it does not express any functional TCR on its surface, or it can be engineered so that it does not express one or more subunits that comprise a functional TCR (e.g., it is engineered so that it does not express (or exhibits reduced expression of) TCR alpha, TCR beta, TCR gamma, TCR delta, TCR epsilon, and / or TCR zeta), or it can be engineered so that it produces only a small amount of functional TCR on its surface. Alternatively, the T cell can express a significantly impaired TCR, for example, by expression of a mutated or truncated form of one or more subunits of the TCR. The term "significantly impaired TCR" means that the TCR does not elicit a harmful immune response in the host.

[0047] The T cells described herein can be engineered, for example, so that they do not express functional HLA on their surface. For example, the T cells described herein can be engineered so that cell surface expression of HLA, such as HLA class I and / or HLA class II, is downregulated. In some embodiments, HLA downregulation can be achieved by reducing or eliminating the expression of beta-2 microglobulin (B2M). In some embodiments, T cells may lack a functional TCR and a functional HLA, such as HLA class I and / or HLA class II. Modified T cells lacking expression of a functional TCR and / or HLA can be obtained by any suitable means, including knockout or knockdown of one or more subunits of the TCR or HLA. For example, T cells can include knockdown of the TCR and / or HLA using siRNA, shRNA, CRISPR, transcription activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN).

[0048] In some embodiments, allogeneic cells can be cells that do not express or express at low levels of inhibitory molecules, such as cells engineered by any method described herein.For example, cells can be cells that do not express or express at low levels of inhibitory molecules that can reduce the ability of CoStAR-expressing cells to initiate immune effector responses.Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, Gal9, adenosine and TGFR beta. Inhibition of inhibitory molecules, for example, by inhibition at the DNA, RNA, or protein level, can optimize the performance of CAR-expressing cells. In embodiments, inhibitory nucleic acids, such as those described herein, can be used, for example, inhibitory nucleic acids, such as dsRNA, for example, siRNA or shRNA, CRISPR, transcription activator-like effector nucleases (TALENs), or zinc finger endonucleases (ZFNs).

[0049] siRNA and shRNA that inhibit endogenous TCR or HLA In some embodiments, TCR expression and / or HLA expression can be inhibited using siRNA or shRNA targeting nucleic acids encoding TCR and / or HLA, and / or inhibitory molecules in T cells described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, Gal9, adenosine, and TGFR beta). The expression of siRNA and shRNA in T cell can be achieved by using any conventional expression system, for example, lentivirus expression system.The exemplary shRNA that downregulates the expression of TCR components is described, for example, in US Patent Application Publication No. 2012 / 0321667.The exemplary siRNA and shRNA that downregulates the expression of HLA class I and / or HLA class II gene is described, for example, in US Patent Application Publication No. 2007 / 0036773.

[0050] CRISPR that inhibits TCR or HLA As used herein, "CRISPR" or "TCR and / or HLA-inhibiting CRISPR" refers to a system that includes a set of CRISPRs or a set of such repeat sequences. As used herein, "Cas" refers to a CRISPR-associated protein. A "CRISPR / Cas" system refers to a system derived from CRISPR and Cas, which can be used to silence or mutate expression of TCR and / or HLA genes, and / or inhibitory molecules described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta). Naturally occurring CRISPR / Cas systems are found in approximately 40% of sequenced eubacterial genomes and 90% of sequenced archaeal genomes. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages, providing a form of adaptive immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845.

[0051] T cell activation and proliferation T cells can generally be activated and expanded using methods described in, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005.

[0052] Generally, T cells of the present invention can be expanded by contacting them with a surface bound with an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. In particular, a population of T cells can be stimulated as described herein, for example, by contact with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. To costimulate accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate for stimulating T cell proliferation. An anti-CD3 antibody and an anti-CD28 antibody can be used to stimulate the proliferation of CD4+ or CD8+ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), and can be used in a similar manner to other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0053] In some embodiments, expansion can be carried out using flasks or vessels, or gas-permeable vessels known to those of skill in the art, and can continue for 7, 8, 9, 10, 11, 12, 13, or 14 days, about 7 to about 14 days, about 8 to about 14 days, about 9 to about 14 days, about 10 to about 14 days, about 11 to about 14 days, about 12 to about 14 days, or about 13 to about 14 days. In some embodiments, the second TIL expansion can continue for about 14 days.

[0054] In certain embodiments, expansion can be performed using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulators can include, for example, about 30 ng / ml of an anti-CD3 antibody such as OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, Calif.), or UHCT-1 (commercially available from BioLegend, San Diego, Calif., USA). CoStAR cells can be grown in vitro by optionally including one or more antigens, such as epitopes, including cancer antigen portions, optionally expressed from a vector, such as human leukocyte antigen A2 (HLA-A2) binding peptides, for example, 0.3 μM MART-1:26-35 (27L) or gpl 00:209-217 (210M), in the presence of a T cell growth factor, for example, 300 IU / ml IL-2 or IL-15. Other suitable antigens can include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigen portions thereof. CoStAR cells can also be rapidly grown by restimulating them with the same cancer antigen pulsed onto antigen-presenting cells expressing HLA-A2. Alternatively, CoStAR cells can be further stimulated, for example, by irradiated autologous lymphocytes or by irradiated HLA-A2+ allogeneic lymphocytes and IL-2.In some embodiments, stimulation occurs as part of proliferation.In some embodiments, proliferation occurs in the presence of irradiated autologous lymphocytes or by irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0055] In certain embodiments, the cell culture medium comprises IL-2. In some embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL IL-2 at about 7500 IU / mL, or about 8000 IU / mL, or 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or 8000 IU / mL.

[0056] In certain embodiments, the cell culture medium comprises an OKT3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, Contains about 100ng / mL, about 200ng / mL, about 500ng / mL, about 1µg / mL, or 0.1ng / mL to 1ng / mL, 1ng / mL to 5ng / mL, 5ng / mL to 10ng / mL, 10ng / mL to 20ng / mL, 20ng / mL to 30ng / mL, 30ng / mL to 40ng / mL, 40ng / mL to 50ng / mL, or 50ng / mL to 100ng / mL of OKT3 antibody. In certain embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is employed as a combination during expansion. In certain embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included during expansion. In some embodiments, a combination of IL-2, IL-15, and IL-21 is employed as a combination during expansion. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof may be included.

[0057] In certain embodiments, expansion can be performed in a supplemented cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells. In certain embodiments, the expansion culture medium contains about 500 IU / mL of IL-15, about 400 IU / mL of IL-15, about 300 IU / mL of IL-15, about 200 IU / mL of IL-15, about 180 IU / mL of IL-15, about 160 IU / mL of IL-15, about 140 IU / mL of IL-15, about 120 IU / mL of IL-15, or about 100 IU / mL of IL-15. / mL IL-15, or about 500 IU / mL to about 100 IU / mL IL-15, or about 400 IU / mL to about 100 IU / mL IL-15, or about 300 IU / mL to about 100 IU / mL IL-15, or about 200 IU / mL IL-15, or about 180 IU / mL IL-15. In some embodiments, the expansion culture medium contains about 20 IU / mL of IL-21, about 15 IU / mL of IL-21, about 12 IU / mL of IL-21, about 10 IU / mL of IL-21, about 5 IU / mL of IL-21, about 4 IU / mL of IL-21, about 3 IU / mL of IL-21, about 2 IU / mL of IL-21, about 1 IU / mL of IL-21, or about 0.5 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21, or about 20 IU / mL to about 0.5 IU / mL of IL-21, or about 15 IU / mL to about 0.5 IU / mL of IL-21, or about 12 IU / mL to about 0.5 IU / mL of IL-21, or about 10 IU / mL to about 0.5 IU / mL of IL-21, or about 5 IU / mL to about 1 IU / mL of IL-21, or about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21, or about 0.5 IU / mL of IL-21.

[0058] In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In one embodiment, the ratio of CoStAR cells to PBMCs and / or antigen-presenting cells during expansion is about 1-25, about 1-50, about 1-100, about 1-125, about 1-150, about 1-175, about 1-200, about 1-225, about 1-250, about 1-275, about 1-300, about 1-325, about 1-350, about 1-375, about 1-400, or about 1-500, or between 1-50 and 1-300, or between 1-100 and 1-200. In certain embodiments, the primary stimulatory signal and the costimulatory signal for T cells can be provided by different protocols. For example, the agents providing each signal can be in solution or linked to a surface. If surface-linked, the agents can be linked to the same surface (i.e., in "cis" formation) or to separate surfaces (i.e., in "trans" formation). Alternatively, one agent can be linked to a surface and the other in solution. In one embodiment, the agent providing the costimulatory signal is bound to a cell surface, and the agent providing the primary activation signal is in solution or linked to a surface. In certain embodiments, both agents can be in solution. In one embodiment, the agent can be in soluble form and crosslinked to a surface, such as a cell, expressing an Fc receptor or antibody or other binding agent that will bind to the agent. In this regard, see, e.g., U.S. Patent Application Publication Nos. 20040101519 and 20060034810, for artificial antigen-presenting cells (aAPCs) intended for use in the activation and expansion of T cells in the present invention.

[0059] In one embodiment, the two agents are immobilized on the same bead, i.e., "cis," or on separate beads, i.e., "trans." By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof. Both agents are co-immobilized on the same bead in equimolar amounts. In one embodiment, a 1:1 ratio of each antibody bound to the beads is used for CD4+ T cell proliferation and T cell growth. In certain embodiments of the invention, a ratio of anti-CD3 antibody:anti-CD28 antibody bound to the beads is used such that an increase in T cell proliferation is observed compared to the proliferation observed using a 1:1 ratio. In certain embodiments, an increase of about 1-fold to about 3-fold is observed compared to the proliferation observed using a 1:1 ratio. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100, and all integer values ​​therebetween. In one embodiment of the invention, more anti-CD28 antibody than anti-CD3 antibody is bound to the particles. That is, the CD3:CD28 ratio is less than 1. In certain embodiments of the invention, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads is greater than 2:1. In one specific embodiment, a 1:100 ratio of CD3:CD28 antibody bound to the beads is used. In one embodiment, a 1:75 ratio of CD3:CD28 antibody bound to the beads is used. In a further embodiment, a 1:50 ratio of CD3:CD28 antibody bound to the beads is used. In one embodiment, a 1:30 ratio of CD3:CD28 antibody bound to the beads is used. In one preferred embodiment, a 1:10 ratio of CD3:CD28 antibody bound to the beads is used. In one embodiment, a 1:3 ratio of CD3:CD28 antibody bound to the beads is used. In yet another embodiment, a 3:1 ratio of CD3:CD28 antibody bound to the beads is used.

[0060] To stimulate T cells or other target cells, particle-to-cell ratios ranging from 1:500 to 500:1 and any integer value therebetween can be used. As those skilled in the art will readily recognize, the particle-to-cell ratio can depend on the size of the particles relative to the target cells. For example, small beads may bind only a few cells, while larger beads may bind many cells. In certain embodiments, the cell-to-particle ratio ranges from 1:100 to 100:1 and any integer value therebetween, and in further embodiments, ratios ranging from 1:9 to 9:1 and any integer value therebetween can also be used to stimulate T cells. While the ratio of anti-CD3 and anti-CD28-linked particles to T cells resulting in T cell stimulation can vary as described above, certain preferred values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with one preferred ratio being at least 1:1 particles per T cell. In one embodiment, a particle-to-cell ratio of 1:1 or less is used. In one particular embodiment, a preferred particle-to-cell ratio is 1:5. In further embodiments, the particle-to-cell ratio can vary depending on the day of stimulation. For example, in one embodiment, the particle to cell ratio is 1:1 to 10:1 on day 1, and then additional particles are added to the cells daily or every other day until day 10, resulting in a final ratio of 1:1 to 1:10 (based on cell count on the day of addition). In one particular embodiment, the particle to cell ratio is 1:1 on day 1 of stimulation and adjusted to 1:5 on days 3 and 5 of stimulation. In one embodiment, particles are added on a daily or every other day basis, resulting in a final ratio of 1:1 on day 1 of stimulation and 1:5 on days 3 and 5 of stimulation. In one embodiment, the particle to cell ratio is 2:1 on day 1 of stimulation and adjusted to 1:10 on days 3 and 5 of stimulation. In one embodiment, particles are added on a daily or every other day basis, resulting in a final ratio of 1:1 on day 1 of stimulation and 1:10 on days 3 and 5 of stimulation. Those of skill in the art will recognize that various other ratios are also suitable for use in the present invention. In particular, the ratio will vary depending on particle size and cell size and type.In one embodiment, the most typical ratios for use are around 1:1, 2:1, and 3:1 on day 1.

[0061] In a further embodiment of the invention, cells, such as T cells, are combined with drug-coated beads, followed by separation of the beads and cells, and then culturing the cells. In an alternative embodiment, the drug-coated beads and cells are not separated prior to culturing, but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0062] Preparation of CoStAR cells Viral and non-viral genetic engineering tools can be used to generate CoStAR T cells and induce permanent or transient expression of therapeutic genes. Retroviral gene delivery is a well-characterized and mature technology that has been used to permanently integrate CARs into the host cell genome (Scholler J., e.g., Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells. Sci. Transl. Med. 2012;4:132ra53; Rosenberg SA et al., Gene transfer into humans—immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N. Engl. J. Med. 1990;323:570-578).

[0063] Non-viral DNA transfection methods can also be used. For example, Singh et al. describe the use of the Sleeping Beauty (SB) transposon system, which has been developed to engineer CAR T cells and has been used in clinical trials (see, for example, ClinicalTrials.gov: NCT00968760 and NCT01653717) (Singh H., et al., Redirecting specificity of T-cell populations for CD19 using the Sleeping Beauty system. Cancer Res. 2008;68:2961-2971). The same technology can be applied to engineer CoStAR cells.

[0064] Various SB enzymes have been used to deliver transgenes. Mates described a hyperactive transposase (SB100X) with an efficiency increase of approximately 100-fold compared with first-generation transposases. SB100X supported 35–50% stable gene transfer in human CD34(+) cells enriched for hematopoietic stem or progenitor cells (Mates L. et al., Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat. Genet. 2009;41:753–761). Multiple transgenes can be delivered from a single multicistronic plasmid (e.g., Thokala R. et al., Redirecting specificity of T cells using the Sleeping Beauty system to express chimeric antigen receptors by mix-and-matching of VL and VH domains targeting CD123+ tumors. PLoS ONE. 2016;11:e0159477) or multiple plasmids (e.g., Hurton LV et al., Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells. Proc. Natl. Acad. Sci. USA. 2016;113:E7788-E7797). Such systems can be used with the CoStAR of the present invention.

[0065] Morita et al. described the piggyBac transposon system for integrating larger transgenes (Morita D. et al., Enhanced expression of anti-CD19 chimeric antigen receptor in piggyBac transposon-engineered T cells. Mol. Ther. Methods Clin. Dev. 2017;8:131-140). Nakazawa et al. described the use of a system to generate EBV-specific cytotoxic T cells expressing a HER2-specific chimeric antigen receptor (Nakazawa Y et al., PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T cells expressing HER2-specific chimeric antigen receptor. Mol. Ther. 2011;19:2133-2143). Manuri et al. used a system to generate CD19-specific T cells. (Manuri PVR et al., piggyBac transposon / transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies. Hum. Gene Ther. 2010;21:427-437). Transposon technology is easy and economical. One potential drawback is that the currently employed long propagation protocols can lead to T cell differentiation, reduced activity, and poor persistence of injected cells. Monjezi et al. have described the development of minicircle vectors that minimize these drawbacks through highly efficient integration (Monjezi R. et al., Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors. Leukemia. 2017;31:186-194). These transposon technologies can be used for CoStAR of the present invention.

[0066] Pharmaceutical Composition The present invention also relates to pharmaceutical compositions comprising the vectors or CoStAR-expressing cells of the present invention together with a pharmaceutically acceptable carrier, diluent, or excipient, and optionally one or more additional pharmaceutically active polypeptides and / or compounds. In some embodiments, provided is a pharmaceutical composition comprising the above-mentioned CoStAR and pharmaceutically acceptable carrier.In some embodiments, provided is a pharmaceutical composition comprising the nucleic acid encoding CoStAR according to any of the above-mentioned embodiments and pharmaceutically acceptable carrier.In some embodiments, provided is a pharmaceutical composition comprising the effector cell that expresses the above-mentioned CoStAR and pharmaceutically acceptable carrier.Such preparations can be in the form suitable for intravenous injection, for example. As used herein, "pharmaceutically acceptable" or "pharmaceutically compatible" means a material that is not biologically or otherwise undesirable, e.g., the material can be incorporated into a pharmaceutical composition administered to a patient without eliciting significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is included. Pharmaceutically acceptable carriers or excipients preferably have passed the necessary standards of toxicity and manufacturing testing and / or are included in the inactive ingredient guides prepared by the U.S. Food and Drug Administration.

[0067] One aspect of the present invention provides a population of modified T cells that express recombinant CoStAR. Suitable populations can be generated by the methods described above. The population of engineered T cells may be for pharmaceutical use. For example, the population of engineered T cells described herein may be used in cancer immunotherapy, such as adoptive T cell therapy. Other aspects of the invention provide use of a population of modified T cells as described herein for the manufacture of a medicament for the treatment of cancer, a population of modified T cells as described herein for the treatment of cancer, and a method of treating cancer that may comprise administering to an individual in need thereof a population of modified T cells as described herein. The population of modified T cells can be autologous, i.e., the modified T cells were originally obtained from the same individual to whom they are subsequently administered (i.e., the donor and recipient individuals are the same). A suitable population of modified T cells for administration to an individual can be generated by a method comprising providing an initial population of T cells obtained from the individual, modifying the T cells to express a cAMP PDE or fragment thereof and an antigen receptor that specifically binds to cancer cells of the individual, and culturing the modified T cells.

[0068] The population of modified T cells can be allogeneic; that is, the modified T cells were originally obtained from an individual different from the individual to whom they are subsequently administered (i.e., the donor and recipient individuals are different). The donor and recipient individuals can be HLA-matched to avoid GVHD and other undesirable immune effects. A suitable population of modified T cells for administration to a recipient individual can be generated by a method comprising providing an initial population of T cells obtained from a donor individual, modifying the T cells to express CoStAR that specifically binds to cancer cells of the recipient individual, and culturing the modified T cells. Following administration of the modified T cells, the recipient individual mounts a T cell-mediated immune response against the recipient individual's cancer cells, which has a beneficial effect on the cancerous condition in the individual.

[0069] Cancerous conditions are characterized by the abnormal growth of malignant cancer cells and can include leukemias such as AML, CML, ALL, and CLL; lymphomas such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma; and solid tumors such as sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, stomach cancer, testicular cancer, gallbladder and bile duct cancer, thyroid cancer, thymus cancer, bone cancer, and brain cancer, and cancer of unknown primary site (CUP). Cancer cells in an individual can be immunologically distinct from the individual's normal somatic cells (i.e., cancer tumors can be immunogenic). For example, cancer cells can induce a systemic immune response in an individual against one or more antigens expressed by the cancer cells. The tumor antigen that induces the immune response can be specific to the cancer cells or can be shared by one or more normal cells of the individual. An individual suitable for the above treatment may be a mammal, such as a rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), primate, ape (e.g., monkey or ape), monkey (e.g., marmoset, baboon), ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or human. In preferred embodiments, the individual is a human. In other preferred embodiments, non-human mammals may be employed, particularly mammals traditionally used as models for demonstrating therapeutic efficacy in humans (e.g., murine, primate, porcine, canine, or lagomorph).

[0070] Treatment methods The term "therapeutically effective amount" refers to an amount of a CoStAR or a composition comprising a CoStAR disclosed herein effective to "treat" a disease or disorder in an individual. In the case of cancer, a therapeutically effective amount of a CoStAR or a composition comprising a CoStAR disclosed herein can reduce the number of cancer cells, reduce the size or mass of a tumor, inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into surrounding organs, inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. A CoStAR or a composition comprising a CoStAR disclosed herein can be cytostatic and / or cytotoxic to the extent that it can prevent the growth and / or kill existing cancer cells. In some embodiments, a therapeutically effective amount is a growth-inhibitory amount. In some embodiments, a therapeutically effective amount is an amount that improves the progression-free survival rate of a patient. In the case of an infectious disease, such as a viral infection, a therapeutically effective amount of CoStAR or a composition comprising CoStAR disclosed herein can reduce the number of cells infected by the pathogen, reduce the production or release of pathogen-induced antigens, inhibit (i.e., slow to some extent, and preferably stop) the spread of the pathogen to uninfected cells, and / or alleviate to some extent one or more symptoms associated with the infection. In some embodiments, a therapeutically effective amount is an amount that prolongs the survival of a patient.

[0071] Cells, including T cells and NK cells, expressing CoStAR for use in the methods of the present invention can be generated ex vivo from the patient's own peripheral blood (autologous), or in the setting of hematopoietic stem cell transplantation from donor peripheral blood (allogeneic), or from peripheral blood from an unrelated donor (allogeneic). Alternatively, T cells or NK cells can be derived by ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells into T cells or NK cells. In these cases, T cells expressing CoStAR and optionally CAR and / or TCR are produced by introducing DNA or RNA encoding CoStAR and optionally CAR and / or TCR by one of many means, including transduction with a viral vector, transfection with DNA or RNA. T cells or NK cells expressing the CoStAR of the present invention, and optionally expressing a TCR and / or CAR, can be used to treat hematological cancers or solid tumors.

[0072] Methods for treating disease involve therapeutic uses of the vectors or cells, including T cells or NK cells, of the present invention. In this regard, the vectors or T cells or NK cells can be administered to a subject with an existing disease or condition to diminish, reduce, or ameliorate at least one symptom associated with the disease and / or to slow, reduce, or prevent the progression of the disease. The methods of the present invention can initiate or promote T cell-mediated cancer cell killing. The vectors or T cells or NK cells of the present invention can be administered to a patient together with one or more additional therapeutic agents. The one or more additional therapeutic agents can be co-administered to a patient. "Co-administration" means administering one or more additional therapeutic agents and the vectors or T cells or NK cells of the present invention sufficiently close in time so that the vectors or T cells or NK cells can enhance the effect of the one or more additional therapeutic agents, or vice versa. In this regard, the vectors or cells can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the vectors or cells and the one or more additional therapeutic agents can be administered simultaneously. One useful co-administered therapeutic agent is IL-2. IL-2 therapy is currently being used to enhance the activity of administered cells in current cell therapies, but is associated with problems of toxicity and resistance.

[0073] As mentioned above, for administration to patients, CoStAR effector cells can be allogeneic or autologous to the patient. In certain embodiments, allogeneic cells are further genetically modified, for example, by gene editing, to minimize or prevent GVHD and / or the patient's immune response to CoStAR cells. CoStAR effector cells are used to treat cancers and neoplastic diseases related to target antigens.Cancers and neoplastic diseases that can be treated using any of the methods described herein include non-vascularized tumors, tumors that are not yet substantially vascularized, and vascularized tumors.Cancers can include non-solid tumors (blood tumors, such as leukemia and lymphoma), or solid tumors.The types of cancers that can be treated by the CoStAR effector cells of the present invention include but are not limited to carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant diseases, such as sarcomas, carcinomas, and melanomas.Adult tumors / cancers and pediatric tumors / cancers are also included. Hematological cancers are cancers of the blood or bone marrow. Cancers of the blood system (or hematopoietic system) include leukemias, including acute leukemia (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroid leukemia), chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive forms), multiple myeloma, plasmacytoma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0074] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. Various types of solid tumors are named for the type of cells that form them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcomas and carcinomas, include adrenocortical carcinoma, cholangiocarcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovial tumors, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, gastric cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, stem cell carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, thyroid cancer (e.g., medullary thyroid cancer and papillary thyroid cancer), pheochromocytoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocarcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer (e.g., cervical carcinoma and pre-invasive cervical dysplasia), colorectal cancer, cancer of the anus, anal canal, or anorectum, vaginal cancer, cancer of the vulva (e.g., squamous cell carcinoma, carcinoma in situ, adenocarcinoma, and fibrosarcoma), penile cancer, oropharyngeal cancer, esophageal cancer Cancer, head cancer (e.g., squamous cell carcinoma), neck cancer (e.g., squamous cell carcinoma), testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriomas, sarcomas, Leydig cell tumors, fibromas, fibroadenomas, adenomatous tumors, and lipomas), bladder cancer, kidney cancer, melanoma, uterine cancer (e.g., endometrial cancer), urothelial cancer (e.g., squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, ureteral cancer, and urinary bladder cancer) cancer), and CNS tumors (e.g., gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germinomas, medulloblastomas, schwannomas, craniopharyngiomas, ependymoma, pinealomas, hemangioblastomas, acoustic neuromas, oligodendroglioma, menangioma, neuroblastomas, retinoblastomas, and brain metastases).

[0075] When an "immunologically effective amount," "antitumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). Pharmaceutical compositions comprising T cells described herein may be administered in amounts up to 10 per kg of body weight, including all integer values ​​within these ranges.4 ~10 9 cells, in some cases 10 per kg of body weight 5 ~10 6 It is generally stated that the T cell composition can be administered in doses of cells. The T cell composition may be administered multiple times at these doses. The cells can be administered using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[0076] Combination Therapy The cells expressing CoStAR described herein may be used in combination with other known drugs and therapies. As used herein, "administered in combination" means that two (or more) different treatments are delivered to a subject during the course of the subject's suffering from a disorder, for example, two or more treatments are delivered after the subject is diagnosed with the disorder and before the disorder is cured or eliminated, or before the treatment is discontinued for other reasons. In some embodiments, the delivery of one treatment is still ongoing when the delivery of the second treatment begins, so there is overlap in administration. This may be referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments, in either case, the treatments are more effective because they are administered in combination. For example, the second treatment is more effective, for example, a lesser amount of the second treatment is required to achieve an equivalent effect, or the second treatment reduces symptoms to a greater extent than would be seen if the second treatment were given without the first treatment, or if a similar situation were seen with the first treatment. In some embodiments, delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed when one treatment is delivered without the other treatment. The effects of the two treatments may be partially additive, fully additive, or greater than additive. Delivery may be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

[0077] The CoStAR expressing cells described herein and at least one additional therapeutic agent can be administered simultaneously, in the same or separate compositions, or sequentially.For sequential administration, the CAR expressing cells described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed. CoStAR therapy and / or other therapeutic agents, procedures, or measures can be administered during periods of active disease or during periods of remission or more inactive disease. CoStAR therapy can be administered before, concurrently with, or after other treatments, or during remission of a disease.

[0078] When administered in combination, the present therapy and additional agents (e.g., second or third agents), or all of them, can be administered in amounts or doses greater than, less than, or equal to the amount or dose of each agent used individually, for example, as monotherapy.In certain embodiments, the amount or dose of the CoStAR therapy, additional agents (e.g., second or third agents), or all of them administered is (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) less than the amount or dose of each agent used individually, for example, as monotherapy.In other embodiments, the amount or dose of the CoStAR therapy, additional agents (e.g., second or third agents), or all of them that produces the desired effect (e.g., cancer treatment) is (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) less than the amount or dose of each agent used individually, for example, as monotherapy, that is required to achieve the same therapeutic effect. In further embodiments, the CoStAR-expressing cells described herein may be used in a therapeutic regimen in combination with surgery, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, or other antibody therapies, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation, peptide vaccines such as those described in Izumoto et al. 2008 J Neurosurg 108:963-971.

[0079] In certain instances, the compounds of the present invention are combined with other therapeutic agents, such as other anti-cancer agents, anti-allergy agents, anti-emetic (or anti-vomiting) agents, pain-relieving agents, cytoprotective agents, and combinations thereof. In one embodiment, the CoStAR-expressing cells described herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab), and surrogates. These include immunomodulatory agents such as vasopressin antagonists (including, for example, folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-induced TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclacinomycin A, gliotoxin, or bortezomib), thalidomide or thalidomide derivatives (e.g., lenalidomide).

[0080] Common chemotherapeutic agents considered for use in combination therapy include busulfan (Myleran®), busulfan injection (Busulfex®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), daunorubicin hydrochloride (Cerubidine®), daunorubicin hydrochloride (Cerubidine®), and daunorubicin hydrochloride (Cerubidine®). These include norubicin citrate liposomal injection (DaunoXome®), dexamethasone, doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), hydroxyurea (Hydrea®), idarubicin (Idamycin®), mitoxantrone (Novantrone®), and gemtuzumab ozogamicin (mylotarg®).

[0081] In embodiments, common chemotherapeutic agents contemplated for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®). , carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (ActinomycinD, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposomal injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluoxetine Lauracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), loxacin (Lyspar®), thiazolinone (Thiazolinone ... These include icovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), Milotarg, paclitaxel (Taxol®), Phoenix (yttrium-90 / MX-DTPA), pentostatin, polifeprosan 20 and carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®). Treatment can be assessed, for example, by tumor regression, reduction in tumor mass or size, time to progression, duration of survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression, and / or activity. Approaches to determining the efficacy of treatment can be employed, including, for example, measuring response by radioimaging.

[0082] The invention is further described in the following non-limiting sections. 1. A chimeric costimulatory receptor (CoStAR) comprising a full-length costimulatory receptor fused to a tumor-associated antigen-specific binding domain, where full-length means the entire mature protein lacking the leader sequence and with deletions or mutations of up to 5 amino acids at the N-extreme terminus of the mature protein, a process which is important for optimal splicing of the costimulatory domain into the chimeric receptor. 2. The CoStAR of clause 1, wherein the costimulatory receptor is selected from the group of CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6. 3. A CoStAR fusion receptor as outlined in paragraphs 1 or 2, wherein the costimulatory domain consists of two or more costimulatory receptor domains fused to a single receptor format, and the binding region is either a direct fusion or includes a linker region, such as a linker up to 50 amino acids in length.

[0083] 4. The antigen-specific binding domain is I. A single-chain antibody fragment that binds to a tumor-associated antigen, including, but not limited to, carcinoembryonic antigen (CEA), 5T4, melanotransferrin (CD228), melanoma-associated chondroitin sulfate proteoglycan (MCSP / CSPG4), CD71, folate receptor, or CA125; or II. Single-chain antibody fragments that bind to tumor-specific peptide (p)-major histocompatibility (MHC) complexes, or III. A tumor-specific pMHC complex antigen-specific single-chain T cell receptor (scTCR), or IV. Naturally occurring antigen-binding polypeptides, such as, but not limited to, transferrin, or V. Domains that bind to antibodies (e.g., Fc binding domains such as, but not limited to, CD16, CD32, or CD64) or other indirect methods of antigen recognition 4. The CoStAR of any one of clauses 1 to 3, derived from

[0084] 5. A fusion protein comprising the polypeptide of any one of clauses 1-4 fused to a protein of interest (POI), such as a marker gene (e.g., DYKDDDDK (SEQ ID NO: 14) epitope tag, CD34, CD19, etc.), a chimeric antigen receptor (CAR), a T cell receptor (TCR), or another receptor for immunotherapy for adoptive cell therapy. 6. The fusion protein of clause 5, comprising a self-cleaving peptide between the polypeptide and the protein of interest. 7. I. A polypeptide as set forth in SEQ ID NO: 1, capable of encoding a polypeptide as set forth in Sections 1 and 2, such as SEQ ID NO: 2, or a variant thereof having at least 80% sequence identity at the protein level; or II. Fusion proteins, such as those set forth in SEQ ID NO: 2 fused to peptides such as those set forth in SEQ ID NO: 3 to 12 or variants thereof having at least 80% sequence identity at the protein level, as described in section 5; Nucleic acid sequences such as: 8. A vector comprising a nucleic acid sequence according to paragraph 7. 9. The vector of clause 8, which also contains a transgene of interest. 10. The vector of clause 9, wherein the transgene of interest encodes a chimeric antigen receptor, a T cell receptor, or another receptor for immunotherapy for adoptive cell therapy, such that when the vector is used to transduce a target cell, the target cell co-expresses the polypeptide of clause 1 and the chimeric antigen receptor, T cell receptor, or another receptor for immunotherapeutic purposes.

[0085] 11. A cell expressing a polypeptide according to paragraph 1. 12. The cell of clause 11, which co-expresses a polypeptide as outlined in clause 1 and a POI at the cell surface. 13. A cell comprising a nucleic acid sequence according to paragraph 7. 14. The cell of any one of clauses 11 to 13, which is a T cell. 15. A method of producing a cell according to any one of clauses 11 to 14, comprising transducing or transfecting a cell with a vector according to any one of clauses 8 to 10. 16. A method for selecting cells expressing a POI, comprising: I. Detecting the expression of the POI epitope on the surface of cells transfected or transduced with the vector described in paragraph 10; and II. Selecting cells identified as expressing the POI epitope A method comprising: 17. A method for preparing a purified population of cells enriched for cells expressing a POI, comprising: 17. A method comprising selecting cells expressing a POI from a population of cells using the method described in clause 16.

[0086] 18. A population of cells isolated from a tissue or cell donor, ex vivo, I. Transducing or transfecting the vector described in section 7, and II. Selecting cells expressing said POI from said population of transduced / transfected cells by the method described in section 16. 18. The method of claim 17, comprising: 19. A cell population enriched for cells expressing a polypeptide according to clause 1, and thus enriched for cells expressing a POI. 20. A method for tracking transduced cells in vivo, comprising detecting expression of a polypeptide according to clause 1 on the surface of said cells. 21. A method of treating a disease in a subject, the method comprising administering to the subject a cell according to any one of clauses 11 to 14 or a cell population according to clause 19. 22. Transducing or transfecting a sample of cells isolated from a subject with a vector described in Section I. 9; and II. Returning the transduced / transfected cells to the patient 22. The method of claim 21, comprising:

[0087] 23. The method of clause 22 for treating cancer. 24. The cell of any one of clauses 11-14 or the cell population of clause 19 for use in adoptive cell transfer. 25. A CoStAR receptor consisting of a tumor-associated antigen-binding domain according to paragraph 4, I. A fusion signaling domain consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. An intracellular domain from human CD137, such as that set forth in SEQ ID NO: 4, or a variant thereof having at least 80% sequence identity at the protein level. CoStAR receptor fused with 26. A CoStAR receptor consisting of a tumor-associated antigen-binding domain according to paragraph 4, I. A fusion signaling domain consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. The intracellular domain from human CD134, such as that set forth in SEQ ID NO: 5, or a variant thereof having at least 80% sequence identity at the protein level. CoStAR receptor fused with

[0088] 27. A CoStAR receptor consisting of a tumor-associated antigen binding domain according to paragraph 4, I. A fusion signaling domain consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. An intracellular domain from human CD2, such as that set forth in SEQ ID NO: 6, or a variant thereof having at least 80% sequence identity at the protein level. CoStAR receptor fused with 28. A CoStAR receptor consisting of a tumor-associated antigen binding domain according to paragraph 4, I. A fusion signaling domain consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. An intracellular domain from human CD29, such as that set forth in SEQ ID NO: 7, or a variant thereof having at least 80% sequence identity at the protein level. CoStAR receptor fused with

[0089] 29. A CoStAR receptor consisting of a tumor-associated antigen binding domain according to paragraph 4, I. A fusion signaling domain consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. An intracellular domain from human GITR, such as that set forth in SEQ ID NO: 8, or a variant thereof having at least 80% sequence identity at the protein level. CoStAR receptor fused with 30. A CoStAR receptor consisting of a tumor-associated antigen binding domain according to paragraph 4, I. A fusion signaling domain consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. An intracellular domain from human IL2Rγ, such as that set forth in SEQ ID NO: 9, or a variant thereof having at least 80% sequence identity at the protein level. CoStAR receptor fused with

[0090] 31. A CoStAR receptor consisting of a tumor-associated antigen binding domain according to paragraph 4, I. A fusion signaling domain consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. An intracellular domain from human CD40, such as that set forth in SEQ ID NO: 10, or a variant thereof having at least 80% sequence identity at the protein level. CoStAR receptor fused with 32. A CoStAR receptor consisting of a tumor-associated antigen binding domain according to paragraph 4, I. A fusion signaling domain consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. An intracellular domain from human CD150, such as that set forth in SEQ ID NO: 11, or a variant thereof having at least 80% sequence identity at the protein level. CoStAR receptor fused with

[0091] 33. A CoStAR receptor consisting of a tumor-associated antigen binding domain according to paragraph 4, I. A fusion signaling domain consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. Intracellular domains from human CD2 and human CD40, such as those set forth in SEQ ID NO: 12 or variants thereof having at least 80% sequence identity at the protein level CoStAR receptor fused with [Example]

[0092] The present invention is further demonstrated in the following non-limiting examples. Example 1 Generation of T cells expressing CoStAR material and method Construct design—MFE23 CoStAR consists of the MFE23-derived single-chain antibody fragment nucleotide sequence with the oncostatin M1 leader sequence fused to the entire human CD28 nucleic acid sequence. The CoStAR nucleotide sequence was codon-optimized, and the gene was synthesized by Genewiz. The construct was cloned into pSF.Lenti (Oxford Genetics) via the XbaI and NheI sites. Lentiviral production - Lentiviral production was performed using a three-plasmid packaging system (Cell Biolabs, San Diego, USA) by mixing 10 μg of each plasmid with 10 μg of the pSF.Lenti lentiviral plasmid containing the transgene in serum-free RPMI containing 50 mM CaCl. The mixture was then transferred to a 75 cm 2 The virus was added dropwise to a 50% confluent monolayer of 293T cells in a flask. At 48 and 72 hours posttransfection, viral supernatants were collected, pooled, and concentrated using LentiPac Lentiviral Supernatant Concentrate (GeneCopoeia, Rockville, Maryland, USA) solution according to the manufacturer's instructions. The lentiviral supernatant was concentrated 10-fold and used to directly infect primary human T cells in the presence of 4 μg / ml polybrene (Sigma-Aldrich, Dorset, UK). Peripheral blood mononuclear cells were isolated from normal healthy donors and then activated with T cell activation and expansion beads (Invitrogen) for 24 hours according to the manufacturer's instructions, after which the lentiviral supernatant was added.

[0093] Cells were examined for transduction 96 hours postinfection using CEA.hFc protein and anti-hFc-PE secondary antibodies with anti-CD34-APCs or anti-CD34-PE alone. Cells were then further expanded using ×10 donor-mismatched irradiated PBMC feeders at a 1:20 to 1:200 ratio in RPMI + 10% FCS supplemented with 1 μg / ml PHA and 200 IU / ml IL-2. After 14 days, cells were stained as before and stored for further assays. Functional assays were performed by mixing CoStAR-positive or -negative cells with wild-type or OKT3-engineered CEA-positive LoVo or LS174T cells. Briefly, T cells were mixed with LoVo cells at various ratios in 96-well plates, and IFNγ or IL-2 was measured by ELISA. The remaining cells were incubated with a 1:10 dilution of WST-1 reagent (Sigma, UK) for 30 minutes, after which absorbance was read at 450 nm. % Cytotoxicity was determined using the following formula: 100 - ((experimental reading - T cells alone) / (tumor alone) x 100).

[0094] First 1×10 7 Proliferation assays were performed by loading T cells at a concentration of 100 cells / ml with 10 μM eFluor 450 proliferation dye (eBioscience, UK) for 10 minutes at 37°C, followed by incubation of the cells with 5 volumes of cold T cell medium on ice for 5 minutes. Cells were then washed extensively to remove unbound dye and added to co-cultures containing tumor cells. On days 2, 6, and 10, cells were removed, a 1:200 dilution of DRAQ7 was added, and the cells were analyzed using a MACSQuant cytometer and MACSQuantify software. Cell counting for proliferation assays was performed by removing cells from the wells and staining them with anti-CD2 PerCP eFluor710 antibody (eBioscience, UK) for 20 min in the dark, followed by staining with DRAQ7, and counting using a MACSQuant analyzer.

[0095] Results—Primary human T cells were isolated from buffy coats obtained from NHSBT. T cells were isolated by Ficoll-mediated isolation and a T cell negative isolation kit (StemCell Technologies). Isolated T cells were activated with human T cell activation and expansion beads (Invitrogen, UK). Cells were incubated with concentrated lentiviral particles and expanded over several days. The lentivirus contained the DNA sequence of the MFE.CoStAR.2A.tCD34 construct (MFE23.scFv fused to full-length human CD28 co-expressed with truncated human CD34 through the 2A cleavage sequence). Successfully transduced cells were further expanded using irradiated feeders as outlined in Materials and Methods. Transduction of donor 1 was measured as 22.69% (17.15 CD34+ / CoStAR+ plus 5.53% CD34- / CoStAR+), donor 2 was measured as 20.73%, and donor 3 was measured as 13.34%. To obtain a T cell population that was >90% CoStAR positive, cells were enriched for CoStAR expression using anti-CD34 antibody. To generate a physiologically relevant in vitro model for testing the effects of CoStAR on T cell activity, untransduced and transduced cells were tested against the CEA+ tumor cell lines LoVo and LS174T. To enable T cell activation in response to unmatched tumor lines and visualize transduced cells using flow cytometry, we engineered tumor cells to express an anti-CD3 single-chain antibody fragment anchored to the cell membrane through a synthetic transmembrane domain using an IRES element and cleaved from a GFP marker gene.

[0096] Single-cell clones of LoVo and LS174T were generated from bulk transfectants. Untransduced and CoStAR-transduced T cells were mixed with wild-type untransduced or OKT3-engineered LS174T or LoVo cells at various effector-to-target ratios. After 24 hours, coculture medium was collected for IL-2 ELISA measurements. Activation-dependent IL-2 secretion was observed from CoStAR+ and CoStAR- T cell populations from three donors in response to OKT3-engineered LS174T cells, while only background IL-2 secretion was observed from transduced and untransduced T cells in response to unengineered tumor cells (Figure 3A-C). CoStAR-enhanced IL-2 secretion in response to OKT3-engineered tumor cells was found in all three donors tested. The effect was most pronounced at E:T ratios of 8:1 and 16:1, but IL-2 secretion was too low to be accurately measured at higher E:T ratios. At lower effector-to-target ratios, IL-2 secretion appeared to be saturated from untransduced cells. These observations were repeated with LoVo cells, and two of the three donors tested against LS174T, with similar results (Fig. 3D and E). To determine the effect of CoStAR on T cell expansion, transduced or untransduced T cells were mixed with wild-type or OKT3-GFP engineered LoVo cells, and total cell numbers were counted after 3 days. CoStAR enhanced the survival and / or proliferation of engineered T cells in response to LoVo-OKT3 but not wild-type LoVo cells, both in the presence (Figure 4A) and absence (Figure 4B) of IL-2. To further investigate this phenomenon, we performed cell proliferation analysis on T cells from two donors using a proliferation dye to count the number of cell cycles each population underwent over a 6-day period (Figure 4C and D). Compared to unengineered cells, a greater proportion of CoStAR-engineered cells underwent 5, 6, or 7 proliferation cycles over a 6-day period in response to LoVo-OKT3, whereas CoStAR-transduced and untransduced cells underwent an average of approximately 2 cycles over the same period in response to wild-type LoVo.

[0097] We generated various fusion receptors consisting of CD28 fused to an additional N-terminal costimulatory domain. We selected costimulatory domains from CD137, CD2, CD29, CD134, CD150, CD40, and GITR, as well as the signaling domain from the IL-2 receptor γ chain (IL2Rγ). Receptors similar to those used in previous studies of inducible costimulation were included. This receptor, designated CD28(IEV), is truncated so that the C-terminal motif of CD28 is the amino acid triplet "IEV." The sequence was newly generated by Genewiz and cloned into a lentiviral vector under the EF1α promoter, along with a CD34 marker gene separated from the fused CoStAR by a 2A self-cleaving peptide. Primary CD8+ T cells were isolated using EasySep beads (StemCell Technologies) and activated with anti-CD3 / anti-CD28 activation / expansion Dynabeads, followed by the addition of lentiviral particles. After a short expansion period, the cells were mixed with LoVo or LoVo-OKT3 cells and incubated with anti-CD107a antibody and brefeldin and monensin. After 16 hours of incubation, the cells were fixed and stained with antibodies against a marker gene (CD34) and antibodies against IL-2, IFNγ, and bcl-xL. Analysis was performed using a MACSQuant analyzer and MACSQuantify software. Figure 5 shows the IL-2 response from CD34- (CoStAR-untransduced) and CD34+ (CoStAR-transduced) cells. Statistical analysis demonstrated that all receptors tested induced a significant increase in the percentage of cells producing IL-2 when containing the mutant CoStAR receptor. Three other readouts were measured simultaneously: IFNγ, a cytokine released under normal signaling conditions but enhanced by costimulation; CD107a, a marker of degranulation; and bcl-xL, an anti-apoptotic protein upregulated by costimulation. Incorporation of CoStAR enhanced all effector functions analyzed to varying degrees, with the CD28.CD2 and CD28.CD40 fusion receptors appearing to elicit the most robust responses of all receptors tested (see Figure 6).

[0098] Example 2 We compared the effects of CD28- and CD28.CD40-based CoStAR on population-based cytokine secretion. Primary T cells from three donors were transduced with CD28(IEV)-truncated CoStAR, full-length CD28 CoStAR, or CD28.CD40 CoStAR (containing full-length CD28 as shown in SEQ ID NO: 10 but lacking the N-terminal N and K residues), or remained untransduced. T cells were enriched for CoStAR expression using the CD34 marker gene, and after expansion, the cells were mixed with LoVo-OKT3 cells and analyzed for IL-2 secretion by ELISA (see Figure 7). Untransduced cells produced an average of 0.80 ng / ml of IL-2, while CD28(IEV) and full-length CD28 CoStAR produced 4.6 and 5.0 ng / ml of IL-2, respectively. However, CD28.CD40 induced an average of 29.0 ng / ml of IL-2 across three donors, thus demonstrating a clear advantage of incorporating CD40 into the basic CD28-based CoStAR.

[0099] Next, we analyzed the effect of CoStAR on T cell expansion. T cells from seven donors were transduced with CD28 or CD28.CD40 CoStAR bearing anti-CA125 (196-14) or folate antagonistic receptor (MOV-19) scFv, or the folate antagonistic receptor peptide (C7) antigen-binding domain. Additional cells were transduced with CD28 CoStAR bearing anti-CEA scFv as a mismatch control. The cells were then mixed with a CA125+ / folate receptor+ / CEA- cell line, OvCAR3, engineered to express membrane-bound OKT3 (OvCAR-OKT3). T cell numbers were counted after 7, 14, and 21 days, and fresh OvCAR-OKT3 was added on days 7 and 14. Limited expansion of cells containing anti-CA125 scFv was observed (mean fold expansion: CD28: 15.1; CD28.CD40: 69.1), whereas cells targeting the folate receptor with scFv expanded in both the CD28 and CD28.CD40 cohorts (mean fold expansion: CD28: 186.7; CD28.CD40: 1295.0). More limited expansion was seen when C7 peptide was used to target the folate receptor (mean fold expansion: CD28: 71.5; CD28.CD40: 28.0). Control CEA-targeted receptors demonstrated limited expansion (mean fold expansion: 28.0).

[0100] To better understand the synergistic effect of signal 1 and signal 2, T cells were engineered with a murine constant domain-modified TCR that recognizes the CEA peptide (691-699) in the context of HLA-A*02, as well as CD28 or CD28.CD40 CoStAR, which target the cell surface CEA protein. As a control, cells were also transduced with CA125-specific CD28 CoStAR. T cells were mixed with HLA-A*02+ / CEA+ H508 cells, and cytokine production was analyzed by intracellular flow cytometry staining. Flow cytometry gating was performed using an antibody against the murine TCRβ constant domain (to mark TCR-engineered cells) and the DYKDDDDK (SEQ ID NO: 14) epitope tag (to mark CoStAR-engineered cells). Thus, it was possible to analyze TCR- / CoStAR-, TCR+ / CoStAR-, TCR- / CoStAR+, and TCR+ / CoStAR+ cells in each coculture well. Next, cytokine production in each subpopulation was plotted for CD4+ or CD8+ T cells (Figure 9). In CD4+ cells, CD28.CD40 CoStAR enhanced CD137 and TNFα production more strongly than TCR stimulation alone, whereas TCR responses in CD4+ cells were inferior due to the dependency of TCR on CD8. IL-2 was a more robust effector activity in CD8+ cells, and CD107a in particular showed stronger induction in the CD28.CD40 CoStAR group. To better compare receptors, effector activity in the TCR+ / CoStAR+ group was plotted for CD4+ and CD8+ cells (Figure 10). In CD4+ cells, induction of CD137 was significantly enhanced by CD28.CD40 compared with CEA or mismatch-targeted CD28 CoStAR. In CD8+ cells, induction of CD137 was significantly increased compared to CEA or mismatch-targeted CD28 CoStAR, whereas induction of CD107a was increased compared to control CoStAR. Thus, CD28.CD40 exhibits enhanced effector activity across a wide range of models and effector activities.

[0101] References JPEG2026035607000002.jpg250158

[0102] array SEQ ID NO: 1: Entire human CD28 mRNA transcript variant transcript, open reading frame highlighted in bold. Locus NM_006139 4900bp mRNA Linear PRI 07-OCT-2016 Definition Homo sapiens CD28 molecule (CD28), transcript variant 1, mRNA. Contract NM_006139 origin

[0103] [ka] [ka] [ka]

[0104] SEQ ID NO: 2: Human CD28 protein sequence: I. The signal peptide is highlighted in bold (this may be deleted from the sequence when used in accordance with the present invention); and II. Transmembrane regions are underlined; and III. The extracellular domain is located between the signal peptide and the transmembrane domain, and the cytoplasmic domain follows the transmembrane domain; and IV. The box region represents the first five amino acids of the mature protein, which may be present, absent, or mutated in any embodiment of the invention. Additionally, one, two, three, four, or five of these residues (i.e., N, K, I, L, or V) may be deleted from the sequence when used in accordance with the present invention.

[0105] [ka]

[0106] SEQ ID NOs: 3-12 below are exemplary sequences of CoStAR signaling domains, which may be fused to human CD28 protein at the SEQ ID NO: 2 box region (which may or may not be mutated, but has been left in its original form for simplicity of illustration): I. The double-underlined region represents the CD28-derived region; and II. The underlined region is from the second costimulatory receptor; and III. The italicized sequence is the region from the third costimulatory receptor. Note that the box region represents the first five amino acids of the mature CD28 protein, which may be present, absent, or mutated in any embodiment of the invention. Additionally, when used in accordance with the present invention, one, two, three, four, or five of these residues (i.e., N, K, I, L, or V) may be deleted from the sequence.

[0107] SEQ ID NO: 3: Truncated cytoplasmic domain CD28 mutant [ka]

[0108] SEQ ID NO: 4: CD28.CD137 fusion [ka]

[0109] SEQ ID NO: 5: CD28.CD134 fusion [ka]

[0110] SEQ ID NO: 6: CD28.CD2 fusion [ka]

[0111] SEQ ID NO: 7: CD28.CD29 fusion [ka]

[0112] SEQ ID NO: 8: CD28.GITR fusion [ka]

[0113] SEQ ID NO: 9: CD28.IL2Rγ fusion [ka]

[0114] SEQ ID NO: 10: CD28.CD40 fusion [ka]

[0115] SEQ ID NO: 11: CD28.CD150 fusion [ka]

[0116] SEQ ID NO: 12: CD28.CD2.CD40 fusion [ka]

[0117] SEQ ID NO: 13: Truncated extracellular domain CD28 (IEV) mutant [ka]

Claims

1. A chimeric costimulatory receptor (CoStAR) comprising an extracellular ligand-binding fragment of a first costimulatory receptor and an intracellular signaling fragment of a second costimulatory receptor fused to a tumor-associated antigen-specific binding domain linked to a signaling domain.

2. 2. The CoStAR of claim 1, comprising a full-length costimulatory receptor fused to a tumor-associated antigen-specific binding domain, wherein the full-length costimulatory receptor comprises the entire mature protein without a leader sequence, and wherein the mature protein has a deletion or mutation of up to 5 amino acids at the N-terminal end of the mature protein.

3. A chimeric costimulatory receptor (CoStAR) comprising a full-length costimulatory receptor fused to a tumor-associated antigen-specific binding domain, wherein the full-length costimulatory receptor comprises the entire mature protein without a leader sequence, and the mature protein has a deletion or mutation of up to 5 amino acids at the extreme N-terminus of the mature protein.

4. The CoStAR of any one of claims 1 to 3, wherein the costimulatory receptor is selected from the group consisting of CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6.

5. 5. The CoStAR of any one of claims 1 to 4, wherein the costimulatory domain comprises or consists of two or more costimulatory receptor domains fused to a single receptor format, and the binding region is either a direct fusion or includes a linker region, such as a linker up to 50 amino acids in length.

6. the antigen-specific binding domain is I. A single chain antibody fragment that binds to a tumor-associated antigen, including, but not limited to, carcinoembryonic antigen (CEA), 5T4, melanotransferrin (CD228), Her2, EGFR, GPC3, melanoma-associated chondroitin sulfate proteoglycan (MCSP / CSPG4), CD71, SM5-1, folate receptor, or CA125; or II. Single-chain antibody fragments that bind to tumor-specific peptide (p)-major histocompatibility (MHC) complexes, or III. A tumor-specific pMHC complex antigen-specific single-chain T cell receptor (scTCR), or IV. A naturally occurring antigen-binding polypeptide, such as, but not limited to, transferrin, or V. Domains that bind to antibodies (e.g., Fc binding domains such as, but not limited to, CD16, CD32, or CD64) or other indirect methods of antigen recognition The CoStAR according to any one of claims 1 to 5, which is selected from the group consisting of

7. 7. A fusion protein comprising the polypeptide of any one of claims 1 to 6 fused to a protein of interest (POI) such as a marker gene (e.g., DYKDDDDK (SEQ ID NO: 14) epitope tag, CD34, CD19, etc.), a chimeric antigen receptor (CAR), a T cell receptor (TCR), or another receptor for immunotherapy for adoptive cell therapy.

8. The fusion protein of claim 8 , comprising a self-cleaving peptide between the polypeptide and the protein of interest.

9. I. A polypeptide according to claims 1 and 2, such as that shown in SEQ ID NO: 1, capable of encoding a polypeptide such as that shown in SEQ ID NO: 2 or a variant thereof having at least 80% sequence identity at the protein level, or II. Fusion proteins, such as those set forth in SEQ ID NOs: 3-12 or variants thereof having at least 80% sequence identity at the protein level; Nucleic acid sequences such as:

10. A vector comprising the nucleic acid sequence of claim 10.

11. The vector of claim 10, which also contains a transgene of interest.

12. 12. The vector of claim 11, wherein the transgene of interest encodes a chimeric antigen receptor, a T-cell receptor, or another receptor for immunotherapy for adoptive cell therapy, such that when the vector is used to transduce a target cell, the target cell co-expresses the polypeptide of claim 1 and the chimeric antigen receptor, T-cell receptor, or another receptor for immunotherapeutic purposes.

13. A cell expressing the polypeptide of claim 1.

14. The cell of claim 13, which co-expresses the polypeptide of claim 1 and a POI on the cell surface.

15. A cell comprising the nucleic acid sequence of claim 10.

16. The cell according to any one of claims 13 to 16, which is a T cell.

17. A method of producing a cell according to any one of claims 13 to 16, comprising the step of transducing or transfecting a cell with a vector according to any one of claims 10 to 12.

18. 1. A method for selecting cells expressing a POI, comprising: I. Detecting the expression of the POI epitope on the surface of cells transfected or transduced with a vector according to any one of claims 10 to 12, and II. Selecting cells found to express the POI epitope A method comprising:

19. A method for preparing a purified population of cells enriched for cells expressing a POI, the method comprising a step of selecting cells expressing a POI from a population of cells using the method of claim 19.

20. A population of cells isolated from a tissue or cell donor is ex vivo I. Transducing or transfecting the vector of any one of claims 10 to 12, and II. Selecting cells expressing the POI from the population of transduced / transfected cells by the method of claim 18.

20. The method of claim 19, comprising:

21. A cell population enriched for cells expressing the polypeptide of claim 1, and therefore enriched for cells expressing the POI.

22. A method for tracking transduced cells in vivo, comprising detecting expression of the polypeptide of claim 1 on the surface of said cells.

23. A method for treating a disease in a subject, the method comprising the step of administering to the subject a cell according to any one of claims 13 to 16 or a cell population according to claim 21.

24. I. Transducing or transfecting a sample of cells isolated from a subject with the vector of any one of claims 10 to 12; and II. Returning the transduced / transfected cells to the patient 25. The method of claim 24, comprising:

25. 25. The method of claim 24 for treating cancer.

26. Cells according to any one of claims 13 to 16 or cell populations according to claim 21 for use in the treatment of disease and / or adoptive cell transfer.

27. A CoStAR receptor comprising the tumor-associated antigen-binding domain of claim 6, I. A fusion signaling domain comprising or consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. An intracellular domain from human CD137, such as that set forth in SEQ ID NO: 4, or a variant thereof having at least 80% or 90% sequence identity at the protein level. The CoStAR receptor fused to

28. A CoStAR receptor comprising the tumor-associated antigen-binding domain of claim 6, I. A fusion signaling domain comprising or consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. An intracellular domain from human CD134, such as that set forth in SEQ ID NO: 5, or a variant thereof having at least 80% or 90% sequence identity at the protein level. The CoStAR receptor fused to

29. A CoStAR receptor comprising the tumor-associated antigen-binding domain of claim 6, I. A fusion signaling domain comprising or consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. The intracellular domain from human CD2, such as that set forth in SEQ ID NO: 6, or a variant thereof having at least 80% or 90% sequence identity at the protein level. The CoStAR receptor fused to

30. A CoStAR receptor comprising the tumor-associated antigen-binding domain of claim 6, I. A fusion signaling domain comprising or consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. An intracellular domain from human CD29, such as that set forth in SEQ ID NO: 7, or a variant thereof having at least 80% or 90% sequence identity at the protein level. The CoStAR receptor fused to

31. A CoStAR receptor comprising the tumor-associated antigen-binding domain of claim 6, I. A fusion signaling domain comprising or consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. An intracellular domain from human GITR, such as that set forth in SEQ ID NO: 8, or a variant thereof having at least 80% or 90% sequence identity at the protein level. The CoStAR receptor fused to

32. A CoStAR receptor comprising the tumor-associated antigen-binding domain of claim 6, I. A fusion signaling domain comprising or consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. An intracellular domain from human IL2Rγ, such as that set forth in SEQ ID NO: 9, or a variant thereof having at least 80% or 90% sequence identity at the protein level. The CoStAR receptor fused to

33. A CoStAR receptor comprising the tumor-associated antigen-binding domain of claim 6, I. A fusion signaling domain comprising or consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. An intracellular domain from human CD40, such as that set forth in SEQ ID NO: 10, or a variant thereof having at least 80% or 90% sequence identity at the protein level. The CoStAR receptor fused to

34. A CoStAR receptor comprising the tumor-associated antigen-binding domain of claim 6, I. A fusion signaling domain comprising or consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. An intracellular domain from human CD150, such as that set forth in SEQ ID NO: 11, or a variant thereof having at least 80% or 90% sequence identity at the protein level. The CoStAR receptor fused to

35. A CoStAR receptor comprising the tumor-associated antigen-binding domain of claim 6, I. A fusion signaling domain comprising or consisting of full-length human CD28, such as that set forth in SEQ ID NO:2, or a variant thereof having at least 80% sequence identity at the protein level; and II. Intracellular domains from human CD2 and human CD40, such as those set forth in SEQ ID NO: 12 or variants thereof having at least 80% or 90% sequence identity at the protein level. The CoStAR receptor fused to

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