Chimeric antigen receptors with a tip co-stimulatory domain

Abstract

Provided herein are systems, kits, compositions, and methods for treating a subject with cancer or other disease with a CAR T-cell expressing a chimeric antigen receptor which comprises a TIP co-stimulatory domain, wherein the TIP co-stimulatory domain comprises a CSKH peptide and / or a SH3 peptide. In certain embodiments, the CAR comprises a binding molecule, transmembrane domain, and an activating domain.

Description

[0001] CHIMERIC ANTIGEN RECEPTORS WITH A TIP CO-STIMULATORY DOMAIN

[0002] The present application claims priority to U.S. Provisional application serial number 63 / 594,211, filed October 30, 2023, which is herein incorporated by reference in its entirety.

[0003] SEQUENCE LISTING

[0004] The text of the computer readable sequence listing filed herewith, titled “CCF_42483_601_SequenceListing.xml”, created October 25, 2024, having a file size of 21,274 bytes, is hereby incorporated by reference in its entirety.

[0005] FIELD OF THE INVENTION

[0006] Provided herein are systems, kits, compositions, and methods for treating a subject with cancer or other disease with a CAR T-cell expressing a chimeric antigen receptor which comprises a TIP co-stimulatory domain ("CAR-TIP"), wherein the TIP co- stimulatory domain comprises a CSKH peptide and / or a SH3B peptide (e.g., for binding to LCK tyrosine kinase). In certain embodiments, the CAR comprises a binding molecule, transmembrane domain, and an activating domain.

[0007] BACKGROUND

[0008] Adoptive cell therapy using Chimeric Antigen Receptor (CAR) T cells, such as Tisagenlecleucel and Axicabtagene Ciloleucel, has revolutionized cancer immunotherapy and become a game-changer in cancer care. However, several challenges, including durable remission and limited expansion after infusion, persist. Recent work has demonstrated that T Cell Receptors (TCRs) are more sensitive than CARs due to the presence of T cell signaling proteins like CD35, CD3s, and CD3y, which facilitate the recruitment of LCK tyrosine kinase to phosphorylate immunoreceptor tyrosine- based activation motifs (ITAMs). Although CARs were designed to mimic TCR signaling, they still lack the full sensitivity.

[0009] SUMMARY OF THE INVENTION

[0010] Provided herein are systems, kits, compositions, and methods for treating a subject with cancer or other disease with a CAR T-cell expressing a chimeric antigen receptor which comprises a TIP co-stimulatory domain, wherein the TIP co-stimulatory domain comprises a CSKH peptide and / or a SH3B peptide. In certain embodiments, the CAR comprises a binding molecule, transmembrane domain, and an activating domain. In some embodiments, provided herein are compositions comprising a fusion protein, wherein the fusion protein comprises: a) a TIP co-stimulatory domain comprising: A) a CSKH peptide comprising at least a portion of a mutated or non-mutated C-terminal Src- related kinase homology (CSKH) motif of tyrosine-protein kinase-interacting protein (TIP) from Herpesvirus saimiri (HVS), wherein the CSKH peptide interacts with the catalytic domain of human lymphocyte-specific protein tyrosine kinase (LCK) (e.g., for its enzymatic activity), and B) an SH3 peptide comprising at least a portion of a mutated or non-mutated SH3 binding domain of TIP HVS, wherein the SH3B peptide binds the SH3 domain of human LCK; and b) at least one of the following: A) a transmembrane domain, and / or B) an activating domain (e.g., T cell activating domain).

[0011] In particular embodiments, provided herein are compositions comprising a CAR T- cell, wherein the CAR T-cell comprises a T cell expressing a chimeric antigen receptor which comprises a TIP co-stimulatory domain, wherein the TIP co-stimulatory domain comprises a CSKH peptide and / or a SH3B peptide. In some embodiments, provided herein are compositions comprising a CAR natural killer cell (CAR-NK), wherein said CAR-NK comprises an NK cell expressing a chimeric antigen receptor which comprises a TIP co- stimulatory domain, wherein said TIP co-stimulatory domain comprises a CSKH peptide and a SH3 peptide.

[0012] In certain embodiments, provided herein are compositions comprising a chimeric antigen receptor (CAR) fusion protein, or a nucleic acid sequence encoding the CAR fusion protein, wherein the CAR fusion protein comprises: i) a binding molecule that binds a tumor antigen or other antigen, wherein the binding molecule is optionally selected from: A) a single-chain variable fragment (scFv) comprising a heavy chain variable domain (VH) and an light chain variable domain (VL), and B) a single variable domain on a heavy chain (VHH); ii) a transmembrane domain; iii) a TIP co-stimulatory domain comprising: A) a CSKH peptide comprising at least a portion of a mutated or non-mutated C-terminal Src- related kinase homology (CSKH) domain of tyrosine-protein kinase-interacting protein (TIP) from Herpesvirus saimiri (HVS), wherein the CSKH peptide interacts with the catalytic domain of human lymphocyte- specific protein tyrosine kinase (LCK), and B) an SH3B peptide comprising at least a portion of a mutated or non-mutated SH3 binding motif of TIP HVS, wherein the SH3B peptide binds human LCK, and iv) an activating domain (e.g., a T cell activating domain).

[0013] In some embodiments, the TIP co-stimulatory domain further comprises a spacer region between the SH3 peptide and the CSKH peptide. In particular embodiments, the spacer region is a mutated or non-mutated spacer sequence of the TIP from HVS. In other embodiments, the spacer region comprises: A) an amino acid sequence shown in SEQ ID NOs:5 or 6; or B) an amino acid sequence with 1, 2, 3, or 4 conservative amino acid changes compared to SEQ ID NOs:5 or 6; or C) an amino acid sequence shown in SEQ ID NOs:5 or 6 with a one, two, or three amino acid deletion at one end or both ends; or D) an amino acid sequence with at least 90% or 95% or 99% sequence identity with SEQ ID NOs: 5 or 6. In other embodiments, the spacer region is a flexible linker amino acid sequence.

[0014] In certain embodiments, the binding molecule binds the tumor antigen, and wherein the tumor antigen is selected from the group consisting of: mesothelin, BCMA, VEGFR-2, CD4, CD5, CD19, CD20, CD30, CD22, CD24, CD25, CD28, CD30, CD33, CD38, CD47, CD52, CD56, CD80, CD81, CD86, CD123, CD138, CD171, CD276, B7H4, CD133, EGFR, GPC3; PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2 / HER-2, ErbB3 / HER3, ErbB4 / HER-4, EphA2, EphlOA, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gpl30, Lewis A, Lewis Y, NGFR, MCAM, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, NY-ESO-1, PSMA, RANK, ROR1, ROR-2, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, L1FRP, LRP5, MUC1, TCRa, TCRp, TLR7, TLR9, PTCHI, WT-1, Robol, a, Frizzled, 0X40, CD79b, and Notch-1-4.

[0015] In some embodiments, the CSKH peptide comprises: A) an amino acid sequence shown in SEQ ID NOs:l or 2; or B) an amino acid sequence with 1 or 2 conservative amino acid changes compared to SEQ ID NOs:l or 2; or C) an amino acid sequence shown in SEQ ID NOs: 1 or 2 with a one or two amino acid deletion at one end or both ends; or D) an amino acid sequence with at least 90% or 95% or 99% sequence identity with SEQ ID NOs: 1 or 2. In particular embodiments, the SH3 peptide comprises: A) an amino acid sequence shown in SEQ ID NOs: 3 or 4; or B) an amino acid sequence with 1 or 2 conservative amino acid changes compared to SEQ ID NOs:3 or 4; or C) an amino acid sequence shown in SEQ ID NOs:3 or 4 with a one or two amino acid deletion at one end or both ends; or D) an amino acid sequence with at least 90% or 95% or 99% sequence identity with SEQ ID NOs: 3 or 4.

[0016] In some embodiments, the tumor antigen is human CD19. In other embodiments, the activating domain comprises CD3 s cytoplasmic domain. In additional embodiments, the fusion protein further comprises: v) an additional co-stimulatory domain. In particular embodiments, the additional co-stimulatory domain is from at least one of the following: CD28, 4-1BB, ICOS-1, CD27, OX-40, GITR, and DAP10.

[0017] In other embodiments, the transmembrane domain comprises human CD28 transmembrane domain or human CD8 transmembrane domain. In additional embodiments, the compositions further buffer, saline solution, and / or water. In particular embodiments, the TIP co-stimulatory domain comprises: A) an amino acid sequence selected from SEQ ID NOs: 7, 8, 9, or 10; or B) an amino acid sequence with 1, 2, 3, or 4 conservative amino acid changes compared to SEQ ID NOs:7, 8, 9, or 10; or C) an amino acid sequence shown in SEQ ID NOs:7, 8, 9, or 10 with a one, two, three, or four amino acid deletion at one end or both ends; or D) an amino acid sequence with at least 90% or 95% sequence identity with SEQ ID NOs: 7, 8, 9, or 10. In further embodiments, the CAR fusion protein comprises: A) an amino acid sequence selected from SEQ ID NOs: l 1, 13, or 15; or B) an amino acid sequence with 1 , 2, 3, or 4 conservative amino acid changes compared to SEQ ID NOs:l 1, 13, or 15; or C) an amino acid sequence shown in SEQ ID NOs:l l, 13, or 15 with a one, two, three, or four amino acid deletion at one end or both ends; or D) an amino acid sequence with at least 90% or 95% or 99% sequence identity with SEQ ID NOs: 11, 13, or 15.

[0018] In some embodiments, the composition comprises the a nucleic acid sequence encoding the CAR fusion protein, wherein the nucleic acid sequence is optionally present in a vector, and wherein optionally the vector comprises a plasmid or viral vector. In further embodiments, the composition further comprises a cell (e.g., human T cell), and wherein the nucleic acid sequence is present in the cell. In other embodiments, the composition comprises the CAR fusion protein. In further embodiments, the composition further comprises a cell with a cell membrane, and wherein the CAR fusion protein spans the cell membrane. In some embodiments, the cell comprises a T cell.

[0019] In certain embodiments, provided herein are methods of treating a patient with cancer or other disease comprising: administering to a subject: i) the nucleic acid sequence encoding the CAR fusion protein described above or herein, and / or ii) a T cell expressing the CAR fusion protein described above or herein. In some embodiments, the subject is a human. In other embodiments, the subject has cancer.

[0020] BRIEF DESCRIPTION OF THE FIGURES

[0021] Figure 1 shows incorporating TIP37 co-stimulation into CAR design. Left: Structure of Chimeric Antigen Receptors (CARs). The 4- IBB CAR, designed with a CD19-specific single-chain variable fragment (scFv), is equipped with intracellular signaling domains in the following order: 4-1BB and CD3^ with or without TIP37. The inclusion of TIP37 as a costimulatory domain is hypothesized to promote the recruitment of LCK to the CAR, thereby enhancing basal CAR-CD3^ phosphorylation, followed by the association of ZAP70 with the CAR. Right: Schematic of the TIP37 Mechanism. TIP37 augments the basal activity of LCK by releasing the kinase domain from the autoinhibitory loop. Figure 2 shows TIP37 co-stimulatory proteins induce LCK-mediated tyrosine phosphorylation. In particular, A) CAR design schemes. Antigen recognition domain (CD 19 scFv): light gray; CD8 transmembrane domain (TM): dark gray; 4-1BB costimulatory domain: green; CD3C, activation domain: purple; TIP37 costimulatory domain (TIP): red. B) Schema illustrating TIP37 Costimulation induces LCK-mediated constitutive phosphorylation of the CAR-CD3^ domain and detection. C) Immunoblot against phosphotyrosine shows that Jurkat T cells expressing TIP / CARs have higher antigen- independent / basal phosphorylation of the CAR-CD3^ (Top), while the mutant Jurkat T cell line missing the functional LCK expression, J.CaM1.6 expressing TIP / CARs attenuates basal phosphorylation of the CAR-CD3^ (Bottom). Relative endogenous GAPDH expression levels were measured.

[0022] Figure 3 shows TIP induces LCK-mediated tyrosine phosphorylation. In particular, A) Schema illustrating the molecular mechanism of TIP37-mediated LCK activation, upon the binding of TIP37, LCK releases its kinase domain from the autoinhibitory loop allowing the assessment of substrates to it. B) Schema of the TIP / CAR constructs in which specific mutations were included to generate CSKH deficient or / and SH3B deficient TIP / CARs. C) Immunoblot against phosphotyrosine shows that Jurkat T cells expressing CSKH deficient or / and SH3B deficient TIP / CARs attenuate basal phosphorylation of CAR-CD3^ (Top).

[0023] Figure 4 shows TIP37 co-stimulation mediates antigen-independent / basal phosphorylation of LCK and ZAP70 and their physical interaction with CARs. A) Schema illustrating TIP37-mediated recruitment of LCK and ZAP70 into the CAR synapse. B) Immunoblot against phosphotyrosine specific to CD3 (pY83-CD3Q, LCK (pY394-LCK), or ZAP70 (pY319 / Y352-ZAP70) show that Jurkat CAR T cells expressing TIP / CARs increase the basal phosphorylation of LCK and ZAP70. C) TIP37 costimulatory domain brings LCK into the CARs. LCK and ZAP70 detection by immunoprecipitation (IP) in CAR molecules pulled down from Jurkat T cells expressing 4-1BB / CAR (WT) or TIP-3 / CAR in the absence of antigen stimulation.

[0024] Figure 5 shows TIP / CAR Jurkat T cells release IL-2 upon antigen stimulation. Schema of the T cell activation. A) Jurkat T cells expressing TIP / CARs are stimulated with CD19-presenting cells (RAJI). 4-1BB / CAR Jurkat T cells are used as a positive control. B) Schema illustrating IL-2 secretion of CD19-CAR T cells mediated by CD19-presenting cells (RAJI). C) IL-2 release measured by IL-2 ELISA shows that TIP / CAR Jurkat T cells produce and release IL-2 upon the CD19-presenting cells stimulation (RAJI), comparable to that of WT / CAR Jurkat T cells, while unstimulated TIP / CAR Jurkat T cells do not release IL-2, (n = 3 for ELISA, data are presented as mean ± SEM). Figure 6 shows A) primary T cell lines (n = 7) expressing TIP / CARs (Top) are immunobloted against phosphotyrosine antibodies showing higher antigen-independent / basal phosphorylation of the CAR-CD3 , compared to that of Primary T cells expressing WT / CARs (middle). Relative endogenous GAPDH expression levels were measured (bottom). B) Schema of TIP37 costimulation domain engineering of LCK and CARs to mediate constitutive phosphorylation of the CAR-CD3^ and recruitment of ZAP70.

[0025] Figures 7A shows the amino acid sequence of TIP-1 CAR (SEQ ID NO: 11). Figure 7B shows the nucleic acid sequence of TTP-1 CAR (SEQ ID NO: 12).

[0026] Figures 8A shows the amino acid sequence of TIP-2 CAR (SEQ ID NO: 13). Figure 8B shows the nucleic acid sequence of TIP-2 CAR (SEQ ID NO: 14).

[0027] Figures 9A shows the amino acid sequence of TIP-3 CAR (SEQ ID NO: 15). Figure 9B shows the nucleic acid sequence of TIP-3 CAR (SEQ ID NO: 16).

[0028] Figure 10 shows expansion of CAR T cells. A) CAR T cells were activated using anti- CD3 / CD28 DynaBeads and cultured in the presence of IL-2. The growth curve shows the total number of expanded CAR T cells. B) Multicolor FACS analysis displays the CD4 / CD8 ratio within subpopulations of WT and TIP-3 CAR T cells. C) FACS quantification further assesses the expression of activation markers CD38 and Ki67 in CD4 and CD8 subpopulations of CAR T cells.

[0029] Figure 11 shows the frequencies of Naive or Stem Cell Memory (N / SCM: CCR7+ / CD45RA+), Central Memory (CM: CCR7+ / CD45RA-), Effector Memory (EM: CCR7- / CD45RA-), and Terminally Differentiated Effector Memory (TEMRA: CCR7- / CD45RA+) subsets within the CD4+ (Fig. 11 A) and CD8+ (Fig. 1 IB) T-cell populations. Data are expressed as the percentage of CD4+ or CD8+ T-cell populations.

[0030] Figure 12 shows antitumor efficacy of TIP-3 CAR-T cells in vivo: NSG mice were intravenously inoculated with RAJI-Luciferase cells. After 7 days, the mice were subcutaneously injected with either WT CAR T cells, TIP-3 CAR T cells, or left untreated. Tumor growth was monitored at specified time points using bioluminescent imaging. Images of the mice are in Figure 12 A, and a graph of plotted results are in Figure 12B.

[0031] DEFINITIONS

[0032] The terms "individual," "host," "subject," and "patient" are used interchangeably herein, and generally refer to a mammal, including, but not limited to, primates, including simians and humans, equines (e.g., horses), canines (e.g., dogs), felines, various domesticated livestock (e.g., ungulates, such as swine, pigs, goats, sheep, and the like), as well as domesticated pets and animals maintained in zoos. In some embodiments, the subject is specifically a human subject.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] Provided herein are systems, kits, compositions, and methods for treating a subject with cancer or other disease with a CAR T-cell expressing a chimeric antigen receptor which comprises a TIP co-stimulatory domain, wherein the TIP co-stimulatory domain comprises a CSKH peptide and / or a SH3 peptide. In certain embodiments, the CAR comprises a binding molecule, transmembrane domain, and an activating domain.

[0035] The present disclosure provides TIP co-stimulatory domains (e.g., TIP37) that enhances T cell persistency and promotes cytokine secretion. The TIP co-stimulatory domains (e.g., TIP37), in some embodiments, comprise a portion of the Herpesvirus saimiri (HVS) tyrosine-protein kinase- interacting protein (TIP). This oncogenic protein induces physical interactions with the major T cell kinase, LCK, and modulates its activation. This interaction appears to be specific to LCK, and likely not other Src kinases. For the exemplary TIP37, it is found to occur through 37 amino acids (146-182) in the central part of TIP comprising two putative motifs, called SH3 binding (SH3B; 174-182) and C-terminal Src- related kinase homology (CSKH; 146-155) motifs, respectively. SH3B is a proline-rich motif that binds to the SH3 domain of LCK, and CSKH is highly homologous to the c-terminal of Src-family kinases that interacts with the catalytic domain of LCK. In TIP37, for example, the 18 amino acids (156-173) between two motifs functions as a spacer required for efficient TIP-LCK interaction. In regard to TIP37, the 37 amino acid peptide fragment by itself is sufficient to form a stable complex with LCK.

[0036] LCK contributes to the early stage of T cell activation not only mediated by native T cell receptor (TCR) stimulation but also induced by CAR-antigen stimulation. Upon antigen- induced T cell activation, the LCK migrates near the TCR or CAR and phosphorylates the tyrosines in the immunoreceptor tyrosine-based activation motifs (IT AMs) in CD3 domains, leading to the docking of ZAP70 kinases to IT AMs of CD3^ and subsequent signaling protein complex formations. The addition of a TIP co-stimulatory domain component to the CAR promotes the LCK-CAR complex formation, which significantly increases the phosphorylation of IT AMs in CARs and downstream signals. Indeed, in work conducted during development of embodiments herein, in vitro experiments utilizing immortalized T cell line, Jurkat cell, show a significant increase in CD3^ phosphorylation within CARs, even in the absence of antigen stimulation, along with subsequent recruitment of the downstream signaling protein ZAP70. This finding was further validated in primary human T cells from seven healthy donors, where TIP37-CAR T cells exhibited dramatically higher levels of CD3^ phosphorylation within CARs at baseline compared to conventional CAR T cells. In an embodiment, the CAR T cell with a TIP co-stimulatory domain (e.g., TIP37 co-stimulatory domain) does not secrete cytokines in unstimulated cells, indicating the enhanced LCK phosphorylation would not allow for the auto-activation of T cells. Thus, TIP co-stimulatory domains introduced in CAR T cells should be safe and induce prolonged immunity. Further characterization of Tip37-CAR in primary T cells from healthy individuals reveals a predominant trend: Tip37-co-stimulated primary CAR T cells exhibit significantly larger percentages of the cytotoxic CD8+ subset, which expresses higher levels of CD38 and Ki67 compared to canonical CAR T cells without Tip37 co-stimulation. Additionally, we observed an expansion of CCR7+CD45RA- central memory subsets in CD4+ CAR19 cells and CCR7- CD45RA- effector memory subsets in CD8+ CAR19 cells. In vivo evaluation demonstrates that TIP37-co-stimulated CAR T cells exhibit enhanced anti-tumor efficacy without signs of premature exhaustion.

[0037] In work conducted during development of embodiments herein, it was hypothesized that enhancing tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motifs (IT AMs) presented on CD3^ in CAR T cells amplifies the CAR-mediated signaling as well as improves sensitivity of CAR co-stimulatory signaling. This was achieved by the development of TIP co-stimulatory domains where, for example, exemplary anti-CD19 CAR T cells co-expressed TIP37, a 37-amino acid segment of the Herpesvirus saimiri (HVS) tyrosine-protein kinase-interacting protein (TIP). TIP co-stimulatory domains interact with the major T cell kinase, LCK, effectively regulating its kinase activation.

[0038] In experimental work conducted during development of embodiments herein we engineered CAR19 T cells co-expressing TIP (TIP-CART19 cells) with a co-stimulatory domain of 4-1BB and CD3C, (TIP-CART19 cells). As a control, we generated CART19 cells lacking the TIP domain (WT CART19 cells). We confirmed that TIP expression induced enhanced CD3 phosphorylation in CART19 T cells.

[0039] Exemplary sequences are provided below:

[0040] TIP CSKH region (146-155): EDLQSFLEKY (SEQ ID NO:1) Alterative TIP CSKH region: EALQAFLEAA (SEQ ID NO:2) TIP SH3B region (176-182): TPPLPPR (SEQ ID NO:3) Alternative TIP SH3B region: TAALAAR (SEQ ID NO:4) TIP spacer region (156-175): PPDFRKPKRDLSATWDPGMP (SEQ ID NO:5) Alternative TIP spacer region: APDFRKPKRDLSATWDPGMP (SEQ ID NO: 6) In some embodiments, the T cells employed here are anti-tumor T cells (e.g., T cells with activity against a tumor (e.g., an autologous tumor) that become activated and expand in response to antigen). Anti-tumor T cells (e.g., useful for adoptive T cell transfer) include, in one embodiment, peripheral blood derived T cells genetically modified with receptors that recognize and respond to tumor antigens. Such receptors are generally composed of extracellular domains comprising a single-chain antibody (scFv) specific for tumor antigen, linked to intracellular T cell signaling motifs (See, e.g., Westwood, J. A. et al, 2005, Proc. Natl. Acad. Sci., USA, 102(52): 19051 - 19056). Other anti-tumor T cells include T cells obtained from resected tumors or tumor biopsies (e.g., tumor infiltrating lymphocytes (TILs). In another embodiment, the T cell is a polyclonal or monoclonal tumor-reactive T cell (e.g., obtained by apheresis, expanded ex vivo against tumor antigens presented by autologous or artificial antigen-presenting cells). In another embodiment, the T cells are engineered to express a T cell receptor of human or murine origin that recognizes a tumor antigen. The invention is not limited by the type of tumor antigen so recognized. Indeed, any T cell containing a receptor that recognizes a tumor antigen finds use in the compositions and methods of the invention. Examples include, but are not limited to, T cells expressing a receptor that recognize an antigen selected from CD 19, CD20, CD22, receptor tyrosine kinase-like orphan receptor 1 (ROR1), disialoganglioside 2 (GD2), Epstein-Barr Virus (EBV) protein or antigen, folate receptor, mesothelin, human carcinoembryonic antigen (CEA), CD33 / IL3Ra, tyrosine protein kinase Met (c-Met) or hepatocyte growth factor receptor (HGFR), prostate-specific membrane antigen (PSMA), Glycolipid F77, epidermal growth factor receptor variant III (EGFRvIII), NY-ESO-1, melanoma antigen gene (MAGE) Family Member A3 (MAGE-A3), melanoma antigen recognized by T cells 1 (MART-1), GP1000, p53, or other tumor antigen described herein.

[0041] In some embodiments, the T cells herein are engineered to express a CAR with a TIP co-stimulatory domain (CAR- TIP). The invention is not limited by the type CAR- TIP. Indeed, any CAR-TIP that binds with specificity to a desired antigen (e.g., tumor antigen) may be employed. In certain embodiments, the CAR- TIP comprises an antigen-binding domain. In certain embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) containing heavy and light chain variable regions that bind with specificity to the desired antigen. In some embodiments, the CAR-TIP further comprises a transmembrane domain (e.g., a T cell transmembrane domain (e.g., a CD28 transmembrane domain)) and a signaling domain comprising one or more immunoreceptor tyrosine-based activation motifs (ITAMs) (e.g., a T cell receptor signaling domain (e.g., TCR chain). In some embodiments, the CAR- TIP comprises one or more co-stimulatory domains (e.g., domains that provide a second signal to stimulate T cell activation). The invention is not limited by the type of co-stimulatory domain. Indeed, any co-stimulatory domain known in the art may be used including, but not limited to, CD28, OX40 / CD134, 4- 1BB / CD137 / TNFRSF9, the high affinity immunoglobulin E receptor-gamma subunit (FcERIy, ICOS / CD278, interleukin 2 subunit beta (ILRP) or CD122, cytokine receptor common subunit gamma (IL-2Ry) or CD 132, and CD40. In one embodiment, the co- stimulatory domain is 4- IBB.

[0042] The CAR-TIP may be engineered to target a tumor antigen of interest by way of engineering a desired antigen binding moiety that specifically binds to an antigen on a tumor cell. As used herein, a “tumor antigen” or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” or “cancer antigen,” refers to antigens that are common to specific hyperproliferative disorders such as cancer. Exemplary antigens mentioned herein are included by way of example. Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. Thus, an antigen binding moiety can be selected based on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen- 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0043] A tumor antigen may comprise one or more antigenic cancer antigens / epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and GP100 in melanoma and prostatic acid phosphatase (PAP) and prostate- specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER-2 / Neu / ErbB-2. Still another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma the tumor- specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. B-cell differentiation antigens such as CD 19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma. The tumor antigen may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA is not unique to a tumor cell and instead is also expressed on some normal cells under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells. Examples of TSA or TAA include, but are not limited to, differentiation antigens such as MART-l / MelanA (MART-1), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor- suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A- PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP- 180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO-1, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alphafetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.291\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0044] The CAR- TIPs of the present invention may comprise a signal peptide N-terminal to the binding molecule (e.g., scFv) so that when the CAR-TIP is expressed inside a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed. The core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases. As an example, the signal peptide may derive from human CD8 or GM-CSF, or a variant thereof having 1 or 2 amino acid mutations provided that the signal peptide still functions to cause cell surface expression of the CAR-TIP.

[0045] The CAR-TIPs of the present invention may comprise a spacer sequence as a hinge to connect antigen binding domain (e.g., scFv) with the transmembrane domain and spatially separate antigen binding domain from the endodomain. A flexible spacer allows to the binding domain to orient in different directions to enable its binding to a tumor antigen. The spacer sequence may, for example, comprise an IgGl Fc region, an IgGl hinge or a CD8 stalk, or a combination thereof, or a human CD28 or CD8 stalk.

[0046] In certain embodiments, provided herein are nucleic acids sequences encoding the CAR-TIPs described herein. The nucleic acid encoding a CAR-TIP can be prepared from an amino acid sequence of the specified CAR-TIP by a conventional method. A base sequence encoding an amino acid sequence can be obtained and the nucleic acid of the present invention can be prepared using a standard molecular biological and / or chemical procedure. For example, based on the base sequence, a nucleic acid can be synthesized, and the nucleic acid of the present invention can be prepared by combining DNA fragments which are obtained from a cDNA library using a polymerase chain reaction (PCR). The nucleic acid encoding the CAR-TIPs of the present invention can be inserted into a vector, and the vector can be introduced into a cell. For example, a virus vector such as a retrovirus vector (including a lentivirus vector, an oncoretrovirus vector, and a pseudo type vector), an adenovirus vector, an adeno- associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector or a Sendai virus vector, an Epstein-Barr virus (EBV) vector, and a HSV vector can be used.

[0047] T cells modified to express the CAR-TIPs herein can be used as a therapeutic agent for a disease. The therapeutic agent comprises the T cells expressing the CAR-TIP as an active ingredient, and may further comprise a suitable excipient. Examples of the excipient include pharmaceutically acceptable excipients known to a person skilled in the art. In certain embodiments, the present invention provides an adoptive cell therapy method for treating cancer, comprising the step of administering CAR- TIP T cells to a subject suffering from cancer.

[0048] In certain embodiments, and additional co-stimulatory domain is employed selected from the group consisting of human CD28, 4-1BB (CD137), ICOS-1, CD27, OX 40 (CD137), DAP10, and GITR (AITR). The cytoplasmic domain, protein binding sites and activation sites (binding motifs) of the above co-stimulatory proteins are shown in Table 1 from US Pat. Pub. US20190023764A1, herein incorporated by reference, particularly for Table 1 and related text. In certain embodiments, the additional co-stimulatory domain is CD28. The fusion proteins may comprise the binding motif YMNM (191-194) immediately repeated 1, 2, 3, or 4 times. In another embodiment, the fusion protein comprises the binding motif PYAP immediately repeated 1, 2, 3, or 4 times. In yet another embodiment, the fusion protein further comprises a mutation from T to P immediately after the last repeat of YMNM. The T to P mutation (T195P) corresponds to amino acid position 195 of the CD28 protein. In yet another embodiment, the fusion protein comprises double mutations, i.e., the binding motifs YMNM and PYAP are both immediately repeated once, optionally further comprises mutation from T to P immediately after the last repeat of YMNM. In another embodiment, the additional co-stimulatory domain is 4-1BB (CD137), ICOS-1, CD27, OX 40 (CD137), DAP 10, or GITR (AITR), and its binding motif can be immediately repeated 1, 2, 3, or 4 times. Further, a mutation site can be created in these co-stimulatory domains to increase the activity.

[0049] Although only a number exemplary embodiments have been described in detail, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this disclosure. Accordingly, all such modifications and alternative are intended to be included within the scope of the invention as defined in the following claims. Those skilled in the art should also realize that such modifications and equivalent constructions or methods do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

Claims

CLAIMSWe claim:

1. A composition comprising a chimeric antigen receptor (CAR) fusion protein, or a nucleic acid sequence encoding said CAR fusion protein, wherein said CAR fusion protein comprises: i) a binding molecule that binds a tumor antigen or other antigen, wherein said binding molecule is optionally selected from:A) a single-chain variable fragment (scFv) comprising a heavy chain variable domain (VH) and an light chain variable domain (VL), andB) a single variable domain on a heavy chain (VHH); ii) a transmembrane domain; iii) a TIP co-stimulatory domain comprising:A) a CSKH peptide comprising at least a portion of a mutated or nonmutated C-terminal Src- related kinase homology (CSKH) domain of tyrosine-protein kinase-interacting protein (TIP) from Herpesvirus saimiri (HVS), wherein said CSKH peptide interacts with the catalytic domain of human lymphocyte-specific protein tyrosine kinase (LCK); andB) an SH3B peptide comprising at least a portion of a mutated or nonmutated SH3 binding domain of TIP HVS, wherein said SH3B peptide binds human LCK, and iv) an activating domain.

2. The composition of claim 1 , wherein said TIP co-stimulatory domain further comprises a spacer region between said SH3 peptide and said CSKH peptide.

3. The composition of claim 2, wherein said spacer region is a mutated or non-mutated spacer sequence of said TIP from HVS.

4. The composition of claim 2, wherein said spacer region comprises:A) an amino acid sequence shown in SEQ ID NOs:5 or 6; orB) an amino acid sequence with 1, 2, 3, or 4 conservative amino acid changes compared to SEQ ID NOs:5 or 6; orC) an amino acid sequence shown in SEQ ID NOs:5 or 6 with a one, two, or three amino acid deletion at one end or both ends; orD) an amino acid sequence with at least 90% or 95% sequence identity with SEQ ID NOs: 5 or 6.

5. The composition of claim 2, wherein said spacer region is a flexible linker amino acid sequence.

6. The composition of claim 1 , wherein said binding molecule binds said tumor antigen, and wherein said tumor antigen is selected from the group consisting of: mesothelin, BCMA, VEGFR-2, CD4, CD5, CD19, CD20, CD30, CD22, CD24, CD25, CD28, CD30, CD33, CD38, CD47, CD52, CD56, CD80, CD81, CD86, CD123, CD138, CD171, CD276, B7H4, CD133, EGFR, GPC3; PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2 / HER-2, ErbB3 / HER3, ErbB4 / HER-4, EphA2, EphlOA, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gpl30, Lewis A, Lewis Y, NGFR, MCAM, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, NY-ESO-1, PSMA, RANK, ROR1, ROR-2, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, L1FRP, LRP5, MUC1, TCRa, TCRp, TLR7, TLR9, PTCHI, WT-1, Robol, a, Frizzled, 0X40, CD79b, and Notch-1-4.

7. The composition of claim 1, wherein said CSKH peptide comprises:A) an amino acid sequence shown in SEQ ID NOs: 1 or 2; orB) an amino acid sequence with 1 or 2 conservative amino acid changes compared to SEQ ID NOs:l or 2; orC) an amino acid sequence shown in SEQ ID NOs: 1 or 2 with a one or two amino acid deletion at one end or both ends; orD) an amino acid sequence with at least 90% or 95% sequence identity with SEQ ID NOs: 1 or 2.

8. The composition of claim 1, wherein said SH3 peptide comprises:A) an amino acid sequence shown in SEQ ID NOs: 3 or 4; orB) an amino acid sequence with 1 or 2 conservative amino acid changes compared to SEQ ID NOs:3 or 4; orC) an amino acid sequence shown in SEQ ID NOs:3 or 4 with a one or two amino acid deletion at one end or both ends; orD) an amino acid sequence with at least 90% or 95% sequence identity with SEQ ID NOs: 3 or 4.

9. The composition of claim 1, wherein said tumor antigen is human CD 19.

10. The composition of claim 1, wherein said activating domain comprises CD3 s cytoplasmic domain11. The composition of claim 1 , wherein said fusion protein further comprises: v) an additional co-stimulatory domain.

12. The composition of claim 1, wherein said additional co-stimulatory domain is from at least one of the following: CD28, 4- IBB, ICOS-1, CD27, OX-40, GITR, and DAP10.

13. The composition of claim 1, wherein said transmembrane domain comprises human CD28 transmembrane domain or human CD8 transmembrane domain.

14. The composition of claim 1, further comprising: buffer, saline solution, and / or water.

15. The composition of claim 1, wherein said TIP co-stimulatory domain comprises:A) an amino acid sequence selected from SEQ ID NOs: 7, 8, 9, or 10; orB) an amino acid sequence with 1, 2, 3, or 4 conservative amino acid changes compared to SEQ ID NOs:7, 8, 9, or 10; orC) an amino acid sequence shown in SEQ ID NOs:7, 8, 9, or 10 with a one, two, three, or four amino acid deletion at one end or both ends; orD) an amino acid sequence with at least 90% or 95% sequence identity with SEQ ID NOs: 7, 8, 9, or 10.

16. The composition of claim 1, wherein said CAR fusion protein comprises:A) an amino acid sequence selected from SEQ ID NOs:l 1, 13, or 15; orB) an amino acid sequence with 1, 2, 3, or 4 conservative amino acid changes compared to SEQ ID NOs:l 1, 13, or 15; orC) an amino acid sequence shown in SEQ ID NOs: 11, 13, or 15 with a one, two, three, or four amino acid deletion at one end or both ends; orD) an amino acid sequence with at least 90% or 95% sequence identity with SEQ ID NOs: 11, 13, or 15.

17. The composition of claim 1, wherein said composition comprises said a nucleic acid sequence encoding said CAR fusion protein, wherein said nucleic acid sequence is optionally present in a vector, and wherein optionally said vector comprises a plasmid or viral vector.

18. The composition of claim 17, wherein said composition further comprises a cell, and wherein said nucleic acid sequence is present in said cell.

19. The composition of claim 1 , wherein said composition comprises said CAR fusion protein.

20. The composition of claim 19, wherein said composition comprises a cell with a cell membrane, and wherein said CAR fusion protein spans said cell membrane.

21. The composition of claim 20, wherein said cell comprises a T cell.

22. A method of treating a patient with cancer or other disease comprising: administering to a subject: i) said nucleic acid sequence encoding said CAR fusion protein of any of claims 1-16, and / or ii) a T cell expressing said CAR fusion protein of any of claims 1-16.

23. The method of claim 22, wherein said subject is a human.

24. The method of claim 22 or 23, wherein said subject has cancer.

25. A composition comprising a fusion protein, wherein said fusion protein comprises: a) a TIP co-stimulatory domain comprising:A) a CSKH peptide comprising at least a portion of a mutated or nonmutated C-terminal Src- related kinase homology (CSKH) domain of tyrosine-protein kinase-interacting protein (TIP) from Herpesvirus saimiri (HVS), wherein said CSKH peptide interacts with the catalytic domain of human lymphocyte-specific protein tyrosine kinase (LCK), andB) an SH3 peptide comprising at least a portion of a mutated or nonmutated SH3 binding domain of TIP HVS, wherein said SH3 peptide binds human LCK, and b) at least one of the following:A) a transmembrane domain, and / orB) an activating domain.

26. The composition of claim 25, wherein said transmembrane domain comprises human CD28 transmembrane domain or human CD8 transmembrane domain.

27. The composition of claim 25, wherein said activating domain comprises CD3^'s cytoplasmic domain28. A composition comprising a CAR T-cell, wherein said CAR T-cell comprises a T cell expressing a chimeric antigen receptor which comprises a TIP co-stimulatory domain, wherein said TIP co-stimulatory domain comprises a CSKH peptide and / or a SH3 peptide.

29. A composition comprising a CAR natural killer cell (CAR-NK), wherein said CAR- NK comprises an NK cell expressing a chimeric antigen receptor which comprises a TIP co- stimulatory domain, wherein said TIP co-stimulatory domain comprises a CSKH peptide and a SH3 peptide.

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