Improved glycan-dependent immunotherapeutic bispecific fusion proteins and chimeric antigen receptors

Abstract

Compositions and methods for treating diseases associated with aberrant glycosylation of cell surface molecules and expression of tumor-associated carbohydrate antigens (TACAs) are provided. Also provided are bispecific fusion proteins and chimeric antigen receptors (CARs) having specificity for tumor associated carbohydrate antigen (TACA-CAR), vectors encoding the TACA-bispecific fusion proteins and CARs, and recombinant cells comprising the TACA-bispecific fusion proteins and CARs.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 351,596, filed June 13, 2022, which is hereby incorporated by reference in its entirety for any and all purposes. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under Grant Nos. U01CA233078, R41CA233111, and R41CA261408 awarded by the National Institutes of Health / National Cancer Institute. The government has certain rights in this invention. Technical Field

[0004] The present disclosure relates generally to the fields of pharmacology and immunology, and specifically to bispecific fusion proteins and chimeric antigen receptors (TACA bispecific fusion proteins; TACA-CARs) targeting tumor-associated carbohydrate antigens and the use of immune cells expressing TACA bispecific fusion proteins and TACA-CARs for treating diseases associated with abnormal glycosylation of cell surface molecules. Background Art

[0005] Antigen-targeted cancer immunotherapies, such as bispecific antibodies (e.g., bispecific T cell engagers) or chimeric antigen receptor T cells (engineered immune cells expressing, for example, chimeric antigen receptors (CARs)), are the most effective immunotherapies known. Both trigger T cell-mediated killing of cancer cells, with complete response rates of CAR T cells as high as approximately 90% in relapsed / refractory B-cell malignancies. Both utilize single-chain variable fragments (scFvs) derived from the variable heavy and 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 an immune cell receptor. Both types of chimeric molecules are genetically expressed in T cells. Both therapies are currently approved for the treatment of CD19 + B-cell malignancies.

[0006] In order to apply bispecific proteins and / or CAR T cells to a variety of cancer types, cell surface cancer antigens that can be safely targeted must first be identified. This is a major challenge, especially for solid cancers. The lack of safe cell surface protein antigens that can be used for targeting makes it impossible for the vast majority of cancer patients to use these emerging immunotherapies. In addition, even if new safe cell surface antigens are identified, different bispecific and / or CAR T cells may need to be developed for each different antigen or cancer. This greatly increases development time and cost. Therefore, there is a need for a better immunotherapy method for targeting antigens that are present in a variety of common cancers but have limited or no expression in normal tissues. The present disclosure meets this unmet need. Summary of the invention

[0007] The present disclosure provides a novel class of immunotherapeutic bispecific fusion proteins and chimeric antigen receptors that effectively target tumor-associated carbohydrate antigens (TACAs) for use in immunotherapy. Specifically, bispecific fusion proteins and CARs are engineered to target TACAs using antigen-binding domains derived from lectins rather than monoclonal antibodies or fragments thereof, regardless of carrier proteins.

[0008] Therefore, one aspect of the present disclosure provides an isolated nucleic acid molecule encoding a bispecific fusion protein, the bispecific fusion protein comprising: (a) an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), and (b) an immune cell recognition domain that specifically binds to a receptor on an immune effector cell. In some embodiments, the antigen binding domain comprises a TACA binding domain derived from a lectin; and the antigen binding domain comprises more than one TACA binding domain.

[0009] Another aspect of the present disclosure provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), the CAR comprising: (a) an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA); (b) a transmembrane domain; (c) a co-stimulatory signaling region; and (d) an intracellular signaling domain. In some embodiments, the antigen binding domain comprises a TACA binding domain derived from a lectin; and the antigen binding domain comprises more than one TACA binding domain.

[0010] In some embodiments, the antigen binding domain of a bispecific fusion protein or CAR disclosed herein comprises two, three, four, five, six, seven, eight, nine, ten or more TACA binding domains.

[0011] In some embodiments, more than one TACA binding domain is operably connected by a linker. In some embodiments, the linker is selected from the group consisting of: a peptide linker, a non-peptide linker, a chemical unit, a hindered cross-linker, and a non-hindered cross-linker. In some embodiments, the linker is a peptide linker. In some embodiments, the length of the peptide linker is 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. In some embodiments, the peptide linker is a glycine-serine linker. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 127. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 131.

[0012] In some embodiments, the antigen binding domain of the bispecific fusion protein or CAR disclosed herein comprises a TACA binding domain derived from a lectin selected from the group consisting of: galactose lectin, sialic acid-binding immunoglobulin-like lectin (siglec), selectin; C-type lectin; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Phaseolus vulgaris leukolectin); E-PHA (Phaseolus vulgaris erythrocyte agglutinin); tomato lectin (Lycopersicon esculentum lectin; LEA); peanut lectin (Arachis hypogaea Agglutinin; PNA); potato lectin (Solanum tuberosum lectin); pokeweed mitogen (Pokeweed lectin); mitogen)(Phytolacca American lectin), wheat germ agglutinin(Triticum Vulgaris lectin); Artocarpus polyphemus lectin(Jacalin letin); Viciavillosa lectin(VVA); Helix pomatia lectin(HPA); Wisteria floribunda lectin(WFA);Sambucus nigra agglutinin (SNA), BC2L-CNt (lectin from the Gram-negative bacterium Burkholderia cenocepacia), Maackia amurensis leukocyte agglutinin (MAL), Psathyrella velutina lectin (PVL), Sclerotium rolfsii lectin (SRL), Eucheuma serra lectin (ESA), CLEC17A (protectin), Aleuria aurantia lectin, Sambucus sieboldiana lectin (SSA), Glechoma hederacea lectin (Gleheda), Morus nigra lectin (Morniga G), Salvia miltiorrhiza var. sclarea lectin, Salvia bogotensis lectin, Salvia horminum lectin, Clerodendrum trichotomum lectin, Moluccella laevis lectin, Griffonia simplicifolia (GsLA4), Psophocarpus tetragonolobus (acidic WBAI), Abrus precatorius lectin, Amaranthus caudatus lectin, Amaranthus leucocarpus lectin, Laelia autumnalis lectin, Artocarpus integrifolia lectin, Maclura pomifera lectin, Artocarpus lakoocha lectin, Dolichos biflorus lectin, Glycine max lectin, and Agaricus bisporus lectin.

[0013] In some embodiments, the galectin is 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 sialic acid-binding immunoglobulin-like lectin is selected from the group consisting of sialic acid-binding immunoglobulin-like lectin-1 (sialoadhesin), sialic acid-binding immunoglobulin-like lectin-2 (CD22), sialic acid-binding immunoglobulin-like lectin-3 (CD33), sialic acid-binding immunoglobulin-like lectin-4 (myelin-associated glycoprotein), sialic acid-binding immunoglobulin-like lectin-5, sialic acid-binding immunoglobulin-like lectin-6, sialic acid-binding immunoglobulin-like lectin-7, sialic acid-binding immunoglobulin-like lectin-8, sialic acid-binding immunoglobulin-like lectin-9, sialic acid-binding immunoglobulin-like lectin-10, sialic acid-binding immunoglobulin-like lectin-11, sialic acid-binding immunoglobulin-like lectin-12, sialic acid-binding immunoglobulin-like lectin-13, sialic acid-binding immunoglobulin-like lectin-14, sialic acid-binding immunoglobulin-like lectin-15, sialic acid-binding immunoglobulin-like lectin-16, sialic acid-binding immunoglobulin-like lectin-17, sialic acid-binding immunoglobulin-like lectin-18, sialic acid-binding immunoglobulin-like lectin-19, sialic acid-binding immunoglobulin-like lectin-20, sialic acid-binding immunoglobulin-like lectin-23, sialic acid-binding immunoglobulin-like lectin-24, sialic acid-binding immunoglobulin-like lectin-25 Sialic acid binding immunoglobulin-like lectin-9, sialic acid binding immunoglobulin-like lectin-10, sialic acid binding immunoglobulin-like lectin-11, sialic acid binding immunoglobulin-like lectin-12, sialic acid binding immunoglobulin-like lectin-13, sialic acid binding immunoglobulin-like lectin-14, sialic acid binding immunoglobulin-like lectin-15, sialic acid binding immunoglobulin-like lectin-16, sialic acid binding immunoglobulin-like lectin-17, sialic acid binding immunoglobulin-like lectin E, sialic acid binding immunoglobulin-like lectin F, sialic acid binding immunoglobulin-like lectin G and sialic acid binding immunoglobulin-like lectin 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 (GALNT 9), 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 of the bispecific fusion protein or CAR disclosed herein 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, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6 sialylated, sialylated, sialyl-Lewis x / a , disialo-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 antigen binding domain selectively targets β1,6GlcNAc-branched N-glycans, Tn epitopes (Tn antigens), sialyl-Tn epitopes (sialyl-Tn antigens), GalNAcα-serine, GalNAcα-threonine, GalNAc or GalNAcβ1.

[0016] In some embodiments, the antigen binding domain of the bispecific fusion protein or CAR disclosed herein comprises the amino acid sequence shown in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152; or The amino acid sequences shown in NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152 have an amino acid sequence with 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.

[0017] In some embodiments, the antigen binding domain comprises an amino acid sequence that is at least 90% homologous to SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152.

[0018] In some embodiments, the isolated nucleic acid comprises an expression vector; and / or in vitro transcribed RNA.

[0019] In some embodiments of the isolated nucleic acid molecules disclosed herein, the isolated nucleic acid molecules encode an amino acid sequence selected from SEQ ID NO: 1-5, 10-34, 39-42, 47-50, 55-58 and 63-66; or a bispecific fusion protein comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-5, 10-34, 39-42, 47-50, 55-58 and 63-66. In some embodiments, the isolated nucleic acid molecules encode a bispecific fusion protein comprising an amino acid sequence selected from SEQ ID NO: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66. In some embodiments, the isolated nucleic acid molecules encode a bispecific fusion protein comprising an amino acid sequence of SEQ ID NO: 32-34, 40-42, 48-50, 56-58 or 64.

[0020] In some embodiments of the isolated nucleic acid molecules disclosed herein, (a) the bispecific fusion protein exhibits enhanced binding to tumor cells expressing Thomsen-nouveau (Tn) antigens when compared to a bispecific fusion protein comprising a flexible linker in the antigen binding domain. In some embodiments, the bispecific fusion protein exhibits enhanced binding to tumor cells expressing β1,6GlcNAc branched N-glycans when compared to a bispecific fusion protein comprising a flexible linker in the antigen binding domain. In these embodiments, the flexible linker is a glycine-serine linker or a linker comprising an amino acid sequence selected from SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127; or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127.

[0021] In some embodiments of the isolated nucleic acid molecules disclosed herein, the immune effector cells targeted by the bispecific fusion protein are selected from the group consisting of: T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, monocytes, dendritic cells and neutrophils. In one embodiment, the immune effector cells are T cells. In one embodiment, the immune effector cells are NK cells. In some embodiments, the receptors on the immune effector cells are selected from the group consisting of: T cell receptor (TCR) α, TCR β, TCR γ, TCR δ, the invariant TCR (invariant TCR) of NKT cells, CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA and CEACAM1. In some embodiments, the receptor on the immune effector cell is: (i) a T cell receptor selected from the group consisting of CD3, CD2, CD28, and CD25; or (ii) a NK cell receptor selected from the group consisting of NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1.

[0022] In some embodiments, the immune cell recognition domain of the bispecific fusion proteins disclosed herein comprises: (i) a peptide, protein, antibody, single domain antibody, nanobody, antibody fragment or single chain variable fragment (scFv) that selectively binds to a receptor on an immune effector cell; (ii) an antibody Fc domain, optionally the Fc region of an IgG molecule; or (iii) the constant region domains CH2 and / or CH3 of an antibody, preferably CH2 and CH3, optionally with or without a hinge region.

[0023] In some embodiments, the immune cell recognition domain comprises: (i) a scFv that selectively binds to CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA and CEACAM1; (ii) an amino acid sequence of SEQ ID NO: 149, 150 or 151; or (iii) an amino acid sequence that has at least 90% sequence identity to an amino acid sequence of SEQ ID NO: 149, 150 or 151.

[0024] In some embodiments, the bispecific fusion protein is an Fc fusion protein comprising an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA) and an Fc domain.

[0025] In some embodiments of the isolated nucleic acid molecules disclosed herein, the transmembrane domain of CAR includes a transmembrane region of a molecule selected from the group consisting of: T cell receptor (TCR) -α, TCR-β, CD3-ζ, CD3-ε, 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 includes a CD8 transmembrane domain. In some embodiments, the transmembrane domain includes SEQ ID NO:148 amino acid sequence.

[0026] In some embodiments of the isolated nucleic acid molecules disclosed herein, the costimulatory domain of CAR is a costimulatory domain of a molecule selected from the group consisting of: CD27, CD28, 4-IBB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD8, LIGHT, NKG2C, B7-H3, a ligand specifically bound to CD83, DAP10, DAP12, Lck, Fas, and a combination thereof. In some embodiments, the costimulatory domain includes: (i) 4-1BB costimulatory domain; (ii) SEQ ID NO: 114 amino acid sequence; (iii) CD28 costimulatory domain; (iv) SEQ ID NO: 113 amino acid sequence; or (v) 4-1BB and CD28 costimulatory domain.

[0027] In some embodiments of the isolated nucleic acid molecules disclosed herein, the intracellular domain of the CAR comprises an intracellular signaling domain of a molecule selected from the group consisting of: T cell receptor (TCR) ζ, FcR-γ, FcR-β, CD3-γ, CD3-δ, CD3-ε, CD3-ζ, CDS, CD5, CD22, CD79a, CD79b and CD66d. In some embodiments, the intracellular signaling domain comprises a CD3 ζ signaling domain; or an amino acid sequence of SEQ ID NO: 115.

[0028] In some embodiments of the isolated nucleic acid molecules disclosed herein, CAR further comprises a hinge domain. In some embodiments, the hinge domain is a protein selected from the group consisting of: CD8 alpha, 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 CD8 alpha hinge domain or wherein the hinge domain comprises an amino acid sequence of SEQ ID NO: 147. In some embodiments, the hinge domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 119, 124, 127, 128, 129, 130, 131, 132, and 147.

[0029] One aspect of the present disclosure provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR encoded by the isolated nucleic acid molecule comprises: (i) an amino acid sequence shown in SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99; or (ii) an amino acid sequence shown in SEQ ID The amino acid sequence shown in NO:71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 has an amino acid sequence with 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.

[0030] One aspect of the present disclosure provides a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA) disclosed herein. In some embodiments, the bispecific fusion protein is encoded by an isolated nucleic acid described herein.

[0031] One aspect of the present disclosure provides a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising: (i) an antigen binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152; or NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152 has an amino acid sequence with 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; and (ii) an immune cell recognition domain that specifically binds to a receptor on an immune effector cell.

[0032] In some embodiments, the immune cell recognition domain comprises: (i) an antibody Fc domain; (ii) an Fc region of an IgG molecule; (iii) a peptide, protein, antibody, single domain antibody, antibody fragment or single chain variable fragment (scFv) that selectively binds to a receptor on an immune effector cell; or (iv) the constant region domains CH2 and / or CH3 of an antibody, preferably CH2 and CH3, optionally with or without a hinge region.

[0033] In some embodiments, the receptor on the immune effector cell is selected from the group consisting of: T cell receptor (TCR) α, TCR β, TCR γ, TCR δ, the invariant TCR of NKT cells, CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA and CEACAM1.

[0034] In some embodiments, the bispecific fusion protein comprises: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, and 63-66; or (b) an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, and 63-66; or (c) an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66; or (d) an amino acid sequence selected from the group consisting of SEQ ID NOs: The amino acid sequence of NO: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66 has an amino acid sequence with at least 90% sequence identity.

[0035] In some embodiments, the bispecific fusion protein selectively targets a TACA selected from the group consisting of: β1,6 branched, β1,6GlcNAc branched N-glycan, T antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6 sialylated, sialylated, sialyl-Lewis x / a , disialo-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 bispecific fusion protein selectively targets Tn antigen or β1,6GlcNAc branched N-glycans.

[0036] In some embodiments, the bispecific fusion protein that selectively targets the Tn antigen comprises an antigen binding domain having an amino acid sequence selected from SEQ ID NO: 103-109, 142-146, or 152; or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 103-109, 142-146, or 152. In some embodiments, the bispecific fusion protein that selectively targets the Tn antigen comprises: (a) an amino acid sequence selected from SEQ ID NO: 26-34, 39-42, 47-50, 55-58, or 63-66; or (b) an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 26-34, 39-42, 47-50, 55-58, or 63-66; (c) an amino acid sequence selected from SEQ ID NO: 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66; or (d) an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66.

[0037] In some embodiments, the bispecific fusion protein that selectively targets β1,6GlcNAc branched N-glycans comprises an antigen binding domain having an amino acid sequence selected from SEQ ID NOs: 100-102 or 133-141; or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 100-102 or 133-141.

[0038] In some embodiments, the bispecific fusion protein that selectively targets β1,6GlcNAc branched N-glycans comprises: (a) an amino acid sequence selected from SEQ ID NOs: 1-5 and 10-25; or (b) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1-5 and 10-25; (c) an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13 or 19-21; or (d) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13 or 19-21.

[0039] One aspect of the present disclosure provides a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising: (i) an antigen binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152; or NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152 has an amino acid sequence with 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; and (ii) an immune cell recognition domain that specifically binds to CD3 on an immune effector cell.

[0040] Another aspect of the present disclosure provides a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising: (i) a TACA binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152; or The amino acid sequence shown in NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, 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; and (ii) a peptide, protein, antibody, single domain antibody, antibody fragment or single chain variable fragment (scFv) that selectively binds to a receptor on an immune effector cell; and / or (iii) the Fc domain of an antibody, optionally the Fc region of an IgG molecule; or (iv) the constant region domains CH2 and / or CH3 of an antibody, preferably CH2 and CH3, optionally with or without a hinge region.

[0041] 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 is encoded by an isolated nucleic acid disclosed herein.

[0042] One aspect of the present disclosure provides a chimeric antigen receptor that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising: (i) an antigen binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152; or The amino acid sequence shown in NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152 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 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; (ii) a CD8 hinge domain; (iii) a CD8 transmembrane domain; (iv) a CD28 co-stimulatory domain and / or a 4-1BB co-stimulatory domain; and (v) a CD3ζ intracellular signaling domain.

[0043] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 72, 88, 89, 91, 92, or 93. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 93, 94, 95, 96, 97, 98, or 99.

[0044] One aspect of the present disclosure provides an expression construct comprising the isolated nucleic acid described herein.

[0045] In some embodiments, the expression construct further comprises a promoter. In this embodiment, the promoter is selected from the group consisting of EF-1α promoter, T cell receptor α (TRAC) promoter, interleukin 2 (IL-2) promoter, or cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, 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, or Rous sarcoma virus promoter.

[0046] 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 one embodiment, the expression construct is a lentiviral vector. In one embodiment, the expression construct is a self-inactivating lentiviral vector.

[0047] In some embodiments, the expression construct comprises an isolated nucleic acid molecule encoding a bispecific fusion protein as described herein and an isolated nucleic acid molecule encoding a CAR as described herein. In some embodiments, the isolated nucleic acid molecule encoding a bispecific fusion protein as described herein and the isolated nucleic acid molecule encoding a CAR as described herein are operably connected by a nucleic acid molecule encoding a self-cleaving 2A peptide selected from P2A, T2A, E2A or F2A.

[0048] One aspect of the present disclosure provides a modified cell comprising an isolated nucleic acid as described herein; a bispecific fusion protein as described herein; a CAR as described herein; or an expression vector as described herein. In some embodiments, the cell is selected from the group consisting of: a T cell, a CD4 + T cells, CD8 + T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs) and regulatory T cells.

[0049] In some embodiments, the modified cell is a T cell. In some embodiments, the modified cell is an autologous cell, a xenogeneic cell, or an allogeneic cell.

[0050] In some embodiments, the modified cell comprises: (a) a bispecific fusion protein disclosed herein and a CAR or a CAR targeting a tumor antigen disclosed herein; or (b) an isolated nucleic acid molecule encoding a bispecific fusion protein disclosed herein and an isolated nucleic acid molecule encoding a CAR or a CAR targeting a tumor antigen disclosed herein; or (c) an expression construct disclosed herein.

[0051] One aspect of the present disclosure provides a composition comprising: (i) an isolated nucleic acid disclosed herein; (ii) a bispecific fusion protein disclosed herein; (iii) a CAR disclosed herein; (iv) an expression vector disclosed herein; or (v) a modified cell disclosed herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0052] One aspect of the present disclosure provides a method for generating a modified cell disclosed herein, the method comprising introducing into a cell an isolated nucleic acid for generating a modified cell, a bispecific fusion protein for generating a modified cell; a CAR for generating a modified cell; or an expression vector for generating a modified cell.

[0053] One aspect of the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition described herein.

[0054] In some embodiments, the composition comprises: (a) an isolated nucleic acid disclosed herein; (b) a bispecific fusion protein disclosed herein; (c) a CAR disclosed herein; (d) a bispecific fusion protein disclosed herein and a CAR disclosed herein; (e) an expression vector disclosed herein; or (f) a modified cell disclosed herein.

[0055] In some embodiments, the cancer is selected from the group consisting of a hematological malignancy, a solid tumor, a primary or metastatic tumor, a leukemia, a carcinoma, a blastoma, a sarcoma, a leukemia, a lymphoid malignancy, a melanoma, and a lymphoma.

[0056] One aspect of the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective composition comprising modified cells, wherein the modified cells comprise a bispecific fusion protein and / or CAR that selectively binds to a tumor-associated carbohydrate antigen (TACA), and wherein the bispecific fusion protein or CAR comprises an antigen binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, and 152; or any of the amino acid sequences shown in NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152 having an amino acid sequence of 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.

[0057] In some embodiments, the bispecific fusion protein further comprises an immune cell recognition domain that specifically binds to a receptor on an immune effector cell. In some embodiments, the immune cell recognition domain specifically binds to CD3. In some embodiments, the immune cell recognition domain is an antibody Fc domain and a domain that specifically binds to CD3.

[0058] In some embodiments of the methods described herein for treating cancer in a subject in need thereof, the bispecific fusion protein comprises: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, and 63-66; or (b) an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, and 63-66; (c) an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66; or (d) an amino acid sequence selected from the group consisting of SEQ ID NOs: The amino acid sequence of NO: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66 has an amino acid sequence with at least 90% sequence identity.

[0059] In some embodiments of the method for treating cancer in a subject in need described herein, CAR further comprises a transmembrane domain; a co-stimulatory domain; and an intracellular signaling domain. In some embodiments, CAR comprises a CD8 transmembrane domain; a CD28 co-stimulatory domain and / or a 4-1BB co-stimulatory domain; and a CD3ζ intracellular signaling domain. In some embodiments, CAR further comprises a hinge domain.

[0060] 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 modified cell population disclosed herein.

[0061] The foregoing summary of the invention and the following description of the drawings and specific embodiments are exemplary and explanatory. They are intended to provide further details of the present disclosure, but should not be construed as limiting. Other purposes, advantages and novel features will be apparent to those skilled in the art from the following specific embodiments of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1A-Figure 1D Schematic representation and features of a glycan-dependent T cell recruitment (GlyTR1) bispecific protein targeting β1,6GlcNAc branched N-glycans are shown.

[0063] Figure 1A The GlyTR1 protein is shown to be a chimeric single polypeptide comprising the variable heavy and light chain domains of an anti-CD3 monoclonal antibody (CD3scFv, OKT3 clone) linked to one or more L-PHA carbohydrate binding domains (CRDs). L-PHA means phytohemagglutinin-L; H(6) means 6x-histidine tag.

[0064] Figure 1B GlyTR1 is shown L-PHAxCD3 Size Exclusion Chromatography (SEC) analysis with protein standard Sigma (Cat. No. MWGF1000-1KT). SEC analysis was performed using a GE Superdex 200 Increase 3.2 / 300 column. Molecular weights were calculated based on trend lines generated from retention times.

[0065] Figure 1C GlyTR1 is shown L-PHAxCD3 and GlyTR1 L-PHAΔ1-5xCD3 Flow cytometric analysis of cell surface binding on Jurkat T cells.

[0066] Figure 1D Flow cytometry analysis of co-culture assays is shown, where carboxyfluorescein succinimidyl ester (CFSE) labeled cancer cells were co-cultured with PBMCs at an E:T of 20:1 for 1 day using the indicated GlyTR1 molecules. Live cancer cells were gated for analysis. Cell death (%) = (100 - number of live cancer cells treated with GlyTR1 / number of live cancer cells not treated with GlyTR1 x 100), where live cells are defined as CFSE + 7AAD - 7-Aminoactinomycin D (7-AAD) is a fluorescent intercalator that undergoes a spectral shift upon association with DNA. thermofisher.com / order / catalog / product / A1310.

[0067] Figure 2A-2D Shown is the improved activity of a GlyTR1 bispecific protein targeting β1,6GlcNAc branched N-glycans and comprising more than one L-PHA carbohydrate binding domain (CRD) when compared to a GlyTR1 comprising one L-PHA carbohydrate binding domain (CRD).

[0068] Figure 2A The results show the use of a GE HiLoad 16 / 600 Superdex 200 pg column for GlyTR1 LPHA(2)xCD3 SEC analysis was performed using protein standard GE (Cat. No. GE28-4038-42). Molecular weights were calculated based on trend lines generated from volume.

[0069] Figure 2B GlyTR1 on Jurkat T cells is shown L-PHA(2)xCD3 Flow cytometric analysis of cell surface binding of monomers and dimers illustrates the presence of dimeric GlyTR1 L-PHA(2)xCD3With monomeric GlyTR1 L-PHA(2)xCD3 showed enhanced binding compared to .

[0070] Figure 2B The monomer GlyTR1L- PHAxCD3 and dimer GlyTR1 L-PHA(2)xCD3 Flow cytometric analysis of cell surface binding illustrates the presence of dimeric GlyTR1 L-PHA(2)xCD3 With monomeric GlyTR1L- PHAxCD3 showed enhanced binding compared to .

[0071] Figure 2D Shown is a line graph illustrating flow cytometry analysis of co-culture assays in which CFSE-labeled cancer cells were co-cultured with PBMCs at an E:T of 20:1 for 1 day using the indicated GlyTR1 molecules; and demonstrating that dimeric GlyTR1 L -PHA(2)xCD3 With monomeric GlyTR1L- PHAxCD3 Compared with the enhancement of cell death. Live cancer cells were gated for analysis. Cell death (%) = (100-number of live cancer cells treated with GlyTR1 / number of live cancer cells not treated with GlyTR1 x 100), where live cells are defined as CFSE + 7AAD - .

[0072] Figure 3A-Figure 3I The dimer GlyTR1 is shown L-PHA(2)xCD3 T cell-dependent cancer killing in various cancer cell lines. Carboxyfluorescein succinimidyl ester (CFSE)-labeled cancer cells (as indicated) were incubated with or without PBMCs ( Figure 3A-3H ) or CD8 T cells ( Fig. 3I ) with a ratio of 1:1( Figure 3A )、10:1( Figure 3B or Figure 3H ) and 20:1( Figure 3C-3G , Fig. 3I ) of E:T co-culture for 1 day ( Figure 3A-3B ) or 3 days ( Figure 3C-Figure 3I ), followed by analysis of cell death by flow cytometry. Cell death % = 100-(living cells treated with GlyTR / living cells not treated with GlyTR) x 100, where living cancer cells are defined as CFSE + 7AAD - (a, b) or CFSE + FVD-eFluor780 - (ci). Fixable reactive dye-eFluor TM-780 is a fixable viability dye that can be used to irreversibly label dead cells prior to cryopreservation, fixation, and / or permeabilization procedures. thermofisher.com / order / catalog / product / 65-2860-40. Data are means ± SEM of triplicate incubations.

[0073] Figure 4A-4B Dimeric GlyTR1 is shown L-PHA(2)xCD3 Reduced non-cancer-induced T cell activation. Figure 4A-4B Shown is the use of GlyTR1 L-PHAxCD3 PBMCs rested for 3 days or treated with Kifunensine ( Figure 4A ) or using dimer GlyTR1 L-PHA(2)xCD3 MDA-MB-231F cancer cells (10 5 cells / ml) of PBMC (10 5 cells / ml)( Figure 4B ) Quantification of flow cytometric analysis of CD69 expression on gated T cells after overnight stimulation. Kifusine is an inhibitor of class I alpha mannosidase, which inhibits glycoprotein processing in the endoplasmic reticulum and Golgi apparatus.

[0074] Figure 5A-5F GlyTR1 is shown L-PHA(2)xCD3 in vivo activity. Figure 5A-Figure 5B GlyTR1 is shown L-PHA(2)xCD3 To induce tumor regression in NSG mice, the mice were injected intraperitoneally with the indicated cancer cell lines on day 0 and then injected 2 or 3 times intraperitoneally every 3-4 days starting from day 6 with 1x10 7 CD8 + T cells were treated (as indicated) with or without twice daily intraperitoneal injection of GlyTR1 LPHA(2)xCD3 Tumor burden quantified by luciferase activity (photons / second (p / s)) was Figure 5C and Figure 5D These data demonstrate that GlyTR1 inhibits ovarian cancer by inhibiting PBS compared with PBS in breast and ovarian cancer models. LPHA(2)xCD3 Induced significant tumor regression. Figure 5E GlyTR1 is shown LPHA(2)xCD3 Accumulation in the lungs with cancer. NSG mice (MDA-MB-231-Fluc) with or without lung metastasis were intravenously injected with or without the fluorophore ( GlyTR1 labeled 680XL (Perkin Elmer LLC) LPHA(2)xCD3 , and the extracted lungs were imaged for luminescence (tumor) and fluorescence (GlyTR1).

[0075] Fig. 5F GlyTR1 is shown LPHA(2)xCD3 Not in non-tumor bearing humanized NSG MI / II - / - Inducing human T cell activation in PBMCs. Humanized NSG-MI / II - / - Mice were injected subcutaneously with GlyTR1 LPHA(2)xCD3 , and after 24 hours, blood T cell activation (CD69 + ).

[0076] Figure 6A-6E The dimer GlyTR1 is shown L-PHA(2)xCD3 in vivo half-life and distribution. Figure 6B The dimer GlyTR1 is shown LPHA(2)xCD3 The serum half-life of GlyTR1 was about 2.7 hours. C57BL / 6 mice (n=2) were injected intravenously with GlyTR1. LPHA (2)xCD3 Blood was collected at the indicated times and serum was analyzed for GlyTR1 by sandwich ELISA L-PHA(2)xCD3 . Fig. 6A and Figure 6C-6E GlyTR1 is shown L-PHA(2)xCD3 Stable in human plasma for up to 21 hours and showed little loss of intact protein, while labeled dimer GlyTR1 L-PHA(2)xCD3 Shows significant accumulation in the liver ( Fig. 6E ). GlyTR1 L-PHA(2)xCD3 use 680XL (Perkin Elmer LLC) was fluorescently labeled and injected intravenously into two C57BL / 6 mice. These mice and a mock-injected mouse were treated with Imaging was performed with a Lumina imager (2 s exposure). Fluorescence in the liver and bladder regions was quantified at each time point and plotted as a percentage of total fluorescence after background subtraction in mock-injected mice ( Figure 6C ). After 8 hours, the mice were sacrificed, and the designated organs were extracted and imaged ( Fig.6D The fluorescence of each organ was quantified and plotted as a percentage of the total fluorescence from all imaged organs after subtracting the background from mock-injected mice ( Fig. 6E ).

[0077] Figure 7Immunohistochemical staining and staining schematics are shown, which demonstrate the expression of L-PHA in normal human tissues. The 'FDA999u' (BioMAx) normal human tissue microarray containing 32 different tissues with replicate samples from 3 different individuals was stained with L-PHA-biotin (0.25ug / ml) for 1 hour and detected by streptavidin-HRP (0.5 hour). The four tissues with the highest staining are highlighted in the box and shown at a higher resolution. The staining of metastatic colon cancer under the same conditions is shown as a positive control. Adr-adrenal gland, Bon-bone marrow, Bre-breast, Ceb-cerebellar tissue, Cer-cervix, Col-colon, Dia-diaphragm, Eso-esophagus, Eye-eye, Hea-heart, Hyp-pituitary gland, Kid-kidney, Lar-larynx, Liv-liver, Lun-lung, Lym-lymph node, Ner-nerve, Ova-ovary, Pan-pancreas, Ple-pleura, Pro-prostate, Sal-salivary gland, Ski-skin, Sma-small intestine, Spl-spleen, Sto-stomach, Str-striated muscle, Tes-testis, Thy-thyroid or thymus, Ton-tonsil, Ute-uterus.

[0078] Fig. 8A - Panel B shows immunohistochemical staining ( Figure 8B ) and staining diagram ( Fig. 8A ), which demonstrated that GlyTR1 LPHA(2)xCD3 Expression in normal human tissues. Specifically, GlyTR1 LPHA(2)xCD3 Low but variable expression was shown in the small intestinal brush border, gastric surface epithelial cells, exocrine pancreas (acinar, intracellular), renal cortex (glomerular, proximal tubules), prostate, and cerebellar molecular layer. However, higher concentrations revealed low but variable staining in the adrenal glands, parotid ducts, thyroid colloid, testes, uterus, spleen, and CNS white matter. A 'FDA999w' (BioMax) normal human tissue microarray containing 32 different tissues with replicate samples from 3 different individuals was performed with / without GlyTR1. LPHA(2)xCD3 (0.5ug / ml) for 1 hour. Detect GlyTR1 with 1ug / ml anti-HIS-HRP antibody LPHA(2)xCD3(0.5 hours). Adr-adrenal gland, Bon-bone marrow, Bre-breast, Ceb-cerebellum, Cer-cervix, Col-colon, Dia-diaphragm, Eso-esophagus, Eye-eye, Hea-heart, Hyp-pituitary, Kid-kidney, Lar-larynx, Liv-liver, Lun-lung, Lym-lymph node, Ner-nerve, Ova-ovary, Pan-pancreas, Per-pericardium, Pro-prostate, Sal-salivary gland, Ske-skeletal muscle, Ski-skin, Sma-small intestine, Spl-spleen, Sto-stomach, Tes-testis, Thy-thymus, Ton-tonsil and Ute-uterus. The higher resolution scale bar is 2 mm.

[0079] Figure 9A-9B Immunohistochemical staining is shown ( Fig. 9B ) and staining diagram ( Fig. 9A ), which demonstrated that GlyTR1 LPHA(2)xCD3 Expression in normal human tissues, such as Figure 8A-8B The 'FDA999-1' (BioLabs) normal human tissue microarray containing 32 different tissues with replicate samples from 3 different individuals as well as prostate cancer and matched normal prostate were analyzed with GlyTR1 LPHA(2)xCD3 (0.67ug / ml) for 1 hour and detected with nti-HIS-HRP at 1ug / ml (0.5 hour). AD-adrenal gland, BO-bone marrow, BRN-brain (cerebrum), BRT-breast, CB-cerebellum tissue, CR-cervix, CO-colon, ES-esophagus, EY-eye, HE-heart, HY-pituitary, KI-kidney, LA-larynx, LI-liver, LU-lung, ME-mesothelium, NE-nerve, OV-ovary, PA-pancreas, PT-parathyroid, PR-prostate, SA-salivary gland, SK-skin, SM-small intestine, SP-spleen, ST-stomach, SM-skeletal muscle, TE-testis, TO-tonsil, TH-thyroid, THM-thymus, TO-tonsil and UT-uterus. The higher resolution scale bar is 10μM.

[0080] Figure 10A-10C GlyTR1 is shown LPHA(2)xCD3 Binding and GlyTR1 in normal primary human renal epithelial cells and hepatocytes LPHA(2)xCD3 Quantification of induced cytotoxicity demonstrated by GlyTR1 LPHA(2)xCD3 Compared with the stable killing of MM1R multiple myeloma cells induced by GlyTR1, normal primary human renal epithelial cells and hepatocytes LPHA(2)xCD3 Insensitivity to T cell-dependent killing. Figure 10A-10C Shown is the effect of GlyTR1 LPHA(2)xCD3 Cell surface binding ( Fig. 10A) and / or GlyTR1 LPHA(2)xCD3 Induced CD8 + T-mediated GlyTR1 LPHA(2)xCD3 Killing of normal primary human renal epithelial cells (SciencCell), normal primary human hepatocytes (ScienCell), and MDA-MB-231 breast cancer cells (MHC-I defective) and / or MM1R multiple myeloma cells (MHC-I defective) ( Figure 10B-Figure 10C Quantification of flow cytometric analysis of 20:1 (E:T=20:1, 2 days). Data are mean ± SEM of three replicate incubations.

[0081] Fig.11 L-PHA immunohistochemical staining of normal human and mouse tissues is shown, demonstrating positive staining of L-PHA in mouse gastric surface epithelial cells, small intestinal brush border, and kidney (tubules > glomeruli). 'FDA999u' (BioMAx) normal human tissue microarrays and mouse (C57BL6, AMS545 (panomics)) tissue microarrays containing 32 and 22 normal tissues, respectively, were stained with L-PHA-biotin (0.5ug / ml) for 1 hour and detected by streptavidin-HRP (0.5 hour). The four most stained human tissues and their mouse counterparts are shown at a higher resolution.

[0082] Fig.12 GlyTR1 is shown L-PHA(2)xCD3 Humanized NSG-MI / II in PBMC - Toxicity assessment in mice demonstrated that GlyTR1 L-PHA(2)xCD3 Treatment did not significantly alter body weight relative to mock-treated mice (Panel b), nor did it affect liver function (AST, ALT, ALP, protein, albumin, total bilirubin), kidney function (BUN, creatinine), electrolytes, glucose, pancreatic function (amylase, precision PSL), thyroid function (total T4, TSH), cholesterol, or muscle (CPK) (Panel c-v), blood levels of hemoglobin, RBC, hematocrit, WBC, WBC differential, or platelets relative to control, CD4 + or CD8 + T cell activation markers CD69 or CD25 in T cells, T cell activation, PD-1 positive CD4 + T and CD8 + Percentage of T cells, total human CD45 + WBC, CD4 + T cells, CD8 + The percentage of T cells, B cells, or regulatory T cells (Treg) or serum hIFNγ or hIL-6 levels. MHC class I and II deficiency (NSG-MI / II- ) were transplanted intravenously (tail vein) with 2x10 7 From day 16, mice were subcutaneously injected with PBS (n=6) or 10ug GlyTR1 twice daily. L -PHA(2)xCD3 (n = 6), for 12 days. The body weight of mice during treatment was assessed (Figure b). One treated mouse lost about 15% of its body weight on the 18th day and recovered after saline injection. One treated mouse had mild hair loss on the head. (Figure c-Figure Q) Mice were euthanized on the 28th day, and blood and major organs were harvested. For clinical biochemistry, blood was equally pooled from 2 mice in the same treatment group to ensure sufficient amount for analysis (Figure c-Figure v); each symbol represents two mice. For TSH, 0.03 data points represent <0.03 (i.e., the detection limit of the assay) (t). A complete blood count was performed for each mouse, and each symbol represents one mouse (Figure w-Figure D). Flow cytometry analysis was performed on a portion of each spleen (Figure E-Figure O), and each symbol represents one mouse (due to technical problems during flow cytometry, for some measurements, n = 5). The plasma of each mouse was used for human IFNγ and IL-6 ELISA, and each symbol represents one mouse (Figure P-Figure Q). All p-values ​​were obtained by Mann-Whitney test (two-tailed).

[0083] Fig.13 GlyTR1 is shown LPHA(2)xCD3 In CD34 + Toxicity assessment in humanized NSG mice demonstrated that GlyTR1 L -PHA(2)xCD3 Treatment did not induce any overt clinical toxicity and did not alter body weight, spleen size / cell count, total human splenic CD4 + and CD8 + T cells, B cells, Treg cells, or T cells positive for CD69, CD25, or PD-1, hemoglobin, RBC, hematocrit, WBC, WBC differential, or platelets relative to control, renal function (BUN, creatinine), liver function (AST, ALT, ALP, protein, albumin, total bilirubin), electrolytes, pancreatic function (amylase, precision PSL), muscle (CPK), thyroid function (TSH), or cholesterol, serum hIFNγ or hIL-6 levels. (Panels A-J) CD34 + Stem cell humanized NSG mice were subcutaneously injected with PBS (n=3), 2.5ug (n=3), 5ug (n=3), or 10ug (n=3) of GlyTR1 twice daily for 32 weeks after irradiation / transplantation. LPHA(2)xCD3, for 10 days. (Figure a) Body weight of mice during treatment. Multiple comparisons were performed by two-way ANOVA and Bonferroni post test correction to obtain P values. *p<0.05. bJ) Mice were euthanized on day 10, and blood and major organs were harvested. Flow cytometry analysis was performed on a portion of each spleen (Figure b-Figure k); each symbol represents one mouse. A complete blood count was performed on the blood, and each symbol represents one mouse (Figure l-Figure p). For clinical biochemistry, blood was equally pooled from 3 mice in the same treatment group to ensure sufficient amount for analysis (Figure q-Figure H); each symbol represents three mice. The plasma of each mouse was used for human IFNγ and IL-6 ELISA, and each symbol represents one mouse (Figure I-Figure J). P values ​​were obtained by multiple comparisons by Kruskal-Wallace test and Dunn's post test correction.

[0084] Figures 14A-14D A schematic diagram of an improved GlyTR2 bispecific protein for targeting Tn antigen comprising multiple carbohydrate binding domains and its improved binding efficiency to various cells is shown. Fig.14A Schematic representations of various GlyTR2 proteins are shown, demonstrating that the GlyTR2 proteins are chimeric single polypeptides comprising the variable heavy and light chains of an anti-CD3 monoclonal antibody (CD3 scFv, OKT3 clone) linked to more than one carbohydrate binding domain (CRD) of CD301 (C-type lectin domain family 10 member A (CLEC10A)). H(6): 6x-histidine tag. Figure 14B-Figure 14D TCRβ with and without inhibitor, Tn antigen, GalNAc, galactose, GalNAc are shown - / - Quantification of flow cytometric analysis of GlyTR2 bispecific protein cell surface binding on Jurkat T cells.

[0085] Figures 15A-15D Improved activity of the GlyTR2 bispecific protein for targeting low-density Tn antigen is shown. Fig.15A A chromatogram is shown, which demonstrates that GlyTR2 CD301(3)xCD3 Mainly composed of large multimers with rigid linker GlyTR2 slCD301(4)xCD3 Mainly monomers. Using GE HiLoad 16 / 600 Superdex 200pg column, GlyTR2 CD301(3)xCD3 and GlyTR2 slCD301(4)xCD3SEC analysis was compared to protein standard GE (Cat. No. GE28-4038-42). Molecular weights were calculated based on trend lines generated from retention volumes. Figure 15B-Figure 15C Comparison of T cell leukemia TCRβ - / - Jurkat and multiple myeloma MM.1R cells ( Fig. 15B ) CD301(3)xCD3 and GlyTR2 slCD301(4)xCD3 Quantification of cell surface binding by flow cytometry analysis and other indicated cancer types using established cell lines is shown for TCRβ - / - The relative binding of Jurkat T cells ( Fig. 15C ). Acute monocytic leukemia (AML): THP-1; ovarian cancer: SKOV3; non-small cell lung cancer NSCLC-1 and NSCLC-2: H1975 and A549, respectively; colorectal cancer: DLD-1; pancreatic cancer: Hs766T; breast cancer: MDA-MB-231F; prostate cancer: PC3. GlyTR2 with 2 repeats containing a rigid linker (SL2) was used slCD301(4)xCD3 . Fig.15D GlyTR2 is shown in the absence or presence of GalNac or GlcNAc. slCD301(4)xCD3 Quantification of flow cytometric analysis of surface binding to MM.1R cells. Data are mean ± SEM of triplicate incubations.

[0086] Figure 16A-16I GlyTR2 is shown slCD301(4)xCD3 Quantification of T cell-dependent cancer killing. Figure 16A-16I In the experiment, carboxyfluorescein succinimidyl ester (CFSE)-labeled cancer cells (as indicated) were incubated with or without CD8 ( Fig.16A , Fig.16I ), T cells ( Fig. 16B ) or PBMC( Figure 16C-16H ) with a ratio of 10:1( Fig.16A , Fig.16H , Fig.16I ) and 20:1( Figure 16B-16G ) were co-cultured with E:T for 3 days, and then cell death was analyzed by flow cytometry. Cell death % = 100-(living cells treated with GlyTR / living cells not treated with GlyTR) x 100, where the living cancer cells are CFSE + FVD-eFluor780 - . Fixable reactive dye - eFluor TM-780 is a fixable viability dye that can be used to irreversibly label dead cells prior to cryopreservation, fixation, and / or permeabilization procedures. thermofisher.com / order / catalog / product / 65-2860-40. Data are means ± SEM of triplicate incubations.

[0087] Figure 17A-17B shows that GlyTR2 slCD301(4)xCD3 Quantification of robust T cell activation induced by GlyTR2 slCD301(4)xCD3 For the presence or absence of SKOV3-Luc + MI - / - Cancer cells (2.5x10 4 cells / ml) of PBMC (5x10 5 After 3 days of stimulation with 500 cells / ml, flow cytometric analysis of CD25 expression on gated T cells was performed.

[0088] Figure 18A-18E GlyTR2 is shown slCD301(4)xCD3 In vivo activity in solid cancers. Figure 18A-18B It is shown that GlyTR2 slCD301(4)xCD3 In vivo tumor regression was induced in NSG mice injected intraperitoneally with the indicated cell lines on day 0, followed by intraperitoneal injections of 1×10 7 CD8 + T cell therapy or subsequent injections of 2x10 every 3-4 days 6 CD8 + T cells (ovarian cancer) (as indicated), with or without twice-daily intraperitoneal injection of GlyTR2sl CD301(4)xCD3 Tumor burden was quantified by luciferase activity (photons / second (p / s)) and Figure 18C-Figure 18D These data demonstrate that GlyTR2 inhibits ovarian cancer compared with PBS treatment in breast and ovarian cancer models. slCD301(4)xCD3 Induced significant tumor regression. Fig.18E GlyTR2 is shown slCD301(4)xCD3 GlyTR2 accumulates in lungs with, but not without, cancer slCD301(4)xCD3 Specificity for cancer cells in vivo. Towards cells with or without lung metastasis (MDA-MB-231-Luc + MI - / - C - / - ) of NSG mice intravenously injected with or without fluorophore ( 680XL) labeled GlyTR2 slCD301(4)xCD3, and the extracted lungs were imaged for luminescence (tumor) and fluorescence (GlyTR2).

[0089] Figure 19A-Figure 19C GlyTR CD301(3)xCD3 Half-life and stability in human plasma. Fig.19A It is shown that when GlyTR CD301(3)xCD3 When GlyTR2 (100 ug) was intravenously injected into C57BL / 6 mice (n=6), CD301(3)xCD3 The serum half-life of is approximately 2 hours. Serum was separated at the indicated times and sampled by sandwich ELISA, captured by anti-CD301 and detected by anti-HIS. Figure 19B-Figure 19C GlyTR CD301(3)xCD3 The stability in human plasma at 37°C is up to 21 hours. CD301(3)xCD3 After incubation in human plasma at 37°C for the indicated times, the proteins were detected and quantified by sandwich ELISA ( Fig.19B ) or by Western blotting using anti-HIS ( Fig.19C ).

[0090] Figures 20A-20F GlyTR2 is shown slCD301xCD3 The biodistribution and safety of GlyTR2 slCD301xCD3 It accumulates in the liver, but is rapidly cleared by the liver, and shows minimal accumulation in the kidney, spleen, lung, and intestine. Figure 20A-20B Fluorescently labeled GlyTR2 is shown slCD301xCD3 Localization in corresponding tissues. slCD301(4)xCD3 Vivo 680XL (Perkin Elmer LLC) was fluorescently labeled and injected intravenously into C57BL / 6 mice along with vehicle. Eight hours later, mice were sacrificed and organs were extracted and imaged. Fig. 20C Shows Fig. 20A Fluorescence was quantified for each organ in the image and plotted as a percentage of the total fluorescence of all imaged organs after background subtraction from the injected vector. Fig. 20C Formalin-fixed paraffin-embedded normal human liver cancer and breast cancer (positive control) and GlyTR2 slCD301(4)xCD3 Co-stained images, followed by detection with anti-HIS-HRP antibody and DAB, demonstrated that GlyTR2 slCD301(4)xCD Does not bind significantly to human or mouse hepatocytes. Fig.20D Shown, with Jurkat TCRβ - / - Compared with leukemia cells and MM.1R multiple myeloma cells, GlyTR2 slCD301(4)xCD Does not bind significantly to human or mouse hepatocytes. Figure 20E-Figure 20FQuantification of flow cytometry analysis is shown, demonstrating that GlyTR2 slCD301(4)xCD3 It does not induce T-cell-dependent killing of human hepatocytes, human renal epithelial cells, or normal T and B cells at concentrations that trigger cancer cell killing. + T cells ( Fig.20E , E:T = 20:1, 2 days) or PBMC ( Fig.20F , E:T = 10:1, 1 day) induced GlyTR2 slCD301(4)xCD3 Jurkat TCRβ - / - leukemia( Fig.20E , Fig.20F ), MM.1R multiple myeloma ( Fig.20E ), normal human hepatocytes or normal lymphocytes (20F) were quantified by flow cytometry analysis. Data are mean ± SEM of 3 incubations.

[0091] Fig.21 GlyTR2 is shown CD301(4)xCD3 Humanized NSG-MI / II in PBMC - Toxicity assessment in mice demonstrated significant changes in body weight, liver function (AST, ALT, ALP, bilirubin, protein, albumin), renal function (urea / creatinine), electrolytes (Na + , Cl - , K + , Ca2 + ), pancreatic function (amylase, precision PSL), thyroid function (total T4, TSH), cholesterol, muscle (CPK), WBC, WBC differential, or platelets, serum hIFNγ or hIL-6 levels, minimal decreases in hemoglobin / RBC / hematocrit relative to controls, human CD45 + The number of white blood cells or CD4 + T cells, CD8 + There were no differences in the number or percentage of T cells, B cells, or regulatory T cells (Treg), and CD4 + or CD8 + There was no difference in T cell activation markers CD69, CD25 or PD-1 in T cells, and no T cell activation. (Figure a) MHC class I and class II deficiency (NSG-MI / II - ) were transplanted intravenously (tail vein) with 2x10 7 From day 16, mice were injected subcutaneously with PBS (n=6) or 100ug GlyTR2 twice daily. CD301(4)xCD3(n=6), continued for 12 days. (Figure b) Body weight of mice during treatment. (Figure c-Figure Q) Mice were euthanized on the 28th day, and blood and major organs were harvested. For clinical biochemistry, blood was equally pooled from 2 mice in the same treatment group to ensure that there was enough for analysis (Figure c-Figure v); each symbol represents two mice. For TSH, 0.03 data points represent <0.03 (i.e., the detection limit of the assay) (Figure t). A complete blood count was performed for each mouse, and each symbol represents a mouse (Figure w-Figure D). Flow cytometry analysis was performed on a portion of each spleen (Figure E-Figure O), and each symbol represents a mouse (due to technical problems during flow cytometry, for some measurements, n=5). The plasma of each mouse was used for human IFNγ and IL-6 ELISA, and each symbol represents a mouse (Figure P-Figure Q). All p values ​​obtained by Mann-Whitney test (two-tailed).

[0092] Figure 22A-22I Schematic diagram of GlyTR-CAR design and GlyTR-CAR T cell expression as well as in vitro and in vivo cytotoxic activity are shown. Fig.22A Three GlyTR-CARs (GlyTR1 LPHA(2) 、GlyTR2 slCD301(4) and mutGlyTR2 slCD301(4) ), which comprises an antigen binding domain with two LPHA or four CD301 domains, a CD8 transmembrane domain, a 41BB co-stimulatory domain, and a CD3ζ intracellular signaling domain. mutGlyTR2 slCD301(4)xCD3 CAR has five point mutations that are critical for sugar and calcium binding in the antigen binding domain (Gln267Gly, Asp269Gly, Glu280Gly, Asn292Gly, and Asp293Gly in SEQ ID NO: 164 (NCBI RefSeq NP_878910.1)). Fig. 22B A schematic diagram illustrating the generation of GlyTR-CAR T cells is shown. To generate CAR T cells, T cells were transduced with lentivirus. Human T-Activator CD3 / CD28 was stimulated for 3 days and then rested from day 4-7. Figure 22C-Figure 22E GlyTR1 is shown LPHA(2) or GlyTR2 slCD301(4) CAR T cells readily killed ovarian cancer cells and breast cancer cells. Fig. 22C Flow cytometric analysis at days 3 and 7 is shown, characterizing the cell size and surface expression of GlyTR-CAR. Figure 22D-Figure 22EQuantification of GlyTR-CAR mediated cell death at day 7 after GlyTR-CAR T cell treatment as shown in the figure. GlyTR-CAR T cells were incubated with the indicated cancer cells at increasing ratios, and active cancer cells were assessed by luminescence after 72 hours. Death % was calculated by [1-(cancer+CAR T / cancer)*100. Figure 22F-22G Compared with non-transduced (NT) T cells ( Fig.22F ) or the absence of cancer cells ( Figure 22G ) compared to the presence of cancer cells, interferon gamma (IFNγ) was produced. The sandwich ELISA assay was used to determine the Fig.22D )and( Fig.22E ) in the supernatant of cultures. Fig.22H Figure 2 shows in vivo killing of breast cancer cells transplanted into mice by the indicated GlyTR2-CAR T cells. Tumor burden quantified by luciferase activity (photons / second (p / s)) was Fig.22I These data demonstrate that GlyTR2-CAR T cells induced significant tumor regression compared to mutGlyTR2-CAR T cells in a breast cancer model. DETAILED DESCRIPTION

[0093] I. Overview

[0094] Current bispecific antibodies (such as Blincyto and AFM11 (CD19 / CD3 TandAb)) induce polyclonal T cell activation in vivo and in vitro in the presence of PBMCs. In CAR T immunotherapy, CAR T cells are activated in vitro before being infused into the patient. This shows that peripheral activation of T cells in vivo is essential for efficient cancer killing. Consistent with this, animal models for other types of immunotherapy have shown that peripheral T cell activation is required for tumor elimination. However, excessive activation can also lead to reduced activity via T cell exhaustion, as reported for standard CAR T cells in animal models. In addition, excessive polyclonal T cell activation can lead to varying degrees of "cytokine release syndrome" in both CAR T and bispecific therapies, a life-threatening complication caused by excessive release of proinflammatory cytokines (e.g., IL-6).

[0095] In order to apply bispecific fusion proteins and / or CAR T cells to multiple cancer types, cell surface cancer antigens that can be safely targeted must first be identified. This is a major challenge, especially for solid cancers. The lack of safe cell surface protein antigens that can be targeted leaves the vast majority of cancer patients without the potential to use these emerging immunotherapies. In addition, even if new safe cell surface antigens are identified, different bispecific and / or CAR T cells may need to be developed for each different antigen / cancer. This greatly increases development time and cost.

[0096] A potential approach to address all of these issues is to target 'tumor-associated carbohydrate antigens' (TACAs), which are overexpressed in many different cancer types and are even higher in metastatic and invasive diseases. Carbohydrates (glycans), as well as glycoproteins and glycolipids, are major cell surface components. Almost all cell surface proteins are glycosylated, with multiple glycans for each protein. Changes in cellular glycosylation are common in cancer due to abnormal expression of glycosyltransferases, glycosidases, and transporters, as well as differences in the abundance of carbohydrate building blocks. These glycosylation changes result in unique antigenic glycans known as tumor-associated carbohydrate antigens (TACAs). TACAs provide the most abundant and widespread cell surface cancer antigens known, with target densities up to about 100-1000 times higher than typical protein antigens. TACAs are overexpressed in many different cancer types and are even higher in metastatic and invasive diseases.

[0097] Altered glycosylation leading to TACAs is a universal feature of cancer. Since nearly all cell surface proteins are glycosylated, with multiple glycans per protein, TACAs provide the most abundant and widespread cell surface cancer antigens known, with target densities up to approximately 100-1000-fold higher than typical protein antigens.

[0098] TACA is not only a marker of cancer, but also often acts as a key driver of tumor growth and metastasis. For example, abnormal overexpression of β1,6GlcNAc branched N-glycans in cancer drives tumor growth, movement, invasion and metastasis. See, for example, Fernandes et al., Cancer research 51:718-723 (1991); Litynska et al., Melanomaresearch 11:205-212 (2001); Lau & Dennis, Glycobiology 18:750-760 (2008); Dennis et al., Science 236:582-585 (1987); and Demetrious et al., J. Cell Biology 130:383-392 (1995). Another related TACA found on tumor cells is the Tn antigen. Although the Tn antigen is not found on the cell surface of normal human tissues, it is expressed in approximately 90% of human cancers and many hematopoietic cancers. In fact, the Tn antigen is one of the most specific human cancer-associated structures known and promotes cell motility, invasion, and metastasis. The Tn antigen is a single N-acetyl-galactosamine (GalNAc) α-O attached to a serine / threonine in proteins such as mucins. Tn is a biosynthetic precursor for O-glycans, which are typically extended with α1,3-linked galactose. The partner protein COSMC, which is required for T synthase to add galactose to GalNAc, is frequently altered in cancer. Mislocalization of the enzyme within the ER / Golgi apparatus may also contribute to aberrant Tn antigen expression in human cancers. Tn antigen can be abnormally extended by sialic acid to form sTn antigen; the sTn antigen is also not typically expressed in normal tissues. Therefore, targeting the TACA epitope may have significant implications for the management of various human cancers.

[0099] Although bispecific proteins and / or CAR T cells targeting TACAs have great therapeutic potential, the lack of high affinity and / or high specificity antibodies against carbohydrate targets is a key limitation to the utilization of glycans as therapeutic targets. Antibodies against carbohydrates are extremely difficult to generate. It has proven challenging to generate monoclonal antibodies with high affinity for complex carbohydrates such as TACAs (e.g., Tn antigens or β1,6GlcNAc branched N-glycans), thus hindering the widespread use of TACAs as cancer-specific antigens. In addition, the affinity of anti-glycan antibodies is generally 1000-100,000-fold lower than that of antibodies against peptide antigens. Anti-carbohydrate antibodies generally also require additional peptide / lipid epitopes for high affinity binding. In addition, antibodies against carbohydrates have low affinity and specificity. In addition, antibody recognition of glycan antigens depends on glycan density, valency, presentation, and flexibility.

[0100] Therefore, there is a need for TACA-specific bispecific fusion proteins and / or TACA-specific CARs to treat diseases associated with aberrant glycosylation of cell surface molecules that are not based on scFvs of antigen-specific monoclonal antibodies.

[0101] In the present disclosure, a new type of immunotherapeutic bispecific fusion protein and CAR are developed to effectively target TACA for immunotherapy. Specifically, bispecific fusion proteins and CARs are engineered to target TACA using antigen binding domains derived from lectins rather than monoclonal antibodies or fragments thereof, regardless of carrier proteins. This new technology is called "glycan-dependent T cell recruitment" or GlyTR (pronounced 'glitter'). One group of GlyTR therapeutic agents is a TACA bispecific fusion protein comprising a TACA binding domain (e.g., a carbohydrate recognition domain) from a lectin, which is operably connected, conjugated or fused to an immune cell recognition domain that specifically binds to a receptor on an immune effector cell. Another group of GlyTR therapeutic agents is a chimeric antigen receptor comprising an antigen binding domain comprising a TACA binding domain derived from a lectin. In all cases, the TACA binding domain specifically binds to TACA expressed on tumor cells, and the TACA binding domain comprises multiple (eg, more than one or at least two) TACA binding domains derived from a lectin.

[0102] A. Summary of Experimental Results

[0103] The present disclosure further provides a novel method for treating a leukemia cell line with enhanced GlyTR binding affinity ( FIGS. 1-2 and 14-15 ), killing activity ( FIGS. 3-5 , 16 , 17-18 and 23 ) and safety ( Figure 12-13 and Figure 20- Fig.21 ) of GlyTR therapeutic agents.

[0104] To drive binding affinity to the high density of TACA present in cancer cells, GlyTR therapeutics were generated with multiple carbohydrate binding domains derived from lectins. Specifically, GlyTR1 was generated. LPHA(2)xCD3 (GlyTR1 LPHAxLPHAxCD3 ; two TACA binding domains) and through three flexible linkers (i.e. (GGGGS) 3 ) Two L-PHA domains connected in series ( Figure 1A SEC revealed that GlyTR1 LPHA(2)xCD About 50%-70% of 3 are dimers, and the rest are monomers (about 30%-40%) or larger polymers (about 10%-20%) ( Figure 2A ). Direct comparison with GlyTR1LPHA(2)xCD3 The monomeric (two L-PHA domains) and dimeric (four L-PHA domains) fractions revealed significantly higher binding of the latter to β1,6GlcNAc-branched N-glycans ( Figure 2B ), further confirming that increasing the number of TACA binding domains in the GlyTR1 protein resulted in higher potency. LPHA(2)xCD3 The monomeric (two L-PHA domains) and dimeric (four L-PHA domains) fractions revealed significantly higher binding of the latter to β1,6GlcNAc-branched N-glycans ( Figure 2B ), further confirming that increasing the number of TACA binding domains in the GlyTR1 protein resulted in higher potency. LPHA(2)xCD3 (four L-PHA domains) bind to target cancer cells significantly better than the original dimer GlyTR1 LPHAxCD3 (two L-PHA domains), resulting in a >3000-fold increase in cancer cell killing activity ( Figure 2C , Figure 2D ). In addition, the dimer GlyTR1 LPHA(2)xCD3 Effectively triggered human T cell-dependent killing of multiple liquid and solid cancer types, EC 50 As low as <100 femtomolar, the liquid and solid cancers include multiple myeloma, T-cell leukemia, acute myeloid leukemia (AML), pancreatic cancer, colon cancer, non-small cell lung cancer, prostate cancer, ovarian cancer and breast cancer ( Figure 3A-Figure 3I These improved GlyTR1 binding domains are safe ( Figure 12-13 and Figure 20- Fig.21 ), stable (Figure 6 and Figure 19) and selectively kill cancer cells (Figures 3-5, Figure 16, Figure 17-18 and Figure 23). In addition, when compared to control cells, engineered cells containing novel and improved TACA bispecific fusion proteins and / or TACA-CARs did not show any T cell-dependent "on-target / off-cancer" toxicity ( Figure 7-Figure 11 The combination of high target density and multiple binding sites results in a clear specificity for high-expressing cancer cells relative to low-expressing normal cells and effectively triggers T cell-mediated killing of the former but not the latter.

[0105] B. Exemplary Benefits of GlyTR Fusion Proteins

[0106] One advantage of targeting TACA for immunotherapy is that almost all cell surface proteins are glycosylated. TACA target density is about 100-1000 times higher than typical protein antigens. Therefore, increasing the number of TACA binding domains in GlyTR can drive cancer cell specificity by enhancing binding affinity. This is in contrast to antibodies, which use high affinity to achieve specificity. In the present disclosure, high affinity binding is achieved by a combination of high-density target expression on tumor cells and the presence of multiple carbohydrate binding domains in engineered GlyTR. This combination of high target density and multiple binding sites enhances the specificity of improved GlyTR to high TACA expressing cells (such as cancer cells) relative to low expressing cells (such as normal cells). Therefore, the specificity of the novel multivalent GlyTR protein (TACA bispecific fusion protein or TACA CAR) to TACA will be determined by the threshold density of target expression specifically detected by GlyTR with multiple TACA binding domains, rather than by the presence or absence of target antigens. The multivalent GlyTR protein disclosed in the present disclosure shows improved specificity for high target expression cancer cells and retains normal tissues with lower expression. As shown in the Examples described herein, the GlyTR-based immunotherapies of the present disclosure induce sufficient T cell activation in vitro and in vivo to maximize cancer killing, but not enough to induce T cell exhaustion or cytokine release syndrome.

[0107] Therefore, the present disclosure provides bispecific fusion proteins and chimeric antigen receptors (CARs) that selectively bind to TACA on target cells; isolated nucleic acid molecules encoding the bispecific fusion proteins and CARs; expression vectors comprising isolated nucleic acid molecules encoding the bispecific fusion proteins and CARs, modified cells comprising isolated nucleic acid molecules and / or expression vectors encoding the bispecific fusion proteins and CARs; compositions comprising modified cells; and methods for treating conditions or diseases associated with tumor-associated carbohydrate antigens (TACAs).

[0108] In one aspect, the present disclosure provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain, optionally a hinge domain.

[0109] In another aspect, the present disclosure provides a kind of nucleic acid molecule encoding a separation of a bispecific fusion protein, the bispecific fusion protein comprises an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA) and an immune cell recognition domain that specifically binds to a receptor on an immune effector cell. In some embodiments, the antigen binding domain of a CAR or bispecific fusion protein comprises multiple (more than one or at least two, three, four or more TACA binding domains derived from lectins).

[0110] In another aspect, the present disclosure provides a chimeric antigen receptor or bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), the chimeric antigen receptor or bispecific fusion protein comprising an antigen binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, or 146; or The amino acid sequences shown in ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146 have an amino acid sequence with 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.

[0111] The chimeric antigen receptor (CAR) or bispecific fusion protein disclosed herein includes a tumor-associated carbohydrate antigen (TACA) binding domain derived from a lectin to target TACA-expressing cells to kill tumor cells, rather than antibodies or antibody fragments. The CAR (TACA-CAR) or bispecific fusion protein disclosed herein is an improvement over the prior art, because CAR or bispecific fusion protein includes a structural modification that enhances the specificity of TACA-CAR or bispecific fusion protein. The first structural modification includes changing the structure of the antigen binding domain by changing the number, connection and sequence of the TACA binding domains derived from lectins. For example, the antigen binding domain of TACA-CAR or bispecific fusion protein includes one or more TACA binding domains derived from lectins. In addition, the joint domain between more than one TACA binding domains is modified.

[0112] In another aspect, the present disclosure provides a composition comprising TACA-CAR or bispecific fusion proteins disclosed herein or a modified cell comprising TACA-CAR or bispecific fusion proteins disclosed herein. The composition of the present disclosure also includes: additional peptides comprising multiple TACA binding domains (at least two), nucleic acid molecules encoding peptides comprising TACA binding domains, cells modified to express peptides comprising multiple TACA binding domains, and a matrix comprising the peptides, nucleic acids, cells, or a combination thereof.

[0113] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising modified cells, the modified cells comprising a chimeric antigen receptor or a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA) disclosed herein. Diseases or disorders that can be treated using the TACA-CAR or bispecific fusion protein disclosed herein are, for example, cancer or any disorder associated with altered protein glycosylation. Cancer may be a hematological malignancy, a solid tumor, a primary or metastatic tumor. In another aspect, the present disclosure provides a method for providing anti-tumor immunity in a mammal, comprising administering to a mammal an effective amount of a modified cell population, the modified cell population comprising a chimeric antigen receptor or a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA) disclosed herein.

[0114] II. Definitions

[0115] 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 the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0116] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Green and Sambrook, eds. (2012) Molecular Cloning: A Laboratory Manual, 4th edition; Ausubel et al., eds. (2015) Current Protocols in Molecular Biology series; Methods in Enzymology series (Academic Press, Inc., NY); MacPherson et al. (2015) PCR 1: A Practical Approach (IRL Press of 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; edited by Hames and Higgins (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; edited by Herdewijn (2005) Oligonucleotide Synthesis: Methods and Applications; edited by Hames and Higgins (1984) Transcription and Translation; edited by Buzdin and Lukyanov (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, ed. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides, ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker, ed. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Lundblad and Macdonald, ed. (2010) Handbook of Biochemistry and Molecular Biology, 4th edition; and Herzenberg et al., ed. (1996) Weir's Handbook of Experimental Immunology, 5th edition. ;

[0117] 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.

[0118] As used herein, the term "about" when referring to a measurable value (such as an amount, duration, etc.) is intended to encompass variations of ±20%, ±10%, ±5%), ±1%), or ±0.1%) from the specified value, as such variations are suitable for performing the disclosed methods.

[0119] As used herein, the term "activated" refers to a state in which a T cell has been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. As used herein, the term "activated T cell" refers, among other things, to a T cell that is undergoing cell division.

[0120] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancer condition. An "anti-tumor effect" can also be manifested as the ability of the peptides, polynucleotides, cells, and antibodies of the present disclosure to prevent tumorigenesis in the first place.

[0121] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is subsequently reintroduced into that individual.

[0122] As used herein, the term "antigen" or "Ag" is defined as a molecule that triggers an immune response. This immune response may involve the production of other antibodies or the activation of specific immune-competent cells or both. It will be understood by the skilled person that any macromolecule, including almost all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. It will be understood by those skilled in the art that any DNA containing a nucleotide sequence or a partial nucleotide sequence encoding a protein that causes an immune response encodes the term "antigen" as used herein. In addition, it will be understood by those skilled in the art that antigens do not need to be encoded only by the full-length nucleotide sequence of a gene. It is obvious that the present disclosure includes but is not limited to the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to trigger a desired immune response. In addition, it will be understood by the skilled person that antigens do not need to be encoded by "genes" at all. It is obvious that antigens can be generated, synthesized, or can be derived from biological samples. Such biological samples can include but are not limited to tissue samples, tumor samples, cells or biological fluids.

[0123] As used herein, the term "allogeneic" refers to a transplant derived from a different animal of the same species.

[0124] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be complete immunoglobulins derived from natural or recombinant sources, or may be immunoreactive portions of complete immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in the present disclosure may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies (scFv) and humanized antibodies. In some embodiments, antibodies refer to such assemblies (e.g., complete antibody molecules, immunoadhesins, or variants thereof) that have significant known specific immunoreactivity to an antigen of interest (e.g., a tumor-associated antigen). Antibodies and immunoglobulins comprise light chains and heavy chains, with or without interchain covalent bonds between the light chains and heavy chains. The basic immunoglobulin structure in vertebrate systems is relatively well understood.

[0125] The term "antibody fragment" refers to a portion of an intact antibody and 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 and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0126] As used herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation.

[0127] As used herein, "antibody light chain," refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation.

[0128] As used herein, the term "antibody variant" includes synthetic and engineered forms of antibodies that are altered so that they do not occur in nature, for example, antibodies comprising at least two heavy chain portions rather than two complete heavy chains (such as domain deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) that are altered to bind to two or more different antigens or to different epitopes on a single antigen); heavy chain molecules linked to scFv molecules, etc. In addition, the term "antibody variant" includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three, four or more copies of the same antigen.

[0129] The term "cancer" as used herein is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0130] As used herein, the terms "cancer-associated antigen" or "tumor antigen" are used interchangeably to refer to molecules (usually proteins, carbohydrates or lipids) expressed in their entirety or as fragments (e.g., MHC / peptides) on the surface of cancer cells, and the molecules can be used to preferentially target agents to cancer cells. In some embodiments, tumor antigens are markers expressed by normal cells and cancer cells (e.g., lineage markers on B cells, such as CD19). In some embodiments, tumor antigens are cell surface molecules overexpressed in cancer cells compared to normal cells, for example, 1-fold overexpression, 2-fold overexpression, 3-fold overexpression or more compared to normal cells. In some embodiments, tumor antigens are cell surface molecules inappropriately synthesized in cancer cells, for example, molecules containing deletions, additions or mutations compared to molecules expressed on normal cells. In some embodiments, tumor antigens will be fully expressed or expressed as fragments (e.g., MHC / peptides) only on the cell surface of cancer cells, and are not synthesized or expressed on the surface of normal cells. In some embodiments, CAR of the present disclosure includes CARs comprising antigen binding domains (e.g., antibodies or antibody fragments) bound to MHC presenting peptides. Typically, peptides derived from endogenous proteins fill the pockets of major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. All nucleated cells constitutively express MHC class I complexes. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy. TCR-like antibodies targeting peptides derived from viruses 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).

[0131] 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, CDS, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD1171, CD276, CEA, claudin 18.2, claudin 24, claudin 31, claudin 40, claudin 50, claudin 60, claudin 70, claudin 80, claudin 90, claudin 100, claudin 110, claudin 120, claudin 130, claudin 140, claudin 150, claudin 160, claudin 170, claudin 180, claudin 160, claudin 180 18.2), c-Met, DLL3, DRS, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, phosphatidylinositol glycan-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-I, Lewis Y, LMPI (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUCI, Nectin4 / FAP, NKG2D-ligand (MIC-A, MIC-B and ULBP 1 to 6), NY-ESO-1, Pl 6, PD-L1, PSCA, PSMA, RORI, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2 and any combination thereof.

[0132] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell receptor that is engineered to express and specifically bind to an antigen on an immune effector cell or its precursor cell. CAR can be used for adoptive cell therapy using adoptive cell transfer. In some embodiments, adoptive cell transfer (or therapy) includes removing T cells from a patient and modifying T cells to express receptors specific for a particular antigen. In some embodiments, CAR is specific to a selected target (e.g., tumor-associated carbohydrate antigen (TACA)). CAR may also include an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region.

[0133] As used herein, the term "co-stimulatory ligand" includes molecules on antigen presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.) that specifically bind to cognate co-stimulatory molecules on T cells, thereby providing signals that mediate 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 the TCR / CD3 complex to the MHC molecule loaded with the peptide. Co-stimulatory ligands may include, but are not limited to, CD2, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory 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. Co-stimulatory ligands also particularly encompass antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited 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 ligands that specifically bind to CD83.

[0134] As used herein, "costimulatory molecules" refer to cognate binding partners on T cells, which specifically bind to costimulatory ligands, so as to mediate the costimulatory response of T cells, such as but not limited to proliferation.Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, which contribute to effective immune responses.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, costimulatory molecules include OX40, CD27, CD2, CD28, ICOS (CD278), and 4-1BB (CD137). Further examples of such co-stimulatory molecules include: CD8, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, 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 (Touch), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.

[0135] As used herein, the term "costimulatory signal" refers to a signal that, in combination with a primary signal (such as TCR / CD3 connection), results in upregulation or downregulation of T cell proliferation and / or key molecules. A costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating 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.

[0136] As used herein, the term "derived from" refers to the relationship between a first molecule and a second molecule. It defines the structural similarity between the first molecule and the second molecule, and does not imply or include a process or source restriction of the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ structure so that it has the desired function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include a restriction to a specific process for producing an intracellular signaling domain, and it does not mean that in order to provide an intracellular signaling domain, one must start with the CD3ζ sequence and delete unwanted sequences or apply mutations to reach the intracellular signaling domain.

[0137] As used herein, "disease" refers to a health state in an animal in which the animal is unable to maintain homeostasis, and in which the animal's health state continues to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but in which the animal's health state is not as good as it would be if the disorder were not present. A disorder will not necessarily result in a further deterioration in the animal's health state if left untreated.

[0138] As used herein, "diseases associated with tumor antigen expression" include, but are not limited to, diseases associated with tumor antigen expression or disorders associated with cells expressing tumor antigens, including, but not limited to, proliferative diseases, such as cancer or malignancies; or precancerous conditions, such as myelodysplasia, myelodysplastic syndrome, or preleukemia; or non-cancer-related indications associated with cells expressing tumor antigens. In some embodiments, the cancer associated with the expression of tumor antigens is a blood cancer. In some embodiments, the cancer associated with the expression of tumor antigens is a solid cancer. Other diseases associated with tumor antigen expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with tumor antigen expression. Non-cancer-related indications associated with tumor antigen expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory conditions (allergies and asthma), and transplantation. In some embodiments, cells expressing tumor antigens express or express mRNA encoding tumor antigens at any time. In some embodiments, cells expressing tumor antigens produce tumor antigen proteins (e.g., wild-type or mutant), and the tumor antigen proteins can be present at normal or reduced levels. In some embodiments, a cell expressing a tumor antigen produces detectable levels of a tumor antigen protein at one time point and subsequently produces substantially no detectable tumor antigen protein.

[0139] As used herein, the term "downregulate" refers to a reduction or elimination of gene expression of one or more genes.

[0140] As used herein, "effective amount" or "therapeutically effective amount" means an amount of a compound, formulation, material, medicament or composition as described herein that is effective to achieve a desired physiological, therapeutic or preventive result in a subject in need thereof. Such results may include, but are not limited to, an amount that, when administered to a mammal, results in a detectable level of immune response compared to the immune response detected in the absence of the composition of the present disclosure. The immune response can be readily assessed by a variety of methods recognized in the art. The skilled artisan will appreciate that the amount of the composition administered herein varies and can be readily determined based on a variety of factors, such as the disease or disorder being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the specific compound being administered, and the like. The effective amount may vary from subject to subject depending on the health and physical condition of the subject to be treated, the taxonomic group of the subject to be treated, the formulation of the composition, the assessment of the medical condition of the subject, and other relevant factors.

[0141] As used herein, the term "coding" refers to the intrinsic properties of a specific sequence of nucleotides in a polynucleotide (such as a gene, cDNA or mRNA) that serves as a template for synthesizing other polymers and macromolecules with a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence in a biological process, and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene encodes the protein. The coding strand (whose nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence table) and the non-coding strand used as a transcription template for a gene or cDNA can be referred to as encoding a protein or other product of the gene or cDNA.

[0142] As used herein, "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0143] As used herein, the term "epitope" is defined as a small chemical molecule on an antigen that can cause an immune response, thereby inducing a B cell and / or T cell response. An antigen can have one or more epitopes. Most antigens have many epitopes; that is, they are multivalent. In general, the size of an epitope is about about 10 amino acids and / or sugars. In certain exemplary embodiments, the 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. It is understood by those skilled in the art that, in general, the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion for antigen specificity, and therefore distinguishes one epitope from another epitope. Based on the present disclosure, the peptide used in the present disclosure can be an epitope. As used herein, the term "exogenous" refers to any substance introduced from outside an organism, cell, tissue, or system or produced outside an organism, cell, tissue, or system.

[0144] As used herein, the term "expand" 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 is increased relative to the number initially present in the culture. In another embodiment, the number of immune cells (e.g., T cells) expanded ex vivo is increased relative to other cell types in the culture.

[0145] As used herein, the term "exogenous" refers to any substance introduced from outside of an organism, cell, tissue, or system or produced outside of an organism, cell, tissue, or system.

[0146] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0147] As used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by a host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0148] As used herein, the term "ex vivo" refers to cells that have been removed from an organism (eg, a human) and propagated outside of the organism (eg, in a culture dish, test tube, or bioreactor).

[0149] As used herein, the term "Fc portion" or "Fc monomer" means in the present disclosure a polypeptide comprising at least one domain having the function of a CH2 domain and at least one domain having the function of a CH3 domain of an immunoglobulin molecule. It is obvious from the term "Fc monomer" that a polypeptide comprising these CH domains is a "polypeptide monomer". An Fc monomer can be a polypeptide comprising at least one immunoglobulin constant region fragment, the fragment excluding the first constant region immunoglobulin domain (CH1) of the heavy chain, but maintaining at least the functional portion of the CH2 domain and the functional portion of the CH3 domain, wherein the CH2 domain is located at the amino terminus of the CH3 domain. In a preferred aspect of this definition, an Fc monomer can be a polypeptide constant region comprising an Ig-Fc hinge region, a CH2 region, and a portion of the CH3 region, wherein the hinge region is located at the amino terminus of the CH2 domain.

[0150] It is envisioned that the hinge region of the present disclosure promotes dimerization. Such Fc polypeptide molecules can be obtained by papain digestion of, for example, but not limited to, immunoglobulin regions (of course, a dimer of two Fc polypeptides will be produced). In another aspect of this definition, an Fc monomer can be a polypeptide region comprising a portion of a CH2 region and a CH3 region. Such Fc polypeptide molecules can be obtained by pepsin digestion of, for example, but not limited to, immunoglobulin molecules. In one embodiment, the polypeptide sequence of an Fc monomer is substantially similar to the following Fc polypeptide sequences: IgG1 Fc region, IgG2 Fc region, IgG3 Fc region, IgG4 Fc region, IgM Fc region, IgAFc region, IgD Fc region, and IgE Fc region. (See, e.g., Padlan, Molecular Immunology, 31 (3), 169-217 (1993)). Because there is some variation among immunoglobulins, and for clarity only, 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.

[0151] The Fc monomer may also include a flexible hinge at the N-terminus of these domains. For IgA and IgM, the Fc monomer may include a J chain. For IgG, the Fc portion comprises immunoglobulin domains CH2 and CH3 and a hinge between the first two domains and CH2. Although the boundaries of the Fc portion may vary, an example of a human IgG heavy chain Fc portion comprising a functional hinge, CH2 and CH3 domains may be defined, for example, as comprising residues SEQ ID NOs 153-158.

[0152] IgG hinge regions can be identified by analogy using Kabat. In one embodiment, the hinge domain / region of the present disclosure comprises amino acid residues corresponding to an 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: 153 or 154).

[0153] In one embodiment, the IgG1 hinge domain / region comprises the amino acid sequence of EPKSCDKTHTCPPCP (SEQ ID NO: 154).

[0154] 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: 155), the IgG3 subtype hinge sequence ELKTPLDTTHTCPRCP (SEQ ID NO: 156) or ELKTPLGDTTHTCPRCP (SEQ ID NO: 157) and / or the IgG4 subtype hinge sequence ESKYGPPCPSCP (SEQ ID NO: 158).

[0155] In some embodiments, the fusion protein further comprises a third domain comprising two polypeptide monomers, wherein each monomer comprises a hinge, a CH2 domain, and a CH3 domain. In one embodiment, the third domain comprises, in order from amino to carboxyl, hinge-CH2-CH3-linker-hinge-CH2-CH3. In one embodiment, the CH2 domain comprises an intradomain cysteine ​​disulfide bridge.

[0156] In another embodiment, the two polypeptide monomers are fused to each other via a peptide linker. However, in another embodiment, the first and second domains 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 an amino acid sequence of GGGS (e.g., Gly4Ser (SEQ ID NO: 128)) or a polymer thereof (e.g., (Gly4Ser) n , wherein n is an integer of 5 or more (e.g., 5, 6, 7, 8, etc. or more)).

[0157] As used herein, the term "homologous" refers to sequence similarity or sequence identity between two peptides or between two nucleic acid molecules. When a certain position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if a certain position in each of the two DNA molecules is occupied by adenine, the molecules are homologous at that position. The homology percentage between the two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared and multiplied by 100. For example, if 6 positions out of 10 positions in the two sequences are matching or homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have a total homology of 50%. In general, when two sequences are aligned to provide maximum homology, a comparison is performed.

[0158] As used herein, the term "identity" refers to the subunit sequence identity between two polymer molecules, in particular between two amino acid molecules, such as between two polypeptide molecules. When two amino acid sequences have the same residue at the same position; for example, if a certain position in each of the two polypeptide molecules is occupied by arginine, the two molecules are identical at that position. The identity or degree of having the same residue at the same position in the alignment of two amino acid sequences is usually expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; for example, if half of the positions in the two sequences (e.g., five positions in a polymer of ten amino acids in length) are identical, the two sequences have 50% identity; if 90% of the positions (e.g., 9 out of 10 positions) are matched or identical, the two amino acid sequences have 90% identity.

[0159] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins used as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. Such proteins include five members, IgA, IgG, IgM, IgD and IgE. IgA is the main antibody present in body secretions (such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and urogenital tracts). IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response of most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation and other antibody reactions, and plays an important role in defending against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function but can serve as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity reactions by causing mast cells and basophils to release mediators after exposure to allergens.

[0160] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify the antigen molecule as foreign and induce antibody formation and / or activate lymphocytes to remove the antigen. The term "immunostimulation" as used herein refers to increasing the overall immune response. The term "immunosuppression" as used herein refers to decreasing the overall immune response.

[0161] As used herein, the term "immune response" is defined as a cellular reaction against an antigen that occurs when lymphocytes recognize the antigen molecule as foreign and induce antibody formation and / or activate lymphocytes to remove the antigen.

[0162] As used herein, the term "immune effector cell" refers to a cell that participates in an immune response (e.g., participates in promoting an immune effector response). Examples of immune effector cells include T cells (e.g., α / ηT cells and γ / δT cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid phagocytes.

[0163] 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, an immune effector function or response refers to the property of a T cell or NK cell to promote the killing of a target cell or inhibit the growth or proliferation of a target cell. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0164] As used herein, "instructional materials" include publications, recordings, diagrams, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present disclosure. The instructional materials of the kits of the present disclosure can be, for example, fixed to a container containing the nucleic acids, peptides, and / or compositions of the present disclosure, or transported together with the container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructional materials can be transported separately from the container so that the recipient can use the instructional materials and the compounds in combination.

[0165] As used herein, the term "isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials in its natural state is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-natural environment, such as, for example, a host cell.

[0166] In the context of the present disclosure, the following abbreviations for common nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0167] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" include 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 also include introns, as long as the nucleotide sequence encoding a protein may contain introns in some versions.

[0168] As used herein, the term "lectin" or "hemagglutinin" refers to a protein or peptide that binds to a carbohydrate structure. It will be understood by those skilled in the art that a lectin is a protein or peptide that is highly specific for binding to a sugar moiety. Lectins are carbohydrate-binding proteins that are highly specific for carbohydrates found on proteins and / or lipids, thereby causing agglutination of specific cells or precipitation of glycoconjugates and polysaccharides. Lectins play a role in recognition at the cellular and molecular levels, and play a variety of roles in biological recognition phenomena involving cells, carbohydrates and proteins. Lectins also mediate the connection and binding of bacteria, viruses and fungi to their intended targets. In one embodiment, "lectin" can be defined as a protein or glycoprotein of a non-immunoglobulin nature that is capable of specific recognition and reversible binding to the carbohydrate moiety of a complex glycoconjugate (protein and / or lipid) without changing the covalent structure of any recognized glycosyl ligand. Lectins are glycoproteins with carbohydrate binding domains that have reversible binding capabilities to specific sugar moieties in glycoproteins or glycolipids and free monosaccharide and polysaccharide structures. Although many organisms express lectins or lectin-like biomolecules, most recently identified lectins of scientific interest have been purified from plant sources.

[0169] As used herein, the term "tumor-associated carbohydrate antigen" or "TACA" refers to carbohydrate structures found in conditions associated with altered glycosylation, such as cancer. Carbohydrate-containing macromolecules (glycans) are ubiquitous in biological systems and are essential for many biological functions. Carbohydrates can be attached to proteins (glycoproteins), lipids (glycolipids), and can also exist in the form of carbohydrate (glycosaminoglycan) chains. Structural changes (glycosylation) in these carbohydrate-containing macromolecules have a significant impact on cancer biology and cancer progression. In fact, altered glycosylation is a common feature of tumor cells and leads to the formation of tumor-associated carbohydrates (TACAs). Cancer cells can often be distinguished from normal cells by the abnormal levels and types of carbohydrate structures displayed on their surfaces.

[0170] Three common changes in carbohydrate-containing macromolecules are associated with cancer: increased expression of truncated or incomplete glycans, increased branching of N-glycans, and increased or altered presence of sialic acid-containing glycans. For example, cancer-associated glycans often show an increase in the amount of sialic acid, and this hypersialylation enhances the activation of sialic acid-binding receptors (such as selectins and sialic acid-binding immunoglobulin-like lectins (Siglecs)), leading to cancer progression. Another of the most common cancer-associated glycosylation changes is the truncation of O-linked carbohydrate chains (truncation of O-glycoproteins), such as mucins. Under normal circumstances, N-acetyl-galactosamine (GalNAc) sugar residues are attached to serine or threonine of glycoproteins (GalNAcα1-O-Ser / Thr, Tn antigens) and are usually extended by T synthase (core 1β3-galactosyltransferase) in the Golgi apparatus, which attaches the galactose residue to the Thomsen-Friedenreich (TF) antigen (Tn antigen). In various cancers, this process is altered and the glycosylation of the Tn antigen or its sialylated form (sialyl-Tn (STn) antigen) is altered, resulting in truncated T antigen, Tn antigen and STn antigen. In addition, an increase in branching of the N-glycoprotein (which stimulates galectin-3) and changes in glycolipids such as, for example, gangliosides (GM3, GM2, CD3 and GD2) are observed. The following TACAs have been observed in various cancers: (i) H / Le in primary non-small cell lung cancer y / ILe a ; (ii) Sialyl-Le in various types of cancer x (SLe x ) and sialyl-Lea (SLea); (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); and (v) globo-H in breast cancer, ovarian cancer and prostate cancer; and (vi) disialylgalactosylgloboside in renal cell carcinoma.

[0171] Thus, the term "tumor-associated carbohydrate antigen (TACA)" encompasses all altered carbohydrate structures on proteins and / or lipids that are expressed on tumor tumor cells, promote cancer metastasis, cancer progression, cancer and non-cancer immunosuppression, and / or promote autoimmune diseases. The skilled person will understand that the carbohydrate structure consists 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.)). The skilled person will understand that the carbohydrate structure can be independent and / or linked to proteins or lipids, referred to as glycoproteins and glycolipids. The skilled person will understand that these carbohydrate structures bind to lectins.

[0172] As used herein, "slow virus" refers to a genus of the Retroviridae family. Slow viruses are unique among retroviruses in being able to infect non-dividing cells; they can deliver large amounts of genetic information to the DNA of host cells, so they are one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of slow viruses. Vectors derived from slow viruses provide a way to achieve significant levels of gene transfer in vivo.

[0173] As used herein, the term "limited toxicity" refers to the peptides, polynucleotides, cells and / or antibodies of the present disclosure that exhibit a lack of significant negative biological effects, anti-tumor effects or significant negative physiological symptoms on healthy cells, non-tumor cells, non-diseased cells, non-target cells or populations of such cells in vitro or in vivo.

[0174] As used herein, the term "flexible polypeptide linker" or "linker" as used in the context of scFv refers to a peptide linker composed of amino acids (such as glycine and / or serine residues) used alone or in combination to connect 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 comprises an amino acid sequence (Gly-Gly-Gly-Ser)n, wherein n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5 and n=6, n=7, n=8, n=9 and n=10 (SEQ ID NO: 6592). In one embodiment, a flexible polypeptide linker includes but is not limited to (Gly4 Ser) 4 or (Gly4Ser) 3 In another embodiment, the linker comprises multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser).

[0175] As used herein, the term "modified" means an altered state or structure of a molecule or cell of the present disclosure. Molecules can be modified in a variety of ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0176] As used herein, the term "modulate" means mediating a detectable increase or decrease in the subject's response level compared to the subject's response level in the absence of the treatment or compound and / or compared to the response level of other identical but untreated subjects. The term encompasses perturbing and / or influencing a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0177] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may contain introns.

[0178] As used herein, the term "operably linked" refers to a functional connection 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 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. In general, operably linked DNA sequences are contiguous and in the same reading frame when it is necessary to connect two protein coding regions.

[0179] As used herein, the term "overexpressed tumor antigen" or "overexpression of a tumor antigen" is intended to mean that the expression level of a tumor antigen in cells of a diseased area (such as a solid tumor) within a particular tissue or organ is abnormal relative to the expression level in normal cells within the patient's tissue or organ. Patients with solid tumors or hematological malignancies characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

[0180] As used herein, the term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection or infusion techniques.

[0181] As used herein, the terms "patient," "subject," and "individual," etc., are used interchangeably herein and refer to any animal or cell thereof (whether in vitro or in situ) suitable for the methods described herein. The subject can be a mammal, such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) or a primate (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, farm animals, sports animals, and pets. Any organism that can cause an immune response can be a subject or patient. In certain exemplary embodiments, the subject is a human.

[0182] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, nucleic acid and polynucleotide as used herein are interchangeable. It is generally known to those skilled in the art that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., using common cloning techniques and PCR. TM etc. by cloning nucleic acid sequences from recombinant libraries or cell genomes, as well as by synthetic means.

[0183] As used herein, the terms "peptide" and "polypeptide" and "protein" are used interchangeably and refer to compounds consisting 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 constitute a protein sequence or a peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and long chains, which are commonly referred to in the art as proteins, and there are many types of proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0184] As used herein, the term "promoter" is defined as a DNA sequence recognized by the cellular synthetic machinery or introduced synthetic machinery, which is required to initiate specific transcription of a polynucleotide sequence.

[0185] As used herein, the term "promoter" or "regulatory sequence" means a nucleic acid sequence required for the expression of a gene product operably linked to the "promoter" or "regulatory sequence". In some cases, this sequence may be a core promoter sequence, while in other cases, this sequence may also include an enhancer sequence and other regulatory elements required for the expression of a gene product. The promoter / regulatory sequence may be, for example, a sequence that expresses a gene product in a tissue-specific manner.

[0186] As used herein, a "constitutive promoter" is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0187] As used herein, an "inducible promoter" is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, results in production of the gene product in a cell essentially only when an inducer corresponding to the promoter is present in the cell.

[0188] As used herein, a "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene, causes the gene product to be produced in a cell substantially only when the cell is a cell of the tissue type corresponding to the promoter.

[0189] As used herein, the term "specific binding" or "selectively binding" for antibodies, antigen binding domains, CARs or bispecific fusion proteins means recognizing a specific antigen (e.g., TACA), but not substantially recognizing or binding other molecules in the sample, antibodies, antigen binding domains, CARs or bispecific fusion proteins. For example, antibodies, antigen binding domains, CARs or bispecific fusion proteins that specifically bind to antigens (e.g., TACA) from one species may also bind to antigens from one or more species. However, this cross-species reactivity itself does not change the specific classification of antibodies. In another example, antibodies, antigen binding domains, CARs or bispecific fusion proteins that specifically bind to antigens (e.g., TACA) may also be combined with different allele forms of antigens (e.g., TACA). However, this cross-reaction itself does not change the specific classification of antibodies, antigen binding domains, CARs or bispecific fusion proteins. In some cases, the term "specific binding" or "specifically binds" can be used to refer to the interaction of an antibody, protein, peptide, antigen binding domain, CAR or bispecific fusion protein with a second chemical species, to mean that the interaction depends on the presence of a specific structure (e.g., antigenic determinant or epitope) on the chemical species; for example, an antibody, antigen binding domain, CAR or bispecific fusion protein recognizes and binds to a specific protein structure (TACA) rather than a general protein. If the antibody, antigen binding domain, CAR or bispecific fusion protein is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody, antigen binding domain, CAR or bispecific fusion protein may reduce the amount of labeled A bound to the antibody.

[0190] As used herein, the term "single-chain antibody" refers to an antibody formed by recombinant DNA technology, in which immunoglobulin heavy and light chain fragments are linked to the Fv region via a stretch of engineered amino acids. A variety of methods for generating single-chain antibodies are known, including 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 334:54454 (1989); Skerra et al., Science 242:1038-1041 (1988) described in those methods.

[0191] As used herein, the term "specificity" refers to the ability to specifically bind (e.g., immune response) to a given target antigen (e.g., a human target antigen). A chimeric antigen receptor can be monospecific and contain one or more binding sites that specifically bind to a target, or a chimeric antigen receptor can be multispecific and contain two or more binding sites that specifically bind to the same or different targets. In certain embodiments, a chimeric antigen receptor is specific for two different (e.g., non-overlapping) portions of the same target. In certain embodiments, a chimeric antigen receptor is specific for more than one target.

[0192] As used herein, the term "specific binding" with respect to an antibody means an antibody or its binding fragment (e.g., scFv) that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to the antigen from one or more species. However, this cross-species reactivity itself does not change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allele forms of the antigen. However, this cross-reactivity itself does not change the specific classification of the antibody. In some cases, the term "specific binding" or "specifically binding" may be used to refer to the interaction of an antibody, protein, chimeric antigen receptor or peptide with a second chemical species to mean that the interaction depends on the presence of a specific structure (e.g., antigenic determinant or epitope) on the chemical species; for example, a chimeric antigen receptor recognizes and binds to a specific protein structure rather than a general protein. If the antibody is specific for epitope "A", then in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0193] As used herein, the term "stimulation" means a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event (such as, but not limited to, signal transduction via a TCR / CD3 complex). Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-β and / or reorganization of cytoskeletal structure, clonal expansion, and differentiation into different subpopulations.

[0194] 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.

[0195] 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.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response of the T cell, including but not limited to activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and particularly encompass MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0196] As used herein, "substantially purified" cells are cells that are substantially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some cases, a substantially purified cell population refers to a homogenous cell population. In other cases, the term refers only to cells that have been separated from cells with which the cells are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0197] As used herein, "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 can specifically bind under conditions sufficient for binding to occur.

[0198] As used herein, the term "T cell receptor" or "TCR" refers to a membrane protein complex that participates in activating T cells in response to antigen presentation. TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR consists of a heterodimer of an alpha (a) chain and a beta (β) chain, and is coupled to three dimer modules CD3δ / CD3ε, CD3γ / CD3ε, and CD3ζ / CD3ζ. In some cells, TCR consists of γ and δ (γ / δ) chains (CD3γ / CD3ε). TCR may exist in α / β and γ / δ forms, which are structurally similar but have different structural positions and functions. Each chain consists of two extracellular domains, a variable domain, and a constant domain. In some embodiments, TCR can be modified on any cell comprising TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδT cells.

[0199] As used herein, the term "therapeutic" means treating and / or preventing. A therapeutic effect is obtained by suppressing, alleviating or eradicating a disease state.

[0200] The term "therapeutically effective amount" or "effective amount" refers to the amount of the subject compound that will elicit a biological or medical response in a tissue, system, or subject that is sought by a researcher, veterinarian, medical doctor, or other clinician. The term "therapeutically effective amount" includes an amount of a compound that, when administered, is sufficient to prevent the development of the condition or disease being treated or to alleviate to some extent one or more signs or symptoms of the condition or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0201] As used herein, the term "therapy" refers to any regimen, method and / or agent (e.g., CAR-T) that can be used to prevent, control, treat and / or improve a disease or its associated symptoms. In some embodiments, the terms "therapies" and "therapy" refer to biological therapy (e.g., adoptive cell therapy), supportive therapy (e.g., lymphocyte clearance therapy) and / or other therapies known to those skilled in the art (such as medical personnel) for preventing, controlling, treating and / or improving a disease or its associated symptoms.

[0202] As used herein, the terms "treat", "treatment" and "treating" refer to the slowing down or improvement of the progression, severity, frequency and / or duration of a disease or its associated symptoms caused by the administration of one or more therapies (including but not limited to CAR-T therapy for the treatment of solid tumors). As used herein, the term "treatment" may also refer to changing the disease process of the subject being treated. The therapeutic effects of treatment include but are not limited to preventing the occurrence and recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the term "treating" disease as used herein means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by the subject.

[0203] As used herein, the term "transfection" or "transformation" or "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0204] As used herein, the phrase "under transcriptional control" or "operably linked" means that the promoter is in the correct location and orientation relative to the polynucleotide to control transcription initiation by RNA polymerase and expression of the polynucleotide.

[0205] As used herein, "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphoteric compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted as including non-plasmid and non-viral compounds that promote nucleic acid transfer into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0206] As used herein, the term "xenogeneic" refers to a transplant derived from an animal of a different species.

[0207] Scope: Throughout this disclosure, various aspects of the disclosure may be presented in range form. It should be understood that the description in range form is merely for convenience and brevity, and should not be interpreted as an immutable rigid limitation on the scope of the disclosure. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and each numerical value within the range. For example, a range description such as 1 to 6 should be considered to have specifically disclosed sub-ranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and each number within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6. This applies regardless of the breadth of the range.

[0208] III. TACA Antigen Binding Domain

[0209] One aspect of the present disclosure provides a bispecific fusion protein or chimeric antigen receptor comprising an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA). In some embodiments, the antigen binding domain comprises a TACA binding domain derived from a lectin. In some embodiments, the antigen binding domain comprises more than one (e.g., multiple) TACA binding domains.

[0210] Malignant transformation of cells is almost universally accompanied by abnormal glycosylation of cell surface proteins or lipids (Kim and Varki, 1997, Glycoconj J, 14: 569-576). In fact, 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 (TACA) (Table 1). This tumor-specific property makes cell surface TACA an excellent target antigen for the production of monoclonal antibodies targeting many common cancers. However, despite decades of efforts, specific antibodies with high affinity for TACA have not yet been obtained due to the difficulty in generating antibodies against carbohydrate antigens.

[0211] Therefore, the inventors of the present disclosure rely on lectins rather than antibodies to engineer bispecific fusion proteins and CARs that selectively target TACA on tumor cells. Lectins and their binding partners are well known in the art. See, for example, functionalglycomics.org / glycomics / publicdata / primaryscreen.jsp. The lectin binding protein disclosed in the present disclosure provides an opportunity for the development of a new class of therapeutic drugs for cancer immunotherapy, and the lectin binding protein has significant advantages over the prior art (e.g., GlyTR chimeric protein). Based on the GlyTR concept and the availability of a variety of different lectins that are specific to many different TACAs, a variety of GlyTRs can be generated by replacing L-PHA with other lectins; or a chimeric protein consisting of a lectin and scFv can be produced, which can recruit other immune effector cells. The function of the lectin domain can be improved by mutation. For example, GlyTR L-PHA x CD3 can be further improved by exchanging the carbohydrate binding domain of E-PHA with that of L-PHA, which resulted in a 20-30 fold increase in binding (Kaneda et al., 2002, J Biol Chem, 277: 16928-16935).

[0212]

[0213]

[0214] The antigen binding domain of the bispecific fusion protein or CAR disclosed herein is designed to specifically target glycoproteins and / or glycolipids (i.e., macromolecules containing carbohydrates) on tumor cells. In some embodiments, the bispecific fusion protein or CAR disclosed herein comprises 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 include any type of protein or its epitope associated with the target cell. For example, CAR or a bispecific fusion protein may include affinity for a target antigen on a target cell indicating a specific state of the target cell.

[0215] In some embodiments, the antigen binding domain comprises multiple (e.g., more than one) TACA binding domains. In some embodiments, the antigen binding domain comprises two, three, four, five, six, seven, eight, nine, ten or more 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.

[0216] A.TACA

[0217] 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.

[0218] 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, α2,6 sialylation, sialylation, sialyl-Lewis^, disialyl-Lewis^, sialyl 6-sulfoLexis^, x 、Lewis-y(Le y ), Lewis Y, Globo H, GD2, GD3, GM3 and fucosyl GM1. In some embodiments, the CAR or bispecific fusion protein selectively targets 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, disialo-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-glycans, GalNAc, Tn antigen, GalNAcα-ser, GalNAc or GalNAcβ1.

[0219] In one embodiment, the TACA binding domain binds to N-glycans. In certain embodiments, the TACA binding domain binds to triantennary and tetraantennary oligosaccharides. In one embodiment, the TACA binding domain binds to β1,6GlcNAc branched N-glycans. In one embodiment, the TACA binding domain binds to a Tn epitope.

[0220] B. Lectin

[0221] 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.

[0222] In some embodiments, the lectin is selected from the group consisting of: galectins, sialic acid-binding immunoglobulin-like lectins, selectins; C-type lectins; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Lycopersicon esculentum lectin); E-PHA (Lycopersicon esculentum lectin); tomato lectin (Lycopersicon esculentum lectin; LEA); peanut lectin (Arachis hypogaea Agglutinin; PNA); potato lectin (Solanum tuberosum lectin); pokeweed mitogen (Phytolacca Americana lectin); wheat germ agglutinin (Wheat germ agglutinin); agglutinin (Triticum Vulgaris lectin); Artocarpus polyphemus lectin (Jacalin letin); Vetch lectin (VVA); Helicoverpa agglutinin (HPA); Wisteria floribunda agglutinin (WFA); Sambucus sambucus agglutinin (SNA); BC2L-CNt (lectin from the Gram-negative bacterium Burkholderia cenocepacia); MAL; PVL; Sclerotium sclerotium agglutinin (SRL); Echinops serrulata agglutinin (ESA); CLEC17A (protectin); Dictyophora aurantifolia agglutinin; S. sessile elderberry agglutinin (SSA); Gleheda; Morniga G; Salvia miltiorrhiza agglutinin; bogotensis agglutinin, Salvia officinalis agglutinin, Changshan Shanhaizhou agglutinin, Concha strychnos nucifera agglutinin, Griffonia suffruticosa (GsLA4), Quaternary bean (acidic WBAI), Abrus precatorius lectin, Amaranthus caudatus lectin, Amaranthus gingkoi lectin, Amaranthus chinensis lectin, Jackfruit agglutinin, Morus alba lectin, Jackfruit agglutinin, Lablab bisporus lectin, Lablab bisporus lectin, Soybean agglutinin, and Agaricus bisporus lectin.

[0223] In some embodiments, the lectin is a galectin that may be 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 sialic acid-binding immunoglobulin-like lectin that can be selected from the group consisting of: sialic acid-binding immunoglobulin-like lectin-1 (sialoadhesin), sialic acid-binding immunoglobulin-like lectin-2 (CD22), sialic acid-binding immunoglobulin-like lectin-3 (CD33), sialic acid-binding immunoglobulin-like lectin-4 (myelin-associated glycoprotein), sialic acid-binding immunoglobulin-like lectin-5, sialic acid-binding immunoglobulin-like lectin-6, sialic acid-binding immunoglobulin-like lectin-7, sialic acid-binding immunoglobulin-like lectin-8, sialic acid-binding immunoglobulin-like lectin-9, sialic acid-binding immunoglobulin-like lectin-10, sialic acid-binding immunoglobulin-like lectin-11, sialic acid-binding immunoglobulin-like lectin-12, sialic acid-binding immunoglobulin-like lectin-13, sialic acid-binding immunoglobulin-like lectin-14, sialic acid-binding immunoglobulin-like lectin-15, sialic acid-binding immunoglobulin-like lectin-16, sialic acid-binding immunoglobulin-like lectin-17, sialic acid-binding immunoglobulin-like lectin-18, sialic acid-binding immunoglobulin-like lectin-19, sialic acid-binding immunoglobulin-like lectin-2 In some embodiments, the TACA binding domain is derived from a selectin or a C-type lectin.

[0224] In some embodiments, the lectin is a polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) which 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). LNT9), ppGalNAc-T10 (GALNT10), ppGalNAc-T12 (GALNT12), ppGalNAc-T13 (GALNT13), ppGalNAc-T14 (GALNT14), ppGalNAc-T15 (GALNT1 5), ppGalNAc-T16 (GALNT16), ppGalNAc-T17 (GALNT17), ppGalNAc-T18 (GALNT18), ppGalNAc-T-like 5 (GALNTL5) and ppGalNAc-T-like 6 (GALNTL6).

[0225] In some embodiments, the antigen binding domain of the CAR or bispecific fusion protein described herein 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, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6 sialylated, sialylated, sialyl-Lewis x / a, disialyl-Lewis x / a, sialyl 6-sulfoLexis x 、Lewis-y(Le y ), Lewis Y, Globo H, GD2, GD3, GM3 and fucosyl GM1.

[0226] C. Chimeric Antigen Receptor

[0227] One aspect of the present disclosure provides compositions and methods for modified immune cells or their precursor cells (e.g., modified T cells), which compositions and methods include chimeric antigen receptors (CARs) with affinity for tumor-associated carbohydrate antigens (TACAs). The subject CAR of the present disclosure includes an antigen binding domain (e.g., tumor-associated carbohydrate antigens (TACAs)), a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain. The subject CAR of the present disclosure may optionally include a hinge domain. In some embodiments, each domain of the subject CAR is separated by a linker. In one aspect, a chimeric antigen receptor selectively binding to a tumor-associated carbohydrate antigen (TACA) is encoded by an isolated nucleic acid disclosed herein.

[0228] One aspect of the present disclosure provides a chimeric antigen receptor (CAR) comprising the amino acid sequence shown in SEQ ID NO:71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. In some embodiments, the CAR comprises 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 shown in SEQ ID NO:71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

[0229] In another aspect of the present disclosure, a chimeric antigen receptor that selectively binds a tumor-associated carbohydrate antigen (TACA) comprises an antigen binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, or 146; or The amino acid sequence shown in NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146 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%, at least 99% sequence identity; a CD8 hinge domain; a CD8 transmembrane domain; a CD28 co-stimulatory domain; and a CD3ζ intracellular signaling domain.

[0230] In another aspect, a chimeric antigen receptor that selectively binds a tumor associated carbohydrate antigen (TACA) comprises an antigen binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, or 146; or The amino acid sequence shown in NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146 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%, at least 99% sequence identity; a CD8 hinge domain; a CD8 transmembrane domain; a 4-1BB co-stimulatory domain; and a CD3ζ intracellular signaling domain.

[0231] In another aspect, a chimeric antigen receptor that selectively binds a tumor associated carbohydrate antigen (TACA) comprises an antigen binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, or 146; or The amino acid sequence shown in NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145 or 146 has at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 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; a CD8 hinge domain; a CD8 transmembrane domain; CD28 and 4-1BB costimulatory domains; and a CD3ζ intracellular signaling domain.

[0232] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 72, 88, 89, 91, 92, or 93. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 93, 94, 95, 96, 97, 98, or 99.

[0233] In some embodiments, the chimeric antigen receptor (CAR) has affinity for: β1,6 branched, β1,6GlcNAc branched N-glycan, T antigen, Tn antigen, sialyl-T epitope, Tn epitope, sialyl-Tn epitope, α2,6 sialylated, sialylated, sialyl-Lewis x / a , disialo-Lewis x / a , Sialyl 6-sulfo Lexis x 、Lewis-y(Le y ), Lewis Y, Globo H, GD2, GD3, GM3 or fucosyl GM1. In some embodiments, the chimeric antigen receptor (CAR) has affinity for β1,6 branched or β1,6GlcNAc branched N-glycans. In some embodiments, the chimeric antigen receptor (CAR) has affinity for Tn antigen or sialyl-Tn epitope.

[0234] 1. Extracellular domain

[0235] The antigen binding domain of CAR is the extracellular region of CAR, for binding to specific target antigens, including proteins, carbohydrates and glycolipids. In some embodiments, CAR comprises affinity for target antigens (e.g., tumor-associated antigens) on target cells (e.g., cancer cells). The target antigen may include any type of protein or its epitope associated with the target cell. For example, CAR may include affinity for target antigens on target cells indicating a specific state of the target cell.

[0236] In some embodiments, the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152. Alternatively, the antigen binding domain comprises 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 shown in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152. In some embodiments, the antigen binding domain comprises an amino acid sequence disclosed in Table 2 or Table 3.

[0237] In some embodiments, the antigen binding domain comprises an amino acid sequence that is at least 90% homologous to SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, or 146.

[0238] The antigen binding domain can be operably connected to another domain of the CAR, such as a transmembrane domain, a costimulatory signaling domain, or an intracellular signaling domain (each domain is described elsewhere herein) for expression in a cell. The antigen binding domain described herein can be combined with any transmembrane domain, any costimulatory signaling domain, any intracellular signaling domain, or any other domain described herein that can be included in the CAR of the present disclosure.

[0239] 2. Connector

[0240] The terms "linker" and "spacer" are used interchangeably herein. Linkers are typically rich in glycine to provide flexibility and rich in serine or threonine to provide solubility. Multiple linkers can be used to connect more than one TACA binding domain. In some embodiments, more than one TACA binding domain can be operably connected 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 hindered cross-linking agent, and a non-hindered cross-linking agent. In one embodiment, the linker is a peptide linker. The peptide linker can be a glycine-serine linker. The length of the peptide linker can be 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.

[0241] Various 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 WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entirety. Those skilled in the art are able to select appropriate linker sequences for the bispecific fusion proteins and / or CARs of the present disclosure.

[0242] In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 127 or the amino acid sequence of SEQ ID NO: 131.

[0243] 3. Transmembrane domain

[0244] Regarding the transmembrane domain, the CAR 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 the subject CAR is a region that can cross the plasma membrane of a cell (e.g., an immune cell or its precursor). The transmembrane domain is used to insert into a cell membrane, such as a eukaryotic cell membrane. In some embodiments, the transmembrane domain is located between the antigen binding domain and the intracellular domain of the CAR.

[0245] In one embodiment, the transmembrane domain is naturally associated with one or more domains in the CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0246] The transmembrane domain may be derived from a natural source or a synthetic source. When the source is natural, the domain may be derived from any membrane-bound protein or transmembrane protein, such as a type I transmembrane protein. When the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates CAR insertion into the cell membrane, for example, an artificial hydrophobic sequence.

[0247] In some embodiments, the chimeric antigen receptor (CAR) comprises a transmembrane domain, which may include a transmembrane region of a molecule selected from the group consisting of: T cell receptor (TCR) -α, TCR-β, CD3-ζ, CD3-ε, 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 includes a CD8 transmembrane domain. Alternatively, the transmembrane domain includes the amino acid sequence of SEQ ID NO: 148.

[0248] In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise primarily hydrophobic residues such as leucine and valine. In certain exemplary embodiments, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0249] The transmembrane domains described herein can be combined with any antigen binding domain described herein, any co-stimulatory signaling domain described herein, any intracellular signaling domain described herein, or any other domain described herein that can be included in a subject's CAR.

[0250] 4. Intracellular domain

[0251] The subject CAR of the present disclosure also includes an intracellular domain. The cellular domain of CAR is responsible for activating at least one effector function of a cell (e.g., an immune cell) expressing CAR. The intracellular domain transduces effector function signals and instructs cells (e.g., immune cells) to perform their specialized functions, such as harming and / or destroying target cells.

[0252] The intracellular domain or cytoplasmic domain of CAR is responsible for activating cells expressing CAR. Examples of intracellular domains for the present invention include, but are not limited to, the cytoplasmic portion of surface receptors, costimulatory molecules, and any synergistic molecules to initiate signal transduction in T cells, as well as any derivatives or variants of these elements and any synthetic sequences with the same functional capabilities. In certain embodiments, the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain.

[0253] Examples of intracellular signaling domains include, but are not limited to, the z chain of the T cell receptor complex or any homolog thereof, syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD3, and CD28. In one embodiment, the intracellular signaling domain may be a human CD3 zeta chain, FcγRin, FcεRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor carrying an immunoreceptor tyrosine activation motif (ITAM), and combinations thereof.

[0254] Other examples of intracellular domains include fragments or domains from one or more molecules or receptors, including but not limited to TCR, CDS ζ, CDS γ, CDS δ, CDS ε, CD86, common FcR γ, FcR β (Fc ε Rib), CD79a, CD79b, Fc γ R1 la, DAP10, DAP12, T cell receptor (TCR), CDS, 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, CDS, ICAM-1, GITR, BAFFR, HVEM ( LIGHTR), SLAMF7, NKpSO(KLRF1), CD127, CD160, CD19, CD4, CDSα, CDSβ, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, C D49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlId, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CD lib, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACA M1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylOS), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKpSO, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other costimulatory molecules described herein, any derivative, variant or fragment thereof, any synthetic sequence of a costimulatory molecule with the same functional capability, and any combination thereof.

[0255] Additional examples of intracellular domains include, but are not limited to, several types of intracellular signaling domains of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins, including CDS, B7 family co-stimulatory proteins, and tumor necrosis factor receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6):651-653). In addition, the intracellular signaling domain may include signaling domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195), such as the signaling domain of NKpSO (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0256] The intracellular signaling domains suitable for the subject CAR of the present disclosure include any desired signaling domains that provide different and detectable signals (e.g., one or more cytokines produced by cells increase; changes in target gene transcription; changes in protein activity; changes in cell behavior, e.g., cell death; cell proliferation; cell differentiation; cell survival; regulation of cell signaling responses; etc.) in response to the activation of CAR (i.e., activated by antigens and dimerizers); In some embodiments, the intracellular signaling domain includes at least one (e.g., one, two, three, four, five, six, 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 linked to the membrane-bound CAR, but diffuses in the cytoplasm.

[0257] The intracellular signaling domains of the subject CARs suitable for the present invention include intracellular signaling polypeptides containing immunoreceptor tyrosine activation motifs (ITAMs). In some embodiments, the ITAM motif is repeated twice in the intracellular signaling domain, wherein the first and second instances of the ITAM motif are separated by 6 to 8 amino acids from each other. In one embodiment, the intracellular signaling domain of the subject CAR comprises 3 ITAM motifs. In some embodiments, the intracellular signaling domain includes a signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine activation motif (ITAM), such as but not limited to FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, FcRL5 (see, e.g., Gillis et al., Front (2014) Immunol.5:254).

[0258] A suitable intracellular signaling domain may be a portion containing an ITAM motif derived from a polypeptide containing a GGAM motif. For example, a suitable intracellular signaling domain may be a domain containing an ITAM motif from any protein containing an ITAM motif. Therefore, a suitable intracellular signaling domain does not need to contain the complete sequence of the complete protein from which it is derived. Examples of suitable polypeptides containing ITAM motifs include, but are not limited to: DAP12, FCER1G (Fcε receptor Iγ chain), CD3δ (CD3δ), CD3ε (CDSε), CD3γ (CDSγ), CD3ζ (CDSζ), and CD79A (antigen receptor complex-associated protein α chain).

[0259] In one embodiment, the intracellular signaling domain is derived from DAP 12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR-associated protein; TYRO protein tyrosine kinase binding protein; killer activated receptor associated protein; killer activated receptor associated protein; etc.). In one embodiment, the intracellular signaling domain is derived from FCεR1G (also known as FCRG; Fcε receptor I γ chain; Fc receptor γ chain; fc-εRI-γ; fcRγ; fceR1γ; high affinity immunoglobulin epsilon receptor subunit γ; immunoglobulin E receptor, high affinity, γ chain; etc.). In one embodiment, the intracellular signaling domain is derived from T cell surface glycoprotein CD3 δ chain (also known as CD3 δ; CD3-DELTA; T3D; CDS antigen, δ subunit; CD3d antigen, δ polypeptide (TiT3 complex); OKT3, δ chain; T cell receptor T3 δ chain; T cell surface glycoprotein CDS δ chain; etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3ε chain (also known as CD3ε, T cell surface antigen T3 / Leu-4ε chain, T cell surface glycoprotein CD3ε chain, AI504783, CDS, CDSε, T3e, etc.).

[0260] In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3γ chain (also known as CD3γ, T cell receptor T3γ chain, CD3-GAMMA, T3G, γ polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CDSζ chain (also known as CD3Z, T cell receptor T3ζ 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 α chain; CD79a antigen (immunoglobulin-associated α); MB-1 membrane glycoprotein; Ig-α; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.). In one embodiment, the intracellular signaling domain suitable for the subject CAR of the present disclosure includes a DAP10 / CD28 type signaling chain. In one embodiment, the intracellular signaling domain suitable for the subject CAR of the present disclosure includes a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CDS ε, CD3, CD22, CD79a, CD79b or CD66d. In one embodiment, the intracellular signaling domain in CAR includes a cytoplasmic signaling domain of human CDS ζ.

[0261] a. Co-stimulatory domain

[0262] In certain embodiments, the intracellular domain includes a co-stimulatory signaling domain. In some embodiments, a chimeric antigen receptor (CAR) includes a co-stimulatory domain, which is a co-stimulatory domain of a molecule selected from the group consisting of: CD27, CD28, 4-IBB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD8, LIGHT, NKG2C, B7-H3, a ligand specifically bound to CD83, DAP10, DAP12, Lck, Fas, and a combination thereof.

[0263] In one embodiment, the costimulatory domain comprises a 4-1BB costimulatory domain or a CD28 costimulatory domain. In one embodiment, the costimulatory domain comprises a 4-1BB costimulatory domain and a CD28 costimulatory domain. In one embodiment, the costimulatory domain comprises an amino acid sequence of SEQ ID NO: 114 or SEQ ID NO: 113. In one embodiment, the costimulatory domain comprises an amino acid sequence of SEQ ID NO: 114 and SEQ ID NO: 113.

[0264] Tolerable variations in the intracellular domain while maintaining a particular activity will be known to those of skill 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 to any of the amino acid sequences set forth in SEQ ID NO: 113 or 114.

[0265] b. Intracellular signaling domain

[0266] In certain embodiments, the intracellular domain comprises an intracellular signaling domain. In some embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor (CAR) comprising an intracellular domain, which may be an intracellular signaling domain of a molecule selected from the group consisting of: T cell receptor (TCR) ζ, FcR-γ, FcR-β, CD3-γ, CD3-δ, CD3-ε, CD3-ζ, CD3, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the intracellular signaling domain comprises a CD3ζ signaling domain; or an amino acid sequence of SEQ ID NO: 115.

[0267] Tolerable variations in the intracellular domain while maintaining a particular activity will be known to those of skill 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 to any amino acid sequence shown in SEQ ID NO: 115.

[0268] 5. Hinge

[0269] The hinge region of CAR is a hydrophilic region between the antigen binding domain and the transmembrane domain. In some embodiments, this domain promotes the correct protein folding of CAR. The hinge region is an optional component of CAR. In some embodiments, the chimeric antigen receptor (CAR) may further include a hinge domain.

[0270] In some embodiments, the hinge domain is a protein selected from the group consisting of 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 one embodiment, the hinge domain is a CD8α hinge domain. In one embodiment, the hinge domain comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the hinge domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 119, SEQ ID NO: 124, 127, 128, 129, 130, 131, 132, and 147.

[0271] In some embodiments, the CAR of the present disclosure includes a hinge region connecting an antigen binding domain and a transmembrane domain, and the transmembrane domain is then connected to an intracellular domain. The hinge region is preferably capable of supporting the antigen binding domain to recognize and bind to a target antigen on a target cell (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3 (2): 125-135). In some embodiments, the hinge region is a flexible domain, so that the antigen binding domain is allowed to have a specific structure and density structure that best recognizes the target antigen on a cell (such as a tumor cell). The flexibility of the hinge region allows the hinge region to adopt many different conformations. 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).

[0272] The length of the hinge region can be from about 4 amino acids to about 50 amino acids, e.g., from about 4 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 40 amino acids, or from about 40 amino acids to about 50 amino acids.

[0273] Suitable hinge regions can be readily selected, and can have any of a number of suitable lengths, such as 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.

[0274] D. Bispecific Fusion Proteins

[0275] One aspect of the present disclosure provides a bispecific fusion protein comprising an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA) and an immune cell recognition domain that specifically binds to a receptor on an immune effector cell.

[0276] Bispecific fusion protein comprises two different binding specificities, and therefore binds to two different antigens. In one embodiment, the bispecific fusion protein comprises a first antigen recognition domain combined with a first antigen (e.g., TACA) and a second antigen recognition domain combined with a second antigen. In one embodiment, the first antigen recognition domain is a TACA binding domain. Examples of TACA are described elsewhere herein, and all of these TACAs can be targeted by the bispecific fusion protein of the present invention. In certain embodiments, the second antigen recognition domain is combined with an immune effector cell. In some embodiments, the antigen binding domain comprises a TACA binding domain derived from a lectin; and the antigen binding domain comprises more than one TACA binding domain as described herein.

[0277] In some embodiments, the bispecific fusion protein comprises an amino acid sequence selected from SEQ ID NO: 1-5, 10-34, 39-42, 47-50, 55-58 or 63-66. Alternatively, the bispecific fusion protein comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 1-5, 10-34, 39-42, 47-50, 55-58 or 63-66. In some embodiments, the amino acid sequence is selected from SEQ ID NO: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66. In some embodiments, the bispecific fusion protein comprises an amino acid sequence selected from SEQ ID NO: 3-5, 10-13, 18-21, 26-34, 39-42, 47-50, 55-58, or 63-66. In some embodiments, the bispecific fusion protein comprises an amino acid sequence disclosed in Table 2 or Table 3. In some embodiments, the bispecific fusion protein comprises an amino acid sequence of SEQ ID NO: 31-34, 39-42, 47-50, 55-58, 63, or 64.

[0278] In some embodiments, the bispecific fusion protein exhibits enhanced binding to tumor cells expressing Thomsen-nouveau (Tn) antigen (Tn antigen) when compared to a bispecific fusion protein comprising a flexible linker in the antigen binding domain. In this embodiment, the antigen binding domain is derived from CD301 (CLEC10A). In this embodiment, the flexible linker is a glycine-serine linker or a linker comprising an amino acid sequence selected from SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127. Alternatively, the flexible linker is a glycine-serine linker or a linker comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127.

[0279] In some embodiments, bispecific fusion protein comprises the immune cell recognition domain that selectively binds the receptor on immune effector cell.In this embodiment, immune effector cell can be selected from the group consisting of: T cell, natural killer (NK) cell, natural killer T (NKT) cell, macrophage, monocyte, dendritic cell and neutrophil.In this embodiment, immune effector cell can be T cell.In another embodiment, immune effector cell can be NK cell.The receptor on immune effector cell can be selected from the group consisting of: T cell receptor (TCR) α, TCR β, TCR γ, TCR δ, constant TCR of NKT cell, CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA and CEACAM1.Or, the receptor on immune effector cell is the T cell receptor selected from the group consisting of CD3, CD2, CD28 and CD25. In some embodiments, the immune effector cell is a NK cell, and the NK cell receptor may be selected from the group consisting of: NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1.

[0280] In some embodiments, the immune cell recognition domain of the bispecific fusion protein comprises a peptide, protein, antibody, single domain antibody, nanobody, antibody fragment or single chain variable fragment (scFv) that selectively binds to a receptor on an immune effector cell. The immune cell recognition domain may comprise an scFv that can selectively bind to CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA and CEACAM1. In some embodiments, the immune cell recognition domain specifically binds to CD3. Alternatively, the immune cell recognition domain may comprise an amino acid sequence of SEQ ID NO: 149, 150 or 151. In some embodiments, the immune cell recognition domain may comprise an amino acid sequence having at least 90% sequence identity to an amino acid sequence of SEQ ID NO: 149, 150 or 151.

[0281] Alternatively, the immune cell recognition domain comprises an antibody Fc domain, optionally an Fc region of an IgG molecule. In one embodiment, the bispecific fusion protein is an Fc fusion protein comprising an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA). In some embodiments, the immune cell recognition domain comprises an antibody Fc domain and a domain that specifically binds to CD3. In another embodiment, the immune cell recognition domain comprises the constant region domain CH2 and / or CH3 of an antibody, preferably CH2 and CH3. The constant region domain CH2 and / or CH3 of an antibody may or may not include a hinge region.

[0282] In some embodiments, the bispecific fusion protein selectively targets a TACA selected from the group consisting of: β1,6 branched, β1,6GlcNAc branched N-glycan, T antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6 sialylated, sialylated, sialyl-Lewis x / a , disialo-Lewis x / a , Sialyl 6-sulfo Lexis x 、Lewis-y(Le y ), Lewis Y, Globo H, GD2, GD3, GM3 and fucosyl GM1.

[0283] In one embodiment, the bispecific fusion protein selectively targets Tn antigen or β1,6GlcNAc branched N-glycans. In some embodiments, the bispecific fusion protein selectively targeting β1,6GlcNAc branched N-glycans comprises an antigen binding domain having an amino acid sequence selected from SEQ ID NO: 100-102 or 133-141; or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 100-102 or 133-141. In another embodiment, the bispecific fusion protein selectively targeting β1,6GlcNAc branched N-glycans comprises an amino acid sequence selected from SEQ ID NO: 1-5 and 10-25; or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-25. In some embodiments, the bispecific fusion protein that selectively targets β1,6GlcNAc branched N-glycans comprises an amino acid sequence selected from SEQ ID NO: 3-5, 11-13, or 19-21; or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 3-5, 11-13, or 19-21. In some embodiments, the bispecific fusion protein exhibits enhanced binding to tumor cells expressing β1,6GlcNAc branched N-glycans when compared to a bispecific fusion protein comprising a flexible linker in the antigen binding domain. In some embodiments, the flexible linker is a glycine-serine linker or a linker comprising an amino acid sequence selected from SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127; or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127.

[0284] Another aspect of the present disclosure provides a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising an antigen binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, and 146; or NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146 has an amino acid sequence with 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; and (ii) an immune cell recognition domain that specifically binds to a receptor on an immune effector cell.

[0285] One aspect of the present disclosure provides a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising an antigen binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, and 146; or NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146 has an amino acid sequence with 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; and an immune cell recognition domain that specifically binds to CD3 on an immune effector cell.

[0286] Another aspect of the present disclosure provides a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising a TACA binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, and 146; or a TACA binding domain selected from the group consisting of: SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, and 146; The amino acid sequence shown in NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115 at least 5%, 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; and peptides, proteins, antibodies, single domain antibodies, antibody fragments, or single chain variable fragments (scFv) that selectively bind to receptors on immune effector cells.

[0287] In some embodiments, the bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA) comprises a TACA binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, and 146; or a TACA binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, and 146; NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146 has an amino acid sequence with 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; and the Fc domain of an antibody. In one embodiment, the domain is the Fc region of an IgG molecule.

[0288] In some embodiments, the bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA) comprises a TACA binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, and 146; or a TACA binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, and 146; NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146 has an amino acid sequence with 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; and the constant region domain CH2 and / or CH3 of an antibody. In one embodiment, the constant region can be CH2 and CH3. In one embodiment, the CH2 and / or CH3 domain comprises a hinge region. In one embodiment, the CH2 and / or CH3 domain does not comprise a hinge region. Another aspect of the present disclosure provides a bispecific fusion protein encoded by an isolated nucleic acid disclosed herein.

[0289] E. Generation of CAR or Bispecific Fusion Proteins

[0290] The bispecific fusion protein, CAR or peptide disclosed herein can be prepared using chemical methods. For example, peptides, bispecific fusion proteins or CARs can be synthesized by solid phase technology (Roberge JY et al. (1995) Science 269: 202-204), cut from the resin, and purified by preparative high performance liquid chromatography. For example, automatic synthesis can be achieved using ABI431A peptide synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

[0291] Peptide, bispecific fusion protein or CAR of the present disclosure can be synthesized by conventional techniques.For example, solid phase peptide synthesis can be used to synthesize peptides or chimeric proteins by chemical synthesis.These methods use solid phase or solution phase synthesis methods (see, for example, about solid phase synthesis technology, JM Stewart and JD Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Co., Rockford 111. (1984) and G. Barany and RBM Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254; and about classical solution synthesis, M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer edited, The Peptides: Analysis, Synthesis, Biology, suprs, Vol. 1). For example, peptides of the invention can be synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid phase chemistry by direct incorporation of phosphothreonine as N-fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivatives.

[0292] The N-terminal or C-terminal fusion protein of the peptide or chimeric protein can be prepared by recombinant technology by fusing the N-terminal or C-terminal of the peptide or chimeric protein with the sequence of the selected protein or selectable marker with the desired biological function to prepare a peptide of the present disclosure, a bispecific fusion protein or a CAR conjugated with other molecules. The resulting fusion protein contains a peptide of the present invention fused with a selected protein or marker protein as described herein. Examples of proteins that can be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA) and truncated myc.

[0293] The peptides or bispecific fusion proteins disclosed herein can be developed using biological expression systems. The use of these systems allows the generation of a large number of random peptide sequence libraries, and the peptide sequences that bind to specific proteins are screened from these libraries. Libraries can be generated by cloning synthetic DNA encoding random peptide sequences into appropriate 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 can also be constructed by simultaneous synthesis of overlapping peptides (see U.S. Patent No. 4,708,871).

[0294] In one aspect, the present disclosure provides any form of peptides, bispecific fusion proteins or CARs having substantial homology to the peptides, bispecific fusion proteins or CARs disclosed herein. Preferably, "substantially homologous" peptides, bispecific fusion proteins or CARs have about 50% homology to the amino acid sequence of the peptides disclosed herein, more preferably about 70% homology, even more preferably about 80% homology, more preferably about 90% homology, even more preferably about 95% homology, and even more preferably about 99% homology. Peptides, bispecific fusion proteins or CARs may alternatively be prepared by recombinant means or by cutting from longer polypeptides.

[0295] According to the variant of the peptide, bispecific fusion protein or CAR disclosed herein, it can be (i) a variant in which one or more amino acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the replaced amino acid residues may or may not be amino acid residues encoded by the genetic code, (ii) a variant in which there are one or more modified amino acid residues (e.g., residues modified by the connection of substituents), (iii) a variant in which the peptide is an alternative splice variant of the peptide disclosed herein, (iv) a fragment of the peptide, and / or (v) a variant in which the peptide is fused to another peptide, the other peptide such as a leader sequence or a secretory sequence or a sequence for purification (e.g., His tag) or for detection (e.g., Sv5 epitope tag). Fragments include peptides generated by proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may be post-translationally modified or chemically modified. According to the teachings herein, such variants are considered to be within the scope of knowledge of those skilled in the art.

[0296] As is known in the art, "similarity" between two peptides is determined by comparing the amino acid sequence of one polypeptide and its conservative amino acid substitutions with the sequence of a second polypeptide. Variants are defined as peptide sequences that are different from the original sequence, preferably, each fragment of interest differs from the original sequence by less than 40% residues, more preferably, each fragment of interest differs from the original sequence by less than 25% residues, more preferably, each fragment of interest differs from the original sequence by less than 10% residues, and most preferably, each fragment of interest differs from the original protein sequence by only a few residues, and at the same time is sufficiently homologous to the original sequence to retain the functionality of the original sequence and / or the ability to bind to TACA. The present invention includes amino acid sequences that have at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90% or 95% similarity or identity to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods well known to those skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm [BLASTanual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S. et al., J. Mol. Biol. 215: 403-410 (1990)].

[0297] The bispecific fusion protein or CAR disclosed herein can be post-translationally modified.For example, post-translational modifications falling within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events need to introduce additional biological mechanisms.For example, processing events such as signal peptide cleavage and core glycosylation are checked by adding canine microsomal membranes or African clawed frog egg extracts (U.S. Patent number 6,103,489) to standard translation reactions.

[0298] In some embodiments, the bispecific fusion proteins or 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 methods can be used to introduce non-natural amino acids during protein translation.

[0299] The peptide, CAR or bispecific fusion protein of the present disclosure can be conjugated with other molecules (such as proteins) to prepare fusion proteins. For example, this can be achieved by synthesizing N-terminal or C-terminal fusion proteins, provided that the resulting fusion protein retains the function of the peptide. The peptide, CAR or bispecific fusion protein of the present disclosure can be phosphorylated using conventional methods (such as Reedijk et al. The EMBO Journal 11 (4): 1365 (1992) described in the method).

[0300] Cyclic derivatives of peptides or bispecific fusion proteins of the present disclosure are also contemplated. Cyclization can allow peptides to present a more favorable conformation in order to associate with other molecules. Cyclization can be achieved using techniques known in the art. For example, a disulfide bond can be formed between two appropriately spaced components with free sulfhydryl groups, or an amide bond can be formed between the amino group of one component and the carboxyl group of another component. Cyclization can also be achieved using amino acids containing azobenzene, 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 one embodiment of the present disclosure, the cyclic peptide can contain a β-turn in the right position. The β-turn can be introduced into the peptide of the present invention by adding the amino acid Pro-Gly at the right position. It may be desirable to produce a cyclic peptide that is more flexible than the cyclic peptide containing peptide bonds connected as described above. A more flexible peptide can be prepared by introducing cysteines at the right and left positions of the peptide and forming a disulfide bridge between the two cysteines. The two cysteines are arranged so that the β-sheet is not deformed and does not rotate. Due to the length of the disulfide bond and the smaller number of hydrogen bonds in the β-sheet portion, the peptide is more flexible. The relative flexibility of the cyclic peptide can be determined by molecular dynamics simulation.

[0301] One aspect of the present disclosure provides a bispecific fusion protein, fusion protein, CAR or peptide fused to or integrated into a target protein, and / or bispecific fusion protein, fusion protein, CAR or peptide can be directed to a desired cell component or cell type or targeting domain of tissue. Bispecific fusion protein, fusion protein, CAR or peptide may also contain additional amino acid sequences or domains. In the sense that various components are from different sources, bispecific fusion protein, fusion protein, CAR or peptide are recombinant, therefore not found together in nature (i.e., heterologous).

[0302] In one embodiment, the targeting domain can be a membrane spanning domain, a membrane binding domain, or a sequence that directs the association of a protein with, for example, a vesicle or a nucleus. In one embodiment, the targeting domain can target the peptide to a specific cell type or tissue. For example, the targeting domain can be an antibody to a cell surface ligand or a cell surface antigen for a target tissue (e.g., bone, regenerated bone, degenerated bone, cartilage). The targeting domain can target the peptide of the present invention to a cellular component.

[0303] III. Nucleic Acids and Expression Vectors

[0304] A. Bispecific Fusion Proteins

[0305] One aspect of the present disclosure provides an isolated nucleic acid molecule encoding a bispecific fusion protein, the bispecific fusion protein comprising an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA) and an immune cell recognition domain that specifically binds to a receptor on an immune effector cell, wherein the antigen binding domain comprises a TACA binding domain derived from a lectin; and the antigen binding domain comprises more than one (e.g., multiple) TACA binding domains.

[0306] In some embodiments, the antigen binding domain comprises two, three, four, five, six, seven, eight, nine, ten or more TACA binding domains. More than one (e.g., multiple) TACA binding domains can be operably connected by a joint, such as a joint selected from the group consisting of: a peptide joint, a non-peptide joint, a chemical unit, a hindered cross-linking agent, a non-hindered cross-linking agent. In one embodiment, the joint is a peptide joint. The peptide joint can be a glycine-serine joint. The length of the peptide joint can be 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.

[0307] In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 127 or the amino acid sequence of SEQ ID NO: 131.

[0308] In some embodiments, the lectin is selected from the group consisting of: galectins, sialic acid-binding immunoglobulin-like lectins, selectins; C-type lectins; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Lycopersicon esculentum lectin); E-PHA (Lycopersicon esculentum lectin); tomato lectin (Lycopersicon esculentum lectin; LEA); peanut lectin (Arachis hypogaea Agglutinin; PNA); potato lectin (Solanum tuberosum lectin); pokeweed mitogen (Phytolacca Americana lectin); wheat germ agglutinin (Wheat germ agglutinin); agglutinin (Triticum Vulgaris lectin); Artocarpus polyphemus lectin (Jacalin letin); Vetch lectin (VVA); Helicoverpa agglutinin (HPA); Wisteria floribunda agglutinin (WFA); Sambucus sambucus agglutinin (SNA); BC2L-CNt (lectin from the Gram-negative bacterium Burkholderia cenocepacia); MAL; PVL; Sclerotium sclerotium agglutinin (SRL); Echinops serrulata agglutinin (ESA); CLEC17A (protectin); Dictyophora aurantifolia agglutinin; S. sessile elderberry agglutinin (SSA); Gleheda; Morniga G; Salvia miltiorrhiza agglutinin; bogotensis agglutinin, Salvia officinalis agglutinin, Changshan Shanhaizhou agglutinin, Concha strychnos nucifera agglutinin, Griffonia suffruticosa (GsLA4), Quaternary bean (acidic WBAI), Abrus precatorius lectin, Amaranthus caudatus lectin, Amaranthus gingkoi lectin, Amaranthus chinensis lectin, Jackfruit agglutinin, Morus alba lectin, Jackfruit agglutinin, Lablab bisporus lectin, Lablab bisporus lectin, Soybean agglutinin, and Agaricus bisporus lectin.

[0309] In some embodiments, the lectin is a galectin that may be 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 sialic acid-binding immunoglobulin-like lectin that can be selected from the group consisting of: sialic acid-binding immunoglobulin-like lectin-1 (sialoadhesin), sialic acid-binding immunoglobulin-like lectin-2 (CD22), sialic acid-binding immunoglobulin-like lectin-3 (CD33), sialic acid-binding immunoglobulin-like lectin-4 (myelin-associated glycoprotein), sialic acid-binding immunoglobulin-like lectin-5, sialic acid-binding immunoglobulin-like lectin-6, sialic acid-binding immunoglobulin-like lectin-7, sialic acid-binding immunoglobulin-like lectin-8, sialic acid-binding immunoglobulin-like lectin-9, sialic acid-binding immunoglobulin-like lectin-10, sialic acid-binding immunoglobulin-like lectin-11, sialic acid-binding immunoglobulin-like lectin-12, sialic acid-binding immunoglobulin-like lectin-13, sialic acid-binding immunoglobulin-like lectin-14, sialic acid-binding immunoglobulin-like lectin-15, sialic acid-binding immunoglobulin-like lectin-16, sialic acid-binding immunoglobulin-like lectin-17, sialic acid-binding immunoglobulin-like lectin-18, sialic acid-binding immunoglobulin-like lectin-19, sialic acid-binding immunoglobulin-like lectin-2 Sialic acid-binding immunoglobulin-like lectin-9, sialic acid-binding immunoglobulin-like lectin-10, sialic acid-binding immunoglobulin-like lectin-11, sialic acid-binding immunoglobulin-like lectin-12, sialic acid-binding immunoglobulin-like lectin-13, sialic acid-binding immunoglobulin-like lectin-14, sialic acid-binding immunoglobulin-like lectin-15, sialic acid-binding immunoglobulin-like lectin-16, sialic acid-binding immunoglobulin-like lectin-17, sialic acid-binding immunoglobulin-like lectin E, sialic acid-binding immunoglobulin-like lectin F, sialic acid-binding immunoglobulin-like lectin G and sialic acid-binding immunoglobulin-like lectin H.

[0310] In some embodiments, the lectin is a polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) which 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). LNT9), ppGalNAc-T10 (GALNT10), ppGalNAc-T12 (GALNT12), ppGalNAc-T13 (GALNT13), ppGalNAc-T14 (GALNT14), ppGalNAc-T15 (GALNT1 5), ppGalNAc-T16 (GALNT16), ppGalNAc-T17 (GALNT17), ppGalNAc-T18 (GALNT18), ppGalNAc-T-like 5 (GALNTL5) and ppGalNAc-T-like 6 (GALNTL6).

[0311] In some embodiments, the antigen binding domain of the bispecific fusion protein described herein selectively targets TACA selected from the group consisting of: β1,6 branched, β1,6GlcNAc branched N-glycan, T antigen, Tn antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6 sialylated, sialylated, sialyl-Lewis x / a, disialyl-Lewis x / a, sialyl 6-sulfoLexis x 、Lewis-y(Le y ), Lewis Y, Globo H, GD2, GD3, GM3 and fucosyl GM1.

[0312] In some embodiments, the antigen binding domain selectively targets β1,6GlcNAc-branched N-glycan, a Tn epitope (Tn antigen), a sialyl-Tn epitope (sialyl-Tn antigen), GalNAc α-serine, GalNAc α-threonine, GalNAc, or GalNAc β1.

[0313] In some embodiments, the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152. Alternatively, the antigen binding domain comprises 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 shown in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152. In some embodiments, the antigen binding domain comprises an amino acid sequence disclosed in Table 2 or Table 3. In some embodiments of the nucleic acid molecules disclosed herein, the antigen binding domain comprises an amino acid sequence having at least 90% homology to SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, or 146.

[0314] In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence selected from SEQ ID NO: 1-5, 10-34, 39-42, 47-50, 55-58 and 63-66. Alternatively, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 1-5, 10-34, 39-42, 47-50, 55-58 and 63-66. In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence selected from SEQ ID NO: 3-5, 10-13, 18-21, 26-34, 39-42, 47-50, 55-58 or 63-66. In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence selected from SEQ ID NO: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66. In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66. In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein disclosed in Table 2 or Table 3. In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence of SEQ ID NO: 31-34, 39-42, 47-50, 55-58, 63 or 64.

[0315] In some embodiments, the bispecific fusion protein exhibits enhanced binding to tumor cells expressing Thomsen-nouveau (Tn) antigen (Tn antigen) when compared to a bispecific fusion protein comprising a flexible linker in the antigen binding domain. In this embodiment, the flexible linker is a glycine-serine linker or a linker comprising an amino acid sequence selected from SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127. Alternatively, the flexible linker is a glycine-serine linker or a linker comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127.

[0316] In some embodiments, the nucleic acid molecule encoding of separation comprises the bispecific fusion protein of the immune cell recognition domain that selectively binds the receptor on immune effector cell.In this embodiment, immune effector cell can be selected from the group consisting of: T cell, natural killer (NK) cell, natural killer T (NKT) cell, macrophage, monocyte, dendritic cell and neutrophil.In this embodiment, immune effector cell can be T cell.In another embodiment, immune effector cell can be NK cell.The receptor on immune effector cell can be selected from the group consisting of: T cell receptor (TCR) α, TCR β, TCR γ, TCR δ, constant TCR of NKT cell, CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA and CEACAM1.Or, the receptor on immune effector cell is the T cell receptor selected from the group consisting of CD3, CD2, CD28 and CD25. In some embodiments, the immune effector cell is a NK cell, and the NK cell receptor may be selected from the group consisting of: NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1.

[0317] In some embodiments, the immune cell recognition domain of the bispecific fusion protein comprises a peptide, protein, antibody, single domain antibody, nanobody, antibody fragment or single chain variable fragment (scFv) that selectively binds to a receptor on an immune effector cell. The immune cell recognition domain may comprise an scFv that can selectively bind to CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA and CEACAM1. In some embodiments, the immune cell recognition domain specifically binds to CD3. Alternatively, the immune cell recognition domain may comprise an amino acid sequence of SEQ ID NO: 149, 150 or 151. In some embodiments, the immune cell recognition domain may comprise an amino acid sequence having at least 90% sequence identity to an amino acid sequence of SEQ ID NO: 149, 150 or 151.

[0318] Alternatively, the immune cell recognition domain comprises an antibody Fc domain, optionally an Fc region of an IgG molecule. In some embodiments, the immune cell recognition domain is an antibody Fc domain and a domain that specifically binds to CD3. In one embodiment, the bispecific fusion protein is an Fc fusion protein comprising an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA). In another embodiment, the immune cell recognition domain comprises an antibody constant region domain CH2 and / or CH3, preferably CH2 and CH3. The antibody constant region domain CH2 and / or CH3 may or may not include a hinge region.

[0319] B. Chimeric Antigen Receptor

[0320] One aspect of the present disclosure provides a kind of nucleic acid molecule encoding a chimeric antigen receptor (CAR) separation, the CAR comprising an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), wherein the antigen binding domain comprises a TACA binding domain derived from a lectin; and the antigen binding domain comprises more than one TACA binding domain; a transmembrane domain; a costimulatory signaling region; and an intracellular signaling domain. In one embodiment, the first nucleic acid sequence encoding the antigen binding domain is operably connected to the second nucleic acid encoding the transmembrane domain, and is further operably connected to the third nucleic acid sequence encoding the costimulatory signaling domain and / or the intracellular signaling domain.

[0321] In some embodiments, the antigen binding domain comprises two, three, four, five, six, seven, eight, nine, ten or more TACA binding domains. More than one (e.g., multiple) TACA binding domains can be operably connected by a joint, such as a joint selected from the group consisting of: a peptide joint, a non-peptide joint, a chemical unit, a hindered cross-linking agent, a non-hindered cross-linking agent. In one embodiment, the joint is a peptide joint. The peptide joint can be a glycine-serine joint. The length of the peptide joint can be 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.

[0322] In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 127 or the amino acid sequence of SEQ ID NO: 131.

[0323] In some embodiments, the lectin is selected from the group consisting of: galectins, sialic acid-binding immunoglobulin-like lectins, selectins; C-type lectins; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Lycopersicon esculentum lectin); E-PHA (Lycopersicon esculentum lectin); tomato lectin (Lycopersicon esculentum lectin; LEA); peanut lectin (Arachis hypogaea Agglutinin; PNA); potato lectin (Solanum tuberosum lectin); pokeweed mitogen (Phytolacca Americana lectin); wheat germ agglutinin (Wheat germ agglutinin); agglutinin (Triticum Vulgaris lectin); Artocarpus polyphemus lectin (Jacalin letin); Vetch lectin (VVA); Helicoverpa agglutinin (HPA); Wisteria floribunda agglutinin (WFA); Sambucus sambucus agglutinin (SNA); BC2L-CNt (lectin from the Gram-negative bacterium Burkholderia cenocepacia); MAL; PVL; Sclerotium sclerotium agglutinin (SRL); Echinops serrulata agglutinin (ESA); CLEC17A (protectin); Dictyophora aurantifolia agglutinin; S. sessile elderberry agglutinin (SSA); Gleheda; Morniga G; Salvia miltiorrhiza agglutinin; bogotensis agglutinin, Salvia officinalis agglutinin, Changshan Shanhaizhou agglutinin, Concha strychnos nucifera agglutinin, Griffonia suffruticosa (GsLA4), Quaternary bean (acidic WBAI), Abrus precatorius lectin, Amaranthus caudatus lectin, Amaranthus gingkoi lectin, Amaranthus chinensis lectin, Jackfruit agglutinin, Morus alba lectin, Jackfruit agglutinin, Lablab bisporus lectin, Lablab bisporus lectin, Soybean agglutinin, and Agaricus bisporus lectin.

[0324] In some embodiments, the lectin is a galectin that may be 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 sialic acid-binding immunoglobulin-like lectin that can be selected from the group consisting of: sialic acid-binding immunoglobulin-like lectin-1 (sialoadhesin), sialic acid-binding immunoglobulin-like lectin-2 (CD22), sialic acid-binding immunoglobulin-like lectin-3 (CD33), sialic acid-binding immunoglobulin-like lectin-4 (myelin-associated glycoprotein), sialic acid-binding immunoglobulin-like lectin-5, sialic acid-binding immunoglobulin-like lectin-6, sialic acid-binding immunoglobulin-like lectin-7, sialic acid-binding immunoglobulin-like lectin-8, sialic acid-binding immunoglobulin-like lectin-9, sialic acid-binding immunoglobulin-like lectin-10, sialic acid-binding immunoglobulin-like lectin-11, sialic acid-binding immunoglobulin-like lectin-12, sialic acid-binding immunoglobulin-like lectin-13, sialic acid-binding immunoglobulin-like lectin-14, sialic acid-binding immunoglobulin-like lectin-15, sialic acid-binding immunoglobulin-like lectin-16, sialic acid-binding immunoglobulin-like lectin-17, sialic acid-binding immunoglobulin-like lectin-18, sialic acid-binding immunoglobulin-like lectin-19, sialic acid-binding immunoglobulin-like lectin-2 Sialic acid-binding immunoglobulin-like lectin-9, sialic acid-binding immunoglobulin-like lectin-10, sialic acid-binding immunoglobulin-like lectin-11, sialic acid-binding immunoglobulin-like lectin-12, sialic acid-binding immunoglobulin-like lectin-13, sialic acid-binding immunoglobulin-like lectin-14, sialic acid-binding immunoglobulin-like lectin-15, sialic acid-binding immunoglobulin-like lectin-16, sialic acid-binding immunoglobulin-like lectin-17, sialic acid-binding immunoglobulin-like lectin E, sialic acid-binding immunoglobulin-like lectin F, sialic acid-binding immunoglobulin-like lectin G and sialic acid-binding immunoglobulin-like lectin H.

[0325] In some embodiments, the lectin is a polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) which 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). LNT9), ppGalNAc-T10 (GALNT10), ppGalNAc-T12 (GALNT12), ppGalNAc-T13 (GALNT13), ppGalNAc-T14 (GALNT14), ppGalNAc-T15 (GALNT1 5), ppGalNAc-T16 (GALNT16), ppGalNAc-T17 (GALNT17), ppGalNAc-T18 (GALNT18), ppGalNAc-T-like 5 (GALNTL5) and ppGalNAc-T-like 6 (GALNTL6).

[0326] In some embodiments, the antigen binding domain of the CAR described herein 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, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6 sialylated, sialylated, 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.

[0327] In some embodiments, the antigen binding domain selectively targets β1,6GlcNAc-branched N-glycan, a Tn epitope (Tn antigen), a sialyl-Tn epitope (sialyl-Tn antigen), GalNAc α-serine, GalNAc α-threonine, GalNAc, or GalNAc β1.

[0328] In some embodiments, the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152. Alternatively, the antigen binding domain comprises 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 shown in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152. In some embodiments, the antigen binding domain comprises an amino acid sequence disclosed in Table 2 or Table 3.

[0329] In some embodiments of the nucleic acid molecules disclosed herein, the antigen binding domain comprises an amino acid sequence having at least 90% homology to SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146.

[0330] In some embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor (CAR) comprising a transmembrane domain, which may include a transmembrane region of a molecule selected from the group consisting of: T cell receptor (TCR) -α, TCR-β, CD3-ζ, CD3-ε, 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 includes a CD8 transmembrane domain. Alternatively, the transmembrane domain includes the amino acid sequence of SEQ ID NO: 148.

[0331] In some embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor (CAR) comprising a costimulatory domain, the costimulatory domain being a costimulatory domain of a molecule selected from the group consisting of: CD27, CD28, 4-IBB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD8, LIGHT, NKG2C, B7-H3, a ligand specifically bound to CD83, DAP10, DAP12, Lck, Fas, and a combination thereof. In one embodiment, the costimulatory domain comprises a 4-1BB costimulatory domain or a CD28 costimulatory domain. In one embodiment, the costimulatory domain comprises a 4-1BB costimulatory domain and a CD28 costimulatory domain. In one embodiment, the costimulatory domain comprises an amino acid sequence of SEQ ID NO:114 or SEQ ID NO:113. In one embodiment, the costimulatory domain comprises the amino acid sequence of SEQ ID NO:114 and SEQ ID NO:113.

[0332] In some embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor (CAR) comprising an intracellular domain, which may be an intracellular signaling domain from a molecule selected from the group consisting of: T cell receptor (TCR) ζ, FcR-γ, FcR-β, CD3-γ, CD3-δ, CD3-ε, CD3-ζ, CDS, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the intracellular signaling domain comprises a CD3ζ signaling domain; or the amino acid sequence of SEQ ID NO: 115.

[0333] In some embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor (CAR) further comprising a hinge domain. In some embodiments, the hinge domain is a protein selected from the group consisting of: 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 one embodiment, the hinge domain is a CD8α hinge domain. In one embodiment, the hinge domain comprises SEQ ID NO:147 amino acid sequence. In some embodiments, the hinge domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:119, SEQ ID NO:124, 127, 128, 129, 130, 131, 132 and 147.

[0334] One aspect of the present disclosure comprises an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising the amino acid sequence set forth in SEQ ID NO:71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. In some embodiments, the CAR comprises 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 shown in SEQ ID NO:71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

[0335] C. Nucleic Acids

[0336] The isolated nucleic acid sequence encoding the bispecific fusion protein or chimeric antigen receptor of the present disclosure can be obtained using any of the many recombinant methods known in the art, such as, for example, by screening a cell library expressing the gene, by delivering the gene from a vector known to include the gene, or by isolating the gene directly from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0337] 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, including, for example, an isolated cDNA molecule, which encodes 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.

[0338] The nucleic acid molecules of the present disclosure can be modified to improve their stability in serum or in a growth medium 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, in order to enhance stability, 3'-residues can be stabilized to prevent degradation, for example, they can be selected so that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides with modified analogs (e.g., substitution of uridine with 2'-deoxythymidine) is tolerable and does not affect the function of the molecule. In one embodiment of the present disclosure, the nucleic acid molecule can contain at least one modified nucleotide analog. For example, the end can be stabilized by incorporating modified nucleotide analogs.

[0339] The limiting examples of nucleotide analogs include sugar and / or backbone modified ribonucleotides (i.e. including modifications to phosphate-sugar backbones). For example, the phosphodiester bond of natural RNA can be modified to include at least one nitrogen or sulfur heteroatom. In preferred backbone modified ribonucleotides, the phosphate group connected to adjacent ribonucleotides is replaced by a modified group (e.g., 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, wherein R is C1-Ce alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

[0340] Examples of other modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides containing at least one non-natural nucleobase in place of a naturally occurring nucleobase. The bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5 position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8 position, such as 8-bromoguanosine; deazanucleotides, such as 7-deaza-adenosine; O-alkylated and N-alkylated nucleotides, such as N6-methyladenosine are suitable. It should be noted that the above modifications may be combined.

[0341] For example, the nucleic acid molecule comprises at least one of the following chemical modifications: 2'-H, 2'-O-methyl or 2'-OH modification of one or more nucleotides. In certain embodiments, the nucleic acid molecules of the present disclosure may have enhanced nuclease resistance. In order to increase nuclease resistance, the nucleic acid molecules may include, for example, 2'-modified ribose units and / or thiophosphate bonds. For example, 2'hydroxyl (OH) may be modified or replaced by a variety of different "oxy" or "deoxy" substituents. In order to increase nuclease resistance, the nucleic acid molecules of the present disclosure may include 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino and / or thiophosphate bonds. Including locked nucleic acid (LNA), ethylene nucleic acid (ENA) (e.g., 2'-4'-ethylene-bridged nucleic acid) and certain nucleobase modifications (such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications) can also increase the binding affinity to the target.

[0342] In one embodiment, the nucleic acid molecule includes 2'-modified nucleotides, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAEOE) or 2'-0-N-methylacetamido (2'-0-MA). In one embodiment, the nucleic acid molecule includes at least one 2'-0-methyl modified nucleotide, and in some embodiments, all nucleotides of the nucleic acid molecule include a 2'-0-methyl modification.

[0343] In some embodiments, the nucleic acid molecules of the present disclosure preferably have one or more of the following properties: The nucleic acid agents discussed herein include unmodified RNA and DNA as well as RNA and DNA that have been modified (e.g., to improve efficacy) and polymers of nucleoside surrogates.

[0344] Unmodified RNA refers to a molecule in which the nucleic acid components (i.e., sugar, base, and phosphate moieties) are identical or substantially identical to naturally occurring nucleic acid components (preferably naturally occurring nucleic acid components in the human body). Rare or uncommon but naturally occurring RNAs are referred to in the art as modified RNAs, see, e.g., Limbach et al., Nucleic Acids Res., 1994, 22: 2183-2196. Such rare or uncommon RNA (commonly referred to as modified RNA) is usually the result of post-transcriptional modification and belongs to the term unmodified RNA as used herein. Modified RNA as used herein refers to a molecule in which one or more components of nucleic acid (i.e., sugar, base and phosphate moiety) are different from naturally occurring nucleic acid components (preferably different from naturally occurring nucleic acid components in the human body). Although they are referred to as "modified RNA", due to modification, they will of course include molecules that are not strictly speaking RNA. Nucleoside substitutes are molecules in which the ribose phosphate backbone is replaced by a non-ribose phosphate construct, which allows the base to exist in the correct spatial relationship, so that hybridization is substantially similar to that seen in the ribose phosphate backbone (e.g., an uncharged mimetic of the ribose phosphate backbone). The modification of the nucleic acid disclosed herein may be present at one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus or nucleobase.

[0345] D. Expression vector

[0346] In one aspect, the present disclosure provides an expression construct comprising an isolated nucleic acid encoding a bispecific fusion protein and / or 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.

[0347] The expression of natural or synthetic nucleic acids encoding peptides of the present invention is usually achieved by operably linking the nucleic acid encoding the peptide or part thereof to a promoter and incorporating the construct into an expression vector. The vector to be used is suitable for replication in eukaryotic cells and optionally integrated. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequence.

[0348] The vectors disclosed herein can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0349] The isolated nucleic acids of the present disclosure can be cloned into various 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. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0350] In addition, the vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. In general, suitable vectors contain replication initiation points, promoter sequences, convenient restriction endonuclease sites and one or more selectable markers (for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent number 6,326,193) that play a role in at least one organism.

[0351] 1. Virus-based systems

[0352] 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 a bispecific fusion protein described herein.

[0353] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro. Various retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Various adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0354] For example, the vector derived from retrovirus (such as slow virus) is a suitable tool for realizing long-term gene transfer, because they allow long-term, stable integration of transgenic and propagation of transgenic in daughter cells.Slow virus vector has additional advantages than the vector derived from oncoretrovirus (such as murine leukemia virus), because they can transduce non-proliferative cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. In one embodiment, the composition includes a vector derived from adeno-associated virus (AAV). Adeno-associated virus (AAV) vector has become a powerful gene delivery tool for treating various diseases. AAV vector has a variety of features that make it ideally suitable for gene therapy, including lack of pathogenicity, minimal immunogenicity and the ability to transduce post-mitotic cells in a stable and efficient manner. By selecting the appropriate combination of AAV serotype, promoter and delivery method, the expression of the specific gene contained in the AAV vector can be specifically targeted to one or more types of cells.

[0355] 2. Control elements

[0356] In some embodiments, the vector also includes conventional control elements, which are operably connected to the transgene in a manner that allows it to be transcribed, translated and / or expressed in cells transfected with a plasmid vector or infected with a virus produced by the present invention. As used herein, "operably connected" sequences include expression control sequences adjacent to the gene of interest and trans or remotely acting to control the expression control sequences of the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product when desired. A large number of expression control sequences known and available in the art include promoters that are natural, constitutive, inducible and / or tissue-specific.

[0357] Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. Typically, they are located in the region 30bp-110bp upstream of the start site, but recently it has been shown that multiple promoters also contain functional elements downstream of the start site. The intervals between promoter elements are often flexible, so promoter function is retained when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the intervals between promoter elements can be increased to 50bp before activity begins to decline. Depending on the promoter, each element seems to work collaboratively or independently to activate transcription.

[0358] 3. Promoter

[0359] In some embodiments, the expression construct further comprises a promoter. The promoter may be selected from the group consisting of an EF-1α promoter, a T cell receptor α (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 leukosis virus promoter, an Epstein-Barr virus immediate early promoter, or a Rous sarcoma virus promoter.

[0360] The immediate early cytomegalovirus (CMV) promoter sequence is an example of a strong constitutive promoter sequence, which can drive the high-level expression of any polynucleotide sequence operably connected thereto. Another example of a suitable promoter is elongated growth factor-1a (EF-1a). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney murine leukemia virus (MoMuLV) promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter and human gene promoter, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter and creatine kinase promoter. In addition, the present invention should not limit the use of constitutive promoters. Inducible promoters are also considered as a part of the present invention. Using inducible promoters provides a kind of molecular switch, which can open the expression of the polynucleotide sequence operably connected thereto when expecting this expression, or close expression when not expecting expression. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0361] Enhancer sequences found on vectors also regulate the expression of genes contained in the vectors. Typically, enhancers are combined with protein factors to enhance the transcription of genes. Enhancers may be located upstream or downstream of the genes they regulate. Enhancers may also be tissue-specific to enhance transcription in specific cells or tissue types. In one embodiment, vectors of the present invention include one or more enhancers to enhance the transcription of genes present in the vector.

[0362] 4. Optional markers

[0363] In order to evaluate the expression of the peptide, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both, to facilitate identification and selection of expressing cells from a cell population that needs to be transfected or infected by a viral vector. In other aspects, selectable markers can be carried on a separate DNA fragment and used for co-transfection procedures. Both selectable markers and reporter genes can be flanked with appropriate regulatory sequences to achieve expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo, etc.

[0364] Reporter gene is used to identify the cells of potential transfection and to evaluate the function of regulatory sequence. In general, reporter gene is a gene that is not present in or is not expressed by a receptor organism or tissue, and the expression of the polypeptide encoded by the reporter gene is shown as some easily detectable properties, for example, enzyme activity. After DNA is introduced into the receptor cell, the expression of the reporter gene is measured at the appropriate time. Suitable reporter gene may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secretory alkaline phosphatase or green fluorescent protein gene (for example, Ui-Tei et al., 2000FEBS Letters 479: 79-82). Suitable expression system is well known and can be prepared or commercially available using known techniques. In general, the construct with the minimum 5' flanking region showing the highest level of reporter gene expression is identified as a promoter. Such promoter region can be connected to the reporter gene and used to assess the ability of the promoter driven by the agent regulation promoter.

[0365] 5. CAR and bispecific fusion protein combination

[0366] In some embodiments, the expression construct comprises an isolated nucleic acid encoding a CAR polypeptide and a bispecific fusion protein polypeptide. In some embodiments, the isolated nucleic acid molecule encoding the bispecific fusion protein and the isolated nucleic acid molecule encoding the CAR are operably connected by a nucleic acid molecule encoding a self-cleaving 2A peptide. As used herein, "self-cleaving peptide" or "2A peptide" refers to an oligopeptide that allows multiple proteins to be encoded as a polymeric protein that dissociates into component proteins after translation. The use of the term "self-cleavage" is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those skilled in the art, including but not limited to those found in members of the Picornaviridae virus family, such as foot-and-mouth disease virus (FMDV), equine rhinitis virus type A (ERAV, Thosea asigna virus (TaV), and porcine tescho virus-1 (PTV-1); and carioviruses, such as Theilovirus and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and "T2A," respectively. Those skilled in the art will be able to select an appropriate self-cleaving peptide for use in the present invention. In one embodiment, the self-cleaving 2A peptide is selected from P2A, T2A, E2A, or F2A.

[0367] In some embodiments, the nucleic acid molecules encoding the separation of bispecific fusion proteins and the nucleic acid molecules encoding the separation of CAR are operably connected by the nucleic acid molecules encoding joints. For the joints disclosed herein (for example, in the context of connecting CAR coding sequences and bispecific fusion protein coding sequences) allow multiple proteins to be encoded by the same nucleic acid sequence (for example, polycistronic or bicistronic sequences), and the protein is translated into a polymeric protein that is dissociated into a separate protein component. For example, the joints in the nucleic acids comprising CAR coding sequences and bispecific fusion protein coding sequences disclosed herein allow CAR and bispecific fusion proteins to be translated into polymeric proteins, and the polymeric proteins are dissociated into separate CAR and bispecific fusion protein components.

[0368] In some embodiments, the linker comprises a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct entry of internal ribosomes into the start codon (such as ATG) of the protein coding region, resulting in cap-independent translation of the gene. Various internal ribosome entry sites are known to those skilled in the art, including but not limited to IRES that can be obtained from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES that can be obtained from, for example, cardiovirus, rhinovirus, foot-and-mouth disease virus (aphthovims), HCV, Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV). Those skilled in the art will be able to select an appropriate IRES for use in the present invention.

[0369] IV. Modified Cells

[0370] One aspect of the present disclosure provides a genetically modified (e.g., engineered) cell, which includes and stably expresses the subject CAR or bispecific fusion protein of the present disclosure. In one aspect, the modified cell includes a nucleic acid encoding a chimeric antigen receptor (CAR) separation, and the CAR includes: an antigen binding domain, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain selectively binding to a tumor-associated carbohydrate antigen (TACA). In some embodiments, the modified cell includes a chimeric antigen receptor selectively binding to a tumor-associated carbohydrate antigen (TACA). In one aspect, the modified cell includes a nucleic acid encoding a separation of a bispecific fusion protein, and the bispecific fusion protein includes an antigen binding domain selectively binding to a tumor-associated carbohydrate antigen (TACA) and an immune cell recognition domain specifically binding to a receptor on an immune effector cell.

[0371] In some embodiments, the modified cell comprises a bispecific fusion protein as described herein and / or a CAR as described herein. In another embodiment, the modified cell comprises an isolated nucleic acid molecule encoding a bispecific fusion protein as described herein and / or an isolated nucleic acid molecule encoding a CAR as described herein. In another embodiment, the modified cell comprises an expression construct as described herein.

[0372] In some embodiments, the modified cell comprises a bispecific fusion protein as described herein. In another embodiment, the modified cell comprises an isolated nucleic acid molecule encoding a bispecific fusion protein as described herein. In another embodiment, the modified cell comprises an expression construct as described herein. In some embodiments, the modified cell comprises a CAR as described herein. In another embodiment, the modified cell comprises an isolated nucleic acid molecule encoding a CAR as described herein. In another embodiment, the modified cell comprises an expression construct as described herein.

[0373] In some embodiments, the modified cell is a genetically modified immune cell (e.g., a T cell) or a precursor thereof, comprising a chimeric antigen receptor (CAR) or a bispecific fusion protein with affinity for a tumor-associated carbohydrate antigen (TACA). In some embodiments, the genetically modified immune cell (e.g., a T cell) or a precursor thereof of the present invention comprises a CAR or a bispecific fusion protein with affinity for: β1,6 branched, β1,6GlcNAc branched N-glycan, T antigen, Tn antigen, sialyl-T epitope, Tn epitope, sialyl-Tn epitope, α2,6 sialylated, sialylated, sialyl-Lewis x / a , disialo-Lewis x / a, Sialyl 6-sulfo Lexis x 、Lewis-y(Le y ), Lewis Y, Globo H, GD2, GD3, GM3 or fucosyl GM1. In some embodiments, the genetically modified immune cells (e.g., T cells) or their precursor cells of the present invention comprise a CAR or bispecific fusion protein with affinity for β1,6 branches or β1,6GlcNAc branched N-glycans. In some embodiments, the genetically modified immune cells (e.g., T cells) or their precursor cells of the present invention comprise a CAR or bispecific fusion protein with affinity for Tn antigen or sialyl-Tn epitope.

[0374] One aspect of the present disclosure provides a modified cell comprising an isolated nucleic acid, fusion protein or expression construct as described herein. In some embodiments, the modified cell (e.g., host cell) is selected from the group consisting of bacterial cells, fungal cells, insect cells or mammalian cells. In some embodiments, the modified cell is a bacterial cell selected from Escherichia coli or Bacillus stearothermophilus. In some embodiments, the modified cell is a fungal cell selected from yeast cells, Saccharomyces cerevisiae or Pichia pastoris. In some embodiments, the modified cell is an insect cell selected from Lepidopteran insect cells or Spodoptera frugiperda. In some embodiments, the modified cell is a mammalian cell selected from the following: Chinese hamster ovary (CHO) cell, baby hamster kidney (BHK) cell, monkey kidney cell, HeLa cell, human hepatocellular carcinoma cell or human embryonic kidney 293 cell. In one embodiment, the modified cell is a CHO cell or a HEK 293 cell.

[0375] A. Modified cells

[0376] In some embodiments, the modified cells are selected from the group consisting of: T cells, CD4 + T cells, CD8 + In some embodiments, the modified cells are T cells. In some embodiments, the genetically modified cells are natural killer (NK) cells. In some embodiments, the genetically modified cells are NKT cells. In some embodiments, the modified cells are autologous cells, xenogeneic cells or allogeneic cells.

[0377] In some embodiments, the modified cells described herein include bispecific fusion proteins described herein and CARs described herein or CARs targeting tumor antigens. In some embodiments, the modified cells described herein include isolated nucleic acid molecules encoding bispecific fusion proteins described herein and isolated nucleic acid molecules encoding CARs described herein or CARs targeting tumor antigens. In some embodiments, the modified cells described herein include expression constructs described herein and expression constructs comprising CARs targeting tumor antigens.

[0378] 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, CDS, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD119, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD119, CD20 71, CD276, CEA, tight junction protein 18.2, c-Met, DLL3, DRS, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, phosphatidylinositol glycan-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-I, Lewis Y, LMPI (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUCI, Nectin4 / FAP, NKG2D-ligands (MIC-A, MIC-B and ULBP 1 to 6), NY-ESO-1, Pl 6. PD-LI, PSCA, PSMA, RORI, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof. In some embodiments, the tumor antigen is TACA.

[0379] In some embodiments, the genetically modified cells are genetically engineered T lymphocytes (T cells), regulatory T cells (Treg), unsensitized 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 producing treatment-related offspring. In some embodiments, the cell is selected from the group consisting of: T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL) and regulatory T cells. In one embodiment, the cell is a T cell. In one embodiment, the modified cell is an autologous cell.

[0380] In some embodiments, the modified cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL) or a regulatory T cell. In this embodiment, a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL) or a regulatory T cell comprises a chimeric antigen receptor (CAR). In some embodiments, a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL) or a regulatory T cell comprises a chimeric antigen receptor (CAR) that selectively or specifically binds to a tumor antigen. 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, CDS, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD119, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD119, CD20 71, CD276, CEA, tight junction protein 18.2, c-Met, DLL3, DRS, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, phosphatidylinositol glycan-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-I, Lewis Y, LMPI (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUCI, Nectin4 / FAP, NKG2D-ligands (MIC-A, MIC-B and ULBP 1 to 6), NY-ESO-1, Pl 6. PD-LI, PSCA, PSMA, RORI, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof. In one embodiment, the tumor antigen is a tumor-associated carbohydrate antigen (TACA). In some embodiments, the modified cell is a CAR T cell. In some embodiments, the modified cell is a CAR T cell that specifically targets a tumor antigen. In some embodiments, the modified cell is a CAR T cell that specifically targets a tumor-associated carbohydrate antigen (TACA).

[0381] In one aspect, the present disclosure provides a modified immune cell population described herein.

[0382] B. Methods of Producing Modified Cells

[0383] 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.

[0384] Modified cells (e.g., cells comprising a subject CAR or a bispecific fusion protein) can be produced by stably transfecting a host cell with an expression vector including a nucleic acid disclosed herein. Additional methods for generating modified cells disclosed herein include, but are not limited to, chemical transformation methods (e.g., using calcium phosphate, dendritic polymers, liposomes, and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical conversion, gene electrotransfer, and / or hydrodynamic delivery), and / or particle-based methods (e.g., piercing transfection, using a gene gun, and / or magnetic transfection). Transfected cells expressing the subject CAR or bispecific fusion proteins disclosed herein can be amplified in vitro.

[0385] In some embodiments, cells are genetically modified by contacting cells with isolated nucleic acids encoding CAR or bispecific fusion proteins as described herein. In some embodiments, nucleic acid sequences are delivered to cells using retroviral or lentiviral vectors. For example, retroviral and lentiviral vectors expressing peptides of the present invention can be delivered to different types of eukaryotic cells and to tissues and whole organisms using transduced cells as carriers or using cell-free local or systemic delivery of encapsulated, bound or naked vectors. The method used may be for any purpose requiring stable expression or stable expression sufficient.

[0386] In other embodiments, the nucleic acid sequence is delivered to the cell using in vitro transcribed mRNA. In vitro transcribed mRNA can be delivered to different types of eukaryotic cells and to tissues and whole organisms using transfected cells as carriers or using cell-free local or systemic delivery of encapsulated, combined or naked mRNA. The method used can be for any purpose requiring transient expression or transient expression is sufficient.

[0387] In certain embodiments, the cell can be any suitable cell type that can express the desired peptide. In certain embodiments, the modified cell is used in the method that the cell is introduced into the receptor. In certain embodiments, with respect to the receptor, the cell is autologous, allogeneic, isogenic or xenogeneic.

[0388] The disclosed compositions and methods can be applied to modulation of T cell activity in basic research and therapy in the areas of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including evaluating the ability of genetically modified T cells to kill target cancer cells.

[0389] Methods for introducing genes into cells and expressing them in cells are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art. For example, the expression vectors can be transferred into host cells by physical, chemical or biological means.

[0390] In some embodiments, the modified cell is a modified host cell. In some embodiments, the modified cell is selected from the group consisting of bacterial cells, fungal cells, insect cells or mammalian cells. In some embodiments, the modified cell is a bacterial cell selected from Escherichia coli or Bacillus stearothermophilus. In some embodiments, the modified cell is a fungal cell selected from yeast cells, Saccharomyces cerevisiae. In some embodiments, the modified cell (e.g., host cell) is an insect cell selected from Lepidoptera insect cells or Spodoptera frugiperda.

[0391] In some embodiments, the modified cell is a mammalian cell selected from the group consisting of Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, monkey kidney cells, HeLa cells, human hepatocellular carcinoma cells, or human embryonic kidney 293 cells. In one embodiment, the modified cell is a CHO cell or a HEK 293 cell.

[0392] 1. Physical methods

[0393] 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.

[0394] 2. Biological methods

[0395] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA vectors and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used methods for inserting genes into mammalian (e.g., human) cells. Other viral vectors can be derived from slow viruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0396] In some embodiments, the nucleic acid encoding the subject CAR of the present disclosure or bispecific fusion protein is introduced into the cell by an expression vector. Provided herein is an expression vector comprising a nucleic acid encoding a subject CAR (e.g., TACA CAR) or a bispecific fusion protein. Suitable expression vectors include lentiviral vectors, gamma retroviral vectors, foamy virus vectors, adeno-associated virus (AAV) vectors, adenoviral vectors, engineered hybrid viruses, naked DNA, including but not limited to transposon-mediated vectors, such as Sleeping Beauty, Piggyback and integrase, such as Phi31. Some other suitable expression vectors include herpes simplex virus (HSV) and retroviral expression vectors.

[0397] Adenovirus expression vector is based on adenovirus, and its ability to be integrated into genomic DNA is low, but the efficiency of transfecting host cells is high. The adenovirus sequence contained in the adenovirus expression vector is sufficient to: (a) support the packaging of the expression vector, and (b) finally express the theme CAR in the host cell. In some embodiments, the adenovirus genome is 36kb, linear, double-stranded DNA, in which foreign DNA sequences (e.g., nucleic acids encoding TACA-CAR or bispecific fusion proteins) can be inserted to replace large fragments of adenovirus DNA, so as to prepare the expression vector of the present invention. See, for example, Danthinne and Imperiale, Gene Therapy 7 (20): 1707-1714 (2000).

[0398] Another expression vector is based on adeno-associated virus, which utilizes an adenovirus coupling system. This AAV expression vector has a high frequency of integration into the host genome. The AAV expression vector can infect non-dividing cells, so the AAV expression vector can be used, for example, in tissue culture or in vivo to deliver genes to mammalian cells. AAV vectors have a wide range of infected hosts. Detailed information on the generation and use of AAV vectors is described in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0399] Retroviral expression vector can be integrated into the host genome, delivering a large amount of foreign genetic material, infecting a wide range of species and cell types, and being packaged in a special cell line. By inserting nucleic acid (for example, nucleic acid encoding TACA-CAR or bispecific fusion protein) into certain positions of the viral genome to produce replication-defective viruses, retroviral vectors are constructed. Although, retroviral vectors can infect a wide variety of cell types, the integration and stable expression of theme CAR or bispecific fusion proteins require the division of host cells.

[0400] Lentiviral vectors are derived from lentiviruses, which are complex retroviruses, and contain other genes with regulatory or structural functions in addition to common retroviral genes gag, pol and env. See, for example, U.S. Patent 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 generated by multiple weakening HIV virulence genes, for example, deleting genes env, vif, vpr, vpu and nef, so that the vector is biologically safe. Lentiviral vectors can infect non-dividing cells, and can be used for in vivo and ex vivo gene transfer and expression of nucleic acids such as encoding theme CAR or bispecific fusion proteins (see, for example, U.S. Patent No. 5,994,136).

[0401] Expression vectors comprising nucleic acids disclosed herein can be introduced into host cells by any means known to those skilled in the art. If desired, the expression vector can include viral sequences for transfection. Alternatively, the expression vector can be introduced by fusion, electroporation, gene gun, transfection, lipofection, etc. Prior to the introduction of the expression vector, the host cells can be grown and amplified in culture, followed by appropriate treatment to introduce and integrate the vector. The host cells are then amplified and can be screened for markers present in the vector.

[0402] Various markers that can be used are known in the art and may include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc. As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably. In some embodiments, the host cell is an immune cell or a precursor thereof, e.g., a T cell, a NK cell or a NKT cell.

[0403] 3. Chemical method

[0404] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0405] In the case of utilizing a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid formulations for introducing nucleic acids into host cells (in vitro, in vitro or in vivo). In another aspect, nucleic acids can be associated with lipids. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, connected to liposomes via a linker molecule associated with liposomes and oligonucleotides, wrapped in liposomes, compounded with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or otherwise associated with lipids. The composition related to lipids, lipid / DNA or lipid / expression vectors is not limited to any specific structure in the solution. For example, they can exist with double-layer structures, micelles or "folding" structures. They can also only be dispersed in the solution, and may form aggregates with uneven size or shape. Lipid is a fatty substance, which can be a naturally occurring lipid or a synthetic lipid. For example, lipids include naturally occurring fat droplets in the cytoplasm as well as the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0406] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dihexadecyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Lipid stock solutions in chloroform or chloroform / methanol can be stored at a temperature of about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term that covers a variety of monolayer and multilayer lipid vehicles formed by generating closed lipid bilayers or aggregates. Liposomes can be characterized by having a vesicle structure having a phospholipid bilayer membrane and an internal aqueous medium. Multilayer 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 encapsulate water and dissolved solutes between the lipid bilayers. Ghosh et al., Glycobiology 5:505-10 (1991). However, compositions having structures different from normal vesicle structures in solution are also contemplated. For example, lipids may present micellar structures or exist only in the form of heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0407] No matter which method is used to introduce exogenous nucleic acid into host cells, various assays can be performed to confirm the presence of recombinant DNA sequences in host cells. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as, for example, by immunological means (ELISA and Western blotting) or by assays described herein to detect the presence or absence of specific peptides to identify agents falling within the scope of the present invention.

[0408] In addition, nucleic acid can be introduced by any means, such as transduction-expanded T cells, transfection-expanded T cells, and electroporation-expanded T cells. One nucleic acid can be introduced by one method, and another nucleic acid can be introduced into T cells by a different method.

[0409] 4. RNA

[0410] RNA has several advantages over more traditional plasmid or viral methods. Gene expression from RNA sources does not require transcription, and protein products are rapidly produced after transfection. In addition, since the RNA only needs to enter the cytoplasm rather than the nucleus, typical transfection methods result in extremely high transfection rates. In addition, plasmid-based methods require that the promoter driving the expression of the gene of interest is active in the cells being studied.

[0411] An advantage of the RNA transfection method of the present invention is that RNA transfection is essentially instantaneous and does not require a vector. RNA transgenes can be delivered to lymphocytes after transient in vitro cell activation and expressed therein as minimal expression cassettes without any additional viral sequences. Under these conditions, transgenes are unlikely to integrate into the host cell genome. Due to the transfection efficiency of RNA and its ability to uniformly modify an entire lymphocyte population, cell cloning is not required.

[0412] Genetic modification of host cells with in vitro transcribed RNA (TVT-RNA) utilizes two different strategies, both of which have been tested in various animal models. Cells are transfected with in vitro transcribed RNA by lipofection or electroporation. It is contemplated that various modifications will be used to stabilize the IVT-RNA in order to achieve prolonged expression of the transferred IVT-RNA. Several IVT vectors are known in the literature that are used as templates for in vitro transcription in a standardized manner and that have been genetically modified to produce stable RNA transcripts.

[0413] The scheme currently used in the art is based on a plasmid vector with the following structure: a 5' RNA polymerase promoter capable of RNA transcription, followed by a gene of interest, its 3' and / or 5' flanking untranslated regions (UTRs), and a 3' polyadenyl 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 polyadenyl cassette (the recognition sequence corresponds to the cleavage site). Therefore, the polyadenyl cassette corresponds to the subsequent poly (A) sequence in the transcript. As a structure of the procedure, some nucleotides remain as part of the restriction site after linearization and extend or mask the poly (A) sequence at the 3' end. It is not clear whether this non-physiological overhang will affect the amount of protein produced by this construct in the cell.

[0414] In one embodiment, the nucleic acid encoding the CAR or bispecific fusion protein of the present disclosure and the separation introduced into the cell of the present disclosure includes RNA. In one embodiment, RNA is mRNA. In one embodiment, RNA is in vitro transcribed (IVT) RNA. 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 into a template by PCR for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.

[0415] In one embodiment, the DNA used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence in an organism's genome. In one embodiment, the DNA is a full-length gene of interest that is a part of a gene. The gene can include some or all of 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 5' and 3' UTRs. Alternatively, the DNA can be an artificial DNA sequence that is not usually expressed in a naturally occurring organism. An exemplary artificial DNA sequence is a sequence containing a gene portion that is linked together to form an open reading frame encoding a fusion protein. The DNA portions that are linked together can be from a single organism or from more than one organism.

[0416] The gene that can be used as the DNA source of PCR includes the gene that encodes the polypeptide that provides treatment or preventive effect for organism or can be used for diagnosing the disease or illness of organism.Preferred gene is the gene that can be used for short-term treatment, or the gene that there is safety problem to the gene of dosage or expression.For example, for the treatment of cancer, autoimmune disease, parasite, virus, bacterium, fungus or other infection, the transgenic to be expressed can encode a kind of polypeptide, it is as the ligand or receptor of immune system cell, or can play the role of stimulating or suppressing the immune system of organism.In some embodiments, it is not expected that the immune system is prolonged and continuously stimulated, and it is not necessary to produce continuous change after successful treatment, because this may cause new problems.For the treatment of autoimmune disease, it may be expected to suppress or block the immune system during the attack, but it cannot be suppressed for a long time, and long-term suppression may cause the patient to be overly sensitive to infection.

[0417] Templates for in vitro mRNA transcription for transfection are generated using PCR. Methods for performing PCR are well known in the art. Primers for PCR are designed to have a region substantially complementary to the DNA region to be used as a PCR template. As used herein, "substantially complementary" refers to a nucleotide sequence, wherein most or all bases in the primer sequence are complementary, or one or more bases are non-complementary or mismatched. Substantially complementary sequences can anneal or hybridize with the expected DNA target under the annealing conditions for PCR. Primers can be designed to be substantially complementary to any part of the DNA template. For example, primers can be designed to amplify gene portions (open reading frames) that are normally transcribed in cells, including 5' and 3' UTR. Primers can also be designed to amplify gene portions encoding specific domains of interest. In one embodiment, primers are designed to amplify the coding region of human cDNA, including all or part of 5' and 3' UTR. Primers that can be used for PCR are generated by synthetic methods well known in the art.

[0418] A "forward primer" is a primer containing a nucleotide region that is substantially complementary to the nucleotides on the DNA template upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to the position of the 5' of the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer containing a nucleotide region that is substantially complementary to the double-stranded DNA template downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to the position of the 3' of the DNA sequence to be amplified relative to the coding strand.

[0419] Any DNA polymerase that can be used for PCR can be used for the method disclosed herein. Reagents and polymerases are commercially available from multiple sources. Chemical structures with the ability to promote stability and / or translation efficiency can also be used. RNA preferably has 5' and 3' UTRs. In one embodiment, the length of the 5' UTR is between zero and 3000 nucleotides. The length of the 5' and 3' UTR sequences to be added to the coding region can be changed by different methods, including but not limited to designing PCR primers that anneal to different UTR regions. Using this method, ordinary technicians can modify the 5' and 3' UTR lengths required for optimal translation efficiency after transfection of the transcribed RNA. The 5' and 3' UTRs can be naturally occurring endogenous 5' and 3' UTRs of the gene of interest. Alternatively, the UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into forward and reverse primers or by making any other modifications to the template. The use of UTR sequences that are not endogenous to the gene of interest can be used to modify the stability and / or translation efficiency of RNA. For example, it is known that the AU-rich elements in the 3' UTR sequence can reduce the stability of mRNA. Therefore, a 3'UTR can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs well known in the art.

[0420] In one embodiment, the 5'UTR may contain the Kozak sequence of an endogenous gene. Alternatively, when a 5'UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5'UTR sequence. The Kozak sequence can increase the translation efficiency of some RNA transcripts, but the Kozak sequence does not seem to be required for all RNA to achieve efficient translation. The need for Kozak sequences in many mRNAs is known in the art. In other embodiments, the 5'UTR may be derived from an RNA virus whose RNA genome remains stable in the cell. In other embodiments, various nucleotide analogs can be used in 3' or 5'UTR to prevent exonuclease degradation of mRNA.

[0421] In order to be able to synthesize RNA from a DNA template without the need for gene cloning, a transcription promoter should be connected to the DNA template upstream of the sequence to be transcribed. When the sequence as a promoter of RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter will be incorporated into the PCR product upstream of the open reading frame to be transcribed. In a preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of T7, T3 and SP6 promoters are known in the art.

[0422] In a preferred embodiment, the mRNA has both a cap on the 5' end and a 3' poly (A) tail, which determines ribosome binding, translation initiation, and stability of the mRNA in the cell. On a circular DNA template (e.g., plasmid DNA), RNA polymerase produces long concatemer products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3' UTR results in normal-sized mRNA, which is ineffective in eukaryotic transfection, even after transcription. On a linear DNA template, bacteriophage T7 RNA polymerase can extend the 3' end of the transcript to the last base beyond the template. Schenborn and Mierendorf, Nuc Acids Res., 13: 6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003).

[0423] The conventional method for incorporating polyA / T stretches into DNA templates is molecular cloning. However, polyA / T sequences integrated into plasmid DNA can lead to plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes the cloning procedure not only laborious and time-consuming, but also often unreliable. This is why it is highly desirable to find a method that allows the construction of DNA templates with polyA / T 3' extensions without the need for cloning.

[0424] The polyA / T fragment of the transcribed DNA template can be generated during PCR by using a reverse primer containing a polyT tail, such as a 100T tail (size can be 50T-5000T), or by any other method after PCR, including but not limited to DNA ligation or in vitro recombination. The poly (A) tail also provides stability to the RNA and reduces RNA degradation. In general, 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 between 100 and 5000 adenosines.

[0425] The Poly (A) tail of the RNA can be further extended after in vitro transcription using a Poly (A) polymerase, such as E. coli Poly (A) polymerase (E-PAP). In one embodiment, the length of the Poly (A) tail is increased from 100 nucleotides to between 300 and 400 nucleotides, resulting in an approximately two-fold increase in the translation efficiency of the RNA. In addition, different chemical groups are connected to the 3' end to increase the stability of the mRNA. This connection may contain modified / artificial nucleotides, aptamers, and other compounds. For example, poly (A) polymerase may be used to incorporate ATP analogs into the poly (A) tail. ATP analogs may further increase the stability of the RNA. The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the method disclosed herein includes a 5' cap. The 5' cap is provided using techniques known in the art and 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)).

[0426] The RNA produced by the method disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any virus, chromosome or artificially designed sequence that initiates the cap-independent ribosome binding with the mRNA and promotes the initiation of translation. Any solute suitable for cell electroporation may be included, and the solute may contain factors that promote cell permeability and vigor, such as sugars, peptides, lipids, proteins, antioxidants and surfactants.

[0427] RNA can be introduced into target cells using any of a variety of different methods, e.g., commercially available methods including, but not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), multifunctional cell electroporator (Eppendort, Hamburg Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or a gene gun particle delivery system, such as a "gene gun" (see, e.g., Nishikawa et al. Hum Gene Ther., 12(8):861-70 (2001).

[0428] In some embodiments, RNA is electroporated into cells, such as RNA transcribed in vitro. The preparation and methodology of electroporating nucleic acid constructs into mammalian cells are taught in, for example, US2004 / 0014645, US2005 / 0052630A1, US2005 / 0070841 A1, US 2004 / 0059285A1, US2004 / 0092907A1. Various parameters (including electric field strength) required for the electroporation of any known cell type are generally known in the relevant research literature and in numerous patents and applications in the field. See, for example, U.S. Patent No. 6,678,556, U.S. Patent No. 7,171,264 and U.S. Patent No. 7,173,116. Devices for the therapeutic application of electroporation are commercially available, such as MedPulser TM DNA electroporation therapy system (Inovio / Genetronics, San Diego, Calif), and described in the following 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; Electroporation can also be used for in vitro cell transfection as described in, for example, US20070128708A1. Electroporation can also be used to deliver nucleic acids to cells in vitro.

[0429] Thus, administration of nucleic acids, including expression constructs, into cells by electroporation-mediated administration using any of the many available devices and electroporation systems known to those of skill in the art presents an exciting new way to deliver RNA of interest to target cells.

[0430] The disclosed methods can be applied to modulate host cell activity in basic research and therapy in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including evaluating the ability of genetically modified host cells to kill target cancer cells. The methods also provide the ability to control expression levels over a wide range by changing, for example, the amount of promoter or input RNA, so that expression levels can be individually regulated. In addition, PCR-based mRNA production technology greatly facilitates the design of mRNAs with different structures and their domain combinations.

[0431] C. Source of immune cells

[0432] Prior to amplification, the immune cell source obtained from the subject is used for ex vivo operation. The target cell source for ex vivo operation may also include, for example, autologous or allogeneic donor blood, umbilical cord blood or bone marrow. For example, the source of immune cells can be from a subject to be treated with the modified immune cells of the present invention, such as blood of the subject, umbilical cord blood of the subject or bone marrow of the subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats and transgenic species thereof. In certain exemplary embodiments, the subject is a human.

[0433] Immune cells can be obtained from a variety of 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, such as 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, such as multipotent stem cells and multipotent stem cells, including induced pluripotent stem cells (iPSC). In some aspects, cells are human cells. With reference to the subject to be treated, cells can be allogeneic and / or autologous. Cells are typically primary cells, such as those directly separated from a subject and / or separated and frozen from a subject.

[0434] In certain embodiments, the immune cell is a T cell, e.g., a CD8 + T cells (e.g., CD8 + Unsensitized T cells, central memory T cells or effector memory T cells), CD4 + T cells, natural killer T cells (NKT cells), regulatory T cells (Treg), 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 cell is a monocyte or a granulocyte, for example, a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil and / or a basophil. In one embodiment, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, for example, an iPS cell generated by a subject, operated to change (e.g., induce mutation) or operate the expression of one or more target genes, and differentiate into, for example, a T cell, for example, a CD8 + T cells (e.g., CD8 + Unsensitized 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.

[0435] In some embodiments, the cells include one or more T cell subsets or other cell types, such as the whole T cell population, CD4 + Cells, CD8 + Cells and their subsets, such as those defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization and / or persistence capacity, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. T cells and / or CD4 + and / or CD8 + The subtype and subpopulation of T cell include unsensitized T (TN) cell, effector T cell (TEFF), memory T cell and its subtype, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM) or terminally differentiated effector memory T cell, tumor infiltrating lymphocyte (TIL), immature T cell, mature T cell, helper T cell, cytotoxic T cell, mucosa-associated invariant T (MAIT) cell, naturally occurring and adaptive regulatory T (Treg) cell, helper T cell, such as TH1 cell, TH2 cell, TH3 cell, TH17 cell, TH9 cell, TH22 cell, follicular helper T cell, α / β T cell and δ / γ T cell. In certain embodiments, any number of T cell lines available in the art can be used.

[0436] In some embodiments, the method includes separating immune cells from a subject, and preparing, processing, culturing and / or engineering them. In some embodiments, the preparation of engineered cells includes one or more culturing and / or preparation steps. As described, cells for engineering can be separated from samples (such as biological samples, for example, samples obtained from subjects or derived from subjects). In some embodiments, the subject from which cells are separated is a subject suffering from a disease or illness or requiring cell therapy or to whom cell therapy will be administered. In some embodiments, the subject is a person who needs a specific therapeutic intervention (such as adoptive cell therapy of separation, processing and / or engineered cells). Therefore, in some embodiments, cells are primary cells, for example, primary human cells. Samples include tissues, liquids and other samples collected directly from subjects, and samples obtained by one or more processing steps (such as separation, centrifugation, genetic engineering (for example, transduced with viral vectors), washing and / or incubation). Biological samples can be samples obtained directly from biological sources or processed samples. 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 samples processed therefrom.

[0437] In some aspects, the sample derived from or separated from the cell is a sample of blood or blood origin or is or is derived from apheresis product or leukocyte apheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, intestinal associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organs and / or cells derived therefrom. In the context of cell therapy (e.g., adoptive cell therapy), sample includes samples from autologous sources and allogeneic sources.

[0438] In some embodiments, the cell is derived from a cell line, such as a T cell line. In some embodiments, the cell is obtained from a xenogeneic source, such as from mice, rats, non-human primates and pigs. In some embodiments, the separation of cells includes one or more preparations and / or cell separation steps based on non-affinity. In some instances, the cell is washed, centrifuged and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, crack or remove cells sensitive to specific reagents. In some instances, cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity and / or resistance to specific components.

[0439] In some instances, for example, cells are obtained from the circulating blood of a subject by apheresis or leukapheresis. The sample contains lymphocytes in some aspects, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and / or platelets, and contains cells except erythrocytes and platelets in some aspects. In some embodiments, the blood cells collected from the subject are washed, for example, to remove the plasma portion and the cells are placed in an appropriate buffer or culture medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some cases, the washing step is completed by tangential flow filtration (TFF) according to the manufacturer's instructions. In certain embodiments, the cells are resuspended in a variety of biocompatible buffers after washing. In certain embodiments, the components of the blood cell sample are removed, and the cells are directly resuspended in culture medium. In some embodiments, the method includes a density-based cell separation method, such as preparing leukocytes from peripheral blood by lysing erythrocytes and by Percoll or Ficoll gradient centrifugation.

[0440] In one embodiment, immune cells are obtained from the circulating blood of an individual by apheresis or leukapheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected by apheresis can be washed to remove the plasma portion, and the cells are placed in an appropriate buffer or culture medium for subsequent processing steps, the buffer or culture medium such as phosphate buffered saline (PBS) or lack of calcium and may lack magnesium or may lack a washing solution of many (if not all) divalent cations. It will be readily appreciated by those of ordinary skill in the art that washing steps can be completed by methods known to those skilled in the art, such as by using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics ...

Claims

1. An isolated nucleic acid molecule encoding a bispecific fusion protein, the bispecific fusion protein comprising: (a) an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), and (b) an immune cell recognition domain that specifically binds to a receptor on an immune effector cell, wherein the antigen binding domain comprises a TACA binding domain derived from a lectin; and wherein the antigen binding domain comprises more than one TACA binding domain.

2. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), the CAR comprising: (a) an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), wherein the antigen binding domain comprises a TACA binding domain derived from a lectin; and wherein the antigen binding domain comprises more than one TACA binding domain; (b) a transmembrane domain; (c) a co-stimulatory signaling region; and (d) an intracellular signaling domain.

3. The isolated nucleic acid molecule of claim 1 or 2, wherein the antigen binding domain comprises two, three, four, five, six, seven, eight, nine, ten or more TACA binding domains.

4. The isolated nucleic acid molecule of any one of claims 1 to 3, wherein the more than one TACA binding domains are operably linked via a linker.

5. The isolated nucleic acid molecule of claim 4, wherein the linker is selected from the group consisting of a peptide linker, a non-peptide linker, a chemical unit, a hindered cross-linker, and a non-hindered cross-linker.

6. The isolated nucleic acid molecule of claim 5, wherein the linker is a peptide linker.

7. The isolated nucleic acid molecule of claim 5 or 6, wherein the peptide linker is 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 in length.

8. The isolated nucleic acid molecule of claim 6 or 7, wherein the peptide linker is a glycine-serine linker.

9. The isolated nucleic acid molecule of any one of claims 4 to 8, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO:

132.

10. The isolated nucleic acid molecule of any one of claims 4 to 9, wherein the linker comprises the amino acid sequence of SEQ ID NO:

127.

11. The isolated nucleic acid molecule of any one of claims 4 to 9, wherein the linker comprises the amino acid sequence of SEQ ID NO:

131.

12. The isolated nucleic acid molecule of any one of claims 1 to 11, wherein the lectin is selected from the group consisting of: galectins, sialic acid-binding immunoglobulin-like lectins, selectins; C-type lectins; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Lycopersicon esculentum lectin); E-PHA (Lycopersicon esculentum lectin); tomato lectin (Lycopersicon esculentum lectin; LEA); peanut lectin (Arachis hypogaea Agglutinin; PNA); potato lectin (Solanum tuberosum lectin); pokeweed mitogen (Phytolacca Americana lectin); wheat germ agglutinin (Wheat germ agglutinin); agglutinin (Triticum Vulgaris lectin); Artocarpus polyphemus lectin (Jacalin letin); Vetch lectin (VVA); Snail lectin (HPA); Wisteria multiflora lectin (WFA); Elderberry lectin (SNA); BC2L-CNt (lectin from the Gram-negative bacterium Burkholderia cenocepacia); MAL; PVL; Sclerotium sclerotium lectin (SRL); Echinops serrulata lectin (ESA); CLEC17A (protectin); Dictyophora aurantifolia lectin; S. sessile elderberry lectin (SSA); Gleheda; Morniga oleracea lectin (MornigaG); Salvia miltiorrhiza lectin; bogotensis agglutinin, Salvia officinalis agglutinin, Changshan Shanhaizhou agglutinin, Concha strychnos nucifera agglutinin, Griffonia suffruticosa (GsLA4), Quaternary bean (acidic WBAI), Abrus precatorius lectin, Amaranthus caudatus lectin, Amaranthus gingkoi lectin, Amaranthus chinensis lectin, Jackfruit agglutinin, Morus alba lectin, Jackfruit agglutinin, Lablab bisporus lectin, Lablab bisporus lectin, Soybean agglutinin, and Agaricus bisporus lectin.

13. The isolated nucleic acid molecule of claim 12, wherein: (i) the galectin is 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) the sialic acid binding immunoglobulin-like lectin is selected from the group consisting of sialic acid binding immunoglobulin-like lectin-1 (sialoadhesin), sialic acid binding immunoglobulin-like lectin-2 (CD22), sialic acid binding immunoglobulin-like lectin-3 (CD33), sialic acid binding immunoglobulin-like lectin-4 (myelin-associated glycoprotein), sialic acid binding immunoglobulin-like lectin-5, sialic acid binding immunoglobulin-like lectin-6, sialic acid binding immunoglobulin-like lectin-7, sialic acid binding immunoglobulin-like lectin-8, sialic acid binding immunoglobulin-like lectin-9, sialic acid binding immunoglobulin-like lectin-10, sialic acid binding immunoglobulin-like lectin-11, sialic acid binding immunoglobulin-like lectin-12, sialic acid binding immunoglobulin-like lectin-13, sialic acid binding immunoglobulin-like lectin-14, sialic acid binding immunoglobulin-like lectin-15, sialic acid binding immunoglobulin-like lectin-16, sialic acid binding immunoglobulin-like lectin-17, sialic acid binding immunoglobulin-like lectin-18, sialic acid binding immunoglobulin-like lectin-19, sialic acid binding immunoglobulin-like lectin-20, sialic acid binding immunoglobulin-like lectin-23, sialic acid binding immunoglobulin-like lectin-24, sialic acid binding immunoglobulin-like lectin-25 9. Sialic acid-binding immunoglobulin-like lectin-10, Sialic acid-binding immunoglobulin-like lectin-11, Sialic acid-binding immunoglobulin-like lectin-12, Sialic acid-binding immunoglobulin-like lectin-13, Sialic acid-binding immunoglobulin-like lectin-14, Sialic acid-binding immunoglobulin-like lectin-15, Sialic acid-binding immunoglobulin-like lectin-16, Sialic acid-binding immunoglobulin-like lectin-17, Sialic acid-binding immunoglobulin-like lectin E, Sialic acid-binding immunoglobulin-like lectin F, Sialic acid-binding immunoglobulin-like lectin G and Sialic acid-binding immunoglobulin-like lectin H.

14. The isolated nucleic acid molecule of claim 12, wherein 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(GAL NT15), ppGalNAc-T16 (GALNT16), ppGalNAc-T17 (GALNT17), ppGalNAc-T18 (GALNT18), ppGalNAc-T-like 5 (GALNTL5), and ppGalNAc-T-like 6 (GALNTL6).

15. The isolated nucleic acid of any one of claims 1 to 14, wherein the antigen binding domain selectively targets a TACA selected from the group consisting of: β1,6 branching, β1,6GlcNAc branched N-glycan, 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 , disialo-Lewis x / a , Sialyl 6-sulfo Lexis x 、Lewis-y(Le y ), Lewis Y, Globo H, GD2, GD3, GM3 and fucosyl GM1.

16. The isolated nucleic acid of any one of claims 1 to 15, wherein 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.

17. The isolated nucleic acid molecule of any one of claims 1 to 16, wherein the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152; or The amino acid sequence shown in NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152 has an amino acid sequence with 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.

18. The isolated nucleic acid of any one of claims 1 to 17, wherein the antigen binding domain comprises an amino acid sequence having at least 90% homology to SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152.

19. The isolated nucleic acid of any one of claims 1 to 18, wherein the isolated nucleic acid comprises an expression vector; and / or in vitro transcribed RNA.

20. An isolated nucleic acid molecule as described in any one of claims 1, 3 to 19, wherein the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58 and 63-66; or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58 and 63-66.

21. The isolated nucleic acid molecule of any one of claims 1, 3 to 20, wherein the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence selected from SEQ ID NO: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66.

22. The isolated nucleic acid molecule of any one of claims 1, 3 to 21, wherein the isolated nucleic acid molecule encodes a bispecific fusion protein comprising the amino acid sequence of SEQ ID NO: 32-34, 40-42, 48-50, 56-58 or 64.

23. The isolated nucleic acid molecule of claim 22, wherein: (a) the bispecific fusion protein exhibits enhanced binding to tumor cells expressing Thomsen-nouveau (Tn) antigen when compared to a bispecific fusion protein comprising a flexible linker in the antigen binding domain; and / or (b) The bispecific fusion protein exhibits enhanced binding to tumor cells expressing β1,6GlcNAc branched N-glycans when compared to a bispecific fusion protein comprising a flexible linker in the antigen binding domain.

24. An isolated nucleic acid molecule as described in claim 23, wherein the flexible linker is a glycine-serine linker or a linker comprising an amino acid sequence selected from SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 129 or SEQ ID NO: 127; or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 129 or SEQ ID NO:

127.

25. The isolated nucleic acid molecule of any one of claims 1 and 3 to 24, wherein the immune effector cells are selected from the group consisting of T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, monocytes, dendritic cells, and neutrophils.

26. The isolated nucleic acid molecule of any one of claims 1 and 3 to 25, wherein the immune effector cell is a T cell.

27. The isolated nucleic acid molecule of any one of claims 1 and 3 to 26, wherein the immune effector cell is a NK cell.

28. The isolated nucleic acid molecule of any one of claims 1 and 3 to 27, wherein the immune cell recognition domain comprises: (i) a peptide, protein, antibody, single domain antibody, nanobody, antibody fragment or single chain variable fragment (scFv) that selectively binds to a receptor on the immune effector cell; (ii) an antibody Fc domain, optionally the Fc region of an IgG molecule; or (iii) the constant region domains CH2 and / or CH3, preferably CH2 and CH3, of an antibody, optionally with or without a hinge region.

29. The isolated nucleic acid molecule of any one of claims 1 and 3 to 28, wherein the receptor on the immune effector cell is selected from the group consisting of: T cell receptor (TCR) α, TCR β, TCR γ, TCR δ, the invariant TCR of NKT cells, CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1.

30. The isolated nucleic acid molecule of any one of claims 1 and 3 to 29, wherein the receptor on the immune effector cell is: (i) a T cell receptor selected from the group consisting of CD3, CD2, CD28 and CD25; or (ii) an NK cell receptor selected from the group consisting of NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA and CEACAM1.

31. The isolated nucleic acid molecule of any one of claims 1 and 3 to 30, wherein the immune cell recognition domain comprises: (i) scFvs that selectively bind to CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1; (ii) the amino acid sequence of SEQ ID NO: 149, 150 or 151; or (iii) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 149, 150 or 151.

32. The isolated nucleic acid molecule of any one of claims 1 and 3 to 31, wherein the bispecific fusion protein is an Fc fusion protein comprising the antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA) and the Fc domain.

33. The isolated nucleic acid molecule of any one of claims 2 and 3 to 19, wherein the membrane spaning domain comprises a membrane spaning region of a molecule selected from the group consisting of: T cell receptor (TCR)-α, TCR-β, CD3-ζ, CD3-ε, 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.

34. The isolated nucleic acid molecule of any one of claims 2 to 19, wherein the transmembrane domain comprises a CD8 transmembrane domain.

35. The isolated nucleic acid molecule of any one of claims 2 to 19, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:

148.

36. An isolated nucleic acid molecule as described in any one of claims 2 to 19, wherein the co-stimulatory domain is a co-stimulatory domain of a molecule selected from the group consisting of: CD27, CD28, 4-IBB (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.

37. The isolated nucleic acid molecule of any one of claims 2 to 19 and 33 to 36, wherein the co-stimulatory domain comprises: (i) 4-1BB co-stimulatory domain; (ii) the amino acid sequence of SEQ ID NO: 114; (iii) CD28 co-stimulatory domain; (iv) the amino acid sequence of SEQ ID NO: 113; or (v) 4-1BB and CD28 co-stimulatory domains.

38. The isolated nucleic acid molecule of any one of claims 2 to 19 and 33 to 37, wherein the intracellular domain comprises an intracellular signaling domain of a molecule selected from the group consisting of: T cell receptor (TCR) ζ, FcR-γ, FcR-β, CD3-γ, CD3-δ, CD3-ε, CD3-ζ, CDS, CD5, CD22, CD79a, CD79b, and CD66d.

39. The isolated nucleic acid molecule of any one of claims 2 to 19 and 33 to 38, wherein the intracellular signaling domain comprises a CD3 zeta signaling domain; or the amino acid sequence of SEQ ID NO:

115.

40. The isolated nucleic acid molecule of any one of claims 2 to 19 and 33 to 39, wherein the CAR further comprises a hinge domain.

41. The isolated nucleic acid molecule of any one of claims 2 to 19 and 33 to 40, wherein the hinge domain is a protein selected from the group consisting of: 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.

42. The isolated nucleic acid molecule of any one of claims 2 to 19 and 33 to 41, wherein the hinge domain is a CD8 alpha hinge domain or wherein the hinge domain comprises the amino acid sequence of SEQ ID NO:

147.

43. An isolated nucleic acid molecule as described in any one of claims 2 to 19 and 33 to 42, wherein the hinge domain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 119, 124, 127, 128, 129, 130, 131, 132 and 147.

44. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: (i) the amino acid sequence shown in SEQ ID NO:71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99; or (ii) an amino acid sequence that has 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 shown in SEQ ID NO:71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

45. A bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), wherein the bispecific fusion protein is encoded by the isolated nucleic acid of any one of claims 1 and 3 to 32.

46. ​​A bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising: (i) an antigen binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152; or amino acid sequences 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 NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152; and (ii) An immune cell recognition domain that specifically binds to a receptor on an immune effector cell.

47. The bispecific fusion protein of claim 46, wherein the immune cell recognition domain comprises: (i) Antibody Fc domain; (ii) Fc region of IgG molecule; (iii) a peptide, protein, antibody, single domain antibody, antibody fragment or single chain variable fragment (scFv) that selectively binds to a receptor on said immune effector cell; or (iv) the constant region domains CH2 and / or CH3, preferably CH2 and CH3, of an antibody, optionally with or without a hinge region.

48. The bispecific fusion protein of claim 46 or 47, wherein the receptor on the immune effector cell is selected from the group consisting of: T cell receptor (TCR) α, TCR β, TCR γ, TCR δ, an invariant TCR from a NKT cell, CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1.

49. The bispecific fusion protein of any one of claims 45 to 48, wherein the bispecific fusion protein comprises: (a) an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 1-5, 10-34, 39-42, 47-50, 55-58 and 63-66; or (b) an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 1-5, 10-34, 39-42, 47-50, 55-58 and 63-66; or (c) an amino acid sequence selected from SEQ ID NO: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66; or (d) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66.

50. The bispecific fusion protein of any one of claims 45 to 49, wherein the bispecific fusion protein selectively targets TACA selected from the group consisting of: β1,6 branched, β1,6GlcNAc branched N-glycan, T antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6 sialylated, sialylated, sialyl-Lewis x / a , disialo-Lewis x / a , Sialyl 6-sulfo Lexis x 、Lewis-y(Le y ), Lewis Y, Globo H, GD2, GD3, GM3 and fucosyl GM1.

51. The bispecific fusion protein of any one of claims 45 to 50, wherein the bispecific fusion protein selectively targets Tn antigen or β1,6GlcNAc branched N-glycan.

52. The bispecific fusion protein of any one of claims 45 to 51, wherein the bispecific fusion protein that selectively targets a Tn antigen comprises an antigen binding domain having an amino acid sequence selected from SEQ ID NOs: 103-109, 142-146, or 152; or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 103-109, 142-146, or 152.

53. The bispecific fusion protein of any one of claims 45 to 52, wherein the bispecific fusion protein that selectively targets the Tn antigen comprises: (a) an amino acid sequence selected from SEQ ID NO: 26-34, 39-42, 47-50, 55-58 or 63-66; or (b) an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 26-34, 39-42, 47-50, 55-58 or 63-66; (c) an amino acid sequence selected from SEQ ID NO: 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66; or (d) an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66.

54. The bispecific fusion protein of any one of claims 45 to 53, wherein the bispecific fusion protein that selectively targets β1,6GlcNAc branched N-glycans comprises an antigen binding domain having an amino acid sequence selected from SEQ ID NOs: 100-102 or 133-141; or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 100-102 or 133-141.

55. The bispecific fusion protein of any one of claims 45 to 54, wherein the bispecific fusion protein that selectively targets β1,6GlcNAc branched N-glycans comprises: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5 and 10-25; or (b) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5 and 10-25; (c) an amino acid sequence selected from SEQ ID NO: 3-5, 11-13 or 19-21; or (d) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 3-5, 11-13 or 19-21.

56. A bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising: (i) an antigen binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152; or amino acid sequences 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 NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152; and (ii) An immune cell recognition domain that specifically binds to CD3 on immune effector cells.

57. A bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising: (i) a TACA binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152; or amino acid sequences 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 NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152; and (ii) a peptide, protein, antibody, single domain antibody, antibody fragment or single chain variable fragment (scFv) that selectively binds to a receptor on said immune effector cell; and / or (iii) the Fc domain of an antibody, optionally the Fc region of an IgG molecule; or (iv) the constant region domains CH2 and / or CH3, preferably CH2 and CH3, of an antibody, optionally with or without a hinge region.

58. A chimeric antigen receptor that selectively binds a tumor-associated carbohydrate antigen (TACA), wherein the CAR is encoded by an isolated nucleic acid as described in any one of claims 2 to 19 and 33 to 44.

59. A chimeric antigen receptor that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising: (i) an antigen binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152; or amino acid sequences 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 NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152; (ii) CD8 hinge domain; (iii) CD8 transmembrane domain; (iv) a CD28 co-stimulatory domain and / or a 4-1BB co-stimulatory domain; and (v) CD3ζ intracellular signaling domain.

60. The chimeric antigen receptor of claim 59, wherein the CAR comprises the amino acid sequence of SEQ ID NO:72, 88, 89, 91, 92 or 93.

61. The chimeric antigen receptor of claim 59, wherein the CAR comprises the amino acid sequence of SEQ ID NO:93, 94, 95, 96, 97, 98 or 99.

62. An expression construct comprising the isolated nucleic acid of any one of claims 1 to 44.

63. The expression construct of claim 62, wherein the expression construct further comprises a promoter.

64. An expression construct as described in claim 62 or 63, wherein the promoter is selected from the group consisting of EF-1α promoter, T cell receptor α (TRAC) promoter, interleukin 2 (IL-2) promoter or cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney murine leukemia virus (MoMuLV) promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter or Rous sarcoma virus promoter.

65. The expression construct of any one of claims 62 to 64, wherein 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.

66. The expression construct of any one of claims 62 to 65, wherein the expression construct is a lentiviral vector.

67. The expression construct of any one of claims 62 to 66, wherein the expression construct is a self-inactivating lentiviral vector.

68. An expression construct as described in any one of claims 62 to 67, wherein the expression construct comprises an isolated nucleic acid molecule encoding the bispecific fusion protein as described in any one of claims 1 and 3 to 32, and an isolated nucleic acid molecule encoding the CAR as described in any one of claims 2 to 19 and 33 to 44.

69. An expression construct as described in claim 68, wherein the isolated nucleic acid molecule encoding the bispecific fusion protein as described in any one of claims 1 and 3 to 32 and the isolated nucleic acid molecule encoding the CAR as described in any one of claims 2 to 19 and 33 to 44 are operably linked by a nucleic acid molecule encoding a self-cleaving 2A peptide selected from P2A, T2A, E2A or F2A.

70. A modified cell comprising the isolated nucleic acid of any one of claims 1 to 44; the bispecific fusion protein of any one of claims 45 to 57; the CAR of any one of claims 58 to 61; or the expression vector of any one of claims 62 to 69.

71. The modified cell of claim 70, wherein the cell is selected from the group consisting of: T cells, CD4 + T cells, CD8 + T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs) and regulatory T cells.

72. The modified cell of claim 70 or 71, wherein the modified cell is a T cell.

73. The modified cell of any one of claims 70 to 71, wherein the modified cell is an autologous cell, a xenogeneic cell, or an allogeneic cell.

74. The modified cell of any one of claims 70 to 73, wherein the modified cell comprises: (a) the bispecific fusion protein of any one of claims 45 to 57, and the CAR or the CAR targeting a tumor antigen of any one of claims 58 to 61; or (b) an isolated nucleic acid molecule encoding a bispecific fusion protein as described in any one of claims 1 and 3 to 32 and an isolated nucleic acid molecule encoding a CAR or a CAR targeting a tumor antigen as described in any one of claims 2 to 19 and 33 to 44; or (c) The expression construct of claim 69.

75. A composition comprising: (i) the isolated nucleic acid of any one of claims 1 to 44; (ii) the bispecific fusion protein of any one of claims 45 to 57; (iii) the CAR of any one of claims 58 to 61; (iv) the expression vector of any one of claims 62 to 69; or (v) The modified cell of any one of claims 70 to 74.

76. The composition of claim 75, further comprising a pharmaceutically acceptable carrier.

77. A method for generating a modified cell as described in any one of claims 70 to 74, the method comprising introducing into a cell an isolated nucleic acid as described in any one of claims 1 to 44, a bispecific fusion protein as described in any one of claims 45 to 57; a CAR as described in any one of claims 58 to 61; or an expression vector as described in any one of claims 62 to 69.

78. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition as described in any one of claims 75 to 76.

79. The method of claim 78, wherein the composition comprises: (a) the isolated nucleic acid of any one of claims 1 to 44; (b) the bispecific fusion protein of any one of claims 45 to 57; (c) the CAR of any one of claims 58 to 61; (d) the bispecific fusion protein of any one of claims 45 to 57 and the CAR or CAR targeting a tumor antigen of any one of claims 58 to 61; (e) the expression vector of any one of claims 62 to 69; or (f) The modified cell of any one of claims 70 to 74.

80. The method of claim 78 or 79, wherein the cancer is selected from the group consisting of a hematological malignancy, a solid tumor, a primary or metastatic tumor, a leukemia, a carcinoma, a blastoma, a sarcoma, a leukemia, a lymphoid malignancy, a melanoma, and a lymphoma.

81. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective composition comprising modified cells, wherein the modified cells comprise a bispecific fusion protein and / or a CAR that selectively binds to a tumor-associated carbohydrate antigen (TACA), and wherein the bispecific fusion protein or the CAR comprises an antigen binding domain selected from the group consisting of: SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, and 152; or any of the amino acid sequences shown in NO:100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152 having an amino acid sequence of 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.

82. The method of claim 81, wherein the bispecific fusion protein further comprises an immune cell recognition domain that specifically binds to a receptor on an immune effector cell.

83. The method of claim 82, wherein the immune cell recognition domain specifically binds CD3.

84. The method of claim 82 or 83, wherein the immune cell recognition domain is an antibody Fc domain and a domain that specifically binds to CD3.

85. The method of any one of claims 81 to 84, wherein the bispecific fusion protein comprises: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58 and 63-66; or (b) an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, and 63-66; (c) an amino acid sequence selected from SEQ ID NO: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66; or (d) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66.

86. The method of claim 81, wherein the CAR further comprises a transmembrane domain; a co-stimulatory domain; and an intracellular signaling domain.

87. A method as described in claim 86, wherein the CAR comprises a CD8 transmembrane domain; a CD28 co-stimulatory domain and / or a 4-1BB co-stimulatory domain; and a CD3ζ intracellular signaling domain.

88. The method of any one of claims 81 and 86 to 87, wherein the CAR further comprises a hinge domain.

89. A method of providing anti-tumor immunity in a mammal, the method comprising administering to the mammal an effective amount of the modified cell population of any one of claims 70 to 74.

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