Improved glycan-dependent chimeric antigen receptor cells

Abstract

Compositions and methods are provided for treating diseases associated with abnormal glycosylation of cell surface molecules and the expression of tumor-associated carbohydrate antigen (TACA). Also provided are a chimeric antigen receptor (CAR) specific for a tumor-associated carbohydrate antigen (TACA-CAR), a vector encoding the TACA-CAR, and a recombinant cell comprising the TACA CAR.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 351,746, filed on June 13, 2022, which is hereby incorporated by reference in its entirety for all purposes.

[0002] Description of Research and Development Sponsored by the Federal Government This invention was made with government support under grant numbers U01CA233078, R41CA233111, and R41CA261408 awarded by the National Institutes of Health / National Cancer Institute. The government has certain rights in this invention.

[0003] This disclosure generally relates to the fields of pharmacology and immunology, and more specifically to chimeric antigen receptors (TACA - CARs) that target tumor - associated carbohydrate antigens, and to the use of immune cells expressing TACA - CARs for treating diseases associated with abnormal glycosylation of cell - surface molecules, as well as methods for preventing or reversing TACA CAR T - cell exhaustion.

Background Art

[0004] Antigen-targeted cancer immunotherapies, such as bispecific antibodies (e.g., bispecific T cell engagers) or chimeric antigen receptor T cells (e.g., engineered immune cells expressing a chimeric antigen receptor (CAR)), are known as the most powerful immunotherapies. Both cause T cell-mediated killing of cancer cells, and the complete response rate of CAR T cells in relapsed / refractory B cell malignancies reaches a high rate of about 90%. Both utilize single-chain variable fragments (scFvs) derived from the variable heavy and variable light chains of monoclonal antibodies to target antigens expressed in cancer. In bispecific antibodies, the antigen-specific scFv is fused to a second scFv specific for CD3, while in CARs, the antigen-specific scFv is fused to a transmembrane domain and one or more cytoplasmic signaling domains derived from immune cell receptors. Both types of chimeric molecules are genetically expressed within T cells. Both therapies are currently approved for the treatment of CD19 + B cell malignancies. To apply bispecific proteins and / or CAR T cells to a wide variety of cancer types, it is first necessary to identify cell surface cancer antigens that can be safely targeted. This is a major challenge, especially for solid tumors. A potential approach to address all aspects of this problem is to target "tumor-associated carbohydrate antigens" (TACAs), which are overexpressed in a wide variety of cancer types and at even higher levels in metastatic and invasive cancers. Carbohydrates (glycans), like glycoproteins and glycolipids, are major cell surface components. Therefore, bispecific proteins and / or chimeric antigen receptor (CAR) T cells for immunotherapy that target cells for killing by T cells rather than antibodies using a lectin-derived tumor-associated carbohydrate antigen (TACA) binding domain have been generated. This new technology is called "glycan-dependent T cell recruiter" or GlyTR (pronounced "glitter").

[0005] However, similar to many useful therapeutic agents and conventional immunotherapies that require manipulation of cells expressing chimeric antigen receptors (e.g., immune cells or stem cells), unregulated (e.g., continuous) chimeric antigen receptor signaling is associated with significant risks. The efficacy of CAR T cells is limited by their early exhaustion. The root cause of T cell exhaustion is continuous antigen exposure that leads to continuous TCR signaling. T cell exhaustion is characterized by marked changes in metabolic function, transcriptional programming, loss of effector functions (e.g., cytokine secretion, killing ability), and co-expression of multiple surface inhibitory receptors. Thus, unregulated signaling can reduce the efficacy of CAR T cells by increasing the susceptibility of the engineered immune cells to exhaustion.

[0006] Accordingly, there is a need for very potent CAR T cells that are less susceptible to exhaustion (e.g., CAR-mediated T cell exhaustion) to target antigens present in multiple common cancers. The present disclosure meets this unmet need. SUMMARY OF THE INVENTION

[0007] One aspect of the present disclosure provides a modified cell comprising an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein (a) the CAR comprises an antigen-binding domain that selectively binds a tumor-associated carbohydrate antigen (TACA) comprising one or more TACA-binding domains, a transmembrane domain, a co-stimulatory domain, and / or an intracellular signaling domain, and (b) the modified cell is less susceptible to tonic signaling by the TACA CAR, thereby reducing exhaustion and / or inflammatory cytokine production in the absence of target cancer cells.

[0008] In some embodiments, the antigen-binding domain comprises a mutation in the TACA-binding domain (TBD, carbohydrate-binding domain) selected from a substitution, deletion, or insertion. In some embodiments, the antigen-binding domain comprises a deletion of the TACA-binding domain (TBD). In some embodiments, the deletion is present in the N-terminal region and / or C-terminal region of the TACA-binding domain (TBD).

[0009] In some embodiments, the deletion is at least about 2 amino acids, at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 36 amino acids, at least about 38 amino acids, at least about 40 amino acids, at least about 45 amino acids, or more. In some embodiments, the deletion is at least about 10 amino acids, at least about 18 amino acids, or at least about 36 amino acids. In some embodiments, the deletion is at least about 36 amino acids.

[0010] In some embodiments, the antigen-binding domain comprises a deletion of the TACA-binding domain (TBD), and the deletion is (a) present at the N-terminus of the TBD and is at least about 18 amino acids, (b) present at the C-terminus and is at least about 10 amino acids, (c) present at the N-terminus of the TBD and is at least about 36 amino acids, or (d) present at the N-terminus of the TBD and is at least about 18 amino acids and present at the C-terminus and is at least about 10 amino acids. In some embodiments, the antigen-binding domain comprises a deletion that removes a disulfide-bond cysteine residue in the TACA-binding domain (TBD). In some embodiments, the expression of the CAR comprising a deletion in the TACA-binding domain (TBD) of the antigen-binding domain is similar to the expression of the CAR comprising the wild-type TBD.

[0011] In some embodiments, (a) modified cells expressing a CAR having a deletion in the tumor-associated carbohydrate antigen (TACA)-binding domain (TBD) of the antigen-binding domain exhibit a decrease in tonic signaling compared to modified cells containing a CAR with a wild-type TBD, (b) modified cells expressing a CAR having a deletion in the TACA-binding domain (TBD) of the antigen-binding domain are less likely to experience exhaustion compared to modified cells containing a CAR with a wild-type TBD, or (c) modified cells expressing a CAR having a deletion in the TACA-binding domain (TBD) of the antigen-binding domain are less likely to experience exhaustion induced by tonic signaling TACA CAR compared to modified cells containing a CAR with a wild-type TBD.

[0012] In some embodiments of the modified cells disclosed herein, the TACA-binding domain is derived from a lectin. In some embodiments, the lectin is selected from galectin, siglec, selectin; C-type lectin; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Phaseolus limensis leukocyte agglutinin); E-PHA (Phaseolus limensis erythrocyte agglutinin); tomato lectin (Lycopersicon esculentum lectin; LEA); peanut lectin (Arachis hypogaea lectin; PNA); potato lectin (Solanum tuberosum lectin), pokeweed mitogen (Phytolacca americana lectin), wheat germ agglutinin (Triticum aestivum germ lectin); Artocarpus polyphyllus lectin (jacalin lectin); hairy vetch agglutinin (VVA); apple snail agglutinin (HPA); fucose lectin (WFA); Sambucus nigra agglutinin (SNA), BC2L-CNt (lectin from the gram-negative bacterium Burkholderia cenocepacia), dog spleen leukocyte agglutinin (MAL), Lentinus edodes (PVL), Sclerotium rolfsii lectin (SRL), Euonymus sieboldianus agglutinin (ESA), CLEC17A (prolectin), Hericium erinaceus lectin, Allium sativum lectin (SSA), Glechoma hederacea lectin (Gleheda), Momordica charantia lectin (Morniga G), Origanum vulgare lectin, Salvia bogotensis lectin, Salvia horminum lectin, Cuscutaceae lectin, Calceolaria integrifolia lectin, Glyphomia simplificifolia (GsLA4), Vicia faba (acidic WBAI), Vigna angularis lectin, Apios americana lectin, Amaranthus leucocarpus lectin, Relhia autumnalis lectin, Paramignya monophylla lectin, Ulex europaeus lectin, Artocarpus lakoocha lectin, Himalayan Vicia faba agglutinin, Himalayan Vicia faba lectin, soybean lectin and mushroom lectin.

[0013] In some embodiments, the lectin is (i) a galectin selected from the group consisting of galectin-1, galectin-2, galectin-3, galectin-4, galectin-5, galectin-6, galectin-7, galectin-8, galectin-9, galectin-10, galectin-11, galectin-12, galectin-13, galectin-14, and galectin-15, and / or (ii) a siglec selected from the group consisting of siglec-1 (sialoadhesin), siglec-2 (CD22), siglec-3 (CD33), siglec-4 (myelin-associated glycoprotein), siglec-5, siglec-6, siglec-7, siglec-8, siglec-9, siglec-10, siglec-11, siglec-12, siglec-13, siglec-14, siglec-15, siglec-16, siglec-17, siglec-E, siglec-F, siglec-G, and siglec-H.

[0014] In some embodiments, the polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) is selected from the group consisting of ppGalNAc-T1 (GALNT1), ppGalNAc-T2 (GALNT2), ppGalNAc-T3 (GALNT3), ppGalNAc-T4 (GALNT4), ppGalNAc-T5 (GALNT5), ppGalNAc-T6 (GALNT6), ppGalNAc-T7 (GALNT7), ppGalNAc-T8 (GALNT8), ppGalNAc-T9 (GALNT9), ppGalNAc-T10 (GALNT10), ppGalNAc-T12 (GALNT12), ppGalNAc-T13 (GALNT13), ppGalNAc-T14 (GALNT14), ppGalNAc-T15 (GALNT15), ppGalNAc-T16 (GALNT16), ppGalNAc-T17 (GALNT17), ppGalNAc-T18 (GALNT18), ppGalNAc-T Like 5 (GALNTL5), and ppGalNAc-T Like 6 (GALNTL6).

[0015] In some embodiments, the antigen-binding domain selectively targets a TACA selected from the group consisting of β1,6-branched, β1,6GlcNAc-branched N-glycans, T antigen, Tn antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6-sialylation, sialylation, sialyl-Lewis x / a , disialyl-Lewis x / a , sialyl 6-sulfo Lexis x , Globo H, GD2, GD3, GM3, and fucosyl GM1. In some embodiments, the antigen-binding domain selectively targets β1,6GlcNAc-branched N-glycan, Tn epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), GalNAcα-serine, GalNAcα-threonine, GalNAc, or GalNAcβ1. In some embodiments, the antigen-binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more TACA-binding domains.

[0016] In some embodiments, the antigen-binding domain comprises a carbohydrate-binding domain (CBD) of a TACA-binding domain that contains a deletion in an amino acid sequence set forth in SEQ ID NOs: 30-54, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence set forth in SEQ ID NOs: 30-54.

[0017] In some embodiments, the antigen-binding domain comprises an amino acid sequence set forth in SEQ ID NOs: 34-39, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the amino acid sequences set forth in SEQ ID NOs: 34-39. In some embodiments, the antigen-binding comprises an amino acid sequence having at least 90% homology to SEQ ID NOs: 34-39.

[0018] In some embodiments of the modified cells disclosed herein, the transmembrane domain comprises the transmembrane region of a molecule selected from the group consisting of T cell receptor (TCR)-alpha, TCR-beta, TCR-gamma, TCR-delta, invariant TCR of NKT cells, CD3-zeta, CD3-epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134 (Ox40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some embodiments, the transmembrane domain comprises the CD8 transmembrane domain or the CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 78 or SEQ ID NO: 87.

[0019] In some embodiments of the modified cells disclosed herein, the co-stimulatory domain is a co-stimulatory domain of a molecule selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD8, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, DAP10, DAP12, Lck, Fas, and combinations thereof. In some embodiments, the co-stimulatory domain comprises (i) a 4-1BB co-stimulatory domain, (ii) the amino acid sequence of SEQ ID NO: 58, (iii) a CD28 co-stimulatory domain, (iv) the amino acid sequence of SEQ ID NO: 88, or (v) a 4-1BB and CD28 co-stimulatory domain.

[0020] In some embodiments of the modified cells disclosed herein, the intracellular domain comprises an intracellular signaling domain of a molecule selected from the group consisting of T cell receptor (TCR) zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, CD3, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain comprises a CD3 zeta signaling domain or the amino acid sequence of SEQ ID NO: 59.

[0021] In some embodiments of the modified cells disclosed herein, the CAR further comprises a hinge domain. In some embodiments, the hinge domain is a protein selected from the group consisting of a CD28 hinge, CD8α, an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, and an artificial spacer sequence. In some embodiments, the hinge domain is (a) a CD8α hinge domain, (b) a CD28 hinge, (c) comprises the amino acid sequence of SEQ ID NO: 77 or 86. In some embodiments, the hinge domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 71-77, and 86.

[0022] Another aspect of the present disclosure provides a modified cell comprising an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising (i) an amino acid sequence set forth in SEQ ID NOs: 23-29, or (ii) an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 23-29.

[0023] In some embodiments of the modified cells disclosed herein, the CAR selectively targets a TACA selected from the group consisting of β1,6-branched, β1,6GlcNAc-branched N-glycan, T antigen, Tn antigen, sialyl-T epitope, Tn epitope, sialyl-Tn epitope, α2,6-sialylation, sialylation, sialyl-Lewisx / a, disialyl-Lewisx / a, sialyl 6-sulfo Lexisx, Globo H, GD2, GD3, GM3, and fucosyl GM1. In some embodiments, the CAR selectively targets β1,6GlcNAc-branched N-glycan, GalNAc, Tn antigen, GalNAcα-ser, GalNAc, or GalNAcβ1.

[0024] In some embodiments of the modified cells disclosed herein, the modified cell comprising the CAR of SEQ ID NOs: 23-29 exhibits reduced tonic signaling compared to the modified cell comprising the CAR of SEQ ID NO: 21 or 22.

[0025] In some embodiments of the modified cells disclosed herein, the cell is selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. In some embodiments, the cell is a T cell. In some embodiments, the cell is an autologous cell.

[0026] In some embodiments of the modified cells disclosed herein, the isolated nucleic acid comprises an expression vector and / or in vitro transcribed RNA.

[0027] One aspect of the present disclosure provides a chimeric antigen receptor expressed in a modified cell described herein.

[0028] Another aspect of the present disclosure is a chimeric antigen receptor that selectively binds to a tumor-associated carbohydrate antigen (TACA), wherein (a) the CAR comprises (i) an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA) and comprises one or more TACA-binding domains derived from a lectin, (ii) a transmembrane domain, (iii) a co-stimulatory signaling region, and (iv) an intracellular signaling domain; and (b) when introduced into a modified cell, the modified cell expressing the CAR is less susceptible to the effects of tonic signaling of the TACA CAR, thereby reducing exhaustion and / or inflammatory cytokine production in the absence of target cancer cells.

[0029] Another aspect of the present disclosure provides a chimeric antigen receptor that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising (a) an antigen-binding domain that contains a deletion and has an amino acid sequence set forth in SEQ ID NOs: 30-54 or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the amino acid sequence set forth in SEQ ID NOs: 30-54; (b) a CD8 or CD28 hinge domain; (c) a CD8 or CD28 transmembrane domain; (d) a CD28 co-stimulatory domain and / or a 4-1BB co-stimulatory domain; and (e) a CD3 zeta intracellular signaling domain.

[0030] In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NOs: 23-29.

[0031] In some embodiments of the CARs disclosed herein, (a) modified cells comprising the CARs of SEQ ID NOs: 23-29 exhibit reduced tonic signaling compared to modified cells comprising the CAR of SEQ ID NOs: 21 or 22, or (b) modified cells expressing a CAR comprising a deletion in the amino acid sequence set forth in SEQ ID NOs: 30-54, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 30-54, are less susceptible to exhaustion compared to modified cells comprising a CAR comprising a wild-type antigen-binding domain, or (c) modified cells expressing a CAR comprising a deletion in the amino acid sequence set forth in SEQ ID NOs: 30-54, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 30-54, are less susceptible to the effects of exhaustion associated with tonic signaling TACA CAR compared to modified cells comprising a CAR comprising a wild-type antigen-binding domain.

[0032] One aspect of the disclosure provides an isolated nucleic acid encoding a CAR expressed by a modified cell described herein, or a CAR described herein.

[0033] One aspect of the present disclosure provides an expression construct comprising an isolated nucleic acid encoding a CAR described herein. In some embodiments, the expression construct further comprises a promoter. In those embodiments, the promoter is selected from an EF-lα promoter, a T cell receptor alpha (TRAC) promoter, an interleukin 2 (IL-2) promoter, or a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a Moloney murine leukemia virus (MoMuLV) promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, or a Rous sarcoma virus promoter.

[0034] In some embodiments, the expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. In some embodiments, the expression construct is a lentiviral vector. In some embodiments, the expression construct is a self-inactivating lentiviral vector.

[0035] One aspect of the present disclosure provides a method of generating a modified cell described herein, the method comprising introducing into the cell an isolated nucleic acid described herein, a chimeric antigen receptor described herein, or an expression construct described herein.

[0036] One aspect of the present disclosure provides a composition comprising (a) a modified cell described herein, (b) a chimeric antigen receptor described herein, (c) a cell or population of cells expressing a nucleic acid described herein, or (d) a cell or population of cells expressing an expression construct described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0037] Another aspect of the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an immunotherapeutic composition comprising (a) the modified cells described herein, (b) the chimeric antigen receptor (CAR) described herein, or (c) the composition described herein.

[0038] In some embodiments, the cancer is selected from the group consisting of hematological malignancies, solid tumors, primary or metastatic tumors, leukemias, carcinomas, blastomas, sarcomas, leukemias, malignant lymphomas, melanomas, and lymphomas.

[0039] Another aspect of the present disclosure is a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective composition comprising modified cells comprising a chimeric antigen receptor that selectively binds to a tumor-associated carbohydrate antigen (TACA), wherein the CAR comprises (i) an amino acid sequence set forth in SEQ ID NOs: 30-54, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 30-54, an antigen-binding domain comprising a deletion, (ii) a CD8 or CD28 hinge domain, (iii) a CD8 or CD28 transmembrane domain, (iv) a CD28 co-stimulatory domain and / or a 4-1BB co-stimulatory domain, and (v) a CD3 zeta intracellular signaling domain. In some embodiments, the modified cells are less susceptible to exhaustion and / or cytokine production in the absence of target cancer cells associated with the tonic signaling TACA CAR.

[0040] One aspect of the present disclosure provides a method of providing anti-tumor immunity in a mammal, the method comprising administering to the mammal an effective amount of a population of modified cells described herein, or a composition described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0041]

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Modes for Carrying Out the Invention

[0042] I. Summary In the co-filed International Application No. PCT / US2023 / 024898, entitled "Bispecific Fusion Proteins and Chimeric Antigen Receptors for Improved Glycan-Dependent Immunotherapy", a new class of bispecific fusion proteins and CARs that effectively target TACAs for immunotherapy have been developed. Specifically, instead of monoclonal antibodies or fragments thereof, antigen-binding domains derived from the lectin's sugar chain recognition domain were used to engineer bispecific fusion proteins and CARs that target TACAs regardless of the carrier protein. This novel technology is referred to as "Glycan-Dependent T Cell Recruiter" or GlyTR (pronounced "glitter"). One set of GlyTR therapeutics is a TACA bispecific fusion protein comprising a sugar chain recognition domain from a lectin operably linked, conjugated, or fused to an immune cell recognition domain that specifically binds to a receptor on immune effector cells. Another set of GlyTR therapeutics is a chimeric antigen receptor comprising an antigen-binding domain that includes a TACA-binding domain (i.e., a sugar chain recognition domain) derived from a lectin. In all cases, the TACA-binding domain (i.e., the sugar chain recognition domain) specifically binds to TACAs expressed on tumor cells, and the TACA-binding domain includes one or more TACA-binding domains derived from a lectin.

[0043] Since GlyTR can target antigens present in multiple common cancers, the GlyTR disclosed in that application represents a significant improvement for immunotherapy. This is because the glycosylation changes that generate TACAs are an almost universal feature of cancer. TACAs provide the most abundant and widespread cell surface cancer antigens known, and their target density can be up to about 100 to 1000 times higher than that of common protein antigens. Furthermore, the TACA target density is about 100 to 1000 times higher than that of common protein antigens. GlyTR also has high avidity binding, which is achieved by the combination of high-density target expression on tumor cells and the presence of multiple sugar chain binding domains of engineered GlyTR. This combination of high target density and multiple binding sites increases the specificity of GlyTR for high-TACA-expressing cells (e.g., cancer cells) compared to low-expressing cells (such as normal cells).

[0044] However, similar to many useful immunotherapies that require the manipulation of cells expressing chimeric antigen receptors (e.g., immune cells or stem cells, etc.), persistent (e.g., unregulated) chimeric antigen receptor signaling is associated with significant risks.

[0045] Many engineered CAR T cells are known to induce antigen-independent effects of the CAR on T cells. Long et al., Nat. Med. 21:581-590 (2015). Several mechanisms by which the CAR delivers antigen-independent signals are conceivable. Tonic CARs still have the ability to induce cytotoxicity in an antigen-dependent manner, but antigen-independent signaling of the CAR has important implications for the clinical use of these chimeric antigen receptors. This is because antigen-independent signaling can lead to premature exhaustion of CAR T cells and, therefore, may limit anti-tumor efficacy in vivo. Tumor-reactive T cell exhaustion is a well-established mechanism of immune evasion in cancer. The underlying cause of T cell exhaustion is persistent antigen exposure leading to continuous TCR signaling. T cell exhaustion is characterized by marked changes in metabolic function, transcriptional programming, loss of effector functions (e.g., cytokine secretion, killing ability), and co-expression of multiple surface inhibitory receptors.

[0046] Thus, unregulated signaling can reduce the efficacy of CAR T cells by increasing their susceptibility to exhaustion of engineered immune cells. The mechanisms leading to exhaustion in the context of tonic T cell signaling are complex and poorly understood, but the CAR structure plays a central role in chronically activating CAR T cells and making them prone to exhaustion.

[0047] In the present disclosure, a novel class of TACA CARs for immunotherapy that cannot induce antigen-independent tonic signaling or has reduced antigen-independent tonic signaling has been designed and developed. The novel CARs of the present disclosure are designed to control, manage, or suppress antigen-independent tonic signaling by CARs containing a lectin-derived TACA-binding domain (i.e., a sugar chain recognition domain). Specifically, the novel CAR design includes (1) manipulation of mutations in the sequence of the antigen-binding domain (i.e., the lectin-derived TACA-binding domain), and / or (2) modification of the transmembrane and intracellular signaling domains (e.g., the 4-1BB signaling domain with or without a costimulatory signaling domain such as CD28), and / or (3) modification of the number and / or linkage of the lectin-derived TACA-binding domains within the antigen-binding domain.

[0048] A. Summary of Experimental Results The present disclosure provides a novel GlyTR CAR comprising an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA) comprising one or more TACA-binding domains, a transmembrane domain, one or more costimulatory domains, and / or an intracellular signaling domain. When expressed in immune cells such as T cells, the novel CARs of the present disclosure do not induce tonic signaling, and modified immune cells expressing the CARs of the present disclosure are less susceptible to tonic signaling by TACA, thereby preventing or reducing exhaustion and / or inflammatory cytokine production in the absence of target cancer cells.

[0049] Similar to known CARs, GlyTR CARs also exhibited antigen-independent tonic CAR signaling. For example, as shown in FIGS. 4A-4B, GlyTR1 LPHA(2) -CAR T cells and GlyTR2 slCD301(4)-CAR T cells exhibited antigen-independent tonic CAR signaling. The tonic signaling effect was prominent when the expression of GlyTR CAR was high but not when it was low, suggesting that tonic signaling was induced by clustering of CARs on the cell surface (Figure 4C). However, all the novel CARs described herein and simultaneously filed in International Application No. PCT / US2023 / 024898, titled "Bispecific Fusion Proteins and Chimeric Antigen Receptors for Improved Glycan-Dependent Immunotherapy," still had the ability to induce potent cytotoxicity in an antigen-dependent manner (Figures 3A-3G). Since antigen-independent signaling may lead to premature exhaustion of CAR T cells and thus limit antitumor efficacy in vivo, and since exhaustion of tumor-reactive T cells is a well-established mechanism of immune evasion in cancer, the present disclosure provides novel and improved GlyTR CARs that reduce antigen-independent tonic signaling to negligible levels. Thereby, improved potent GlyTR CARs and GlyTR CAR T cells that are less susceptible to the effects of exhaustion are provided.

[0050] As shown in the following examples, GlyTR tonic signaling was eliminated and reduced by engineering a GlyTR CAR that cannot multimerize or cluster at the cell membrane. As shown in Figure 5B, GlyTR tonic signaling was in part caused by CAR clustering. Thus, one aspect of preventing or reducing GlyTR CAR T cell exhaustion involves introducing mutations into the carbohydrate recognition domain (CRD) of the TACA-binding domain. For example, tonic signaling was reduced by engineering one or more deletions in the TACA-binding domain selected from the group consisting of deletion of the first 36 N-terminal amino acids, deletion of the first 18 N-terminal amino acids, deletion of the first two disulfide bond cysteine residues, deletion of the last 10 C-terminal amino acids, and combinations of those deletions. In particular, a method of preventing GlyTR exhaustion in terms of tonic signaling involves deleting the first two disulfide bond cysteine residues within the CRD of the TACA-binding domain.

[0051] Furthermore, GlyTR tonic signaling was eliminated and reduced in the following additional manner. First, adding an additional co-stimulatory domain to the second-generation GlyTR CAR reduced tonic CAR signaling. In an exemplary embodiment, adding the CD28 co-signaling domain to the intracellular domain of a GlyTR CAR comprising the 4-1BB co-stimulatory domain and the CD3 zeta intracellular domain to generate a GlyTR-CD28-4-1BB CAR was sufficient to reduce tonic signaling.

[0052] Second, the length and rigidity of the linker separating the TACA-binding domains (e.g., L-PHA domains) within the GlyTR-CAR (Figures 1A and 2A) that contain more than one TACA-binding domain were altered. Third, GlyTR-CAR tonic signaling was decreased by preventing dimerization of the TACA-binding domains. For example, deletion of the first 5 amino acids of one or both of the two L-PHA domains of GlyTR1-CAR decreased CAR dimerization / tonic signaling. Preventing dimerization of GlyTR1-CAR by deletion of the first 5 amino acids of L-PHA also decreased the binding avidity to β1,6GlcNAc-branched N-glycans. This is because GlyTR1 LPHAΔ1-5xCD3 Binding / killing by the bispecific protein was reduced compared to the parental GlyTR1 LPHAxCD3 as shown by the decrease. See International Application No. PCT / US2023 / 024898, filed concurrently, entitled "Bispecific Fusion Proteins and Chimeric Antigen Receptors for Improved Glycan-Dependent Immunotherapy". This application is incorporated by reference in its entirety.

[0053] Accordingly, the present disclosure provides a GlyTR-CAR that induces sufficient T cell activation in vitro and in vivo to maximize the cancer killing effect, but that cannot induce T cell exhaustion or cytokine release syndrome. The present disclosure further provides GlyTR therapeutics having enhanced GlyTR binding avidity, killing activity, and safety.

[0054] B. Exemplary Advantages of TACA CARs Prevention or restoration of T cell exhaustion has long been sought as a means to enhance the effectiveness of T cells (e.g., in cancer patients or patients with chronic infections). Tonic signaling by CARs is a common design problem that can aid in killing cancer cells but also increase the risk of toxicity due to overactivation of T cells. Both intracellular and extracellular domains can drive tonic signaling. Existing technologies for preventing T cell exhaustion from the perspective of the tonic signaling system have problems with limited dynamic range and basal ("leaky") activity. Furthermore, the solutions currently found in the prior art focus on modifying co-stimulatory domains that enable fine-tuning and optimization of the response profile of the transferred immune cells, or on the manipulation of regulatable or inducible CARs (e.g., addition of a regulatable destabilizing domain (RDD), a protease cleavage site to the CAR, or an inducible domain). In the case of GlyTR CAR T cells, tonic signaling is likely driven by both the extracellular and intracellular domains. Thus, elimination / reduction of tonic signaling in GlyTR CAR T cells was achieved by manipulating the extracellular domain to prevent multimerization and / or by incorporating additional (e.g., one or more) intracellular co-stimulatory domains.

[0055] Accordingly, the present disclosure provides chimeric antigen receptors (CARs), modified cells, compositions, and methods for treating conditions or diseases associated with tumor-associated carbohydrate antigen (TACA). The CARs of the present disclosure provide a novel CAR design, which combines an engineered antigen-binding domain (i.e., a TACA-binding domain derived from a lectin containing one or more mutations in the carbohydrate recognition domain (CRD)) with (1) linker flexibility, (2) modulation of intra- and intermolecular binding / multimerization, and (3) and / or alteration of the transmembrane and intracellular signaling domains, resulting in improvements over the prior art. The combination of these elements has provided GlyTR with unexpected properties. Examples thereof include the ability to target antigens present in multiple common cancers, the ability to induce a potent cytotoxic effect against multiple common cancers, and the ability to be less affected by tonic signaling.

[0056] One aspect of the present disclosure includes a modified cell that is less affected by tonic signaling and includes an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR). The CAR includes an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA) and includes one or more TACA-binding domains, a transmembrane domain, a co-stimulatory domain, and / or an intracellular signaling domain. The modified cell is less affected by tonic signaling by the TACA CAR, thereby reducing exhaustion and / or inflammatory cytokine production in the absence of target cancer cells.

[0057] In some embodiments, the antigen-binding domain of the CAR comprises a mutation in the tumor-associated carbohydrate antigen (TACA)-binding domain (TBD) selected from a substitution, deletion, or insertion. In one embodiment, the antigen-binding domain comprises a deletion of the TACA-binding domain (TBD). In that embodiment, the deletion is present in the N-terminal region and / or the C-terminal region of the TACA-binding domain (TBD). In some embodiments, the deletion is at least about 2 amino acids, at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 36 amino acids, at least about 38 amino acids, at least about 40 amino acids, at least about 45 amino acids, or more.

[0058] Another aspect of the present disclosure provides a chimeric antigen receptor that selectively binds to a tumor-associated carbohydrate antigen (TACA) and comprises a deletion in the CBD of the antigen-binding domain, the deletion comprising an amino acid sequence set forth in SEQ ID NOs: 30-54 or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 30-54.

[0059] The chimeric antigen receptor (TACA-CAR) of the present disclosure includes structural modifications that control the non-antigen-dependent tonic signaling of TACA-CAR, and thus is improved over the prior art. The first structural modification includes altering the structure of the antigen-binding domain by changing the number, linkage, and sequence of the TACA-binding domains derived from lectins. For example, the antigen-binding domain of TACA-CAR includes one or more TACA-binding domains derived from lectins. The second structural modification includes altering the structure of the transmembrane domain and / or the intracellular signaling domain. For example, the TACA-CAR of the present disclosure includes one or more intracellular domains derived from co-stimulatory molecules (e.g., 41BB signaling domain and / or CD28). The disease or condition is, for example, cancer or any condition associated with changes in protein glycosylation. The cancer may be a hematological malignancy, a solid tumor, a primary tumor, or a metastatic tumor.

[0060] Another aspect of the present disclosure provides a modified cell comprising an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising (i) an amino acid sequence set forth in SEQ ID NOs: 23-29, or (ii) an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence set forth in SEQ ID NOs: 23-29.

[0061] Another aspect of the present disclosure provides a chimeric antigen receptor, an isolated nucleic acid encoding a CAR, and / or an expression construct comprising the isolated nucleic acid, wherein the CAR selectively binds to a tumor-associated carbohydrate antigen (TACA), and (a) the CAR comprises an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA) and that comprises one or more TACA-binding domains derived from a lectin, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain; and (b) when introduced into a modified cell, the modified cell expressing the CAR is less susceptible to the effects of tonic signaling by the TACA CAR, thereby reducing exhaustion and / or inflammatory cytokine production in the absence of target cancer cells.

[0062] Another aspect of the present disclosure provides a chimeric antigen receptor, an isolated nucleic acid encoding a CAR, and / or an expression construct comprising the isolated nucleic acid, wherein the CAR selectively binds to a tumor-associated carbohydrate antigen (TACA), and (a) an antigen-binding domain that comprises the amino acid sequence set forth in SEQ ID NOs: 30-54, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the amino acid sequence set forth in SEQ ID NOs: 30-54, and that comprises deletions; (b) a CD8 or CD28 hinge domain; (c) a CD8 or CD28 transmembrane domain; (d) a CD28 co-stimulatory domain and / or a 4-1BB co-stimulatory domain; and (e) a CD3 zeta intracellular signaling domain.

[0063] Another aspect of the present disclosure provides a composition comprising the TACA-CAR disclosed herein, a method of generating the modified cells disclosed herein, and a method of treating a disease (e.g., cancer) in a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the modified cells described herein.

[0064] II. Definitions 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. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, but the preferred methods and materials are described below. It is also to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0065] In the practice of the present disclosure, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the skill of those of ordinary skill in the art are employed. For example, Green and Sambrook eds. (2012) Molecular Cloning: A Laboratory Manual, 4th edition; the series Ausubel et al. eds. (2015) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (2015) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; McPherson et al. (2006) PCR: The Basics (Garland Science); Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Greenfield ed. (2014) Antibodies, A Laboratory Manual; Freshney (2010) Culture of Animal Cells: A Manual of Basic Technique, 6th edition; Gait ed. (1984) Oligonucleotide Synthesis; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Herdewijn ed. (2005) Oligonucleotide Synthesis: Methods and Applications; Hames and Higgins eds. (1984) Transcription and Translation; Buzdin and Lukyanov ed.(2007) Nucleic Acids Hybridization: Modern Applications; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi ed. (2007) In Vitro Transcription and Translation Protocols, 2nd edition; Guisan ed. (2006) Immobilization of Enzymes and Cells; Perbal (1988) A Practical Guide to Molecular Cloning, 2nd edition; Miller and Calos eds., (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Lundblad and Macdonald eds. (2010) Handbook of Biochemistry and Molecular Biology, 4th edition; and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology, 5th edition. See also.

[0066] As used herein, each of the following terms has the meaning associated with it in this section. As used herein, the articles “a” and “an” refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, “an element” means one element or more than one element.

[0067] As used herein, each of the following terms has the meaning associated with it in this section. As used herein, the articles "a" and "an" refer to one or more than one (i.e., at least one) of the objects of the grammatical article. For example, "an element" means one element or more than one element.

[0068] As used herein, the term "about", when referring to a measurable value such as an amount, a temporal duration, etc., means an inclusion of a variation of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, and such variations are appropriate for carrying out the disclosed methods.

[0069] As used herein, the term "activation" refers to the state of a T cell that has received a stimulus sufficient to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector functions. As used herein, the term "activated T cell" refers, inter alia, to a T cell that is undergoing cell division.

[0070] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in mean survival, or an improvement in various physiological symptoms associated with the cancer condition. The "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present disclosure to prevent the occurrence of tumors in the first place.

[0071] As used herein, the term "autologous" is intended to refer to any material that is derived from the same individual and is later reintroduced into that individual.

[0072] As used herein, the terms "antigen" or "Ag" are defined as molecules that elicit an immune response. This immune response may involve the production of other antibodies, and / or the activation of specific immunocompetent cells, or both. One of skill in the art will understand that virtually any macromolecule, including substantially all proteins or peptides, can serve as an antigen. Additionally, an antigen can be derived from recombinant DNA or genomic DNA. One of skill in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will thus "encode an antigen" in the meaning of the term as used herein. Further, one of skill in the art will understand that an antigen need not necessarily be encoded by only the full-length nucleotide sequence of a gene. The present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and it will be readily apparent that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Moreover, one of skill in the art will understand that an antigen need not be encoded by a "gene" at all. It is visually readily understandable that an antigen can be produced synthetically or can be derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

[0073] As used herein, the term "allogeneic" refers to a graft derived from different animals of the same species.

[0074] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from a natural source or a recombinant source, or it can be an immunoreactive portion of an intact immunoglobulin. Antibodies are generally tetramers of immunoglobulin molecules. The antibodies in the present disclosure may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, F(ab)2, single-chain antibodies (scFv), and humanized antibodies. In some embodiments, an antibody refers to an assembly (e.g., an intact antibody molecule, an immunoadhesin, or a variant thereof) that has significant known specific immunoreactive activity against a target antigen (e.g., a tumor-associated antigen). Antibodies and immunoglobulins contain light and heavy chains, which may or may not have interchain disulfide bonds. The basic immunoglobulin structure in the vertebrate system is relatively well understood.

[0075] The term "antibody fragment" refers to a part of an intact antibody and also refers to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0076] As used herein, the term "antibody heavy chain" refers to the larger of the two polypeptide chains that exist in the native conformation in all antibody molecules.

[0077] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains that exist in the native conformation in all antibody molecules.

[0078] As used herein, the term "antibody variant" includes synthetic and engineered forms of an antibody that have been altered such that they do not occur naturally, examples of which include antibodies that contain at least two heavy chain moieties but do not contain two full-length heavy chains (such as domain-deleted antibodies or minibodies), multispecific forms of antibodies that have been altered to bind two or more different antigens or to bind different epitopes on a single antigen (e.g., bispecific, trispecific, etc.), heavy chain molecules conjugated to scFv molecules, and the like. Further, the term "antibody variant" includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind three, four, or more copies of the same antigen).

[0079] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells may spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colon cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

[0080] As used herein, the terms “cancer-related antigen” or “tumor antigen” interchangeably refer to a molecule (typically a protein, carbohydrate or lipid) that is expressed on the surface of cancer cells either as a whole or as a fragment (e.g., MHC / peptide) and that is useful for the preferential targeting of an agent with pharmacological activity to those cancer cells. In some embodiments, the tumor antigen is a marker expressed by both normal and cancer cells (e.g., a lineage marker such as CD19 on B cells). In some embodiments, the tumor antigen is a cell surface molecule that is overexpressed in cancer cells as compared to normal cells (e.g., 1-fold overexpression, 2-fold overexpression, 3-fold or greater overexpression as compared to normal cells). In some embodiments, the tumor antigen is a cell surface molecule that is inappropriately synthesized within cancer cells and contains, for example, deletions, additions or mutations as compared to the molecule expressed on normal cells. In some embodiments, the tumor antigen is expressed exclusively on the cell surface of cancer cells, either as a whole or as a fragment (e.g., MHC / peptide), and is not synthesized or expressed on the surface of normal cells. In some embodiments, the CARs of the present disclosure include CARs that include an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide. Peptides derived from endogenous proteins typically fill the pockets of major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8 + T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets in immunotherapy. TCR-like antibodies that target peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described. For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage display libraries.

[0081] In some embodiments, the tumor antigen is selected from the group consisting of tumor-associated carbohydrate antigen (TACA), alpha-fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-IX, CD2, CD3, CD4, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, Claudin 18.2, c-Met, DLL3, DRS, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRα) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Rα, IL13Rα2, LAGE-I, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin 4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP 1-6), NY-ESO-1, p16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof.

[0082] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell receptor that is expressed on an immune effector cell or its progenitor cell and is engineered to specifically bind an antigen. The CAR may be used in adoptive cell therapy by adoptive cell transfer. In some embodiments, adoptive cell transfer (or therapy) includes removing T cells from a patient and modifying the T cells to express a receptor specific for a particular antigen. In some embodiments, the CAR is specific for a selected target, such as a tumor-associated carbohydrate antigen (TACA). The CAR may also include an extracellular domain that includes an antigen-binding region, a transmembrane domain, and an intracellular activation domain.

[0083] As used herein, the term "costimulatory ligand" includes molecules on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.), which specifically bind to cognate costimulatory molecules on T cells, thereby providing a signal that mediates T cell responses including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, the binding of a peptide-loaded MHC molecule to the TCR / CD3 complex. Costimulatory ligands can include, but are not limited to, CD2, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands can also include, inter alia, antibodies that specifically bind to costimulatory molecules present on T cells, examples of which include, but are not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0084] As used herein, "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by that T cell, examples of which include, but are not limited to, proliferation. Costimulatory molecules are cell surface molecules that are neither antigen receptors nor their ligands and contribute to an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, Toll ligand receptors, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS (CD278), NKG2C, B7-H3 (CD276), and the intracellular domain derived from killer immunoglobulin-like receptors (KIR). In some embodiments, the costimulatory molecules include OX40, CD27, CD2, CD28, ICOS (CD278), and 4-1BB (CD137).Further examples of such costimulatory molecules include ligands that specifically bind to CD8, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0085] As used herein, the term "costimulatory signal" refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules. A costimulatory intracellular signaling domain may be the intracellular portion of a costimulatory molecule. Costimulatory molecules can be represented by the following protein families. They are TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activated NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD8, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and ligands that specifically bind to CD83, etc.

[0086] As used herein, the term "derived from" refers to the relationship between a first molecule and a second molecule. This defines the structural similarity between the first molecule and the second molecule and implies or includes no limitation on the process or source for the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from the CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure to have the required function (i.e., the ability to generate a signal under appropriate conditions). This implies or includes no limitation on the specific process for producing the intracellular signaling domain. It does not mean that one needs to start from the CD3 zeta sequence, delete unnecessary sequences, or introduce mutations to reach the intracellular signaling domain in order to provide the intracellular signaling domain.

[0087] As used herein, "disease" refers to the health state of an animal in which the animal is unable to maintain homeostasis and the health of the animal continues to deteriorate if the disease is not improved. In contrast, an "ailment" of an animal is a health state in which the animal can maintain homeostasis, but the health state of the animal is less favorable than in the absence of the ailment. The health state of the animal is not necessarily further reduced by the ailment even if left untreated.

[0088] As used herein, "disease associated with the expression of a tumor antigen" includes, but is not limited to, diseases associated with the expression of a tumor antigen or conditions associated with cells expressing the tumor antigen, including, but not limited to, proliferative diseases such as cancer or malignancy, or pre-cancerous conditions such as myelodysplasia, myelodysplastic syndrome or pre-leukemia; or non-cancer-related indications associated with cells expressing the tumor antigen. In some embodiments, the cancer associated with the expression of the tumor antigen is a blood cancer. In some embodiments, the cancer associated with the expression of the tumor antigen is a solid cancer. Further diseases associated with the expression of the tumor antigen include, but are not limited to, non-conventional and / or atypical cancers, malignancies, pre-cancerous conditions, or proliferative diseases associated with the expression of the tumor antigen. Non-cancer-related indications associated with the expression of the tumor antigen include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, the tumor antigen-expressing cells express mRNA encoding the tumor antigen or express at any point in time. In some embodiments, the tumor antigen-expressing cells produce a tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal or reduced levels. In some embodiments, the tumor antigen-expressing cells produce a detectable level of the tumor antigen protein at one point in time and then substantially no longer produce a detectable tumor antigen protein.

[0089] In some embodiments, the disease associated with abnormal glycosylation is cancer or an autoimmune disease. As used herein, the term "autoimmune disease" is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type I), dystrophic epidermolysis bullosa, orchitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis.

[0090] As used herein, the term "downregulation" refers to a decrease or elimination of gene expression of one or more genes.

[0091] As used herein, "effective amount" or "therapeutically effective amount" means an amount of a compound, formulation, material, pharmaceutical agent, or composition as described herein effective to achieve the desired physiological, therapeutic, or prophylactic result in a subject in need of such result. Such results can include, but are not limited to, an amount that causes a detectable level of immune response as compared to an immune response detected in the absence of the composition of the present disclosure when administered to a mammal. The immune response can be readily evaluated by a variety of methods recognized in the art. One of ordinary skill in the art will understand that the amount of the composition administered herein will vary and can be readily determined based on multiple factors such as the disease or condition being treated, the age of the mammal being treated, as well as the health and physical condition, the severity of the disease, the particular compound being administered, etc. The effective amount may vary for each subject depending on the health and physical condition of the subject being treated, the taxonomic group of the subject being treated, the formulation of the composition, the evaluation of the medical condition of the subject, and other relevant factors.

[0092] As used herein, the term "encode" refers to the inherent property of a specific sequence of nucleotides within a polynucleotide such as a gene, cDNA or mRNA, and serves as a template for the synthesis of other polymers and macromolecules in a biological process having either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, when a protein is produced in a cell or other biological system by transcription and translation of mRNA corresponding to a gene, that gene encodes the protein. Both the coding strand (the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (used as a template for transcription of the gene or cDNA) can be said to encode the protein or other product of that gene or cDNA.

[0093] As used herein, the term "endogenous" refers to any substance that is from within or produced within a living organism, cell, tissue or system.

[0094] As used herein, the term "epitope" is defined as a small chemical moiety on an antigen that can elicit an immune response and induce a B cell response and / or a T cell response. An antigen can have one or more epitopes. Most antigens have multiple epitopes; that is, they are multivalent. Generally, an epitope is on the order of approximately 10 amino acids and / or sugars in size. In certain exemplary embodiments, an epitope is about 4-18 amino acids, about 5-16 amino acids, about 6-14 amino acids, about 7-12 amino acids, or about 8-10 amino acids. Generally, the overall three-dimensional structure rather than the specific linear sequence of the molecule is the primary criterion for antigen specificity, and thus one of ordinary skill in the art understands that this overall three-dimensional structure distinguishes one epitope from another. Based on the present disclosure, the peptides used in the present disclosure can be used as epitopes. As used herein, the term "exogenous" refers to any substance that is introduced from outside of an organism, cell, tissue, or system or is produced outside of them.

[0095] As used herein, the term "expansion" as used herein refers to an increase in number, such as an increase in the number of immune cells (e.g., T cells). In some embodiments, the number of immune cells (e.g., T cells) expanded ex vivo increases compared to the number originally present in the culture. In another embodiment, the number of immune cells (e.g., T cells) expanded ex vivo increases compared to other cell types in the culture.

[0096] As used herein, the term "exogenous" refers to any substance that is introduced from outside of an organism, cell, tissue, or system or is produced outside of them.

[0097] As used herein, the term "expression" as used herein is defined as the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0098] As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains cis-acting elements sufficient for expression and other elements for expression can be supplied by the host cell or an in vitro expression system. Expression vectors include all those known in the art, examples of which include cosmids, plasmids (e.g., naked or contained in liposomes), and viruses incorporating recombinant polynucleotides (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0099] As used herein, the term "ex vivo" refers to cells removed from a living body (e.g., a human) and grown outside the living body (e.g., in a culture dish, test tube, or bioreactor).

[0100] As used herein, the term "Fc portion" or "Fc monomer" means, in the context of the present disclosure, a polypeptide comprising at least one domain having the function of the CH2 domain of an immunoglobulin molecule and at least one domain having the function of the CH3 domain. As is apparent from the term "Fc monomer", the polypeptide comprising these CH domains is a "polypeptide monomer". The Fc monomer can be a polypeptide comprising at least a fragment of the constant region of an immunoglobulin that excludes the first constant region immunoglobulin domain (CH1) of the heavy chain and maintains a functional portion of at least one CH2 domain and a functional portion of one CH3 domain (the CH2 domain being the amino terminus of the CH3 domain). In a preferred embodiment of this definition, the Fc monomer can be a polypeptide constant region comprising the CH2 region and the CH3 region, which are part of the Ig-Fc hinge region, and the hinge region is the amino terminus of the CH2 domain.

[0101] The hinge region of the present disclosure is assumed to promote dimerization. Such Fc polypeptide molecules can be obtained, for example, by papain digestion of the immunoglobulin region (of course, a dimer of two Fc polypeptides is formed). However, this is an example and not a limitation. In another aspect of this definition, the Fc monomer can be a polypeptide region comprising a part of the CH2 region and the CH3 region. Such Fc polypeptide molecules can be obtained by pepsin digestion of immunoglobulin molecules. However, this is an example and not a limitation. In one embodiment, the polypeptide sequence of the Fc monomer is substantially similar to the Fc polypeptide sequences of the IgG1 Fc region, IgG2 Fc region, IgG3 Fc region, IgG4 Fc region, IgM Fc region, IgA Fc region, IgD Fc region, and IgE Fc region. (See, for example, Padlan, Molecular Immunology, 31(3), 169-217(1993)). Since there are some variations among immunoglobulins, for simplicity, the Fc monomer refers to the last two heavy chain constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three heavy chain constant region immunoglobulin domains of IgE and IgM.

[0102] The Fc monomer can also include a flexible hinge at the N-terminus of these domains. In the case of IgA and IgM, the Fc monomer may include a J chain. In the case of IgG, the Fc portion includes the immunoglobulin domains CH2 and CH3, and a hinge between the first two domains and CH2. Although the boundaries of the Fc portion can vary, an example of the human IgG heavy chain Fc portion comprising a functional hinge, CH2, and CH3 domains can be defined to include the amino acid sequences of SEQ ID NOs: 79-84.

[0103] The IgG hinge region can be identified by analogy using Kabat. In one embodiment, the hinge domain / region of the present disclosure comprises amino acid residues corresponding to the IgG1 sequence stretch from D234 to P243 according to Kabat numbering. In some embodiments, the hinge domain / region of the present disclosure comprises or consists of the IgG1 hinge sequence DKTHTCPPCP (SEQ ID NO: 79 or 80). In one embodiment, the IgG1 hinge domain / region comprises the amino acid sequence of EPKSCDKTHTCPPCP (SEQ ID NO: 80). In a further embodiment of the present disclosure, the hinge domain / region comprises or consists of the IgG2 subtype hinge sequence ERKCCVECPPCP (SEQ ID NO: 81), the IgG3 subtype hinge sequence ELKTPLDTTHTCPRCP (SEQ ID NO: 82) or ELKTPLGDTTHTCPRCP (SEQ ID NO: 83), and / or the IgG4 subtype hinge sequence ESKYGPPCPSCP (SEQ ID NO: 84). In a further embodiment of the present disclosure, the IgG hinge domain / region comprises or consists of the hinge amino acid sequences disclosed in Table 2 or Table 3. In some embodiments, the fusion protein further comprises a third domain comprising two polypeptide monomers, each monomer comprising a hinge, a CH2 domain and a CH3 domain. In one embodiment, the third domain comprises, in amino to carboxyl order, hinge-CH2-CH3-linker-hinge-CH2-CH3. In one embodiment, the CH2 domain comprises an intra-domain cysteine disulfide bridge.

[0104] In another embodiment, two polypeptide monomers are fused to each other via a peptide linker. In yet another embodiment, the first domain and the second domain are fused to the third domain via a peptide linker. In some embodiments, the peptide linker of the fusion protein of the present disclosure comprises the amino acid sequence of GGGGS, i.e., Gly4Ser (SEQ ID NO: 72), or a polymer thereof, i.e., (Gly4Ser)n, where n is an integer of 5 or more (e.g., 5, 6, 7, 8, etc., or more). In some embodiments, the peptide linker of the fusion protein of the present disclosure comprises the amino acid sequences of SEQ ID NOs: 60-76.

[0105] As used herein, the term "homologous" refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When the positions of both of the two sequences being compared are occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is correlated with the value obtained by dividing the number of matching or homologous positions shared by the two sequences by the number of positions compared × 100. For example, if 6 out of 10 positions of two sequences are matching or homologous, the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, the comparison is made when the two sequences are aligned to obtain the maximum homology.

[0106] As used herein, the term "identity" refers to subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, such as between two polypeptide molecules. Two amino acid sequences are identical at a position if they have the same residue at the same position; for example, if each position in two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position within an alignment is often expressed as a percentage. The identity between two amino acid sequences is directly correlated with the number of matching or identical positions. For example, if half of the positions within two sequences (e.g., 5 positions within a polymer that is 10 amino acids in length) are identical, the two sequences are 50% identical, and if 90% of the positions (e.g., 9 out of 10) match or are identical, the two amino acid sequences are 90% identical.

[0107] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the major antibody present in body secretions, examples of which include saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and urogenital tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important in defense against bacteria and viruses. IgD is an immunoglobulin whose antibody function is not known but which may serve as an antigen receptor. IgE is an immunoglobulin that causes the release of mediators from mast cells and basophils upon exposure to allergens, resulting in immediate hypersensitivity.

[0108] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify an antigen molecule as foreign, induce the formation of antibodies, and / or activate lymphocytes to eliminate the antigen. As used herein, the term "immunostimulation" is used to refer to an increase in the overall immune response. As used herein, the term "immunosuppression" is used to refer to a decrease in the overall immune response.

[0109] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify an antigen molecule as foreign, induce the formation of antibodies, and / or activate lymphocytes to eliminate the antigen.

[0110] As used herein, the term "immune effector cell" refers to a cell that is involved in an immune response (e.g., in promoting an immune effector response). Examples of immune effector cells include T cells (e.g., alpha / beta T cells and gamma / delta T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0111] As used herein, the term "immune effector function" or "immune effector response" refers to a function or response that enhances or promotes an immune attack on a target cell. In some embodiments, the immune effector function or response refers to the property of T cells or NK cells that promotes the killing of target cells or the inhibition of growth or proliferation. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0112] As used herein, "explanatory materials" include publications, records, charts, or any other medium of expression that can be used to convey the usefulness of the disclosed compositions and methods. The explanatory materials of the kits of the present disclosure may, for example, be affixed to a container containing the nucleic acids, peptides, and / or compositions of the present disclosure, or shipped together with a container containing the nucleic acids, peptides, and / or compositions of the present disclosure. Alternatively, the explanatory materials may be shipped separately from the container, provided that the recipient is intended to use the explanatory materials in concert with the compound.

[0113] As used herein, the term "isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from the substances that coexist in the natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-natural environment such as, for example, a host cell.

[0114] In the context of the present disclosure, the following abbreviations are used for commonly occurring nucleobases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0115] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may include introns, provided that the nucleotide sequence encoding the protein may contain introns (s) in some version.

[0116] As used herein, the terms "lectin" or "hemagglutinin" refer to proteins or peptides that bind to sugar chain structures. A skilled artisan will understand that lectins are proteins or peptides that are highly specific for binding to sugar moieties. Lectins are sugar chain-binding proteins that are highly specific for sugar chains present on proteins and / or lipids, and cause aggregation of specific cells or precipitation of glycoconjugates and polysaccharides. Lectins play a role in recognition at the cellular and molecular levels and play numerous roles in biological recognition phenomena involving cells, sugar chains, and proteins. Lectins also mediate the attachment and binding of bacteria, viruses, and fungi to their targets. In one embodiment, a "lectin" can be defined as a non-immunoglobulin protein or glycoprotein that can specifically recognize and reversibly bind to the sugar chain portion of a complex glycoconjugate (protein and / or lipid) without altering any covalent structure of the recognized glycosyl ligand. Lectins are glycoproteins having a sugar chain-binding domain with the ability to reversibly bind to glycoproteins or glycolipids, as well as to specific sugar moieties within free monosaccharides and glycan structures. Many organisms express lectins or lectin-like biomolecules, but most of the scientifically important lectins identified recently have been purified from plant sources.

[0117] As used herein, the term "tumor-associated carbohydrate antigen" or "TACA" refers to sugar chain structures found in disorders associated with altered glycosylation (e.g., cancer). Carbohydrate-containing macromolecules (glycans) are ubiquitous in biological systems and are essential for many biological functions. Sugar chains can attach to proteins (glycoproteins), lipids (glycolipids), and exist as chains of sugar chains (glycosaminoglycans). Changes in the structure of these carbohydrate-containing macromolecules (glycosylation) have a major impact on cancer biology and cancer progression. Indeed, altered glycosylation is a common feature of tumor cells and leads to the formation of tumor-associated carbohydrate antigens (TACAs). Tumor cells can often be distinguished from normal cells because they display abnormal levels and types of sugar chain structures on their surface.

[0118] Three common changes in glycoprotein-containing polymers, namely increased expression of truncated or incomplete glycans, increased branching of N-glycans, and increased or altered presence of sialic acid-containing glycans, are associated with cancer. For example, in cancer-related glycans, the amount of sialic acid is often increased, and this hyper-sialylation enhances the activation of sialic acid-binding receptors such as selectins and siglecs, leading to cancer progression. Another of the most common cancer-related changes in glycosylation is the cleavage of O-linked glycan chains such as mucins (O-glycoprotein cleavage). Under normal conditions, N-acetylgalactosamine (GalNAc) sugar residues attach to serine or threonine of glycoproteins (GalNAcα1-O-Ser / Thr, Tn antigen), and are usually elongated by T-synthase (core 1 β3-galactosyltransferase) in the Golgi apparatus that attaches galactose residues to the Thomsen-Friedenreich (TF) antigen (Tn antigen). In various cancers, this process changes, the glycosylation of the Tn antigen or its sialylated form (sialyl-Tn (STn) antigen) changes, and truncated T, Tn, and STn antigens are produced. Furthermore, increased branching of N-glycoproteins that stimulate galectin-3, and changes in glycolipids such as gangliosides (GM3, GM2, CD3, and GD2) have also been observed.

[0119] The following TACAs have been observed in various cancers. That is, (i) H / Le y / ILe a in primary non-small cell lung carcinomas, (ii) sialyl-Le x (SLe x ) and sialyl-Lea (SLea) in various types of cancers, (iii) Tn and sialyl-Tn in colorectal cancer, lung cancer, breast cancer, and many other cancers, (iv) GM2, GD2, and GD3 gangliosides in neuroectodermal tumors (melanoma and neuroblastoma), (v) globo-H in breast cancer, ovarian cancer, and prostate cancer, (vi) disialyl galactosyl globoside in renal cell carcinomas.

[0120] Accordingly, the term "tumor-associated carbohydrate antigen (TACA)" encompasses all altered glycan structures on proteins and / or lipids that are expressed on tumor cells and promote cancer metastasis, cancer progression, cancer and non-cancer immunosuppression, and / or promote autoimmune disorders. A skilled artisan will understand that a glycan structure is composed of one or more linked sugars or monosaccharides. See, for example, Mantuano et al, J. Immunotherapy Cancer 8(2): 8:e001222 (2020); Hakomori, Si. (2001). Tumor-Associated Carbohydrate Antigens Defining Tumor Malignancy: Basis for Development of Anti-Cancer Vaccines, in The Molecular Immunology of Complex Carbohydrates-2. Advances in Experimental Medicine and Biology, vol 491. Springer, Boston, MA (Wu et al (eds)). A skilled artisan will understand that a glycan structure can exist independently of and / or attach to proteins or lipids known as glycoproteins and glycolipids. A skilled artisan will understand that these glycan structures bind to lectins.

[0121] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells and can deliver large amounts of genetic information into the DNA of host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means of achieving significant levels of gene transfer in vivo.

[0122] As used herein, the term "limited toxicity" means that the peptides, polynucleotides, cells and / or antibodies of the present disclosure do not exert a substantially negative biological effect, anti-tumor effect, or substantially negative physiological symptom on healthy cells, non-tumor cells, non-disease cells, non-target cells or populations of such cells, either in vitro or in vivo.

[0123] As used herein, the term "flexible polypeptide linker" or "linker" when used in the context of an scFv refers to a peptide linker composed of amino acids such as glycine and / or serine residues used alone or in combination to link the variable heavy chain region and the variable light chain region together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and includes the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer of 1 or more. For example, n = 1, n = 2, n = 3, n = 4, n = 5, n = 6, n = 7, n = 8, n = 9, n = 10 (SEQ ID NO: 6592). In one embodiment, the flexible polypeptide linker includes, but is not limited to, (Gly4 Ser)4 or (Gly4 Ser)3. In another embodiment, the linker includes multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser).

[0124] As used herein, the term "modification" means an altered state or structure of a molecule or cell of the present disclosure. A molecule may be modified in many ways, including chemical, structural and functional modifications. A cell may be modified by introduction of nucleic acids.

[0125] As used herein, the term "modulate" means to mediate a detectable increase or decrease in the response level in a subject as compared to the response level in the subject in the absence of treatment or compound and / or as compared to the response level in an otherwise identical but untreated subject. This term encompasses disrupting and / or affecting the original signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0126] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may include introns.

[0127] As used herein, the term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, are in the same reading frame.

[0128] As used herein, the term "overexpressed tumor antigen" or "overexpression of a tumor antigen" is intended to indicate that the expression level of a tumor antigen in cells of a disease area, such as a solid tumor in a patient's specific tissue or organ, is abnormal as compared to the expression level in normal cells of that tissue or organ. A patient having a solid tumor or hematological malignancy characterized by overexpression of a tumor antigen can be determined by standard assays known in the art.

[0129] As used herein, the term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.) injection, intravenous (i.v.) injection, intramuscular (i.m.) injection or intrasternal injection, or infusion techniques.

[0130] As used herein, terms such as "patient", "subject" and "individual" are used interchangeably herein and refer to any animal or its cells suitable for the methods described herein, whether in vitro or in situ. The subject can be a mammal, examples of which include non - primates (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or primates (e.g., monkeys and humans). In certain embodiments, the term "subject" as used herein refers to a vertebrate such as a mammal. Mammals include, but are not limited to, humans, non - human primates, wild animals, feral animals, livestock, sport animals, pets, etc. Any organism capable of eliciting an immune response may also be a subject or patient. In certain exemplary embodiments, the subject is a human.

[0131] As used herein, the term "polynucleotide" as used herein is defined as a chain of nucleotides. Further, a nucleic acid is a polymer of nucleotides. Thus, the nucleic acids and polynucleotides as used herein are interchangeable. One of ordinary skill in the art has the general knowledge that a nucleic acid is a polynucleotide and can be hydrolyzed to monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., using conventional cloning techniques and PCR™, etc., and synthetic means, including cloning of nucleic acid sequences from recombinant libraries or cell genomes, but are not limited thereto.

[0132] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can be included in the sequence of the protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, this term refers to both short chains, which in the art are generally also referred to as peptides, oligopeptides and oligomers, and long chains, which in the art are generally referred to as proteins and which come in many types. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0133] As used herein, the term "promoter" as used herein is defined as a DNA sequence recognized by the cell's synthetic apparatus, or an introduced synthetic apparatus, necessary to initiate specific transcription of a polynucleotide sequence.

[0134] As used herein, the term "promoter" or " / regulatory sequence" means a nucleic acid sequence necessary for the expression of a gene product operably linked to the "promoter" or "regulatory sequence". In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include enhancer sequences and other regulatory elements necessary for the expression of the gene product. The promoter / regulatory sequence may, for example, express the gene product in a tissue-specific manner.

[0135] As used herein, a "constitutive promoter" is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced intracellularly under most or all physiological conditions of the cell.

[0136] As used herein, an "inducible promoter" is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced intracellularly only when an inducer substantially corresponding to the promoter is present intracellularly.

[0137] As used herein, a "tissue-specific promoter" is a nucleotide sequence that, when operably linked to a polynucleotide encoded or specified by a gene, causes the gene product to be produced intracellularly only when the cell is substantially a cell of the tissue type corresponding to the promoter.

[0138] As used herein with respect to an antibody, antigen-binding domain, CAR or bispecific fusion protein, the terms "specifically binds" or "selectively binds" mean an antibody, antigen-binding domain, CAR or bispecific fusion protein that recognizes a specific antigen (e.g., TACA) but does not substantially recognize or bind other molecules in a sample. For example, an antibody, antigen-binding domain, CAR or bispecific fusion protein that specifically binds to a particular antigen (e.g., TACA) may also bind to one or more species of that antigen. However, such cross-reactivity per se does not change the classification of the antibody as specific. In another example, an antibody, antigen-binding domain, CAR or bispecific fusion protein that specifically binds to an antigen (e.g., TACA) may also bind to different allelic forms of that antigen (e.g., TACA). However, such cross-reactivity per se does not change the classification of the antibody, antigen-binding domain, CAR or bispecific fusion protein as specific. In some instances, the terms "specific binding" or "specifically binds" are used with respect to the interaction of an antibody, protein, peptide, antigen-binding domain, CAR or bispecific fusion protein with a second chemical species and can mean that the interaction depends on the presence of a particular structure (e.g., antigen determinant or epitope) with respect to the chemical species. For example, an antibody, antigen-binding domain, CAR or bispecific fusion protein generally recognizes and binds a specific protein structure (TACA) rather than a protein per se. If an antibody, antigen-binding domain, CAR or bispecific fusion protein is specific for epitope "A", in a reaction containing label "A" and the antibody, antigen-binding domain, CAR or bispecific fusion protein, the amount of labeled A bound to the antibody may decrease if a molecule containing epitope A (or free, unlabeled A) is present.

[0139] As used herein, the term "single-chain antibody" refers to an antibody formed by recombinant DNA techniques in which immunoglobulin heavy and light chain fragments are linked to the Fv region via an engineered range of amino acids. A variety of methods for generating single-chain antibodies are known, including those described in U.S. Patent No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Ward et al., Nature 334:544-54 (1989); Skerra et al., Science 242:1038-1041 (1988).

[0140] As used herein, the term "specificity" refers to the ability to specifically bind (e.g., immunoreact with) a given target antigen (e.g., a human target antigen). A chimeric antigen receptor can be monospecific, containing one or more binding sites that specifically bind a target, or a chimeric antigen receptor can be multispecific, containing two or more binding sites that specifically bind the same or different targets. In certain embodiments, the chimeric antigen receptor is specific for two different (e.g., non-overlapping) portions of the same target. In certain embodiments, the chimeric antigen receptor is specific for more than one target.

[0141] As used herein, the term "specifically binds" with respect to an antibody means an antibody or a binding fragment thereof (e.g., scFv) that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to a certain antigen may also bind to that antigen of one or more species. However, such cross-reactivity per se does not change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity per se does not change the classification of the antibody as specific. In some instances, the terms "specific binding" or "specifically binds" are used with respect to the interaction of an antibody, protein, chimeric antigen receptor or peptide with a second chemical species, and can mean that the interaction depends on the presence of a particular structure (e.g., antigen determinant or epitope) with respect to the chemical species. For example, a chimeric antigen receptor generally recognizes and binds a specific protein structure rather than a protein. If an antibody is specific for epitope "A", in a reaction containing label "A" and the antibody, the amount of labeled A bound to the antibody will decrease if a molecule containing epitope A (or free unlabeled A) is present.

[0142] As used herein, the term "stimulate" means a primary response induced by binding a stimulatory molecule (e.g., TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, examples of which include, but are not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate a change in the expression of specific molecules, examples of which include downregulation of TGF-β, and / or rearrangement of the cytoskeletal structure, clonal expansion, and differentiation into different subsets.

[0143] As used herein, the term "stimulatory molecule" means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0144] As used herein, the term "stimulatory ligand" means a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), specifically binds to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby being able to mediate a primary response by the T cell, which primary response includes, but is not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, inter alia, MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0145] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cell types with which it is normally associated in its natural state. In some instances, a population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to cells that have been separated from the cell, which are naturally associated with the cell in its natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0146] As used herein, a "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.

[0147] As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. The TCR plays a role in recognizing antigens bound to major histocompatibility complex molecules. The TCR is composed of a heterodimer of an alpha (α) chain and a beta (β) chain coupled to three dimeric modules, CD3δ / CD3ε, CD3γ / CD3ε, and CD3ζ / CD3ζ. In some cells, the TCR is composed of a gamma chain and a delta (γ / δ) chain (CD3γ / CD3ε). The TCR may exist in alpha / beta and gamma / delta forms, which are structurally similar but have different anatomical locations and functions. Each chain is composed of two extracellular domains, namely a variable domain and a constant domain. In some embodiments, the TCR may be modified on any cell containing the TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells.

[0148] As used herein, the term "T cell exhaustion" or "exhaustion" refers to a decrease in T cell function that can occur as a result of infection (e.g., chronic infection) or disease. T cell exhaustion is associated with increased expression of PD-1, TIM-3, and LAG-3, apoptosis, and decreased cytokine secretion. Thus, terms such as "ameliorating T cell exhaustion," "suppressing T cell exhaustion," "reducing T cell exhaustion," etc. refer to a state in which the functionality of T cells is restored, which functionality is characterized by a decrease in expression and / or one or more levels of PD-1, TIM-3, and LAG-3; an increase in the formation of memory cells and / or the maintenance of memory markers (e.g., CD62L); prevention of apoptosis; an increase in the production and / or secretion of cytokines (e.g., IL-2) induced by antigen; enhancement of cytotoxic / killing ability; increased recognition of tumor targets with low surface antigens; and enhanced proliferation in response to antigen.

[0149] As used herein, the term "treatment" means treatment and / or prevention. A therapeutic effect is obtained by suppression, remission or eradication of a medical condition.

[0150] The term "therapeutically effective amount" or "effective amount" refers to the amount of a subject compound that induces a biological or medical response in a tissue, system or subject sought by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of a compound that, when administered, is sufficient to prevent or to some extent alleviate the onset of one or more signs or symptoms of a disorder or disease in a subject being treated. A therapeutically effective amount will vary depending on the compound, the disease and its severity, the age, weight, etc. of the patient being treated.

[0151] As used herein, the term "treatment" refers to any protocol, method and / or agent (e.g., CAR-T) that can be used for the prevention, management, treatment and / or amelioration of a disease or symptoms associated therewith. In some embodiments, the terms "therapy" and "therapies" refer to biological therapies (e.g., adoptive cell therapy), supportive therapies (e.g., lymphodepletion therapy), and / or other therapies known to those of skill in the art, such as medical personnel, useful for the prevention, management, treatment and / or amelioration of a disease or symptoms associated therewith.

[0152] As used herein, the terms "treating," "treatment," and "treatment thereof" refer to a decrease or improvement in the progression, severity, frequency, and / or duration of a disease or a symptom associated therewith that results from the administration of one or more therapies (including, but not limited to, CAR-T therapy directed to the treatment of solid tumors). The term "treatment" as used herein can also refer to changing the course of a disease in a subject being treated. The therapeutic effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of a disease, alleviation of symptoms, reduction of direct or indirect pathological consequences of a disease, reduction in the rate of disease progression, improvement of a medical condition or pain relief, and remission or improvement of prognosis. In some embodiments, "treatment" of a disease as the term is used herein means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0153] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is introduced or transferred into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. This cell includes primary subject cells and their progeny.

[0154] As used herein, the phrases "under transcriptional control" or "operably linked" mean that a promoter is in the correct position and orientation with respect to a polynucleotide and controls the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0155] As used herein, "vector" is a composition of substances that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell. A number of vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes self-replicating plasmids or viruses. This term should also be construed to include non-plasmid and non-viral compounds that facilitate the entry of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0156] As used herein, the term "heterologous" refers to a graft derived from animals of different species.

[0157] Range: Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, a description of a range should be considered to specifically disclose not only the individual numerical values within that range but also all possible sub-ranges. For example, a description of a range such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and further individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0158] III. TACA Antigen-Binding Domain One aspect of the present disclosure provides a chimeric antigen receptor that selectively binds to a tumor-associated carbohydrate antigen (TACA). In some embodiments, the CAR comprises an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA) and that includes one or more TACA-binding domains derived from a lectin, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain. In some embodiments, when the CAR is introduced into a modified cell, the modified cell expressing the CAR is less susceptible to the effects of CAR-induced tonicity, thereby reducing exhaustion and / or inflammatory cytokine production in the absence of target cancer cells.

[0159] Another aspect of the present disclosure provides a chimeric antigen receptor that selectively binds to a tumor-associated carbohydrate antigen (TACA), the chimeric antigen receptor comprising an antigen-binding domain that has a deletion and that has an amino acid sequence set forth in SEQ ID NOs: 30-54 or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the amino acid sequence set forth in SEQ ID NOs: 30-54, a CD8 or CD28 hinge domain, a CD8 or CD28 transmembrane domain, a CD28 co-stimulatory domain and / or a 4-1BB co-stimulatory domain, and a CD3 zeta intracellular signaling domain.

[0160] Malignant transformation of cells is almost invariably associated with abnormal glycosylation of cell surface proteins or lipids (Kim and Varki, 1997, Glycoconj J, 14:569-576). Indeed, changes in cell surface glycosylation have been observed in all types of experimental and human cancers (Hakomori, 2002, PNAS USA, 99:10231-10233), and these altered sugar structures are called tumor-associated carbohydrate antigens (TACAs) (Table 1). Due to this tumor-specific property, cell surface TACAs are excellent target antigens for the production of monoclonal antibodies targeting many common cancers. However, despite decades of effort, specific antibodies with high affinity for TACAs are not yet available because it is difficult to generate antibodies against carbohydrate antigens.

[0161] Therefore, the inventors of the present disclosure engineered therapeutic bispecific fusion proteins and CARs that selectively target TACAs on tumor cells, relying on lectins rather than antibodies. Lectins and their binding partners are well known in the art. See, for example, functionalglycomics.org / glycomics / publicdata / primaryscreen.jsp. The lectin-binding proteins of the present disclosure have significant advantages over existing technologies (e.g., GlyTR chimeric proteins) and provide opportunities for the development of a new class of therapeutics for cancer immunotherapy. Based on the concept of GlyTR and the availability of numerous different lectins specific for a multitude of different TACAs, multiple GlyTRs can be generated by replacing L-PHA with other lectins. Alternatively, chimeric proteins composed of lectins and scFvs that can recruit other immune effector cells can also be produced. The functionality of the lectin domain can be improved by mutagenesis. For example, GlyTR L-PHA x CD3 can be further improved by exchanging the sugar-binding domain of E-PHA with L-PHA, resulting in a 20- to 30-fold increase in binding (Kaneda et al., 2002, J Biol Chem, 277:16928-16935).

Table 1

[0162] The antigen-binding domain of the CAR disclosed in this specification is designed to specifically target glycoproteins and / or glycolipids (i.e., carbohydrate-containing macromolecules) on tumor cells. In some embodiments, the CAR of the present disclosure comprises an affinity for a target antigen (e.g., a tumor-associated carbohydrate antigen) on a target cell (e.g., a cancer cell). The target antigen may comprise any type of protein or epitope thereof that associates with the target cell. For example, the CAR may comprise an affinity for a target antigen on a target cell that indicates a particular state of the target cell.

[0163] In some embodiments, the antigen-binding domain comprises a plurality of (e.g., more than one) TACA-binding domains. In some embodiments, the antigen-binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more TACA-binding domains. In one embodiment, the antigen-binding domain comprises one TACA-binding domain. In one embodiment, the antigen-binding domain comprises two TACA-binding domains. In one embodiment, the antigen-binding domain comprises three TACA-binding domains. In one embodiment, the antigen-binding domain comprises four TACA-binding domains.

[0164] A. TACA In some embodiments, the TACA-binding domain is derived from a lectin. The TACA-binding domain may comprise any peptide, protein, lectin, lectin fragment, antibody, antibody fragment, small molecule, nucleic acid, etc. that can specifically bind to TACA. In some embodiments, the antigen-binding domain selectively targets β1,6GlcNAc-branched N-glycan, Tn epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), GalNAcα-serine, GalNAcα-threonine, GalNAc or GalNAcβ1.

[0165] Exemplary TACAs and their binding partners are listed in Table 1. Exemplary TACAs include, but are not limited to, β1,6 branching, T antigen, sialyl-T epitope, Tn epitope, sialyl-Tn epitope, a2,6 sialylation, sialylation, sialyl-Lewis x , Lewis-y(Le y ), Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GM1. In some embodiments, the CAR or bispecific fusion is selectively targeted to a TACA selected from the group consisting of β1,6 branching, β1,6GlcNAc-branched N-glycan, T antigen, Tn antigen, sialyl-T epitope, Tn epitope, sialyl-Tn epitope, α2,6 sialylation, sialylation, sialyl-Lewis x / a , disialyl-Lewis x / a , sialyl 6-sulfo Lexis x , Lewis-y(Le y ), Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GM1. In some embodiments, the CAR selectively targets β1,6GlcNAc-branched N-glycan, GalNAc, Tn antigen, GalNAcα-ser, GalNAc, or GalNAcβ1.

[0166] In one embodiment, the TACA binding domain binds to N-glycan. In certain embodiments, the TACA binding domain binds to tri- and tetra-branched oligosaccharides. In one embodiment, the TACA binding domain binds to β1,6GlcNAc-branched N-glycan. In one embodiment, the TACA binding domain binds to the Tn epitope.

[0167] B. Lectin In certain embodiments, the TACA binding domain is a peptide sequence derived from a lectin protein. In certain embodiments, the lectin is selected from the group consisting of mammalian lectins, human lectins, plant lectins, bacterial lectins, viral lectins, fungal lectins, and protozoan lectins. In some embodiments, the antigen-binding domain comprises a TACA binding domain derived from a lectin. In some embodiments, the antigen-binding domain comprises at least two TACA binding domains from a lectin.

[0168] In some embodiments, the lectin is selected from the group consisting of galectin, siglec, selectin; C-type lectin; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Phaseolus vulgaris leukocyte agglutinin); E-PHA (Phaseolus vulgaris erythroagglutinin); tomato lectin (Lycopersicon esculentum lectin; LEA); peanut lectin (Arachis hypogaea lectin; PNA); potato lectin (Solanum tuberosum lectin), pokeweed mitogen (Phytolacca americana lectin), wheat germ agglutinin (Triticum aestivum germ lectin); Artocarpus polyphyllus lectin (jacalin lectin); hairy bittercress lectin (VVA); apple snail lectin (HPA); fucose lectin (WFA); Sambucus nigra lectin (SNA), BC2L-CNt (lectin from the Gram-negative bacterium Burkholderia cenocepacia), dogwood leukocyte agglutinin (MAL), Pleurotus ostreatus (PVL), Sclerotium rolfsii lectin (SRL), Echinops sphaerocephalus lectin (ESA), CLEC17A (prolectin), Hericium erinaceus lectin, Allium sativum lectin (SSA), Glechoma hederacea lectin (Gleheda), Momordica charantia lectin (Morniga G), Salvia officinalis lectin, Salvia bogotensis lectin, Salvia horminum lectin, Cuscutaceae lectin, Salvia virgata lectin, Glyphomia simplificifolia (GsLA4), Vicia faba (acidic WBAI), Vigna angularis lectin, Apios americana lectin, Amaranthus leucocarpus lectin, Ralstonia autumnalis lectin, Paramicia lectin, Utricularia vulgaris lectin, Artocarpus lakoocha lectin, Himalayan Vicia faba agglutinin, Himalayan Vicia faba lectin, soybean lectin and mushroom lectin.

[0169] In some embodiments, the lectin can be a galectin selected from the group consisting of galectin-1, galectin-2, galectin-3, galectin-4, galectin-5, galectin-6, galectin-7, galectin-8, galectin-9, galectin-10, galectin-11, galectin-12, galectin-13, galectin-14, and galectin-15. In some embodiments, the lectin can be a siglec selected from the group consisting of siglec-1 (sialoadhesin), siglec-2 (CD22), siglec-3 (CD33), siglec-4 (myelin-associated glycoprotein), siglec-5, siglec-6, siglec-7, siglec-8, siglec-9, siglec-10, siglec-11, siglec-12, siglec-13, siglec-14, siglec-15, siglec-16, siglec-17, siglec-E, siglec-F, siglec-G, and siglec-H. In some embodiments, the TACA-binding domain is derived from a selectin or a C-type lectin.

[0170] In some embodiments, the lectin is a polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) that can be selected from the group consisting of ppGalNAc-T1 (GALNT1), ppGalNAc-T2 (GALNT2), ppGalNAc-T3 (GALNT3), ppGalNAc-T4 (GALNT4), ppGalNAc-T5 (GALNT5), ppGalNAc-T6 (GALNT6), ppGalNAc-T7 (GALNT7), ppGalNAc-T8 (GALNT8), ppGalNAc-T9 (GALNT9), ppGalNAc-T10 (GALNT10), ppGalNAc-T12 (GALNT12), ppGalNAc-T13 (GALNT13), ppGalNAc-T14 (GALNT14), ppGalNAc-T15 (GALNT15), ppGalNAc-T16 (GALNT16), ppGalNAc-T17 (GALNT17), ppGalNAc-T18 (GALNT18), ppGalNAc-T Like 5 (GALNTL5), and ppGalNAc-T Like 6 (GALNTL6).

[0171] In some embodiments, the antigen-binding domains of the CARs or bispecific fusion proteins described herein are β1,6-branched, β1,6GlcNAc-branched N-glycans, T antigen, Tn antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6-sialylation, sialylation, sialyl-Lewisx / a, disialyl-Lewisx / a, sialyl 6-sulfo Lexis x , Lewis-y (Le y ), Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GM1, and selectively target TACAs selected from the group consisting of.

[0172] C. Chimeric Antigen Receptor One aspect of the present disclosure provides chimeric antigen receptors (CARs) having affinity for tumor-associated carbohydrate antigens (TACAs), modified cells comprising CARs, modified immune cells or progenitor cells thereof, and compositions and methods for using the same. The CARs of interest in the present disclosure include an antigen-binding domain (e.g., a tumor-associated carbohydrate antigen (TACA)), a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain. The CARs of interest in the present disclosure may optionally include a hinge domain. Thus, the CARs of interest in the present disclosure include an antigen-binding domain (e.g., a TACA-binding domain), a hinge domain, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain. In some embodiments, each domain of the CAR of interest is separated by a linker. The CARs of interest in the present disclosure are mutated to prevent CAR-induced T cell exhaustion caused by T cell exhaustion. In some embodiments, the antigen-binding domain of the CAR is mutated to regulate CAR signaling.

[0173] In some embodiments, the CAR comprises the amino acid sequences of SEQ ID NOs: 23-29. In some embodiments, modified cells comprising the CARs of SEQ ID NOs: 23-29 exhibit reduced tonic signaling as compared to modified cells comprising the CARs of SEQ ID NO: 21 or 22. In some embodiments, modified cells expressing a CAR comprising a deletion in the amino acid sequence set forth in SEQ ID NOs: 30-54, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 30-54 are less susceptible to exhaustion as compared to modified cells comprising a CAR comprising a wild-type antigen-binding domain. In some embodiments, modified cells expressing a CAR comprising a deletion in the amino acid sequence set forth in SEQ ID NOs: 30-54, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 30-54 are less susceptible to exhaustion associated with tonic signaling TACA CAR as compared to modified cells comprising a CAR comprising a wild-type antigen-binding domain. Another aspect of the present disclosure provides a chimeric antigen receptor expressed in the modified cells disclosed herein.

[0174] In some embodiments, the chimeric antigen receptor (CAR) is β1,6 branched, β1,6GlcNAc branched N-glycan, T antigen, Tn antigen, sialyl-T epitope, Tn epitope, sialyl-Tn epitope, α2,6 sialylation, sialylation, sialyl-Lewis x / a , disialyl-Lewis x / a , sialyl 6-sulfo Lexis x , Globo H, GD2, GD3, GM3 or fucosyl GM1. In some embodiments, the chimeric antigen receptor (CAR) has an affinity for β1,6 branched or β1,6GlcNAc branched N-glycan. In some embodiments, the chimeric antigen receptor (CAR) has an affinity for Tn antigen or sialyl-Tn epitope.

[0175] 1. Antigen-binding domain The antigen-binding domain of the CAR is the extracellular region of the CAR for binding to a specific target antigen including proteins, glycans and glycolipids. In some embodiments, the CAR includes an affinity for a target antigen (e.g., a tumor-associated antigen) on a target cell (e.g., a cancer cell). The target antigen may include any type of protein or its epitope that associates with the target cell. For example, the CAR may include an affinity for a target antigen on a target cell that indicates a specific state of the target cell.

[0176] In some embodiments, the antigen-binding domain includes more than one TACA-binding domain. In some embodiments, the antigen-binding domain includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 TACA-binding domains. In one embodiment, the antigen-binding domain includes two TACA-binding domains. In one embodiment, the antigen-binding domain includes three TACA-binding domains. In one embodiment, the antigen-binding domain includes four TACA-binding domains. The TACA-binding domain may include any peptide, protein, lectin, lectin fragment, antibody, antibody fragment, small molecule, nucleic acid, etc. that can specifically bind to TACA.

[0177] In some embodiments, the antigen-binding domain comprises a mutation in the tumor-associated carbohydrate antigen (TACA)-binding domain (TBD) selected from a substitution, deletion, or insertion.

[0178] In some embodiments, the antigen-binding domain comprises a deletion in the TACA-binding domain (TBD). In one embodiment, the deletion is present in the N-terminal region and / or C-terminal region of the TACA-binding domain (TBD). In one embodiment, the deletion is at least about 2 amino acids, at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 36 amino acids, at least about 38 amino acids, at least about 40 amino acids, at least about 45 amino acids, or more. In one embodiment, the deletion is at least about 10 amino acids, at least about 18 amino acids, or at least about 36 amino acids. In one embodiment, the deletion is at least about 36 amino acids.

[0179] In some embodiments, the antigen-binding domain comprises a deletion in the TACA-binding domain (TBD), the deletion is present at the N-terminus of the TBD and is at least about 18 amino acids. In some embodiments, the antigen-binding domain comprises a deletion in the TACA-binding domain (TBD), the deletion is present at the C-terminus of the TBD and is at least about 10 amino acids. In some embodiments, the antigen-binding domain comprises a deletion in the TACA-binding domain (TBD), the deletion is present at the N-terminus of the TBD and is at least about 36 amino acids. In some embodiments, the antigen-binding domain comprises a deletion in the TACA-binding domain (TBD), the deletion is present at the N-terminus of the TBD and is at least about 18 amino acids and is present at the C-terminus and is at least about 10 amino acids.

[0180] In some embodiments, the antigen-binding domain comprises a deletion that removes a disulfide-bond cysteine residue in the tumor-associated carbohydrate antigen (TACA)-binding domain (TBD). In some embodiments, the expression of the chimeric antigen receptor (CAR) comprising a deletion in the TACA-binding domain (TBD) of the antigen-binding domain is similar to the expression of a CAR comprising a wild-type TBD.

[0181] In some embodiments, the antigen-binding domain comprises a deletion in the carbohydrate-binding domain of the TACA-binding domain, wherein the antigen-binding domain has an amino acid sequence set forth in SEQ ID NOs: 30-54, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 sequence set forth in SEQ ID NOs: 30-54.

[0182] In some embodiments, the antigen-binding domain comprises an amino acid sequence set forth in SEQ ID NOs: 34-39, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 sequence set forth in SEQ ID NOs: 34-39.

[0183] In some embodiments, the antigen-binding comprises an amino acid sequence having at least 90% homology with SEQ ID NOs: 34-39. In some embodiments, the antigen-binding domain comprises an amino acid sequence disclosed in Table 2 or Table 3.

[0184] The antigen-binding domain may be operably linked to another domain of the CAR, such as a transmembrane domain, a co-stimulatory signaling domain, or an intracellular signaling domain (each described elsewhere in this specification), for expression within a cell. The antigen-binding domains described herein can be combined with any of the transmembrane domains, any of the co-stimulatory signaling domains, any of the intracellular signaling domains, or any of the other domains described herein and that can be included in the CARs of the present disclosure.

[0185] 2. Linker As used herein, the terms “linker” and “spacer” are used interchangeably. Linkers generally contain a high abundance of glycine to increase flexibility and a high abundance of serine or threonine to increase solubility. Multiple linkers may be used to connect more than one TACA-binding domain. In some embodiments, for example, more than one TACA-binding domain can be operably linked by a linker such as a linker that can be selected from the group consisting of a peptide linker, a non-peptide linker, a chemical unit, a disulfide crosslinking linker, a non-disulfide crosslinking linker. In one embodiment, the linker is a peptide linker. The peptide linker can be a glycine-serine linker. The peptide linker can have a length of at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, or at least about 15 amino acids.

[0186] A variety of linker sequences are known in the art, and non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917(2008) and WO2014 / 087010, the contents of which are hereby incorporated by reference in their entirety. One of ordinary skill in the art will be able to select an appropriate linker sequence for use in the bispecific fusion proteins and / or CARs of the present disclosure.

[0187] In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO:71, or the amino acid sequence of SEQ ID NO:75.

[0188] 3. Transmembrane domain Regarding the transmembrane domain, the CARs of the present disclosure (e.g., TACA CAR) can be designed to include a transmembrane domain that connects the antigen-binding domain of the CAR to the intracellular domain. The transmembrane domain of a target CAR is a region that can span the cell membrane of a cell (e.g., an immune cell or its precursor). The transmembrane domain is for the purpose of insertion into the cell membrane (e.g., a eukaryotic cell membrane). In some embodiments, the transmembrane domain intervenes between the antigen-binding domain and the intracellular domain of the CAR.

[0189] In one embodiment, the transmembrane domain is naturally associated with one or more of the domains within the CAR. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid the binding of such a domain to the transmembrane domain of the same or different surface membrane proteins and minimize interactions with other members of the receptor complex.

[0190] The transmembrane domain can be derived from either a natural source or a synthetic source. If the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein (e.g., a type I transmembrane protein). If the source is synthetic, the transmembrane domain can be any artificial sequence that facilitates the insertion of the CAR into the cell membrane, such as an artificial hydrophobic sequence.

[0191] In some embodiments, the chimeric antigen receptor (CAR) comprises a transmembrane domain, which transmembrane domain may comprise the transmembrane region of a molecule selected from the group consisting of T cell receptor (TCR)-alpha, TCR-beta, CD3-zeta, CD3-epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134 (Ox40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In one embodiment, the transmembrane domain comprises the CD8 transmembrane domain.

[0192] In some embodiments, the transmembrane domain comprises the CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 78 or SEQ ID NO: 87.

[0193] In some embodiments, the transmembrane domain may be a synthetic compound, in which case the transmembrane domain will mainly comprise hydrophobic residues such as leucine and valine. In certain exemplary embodiments, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0194] The transmembrane domains described herein can be combined with any of the antigen-binding domains described herein, any of the co-stimulatory signaling domains described herein, any of the intracellular signaling domains described herein, or any of the other domains described herein and that may be included in the subject CAR.

[0195] 4. Intracellular Domain The CARs of the present disclosure also include an intracellular domain. The intracellular domain of the CAR is responsible for activating at least one of the effector functions of the cell (e.g., immune cell) in which the CAR is expressed. The intracellular domain causes transduction of an effector function signal, leading the cell (e.g., immune cell) to perform special functions such as damaging and / or destroying the target cell.

[0196] The intracellular domain of the CAR or otherwise the cytoplasmic domain is responsible for activating the cell in which the CAR is expressed. Examples of intracellular domains used in the present invention include the cytoplasmic portion of a surface receptor, a co-stimulatory molecule, and any molecule that acts in concert to initiate signal transduction within a T cell, as well as any derivative or variant of these elements, and any synthetic sequence having the same functional capacity, but are not limited thereto. In certain embodiments, the intracellular domain includes a co-stimulatory signal transduction domain and an intracellular signal transduction domain.

[0197] Examples of intracellular signal transduction domains include, but are not limited to, either the zeta chain of the T cell receptor complex or its homolog, syk family tyrosine kinases (such as Syk, ZAP 70, etc.), src family tyrosine kinases (such as Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction (such as CD2, CD3, and CD28). In one embodiment, the intracellular signal transduction domain may be the human CD3 zeta chain, FcγRin, FcεRI, the cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) having a cytoplasmic receptor, and combinations thereof.

[0198] Other examples of intracellular domains include fragments or domains from one or more molecules or receptors, where the molecule or receptor specifically binds to TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma R1 la, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CD3, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp50 (KLRF1), CD127, CD160, CD19, CD4, CD3 alpha, CD3 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp50, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other stimulatory molecules described herein, any derivatives, variants, or fragments thereof, any synthetic sequences of stimulatory molecules having the same functional capacity, and any combinations thereof, including but not limited to these.

[0199] Further examples of intracellular domains include intracellular signaling domains of several types of various other immune signaling receptors, which intracellular signaling domains include first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory factors, and tumor necrosis factor receptor (TNFR) superfamily receptors, but are not limited thereto (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6):651-653). Further, the intracellular signaling domain may include signaling domains used by NK cells and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6:195), examples of which include NKpSO (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5):2290-2299), and DAP12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7):3201-3212), the signaling domains of NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0200] Intracellular signaling domains suitable for use in the CARs of the present disclosure include any desired signaling domain that provides a distinct and detectable signal (e.g., an increase in the production of one or more cytokines by the cell, a change in the transcription of a target gene, a change in the activity of a protein, a change in cell behavior (e.g., cell death), cell proliferation, cell differentiation, cell survival, regulation of a cell signaling response, etc.) in response to activation of the CAR (i.e., activation by an antigen and a dimerization agent). In some embodiments, the intracellular signaling domain includes at least one (e.g., 1, 2, 3, 4, 5, 6, etc.) GGAM motif, as described below. In some embodiments, the intracellular signaling domain includes a DAP10 / CD28 type signaling chain. In some embodiments, the intracellular signaling domain is not covalently bound to the membrane-bound CAR and instead diffuses within the cytoplasm.

[0201] Intracellular signaling domains suitable for use in the subject CARs include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, the ITAM motif is repeated twice within the intracellular signaling domain, and the first and second instances of the ITAM motif are separated from each other by 6-8 amino acids. In one embodiment, the intracellular signaling domain of the subject CAR comprises three ITAM motifs. In some embodiments, the intracellular signaling domain comprises the signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine-based activation motif (ITAM), examples of which include, but are not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5 (see, e.g., Gillis et al., Front (2014) Immunol. 5:254).

[0202] A suitable intracellular signaling domain can be an ITAM motif-containing portion derived from a polypeptide containing a GGAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3δ (CD3 delta), CD3ε (CD3 epsilon), CD3γ (CD3 gamma), CD3ζ (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0203] In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP, TYRO protein tyrosine kinase-binding protein, KARAP, PLOSL, DNAX activation protein 12, KAR-associated protein, TYRO protein tyrosine kinase-binding protein, killer activation receptor-associated protein, killer activation receptor-associated protein, etc.). In one embodiment, the intracellular signaling domain is derived from FCεR1G (also known as FCRG, Fc epsilon receptor I gamma chain, Fc receptor gamma chain, fc-epsilonRI-gamma, fcR gamma, fceRl gamma, high-affinity immunoglobulin epsilon receptor subunit gamma, immunoglobulin E receptor, high-affinity, gamma chain, etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3δ, CD3-DELTA, CD3 antigen, delta subunit, CD3d antigen, delta polypeptide (TiT3 complex), OKT3, delta chain, T cell receptor T3 delta chain, T cell surface glycoprotein CD3 delta chain, etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3ε, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.).

[0204] In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3γ, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signaling domain is derived from CD79A (also known as B cell antigen receptor complex-associated protein alpha chain, CD79a antigen (immunoglobulin-related alpha), MB-1 membrane glycoprotein, Ig-α, membrane-bound immunoglobulin-related protein, surface IgM-related protein, etc.). In one embodiment, the intracellular signaling domain suitable for use in the CARs of the present disclosure comprises a DAP10 / CD28 type signaling chain. In one embodiment, the intracellular signaling domain suitable for use in the CARs of the present disclosure comprises a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3, CD22, CD79a, CD79b or CD66d. In one embodiment, the intracellular signaling domain within the CAR comprises the cytoplasmic signaling domain of human CD3 zeta.

[0205] a. Co-stimulatory domain In certain embodiments, the intracellular domain comprises a co-stimulatory signaling domain. In some embodiments, the chimeric antigen receptor (CAR) comprises a co-stimulatory domain, and this co-stimulatory domain is a co-stimulatory domain of a molecule selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD8, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, DAP10, DAP12, Lck, Fas, and combinations thereof. In some embodiments, the co-stimulatory domain comprises a 4-1BB co-stimulatory domain or the amino acid sequence of SEQ ID NO: 58. In some embodiments, the co-stimulatory domain comprises a CD28 co-stimulatory domain or the amino acid sequence of SEQ ID NO: 88. In some embodiments, the co-stimulatory domain comprises a 4-1BB co-stimulatory domain and a CD28 co-stimulatory domain.

[0206] b. Intracellular signaling domain In certain embodiments, the intracellular domain comprises an intracellular signaling domain. In some embodiments, an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprises an intracellular domain that may be from the intracellular signaling domain of a molecule selected from the group consisting of T cell receptor (TCR) zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, CD3, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the intracellular signaling domain comprises a CD3 zeta signaling domain, or the amino acid sequence of SEQ ID NO: 59.

[0207] Tolerable variations of the intracellular domain while maintaining specific activity are known to those skilled in the art. For example, in some embodiments, the intracellular domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any of the amino acid sequences set forth in SEQ ID NO: 59.

[0208] 5. Hinge The hinge region of the CAR is a hydrophilic region located between the antigen-binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding of the CAR. The hinge region is an optional component of the CAR. In some embodiments, the chimeric antigen receptor (CAR) may further include a hinge domain.

[0209] In some embodiments, the hinge domain is a protein selected from the group consisting of CD8α, CD28 hinge, the Fc fragment of an antibody, the hinge region of an antibody, the CH2 region of an antibody, the CH3 region of an antibody, and an artificial spacer sequence. In one embodiment, the hinge domain is the CD8α hinge domain. In one embodiment, the hinge domain is the CD28 hinge domain. In one embodiment, the hinge domain comprises the amino acid sequence of SEQ ID NO: 77 or 86. In some embodiments, the hinge domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, SEQ ID NO: 68, 71-77, and 86.

[0210] In some embodiments, the CAR of the present disclosure includes a hinge region that connects the antigen-binding domain to the transmembrane domain, and then the transmembrane domain connects to the intracellular domain. The hinge region preferably can assist the antigen-binding domain in recognizing and binding to a target antigen on the target cell (see, for example, Hudecek et al., Cancer Immunol. Res. (2015) 3(2):125-135). In some embodiments, the hinge region is a flexible domain. Thus, the antigen-binding domain can have a structure that optimally recognizes the specific structure and density of the target antigen on cells such as tumor cells. Due to the flexibility of the hinge region, the hinge region can adopt a number of different three-dimensional structures. In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a hinge region derived from CD8 or CD28).

[0211] The hinge region can have a length of about 4 amino acids to about 50 amino acids, for example, about 4 amino acids to about 10 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 20 amino acids, about 20 amino acids to about 25 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 40 amino acids, or about 40 amino acids to about 50 amino acids.

[0212] A suitable hinge region can be easily selected and can be any of a plurality of suitable lengths, examples of which include 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids (including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids), and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0213] E. Generation of CAR The CARs of the present disclosure may be manufactured using chemical methods. For example, the CAR can be synthesized by solid-phase techniques (Roberge J Y et al (1995) Science 269:202-204), cut out from the resin, and purified by preparative high-performance liquid chromatography. Automated synthesis can be achieved using, for example, an ABI 431 A peptide synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

[0214] The CARs of the present disclosure may be synthesized by conventional techniques. For example, the CAR may be synthesized by chemical synthesis using solid-phase peptide synthesis methods. In these methods, either solid-phase synthesis or liquid-phase synthesis is employed (for example, for solid-phase synthesis techniques, see J. M. Stewart, and J. D. Young, Solid Phase Peptide Synthesis, 2 ndEd., Pierce Chemical Co., Rockford 111. (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254; for classical solution synthesis, see M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, suprs, Vol 1). As an example, the CARs of the present invention may be synthesized by directly incorporating phosphothreonine as an N-fluorenylmethoxycarbonyl-O-benzyl-L-phosphothreonine derivative using 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase chemistry.

[0215] An N-terminal fusion protein or C-terminal fusion protein comprising a CAR of the present disclosure conjugated to another molecule may be prepared by recombinant techniques by fusing the N-terminus or C-terminus of the chimeric protein with the sequence of a selected protein or selectable marker having a desired biological function. The resulting fusion protein contains a CAR of the present disclosure fused to the selected protein or marker protein as described herein. Examples of proteins that may be used in the preparation of the fusion protein include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.

[0216] The CARs of the present disclosure may be developed using biological expression systems. By using these systems, it is possible to produce large libraries of random peptide sequences and screen these libraries for peptide sequences that bind to specific proteins. Libraries may be produced by cloning synthetic DNA encoding random peptide sequences into suitable expression vectors (see Christian et al 1992, J. Mol. Biol. 227:711; Devlin et al, 1990 Science 249:404; Cwirla et al 1990, Proc. Natl. Acad, Sci. USA, 87:6378). Libraries may also be constructed by simultaneous synthesis of overlapping peptides (see U.S. Patent No. 4,708,871).

[0217] In one aspect, the present disclosure provides any form of CAR that has substantial homology to the CARs disclosed herein. Preferably, a CAR that is "substantially homologous" is about 50% homologous to the amino acid sequence of the peptides disclosed herein, more preferably about 70% homologous, even more preferably about 80% homologous, more preferably about 90% homologous, even more preferably about 95% homologous, and even more preferably about 99% homologous. The CARs may alternatively be produced by recombinant means or by cleavage from a longer polypeptide.

[0218] Variants of the CAR according to the present disclosure are those in which (i) one or more of the amino acid residues are replaced with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), and such replaced amino acid residues may or may not be encoded by the genetic code, (ii) those in which there are one or more modified amino acid residues (e.g., residues modified by the attachment of substituents), (iii) those in which the peptide is an alternative splice variant of the peptide of the present disclosure, (iv) fragments of the peptide, and / or (v) those in which the peptide may be fused to another peptide such as a leader or secretion sequence, or a sequence employed for purification (e.g., His tag) or detection (e.g., Sv5 epitope tag). Fragments include peptides generated by proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may be subject to post-translational or chemical modifications. Such variants are determined to be within the scope of those skilled in the art according to the teachings herein.

[0219] As is known in the art, the "similarity" between two peptides is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions to the sequence of a second polypeptide. A variant is defined as including a peptide sequence that differs from the original sequence, i.e., preferably less than 40% of the residues per targeted segment differ from the original sequence, more preferably less than 25% of the residues per targeted segment differ from the original sequence, more preferably less than 10% of the residues per targeted segment differ from the original sequence, and most preferably only a very few residues per targeted segment differ from the original protein sequence, while at the same time being homologous enough to maintain the functionality of the original sequence and / or the ability to bind to TACA. The present disclosure includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90% or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods well known to those of skill in the art. The identity between two amino acid sequences is preferably determined using the BLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)].

[0220] The CARs of the present disclosure can be post-translationally modified. For example, post-translational modifications included within the scope of the present invention include cleavage of the signal peptide, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding, proteolytic processing, and the like. Some modification or processing events require the introduction of additional biological machinery. For example, processing events such as cleavage of the signal peptide and core glycosylation are examined by adding dog microsomal membranes or Xenopus laevis oocyte extracts (U.S. Patent No. 6,103,489) to the standard translation reaction.

[0221] In some embodiments, the CARs of the present disclosure may include non-natural amino acids formed by post-translational modification or by introducing non-natural amino acids during translation. Various approaches are available for introducing non-natural amino acids during protein translation.

[0222] The CARs of the present disclosure may be conjugated with other molecules such as proteins to prepare fusion proteins. This can be achieved, for example, by synthesizing N-terminal or C-terminal fusion proteins. However, it is necessary that the resulting fusion protein retains the functionality of the peptide. The CARs of the present disclosure may be phosphorylated using conventional methods, examples of which include the methods described in Reedijk et al. The EMBO Journal 11(4):1365(1992).

[0223] Cyclic derivatives of the CARs of the present disclosure are also contemplated. Cyclization may enable the peptide to adopt a preferred conformation upon association with other molecules. Cyclization may be achieved using techniques known in the art. For example, a disulfide bond may be formed between two components having free sulfhydryl groups and appropriately spaced apart, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component. Also, cyclization may be achieved using azobenzene-containing amino acids as described in Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The components forming the bond may be side chains of amino acids, non-amino acid components, or a combination of the two. In embodiments of the present disclosure, the cyclic peptide may contain a β-turn in the correct position. A β-turn may be introduced into the peptides of the present invention by adding the amino acids Pro-Gly in the correct position. It may be desirable to produce a cyclic peptide that is more flexible than a cyclic peptide containing a peptide bond linkage as described above. A more flexible peptide may be prepared by introducing cysteine at the right and left positions of the peptide and forming a disulfide bridge between the two cysteines. The two cysteines are arranged so as not to distort or rotate the β-sheet. As a result of the shorter disulfide linkage and fewer hydrogen bonds in the β-sheet portion, the peptide becomes more flexible. The relative flexibility of the cyclic peptide can be determined by molecular dynamics simulations.

[0224] One aspect of the present disclosure provides a CAR that is fused or integrated with a target protein and / or a targeting domain that can direct the CAR to a desired cellular component or cell type or tissue. The CAR may also contain additional amino acid sequences or domains. The CAR is recombinant in the sense that the various components are from various sources and thus are not found together in nature (i.e., are heterologous).

[0225] In one embodiment, the target domain can be a transmembrane domain, a membrane-binding domain, or a sequence that directs a protein to associate with, for example, a vesicle or a nucleus. In one embodiment, the target domain can enable the peptide to reach a specific cell type or tissue. For example, the target domain can be a cell surface ligand or antibody against a cell surface antigen of a target tissue (e.g., bone, regenerated bone, degenerated bone, cartilage). The target domain may enable the peptide of the present invention to reach a cellular component.

[0226] IV. Nucleic Acids and Expression Vectors One aspect of the present disclosure relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), a hinge domain, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain.

[0227] One aspect of the present disclosure provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), the antigen-binding domain comprising a TACA-binding domain derived from a lectin, and the antigen-binding domain comprising one or more TACA-binding domains, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain. In one embodiment, a first nucleic acid sequence encoding the antigen-binding domain is operably linked to a second nucleic acid sequence encoding the transmembrane domain and further operably linked to a third nucleic acid sequence encoding the co-stimulatory signaling domain and / or the intracellular signaling domain.

[0228] Another aspect of the disclosure provides a CAR expressed by a modified cell disclosed herein, or an isolated nucleic acid encoding a polypeptide of a CAR disclosed herein. In some embodiments, the CAR encoded by the nucleic acid does not induce tonic signaling. In some embodiments, a modified cell expressing a nucleic acid encoding a CAR disclosed herein is less susceptible to the effects of tonic signaling by the CAR, thereby reducing exhaustion and / or inflammatory cytokine production in the absence of target cancer cells.

[0229] In some embodiments, the nucleic acid encodes an antigen-binding domain comprising one or more TACA-binding domains. In some embodiments, the antigen-binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 TACA-binding domains. In one embodiment, the antigen-binding domain comprises two TACA-binding domains. In one embodiment, the antigen-binding domain comprises three TACA-binding domains. In one embodiment, the antigen-binding domain comprises four TACA-binding domains. The TACA-binding domain may include any peptide, protein, lectin, lectin fragment, antibody, antibody fragment, small molecule, nucleic acid, etc. that can specifically bind to TACA. In some embodiments, the antigen-binding domain comprises a mutation in the TACA-binding domain (TBD) selected from substitutions, deletions, or insertions.

[0230] In some embodiments, the antigen-binding domain comprises a deletion of the tumor-associated carbohydrate antigen (TACA)-binding domain (TBD). In one embodiment, the deletion is present in the N-terminal region and / or C-terminal region of the TACA-binding domain (TBD). In one embodiment, the deletion is at least about 2 amino acids, at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 36 amino acids, at least about 38 amino acids, at least about 40 amino acids, at least about 45 amino acids, or more. In one embodiment, the deletion is at least about 10 amino acids, at least about 18 amino acids, or at least about 36 amino acids. In one embodiment, the deletion is at least about 36 amino acids.

[0231] In some embodiments, the antigen-binding domain comprises a deletion in the TACA-binding domain (TBD), the deletion is present at the N-terminus of the TBD, and is at least about 18 amino acids. In some embodiments, the antigen-binding domain comprises a deletion in the TACA-binding domain (TBD), the deletion is present at the C-terminus of the TBD, and is at least about 10 amino acids. In some embodiments, the antigen-binding domain comprises a deletion in the TACA-binding domain (TBD), the deletion is present at the N-terminus of the TBD, and is at least about 36 amino acids. In some embodiments, the antigen-binding domain comprises a deletion in the TACA-binding domain (TBD), the deletion is present at the N-terminus of the TBD and is at least about 18 amino acids and is present at the C-terminus and is at least about 10 amino acids.

[0232] In some embodiments, the antigen-binding domain comprises a deletion that removes a disulfide-bond cysteine residue in the TACA-binding domain (TBD). In some embodiments, the expression of the chimeric antigen receptor (CAR) comprising a deletion in the TACA-binding domain (TBD) of the antigen-binding domain is similar to the expression of a CAR comprising a wild-type TBD.

[0233] In some embodiments, the antigen-binding domain comprises a carbohydrate-binding domain of a TACA-binding domain that contains a deletion and has an amino acid sequence set forth in SEQ ID NOs: 30-54 or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 sequence set forth in SEQ ID NOs: 30-54.

[0234] In some embodiments, the antigen-binding domain comprises an amino acid sequence set forth in SEQ ID NOs: 34-39 or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 sequence set forth in SEQ ID NOs: 34-39.

[0235] In some embodiments, the antigen-binding comprises an amino acid sequence having at least 90% homology with SEQ ID NOs: 34-39. In some embodiments, the antigen-binding domain comprises an amino acid sequence disclosed in Table 2 or Table 3.

[0236] For intracellular expression, the antigen-binding domain may be operably linked to another domain of the CAR, such as a transmembrane domain, a co-stimulatory signaling domain, or an intracellular signaling domain (each described elsewhere herein). The antigen-binding domains described herein can be combined with any of the transmembrane domains, any of the co-stimulatory signaling domains, any of the intracellular signaling domains, or any of the other domains described herein and that can be included in the CARs of the present disclosure.

[0237] A. Nucleic acid The isolated nucleic acid sequences encoding the chimeric antigen receptors of the present disclosure can be obtained using any of a number of recombinant methods known in the art, examples of which include, for example, screening a library from cells that express the gene, deriving the gene from a vector known to contain the gene, or isolating directly from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced by synthesis rather than cloning.

[0238] The isolated nucleic acid may comprise any type of nucleic acid, including but not limited to DNA and RNA. For example, in one embodiment, the composition comprises an isolated DNA molecule, which includes, for example, an isolated cDNA molecule encoding a peptide of the present disclosure, or a functional fragment thereof. In one embodiment, the composition comprises an isolated RNA molecule encoding a peptide of the present disclosure, or a functional fragment thereof.

[0239] The nucleic acid molecules of the present disclosure can be modified to improve their stability in serum or in growth media for cell culture. Modifications can be added to enhance stability, functionality, and / or specificity, and to minimize the immunostimulatory properties of the nucleic acid molecules of the present disclosure. For example, to enhance stability, the 3' residue may be stabilized against degradation; for example, the 3' residue may be selected such that it is composed of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution with modified analogs of pyrimidine nucleotides, for example substitution of uridine with 2'-deoxythymidine, is tolerated and does not affect the function of the molecule. In one embodiment of the present disclosure, the nucleic acid molecule may contain at least one modified nucleotide analog. For example, the termini may be stabilized by incorporating modified nucleotide analogs.

[0240] Non-limiting examples of nucleotide analogs include ribonucleotides that are sugar-modified and / or backbone-modified (i.e., including modifications to the phosphate-sugar backbone). For example, the phosphodiester bond of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In preferred backbone-modified ribonucleotides, the phosphate ester group that binds to adjacent ribonucleotides is replaced by a modified group such as a phosphorothioate group. In preferred sugar-modified ribonucleotides, the 2' OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or ON, where R is Ci-Ce alkyl, alkenyl, or alkynyl, and halo is F, CI, Br, or I.

[0241] Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides that contain at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. The base may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include uridine and / or cytidine modified at the 5-position (e.g., 5-(2-amino)propyluridine, 5-bromouridine), adenosine and / or guanosine modified at the 8-position (e.g., 8-bromoguanosine), deazanucleotides (e.g., 7-deazaadenosine), O- and N-alkylated nucleotides (e.g., N6-methyladenosine is preferred), but are not limited thereto. It should be noted that the above modifications may be combined.

[0242] For example, a nucleic acid molecule may include at least one of the following chemical modifications, i.e., modification of the 2'-H, 2'-O-methyl, or 2'-OH of one or more nucleotides. In certain embodiments, the nucleic acid molecules of the present disclosure can have enhanced resistance to nucleases. To improve nuclease resistance, the nucleic acid molecule can include, for example, 2'-modified ribose units and / or phosphorothioate linkages. For example, the 2'-hydroxyl group (OH) can be modified or replaced with a plurality of different "oxy" substituents or "deoxy" substituents. To improve nuclease resistance, the nucleic acid molecules of the present disclosure can include 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. Also, specific nucleobase modifications such as locked nucleic acid (LNA), ethylene nucleic acid (ENA) (e.g., 2'-4'-ethylene-bridged nucleic acid), and 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modification can improve the binding affinity to the target.

[0243] In one embodiment, the nucleic acid molecule comprises a 2'-modified nucleotide, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamide (2'-O-MA). In one embodiment, the nucleic acid molecule comprises at least one 2'-O-methyl modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule comprise a 2'-O-methyl modification.

[0244] In some embodiments, the nucleic acid molecules of the present disclosure preferably have one or more of the following properties, namely that the nucleic acid agents discussed herein include unmodified RNA and DNA in other respects, as well as RNA and DNA modified, for example, to improve efficacy, and polymers of nucleoside analogs.

[0245] Unmodified RNA refers to a molecule in which the components of the nucleic acid (i.e., the sugar, base, and phosphate moieties) are the same as or essentially the same as those found in nature, preferably those naturally occurring in the human body. In the art, rare or abnormal but naturally occurring RNAs are referred to as modified RNAs. See, for example, Limbach et al., Nucleic Acids Res., 1994, 22:2183-2196. Such rare or abnormal RNAs are often referred to as modified RNAs and are generally the result of post-transcriptional modification and are within the scope of the term unmodified RNA as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid (i.e., the sugar, base, and phosphate moieties) are different from those found in nature, preferably different from those present in the human body. These are called "modified RNAs" and, because they are modified, include molecules that are not strictly speaking RNAs. Nucleoside analogs are molecules in which the ribophosphate backbone is replaced by a non-ribophosphate construct, which allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to that seen with a ribophosphate backbone (e.g., an uncharged mimic of a ribophosphate backbone). Modifications to the nucleic acids of the present disclosure may be present in one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobase.

[0246] B. Expression Vector In one aspect, the present disclosure provides an expression construct comprising an isolated nucleic acid encoding a CAR disclosed herein. In some embodiments of the present disclosure, the isolated nucleic acid described herein comprises an expression vector and / or in vitro transcribed RNA. In another embodiment, the expression construct comprises an isolated nucleic acid encoding a CAR described herein.

[0247] The expression of natural or synthetic nucleic acids encoding the CAR of the present invention is generally achieved by operably linking a nucleic acid encoding a peptide or a part thereof to a promoter and incorporating the construct into an expression vector. The vectors used are suitable for replication and optionally integration into eukaryotic cells. Typical vectors contain transcription terminators and translation terminators, initiation sequences and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0248] Also, the vectors of the present disclosure may be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466. These patents are hereby incorporated by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0249] The isolated nucleic acids of the present disclosure can be cloned into a plurality of types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0250] Furthermore, the vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), as well as in other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0251] 1. Virus-based systems In some embodiments, the expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. In some embodiments, the expression construct is a lentiviral vector. In some embodiments, the expression construct is a self-inactivating lentiviral vector. In some embodiments, the expression construct comprises an isolated nucleic acid encoding the CAR described herein.

[0252] Multiple virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector using techniques known in the art and packaged into retroviral particles. The recombinant virus can then be isolated and delivered to the target cells either in vivo or ex vivo. Multiple retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Multiple adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0253] For example, vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow for stable integration of the transgene over time and its propagation in daughter cells. Lentiviral vectors have the additional advantage of being able to transduce non-proliferating cells such as hepatocytes compared to vectors derived from oncoviruses such as murine leukemia virus. They also have the additional advantage of low immunogenicity. In one embodiment, the composition comprises a vector derived from adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors have multiple characteristics that make them ideally suited for gene therapy, including lack of pathogenicity, minimal immunogenicity, and the ability to transduce post-mitotic cells in a stable and efficient manner. The expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by selecting an appropriate combination of AAV serotype, promoter, and delivery method.

[0254] 2. Regulatory Elements In some embodiments, the vector also includes conventional control elements that are operably linked to the transgene in a manner that enables transcription, translation, and / or expression in cells transfected with the plasmid vector or in cells infected with the virus produced by the present invention. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation sequences, transcription termination sequences, promoter sequences, and enhancer sequences; efficient RNA processing signals such as splicing signals and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, optionally, sequences that enhance the secretion of the encoded product. A number of expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be utilized.

[0255] Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. Generally, these are located in the region 30 - 110 bp upstream of the start site, although recently it has been shown that multiple promoters also contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is maintained even when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased up to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription.

[0256] 3. Promoter In some embodiments, the expression construct further comprises a promoter. The promoter may be selected from an EF-lα promoter, a T cell receptor alpha (TRAC) promoter, an interleukin 2 (IL-2) promoter, or a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a Moloney murine leukemia virus (MoMuLV) promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, or a Rous sarcoma virus promoter.

[0257] The immediate early cytomegalovirus (CMV) promoter sequence is an example of a strong constitutive promoter sequence capable of driving high-level expression of any operably linked polynucleotide sequence. Another example of a suitable promoter is elongation growth factor-1a (EF-1a). However, other constitutive promoter sequences may 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, Moloney murine leukemia virus (MoMuLV) promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and even human gene promoters (examples include, but are not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter). Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of an operably linked polynucleotide sequence when such expression is desired or turn off the expression when it is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0258] Enhancer sequences present on the vector also regulate the expression of the genes contained therein. Generally, enhancers bind to protein factors to enhance gene transcription. The enhancer may be placed upstream or downstream of the gene it regulates. Also, the enhancer may be made tissue-specific to enhance transcription in specific cell or tissue types. In one embodiment, the vector of the present invention includes one or more enhancers to promote the transcription of genes present within the vector.

[0259] 4. Selectable Marker To evaluate peptide expression, the expression vector introduced into the cells may also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the population of cells to be transfected or infected by the viral vector. In other embodiments, the selectable marker may be carried on a separate DNA fragment and used in a co-transfection procedure. Appropriate regulatory sequences may be placed for both the selectable marker and the reporter gene to allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

[0260] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and encodes a polypeptide whose expression appears as some easily detectable property, such as enzyme activity. Expression of the reporter gene is assayed at a suitable time point after the DNA has been introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, a construct having a minimal 5' flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such a promoter region may be ligated to the reporter gene and used to evaluate an agent for its ability to regulate promoter drive.

[0261] V. Modified Cells A. CAR T Cell Exhaustion The underlying cause of T cell exhaustion is continuous antigen exposure leading to continuous TCR signaling. The present disclosure provides CAR T cells that do not experience antigen-induced tonic signaling and exhaustion in the context of treating a disease or condition through selective regulation (e.g., reduction) of CAR cell surface expression (preventing clustering). Thus, the CAR T cells of the present disclosure will maintain, recover, or enhance functionality. "T cell exhaustion" refers to a decline in T cell function that can occur as a result of infection (e.g., chronic infection) or disease. T cell exhaustion is associated with increased expression of PD-1, TIM-3, and LAG-3, apoptosis, and decreased cytokine secretion. Thus, terms such as "ameliorating T cell exhaustion", "suppressing T cell exhaustion", "decreasing T cell exhaustion" refer to a state in which T cell functionality is restored, which functionality is characterized by one or more of a decrease in expression, and / or levels of one or more of PD-1, TIM-3, and LAG-3; an increase in the formation of memory cells and / or maintenance of memory markers (e.g., CD62L); prevention of apoptosis; an increase in the production and / or secretion of antigen-induced cytokines (e.g., IL-2); enhancement of cytotoxic / killing ability; an increase in the recognition of tumor targets with low surface antigens; and enhancement of proliferation in response to antigen.

[0262] Modified cells expressing a CAR are subject to tonic antigen-independent signaling by receptor clustering, as well as high levels of PD-1, TIM-3, and LAG-3 expression, reduced antigen-induced cytokine production, and excessive programmed cell death, recapitulating the basic biology of T cell exhaustion as shown. Since tonic signaling depends largely on the CAR receptor levels within CAR T cells, the level of tonic signaling can be regulated using control of CAR expression levels (e.g., in vitro or in vivo). Since tonic signaling depends largely on the levels of the CAR receptor, precise control of the CAR expression level precisely regulates the level of tonic signaling. Accordingly, the present disclosure provides a CAR with a mutated antigen-binding domain that prevents clustering of the TACA CAR at the cell membrane, thereby preventing tonic signaling. Accordingly, the structural modifications engineered with the CARs of the present disclosure regulate the surface expression of the chimeric receptor in modified cells, thereby preventing or reversing CAR T cell exhaustion and restoring the function of the modified CAR T cells.

[0263] The present disclosure is not limited by the type of functionality of CAR T cells that is maintained, restored, or enhanced (e.g., extended). Indeed, compositions comprising CAR T cells modified to express a CAR comprising a modified TACA-binding domain and methods of using the same may be used to maintain, restore, or enhance the functionality of CAR T cells, which functionality includes cytotoxic activity against tumor cells; promotion of the survival and function of CAR T cells; induction of cytokine expression such as expression of interleukin-2 (IL-2) to promote the survival of T cells, induction of apoptosis of tumor cells, and / or induction of interferon (IFN)-gamma to activate the innate immune response (e.g., against cancer) through the expression of Fas ligand (FasL) and / or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL); and / or enhancement of induction of cell cycle arrest and / or apoptosis, including but not limited to these. In some embodiments, the CAR T cells of the present disclosure confer sensitivity to induction of cell cycle arrest and / or apoptosis on cancer cells, including cells that are normally resistant to such inductive stimuli.

[0264] One aspect of the present disclosure provides a genetically modified (e.g., engineered) cell that stably expresses and comprises the subject CAR of the present disclosure. In one aspect, the modified cell comprises an isolated nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain that selectively binds a tumor-associated carbohydrate antigen (TACA), a hinge domain, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain. In some embodiments, the modified cell comprises a chimeric antigen receptor that selectively binds a tumor-associated carbohydrate antigen (TACA).

[0265] One aspect of the present disclosure provides a modified cell comprising an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain that selectively binds a tumor-associated carbohydrate antigen (TACA) comprising one or more TACA-binding domains, a transmembrane domain, a co-stimulatory domain, and / or an intracellular signaling domain, and the modified cell is less susceptible to tonic signaling by the TACA CAR, thereby reducing exhaustion and / or inflammatory cytokine production in the absence of target cancer cells.

[0266] In some embodiments, the antigen-binding domain comprises a mutation in the TACA-binding domain (TBD) selected from a substitution, deletion, or insertion. In some embodiments, the antigen-binding domain comprises a deletion of the TACA-binding domain (TBD). In some embodiments, the deletion is present in the N-terminal region and / or the C-terminal region of the TACA-binding domain (TBD).

[0267] In some embodiments, the deletion is at least about 2 amino acids, at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 36 amino acids, at least about 38 amino acids, at least about 40 amino acids, at least about 45 amino acids, or more. In some embodiments, the deletion is at least about 10 amino acids, at least about 18 amino acids, or at least about 36 amino acids. In some embodiments, the deletion is at least about 36 amino acids.

[0268] In some embodiments, the antigen-binding domain comprises a deletion in the tumor-associated carbohydrate antigen (TACA)-binding domain (TBD), the deletion is present at the N-terminus of the TBD, and is at least about 18 amino acids. In some embodiments, the antigen-binding domain comprises a deletion at the C-terminus of the TACA-binding domain (TBD), and is at least about 10 amino acids. In some embodiments, the antigen-binding domain comprises a deletion at the N-terminus of the TACA-binding domain (TBD) and the TBD, and is at least about 36 amino acids. In some embodiments, the antigen-binding domain comprises a deletion at the N-terminus of the TACA-binding domain (TBD), and is at least about 18 amino acids, and is present at the C-terminus, and is at least about 10 amino acids.

[0269] In some embodiments, the antigen-binding domain comprises a deletion that removes a disulfide bond cysteine residue in the TACA-binding domain (TBD). In some embodiments, the expression of the chimeric antigen receptor (CAR) comprising a deletion in the TACA-binding domain (TBD) of the antigen-binding domain is similar to the expression of a CAR comprising a wild-type TBD. In some embodiments, modified cells expressing a CAR having a deletion in the TACA-binding domain (TBD) of the antigen-binding domain exhibit a decrease in tonic signaling compared to modified cells comprising a CAR comprising a wild-type TBD.

[0270] In some embodiments, modified cells expressing a CAR having a deletion in the TACA-binding domain (TBD) of the antigen-binding domain are less likely to experience exhaustion compared to modified cells comprising a CAR comprising a wild-type TBD. In some embodiments, modified cells expressing a CAR having a deletion in the TACA-binding domain (TBD) of the antigen-binding domain are less likely to experience exhaustion induced by tonic signaling of the TACA CAR compared to modified cells comprising a CAR comprising a wild-type TBD.

[0271] In some embodiments, the antigen-binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more TACA-binding domains. In some embodiments, the TACA-binding domain is derived from a lectin. In those embodiments, the lectin is selected from galectin, siglec, selectin; C-type lectin; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Phaseolus vulgaris leukocyte agglutinin); E-PHA (Phaseolus vulgaris erythroagglutinin); tomato lectin (Lycopersicon esculentum lectin; LEA); peanut lectin (Arachis hypogaea lectin; PNA); potato lectin (Solanum tuberosum lectin), pokeweed mitogen (Phytolacca americana lectin), wheat germ agglutinin (Triticum aestivum germ lectin); Artocarpus polyphyllus lectin (jacalin lectin); hairy vetch lectin (VVA); apple snail lectin (HPA); fucose lectin (WFA); Sambucus nigra lectin (SNA), BC2L-CNt (lectin from the gram-negative bacterium Burkholderia cenocepacia), dog spleen leukocyte lectin (MAL), Pleurotus ostreatus (PVL), Sclerotium rolfsii lectin (SRL), Erythrina speciosa lectin (ESA), CLEC17A (prolectin), Hericium erinaceus lectin, Helix pomatia lectin (SSA), Glechoma hederacea lectin (Gleheda), Momordica charantia lectin (Morniga G), Ononis spinosa lectin, Salvia bogotensis lectin, Salvia horminum lectin, Cuscutaceae lectin, Calceolaria integrifolia lectin, Glyphomia simplifolia (GsLA4), Canavalia gladiata (acidic WBAI), Vigna angularis lectin, Apios americana lectin, Amaranthus leucocarpus lectin, Relya autumnalis lectin, Paramignya monophylla lectin, Ulex europaeus lectin, Artocarpus lakoocha lectin, Himalayan apple bean lectin, Himalayan apple bean lectin, soybean lectin and mushroom lectin.

[0272] In some embodiments, the lectin is a galectin selected from the group consisting of galectin-1, galectin-2, galectin-3, galectin-4, galectin-5, galectin-6, galectin-7, galectin-8, galectin-9, galectin-10, galectin-11, galectin-12, galectin-13, galectin-14 and galectin-15. In some embodiments, the lectin is a siglec selected from the group consisting of siglec-1 (sialoadhesin), siglec-2 (CD22), siglec-3 (CD33), siglec-4 (myelin-associated glycoprotein), siglec-5, siglec-6, siglec-7, siglec-8, siglec-9, siglec-10, siglec-11, siglec-12, siglec-13, siglec-14, siglec-15, siglec-16, siglec-17, siglec-E, siglec-F, siglec-G and siglec-H.

[0273] In some embodiments, the lectin is a polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) selected from the group consisting of ppGalNAc-T1 (GALNT1), ppGalNAc-T2 (GALNT2), ppGalNAc-T3 (GALNT3), ppGalNAc-T4 (GALNT4), ppGalNAc-T5 (GALNT5), ppGalNAc-T6 (GALNT6), ppGalNAc-T7 (GALNT7), ppGalNAc-T8 (GALNT8), ppGalNAc-T9 (GALNT9), ppGalNAc-T10 (GALNT10), ppGalNAc-T12 (GALNT12), ppGalNAc-T13 (GALNT13), ppGalNAc-T14 (GALNT14), ppGalNAc-T15 (GALNT15), ppGalNAc-T16 (GALNT16), ppGalNAc-T17 (GALNT17), ppGalNAc-T18 (GALNT18), ppGalNAc-T Like 5 (GALNTL5) and ppGalNAc-T Like 6 (GALNTL6).

[0274] In some embodiments, the antigen-binding domain selectively targets a TACA selected from the group consisting of β1,6-branched, β1,6GlcNAc-branched N-glycans, T antigen, Tn antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6-sialylation, sialylation, sialyl-Lewisx / a, disialyl-Lewisx / a, sialyl 6-sulfo Lexisx, Globo H, GD2, GD3, GM3, and fucosyl GM1.

[0275] In some embodiments, the antigen-binding domain selectively targets β1,6GlcNAc-branched N-glycans, Tn epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), GalNAcα-serine, GalNAcα-threonine, GalNAc, or GalNAcβ1.

[0276] In some embodiments, the antigen-binding domain comprises a TACA-binding domain of the antigen-binding domain that contains a deletion in an amino acid sequence set forth in SEQ ID NOs: 30-54, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 30-54.

[0277] In some embodiments, the antigen-binding domain comprises an amino acid sequence set forth in SEQ ID NOs: 34-39, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the amino acid sequences set forth in SEQ ID NOs: 34-39. In some embodiments, the antigen-binding comprises an amino acid sequence having at least 90% homology to SEQ ID NOs: 34-39.

[0278] In some embodiments, the chimeric antigen receptor (CAR) comprises a transmembrane domain, which may comprise the transmembrane region of a molecule selected from the group consisting of T cell receptor (TCR)-alpha, TCR-beta, CD3-zeta, CD3-epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134 (Ox40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In one embodiment, the transmembrane domain comprises the CD8 transmembrane domain. In some embodiments, the transmembrane domain comprises the CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 78 or SEQ ID NO: 87. In some embodiments, the transmembrane domain may be a synthetic compound, in which case the transmembrane domain will primarily comprise hydrophobic residues such as leucine and valine. In certain exemplary embodiments, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain.

[0279] In certain embodiments, the intracellular domain comprises a co-stimulatory signaling domain. In some embodiments, the chimeric antigen receptor (CAR) comprises a co-stimulatory domain, and this co-stimulatory domain is a co-stimulatory domain of a molecule selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD8, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, DAP10, DAP12, Lck, Fas, and combinations thereof.

[0280] In some embodiments, the co-stimulatory domain comprises a 4-1BB co-stimulatory domain or the amino acid sequence of SEQ ID NO: 58. In some embodiments, the co-stimulatory domain comprises a CD28 co-stimulatory domain or the amino acid sequence of SEQ ID NO: 88. In some embodiments, the co-stimulatory domain comprises a 4-1BB co-stimulatory domain and a CD28 co-stimulatory domain.

[0281] In certain embodiments, the intracellular domain comprises an intracellular signaling domain. In some embodiments, the isolated nucleic acid molecule encoding the chimeric antigen receptor (CAR) comprises an intracellular domain, and this intracellular domain may be from the intracellular signaling domain of a molecule selected from the group consisting of T cell receptor (TCR) zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, CD3, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the intracellular signaling domain comprises a CD3 zeta signaling domain or the amino acid sequence of SEQ ID NO: 59.

[0282] Tolerable variations of the intracellular domain while maintaining specific activity are known to those skilled in the art. For example, in some embodiments, the intracellular domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any of the amino acid sequences set forth in SEQ ID NO: 59.

[0283] The hinge region of the CAR is a hydrophilic region located between the antigen-binding domain and the transmembrane domain. In some embodiments, this domain promotes proper protein folding of the CAR. The hinge region is an optional component of the CAR. In some embodiments, the chimeric antigen receptor (CAR) may further comprise a hinge domain.

[0284] In some embodiments, the hinge domain is a protein selected from the group consisting of CD8α, CD28 hinge, the Fc fragment of an antibody, the hinge region of an antibody, the CH2 region of an antibody, the CH3 region of an antibody, and an artificial spacer sequence. In one embodiment, the hinge domain is the CD8α hinge domain. In one embodiment, the hinge domain is the CD28 hinge domain. In one embodiment, the hinge domain comprises the amino acid sequence of SEQ ID NO: 77 or 86. In some embodiments, the hinge domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, SEQ ID NO: 68, 71-77, and 86.

[0285] One aspect of the present disclosure provides a modified cell comprising an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising an amino acid sequence set forth in SEQ ID NOs: 23-29, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence set forth in SEQ ID NOs: 23-29. In some embodiments, the isolated nucleic acid expressed in the modified cell comprises an expression vector and / or in vitro transcribed RNA.

[0286] In some embodiments, the CAR selectively targets a TACA selected from the group consisting of β1,6-branched, β1,6GlcNAc-branched N-glycan, T antigen, Tn antigen, sialyl-T epitope, Tn epitope, sialyl-Tn epitope, α2,6 sialylation, sialylation, sialyl-Lewisx / a, disialyl-Lewisx / a, sialyl 6-sulfo Lexisx, Globo H, GD2, GD3, GM3, and fucosyl GM1. In some embodiments, the CAR selectively targets β1,6GlcNAc-branched N-glycan, GalNAc, Tn antigen, GalNAcα-ser, GalNAc or GalNAcβ1. In some embodiments, the modified cell comprising the CAR of SEQ ID NOs: 23-29 exhibits reduced tonic signaling compared to the modified cell comprising the CAR of SEQ ID NO: 21 or 22.

[0287] In some embodiments, the genetically modified cells are genetically engineered T lymphocytes (T cells), regulatory T cells (Tregs), naive T cells (TN), memory T cells (e.g., central memory T cells (TCM), effector memory cells (TEM)), natural killer cells (NK cells), natural killer T cells (NKT cells), and macrophages capable of giving rise to progeny relevant to the treatment. In some embodiments, the cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. In one embodiment, the cells are T cells. In one embodiment, the modified cells are autologous cells.

[0288] The modified cells (e.g., including the subject CAR) may be produced by stably transfecting host cells with an expression vector containing the nucleic acids of the present disclosure. Additional methods for generating the modified cells of the present disclosure include chemical conversion methods (e.g., using calcium phosphate, dendrimers, liposomes, and / or cationic polymers), non-chemical conversion methods (e.g., electroporation, optical conversion, gene electrotransfer, and / or hydrodynamic delivery), and / or particle-based methods (e.g., impalefection using a gene gun and / or magnetofection), but are not limited thereto. The transfected cells expressing the subject CAR of the present disclosure may be expanded ex vivo.

[0289] In some embodiments, the cells are genetically modified by contacting the cells with an isolated nucleic acid encoding a TACA CAR as described herein. In some embodiments, the nucleic acid sequence is delivered to the cells using a retroviral or lentiviral vector. For example, retroviral and lentiviral vectors expressing the peptides of the invention can be used with transduced cells as carriers or with cell-free local or systemic delivery of conjugated or naked encapsulated vectors to deliver to various types of eukaryotic cells, as well as to tissues and whole organisms. This method used can be used for any purpose where stable expression is required or sufficient.

[0290] In other embodiments, the nucleic acid sequence is delivered to the cells using in vitro transcribed mRNA. In vitro transcribed mRNA can be delivered to various types of eukaryotic cells, as well as to tissues and whole organisms using transfected cells as carriers or with cell-free local or systemic delivery of conjugated or naked encapsulated mRNA. This method used can be used for any purpose where transient expression is required or sufficient.

[0291] In certain embodiments, the cells may be any suitable cell type capable of expressing the desired peptide. In certain embodiments, the modified cells are used in a manner in which the cells are introduced into a recipient. In certain embodiments, the cells are autologous, allogeneic, syngeneic or xenogeneic cells to the recipient.

[0292] The disclosed compositions and methods can be applied to the regulation of T cell activity in basic research and treatment in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including the evaluation of the ability of genetically modified T cells to kill target cancer cells.

[0293] B. Methods for generating modified cells In one aspect, the present disclosure provides a method for generating a modified cell disclosed herein, the method comprising introducing into a cell an isolated nucleic acid encoding a CAR or a bispecific fusion protein, or an expression construct of the present disclosure.

[0294] Modified cells (e.g., comprising a subject CAR or bispecific fusion protein) may be produced by stably transfecting a host cell with an expression vector comprising a nucleic acid of the present disclosure. Additional methods for generating the modified cells of the present disclosure include chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer and / or hydrodynamic delivery), and / or particle-based methods (e.g., impalefection using a gene gun and / or magnetofection), but are not limited thereto. The transfected cells expressing the subject CAR or bispecific fusion protein may be expanded ex vivo.

[0295] In some embodiments, the cell is genetically modified by contacting the cell with an isolated nucleic acid encoding a CAR or a bispecific fusion protein as described herein. In some embodiments, the nucleic acid sequence is delivered to the cell using a retroviral or lentiviral vector. For example, retroviral and lentiviral vectors expressing the peptides of the invention can be used to transduce cells as carriers, or for cell-free local or systemic delivery of conjugated or naked encapsulated vectors to various types of eukaryotic cells, as well as tissues and whole organisms. This method used can be used for any purpose where stable expression is required or sufficient.

[0296] In other embodiments, the nucleic acid sequence is delivered to cells using in vitro transcribed mRNA. The in vitro transcribed mRNA can be delivered to various types of eukaryotic cells, as well as tissues and whole organisms, using transfected cells as carriers or cell-free local or systemic delivery of conjugated or naked encapsulated mRNA. This method used can be used for any purpose where transient expression is required or sufficient.

[0297] In certain embodiments, the cells can be any suitable cell type capable of expressing the desired peptide. In certain embodiments, the modified cells are used in the manner in which the cells are introduced into the recipient. In certain embodiments, the cells are autologous, allogeneic, syngeneic or xenogeneic cells to the recipient.

[0298] The disclosed compositions and methods can be applied to the regulation of T cell activity in basic research and treatment in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including the evaluation of the ability of genetically modified T cells to kill target cancer cells.

[0299] Methods for introducing and expressing genes into cells are known in the art. From the perspective of expression vectors, the vectors can be easily introduced into host cells by any method in the art, examples of which include, for example, mammalian cells, bacterial cells, yeast cells or insect cells. For example, the expression vector can be transferred into host cells by physical means, chemical means or biological means.

[0300] 1. Physical methods Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0301] 2. Biological methods Biological methods for introducing a target polynucleotide into a host cell include the use of DNA vectors and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0302] In some embodiments, the nucleic acid encoding the subject CAR or bispecific fusion protein of the present disclosure is introduced into cells by an expression vector. Expression vectors containing nucleic acids encoding a subject CAR (e.g., a TACA CAR) or bispecific fusion protein are provided herein. Suitable expression vectors include lentiviral vectors, gammaretroviral vectors, foamy viral vectors, adeno-associated virus (AAV) vectors, adenoviral vectors, engineered hybrid viruses, naked DNA, and further include transposon-mediated vectors such as sleeping beauty, piggyback, and integrases such as Phi31, but are not limited thereto. Some other suitable expression vectors include herpes simplex virus (HSV) and retroviral expression vectors.

[0303] The adenovirus expression vector is based on adenovirus, which has a low ability to integrate into genomic DNA but a high efficiency of transfection into host cells. The adenovirus expression vector contains (a) sequences that support the packaging of the expression vector and (b) adenovirus sequences sufficient to ultimately express the target CAR in the host cell. In some embodiments, the adenovirus genome is a 36 kb linear double-stranded DNA, and to produce the expression vector of the present invention, a foreign DNA sequence (e.g., a nucleic acid encoding TACA-CAR or a bispecific fusion protein) may be inserted to replace a large piece of the adenovirus DNA. See, for example, Danthinne and Imperiale, Gene Therapy 7(20):1707-1714(2000).

[0304] Another expression vector is based on adeno-associated virus that utilizes an adenovirus coupling system. This AAV expression vector has a high frequency of integration into the host genome. Since it can also infect non-dividing cells, it is useful for delivering genes to mammalian cells, for example, in tissue culture or in vivo. In terms of infectivity, the AAV vector has a broad host range. Details regarding the production and use of AAV vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797,368.

[0305] Retrovirus expression vectors can be integrated into the host genome, deliver large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specific cell lines. Retroviral vectors are constructed by inserting a nucleic acid (e.g., a nucleic acid encoding TACA-CAR or a bispecific fusion protein) at a specific position in the viral genome to produce a replication-defective virus. Although retroviral vectors can infect a wide variety of cell types, the integration and stable expression of the target CAR or bispecific fusion protein require the division of the host cell.

[0306] Lentiviral vectors are derived from lentiviruses, which are complex retroviruses that contain, in addition to the gag, pol, and env genes, which are common retroviral genes, other genes with regulatory or structural functions. See, for example, U.S. Pat. Nos. 6,013,516 and 5,994,136. Some examples of lentiviruses include human immunodeficiency virus (HTV-1, HTV-2) and simian immunodeficiency virus (SIV). Lentiviral vectors are produced by complex attenuation of the HIV virulence genes, for example, by deleting the env, vif, vpr, vpu, and nef genes, rendering the vector biologically safe. Lentiviral vectors can infect non-dividing cells and can be used for gene transfer and expression, both in vivo and ex vivo, of nucleic acids encoding, for example, a subject CAR or bispecific fusion protein (see, for example, U.S. Pat. No. 5,994,136).

[0307] An expression vector containing the nucleic acids of the present disclosure can be introduced into a host cell by any means known to those skilled in the art. The expression vector may optionally contain viral sequences for transfection. Alternatively, the expression vector may be introduced by fusion, electroporation, biolistics, transfection, lipofection, etc. The host cell can be grown and expanded in culture prior to introduction of the expression vector, followed by appropriate treatment for vector introduction and integration. The host cell can then be expanded and screened by a marker present in the vector.

[0308] A variety of markers that can be used are known in the art and include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, and the like. As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. In some embodiments, the host cell is an immune cell or a progenitor cell thereof, examples of which include T cells, NK cells, or NKT cells.

[0309] 3. Chemical methods Chemical means for introducing polynucleotides into host cells include colloidal dispersions such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems used as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).

[0310] When using non-viral delivery systems, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule associated with both the liposome and the oligonucleotide, incorporated into a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in a micelle or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they may exist as micelles within a bilayer structure or in a "disrupted" structure. They may also simply be dispersed in solution and may form aggregates that are not uniform in size or shape. Lipids are fatty substances and may be naturally occurring lipids or synthetic lipids. For example, lipids include, in addition to lipid droplets that naturally occur within the cytoplasm, a class of compounds that contains long-chain aliphatic hydrocarbons and their derivatives, examples of which include fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0311] Suitable lipids can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") can be obtained from Sigma (St. Louis, Missouri), dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, New York), cholesterol ("Choi") can be obtained from Calbiochem-Behring, and dimyristoyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Alabama). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Since chloroform evaporates more readily than methanol, it is used as the sole solvent. "Liposome" is a general term encompassing various single and multi-layer lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of aqueous solution, they form spontaneously. The lipid components undergo self-rearrangement before forming a closed structure and take up water and dissolved solutes between the lipid bilayers. Ghosh et al., Glycobiology 5:505-10 (1991). However, compositions having structures different from the normal vesicular structure in solution are also included. For example, the lipids may assume a micellar structure or simply exist as a heterogeneous aggregate of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0312] Regardless of the method used to introduce exogenous nucleic acids into a host cell, various assays may be performed to confirm the presence of a recombinant DNA sequence within the host cell. Such assays include, for example, "molecular biology" assays well known to those of skill in the art (such as Southern blotting, Northern blotting, RT-PCR, and PCR), and "biochemical" assays (such as detecting the presence or absence of a specific peptide by immunological means (ELISA and Western blot), or by the assays described herein for identifying agents within the scope of the present invention).

[0313] Moreover, the nucleic acid may be introduced by any means, examples of which include causing transduction in expanded T cells, transfecting expanded T cells, and performing electroporation on expanded T cells. One nucleic acid may be introduced into T cells by one method, and another nucleic acid may be introduced into T cells by a different method.

[0314] 4.RNA RNA has several advantages over more traditional plasmid or viral approaches. Gene expression from an RNA source does not require transcription, and protein products are rapidly produced after transfection. Furthermore, RNA only needs to access the cytoplasm rather than the nucleus, and thus extremely high transfection rates occur with common transfection methods. Additionally, plasmid-based approaches require that the promoter driving the expression of the gene of interest be active within the cells under study.

[0315] One of the advantages of the RNA transfection method of the present invention is that RNA transfection is essentially transient and does not require a vector. The RNA transgene can be delivered to and expressed in lymphocytes after a short period of in vitro cell activation as a minimal expression cassette without the need for any additional viral sequences. Under these conditions, the probability that the transgene integrates into the genome of the host cell is low. Due to the efficiency of RNA transfection and the ability to uniformly modify the entire lymphocyte population, cell cloning is not necessary.

[0316] Gene modification of host cells with in vitro transcribed RNA (TVT-RNA) uses two different strategies, both of which are being continuously tested in various animal models. Cells are transfected with in vitro transcribed RNA by means such as lipofection or electroporation. To achieve long-term expression of the transferred IVT-RNA, it is desirable to stabilize the IVT-RNA using various modifications. Several IVT vectors are known in the literature that are utilized in a standardized manner as templates for in vitro transcription and are genetically modified to produce stabilized RNA transcripts.

[0317] Currently, the protocols used in this technical field are based on the following structure, namely, a 5' RNA polymerase promoter that enables RNA transcription, followed by a gene of interest flanked by untranslated regions (UTRs) on either the 3' and / or 5', and a plasmid vector having a 3' polyadenylation cassette containing 50 - 70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized by a type II restriction enzyme downstream of the polyadenylation cassette (the recognition sequence corresponds to the cleavage site). Thus, the polyadenylation cassette corresponds to the late poly(A) sequence within the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization, extending or masking the poly(A) sequence at the 3' end. Whether this non-physiological overhang affects the amount of protein produced intracellularly from such constructs is not clear.

[0318] In one embodiment, the isolated nucleic acid encoding the CAR or bispecific fusion protein of the present disclosure and introduced into the cells of the present disclosure contains RNA. In one embodiment, the RNA is mRNA. In one embodiment, the RNA is in vitro transcribed (IVT) RNA. The RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). DNA of interest from any source can be directly converted to a template for in vitro mRNA synthesis by PCR using appropriate primers and RNA polymerase. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable DNA source.

[0319] In one embodiment, the DNA used for PCR contains an open reading frame. The DNA can be from a DNA sequence that naturally occurs from the genome of an organism. In one embodiment, the DNA is a full-length gene for the purpose of a part of a gene. The gene can include some or all of the 5' and / or 3' untranslated regions (UTRs). The gene can include exons and introns. In one embodiment, the DNA used for PCR is a human gene. In another embodiment, the DNA used for PCR is a human gene that includes a 5' UTR and a 3' UTR. Alternatively, the DNA can be an artificial DNA sequence that is not normally expressed in naturally occurring organisms. An exemplary artificial DNA sequence is a sequence containing portions of genes ligated together to form an open reading frame encoding a fusion protein. The portions of DNA ligated together can be from a single organism or from more than one organism.

[0320] Genes that can be used as a source of DNA for PCR include genes encoding polypeptides that provide a therapeutic or prophylactic effect to an organism, or genes that can be used to diagnose a disease or disorder of an organism. Preferred genes are genes useful for short-term treatment or genes with safety concerns regarding dosage or the expressed gene. For example, in the treatment of cancer, autoimmune disorders, parasites, viruses, bacteria, fungi, or other infectious diseases, the introduced gene(s) to be expressed can encode a polypeptide that functions as a ligand or receptor for cells of the immune system, or can function to stimulate or inhibit the immune system of the organism. In some embodiments, it is not desirable to continuously stimulate the immune system over a long period of time, nor is it necessary to create a change that persists after treatment has been successful. This is because this can potentially cause new problems. In the treatment of autoimmune disorders, it may be desirable to inhibit or suppress the immune system while symptoms are rapidly worsening, but it is not preferable to continue for a long time as the patient can become overly sensitive to infectious diseases.

[0321] Using PCR, a template for in vitro transcription of mRNA used for transfection is generated. Methods for performing PCR are well known in the art. Primers used in PCR are designed to have regions that are substantially complementary to regions of the DNA that is used as the template for PCR. As used herein, "substantially complementary" when used herein refers to a nucleotide sequence in which most or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary or mismatched. A substantially complementary sequence can anneal or hybridize to the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify portions of genes (open reading frames) that are normally transcribed intracellularly, including the 5'UTR and 3'UTR. Also, primers can be designed to amplify a portion of a gene that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of human cDNA, including all or part of the 5'UTR and 3'UTR. Primers useful for PCR are generated by synthetic methods well known in the art.

[0322] A "forward primer" is a primer that contains a region of nucleotides that is substantially complementary to the nucleotides on the DNA template that are upstream of the DNA sequence to be amplified. As used herein, "upstream" is used to refer to the location on the 5' side of the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that contains a nucleotide region that is substantially complementary to the double-stranded DNA template that is downstream of the DNA sequence to be amplified. As used herein, "downstream" is used to refer to the location on the 3' side of the DNA sequence to be amplified relative to the coding strand.

[0323] Any DNA polymerase useful for PCR can be used by the methods disclosed herein. Reagents and polymerases are commercially available from multiple sources. Chemical structures having the ability to promote stability and / or translation efficiency may also be used. RNA preferably has a 5'UTR and a 3'UTR. In one embodiment, the length of the 5'UTR is from 0 to 3000 nucleotides. The lengths of the 5'UTR sequence and the 3'UTR sequence added to the coding region can be altered by various methods, including, but not limited to, the design of PCR primers that anneal to various regions of the UTR. Using this approach, one of ordinary skill in the art can alter the lengths of the 5'UTR and 3'UTR necessary to achieve optimal translation efficiency after transfection of the transcribed RNA. The 5'UTR and 3'UTR can be the naturally occurring endogenous 5'UTR and 3'UTR of the gene of interest. Alternatively, a UTR sequence that is not endogenous to the gene of interest can be added by incorporating the UTR sequence into forward and reverse primers or by altering the template in any other way. The use of a UTR sequence that is not endogenous to the gene of interest can be useful for altering the stability and / or translation efficiency of the RNA. For example, AU-rich elements within the 3'UTR sequence are known to potentially decrease mRNA stability. Thus, based on the properties of UTRs well known in the art, the 3'UTR can be selected or designed to improve the stability of the transcribed RNA.

[0324] In one embodiment, the 5’UTR can contain the Kozak sequence of an endogenous gene. Alternatively, as described above, when a non-endogenous 5’UTR is added to the gene of interest by PCR, the consensus Kozak sequence can be redesigned by adding the 5’UTR sequence. The Kozak sequence can improve the translation efficiency of some RNA transcripts, but it is not thought to be necessary for all RNAs to enable efficient translation. It is known in the art that many mRNAs require the Kozak sequence. In other embodiments, the 5’UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3’UTR or 5’UTR to inhibit exonucleolytic degradation of the mRNA.

[0325] To enable synthesis of RNA from a DNA template without the need for gene cloning, it is necessary to attach a transcription promoter to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for RNA polymerase is added to the 5’ end of the forward primer, the promoter for RNA polymerase is incorporated into the PCR product upstream of the open reading frame to be transcribed. In a preferred embodiment, the promoter is the T7 polymerase promoter as described elsewhere herein. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of the T7 promoter, T3 promoter, and SP6 promoter are known in the art.

[0326] In a preferred embodiment, the mRNA has both a 5' end cap and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability in the cell. On circular DNA templates (e.g., plasmid DNA), RNA polymerase produces long-chain products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3' UTR yields a normally sized mRNA that is not effective for eukaryotic transfection, even post-transcriptionally. With a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template. Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65(2003).

[0327] The conventional method of integrating a poly A / T stretch into a DNA template is molecular cloning. However, poly A / T sequences integrated into plasmid DNA can cause plasmid instability, so plasmid DNA templates obtained from bacterial cells often contain a high degree of deletions and other abnormalities. For this reason, the cloning procedure is not only cumbersome and time-consuming, but often unreliable. Therefore, a method that can construct a DNA template with a poly A / T 3' stretch without cloning is highly desirable.

[0328] The poly A / T segment of the transcribed DNA template can be produced during PCR by using a reverse primer containing a poly T tail, such as a 100T tail (the size can range from 50 to 5000T), or can also be produced after PCR by any other method (including but not limited to DNA ligation or in vitro recombination). The poly(A) tail also provides stability to RNA and reduces RNA degradation. Generally, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100 to 5000 adenosines.

[0329] The poly(A) tail of RNA can be further extended after in vitro transcription using a poly(A) polymerase such as Escherichia coli poly A polymerase (E-PAP). In one embodiment, by increasing the length of the poly(A) tail from 100 nucleotides to 300 - 400 nucleotides, the translation efficiency of RNA increases by approximately twofold. Additionally, attaching different chemical groups to the 3' end can improve mRNA stability. Such attachments can contain modified / artificial nucleotides, aptamers, and other compounds. For example, an ATP analog can be incorporated into the poly(A) tail using a poly(A) polymerase. The ATP analog can further improve the stability of RNA. The 5' cap also confers stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein contains a 5' cap. The 5' cap is known in the art and is provided using the techniques described herein (Cougot, et al., Trends in Biochem. Sci., 29:436 - 444 (2001); Stepinski, et al., RNA, 7:1468 - 95 (2001); Elango, et al., Biochim. Biophys. Res. Commun, 330:958 - 966 (2005)).

[0330] The RNA produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates the binding of cap-independent ribosomes to mRNA and promotes the initiation of translation. Any solute suitable for cell electroporation that can contain factors that promote cell permeability and viability can be included, examples of which include sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0331] The RNA can be introduced into target cells using any of a plurality of different methods. For example, commercially available methods include electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), ECM830 (BTX) (Harvard Instruments, Boston, MA), or Gene Pulser II (BioRad, Denver, CO), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or a biolistic particle delivery system such as a "gene gun" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70(2001)), but are not limited thereto.

[0332] In some embodiments, the RNA is introduced into the cell by electroporation, examples of which include in vitro transcribed RNA. The formulation and methodology of electroporation of nucleic acid constructs into mammalian cells are taught, for example, in US2004 / 0014645, US2005 / 0052630A1, US2005 / 0070841Al, US2004 / 0059285A1, US2004 / 0092907A1. Various parameters, including the electric field strength, required for electroporation of any known cell type are generally known in the relevant research literature and in numerous patents and applications in this field. See, for example, U.S. Patent No. 6,678,556, U.S. Patent No. 7,171,264, U.S. Patent No. 7,173,116. Devices for the therapeutic use of electroporation are commercially available (e.g., MedPulser® DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.)) and are described in patents such as U.S. Patent No. 6,567,694, U.S. Patent No. 6,516,223, U.S. Patent No. 5,993,434, U.S. Patent No. 6,181,964, U.S. Patent No. 6,241,701, and U.S. Patent No. 6,233,482. Also, electroporation may be used for in vitro transfection of cells as described, for example, in US20070128708A1. Also, electroporation may be utilized to deliver nucleic acids to cells in vitro.

[0333] Thus, administering nucleic acids, including expression constructs, to cells by electroporation using any of a number of available devices and electroporation systems known to those of skill in the art provides an exciting new means for delivering the desired RNA to target cells.

[0334] The disclosed method can be applied to the regulation of host cell activity in basic research and treatment in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including the evaluation of the ability of genetically modified host cells to kill target cancer cells. This method also provides the ability to control the level of expression over a wide range, for example, by altering the promoter or the amount of input RNA, allowing the individual regulation of the expression level. Furthermore, PCR-based mRNA production techniques greatly facilitate the design of mRNAs having various structures and combinations of their domains.

[0335] C. Source of immune cells Prior to expansion, a source of immune cells is obtained from a subject for ex vivo manipulation. Sources of target cells for ex vivo manipulation may also include, for example, blood, cord blood, or bone marrow from an autologous donor or an allogeneic donor. For example, the source of immune cells may be from a subject to be treated with the modified immune cells of the present invention, such as the subject's blood, the subject's cord blood, or the subject's bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. In a particular exemplary embodiment, the subject is human.

[0336] Immune cells can be obtained from multiple sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, examples of which include cells of innate or adaptive immunity, such as myeloid cells or lymphoid cells (including lymphocytes, typically T cells and / or NK cells and / or NKT cells). Other exemplary cells include stem cells, examples of which include multipotent stem cells and pluripotent stem cells (including induced pluripotent stem cells (iPSCs)). In certain embodiments, the cells are human cells. With respect to the subject to be treated, the cells may be allogeneic and / or autologous. Generally, the cells are primary cells, examples of which include cells isolated directly from a subject and / or cells isolated from a subject and cryopreserved.

[0337] In certain embodiments, the immune cells are T cells, such as CD8 + T cells (such as CD8 + naïve T cells, central memory T cells, or effector memory T cells), CD4 + T cells, natural killer T cells (NKT cells), regulatory T cells (Tregs), stem cell memory T cells, lymphoid progenitor cells, hematopoietic stem cells, natural killer cells (NK cells), natural killer T cells (NK cells) or dendritic cells. In some embodiments, the cells are monocytes or granulocytes, examples of which include myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and / or basophils. In one embodiment, the target cells are induced pluripotent stem (iPS) cells, or cells derived from iPS cells, examples of which are generated from a subject and are engineered to alter (e.g., induce mutations in) or manipulate the expression of one or more target genes, such as T cells, such as CD8 + T cells (such as CD8 + naïve T cells, central memory T cells or effector memory T cells), CD4 + T cells, stem cell memory T cells, lymphoid progenitor cells or hematopoietic stem cells into which the iPS cells have differentiated.

[0338] In some embodiments, the cells comprise one or more subsets of T cells or other cell types, examples of which include the entire T cell population, CD4 + cells, CD8 + cells, and subpopulations thereof, examples of which are defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization and / or persistence capacity, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. T cells and / or CD4 + T cells and / or CD8 +Among the subtypes and subpopulations of T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells, and their subtypes (such as stem cell memory T cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM) or terminally differentiated effector memory T cells, etc.), tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells (such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells and delta / gamma T cells, etc.). In certain embodiments, any number of T cell lines available in the art may be used.

[0339] In some embodiments, the method includes isolating immune cells from a subject, preparing them, treating them, culturing them, and / or manipulating them. In some embodiments, the preparation of the manipulated cells includes one or more culturing steps and / or preparation steps. The cells for the manipulation described may be isolated from a sample, such as a biological sample obtained from or derived from a subject, for example. In some embodiments, the subject from whom the cells are isolated has a disease or condition, or requires cell therapy, or is a subject to whom cell therapy is administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, examples of which include adoptive cell therapy in which cells are isolated, treated, and / or manipulated. Thus, the cells in some embodiments are primary cells, such as primary human cells. The sample includes tissues, fluids, and other samples taken directly from the subject, as well as samples obtained from one or more processing steps, examples of which include separation, centrifugation, genetic manipulation (e.g., transduction by a viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a processed sample. Biological samples include, but are not limited to, body fluids (such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), tissue and organ samples (including processed samples derived therefrom).

[0340] In certain aspects, the sample from which the cells are derived or isolated is blood, or a blood-derived sample, or is derived from an apheresis product or a leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organs, and / or cells derived therefrom. The sample includes samples from autologous and allogeneic sources in the context of cell therapy (e.g., adoptive cell therapy).

[0341] In some embodiments, the cells are derived from a cell line, such as a T cell line. The cells in some embodiments are obtained from a heterologous source, such as from mice, rats, non-human primates, and pigs. In some embodiments, the isolation of the cells includes one or more preparation and / or cell separation steps based on non-affinity. In some examples, the cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, concentrate desired components, and lyse or remove cells sensitive to specific reagents. In some examples, the cells are separated based on one or more characteristics, examples of which include density, adhesion properties, size, sensitivity, and / or resistance to specific components.

[0342] In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. In certain embodiments, the sample contains lymphocytes (including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets), and in certain embodiments, contains cells other than red blood cells and platelets. In some embodiments, the blood cells collected from the subject are washed, for example, to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some specific embodiments, the washing step is achieved by tangential flow filtration (TFF) according to the manufacturer's instructions. In certain embodiments, the cells are resuspended in various biocompatible buffers after washing. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in the culture medium. In some embodiments, the method includes a density-based cell separation method, examples of which include the preparation of white blood cells from peripheral blood by lysing red blood cells and centrifuging through a Percoll gradient or a Ficoll gradient.

[0343] In one embodiment, immune cells are obtained from an individual's circulating blood by apheresis or leukapheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing steps, examples of which include phosphate-buffered saline (PBS) or a wash solution, which may be calcium-deficient, magnesium-deficient, or may lack many divalent cations, if not all. As will be readily understood by those skilled in the art, the washing step may be accomplished by methods known to those skilled in the art, examples of which include the use of a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in various biocompatible buffers, examples of which include, for example, Ca 2+ -free and Mg 2+ -free PBS, PlasmaLyte A, or another saline solution regardless of the presence or absence of buffer. In some embodiments, undesirable components of the apheresis sample may be removed and the cells may be resuspended directly in the culture medium.

[0344] In some embodiments, the isolation method involves separating various cell types based on the intracellular expression or presence of one or more specific molecules, examples of such specific molecules including surface markers (e.g., surface proteins), intracellular markers, or nucleic acids. In some embodiments, any known separation method based on such markers may be used. In some embodiments, the separation is an affinity or immunoaffinity-based separation. For example, in certain aspects, the isolation involves separating cells and cell populations based on the expression or expression level of one or more markers (typically cell surface markers) in the cells, for which purpose, for example, incubation with an antibody or binding partner that specifically binds to such a marker is carried out, followed by generally a washing step and separation of the cells to which the antibody or binding partner has bound from the cells that have not bound to the antibody or binding partner. Such separation steps can be based on positive selection in which the cells to which the reagent has bound are retained for further use, and / or negative selection in which the cells that have not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use.

[0345] In certain embodiments, negative selection can be particularly useful where antibodies that specifically identify cell types within a heterogeneous population are not available, and separation can be most effectively performed based on markers expressed by cells other than the desired population. As a result of this separation, it is not necessary to achieve 100% enrichment or depletion of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cells, such as cells expressing a marker, refers to increasing the number or proportion of such cells, but it is not necessary for cells that do not express the marker to be completely absent. Similarly, negative selection, removal or depletion of a particular type of cells, such as cells expressing a marker, refers to decreasing the number or proportion of such cells, but it is not necessary for all such cells to be completely removed. In certain exemplary embodiments, multiple rounds of separation steps are performed, where a fraction positively selected or negatively selected from one step is then subjected to another separation step such as positive selection or negative selection.

[0346] In certain exemplary embodiments, a single separation step can deplete cells that simultaneously express multiple markers, for example, by incubating the cells with multiple antibodies or binding partners that are each specific for a marker that is a target for negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating the cells with multiple antibodies or binding partners that are expressed in the various cell types.

[0347] In some embodiments, in one or more of the T cell populations, cells that are positive (marker i+) or express them at a high level (marker high) for one or more specific markers (such as surface markers), or cells that are negative (marker -) or express them at a relatively low level (marker low) for one or more markers, are enriched or depleted. For example, in certain embodiments, one or more surface markers (e.g., CD28 + , CD62L+ 、CCR7 + 、CD27 + 、CD127 + 、CD4 + 、CD8 + 、CD45RA + and / or CD45RO + A specific subpopulation of T cells that is positive for, or expresses them at high levels, such as cells that are positive for (e.g., CD8 + cells or T cells, e.g., CD3 + cells) for CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L, or cells that express them at high surface levels are enriched (i.e., positively selected), and / or cells that are positive for CD45RA, or express them at high surface levels are depleted (e.g., negatively selected). In some embodiments, for cells, cells that are positive for, or express them at high surface levels, for CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127) are enriched or depleted. In a particular exemplary embodiment, for CD8 + T cells, cells that are positive for CD45RO (or negative for CD45RA) and positive for CD62L are enriched. For example, CD3 + 、CD28 + T cells can be positively selected using magnetic beads conjugated with CD3 / CD28 (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0348] In some embodiments, T cells are isolated from a PBMC sample by negative selection of markers expressed on non-T cells, examples of which non-T cells include B cells, monocytes, or other white blood cells (such as CD14). In certain aspects, CD4 + or CD8 + selection steps are used to isolate CD4 + helper T cells and CD8 + cytotoxic T cells. Such CD4 + and CD8 + populations can be further classified into subpopulations that express, or express to a relatively high degree, one or more of naive T cells, memory T cells and / or effector T cells, or by positive or negative selection for markers that are expressed. In some embodiments, in CD8+ cells, naive stem cells, central memory stem cells, effector memory stem cells and / or central memory stem cells are either further enriched or depleted, which is by, for example, positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is performed to improve efficacy, for example, to improve long-term survival, expansion and / or engraftment after administration, which in certain aspects is particularly potent in such subpopulations.

[0349] In some embodiments, the efficacy is further enhanced when CD8 + T cells rich in TCM are combined with CD4 + T cells. In some embodiments, memory T cells are present in both the CD62L + subset and the CD62L- subset of peripheral blood lymphocytes. In PBMCs, the CD62L-CD8 + and / or CD62L + CD8 + fractions can be enriched or depleted, which is by, for example, using anti-CD8 antibodies and anti-CD62L antibodies. In some embodiments, CD4 + T cell populations and / or CD8 + T cell populations+ In the T cell population, central memory (TCM) cells are enriched. In some embodiments, the enrichment for central memory T (TCM) cells is based on being positive for or having high surface expression of CD45RO, CD62L, CCR7, CD28, CD8 and / or CD127. In certain embodiments, the enrichment is based on negative selection against cells that express or highly express CD45RA and / or granzyme B. In certain embodiments, the CD8 population enriched for TCM cells + is isolated by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one embodiment, the enrichment for central memory T (TCM) cells is carried out starting from the negative fraction of cells selected based on CD4 expression, and this negative fraction undergoes negative selection based on expression of CD14 and CD45RA and positive selection based on CD62L. Such selections are carried out simultaneously in certain embodiments and sequentially in any order in other embodiments. In some particular methods, the CD8 + The same CD4 expression-based selection step used to prepare the cell population or subpopulation is used for the CD4 + cell population or subpopulation as well, so that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, optionally followed by one or more additional positive or negative selection steps.

[0350] CD4 + T helper cells are classified into naive cells, central memory cells, and effector cells by identifying cell populations having cell surface antigens. CD4 + lymphocytes can be obtained by standard methods. In some embodiments, naive CD4 + T lymphocytes are CD45RO - , CD45RA + , CD62L + , CD4 +It is a T cell. In some embodiments, the central memory CD4 + cells are CD62L + and CD45RO + In some embodiments, the effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich CD4 + cells by negative selection, the monoclonal antibody mixture generally includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix such as magnetic beads or paramagnetic beads, enabling the separation of cells for positive selection and / or negative selection.

[0351] In some embodiments, the cells are incubated and / or cultured before or in connection with genetic manipulation. The incubation step can include culturing, cultivating, stimulating, activating and / or proliferating. In some embodiments, the composition or cells are incubated in the presence of a stimulating condition or a stimulating agent. Such conditions include conditions designed to induce the growth, expansion, activation and / or survival of cells in the population, mimic antigen exposure, and / or prime the cells for genetic manipulation (such as the introduction of a recombinant antigen receptor). Such conditions can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agent (such as nutrients, amino acids, antibiotics, ions and / or stimulants), examples of which include cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agent designed to activate the cells. In some embodiments, the stimulating condition or stimulating agent includes one or more agents, such as a ligand, that can activate the intracellular signaling domain of the TCR complex.

[0352] In certain embodiments, the agent activates or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such an agent can include an antibody and / or one or more cytokines. Examples of such antibodies include antibodies specific for TCR components and / or co-stimulatory receptors, examples of which include anti-CD3 and anti-CD28 bound to a solid support such as beads, for example. Optionally, the expansion method may further comprise the step of adding an anti-CD3 antibody and / or an anti-CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulating agent includes IL-2 and / or IL-15. For example, the IL-2 concentration is at least about 10 units / mL.

[0353] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example, by centrifugal isolation using a Percoll™ gradient. Alternatively, T cells can be isolated from umbilical cord. In any case, a specific subpopulation of T cells can be further isolated by positive selection techniques or negative selection techniques. In umbilical cord blood mononuclear cells isolated in this way, cells expressing certain antigens may be depleted, including but not limited to CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be achieved using isolated antibodies, biological samples containing antibodies such as ascites, antibodies bound to a physical support, and antibodies bound to cells.

[0354] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugal isolation using a Percoll™ gradient or by counterflow centrifugal elutriation. CD3 + , CD28 + , CD4 + , CD8 + , CD45RA + and CD45RO +Specific subpopulations of T cells, such as T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubating for a period sufficient for positive selection of the desired T cells using beads conjugated with anti-CD3 / anti-CD28 (i.e., 3x28), such as DYNABEADS® M-450 CD3 / CD28 T. In one embodiment, that period is about 30 minutes. In a further embodiment, the period ranges from 30 minutes to 36 hours or more and all integer values in between. In a further embodiment, the period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the period is 10 - 24 hours. In a preferred embodiment, the incubation period is 24 hours. In the case of isolating T cells from a patient with leukemia, the use of a longer incubation time, such as 24 hours, can improve cell yield. Using a longer incubation time, for example, when isolating tumor-infiltrating lymphocytes (TIL) from tumor tissue or an immunocompromised individual, T cells may be isolated under any circumstances where there are only a few T cells compared to other cell types. Furthermore, by using a longer incubation time, the capture efficiency of CD8 + T cells can be improved. Thus, by simply shortening or lengthening the time during which T cells can bind to CD3 / CD28 beads and / or (as further described herein) by increasing or decreasing the ratio of beads to T cells, it is possible to preferentially select whether to include or not include subpopulations of T cells at the start of culture or at other times during processing. Furthermore, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, it is possible to preferentially select whether to include or not include subpopulations of T cells at the start of culture or at other desired times. One skilled in the art will recognize that multiple rounds of selection can also be used in the context of the present invention.

[0355] In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells for activation and expansion processes. "Unselected" cells may also be subject to further rounds of selection. Enrichment of a T cell population by negative selection can be achieved using a combination of antibodies that target unique surface markers on the negatively selected cells. Exemplary methods include negative magnetic immunoadhesion or flow cytometry cell sorting and / or cell selection using a mixture of monoclonal antibodies that target cell surface markers present on the negatively selected cells. For example, to enrich for CD4 + cells by negative selection, the mixture of monoclonal antibodies typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0356] In certain embodiments, it may be desirable to enrich or positively select for regulatory T cells that typically express CD4 + , CD25 + , CD62L M , GITR + , and FoxP3 + .

[0357] Alternatively, in certain embodiments, regulatory T cells are depleted by beads conjugated with anti-C25 or other similar selection methods. The concentration of cells and surfaces (e.g., particles such as beads) can be varied to isolate a desired population of cells by positive or negative selection. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the beads and the cells. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In yet another embodiment, a concentration of more than 100 million cells / ml is used. In yet another embodiment, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells / ml are used. In yet another embodiment, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million or 1 billion cells / ml are used. In further embodiments, concentrations of 125 million or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation and cell expansion. Further, using high cell concentrations enables more efficient capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells, or cells from samples with many tumor cells (i.e., leukemia blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and may be desirable to obtain. For example, using high concentrations of cells enables more efficient selection of CD8 + T cells that typically have weaker CD28 expression.

[0358] In related embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surfaces (e.g., particles such as beads), the interaction between the particles and the cells is minimized. This results in the selection of cells that express a large amount of the desired antigen to be bound to the particles. For example, CD4 + T cells express a higher level of CD28 and diluted concentrations of CD8 +It is captured more efficiently than T cells. In one embodiment, the concentration of the cells used is 5×10 6 / ml. In other embodiments, the concentration used can be from about 1×10 5 / ml to 1×10 6 / ml, and any integer value therebetween.

[0359] In other embodiments, the cells may be incubated on a rotator at various speeds, for various lengths of time, at either 2-10°C or room temperature. The T cells for stimulation can also be frozen after the washing step. Without being bound by theory, the freezing step and subsequent thawing step provide a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. Many freezing solutions and parameters are known in the art and useful in this context, but one method involves using PBS containing 20% DMSO and 8% human serum albumin, or a culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or PlasmaLyte A at 31.25%, dextrose 5% at 31.25%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 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° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods, or even methods of uncontrolled freezing immediately at -20°C or in liquid nitrogen, may be used. In certain embodiments, the cryopreserved cells are thawed and washed as described herein and allowed to stand at room temperature for 1 hour prior to activation using the methods of the present invention.

[0360] Also, in the context of the present disclosure, it is also contemplated to collect a blood sample or an apheresis product from a subject during a period prior to the time when expanded cells as described herein are required. Thus, the source of the cells to be expanded can be collected at any desired time, isolate the desired cells such as T cells, and freeze them for later use in T cell therapy for any number of diseases or conditions (such as those described herein) that would benefit from T cell therapy. In one embodiment, a blood sample or apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or apheresis is taken from a generally healthy subject who is at risk of developing a disease but has not yet developed the disease. The cells of interest are isolated and frozen for later use. In certain embodiments, the T cells may be expanded, frozen, and used at a later time. In certain embodiments, the sample is collected from the patient shortly after diagnosis of a particular disease as described herein, but before any treatment is administered. In a further embodiment, the cells are isolated from a blood sample or apheresis from the subject prior to any number of related treatment modalities, including treatments using agents (examples of which include natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents (such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506), antibodies), or other immune-depleting agents (examples of which include CAMPATH, anti-CD3 antibody, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation), but are not limited thereto. These drugs either inhibit calcineurin, a calcium-dependent phosphatase (cyclosporine and FK506), or inhibit p70S6 kinase, which is important for growth factor-induced signal transduction (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993).In a further embodiment, the cells are isolated for the patient and cryopreserved for later use in conjunction with (e.g., before, simultaneously, or after) bone marrow transplantation or stem cell transplantation, any chemotherapeutic agent (such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, etc.), T cell depletion therapy using an antibody (such as OKT3 or CAMPATH), or B cell depletion therapy using an agent that reacts with CD20 (such as rituxan). In another embodiment, the cells can be isolated prior to B cell depletion therapy using an agent that reacts with CD20 (such as rituxan) and cryopreserved for later use for treatment after B cell depletion therapy.

[0361] In a further embodiment of the present disclosure, the T cells are obtained from the patient immediately after treatment. In this regard, in certain treatments using drugs that damage the immune system following treatment of a particular cancer, it has been observed that shortly after treatment, during the period when the patient would normally be recovering from the treatment, the quality of the T cells obtained can be optimized or improved with respect to their ability to expand ex vivo. Similarly, after ex vivo manipulation using the methods described herein, these cells can be in a state favorable for enhanced engraftment and in vivo expansion. Accordingly, collection of blood cells, including T cells, dendritic cells, or other cells of the hematopoietic system, at this recovery stage is contemplated within the context of the present invention. Further, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create a state favorable for the repopulation, recirculation, regeneration, and / or expansion of certain cell types, particularly during a determined period after treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0362] It is also possible to freeze the T cells after the washing step, in which case the monocyte removal step is not necessary. Without wishing to be bound by theory, the freezing step and subsequent thawing step remove granulocytes in the cell population and to some extent remove monocytes, providing a more uniform product. After the washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. Many freezing solutions and parameters are known in the art and useful in this context, but in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen at a rate of 1°C per minute to -80°C and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods, or even methods of uncontrolled freezing immediately at -20°C or in liquid nitrogen, may be used.

[0363] In one embodiment, the population of T cells is contained intracellularly and examples thereof include peripheral blood mononuclear cells, cord blood cells, a population of purified T cells, and T cell lines. In another embodiment, the peripheral blood mononuclear cells contain the population of T cells. In yet another embodiment, the purified T cells contain the population of T cells.

[0364] D. Expansion of Immune Cells The cells can be activated and expanded in number, either before or after modifying the cells to express the subject CAR or bispecific fusion protein, using methods such as those 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. Publication No. 20060121005.

[0365] For example, the immune cells of the present disclosure may be expanded by contacting them with a surface to which an agent that stimulates CD3 / TCR complex-related signals and a ligand that stimulates co-stimulatory molecules on the surface of the immune cells are attached. In particular, an immune cell population may be stimulated 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., biyostatin) in combination with a calcium ionophore. A ligand that binds to an accessory molecule is used to co-stimulate the accessory molecule on the surface of the immune cells. For example, under conditions suitable for stimulating the proliferation of immune cells, the immune cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Buzanson, France), and these can be used in the present invention as well as other methods and reagents known in the art. See, for example, ten Berge et al., Transplant Proc. 30(8):3975-3977 (1998); Haanen et al., J. Exp. Med. 190(9):1319-1328 (1999); and Garland et al., J. Immunol. Methods 227(1-2):53-63 (1999).

[0366] The expansion of immune cells by the methods disclosed herein can be increased by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more, and can be any whole or partial integer therebetween. In one embodiment, the immune cells expand in the range of about 20-fold to about 50-fold.

[0367] After culturing, the immune cells can be incubated in the cell culture medium in the culture device for a certain period or until the cells reach a confluency or high cell density suitable for optimal passage, and then the cells can be transferred to another culture device. The culture device can be any culture device commonly used for culturing cells in vitro. In certain exemplary embodiments, the confluency level before transferring the cells to another culture device is 70% or more. In particularly exemplary embodiments, the confluency level is 90% or more. The period can be any time suitable for culturing cells in vitro. The immune cell culture medium can be replaced at any time during the culture of the immune cells. In certain exemplary embodiments, the immune cell culture medium is replaced approximately every 2 to 3 days. The immune cells can then be harvested from the culture device and either used immediately or cryopreserved and stored for later use. In one embodiment, the present invention includes cryopreserving the expanded immune cells. The cryopreserved immune cells are thawed before introducing nucleic acid into the immune cells.

[0368] In another embodiment, the method includes isolating immune cells and expanding the immune cells. In another embodiment, the present invention further includes cryopreserving the immune cells before expansion. In yet another embodiment, the cryopreserved immune cells are thawed for electroporation using RNA encoding a chimeric membrane protein.

[0369] Another procedure for ex vivo expanded cells is described in U.S. Patent No. 5,199,942 (incorporated herein by reference). The expansion as described in U.S. Patent No. 5,199,942 can be an alternative or additional method to other expansion methods described herein. Briefly, ex vivo culture and expansion of immune cells involves addition of cell growth factors such as those described in U.S. Patent No. 5,199,942, or other factors such as flt3-L, IL-1, IL-3, and c-kit ligand. In one embodiment, expansion of immune cells involves culturing the immune cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand. The culturing step (after contact with an agent or electroporation as described herein) can be made very short, for example, less than 24 hours, examples of which include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step (contact with an agent as described herein) can be made longer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more, as further described herein.

[0370] Various terms are used to describe cells in culture. Cell culture generally refers to cells taken from a living body and grown under controlled conditions. Primary cell culture is the culture of cells, tissues, or organs taken directly from an organism and performed prior to the first subculture. Cells are placed in a growth medium under conditions that promote cell growth and / or cell division and expand during culture, resulting in a larger population of cells. When cells expand in culture, the rate of cell proliferation is generally measured by the time required for the cell number to double (otherwise known as the doubling time).

[0371] Each round of subculture is called a passage. When cells are subcultured, they are said to have been passaged. A specific population of cells, i.e., a cell line, may be called or characterized by the number of passages. For example, a cultured cell population that has been passaged 10 times may be called a P10 culture. The primary culture, i.e., the first culture after isolating cells from tissue, is designated P0. Following the first subculture, the cells are described as a secondary culture (P1 or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. Those skilled in the art will understand that the number of cells may double many times during the passage period. Thus, the number of cell doublings in a culture will be greater than the passage number. The expansion of cells (i.e., the number of cell doublings) during the period between passages depends on many factors, including, but not limited to, seeding density, substrate, medium, and the time between passages.

[0372] In certain embodiments, primary stimulation signaling and co-stimulation signals to T cells may be provided by various protocols. For example, the agents providing each signal may be present in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in a "cis" configuration) or to a different surface (i.e., in a "trans" configuration). Alternatively, one agent may be coupled to a surface and the other agent may be present in solution. In one embodiment, the agent providing the co-stimulation signal is bound to the cell surface and the agent providing the primary activation signal is present in solution or coupled to a surface. In certain embodiments, both agents may be present in solution. In another embodiment, the agent may be in a soluble form and then cross-linked to a surface, examples of which include cells expressing Fc receptors or antibodies, or other binding agents that bind to the agent. In this regard, for artificial antigen-presenting cells (aAPCs) contemplated for use in activating and expanding T cells in the present invention, see, for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810.

[0373] In one embodiment, the two agents are immobilized on beads either on the same bead (i.e., "cis") or on a different bead (i.e., "trans"). As an example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the co-stimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof. Both agents are co-immobilized on the same bead with an equivalent molecular weight. In one embodiment, a ratio of 1:1 of each antibody bound to the bead is used for the expansion of CD4+ T cells and the growth of T cells. In a particular aspect of the invention, the ratio of anti-CD3:CD28 antibodies bound to the bead is used such that an increase in the expansion of T cells is observed compared to the expansion observed when using a 1:1 ratio. In a particular embodiment, an increase of about 1-fold to about 3-fold is observed compared to the expansion observed when using a 1:1 ratio. In one embodiment, the ratio of CD3:CD28 antibodies bound to the bead ranges from 100:1 to 1:100 and includes all integer values therebetween. In one aspect of the invention, more anti-CD28 antibodies are bound to the particle than anti-CD3 antibodies. That is, the CD3:CD28 ratio is less than 1. In a particular embodiment of the invention, the ratio of anti-CD28 antibody bound to the bead to anti-CD3 antibody is greater than 2:1. In a particular embodiment, a CD3:CD28 ratio of 1:100 of the antibody bound to the bead is used. In another embodiment, a CD3:CD28 ratio of 1:75 of the antibody bound to the bead is used. In a further embodiment, a CD3:CD28 ratio of 1:50 of the antibody bound to the bead is used. In another embodiment, a CD3:CD28 ratio of 1:30 of the antibody bound to the bead is used. In a preferred embodiment, a CD3:CD28 ratio of 1:10 of the antibody bound to the bead is used. In another embodiment, a CD3:CD28 ratio of 1:3 of the antibody bound to the bead is used. In yet another embodiment, a CD3:CD28 ratio of 3:1 of the antibody bound to the bead is used.

[0374] Ratios of particles to cells from 1:500 to 500:1, and any integer value therebetween, may be used to stimulate T cells, or other target cells. As will be readily understood by those skilled in the art, the ratio of particles to cells may depend on the particle size relative to the target cells. For example, small-sized beads can bind to only a few cells, while larger beads can bind to more cells. In certain embodiments, the ratio of cells to particles ranges from 1:100 to 100:1, and any integer value therebetween, and in further embodiments, the ratio includes 1:9 to 9:1, and any integer value therebetween, and these can also be used to stimulate T cells. The ratio of anti-CD3-coupled particles and anti-CD28-coupled particles to T cells that results in T cell stimulation can vary as shown above, but 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, and one preferred ratio is at least 1:1 particles per T cell. In one embodiment, a ratio of 1:1 or less of particles to cells is used. In one particular embodiment, a preferred particle:cell ratio is 1:5. In further embodiments, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is 1:1 to 10:1 on the first day, and additional particles are added to the cells daily or every other day for up to 10 days, and the final ratio is 1:1 to 1:10 (based on the number of cells on the day of addition). In one particular embodiment, the ratio of particles to cells is 1:1 on the first day of stimulation and is adjusted to 1:5 on the 3rd and 5th days of stimulation. In another embodiment, particles are added daily or every other day such that the final ratio is 1:1 on the first day of stimulation and 1:5 on the 3rd and 5th days of stimulation. In another embodiment, the ratio of particles to cells is 2:1 on the first day of stimulation and is adjusted to 1:10 on the 3rd and 5th days of stimulation. In another embodiment, particles are added daily or every other day such that the final ratio is 1:1 on the first day of stimulation and 1:10 on the 3rd and 5th days of stimulation.One skilled in the art will understand that other various ratios may be suitable for use in the present invention. In particular, the ratios vary depending on the particle size, as well as depending on the cell size and cell type. In a further embodiment of the present invention, cells such as T cells are mixed with beads coated with an agent, then the beads and the cells are separated, and then the cells are cultured. In an alternative embodiment, before culturing, the beads coated with the agent and the cells are not separated and are cultured together. In a further embodiment, the beads and the cells are first concentrated by applying a force such as a magnetic force, resulting in an increase in the ligation of cell surface markers, thereby inducing cell stimulation.

[0375] As an example, cell surface proteins may be ligated by enabling paramagnetic beads (3x28 beads) attached with anti-CD3 and anti-CD28 to contact T cells. In one embodiment, cells (e.g., 104 to 109 T cells) and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads at a ratio of 1:1) are mixed in a buffer, preferably PBS (without divalent cations such as calcium and magnesium). Here too, those skilled in the art can readily understand that any cell concentration may be used. For example, the target cells may be very dilute in the sample and contain only 0.01% of the sample, or the entire sample (i.e., 100%) may contain the target cells of interest. Thus, any number of cells is within the context of the present invention. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between the cells and the particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In another embodiment, more than 100 million cells / ml is used. In a further embodiment, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells / ml are used. In yet another embodiment, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million or 100 million cells / ml are used. In further embodiments, concentrations of 125 million or 150 million cells / ml can be used. Using a high concentration can result in an increase in cell yield, cell activation and cell expansion. Furthermore, using a high cell concentration enables more efficient capture of cells that may weakly express a target antigen such as CD28-negative T cells. Such a population of cells may have therapeutic value and in certain embodiments it is desirable to obtain them. For example, using a high concentration of cells enables more efficient selection of CD8+ T cells that typically have weaker CD28 expression.

[0376] In one embodiment, the cells may be cultured for several hours (about 3 hours) to about 14 days, or for any integer value of time unit therebetween. Conditions suitable for immune cell culture include a suitable medium (e.g., Minimal Essential Medium or RPMI Medium 1640 or X-vivo15 (Lonza)) that may contain factors necessary for growth and viability, among which are serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, TNF-α, or any other optional additive for cell growth known to skilled artisans. Other additives for cell growth include, but are not limited to, surfactants, plasmanate, and reducing agents (such as N-acetyl-cysteine and 2-mercaptoethanol). The medium can include RPMI1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, to which amino acids, sodium pyruvate, and vitamins are added, and which either does not contain serum, or is supplemented with an appropriate amount of serum (or plasma), or a defined set of hormones, and / or a sufficient amount of cytokine(s) for the growth and expansion of immune cells. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures and not in cultures of cells to be injected into a subject. The target cells are maintained under conditions necessary to support growth, examples of which include a suitable temperature (e.g., 37 °C) and atmosphere (e.g., air + 5% CO2).

[0377] The medium used for culturing immune cells may contain an agent that can co-stimulate the immune cells. For example, the agent that can stimulate CD3 is an antibody against CD3, and the agent that can stimulate CD28 is an antibody against CD28. This is because the cells isolated by the methods disclosed herein can be expanded by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more, as demonstrated by the data disclosed herein. In one embodiment, by culturing the electroporated population, the immune cells expand in the range of about 2-fold to about 50-fold or more. In one embodiment, human regulatory T cells are expanded via KT64.86 artificial antigen-presenting cells (aAPCs) coated with anti-CD8 antibody. Methods for expanding and activating immune cells are described in U.S. Patent Nos. 7,754,482, 8,722,400, and 9,555,105, the contents of which are incorporated herein by reference in their entirety.

[0378] In one embodiment, the method of expanding immune cells can further include isolating the expanded immune cells for further use. In another embodiment, the method of expanding can further include subsequently electroporating the expanded immune cells and then culturing them. The subsequent electroporation may include introducing a nucleic acid encoding an agent that further stimulates the immune cells into the population of expanded immune cells (such as causing transduction of the expanded immune cells, transfecting the expanded immune cells, or introducing nucleic acid into the expanded immune cells by electroporation). The agent may stimulate the immune cells, such as by stimulating further expansion, effector function, or another immune cell function.

[0379] T cells exposed to different stimulation times may exhibit different characteristics. For example, common blood, or apheresis-derived peripheral blood mononuclear cell products, have a larger helper T cell population (TH, CD4 + ) than cytotoxic or suppressor T cell populations (Tc, CD8 ) than cytotoxic or suppressor T cell populations (Tc, CD8 + ). By expanding T cells ex vivo by stimulating the CD3 receptor and CD28 receptor, a population of T cells mainly composed of TH cells is produced before about 8-9 days, but after about 8-9 days, the T cell population comes to include an increasingly large population of Tc cells. Therefore, depending on the treatment purpose, it may be advantageous to inject a T cell population mainly containing TH cells. Similarly, when a subset of antigen-specific Tc cells has been isolated, it may be beneficial to expand this subset to a greater extent.

[0380] Furthermore, in addition to the CD4 and CD8 markers, other phenotypic markers change significantly, but in most cases, they change reproducibly during the cell expansion process. Therefore, such reproducibility enables the ability to adjust T cell products activated for specific purposes.

[0381] E. Scaffold In another aspect, the present disclosure provides a scaffold or substrate composition, which comprises a peptide containing a TACA binding domain, a nucleic acid molecule encoding a peptide containing a TACA binding domain, a cell modified to express a peptide containing a TACA binding domain, or a combination thereof. For example, in one embodiment, a peptide containing a TACA binding domain, a nucleic acid molecule encoding a peptide containing a TACA binding domain, a cell modified to express a peptide containing a TACA binding domain, or a combination thereof is present within the scaffold. In another embodiment, a peptide containing a TACA binding domain, a nucleic acid mole...

Claims

1. Modified cells containing isolated nucleic acid molecules encoding chimeric antigen receptors (CARs), (a) The CAR comprises an antigen-binding domain, a transmembrane domain, a costimulatory domain and / or an intracellular signaling domain that selectively binds tumor-associated glycogen antigens (TACAs), including one or more TACA-binding domains, wherein the TACA-binding domain is derived from a lectin, (b) The modified cells become less susceptible to the effects of TACA CAR-mediated tonic signaling, thereby reducing exhaustion, pro-inflammatory cytokine production, or both, in the absence of target cancer cells. The modified cells.

2. (a) The antigen-binding domain comprises one, two, three, four, five, six, seven, eight, nine, ten, or more TACA-binding domains, (b) The antigen-binding domain includes a mutation in the TACA-binding domain (TBD), selected from substitution, deletion, or insertion, or (c) The antigen-binding domain includes a deletion in the TACA-binding domain (TBD), or (d) The antigen-binding domain includes a deletion in the N-terminal region, the C-terminal region, or both of the TACA-binding domain (TBD). The modified cell according to claim 1.

3. The antigen-binding domain includes a deletion in the TACA-binding domain (TBD), and the deletion is (a) At least about 18 amino acids present at the N-terminus of the TBD, (b) At the C-terminus, there are at least about 10 amino acids, (c) At least about 36 amino acids present at the N-terminus of the TBD, or (d) At least about 18 amino acids present at the N-terminus of the TBD, and at least about 10 amino acids present at the C-terminus, The modified cell according to claim 1.

4. (a) The modified cells expressing a CAR having a deletion in the TACA-binding domain (TBD) of the antigen-binding domain show reduced tonic signaling compared to modified cells containing a CAR containing the wild-type TBD, (b) The modified cells expressing a CAR having a deletion in the TACA-binding domain (TBD) of the antigen-binding domain are less likely to experience exhaustion compared to modified cells containing a CAR containing the wild-type TBD, or (c) Modified cells expressing a CAR having a deletion in the TACA-binding domain (TBD) of the antigen-binding domain are less likely to experience exhaustion induced by tonic signaling TACA CAR compared to modified cells containing a CAR containing the wild-type TBD. The modified cell according to claim 1.

5. The modified cell according to claim 1, wherein the antigen-binding domain comprises a deletion in the CBD of the TACA-binding domain, which includes an amino acid sequence described in any one of SEQ ID NOs: 30 to 54, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 an amino acid sequence described in any one of SEQ ID NOs: 30 to 54.

6. The modified cell according to claim 1, wherein the antigen-binding domain comprises an amino acid sequence described in any one of SEQ ID NOs: 34 to 39, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 an amino acid sequence described in any one of SEQ ID NOs: 34 to 39.

7. (a) The transmembrane domain includes a transmembrane region of a molecule selected from the group consisting of T cell receptor (TCR)-alpha, TCR-beta, TCR-gamma, TCR-delta, NKT cell invariant TCR, CD3-zeta, CD3-epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134 (Ox40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9, (b) The costimulatory domain is a costimulatory domain of a molecule selected from the group consisting of ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD8, LIGHT, NKG2C, B7-H3, CD83, DAP10, DAP12, Lck, Fas, and combinations thereof, (c) The intracellular domain includes an intracellular signaling domain of a molecule selected from the group consisting of T cell receptor (TCR) zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, CD3, CD5, CD22, CD79a, CD79b, and CD66d, or (d) The CAR further comprises a hinge domain, The modified cell according to claim 1.

8. (a) The transmembrane domain comprises the amino acid sequence of SEQ ID NO: 78 or SEQ ID NO: 87, (b) The co-stimulatory domain is (i) 4-1BB co-stimulatory domain, (ii) Amino acid sequence of sequence number 58, (iii) CD28 co-stimulation domain, (iv) The amino acid sequence of SEQ ID NO: 88, or (v) 4-1BB and CD28 co-stimulatory domains Does it include, (c) The intracellular signaling domain comprises a CD3 zeta signaling domain or the amino acid sequence of Sequence ID No. 59, or (d) The hinge domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 71-77 and 86. The modified cell according to claim 7.

9. The CAR is (a) The amino acid sequence described in any one of Sequence IDs 23 to 29, or (b) An amino acid sequence having 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 any one of the amino acid sequences described in Sequence ID No. 23 to 29, The modified cells according to claim 1, comprising:

10. The modified cell according to claim 9, wherein the modified cell containing any one of the CARs of SEQ ID NOs. 23 to 29 exhibits reduced tonic signaling compared to the modified cell containing the CAR of SEQ ID NOs. 21 or 22.

11. The modified cells according to claim 1, wherein the cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells.

12. A chimeric antigen receptor (CAR) that selectively binds tumor-associated glycan antigens (TACAs), (a) an antigen-binding domain containing a deletion in an amino acid sequence that has at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 an amino acid sequence that has (b) CD8 or CD28 hinge domain, (c) CD8 or CD28 transmembrane domain, (d) CD28 costimulatory domain, 4-1BB costimulatory domain, or both, (e) CD3 zeta intracellular signaling domain and The chimeric antigen receptor, including the aforementioned.

13. (a) The CAR comprises an amino acid sequence described in any one of Sequence IDs 23 to 29, (b) Modified cells containing any one of the CARs in SEQ ID NOs. 23-29 show reduced tonic signaling compared to modified cells containing the CAR in SEQ ID NOs. 21 or 22, (c) Modified cells expressing a CAR containing a deletion in an amino acid sequence described in any one of SEQ ID NOs. 30 to 54, or in an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 an amino acid sequence described in any one of SEQ ID NOs. 30 to 54, are less susceptible to exhaustion compared to modified cells containing a CAR containing a wild-type antigen-binding domain, or (d) Modified cells expressing a CAR containing a deletion in an amino acid sequence that has at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 an amino acid sequence that has at least 99% sequence identity with an amino acid sequence that has at least 96%, at least 97%, at least 98%, or at least 99% are less susceptible to exhaustion associated with tonic signaling TACA CAR compared to modified cells containing a CAR containing a wild-type antigen-binding domain. The chimeric antigen receptor according to claim 12.

14. An isolated nucleic acid encoding the CAR according to claim 12.

15. An expression construct comprising the isolated nucleic acid described in claim 14.

16. A composition comprising the modified cells described in Claim 1.

17. An immunotherapy composition comprising the modified cells according to claim 1 for treating cancer in a subject that needs to be treated for cancer.

18. The immunotherapy composition according to claim 17, wherein the cancer is selected from the group consisting of hematological malignancies, solid tumors, primary or metastatic tumors, leukemia, carcinoma, blastoma, sarcoma, malignant lymphoma, melanoma, and lymphoma.

19. A therapeutically effective composition for treating cancer in a subject requiring treatment of cancer, comprising modified cells containing a chimeric antigen receptor (CAR) that selectively binds tumor-associated glycosylation antigens (TACAs), wherein the CAR is (a) an antigen-binding domain comprising an amino acid sequence described in any one of SEQ ID NOs: 30 to 54, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 an amino acid sequence described in any one of SEQ ID NOs: 30 to 54, with a deletion. (b) CD8 or CD28 hinge domain, (c) CD8 or CD28 transmembrane domain, (d) CD28 costimulatory domain, 4-1BB costimulatory domain, or both, (e) CD3 zeta intracellular signaling domain and The therapeutically effective composition comprising the above.

20. The therapeutically effective composition according to claim 19, wherein the modified cells are less susceptible to exhaustion, cytokine production, or both, in the absence of target cancer cells associated with the tonic signaling pathway TACA CAR.

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