Improved glycan-dependent immunotherapeutic bispecific proteins with longer half-life

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 fusion proteins having specificity for tumor-associated carbohydrate antigen (TACA) comprising a half-life extender molecule, vectors encoding the TACA-fusion proteins, and recombinant cells comprising the TACA-specific fusion proteins.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 351,634, filed June 13, 2022, which is hereby incorporated by reference in its entirety for any and all purposes.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] 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

[0005] The present disclosure relates generally to the fields of pharmacology and immunology, and in particular to bispecific fusion proteins targeting tumor-associated carbohydrate antigens (TACA bispecific fusion proteins) with increased half-life and the use of immune cells expressing TACA bispecific fusion proteins for treating diseases associated with abnormal glycosylation of cell surface molecules. Background Art

[0006] 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 reaching 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. To apply bispecific proteins and / or CAR T cells to a variety of cancer types, it is necessary to first identify cell-surface cancer antigens that can be safely targeted. This is a major challenge, especially for solid cancers.

[0007] One 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 disease. Carbohydrates (glycans), along with glycoproteins and glycolipids, are major cell surface components. Consequently, immunotherapeutic bispecific proteins and chimeric antigen receptor (CAR) T cells have been generated that use TACA-binding domains derived from lectins to target cells so that they are killed by T cells rather than antibodies. This new technology is called 'glycan-dependent T cell recruitment' or GlyTR (pronounced 'glitter'). However, it is not compatible with many useful therapeutic approaches and traditional immunotherapies such as bispecific T cell engagers. Tandem diabodies (TandAbs) or dual-affinity retargeting proteins Similarly, GlyTR is rapidly eliminated from the body when administered to a subject. This rapid clearance requires that large or multiple doses of GlyTR be administered to the subject in order to achieve the desired therapeutic effect.

[0008] Therefore, there is a need for improved immunotherapy approaches with improved pharmacokinetic properties to target antigens present in a variety of common cancers. The present disclosure addresses this unmet need. Summary of the Invention

[0009] One embodiment relates to a new class of immunotherapeutic fusion proteins (trispecific fusion proteins) with enhanced serum half-life for treating diseases associated with aberrant glycosylation of cell surface molecules and / or expression of tumor-associated carbohydrate antigens (TACAs); isolated nucleic acids and vectors encoding TACA fusion proteins, recombinant (i.e., modified or host) cells comprising TACA-specific fusion proteins; and compositions and methods comprising the fusion proteins for use in immunotherapy.

[0010] One aspect of the present disclosure provides an isolated nucleic acid molecule encoding a fusion protein comprising: (i) an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA); (ii) an immune cell recognition domain that specifically binds to a receptor on an immune effector cell; and (iii) a half-life extension domain, wherein the half-life extension domain is a polypeptide capable of extending the half-life of the fusion protein.

[0011] In some embodiments, the half-life extension domain is located at the N-terminus or C-terminus of the fusion protein. In some embodiments, the half-life extension domain comprises a molecule selected from the group consisting of: a polypeptide capable of binding to albumin, albumin, serum albumin, the Fc domain of an antibody, a polyethylene glycol moiety (PEG), a poly(lactic-co-glycolic acid) (PLGA) copolymer, a polymer hydrogel, a nanoparticle, a fatty acid chain, an acyl group, a myristate group, a palmitoylated group, and a steryl group. In one embodiment, the half-life extension domain comprises an Fc domain of an antibody selected from the Fc region of IgG1, IgG2, IgG3, or IgG4. In one embodiment, the half-life extension domain comprises a PEG moiety. In this embodiment, the PEG moiety is less than about 0.5k, less than about 1.0k, less than about 2.0k, less than about 3.0k, less than about 4.0k, less than about 5.0k, less than about 6.0k, less than about 7.0k, less than about 6.0k, less than about 7.0k, less than about 8.0k, less than about 10.0k, less than about 12.0k, less than about 14.0k, less than about 16.0k, less than about 18.0k, or less than about 20.0k.

[0012] In some embodiments, the half-life extension domain comprises a molecule capable of binding to serum albumin. In some embodiments, the half-life extension domain comprises a polypeptide comprising the amino acid sequence of D-Xaa-CLP-Xaa-WGCLW (SEQ ID NO: 70), QGLIGDICLPRWGCLWGDSVK (SEQ ID NO: 71), RLIEDICLPRWGCLWEDD (SEQ ID NO: 72), or EDICLPRWGCLWED (SEQ ID NO: 73). In this embodiment, Xaa is any amino acid. In some embodiments, the half-life extension domain comprises a fatty acid chain conjugated polypeptide. In this embodiment, the fatty acid chain is selected from a C-16 fatty acid chain or a C-18 fatty acid chain. In some embodiments, the half-life extension domain comprises a C-16 fatty acid conjugated molecule. In some embodiments, the half-life extension domain comprises an antibody fragment, optionally a single domain antibody, a CDR of a single domain antibody, or a single chain variable fragment (scFv) that selectively binds to serum albumin.

[0013] In some embodiments, the half-life extension domain comprises a serum albumin polypeptide. In this embodiment, the serum albumin is human serum albumin. In some embodiments, the half-life extension domain comprises the amino acid sequence of SEQ ID NO: 57.

[0014] In some embodiments, the half-life of the fusion protein is enhanced by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 16-fold, at least about 18-fold, or at least about 20-fold compared to a fusion protein lacking the half-life extension domain. In some embodiments, the half-life extension is based on the mean plasma residence time of the fusion protein.

[0015] In some embodiments, the antigen binding domain comprises more than one TACA binding domain. In some embodiments, the antigen binding domain comprises two, three, four, five, six, seven, eight, nine, or ten TACA binding domains. In this embodiment, the TACA binding domains are operably connected via 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 an unhindered cross-linker.

[0016] 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: GGGGS (SEQ ID NO: 86), GGGGSGGGGS (SEQ ID NO: 87), GGGGSGGGGSGGGGS (SEQ ID NO: 85), AEAAAKA (SEQ ID NO: 88), AEAAAKAAEAAAKA (SEQ ID NO: 89) and AEAAAKAAEAAAKAAEAAAKA (SEQ ID NO: 90). In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 85. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 89.

[0017] 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, wherein the TACA binding domains are from a lectin selected from the group consisting of: galectin, 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); tomatolectin (Lycopersicon esculentum lectin; LEA); peanutlectin (Arachis hypogaea Agglutinin; PNA); potatolectin (Solanum tuberosum lectin); lectin), pokeweed mitogen (Phytolacca Americana lectin), wheat germ agglutinin (Triticum Vulgaris lectin); Artocarpus polyphemus lectin (Jacalin letin); Vicia villosa lectin (VVA); Helix pomatia agglutinin (HPA); Wisteria floribunda agglutinin (WFA);Sambucus nigra agglutinin (SNA), BC2L-CNt (lectin from the Gram-negative bacterium Burkholderia cenocepacia), Maackia amurensis leukolectin (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 sclarea agglutinin, Salvia bogotensis agglutinin, Salvia horminum agglutinin, Clerodendrum trichotomum agglutinin, Moluccella laevis agglutinin, Griffonia simplicifolia (GsLA4), Psophocarpus tetragonolobus (acidic WBAI), Abrus precatorius agglutinin, Amaranthus caudatus agglutinin, Amaranthus leucocarpus agglutinin, Laelia autumnalis agglutinin, Artocarpus integrifolia agglutinin, Maclurapomifera agglutinin, Artocarpus Lakoocha lectin, Dolichos biflorus agglutinin, Dolichos biflorus lectin, Glycinemax soybean lectin, and Agaricus bisporus agglutinin.

[0018] In some embodiments, the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO:33-56. In some embodiments, the antigen binding domain comprises an amino acid sequence having at least 90% homology to SEQ ID NO:33-56.

[0019] In some embodiments, the immune effector cell is 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 cell is a T cell. In one embodiment, the immune effector cell is a NK cell.

[0020] In some embodiments, the immune cell recognition domain comprises: (i) an antibody Fc domain, optionally an Fc domain of an IgG molecule; (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 constant region domains CH2 and / or CH3 of an antibody, preferably CH2 and CH3, optionally with or without a hinge region.

[0021] In some embodiments, the receptor on the immune effector cell is selected from the group consisting of: T cell receptor (TCR) α, TCR β, CD3, TCR γ, TCR δ, the invariant TCR of NKT cells, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA and CEACAM1. In one embodiment, the receptor on the immune effector cell is the T cell receptor selected from the group consisting of CD3, CD2, CD28 and CD25. In another embodiment, the receptor on the immune effector cell is the 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 comprises a scFv that selectively binds to CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1. In one embodiment, the immune cell recognition domain comprises the amino acid sequence of SEQ ID NO: 59, 60, or 61. In one embodiment, the immune cell recognition domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 59, 60, or 61.

[0023] In some embodiments, the encoded 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. In this embodiment, the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 69 or 91-94.

[0024] In some embodiments of the present disclosure, the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-32; or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-32. In some embodiments, the encoded 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 antigen or epitope, α2,6 sialylation, sialylation, sialyl-Lewis x / a , disialo-Lewis x / a , sialyl 6-sulfo Lexis x 、Lewis-y(Le y ), Globo H, GD2, GD3, GM3 and fucosyl GM1. In some embodiments, the encoded fusion protein selectively targets β1,6GlcNAc branched N-glycans, GalNAc, Tn antigen, GalNAcα-ser, GalNAcα-thr, GalNAc or GalNAcβ1.

[0025] In some embodiments, the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 1-12. In some embodiments, the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 13-32. In some embodiments, the encoded fusion protein binds to tumor cells expressing β1,6GlcNAc branched N-glycans compared to bispecific fusion proteins comprising a flexible linker in the antigen binding domain. In some embodiments, the encoded fusion protein binds to tumor cells expressing the Thomsen-nouveau (Tn) antigen compared to fusion proteins 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: 86, SEQ ID NO: 87, or SEQ ID NO: 85; or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 85.

[0026] In some embodiments, the isolated nucleic acid comprises an expression vector; and / or in vitro transcribed RNA.

[0027] Another aspect of the present disclosure provides a fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA).In some embodiments, the fusion protein is encoded by an isolated nucleic acid described herein.

[0028] Another aspect of the present disclosure provides a 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 NOs: 33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NOs: 33-56; (ii) an immune cell recognition domain that specifically binds to a receptor on an immune effector cell; and (iii) a half-life extending domain, wherein the half-life extending domain is a polypeptide that is capable of extending the half-life of the fusion protein.

[0029] In some embodiments, the half-life extension domain is located at the N-terminus or C-terminus of the fusion protein. In some embodiments, the half-life extension domain comprises a molecule selected from the group consisting of a polypeptide capable of binding to albumin, albumin, serum albumin, an Fc domain of an antibody, a polyethylene glycol moiety (PEG), a poly(lactic-co-glycolic acid) (PLGA) copolymer, a polymer hydrogel, a nanoparticle, a fatty acid chain, an acyl group, a myristate group, a palmitoylated group, and a steryl group.

[0030] In some embodiments, the half-life extending domain comprises an Fc domain of an antibody selected from an IgG1, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the half-life extending domain comprises a PEG moiety. In this embodiment, the PEG moiety is less than about 0.5 kJ, less than about 1.0 kJ, less than about 2.0 kJ, less than about 3.0 kJ, less than about 4.0 kJ, less than about 5.0 kJ, less than about 6.0 kJ, less than about 7.0 kJ, less than about 6.0 kJ, less than about 7.0 kJ, less than about 8.0 kJ, less than about 10.0 kJ, less than about 12.0 kJ, less than about 14.0 kJ, less than about 16.0 kJ, less than about 18.0 kJ, or less than about 20.0 kJ.

[0031] In some embodiments, the half-life extension domain comprises a molecule capable of binding to serum albumin. In some embodiments, the half-life extension domain comprises a polypeptide comprising the amino acid sequence of D-Xaa-CLP-Xaa-WGCLW (SEQ ID NO: 70), QGLIGDICLPRWGCLWGDSVK (SEQ ID NO: 71), RLIEDICLPRWGCLWEDD (SEQ ID NO: 72), or EDICLPRWGCLWED (SEQ ID NO: 73). In this embodiment, Xaa is any amino acid. In some embodiments, the half-life extension domain comprises a fatty acid chain conjugated polypeptide, wherein the fatty acid chain is selected from a C-16 fatty acid chain or a C-18 fatty acid chain. In some embodiments, the half-life extension domain comprises a C-16 fatty acid conjugated molecule. In some embodiments, the half-life extension domain comprises an antibody fragment, optionally a single domain antibody, a CDR of a single domain antibody, or a single chain variable fragment (scFv) that selectively binds to serum albumin.

[0032] In some embodiments, the half-life extension domain comprises a serum albumin polypeptide. In this embodiment, the serum albumin is human serum albumin. In some embodiments, the half-life extension domain comprises the amino acid sequence of SEQ ID NO: 57.

[0033] In some embodiments, the half-life of the fusion protein is enhanced by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 16-fold, at least about 18-fold, or at least about 20-fold compared to a fusion protein lacking the half-life extending domain.

[0034] In some embodiments, half-life extension is based on the average plasma retention time of fusion protein. In some embodiments, immune cell recognition domain comprises antibody Fc domain. In one embodiment, immune cell recognition domain comprises the Fc domain of IgG molecule. In some embodiments, immune cell recognition domain comprises peptide, protein, antibody, single domain antibody, antibody fragment or single chain variable fragment (scFv) that selectively binds to receptor on immune effector cell; and / or immune cell recognition domain comprises constant region domain CH2 and / or CH3 of antibody, preferably CH2 and CH3, optionally with or without hinge region.

[0035] In some embodiments, the receptor on the immune effector cell is selected from the group consisting of: T cell receptor (TCR) alpha, TCR beta, CD3, TCR gamma, TCR delta, invariant TCR from NKT cells, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1.

[0036] In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-32; or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-32. In some embodiments, the 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 antigen or epitope, α2,6 sialylation, sialylation, sialyl-Lewis x / a , disialo-Lewis x / a , sialyl 6-sulfo Lexis x 、Lewis-y(Le y ), Globo H, GD2, GD3, GM3 and fucosyl GM1.

[0037] In some embodiments, the fusion protein selectively targets a Tn antigen or a β1,6GlcNAc branched N-glycan. In some embodiments, the fusion protein that selectively targets a Tn antigen comprises an antigen binding domain having an amino acid sequence selected from SEQ ID NOs: 36-42, 52-56, or 62; or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 36-42, 52-56, or 62. In some embodiments, the fusion protein that selectively targets a Tn antigen comprises an amino acid sequence selected from SEQ ID NOs: 13-32; or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 13-32.

[0038] In some embodiments, the 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: 33-35 or 43-51, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 33-35 or 43-51. In some embodiments, the fusion protein that selectively targets β1,6GlcNAc branched N-glycans comprises an amino acid sequence selected from SEQ ID NOs: 1-12, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-12.

[0039] Another aspect of the present disclosure provides a 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 NOs: 33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NOs: 33-56; (ii) an immune cell recognition domain that specifically binds to CD3 on immune effector cells; and (iii) a half-life extending domain. In some embodiments, the half-life extending domain is a polypeptide that is capable of extending the half-life of the fusion protein. In some embodiments, the half-life extension domain comprises the amino acid sequence of human serum albumin or SEQ ID NO:57.

[0040] Another aspect of the present disclosure provides a 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 NOs: 33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NOs: 33-56; and (ii) an Fc domain of an antibody. In some embodiments, the Fc domain is a half-life extending domain. In some embodiments, the Fc domain is an IgG molecule; or the Fc domain comprises amino acids of SEQ ID NOs: 69 or 91-94.

[0041] One aspect of the present disclosure provides an expression construct comprising the isolated nucleic acid disclosed herein. In some embodiments, the expression construct further comprises a promoter. In some embodiments, the expression construct further comprises a promoter selected from: 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, MoMuLV promoter, avian leukemia virus (avianleukemia virus) promoter, Epstein-Barr virus (Epstein-Barr virus) immediate early promoter or Rous sarcoma virus (Rous sarcoma virus) promoter. In some embodiments, the expression construct is a viral vector selected from the group consisting of: retroviral vector, lentiviral vector, adenoviral vector and 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.

[0042] One aspect of the present disclosure provides a kind of cell of modification, it comprises the nucleic acid of separation as herein described, fusion protein or expression construct.In some embodiments, the cell (for example, host cell) of modification is selected from the group consisting of bacterial cell, fungal cell, insect cell or mammalian cell.In some embodiments, the cell of modification is the bacterial cell selected from Escherichia coli (Escherichia coli) or Bacillus stearothermophilus (Bacillus stearothermophilus).In some embodiments, the cell of modification is the fungal cell selected from yeast cell, Saccharomyces cerevisiae (Saccharomyces cerevisiae) or Pichia pastoris (Pichia pastoris).In some embodiments, the cell of modification is the insect cell selected from Lepidopteran (lepidopteran) insect cell or Spodoptera frugiperda (Spodoptera frugiperda).In some embodiments, the cell of modification is selected from following mammalian cell: Chinese hamster ovary (CHO) cell, baby hamster kidney (BHK) cell, monkey kidney cell, HeLa cell (HeLa cell), human hepatocellular carcinoma cell or human embryonic kidney 293 cell. In one embodiment, the modified cells are CHO cells or HEK 293 cells.

[0043] In some embodiments, the modified cells are T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL) or regulatory T cells. In this embodiment, T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL) or regulatory T cells include chimeric antigen receptors (CAR). In some embodiments, T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL) or regulatory T cells include chimeric antigen receptors (CAR) that selectively or specifically bind to tumor antigens. 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, CD11 71, CD276, CEA, claudin 18.2, c-Met, DLL3, DRS, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, 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).

[0044] Another aspect of the present disclosure provides a method for producing a modified cell comprising a fusion protein described herein, the method comprising: (a) introducing an isolated nucleic acid; a fusion protein; or an expression construct described herein into a cell; (b) culturing the cell in a culture medium under conditions that induce expression of the fusion protein, the fusion protein encoded by the nucleic acid, or the fusion protein encoded by the expression construct; and (c) recovering the fusion protein from the cell mass or the culture medium.

[0045] Another aspect of the present disclosure provides a composition comprising: (a) a fusion protein encoded by an isolated nucleic acid as described herein; (b) a fusion protein as described herein; (c) a modified cell as described herein; or (d) a fusion protein encoded by an expression construct as described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. 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, the T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL) or a regulatory T cell comprises a chimeric antigen receptor (CAR) targeting a tumor antigen. In some embodiments, the modified cell is a CART cell. In some embodiments, the modified cell is a CAR T cell specifically targeting 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, CD11 71, CD276, CEA, claudin 18.2, c-Met, DLL3, DRS, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, 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-L1, PSCA, PSMA, RORI, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof.

[0046] Another aspect of the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising administering an immunotherapy composition to the subject, the immunotherapy composition comprising: (a) a fusion protein encoded by an isolated nucleic acid as described herein; (b) a fusion protein as described herein; (c) a modified cell as described herein; or (d) a composition as described herein. 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, the T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), or a regulatory T cell comprises a chimeric antigen receptor (CAR). In one embodiment, 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, the 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 modified cell is a CAR T cell. In some embodiments, the modified cells are CAR T cells that specifically target a tumor antigen.

[0047] 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, CD11 71, CD276, CEA, claudin 18.2, c-Met, DLL3, DRS, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, 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-L1, PSCA, PSMA, RORI, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof. In some embodiments, tumor-associated carbohydrate antigen (TACA).

[0048] 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.

[0049] Another 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 a modified cell comprising a fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), wherein the fusion protein comprises: (i) an antigen binding domain selected from the group consisting of: SEQ ID NO: 33-56; or a fusion protein comprising: any of the amino acid sequences shown in NOs:33-56 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; (ii) an immune cell recognition domain that specifically binds to a receptor on an immune effector cell; and (iii) a half-life extending domain, wherein the half-life extending domain is a polypeptide that is capable of extending the half-life of the fusion protein.

[0050] 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. In some embodiments, the immune cell recognition domain is an antibody Fc domain and a domain that specifically binds to CD3. In some embodiments, the immune cell recognition domain is an antibody Fc domain.

[0051] In some embodiments, the half-life extension domain comprises human serum albumin or the amino acid sequence of SEQ ID NO: 57. In some embodiments, the half-life extension domain comprises the Fc domain of an IgG molecule; or the amino acid sequence of SEQ ID NO: 69 or 91-94. In some embodiments, the fusion protein comprises an amino acid sequence selected from SEQ ID NO: 1-32.

[0052] Another 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 therapeutically effective composition comprising a modified cell comprising a fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), wherein the fusion protein comprises: (i) an antigen binding domain selected from the group consisting of SEQ ID NOs: 33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 33-56; and (ii) an Fc domain of an antibody. In some embodiments, the Fc domain is an IgG molecule. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 69 or 91-94.

[0053] Another aspect of the present disclosure provides a method for providing anti-tumor immunity in a mammal, comprising administering to a mammal a therapeutically effective amount of: (a) a fusion protein encoded by an isolated nucleic acid as described herein; (b) a fusion protein with enhanced serum half-life as described herein; (c) a modified cell population as described herein; or (d) a composition as described herein. In some embodiments, the modified cells are T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs) or regulatory T cells. In this embodiment, T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs) or regulatory T cells comprise 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, CD11 71, CD276, CEA, claudin 18.2, c-Met, DLL3, DRS, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, 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 a tumor-associated carbohydrate antigen (TACA). In some embodiments, the modified cells are CAR T cells. In some embodiments, the modified cells are CAR T cells that specifically target tumor antigens. In some embodiments, the modified cells are CAR T cells that specifically target tumor-associated carbohydrate antigens (TACA). BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1AThe 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.

[0055] Figure 1B GlyTR1 is shown L-PHAxCD3 Size exclusion chromatography (SEC) analysis was performed using a GE Superdex 200 Increase 3.2 / 300 column and a protein standard (Sigma, catalog number MWGF1000-1KT). Molecular weights were calculated based on a trend line generated from retention times. Figure 1C GlyTR1 is shown L-PHAxCD3 and GlyTR1 L-PHAΔ1-5xCD3 Flow cytometric analysis of cell surface binding on Jurkat T cells.

[0056] Figure 1D Figure 2 shows flow cytometric analysis of a co-culture assay in which carboxyfluorescein succinimidyl ester (CFSE)-labeled cancer cells were co-cultured with PBMCs at an E:T ratio 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-labeled cells. + 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

[0057] Figure 2A-2D Improved activity of the GlyTR1 bispecific protein targeting β1,6GlcNAc branched N-glycans and comprising more than one L-PHA carbohydrate binding domain (CRD) is shown when compared to GlyTR1 comprising one L-PHA carbohydrate binding domain (CRD).

[0058] Figure 2A The results show the cleavage of GlyTR1 using a GE HiLoad 16 / 600 Superdex 200 pg column. LPHA(2)xCD3 SEC analysis was performed using protein standard GE (Cat. No. GE28-4038-42). Molecular weights were calculated based on a trend line generated from volume. Figure 2BGlyTR1 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)xCD3 With monomeric GlyTR1 L-PHA(2)xCD3 showed enhanced binding compared to .

[0059] Figure 2C Shown is 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 GlyTR1 L-PHAxCD3 showed enhanced binding compared to .

[0060] Figure 2D Shown is a line graph illustrating flow cytometric analysis of co-culture assays in which CFSE-labeled cancer cells were co-cultured with PBMCs at an E:T ratio of 20:1 for 1 day using the indicated GlyTR1 molecules; and demonstrating that dimeric GlyTR1 L -PHA(2)xCD3 With monomeric GlyTR1 L-PHAxCD3 Compared with the control group, the 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 - .

[0061] Figure 3A-Figure 3I Dimeric 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 ( Figure 3I ) with a ratio of 1:1( Figure 3A )、10:1( Figure 3B or Figure 3H ) and 20:1( Figure 3C-3G 、 Figure 3I ) of E:T co-culture for 1 day ( Figure 3A-3B ) or 3 days ( Figure 3C-Figure 3I ), 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 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.

[0062] Figure 4A-4B Dimeric GlyTR1 is shown L-PHA(2)xCD3 Reduced non-cancer-induced T cell activation. Figure 4A-4B Shown with GlyTR1 L-PHAxCD3 PBMCs rested for 3 days or treated with Kifunensine ( Figure 4A ) or using dimer GlyTR1 L-PHA(2)xCD3 The expression of 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. Kifunine is an inhibitor of class I α-mannosidase, which inhibits glycoprotein processing in the endoplasmic reticulum and Golgi apparatus.

[0063] Figure 5A - Panel F shows GlyTR1 L-PHA(2)xCD3 in vivo activity. Figure 5A-5B GlyTR1 is shown L-PHA(2)xCD3 Tumor regression was induced in NSG mice by intraperitoneal injection of the indicated cancer cell lines on day 0, followed by two or three intraperitoneal injections of 1×10 7 CD8 + T cells were treated (as indicated) with or without twice-daily intraperitoneal injection of GlyTR1 LPHA(2)xCD3 Tumor burden was quantified by luciferase activity (photons / second (p / s)) in Figure 5C and Figure 5D These data demonstrate that GlyTR1 inhibits the expression of PBS in both breast and ovarian cancer models. LPHA(2)xCD3 Induced significant tumor regression. Figure 5E GlyTR1 is shown LPHA(2)xCD3 Accumulation in the lungs of cancer-bearing mice. NSG mice (MDA-MB-231-Fluc) with or without lung metastasis were intravenously injected with or without the fluorophore (Vivo GlyTR1 labeled 680XL (Perkin Elmer LLC) LPHA(2)xCD3 , and the extracted lungs were imaged for luminescence (tumor) and fluorescence (GlyTR1).

[0064] Figure 5F GlyTR1 is shown LPHA(2)xCD3 Not in non-tumor bearing humanized NSG MI / II - / - Induce human T cell activation in PBMC. Humanized NSG-MI / II - / - Mice were injected subcutaneously with GlyTR1 LPHA(2)xCD3 , and 24 hours later, blood T cell activation (CD69 + ).

[0065] Figures 6A-6E Dimeric GlyTR1 is shown L-PHA(2)xCD3 in vivo half-life and distribution. Figure 6B Dimeric GlyTR1 is shown LPHA(2)xCD3 The serum half-life of GlyTR1 is about 2.7 hours. GlyTR1 was injected intravenously into C57BL / 6 mice (n=2). LPHA (2)xCD3 Blood was collected at the indicated times, and serum GlyTR1 was analyzed by sandwich ELISA L-PHA(2)xCD3 . Figure 6A and Figure 6C-6E GlyTR1 is shown L-PHA(2)xCD3 Stable in human plasma for up to 21 hours and showing little loss of intact protein, while labeled dimer GlyTR1 L-PHA(2)xCD3 Shows significant accumulation in the liver ( Figure 6E ). GlyTR1 L-PHA(2)xCD3 Vivo 680XL (Perkin Elmer LLC) was fluorescently labeled and injected intravenously into two C57BL / 6 mice. These mice and one mock-injected mouse were treated with Lumina imager (2s exposure) was used for imaging. Fluorescence in the liver and bladder regions was quantified at each time point and plotted as a percentage of total fluorescence after subtracting the background in mock-injected mice ( Figure 6C ). After 8 hours, the mice were sacrificed, and the designated organs were extracted and imaged ( Figure 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 ( Figure 6E ).

[0066] Figure 7Immunohistochemical staining and staining schematics are shown, demonstrating L-PHA expression in normal human tissues. 'FDA999u' (BioMAx) normal human tissue microarrays containing 32 different tissues with replicate samples from three different individuals were stained with L-PHA-biotin (0.25 μg / 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 higher resolution. 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.

[0067] Figure 8A - Panel B shows immunohistochemical staining ( Figure 8B ) and staining diagram ( Figure 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 brush border of the small intestine, gastric surface epithelial cells, exocrine pancreas (acinar, intracellular), renal cortex (glomeruli, proximal tubules), prostate, and cerebellar molecular layer. However, higher concentrations revealed low but variable staining in the adrenal gland, parotid ducts, thyroid colloid, testis, 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 and without GlyTR1. LPHA(2)xCD3 (0.5ug / ml) for 1h. GlyTR1 was detected 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 gland, 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. Scale bar for higher resolution is 2 mm.

[0068] Figure 9A-9B Immunohistochemical staining is shown ( Figure 9B ) and staining diagram ( Figure 9A ), which demonstrated that GlyTR1 LPHA(2)xCD3 In such 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 (0.67 μg / ml) for 1 hour and detected with nti-HIS-HRP at 1 μg / ml (0.5 hour). AD-adrenal gland, BO-bone marrow, BRN-brain (cerebrum), BRT-breast, CB-cerebellum, 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. Scale bar for higher resolution is 10 μM.

[0069] Figures 10A-10C GlyTR1 is shown LPHA(2)xCD3 Binding to GlyTR1 in normal primary human renal epithelial cells and hepatocytes LPHA(2)xCD3 Quantification of induced cytotoxicity, which was demonstrated with 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 showed a strong LPHA(2)xCD3 Insensitive to T cell-dependent killing. Figures 10A-10C Shown is the GlyTR1 LPHA(2)xCD3 Cell surface binding ( Figure 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-10C Quantification of flow cytometric analysis of 577 cells (E:T=20:1, 2 days). Data are mean ± SEM of three replicate incubations.

[0070] Figure 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 (Pan-omics)) tissue microarrays containing 32 and 22 normal tissues, respectively, were stained with L-PHA-biotin (0.5 ug / 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.

[0071] Figure 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), renal function (BUN, creatinine), electrolytes, glucose, pancreatic function (amylase, precision PSL), thyroid function (total T4, TSH), cholesterol, or muscle (CPK) (panels c-v), blood levels of hemoglobin, RBC, hematocrit, WBC, WBC differential, or platelets relative to controls, CD4 + or CD8 + T cell activation markers CD69 or CD25, 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 Human PBMC (Fig. a) Starting from day 16, mice were subcutaneously injected with PBS (n=6) or 10 μg 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 developed 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 derived by Mann-Whitney test (two-tailed).

[0072] Figure 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 significant 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), 2.5 μg (n=3), 5 μg (n=3), or 10 μg (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 using a two-way analysis of variance (2-way ANOVA) and Bonferroni post-test correction to derive 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 (Figures b-k); each symbol represents one mouse. A complete blood count was performed on the blood, with each symbol representing one mouse (Figures l-p). For clinical biochemistry, blood was equally pooled from three mice in the same treatment group to ensure sufficient amount for analysis (Figures q-h); each symbol represents three mice. Plasma from each mouse was used for human IFNγ and IL-6 ELISA, with each symbol representing one mouse (Figures I-J). Multiple comparisons were performed using the Kruskal-Wallace test and Dunn's post-test correction to derive P values.

[0073] Figures 14A-14D Shown is 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. Figure 14A Schematic diagrams 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. Figures 14B-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.

[0074] Figures 15A-15D Improved activity of the GlyTR2 bispecific protein for targeting low-density Tn antigen is shown. Figure 15A A chromatogram is shown, which illustrates that GlyTR2 CD301(3)xCD3 Using a GE HiLoad 16 / 600 Superdex 200pg column, GlyTR2 CD301(3)xCD3 and GlyTR2s lCD301(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-15C Shown is a comparison of T cell leukemia TCRβ - / - Jurkat and multiple myeloma MM.1R cells ( Figure 15B ) on GlyTR2 CD301(3)xCD3 and GlyTR2sl CD301(4)xCD3 Quantification of cell surface binding by flow cytometric analysis and other indicated cancer types using established cell lines is shown for TCRβ - / - The relative binding of Jurkat T cells ( Figure 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 two repeats containing a rigid linker (SL2) was used. slCD301(4)xCD3 . Figure 15D Shown are the GlyTR2 in the absence or presence of GalNac or GlcNAc. slCD301(4)xCD3 Quantification of surface binding to MM.1R cells by flow cytometry. Data are means ± SEM of triplicate incubations.

[0075] Figures 16A-16I GlyTR2 is shown slCD301(4)xCD3 Quantification of T cell-dependent cancer killing. Figures 16A-16I In the experiment, carboxyfluorescein succinimidyl ester (CFSE)-labeled cancer cells (as indicated) were incubated with or without CD8 ( Figure 16A 、 Figure 16I ), T cells ( Figure 16B ) or PBMC( Figures 16C-16H ) with 10:1( Figure 16A 、 Figure 16H 、 Figure 16I ) and 20:1( Figures 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 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.

[0076] Figures 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.

[0077] Figures 18A-18E GlyTR2 is shown slCD301(4)xCD3 In vivo activity in solid cancers. Figures 18A-18B GlyTR2 is shown slCD301(4)xCD3 In vivo tumor regression was induced in NSG mice that were 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 cells every 3-4 days 6 CD8 + T cells (ovarian cancer) (as indicated), with and 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 the expression of PBS in breast and ovarian cancer models. slCD301(4)xCD3 Induced significant tumor regression. Figure 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. To the presence or absence of lung metastasis (MDA-MB-231-Luc + MI - / - C - / - ) of NSG mice intravenously injected with or without fluorophore (Vivo 680XL) labeled GlyTR2 slCD301(4)xCD3, and the extracted lungs were imaged for luminescence (tumor) and fluorescence (GlyTR2).

[0078] Figures 19A-19C GlyTR is shown CD301(3)xCD3 Half-life and stability in human plasma. Figure 19A It is shown that when GlyTR CD301(3)xCD3 When GlyTR2 (100 μg) 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 is shown CD301(3)xCD3 Stability in human plasma at 37°C for 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 ( Figure 19B ) or by Western blotting using anti-HIS ( Figure 19C ).

[0079] 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-Figure 20B Fluorescently labeled GlyTR2 is shown slCD301xCD3 Localization of GlyTR 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, the mice were sacrificed and their organs were harvested and imaged. Figure 20C Shown Figure 20A Fluorescence was quantified for each organ in the WT and plotted as a percentage of the total fluorescence of all imaged organs after background subtraction from the injected vector. Figure 20C Shown are the normal human liver cancer and breast cancer (positive control) and GlyTR2 in formalin-fixed paraffin-embedded tissues. slCD301(4)xCD3 Co-stained images, followed by detection with anti-HIS-HRP antibody and DAB, demonstrated that GlyTR2 slCD301(4)xCD Does not significantly bind to human or mouse hepatocytes. Figure 20D It is shown that when combined with Jurkat TCRβ - / - When leukemia cells and MM.1R multiple myeloma cells were compared, GlyTR2 slCD301(4)xCD Does not significantly bind 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 ( Figure 20E , E:T=20:1, 2 days) or PBMC ( Figure 20F , E:T=10:1, 1 day) induced GlyTR2 slCD301(4)xCD3 Jurkat TCRβ - / - leukemia( Figure 20E 、 Figure 20F ), MM.1R multiple myeloma ( Figure 20E ), normal human hepatocytes, or normal lymphocytes (20F) were quantified by flow cytometry analysis. Data are the mean ± SEM of three incubations.

[0080] Figure 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 + White blood cell count 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, indicating no T cell activation. (Figure a) MHC class I and class II deficiency (NSG-MI / II - ) were transplanted intravenously (tail vein) with 2x10 7 Human PBMCs. Starting from day 16, mice were subcutaneously injected with PBS (n=6) or 100 μg 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 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) (Figure 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 ​​obtained by Mann-Whitney test (two-tailed).

[0081] Figures 22A-22E It is shown that when compared with the parent GlyTR slCD301(4)xCD3 In comparison, GlyTR slCD301(4)xCD3 Human serum albumin (HSA) was added to GlyTR slCD301(4)xCD3 The half-life was extended fivefold. Figures 22A-22B It was shown that when compared to the parent, HSA-GlyTR slCD301(4)xCD3 Human serum albumin (HSA) was synthesized by gene synthesis and GlyTR CD301(4)xCD3 N-terminal genetic fusion (HSA-GlyTR slCD301(4)xCD3) , by suspending ExpiCHO TM The HSA-GlyTR was produced by transient transfection of cells and purified by metal ion affinity chromatography and size exclusion chromatography. slCD301(4)xCD3 With the parent GlyTR slCD301(4)xCD3 cell surface binding. Figure 22C-Figure 22D It is shown that compared with the parent GlyTR slCD301(4)xCD3 , HSA-GlyTR slCD301(4)xCD3 Breast cancer cells labeled with carboxyfluorescein succinimidyl ester (CFSE) were stained with or without CD8 + T cells were co-cultured and then analyzed for cancer cell death by flow cytometry ( Figure 22C ) and T cell activation ( Figure 22D ) and quantified. % cell death was calculated as in Figure 5. Data are mean ± SEM of three replicate incubations. Figure 22E Shown two hours after intravenous injection, HSA-GlyTR slCD301(4)xCD3 The plasma concentration of parent GlyTR slCD301(4)xCD3This is approximately 5-fold higher, indicating an enhanced half-life. slCD301(4)xCD3 and parental GlyTR slCD301(4)xCD3 The cells were injected intravenously into C57BL / 6 mice (n=2) and the plasma was analyzed for both proteins by sandwich ELISA at 2 hours. Data are mean ± SEM. DETAILED DESCRIPTION

[0082] I. Overview

[0083] In international application number PCT / US2023 / 024898 (titled “Improved Glycan-Dependent Immunotherapeutic Bi-Specific Fusion Proteins and Chimeric Antigen Receptors”), filed concurrently, a new class of immunotherapeutic bispecific fusion proteins and CARs were developed to effectively target TACAs for immunotherapy. Specifically, bispecific fusion proteins and CARs were engineered to target TACAs using antigen binding domains derived from lectins rather than monoclonal antibodies or fragments thereof, regardless of carrier protein. This new technology is called “glycan-dependent T cell recruitment” or GlyTR (pronounced ‘glitter’). One group of GlyTR therapeutics are TACA bispecific fusion proteins that comprise a carbohydrate recognition domain (e.g., a TACA binding domain) from a lectin operably linked, conjugated, or fused to an immune cell recognition domain that specifically binds to a receptor on immune effector cells. Another group of GlyTR therapeutics are chimeric antigen receptors that comprise 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 one or more TACA binding domains derived from a lectin.

[0084] The GlyTR disclosed in this application represents a major improvement in immunotherapy because GlyTR can target antigens present in a variety of common cancers. This is because altered glycosylation that generates TACAs is a common feature of cancer. TACAs provide the most abundant and widespread cell surface cancer antigens known, with a target density that is up to about 100-1000 times higher than typical protein antigens. In addition, the TACA target density is about 100-1000 times higher than typical protein antigens. GLyTR also has high-affinity binding, which is achieved by a combination of high-density targeted expression on tumor cells and the presence of multiple carbohydrate binding domains of the engineered GlyTR. This combination of high target density and multiple binding sites enhances the specificity of GlyTR for high TACA expressing cells (e.g., cancer cells) relative to low expressing cells (e.g., normal cells).

[0085] However, compared with many useful therapeutic approaches and traditional immunotherapies (e.g., bispecific T cell engagers Tandem diabodies (TandAbs) or dual-affinity retargeting proteins Similarly, GlyTR is rapidly eliminated from the body when administered to a subject. Therefore, there is a need for TACA-specific bispecific fusion proteins with enhanced serum half-lives to allow for increased therapeutic potential, such as low-dose drug formulations, reduced regular administration, and / or novel pharmaceutical compositions.

[0086] In the present disclosure, a new type of immunotherapeutic trispecific fusion protein with enhanced serum half-life has been developed to effectively target TACA for immunotherapy. The new TACA trispecific fusion protein comprises a first domain, which is an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA); a second domain, which is an immune cell recognition domain that selectively binds to a receptor (e.g., CD3) in an immune cell; and a third domain, which is a half-life extension domain. The half-life extension domain is a polypeptide that can extend the half-life of the fusion protein. The three domains can be recombined in different ways as described below. For example, the half-life extension domain may be located at the N-terminus, middle, or C-terminus of the trispecific fusion protein.

[0087] A. Summary of Experimental Results

[0088] The novel trispecific fusion proteins disclosed herein have serum half-lives that are at least five-fold greater than those of the parent bispecific fusion proteins, while maintaining substantially similar binding, killing, and T cell activation to TACA-positive cancer cells relative to the parent bispecific fusion proteins. Figure 6B As shown, the dimer GlyTR1 LPHA(2)xCD3 The serum half-life is approximately 2.7 hours ( Figure 6B ). GlyTR1 LPHA(2)xCD3It accumulates primarily in the liver and to much lesser extent in the spleen and kidneys ( Figure 6A , Figure 6C-6E ). GlyTR2 CD301 (3)xCD3 The serum half-life of GlyTR1 LPHA(2)xCD3 The serum half-life of Figure 19A ). GlyTR2 slCD301(4)xCD3 It also accumulates in the liver and, to a lesser extent, in the kidneys, spleen, lungs, and intestines ( Figure 20A 、 Figure 20B However, hepatic accumulation does not indicate binding to GalNAc-containing glycans on hepatocytes because GlyTR2 slCD301(4)xCD3 No significant binding to human or mouse hepatocytes ( Figure 20C 、 Figure 20D , and data not shown). These observations suggest that GlyTRs molecules are rapidly cleared and degraded by the liver, which may reduce the therapeutic effectiveness of GlyTR molecules. The observed half-life of GlyTR fusion proteins is comparable to that of FDA-approved therapeutics. The observed half-life of dapoxetine (blinatumomab) was similar. A protein that binds CD3 and CD19 Immunotherapy, and due to its short half-life, it requires continuous intravenous infusion in two 28-day treatment regimens. In order to avoid this cumbersome regimen and extend the half-life of GlyTR, the present inventors have generated a novel and improved GlyTR, which further comprises a half-life extension domain capable of extending the half-life of the fusion protein. Figure 22E As shown, containing GlyTR slCD301(4)xCD3 The half-life of the trispecific fusion protein of GlyTR and human serum albumin (HSA) domains was significantly longer than that of the parent GlyTR after two hours of intravenous injection. slCD301(4)xCD3 In addition, compared with the parent GlyTR slCD301(4)xCD3 In comparison, the novel trispecific fusion protein HSA-GlyTR slCD301(4)xCD3 Shows similar binding and killing to Tn antigen-positive cancer cells ( Figures 22A-22C Despite the presence of additional domains, the novel trispecific fusion protein HSA-GlyTR slCD301(4)xCD3 Compared with the parent GlyTR slCD301(4)xCD3 Compared with the control group, the T cell activation levels were similar ( Figure 22D ).

[0089] B. Exemplary Benefits of GlyTR Fusion Proteins

[0090] GlyTR tri-specific fusion proteins as described herein are designed to specifically and selectively target cells expressing any TACA by raising cytotoxic T cells. By combining with receptors (e.g., CD3) on immune effector cells, GlyTR tri-specific fusion proteins can cross-link cytotoxic T cells with cells expressing TACA in a highly specific manner, thereby guiding the cytotoxic potential of T cells to target cells. GlyTR tri-specific fusion proteins as described herein are combined with cytotoxic T cells via combining with surface-expressed immune receptors (e.g., CD3 proteins forming a part for TCR). Several GlyTR tri-specific fusion proteins are combined with CD3 and with the TACA expressed on the surface of specific cells simultaneously, causing T cell activation and mediating the subsequent cracking of specific TACA expressing cells. Therefore, GlyTR tri-specific fusion proteins are considered to demonstrate strong, specific and effective target cell killing.

[0091] An additional advantage of the GlyTR trispecific fusion proteins described herein is their extended half-life compared to traditional monoclonal antibodies and other smaller bispecific molecules. In general, the effectiveness of recombinant protein drugs depends largely on the intrinsic pharmacokinetics of the protein itself. Here, the main benefit of the GlyTR trispecific fusion proteins described herein is that they have an extended pharmacokinetic elimination half-life by virtue of the half-life extension domain (e.g., a domain specific for HSA).

[0092] Fusion proteins lacking a half-life extension domain (e.g., GlyTR bispecific fusion proteins, or other immunotherapeutic molecules known in the art, such as bispecific T cell engagers) Tandem diabodies (TandAbs) or dual-affinity retargeting proteins The serum elimination half-life of the GlyTR tri-specific fusion proteins described herein is extended by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 16-fold, at least about 18-fold, or at least about 20-fold compared to a GlyTR tri-specific fusion protein (a GlyTR tri-specific fusion protein). or Molecules with relatively short elimination half-lives (e.g. It is rapidly cleared from the circulation with a short elimination half-life of 1.25 h ± 0.63 h (mean ± SD). For these reasons, for example, CD19×CD3 bispecific fusion molecules need to be administered continuously at high concentrations (15μg-28μg / day) to recruit and activate a large number of suboptimal T cells to achieve half-maximal target cell lysis. CD19×CD3 was administered as a continuous intravenous infusion (iv) for 4 weeks to maintain adequate therapeutic serum concentrations.

[0093] Thus, the longer intrinsic half-life of the GlyTR tri-specific fusion proteins described herein addresses the short half-life problem, allowing for increased therapeutic potential, such as, for example, lower-dose drug formulations, reduced regular administration, and / or novel pharmaceutical compositions.

[0094] In one aspect, the present disclosure provides an isolated nucleic acid molecule encoding a fusion protein (e.g., a tri-specific fusion protein), the fusion protein comprising an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA); an immune cell recognition domain that specifically binds to a receptor on an immune effector cell; and a half-life extension domain. In some embodiments, the half-life extension domain is a polypeptide that can extend the half-life of the fusion protein. In some embodiments, the half-life extension domain is located at the N-terminus, middle, or C-terminus of the fusion protein.

[0095] In another aspect, the present disclosure provides a fusion protein (e.g., a trispecific fusion protein) that selectively binds to a tumor-associated carbohydrate antigen (TACA) and is encoded by an isolated nucleic acid molecule disclosed herein. The antigen binding domain of the fusion protein disclosed herein is selected from the group consisting of: SEQ ID NO: 33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 33-56; an immune cell recognition domain that specifically binds to a receptor on an immune effector cell; and a half-life extension domain. The half-life extension domain is a polypeptide capable of extending the half-life of the fusion protein (e.g., a polypeptide capable of binding to albumin, albumin, serum albumin, the Fc domain of an antibody, a polyethylene glycol moiety (PEG), a poly(lactic-co-glycolic acid) (PLGA) copolymer, a polymer hydrogel, a nanoparticle, a fatty acid chain, an acyl group, a myristate group, a palmitoylated group, and a steryl group). In some embodiments, the half-life extension domain comprises human serum albumin or the amino acid sequence of SEQ ID NO: 110.

[0096] In one aspect, the present disclosure provides a modified cell comprising an isolated nucleic acid molecule disclosed herein, a fusion protein disclosed herein. For example, the cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell. In another aspect, the present disclosure provides an expression construct comprising an isolated nucleic acid and / or promoter disclosed herein. A composition is also provided, comprising an isolated nucleic acid disclosed herein; a fusion protein; a modified cell; or an expression construct. In one aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, comprising administering an immunotherapeutic composition disclosed herein to the subject.

[0097] II. Definitions

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. 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 particular embodiments only and is not intended to be limiting.

[0099] 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 ed.; 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 ed.; Gait, ed. (1984) Oligonucleotide Synthesis; edited by Hames and Higgins (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic AcidHybridization; 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; ImmobilizedCells and Enzymes (IRL Press (1986));Grandi, ed. (2007) In Vitro Transcription and Translation Protocols, 2nd ed.; Guisan, ed. (2006) Immobilization of Enzymes and Cells; Perbal, ed. (1988) A Practical Guide to Molecular Cloning, 2nd ed.; 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 ed.; and Herzenberg et al., ed. (1996) Weir's Handbook of Experimental Immunology, 5th ed.;

[0100] 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.

[0101] 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.

[0102] As used herein, the term "activated" refers to a state in which T cells have 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 cells" refers, among other things, to T cells that are undergoing cell division.

[0103] As used herein, the term "affinity" refers to the strength of the interaction between the bispecific fusion proteins described herein and TACA at a single antigenic site. Within each antigenic site, the TACA binding domain derived from the lectin interacts with the antigen at multiple sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0104] As used herein, the term "avidity" refers to an informative measure of the overall stability or strength of a bispecific fusion protein-TACA antigen complex. It is governed by three primary factors: the affinity of the lectin-derived TACA binding domain; the valency of both the TACA and TACA binding domain; and the structural arrangement of the interacting moieties. Ultimately, these factors define the specificity of the bispecific fusion protein, i.e., the likelihood that a particular bispecific fusion protein will bind to a precise antigenic epitope.

[0105] 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 the 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.

[0106] 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.

[0107] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may involve the production of other antibodies or the activation of specific immunocompetent cells, or both. It will be understood by those skilled in the art 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 comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes the term "antigen" as used herein. In addition, it will be understood by those skilled in the art that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. In addition, it will be understood by those skilled in the art that an antigen need not be encoded by a "gene" at all. It will be apparent that an antigen can be generated, synthesized, or can be derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0108] As used herein, the term "allogeneic" refers to a transplant derived from a different animal of the same species.

[0109] 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 they may be immunoreactive portions of complete immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The 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 and heavy chains, with or without interchain covalent bonds between the light and heavy chains. The basic immunoglobulin structure in vertebrate systems is relatively well understood.

[0110] As used herein, 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.

[0111] 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.

[0112] 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.

[0113] 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).

[0114] As used herein, the term "cancer" is defined as a disease characterized by the rapid, 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, and the like.

[0115] As used herein, the terms "cancer associated antigen" or "tumor antigen" are used interchangeably to refer to molecules (typically proteins, carbohydrates, or lipids) expressed in their entirety on the surface of cancer cells or as fragments (e.g., MHC / peptides), 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 CAR comprising an antigen binding domain (e.g., an antibody or antibody fragment) bound to an MHC presenting peptide. Under normal circumstances, peptides derived from endogenous proteins fill the pockets of major histocompatibility complex (MHC) class I molecules and are recognized by the T cell receptor (TCR) 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 class of unique 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).

[0116] 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, CD11 71, CD276, CEA, claudin 18.2, c-Met, DLL3, DRS, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, 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-L1, PSCA, PSMA, RORI, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof.

[0117] 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 the patient and modifying the 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.

[0118] 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 any limitation on the process or source of the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta 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 any limitation on a particular process for producing the intracellular signaling domain, nor does it mean that in order to provide the intracellular signaling domain, one must start with the CD3 zeta sequence and delete unwanted sequences or apply mutations to arrive at the intracellular signaling domain.

[0119] As used herein, "disease" refers to a state of health in an animal in which the animal is unable to maintain homeostasis and in which the animal's health continues to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is not as good as it would be in the absence of the disorder. If left untreated, a disorder does not necessarily lead to a further deterioration in the animal's health.

[0120] 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 malignant tumors; 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, malignant tumors, 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 at any time mRNA encoding a tumor antigen. 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 levels or reduced levels. In some embodiments, a cell expressing a tumor antigen produces detectable levels of the tumor antigen protein at one time point and subsequently produces substantially no detectable tumor antigen protein.

[0121] As used herein, the term "downregulate" refers to the reduction or elimination of gene expression of one or more genes.

[0122] As used herein, an "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 outcome in a subject in need thereof. Such an outcome may include, but is 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. Immune response can be readily assessed by a variety of art-recognized methods. One of skill 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 condition 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 being treated, the taxonomic group of the subject being treated, the formulation of the composition, an assessment of the subject's medical condition, and other relevant factors.

[0123] As used herein, the term "encoding" 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, as well as the biological properties resulting therefrom. Thus, if transcription and translation of the mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene encodes a protein. The coding strand (its 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.

[0124] As used herein, "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0125] 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 approximately 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 primary criterion for antigen specificity and therefore distinguishes one epitope from another. Based on the present disclosure, the peptides used in the present disclosure can be epitopes. 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.

[0126] 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.

[0127] As used herein, the term "exogenous" refers to any substance that is introduced from outside of an organism, cell, tissue, or system or that is produced outside of an organism, cell, tissue, or system.

[0128] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0129] 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. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0130] 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).

[0131] As used herein, the term "Fc portion" or "Fc monomer" in the present disclosure refers to 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. As is apparent from the term "Fc monomer," 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 retaining at least one functional portion of a CH2 domain and one functional portion of a 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 a CH3 region, wherein the hinge region is located at the amino terminus of the CH2 domain.

[0132] It is envisioned that the hinge region of the present disclosure promotes dimerization. Such Fc polypeptide molecules can be obtained by, for example, but not limited to, papain digestion of an immunoglobulin region (which will of course produce a dimer of two Fc polypeptides). 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, for example, but not limited to, pepsin digestion of an immunoglobulin molecule. In one embodiment, the polypeptide sequence of the 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, for example, 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.

[0133] The Fc monomer may also include a flexible hinge located 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 the 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 can be defined as comprising the amino acid sequence of SEQ ID NOs 63-68.

[0134] 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 the IgG1 sequence stretch from D234 to P243 according to Kabat numbering. In some embodiments, the hinge domain / region of the present disclosure comprises or consists of the IgG1 hinge sequence DKTHTCPPCP (SEQ ID NO: 63 or 64).

[0135] In one embodiment, the IgG1 hinge domain / region comprises the amino acid sequence of EPKSCDKTHTCPPCP (SEQ ID NO: 64).

[0136] In a further embodiment of the present disclosure, the hinge domain / region comprises or consists of the following: IgG2 subtype hinge sequence ERKCCVECPPCP (SEQ ID NO: 65), IgG3 subtype hinge sequence ELKTPLDTTHTCPRCP (SEQ ID NO: 66) or ELKTPLGDTTHTCPRCP (SEQ ID NO: 67) and / or IgG4 subtype hinge sequence ESKYGPPCPSCP (SEQ ID NO: 68). In a further embodiment of the present disclosure, the IgG hinge domain / region comprises or consists of the hinge amino acid sequence disclosed in Table 2 or Table 3.

[0137] In some embodiments, the fusion protein further comprises a third domain comprising two polypeptide monomers, each of which comprises a hinge, a CH2 domain, and a CH3 domain. In one embodiment, the third domain comprises, in amino to carboxyl order: hinge-CH2-CH3-linker-hinge-CH2-CH3. In one embodiment, the CH2 domain comprises an intradomain cysteine ​​disulfide bridge.

[0138] In another embodiment, 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 includes GGGGS (e.g., Gly4Ser (SEQ ID NO:86)) amino acid sequence or its polymer (e.g., (Gly4Ser) n, wherein n is 5 or larger integer (e.g., 5,6,7,8 etc. or larger)). In some embodiments, the peptide linker of the fusion protein of the present disclosure includes SEQ ID NO:74-90 amino acid sequence.

[0139] As used herein, the term "half-life extending molecule" means a biological or chemical entity that confers additional functionality to the molecule to which it is attached. In certain embodiments, the half-life extender is a polypeptide, e.g., human serum albumin (HSA), or a chemical entity, e.g., polyethylene glycol (PEG), that increases the half-life of the bispecific fusion protein disclosed herein. The half-life extending molecule can increase the half-life of the bispecific fusion protein by at least about 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 25 hours, at least 30 hours, at least 35 hours, at least 40 hours, or longer, as determined by an assay disclosed herein.

[0140] 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 percentage homology 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 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, a comparison is made when two sequences are aligned to provide maximum homology.

[0141] 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 position in each of two polypeptide molecules is occupied by arginine, the two molecules are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment 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 (e.g., five positions in a polymer of ten amino acids in length) of the positions in the two sequences 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.

[0142] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that act as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. This class of proteins includes 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 act 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.

[0143] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes recognize 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.

[0144] 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.

[0145] 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-derived phagocytes.

[0146] 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.

[0147] 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, for example, be affixed to a container containing the nucleic acids, peptides, and / or compositions of the present disclosure, or can be shipped together with the container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructional materials can be shipped separately from the container so that the recipient can use the instructional materials and the compounds together.

[0148] As used herein, the term "isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally occurring 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 can exist in a substantially purified form or can exist in a non-natural environment, such as, for example, a host cell.

[0149] In the context of this 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.

[0150] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that 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.

[0151] As used herein, the term "lectin" or "hemagglutinin" refers to a protein or peptide that binds to a carbohydrate structure. Those skilled in the art will understand that a lectin is a protein or peptide that is highly specific for binding to a carbohydrate moiety. Lectins are carbohydrate-binding proteins that are highly specific for carbohydrates found on proteins and / or lipids, thereby causing the aggregation of specific cells or the 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, a "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 a carbohydrate binding domain that have the ability to reversibly bind to specific sugar moieties in glycoproteins or glycolipids, as well as free monosaccharide and polysaccharide structures. Although many organisms express lectins or lectin-like biomolecules, most of the recently identified lectins of scientific interest have been purified from plant sources.

[0152] As used herein, the term "tumor-associated carbohydrate antigens" or "TACAs" 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.

[0153] 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 antigen) 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, increased N-glycoprotein branching (which stimulates galectin-3) and changes in glycolipids (such as, for example, gangliosides (GM3, GM2, CD3, and GD2)) are observed.

[0154] 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, lung, breast, and many other cancers; (iv) GM2, GD2, and GD3 gangliosides in neuroectodermal tumors (melanoma and neuroblastoma); and (v) globo-H in breast, ovarian, and prostate cancers; and (vi) disialylgalactosylgloboside in renal cell carcinoma.

[0155] 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, promoting cancer metastasis, cancer progression, cancer and non-cancer immunosuppression, and / or promoting autoimmune diseases. A skilled person will understand that a carbohydrate structure is composed of one or more linked sugars or monosaccharides. See, for example, Mantuano et al., J. Immunotherapy Cancer 8(2):8:e001222(2020); Hakomori, Si. (2001). Tumor-Associated Carbohydrate Antigens Defining Tumor Malignancy:Basis for Development of Anti-Cancer Vaccines, in The Molecular Immunology of Complex Carbohydrates—2. Advances in Experimental Medicine and Biology, Vol. 491. Springer, Boston, MA (Wu et al. (eds.)). A skilled person will understand that a carbohydrate structure can be independent and / or attached to a protein or lipid, referred to as a glycoprotein and glycolipid. A skilled person will understand that these carbohydrate structures bind to lectins.

[0156] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver large amounts of genetic information into the host cell's DNA, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means of achieving significant levels of gene transfer in vivo.

[0157] 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.

[0158] 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 the amino acid sequence (Gly-Gly-Gly-Gly-Ser)n, where 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:80-87). In one embodiment, the flexible polypeptide linker includes but is not limited to (Gly4Ser)4 or (Gly4Ser)3. In another embodiment, the linker includes multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser).

[0159] As used herein, the term "modified" refers to an altered state or structure of a molecule or cell of the present disclosure. A molecule can be modified in a variety of ways, including chemically, structurally, and functionally. A cell can be modified by the introduction of a nucleic acid.

[0160] As used herein, the term "modulate" means mediating a detectable increase or decrease in the level of a subject's response compared to the subject's response in the absence of the treatment or compound and / or compared to the response of an otherwise identical but untreated subject. The term encompasses perturbing and / or influencing a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0161] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may contain introns.

[0162] As used herein, the term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that results in 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. Generally, operably linked DNA sequences are contiguous and, when necessary to link two protein coding regions, are in the same reading frame.

[0163] 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 a tumor antigen can be determined by standard assays known in the art.

[0164] 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.

[0165] 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 elicit an immune response can be a subject or patient. In certain exemplary embodiments, the subject is a human.

[0166] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, nucleic acid and polynucleotide 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 conventional cloning techniques and PCR. TM etc. by cloning nucleic acid sequences from recombinant libraries or cell genomes, as well as by synthesis.

[0167] As used herein, the terms "peptide" and "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that can constitute a protein sequence or 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 terms refer 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, and the like. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0168] As used herein, the term "promoter" is defined as a DNA sequence recognized by the cellular synthetic machinery, or introduced synthetic machinery, required to initiate specific transcription of a polynucleotide sequence.

[0169] As used herein, the term "promoter" or "regulatory sequence" refers to a nucleic acid sequence required for 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 expression of the gene product. The promoter / regulatory sequence may be, for example, a sequence that expresses a gene product in a tissue-specific manner.

[0170] 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.

[0171] As used herein, an "inducible promoter" is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell essentially only when an inducer corresponding to the promoter is present in the cell.

[0172] 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.

[0173] As used herein, the term "specific binding" or "selectively binding" for antibodies, antigen-binding domains, CARs or bispecific fusion proteins refers to antibodies, antigen-binding domains, CARs or bispecific fusion proteins that recognize specific antigens (e.g., TACAs), but does not substantially recognize or bind to other molecules in the sample. For example, antibodies, antigen-binding domains, CARs or bispecific fusion proteins that specifically bind to antigens (e.g., TACAs) from one species can also be combined with antigens from one or more species. However, this cross-species reactivity itself does not change the specific classification of the antibody. In another example, antibodies, antigen-binding domains, CARs or bispecific fusion proteins that specifically bind to antigens (e.g., TACAs) can also be combined with different allele forms of antigens (e.g., TACAs). 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.

[0174] 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. Various 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).

[0175] As used herein, the term "specificity" refers to the ability to specifically bind (e.g., immunoreact) 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.

[0176] As used herein, the term "specific binding" or "selective 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 the 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 allelic forms of the antigen. However, this cross-reactivity itself does not change the specific classification of the antibody.

[0177] As used herein, the term "specific binding" or "specifically binds" 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 is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, a chimeric antigen receptor recognizes and binds to a specific protein structure rather than proteins in general. If the antibody is specific for epitope "A," then in a reaction containing labeled "A" and the antibody, the presence of molecules containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody. In some embodiments, the term "specific binding" between an antibody, protein, chimeric antigen receptor or peptide and a second chemical species means that the binding is at least about 10 2 M -1 , at least about 5x10 2 M -1 , at least about 10 3 M -1 , at least about 5x10 3 M -1 , at least about 10 4 M -1 , at least about 5x10 4 M -1 , at least about 10 5 M -1 , at least about 5x10 5 M -1 , at least about 10 6 M-1 , at least about 5x10 6 M -1 , at least about 10 7 M -1 , at least about 5x10 7 M -1 , at least about 10 8 M -1 , at least about 5x10 8 M -1 , at least about 10 9 M -1 , at least about 5x10 9 M -1 , at least about 10 10 M -1 , at least about 5x10 10 M -1 , at least about 10 11 M -1 , at least about 5x10 11 M -1 , at least about 10 12 M -1 , at least about 5x10 12 M -1 , at least about 10 13 M -1 , at least about 5x10 13 M -1 , at least 10 14 M -1 , at least about 5x10 14 M -1 , at least about 10 15 M -1 Or at least about 5x10 15 M -1 The equilibrium constant (K A )(k on / k off )combined.

[0178] In some embodiments, the term "specifically binds" or "selectively binds" refers to a binding reaction between a TACA bispecific fusion protein and its cognate antigen (e.g., TACA on a tumor cell) that determines the presence of the cognate antigen in a heterogeneous population of proteins and other biologics.

[0179] In addition to the above equilibrium constant (K A ), the TACA bispecific fusion proteins disclosed herein may typically have a kinase activity of less than 5×10 -2 M, less than 10 -2 M, less than 5×10 -3 M, less than 10 -3M, less than 5×10 -4 M, less than 10 -4 M, less than 5×10 -5 M, less than 10 -5 M, less than 5×10 -6 M, less than 10 -6 M, less than 5×10 -7 M, less than 10 -7 M, less than 5×10 -8 M, less than 10 -8 M, less than 5×10 -9 M, less than 10 -9 M, less than 5×10 -10 M, less than 10 -10 M, less than 5×10 -11 M, less than 10 -11 M, less than 5×10 -12 M, less than 10 -12 M, less than 5×10 -13 M, less than 10 -13 M, less than 5×10 -14 M, less than 10 -14 M, less than 5×10 -15 M or less than 10 -15 M or smaller dissociation rate constant (K D ), and the affinity of binding to TACA is at least two-fold higher than the affinity of binding to a nonspecific antigen. In one embodiment, the TACA bispecific fusion protein disclosed herein has a dissociation constant (K) of less than 3000 pM, less than 2500 pM, less than 2000 pM, less than 1500 pM, less than 1000 pM, less than 750 pM, less than 500 pM, less than 250 pM, less than 200 pM, less than 150 pM, less than 100 pM, less than 75 pM, less than 10 pM, less than 1 pM. d ), as assessed using methods described herein or known to those skilled in the art (e.g., BIAcore assay, ELISA, FACS, SET) (Biacore International AB, Uppsala, Sweden).

[0180] As used herein, the term "K Assoc ” or “K A " refers to the association rate of a particular bispecific fusion protein-TACA interaction. As used herein, the term "K dis ” or “K d " refers to the dissociation rate of a particular bispecific fusion protein-TACA interaction. As used herein, the term "K D ” refers to the dissociation constant, which is given by Kd With K a The ratio (K d / K a ) and expressed as a molar concentration (M). The K of the bispecific fusion protein disclosed herein can be determined using established methods in the art. D A K value for measuring antibodies D The methods are by using surface plasmon resonance or using biosensor systems such as system.

[0181] As used herein, in the context of two or more nucleic acids or peptide sequences, phrases "percentage identity" or "percentage identity" refer to two or more identical sequences or subsequences. When comparing and alignment to obtain maximum correspondence in a comparison window or a specified region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, if two sequences have a specified percentage of identical amino acid residues or nucleotides (that is, in a specified region, or when not specified, within the entire sequence, 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity), then two sequences are "substantially identical". Optionally, identity is present in a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably in a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0182] For sequence comparison, usually a sequence serves as a reference sequence for comparison with a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified (if necessary), and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0183] As used herein, the term "comparison window" includes a segment with reference to any one of the number of contiguous positions selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are well known in the art. For example, optimal alignment of sequences for comparison can be performed by, e.g., the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search similarity method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology).

[0184] Two examples of algorithms suitable for determining percentages of sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25: 3389-3402; and Altschul et al. (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. Word hits are extended in both directions along each sequence until the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word match in each direction is halted when: the cumulative alignment score falls by the amount X from its maximum achieved value; the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915), alignments (B) 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0185] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0186] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol, Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0187] In addition to the above-mentioned percentages of sequence identity, another indicator that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, for example, where the two peptides differ only in conservative substitutions, the polypeptide is typically substantially identical to the second polypeptide. Another indicator that two nucleic acid sequences are substantially identical is that the two molecules or their complementary sequences hybridize to each other under stringent conditions, as described below. Yet another indicator that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0188] As used herein, the term "stimulation" refers to 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 the 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.

[0189] 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.

[0190] 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 peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0191] 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 population of cells. In other cases, the term refers only to cells that have been separated from cells with which they 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.

[0192] 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.

[0193] 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.

[0194] As used herein, the term "therapeutic" means treating and / or preventing. A therapeutic effect is achieved by suppressing, alleviating, or eradicating a disease state.

[0195] The term "therapeutically effective amount" or "effective amount" refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the 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 being treated.

[0196] 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 therapies (e.g., adoptive cell therapy), supportive therapies (e.g., lymphocyte depletion 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.

[0197] 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 course of the disease in 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" a disease as used herein means reducing the frequency or severity of at least one sign or symptom of a disease or condition experienced by a subject.

[0198] 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. Such cells include the primary subject cell and its progeny.

[0199] 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.

[0200] As used herein, a "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 to include non-plasmid and non-viral compounds that promote the transfer of nucleic acids 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.

[0201] As used herein, the term "xenogeneic" refers to a transplant derived from an animal of a different species.

[0202] Range: Throughout this disclosure, various aspects of the disclosure may be presented in range format. It should be understood that descriptions in range format are provided for convenience and brevity only and should not be construed as immutable, rigid limitations on the scope of the disclosure. Therefore, descriptions of ranges should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within the ranges. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within the ranges, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0203] III. Fusion proteins with extended half-lives

[0204] One aspect of the present disclosure provides a fusion protein (e.g., a trispecific fusion protein) with enhanced half-life, comprising an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA); an immune cell recognition domain that specifically binds to a receptor on an immune effector cell; and a half-life extension domain. The half-life extension domain is a polypeptide that can extend the half-life of the entire fusion protein. The three domains of the fusion protein can be formulated in any order. For example, the half-life extension domain can be located at the N-terminus, middle, or C-terminus of the fusion protein.

[0205] Specifically, the TACA fusion protein (e.g., a trispecific fusion protein) of the present disclosure may comprise a first domain (I), which is an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA); a second domain (II), which is an immune cell recognition domain that specifically binds to a receptor on an immune cell (e.g., CD3); and a third domain (III), which is a half-life extension domain. In some embodiments, the TACA fusion protein (e.g., a trispecific fusion protein) may comprise a first domain (I), which is an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA); a second domain (II), which is a half-life extension domain; and a third domain (III), which is an immune cell recognition domain that specifically binds to a receptor on an immune cell (e.g., CD3). In some embodiments, the TACA trispecific fusion protein comprises a first domain (I), which is an immune cell recognition domain that specifically binds to a receptor in an immune cell (e.g., CD3); a second domain (II), which is an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA); and a third domain (III), which is a half-life extension domain.

[0206] In some embodiments, a TACA fusion protein (e.g., a trispecific fusion protein) comprises a first domain (I), which is an immune cell recognition domain that specifically binds to a receptor in an immune cell (e.g., CD3); a second domain (II), which is a half-life extension domain; and a third domain (III), which is an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA). In some embodiments, a TACA fusion protein (e.g., a trispecific fusion protein) comprises a first domain (I), which is a half-life extension domain; (b) a second domain (II), which is an immune cell recognition domain that specifically binds to a receptor in an immune cell (e.g., CD3); and (c) a third domain (III), which is an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA). In some embodiments, a TACA fusion protein (e.g., a trispecific fusion protein) comprises a first domain (I) that is a half-life extension domain; (b) a second domain (II) that is an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA); and (c) a third domain (III) that is an immune cell recognition domain that specifically binds to a receptor (e.g., CD3) in an immune cell. In some embodiments, the first, second, and third domains are connected by a linker.

[0207] A. Half-life extension domain

[0208] The half-life extension domain extends the elimination half-life of the protein of interest. The half-life extension domain also changes the pharmacodynamic properties of the protein, including changes in the tissue distribution, penetration and diffusion of the trispecific antigen-binding protein. In some embodiments, compared to proteins without the half-life extension domain, the half-life extension domain provides improved tissue (including tumor) targeting, tissue distribution, tissue penetration, diffusion within the tissue and enhanced efficacy. The treatment methods including the TACA fusion proteins (e.g., trispecific fusion proteins) with half-life extension domains disclosed herein effectively and efficiently utilize the GlyTR fusion proteins of reduced amounts, thereby resulting in reduced side effects, such as reduced cytotoxicity of non-tumor cells. Therefore, the half-life extension domain enhances the therapeutic utility of GlyTR.

[0209] In addition, the binding affinity of the half-life extension domain can be selected to target a specific elimination half-life in a specific trispecific antigen-binding protein. Therefore, in some embodiments, the half-life extension domain has a high binding affinity. In other embodiments, the half-life extension domain has a moderate binding affinity. In other embodiments, the half-life extension domain has a low or marginal binding affinity. Exemplary binding affinities include 10nM or lower (high), between 10nM and 100nM (medium), and a KD concentration greater than 100nM (low). As described above, the binding affinity of the half-life extension domain (e.g., serum albumin) can be determined by known methods such as surface plasmon resonance (SPR).

[0210] 1. Molecules that enhance serum half-life

[0211] Several approaches can be taken to improve the serum half-life of the fusion proteins disclosed herein. In some embodiments, the half-life extension domain comprises a molecule selected from the group consisting of: a polypeptide capable of binding to albumin, albumin, serum albumin, an Fc domain of an antibody, a polyethylene glycol moiety (PEG), a poly(lactic-co-glycolic acid) (PLGA) copolymer, a polymer hydrogel, a nanoparticle, a fatty acid chain, an acyl group, a myristate group, a palmitoylated group, and a steryl group. In some embodiments, the half-life extension domain binds to human serum albumin.

[0212] Typically, polypeptides that increase serum half-life in vivo are naturally occurring polypeptides in vivo and / or polypeptides that are resistant to degradation or removal by endogenous mechanisms that remove unwanted substances from an organism (e.g., a mammal or a human). For example, polypeptides that enhance serum half-life in vivo can be selected from proteins from the extracellular matrix, proteins present in the blood, proteins present at the blood-brain barrier or neural tissue, proteins localized to the kidney, liver, lung, heart, skin or bone, stress proteins, disease-specific proteins, or proteins involved in Fc transport.

[0213] In some embodiments, suitable polypeptides comprising a half-life extending domain that enhances serum half-life in vivo include, for example, transferrin receptor-specific ligand-neuropharmaceutical fusion protein (see, U.S. Pat. No. 5,977,307, the teachings of which are incorporated herein by reference), brain capillary endothelial cell receptor, transferrin, transferrin receptor (e.g., soluble transferrin receptor), insulin, insulin-like growth factor 1 (IGF 1) receptor, insulin-like growth factor 2 (IGF 2) receptor, insulin receptor, coagulation factor X, alpha 1-antitrypsin, and HNF 1α. Suitable polypeptides that enhance serum half-life also include alpha-1 glycoprotein (seromucoid; AAG), alpha-1 antichymotrypsin (ACT), alpha-1 microglobulin (protein HC; AIM), antithrombin III (AT III), apolipoprotein A-1 (Apo A-1), apolipoprotein B (Apo B), ceruloplasmin (Cp), complement component C3 (C3), complement component C4 (C4), C1 esterase inhibitor (C1 INH), C-reactive protein (CRP), ferritin (FER), hemopexin (HPX), lipoprotein (a) (Lp(a)), mannose binding protein (MBP), myoglobin (Myo), prealbumin (transthyretin; PAL), retinol binding protein (RBP), and rheumatoid factor (RF).

[0214] In some embodiments, suitable polypeptides comprising a half-life extension domain that enhances serum half-life in vivo include proteins from the extracellular matrix, including, for example, collagen, laminin, integrin, and fibronectin. Collagen is the main protein of the extracellular matrix. There are about 15 types of collagen molecules currently known, which are present in different parts of the human body, for example, type I collagen (accounting for 90% of human collagen) is present in bones, skin, tendons, ligaments, corneas, and internal organs, or type II collagen is present in cartilage, intervertebral discs, spinal cords, and vitreous humor of the eye.

[0215] In some embodiments, suitable polypeptides comprising a half-life extending domain that enhances serum half-life in vivo include proteins from the blood, including, for example, plasma proteins (e.g., fibrin, alpha-2 macroglobulin, serum albumin, fibrinogen (e.g., fibrinogen A, fibrinogen B), serum amyloid A, haptoglobin, profilin, ubiquitin, uteroglobulin, and beta-2-microglobulin), enzymes and enzyme inhibitors (e.g., plasminogen, lysozyme, cystatin C, alpha-1-antitrypsin, and pancreatic trypsin inhibitor), proteins of the immune system, such as immunoglobulins (e.g., IgA, IgD, IgE, IgG, IgM, immunoglobulin light chains (κ / λ)), transporters (e.g., retinol binding protein, alpha-1 microglobulin), defensins (e.g., beta-defensin 1, neutrophil defensin 1, neutrophil defensin 2, and neutrophil defensin 3), and the like. Suitable proteins present at the blood-brain barrier or neural tissue include, for example, melanocortin receptors, myelin, ascorbic acid transporter, and the like.

[0216] In some embodiments, suitable polypeptides comprising a half-life extending domain that enhances serum half-life in vivo include proteins localized to the kidney (e.g., polycystin, type IV collagen, organic anion transporter K1, Heymann's antigen), proteins localized to the liver (e.g., alcohol dehydrogenase, G250), proteins localized to the lung (e.g., secretory component, which binds IgA), proteins localized to the heart (e.g., HSP 27, which is associated with dilated cardiomyopathy), proteins localized to the skin (e.g., keratin), bone-specific proteins such as morphogenetic proteins (BMPs), which are a subset of the transforming growth factor beta protein superfamily that exhibit osteogenic activity (e.g., BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8), tumor-specific proteins (e.g., trophoblast antigen, herceptin receptor, estrogen receptor, cathepsins (e.g., cathepsin B, which can be found in the liver and spleen)).

[0217] In some embodiments, disease-specific proteins that can extend the serum half-life of proteins include, for example, antigens expressed only on activated T cells, including LAG-3 (lymphocyte activation gene), osteoprotegerin ligand (OPGL; see Nature 402, 304-309 (1999)), OX40 (a member of the TNF receptor family, expressed on activated T cells and specifically upregulated in human T-cell leukemia virus type 1 (HTLV-I) producing cells; see Immunol. 165(1):263-70 (2000)). Suitable disease-specific proteins also include, for example, metalloproteinases (associated with arthritis / cancer), including Drosophila CG6512, human paraplegin, human FtsH, human AFG3L2, murine ftsH; and angiogenic growth factors, including acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), vascular endothelial growth factor / vascular permeability factor (VEGF / VPF), transforming growth factor-α (TGFα), tumor necrosis factor-α (TNF-α), angiopoietins, interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derived endothelial growth factor (PD-ECGF), placental growth factor (P1GF), midkine platelet-derived growth factor-BB (PDGF), and chemokines.

[0218] In some embodiments, suitable polypeptides comprising a half-life extension domain that enhances serum half-life in vivo include stress proteins, such as heat shock proteins (HSPs). HSPs are typically found intracellularly. When they are found extracellularly, this indicates that the cell has died and overflowed its contents. This unprogrammed cell death (necrosis) occurs when extracellular HSPs trigger an immune system response due to trauma, disease, or injury. Binding to extracellular HSPs can result in the compositions of the present disclosure being localized to the site of disease.

[0219] 2. Human serum albumin

[0220] In some embodiments, the half-life extension domain comprises human serum albumin or a molecule capable of binding to serum albumin. Human serum albumin (HSA) (molecular mass of about 67 kDa) is the most abundant protein in plasma. The mature form of HSA comprises about 585 amino acids. HSA is used to maintain plasma pH, promote colloid blood pressure, is used as a carrier of many metabolites and fatty acids, and is used as the main drug transporter in plasma. HSA is responsible for a significant part of serum osmotic pressure and is also used as a carrier for endogenous and exogenous ligands. HSA is present in plasma at a concentration of about 50 mg / ml (600 μM) and has a half-life of about 20 days in the human body.

[0221] The role of albumin as a carrier molecule and its inert properties are desirable properties for use as a carrier and transporter for polypeptides in vivo. The use of albumin as a component of albumin fusion proteins as a carrier for various proteins is known to those skilled in the art. See, for example, WO 1993 / 15199, WO 1993 / 15200, and EP 413 622. In some embodiments, fragments of HSA can be used in the fusion proteins of the present disclosure (e.g., tri-specific fusion proteins). The HSA fragment can be an N-terminal fragment. Therefore, by genetic or chemical methods, the fusion proteins described herein are fused or conjugated to human albumin to stabilize or extend the serum half-life and / or maintain the activity of the molecule in solution, in vitro, and / or in vivo for an extended period of time.

[0222] In some embodiments, the HSA is wild-type HSA having the amino acid sequence of SEQ ID NO: 57. In some embodiments, the fusion protein has the amino acid sequence shown in Table 2. In some embodiments, the fusion protein comprises the amino acid sequence shown in Table 2 or Table 3.

[0223] a. Modified HSA

[0224] In some embodiments, HSA is mutated. Mutant HSA may have two amino acid substitutions relative to wild-type HSA. For example, the cysteine ​​at position 34 may be substituted with serine ("C34S") and / or the aspartic acid at position 503 may be substituted with glutamine "N503Q". In some embodiments, the cysteine ​​residue at position 34 (i.e., C34) may be mutated to any amino acid residue other than cysteine ​​(e.g., serine, threonine, or alanine). Similarly, the asparagine residue at position 503 may be mutated to any amino acid residue other than asparagine (e.g., glutamine, serine, histidine, or alanine). Specifically, replacing cysteine ​​with serine at amino acid residue 34 may result in reduced oxidation and protein heterogeneity of HSA. In wild-type HSA, the asparagine at amino acid residue 503 is sensitive to deamination, which may result in a shortened pharmacological half-life. Substituting glutamine for asparagine at amino acid residue 503 can result in an increase in the pharmacological half-life of HSA, and correspondingly increase the pharmacological half-life of the bispecific fusion proteins disclosed herein, or cells, tissues, or organs thereof.

[0225] In some embodiments, the half-life extension domain comprises a truncated wild-type HSA polypeptide operably linked to a bispecific fusion protein with or without a connecting linker. In some embodiments, the truncated HSA is a wild-type HSA polypeptide lacking 1, 2, 3, 4, 5, 10, 15, 20, 50, 100, 200 or more amino acids of the full-length wild-type HSA amino acid sequence. The truncation can occur at one or both ends of the HSA or can include the deletion of internal residues. Truncations of more than one amino acid residue do not need to be linear (i.e., continuous).

[0226] The half-life extension domain (e.g., HSA) can (but need not) be modified by site-specific chemical modification of amino acid residues in HSA. The properly folded tertiary structure of HSA displays certain amino acid residues on the external surface of the protein. These surface-exposed residues can be substituted with chemically reactive amino acid residues (e.g., cysteine) to allow site-specific conjugation of other agents.

[0227] Alternatively or in addition, the half-life extension domain (e.g., HSA) can be optionally modified by adding or removing asparagine, serine, or threonine residues from the HSA sequence to alter the glycosylation of these amino acid residues. The glycosylation sites added to HSA can be surface exposed. The glycosyl or other carbohydrate moiety introduced into HSA can be directly conjugated to a diagnostic, therapeutic, or cytotoxic agent.

[0228] The surface-exposed amino acid residues of the half-life extension domain (e.g., HSA) can be replaced by cysteine ​​residues to allow chemical conjugation of diagnostic agents, therapeutic agents, or cytotoxic agents. The cysteine ​​residues exposed on the surface of HSA (when folded into their native tertiary structure) allow specific conjugation of diagnostic agents, therapeutic agents, or cytotoxic agents to thiol-reactive groups (such as maleimide or haloacetyl). The nucleophilic reactivity of the thiol functional group of the cysteine ​​residue with the maleimide group is about 1000 times higher than that of any other amino acid functional group in the protein (such as the amino group or N-terminal amino group of a lysine residue). The thiol-specific functional groups in iodoacetyl and maleimide reagents can react with amine groups, but require higher pH values ​​(>9.0) and longer reaction times (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London). The amount of free thiols in proteins can be estimated using a standard Ellman's assay. In some cases, reduction of disulfide bonds with reagents such as dithiothreitol (DTT) or selenol (Singh et al., Anal. Biochem. 304: 147-156 (2002)) is required to generate reactive free thiols.

[0229] The site of cysteine ​​substitution can be identified by analyzing the surface accessibility of HSA. Surface accessibility can be expressed as the surface area (e.g., square angstroms) that can be contacted by solvent molecules (e.g., water). The space occupied by water is approximately a sphere with a radius of 1.4 angstroms. The software for calculating the surface accessibility of each amino acid of a protein is freely available or requires permission. For example, the CCP4 crystallographic program suite uses an algorithm to calculate the surface accessibility of each amino acid in a protein with known x-ray crystallography-derived coordinates ("The CCP4 Suite:Programs for ProteinCrystallography" Acta.Cryst.D50:760-763 (1994); ccp4.ac.uk / dist / html / INDEX.html). Solvent accessibility can also be assessed using the free software DeepView Swiss PDBViewer downloaded from the Swiss Institute of Bioinformatics. The cysteine ​​substitution of surface-exposed sites allows reactive cysteine ​​to be conjugated to the thiol-reactive group connected to a diagnostic agent or therapeutic agent.

[0230] b. Glycosylated HSA

[0231] In addition, altered serum clearance rates can be achieved by engineering glycosylation sites into half-life extension domains (e.g., HSA). In certain embodiments, HSA can be glycosylated. Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the connection of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (wherein X represents any amino acid except proline) are recognition sequences for the enzymatic connection of a carbohydrate moiety to an asparagine side chain. Therefore, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the connection of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, the most common of which is serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0232] Addition or deletion of glycosylation sites in HSA is conveniently achieved by altering the amino acid sequence, thereby generating one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Alterations can also be made by addition, deletion, or substitution of one or more serine or threonine residues in the HSA sequence (for O-linked glycosylation sites). The resulting carbohydrate structure on HSA can also be used for site-specific conjugation of cytotoxic agents, immunomodulators, or cytostatics. In some embodiments, human serum albumin is glycosylated.

[0233] c. Production of HSA fusion protein

[0234] Fusion of albumin to another protein can be achieved by genetic manipulation such that the DNA encodes HSA or a fragment thereof. A suitable host is then transformed or transfected with the fused nucleotide sequence and arranged on a suitable plasmid to express the fusion polypeptide. Expression can be achieved in vitro, for example, from prokaryotic or eukaryotic cells, or in vivo (e.g., from a transgenic organism). Additional methods related to HSA fusions are described, for example, in WO 2001 / 077137 and WO 2003 / 06007 (incorporated herein by reference).

[0235] The non-covalent association between the protein of interest and HSA extends the elimination half-life of this short-lived protein. For example, when administered intravenously to mice, the recombinant fusion of HSA and GlyTR of the present disclosure resulted in at least a 5-fold increase in half-life compared to administration of GlyTR alone. Figures 22A-22EIn some embodiments, the half-life of the fusion protein is enhanced by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 16-fold, at least about 18-fold, at least about 20-fold, or at least about 30-fold compared to a fusion protein lacking a half-life extension domain. In some embodiments, the half-life extension is based on the mean plasma residence time of the fusion protein. The serum clearance rate of a fusion protein comprising HSA can be optimized by testing conjugates containing truncated wild-type HSA.

[0236] A molecule capable of extending the half-life of another molecule (such as HSA) can be incorporated into the fusion protein by being directly or indirectly connected to the fusion protein. The term "direct" connection refers to that the fusion protein is immediately bound to the half-life extension domain so that there is no gap between the fusion protein and the half-life extension domain. In some embodiments, the fusion protein is a bispecific fusion protein. The term "indirect" connection refers to that the bispecific fusion protein is not immediately bound to the half-life extension domain, but is bound by an amino acid "connection linker" between the bispecific fusion protein and the half-life extension domain. Examples of amino acid connection linkers include, but are not limited to, linkers consisting entirely of glycine, alanine, serine, glutamine, leucine or valine residue linkers or any combination of these residues. The length of these amino acid connection linkers can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids and provide a flexible tether between the fusion protein (e.g., TACA bispecific fusion protein) and the half-life extension domain. The amino acid-linked linker can be attached (e.g., covalently (e.g., a peptide bond), ionically or hydrophobically, or via a high-affinity protein-protein binding interaction (e.g., biotin and avidin)) to the C-terminus or N-terminus of the half-life extension domain (such as HSA or a mutant HSA).

[0237] d. Molecules that can bind to serum albumin

[0238] In some embodiments, the half-life extension domain comprises a molecule capable of binding to serum albumin. For example, a peptide ligand that binds to human serum albumin may comprise the amino acid sequence D-Xaa-CLP-Xaa-WGCLW (SEQ ID NO: 70) and, when administered to a subject, may be fused to a fusion protein disclosed herein (e.g., a bispecific GlyTR) to recruit HSA. In some embodiments, Xaa can be any amino acid. Peptide ligands that bind to mammalian serum albumin can be identified by their ability to compete with the peptide ligand for binding to human serum albumin in an in vitro assay. In some embodiments, the peptide ligand that binds to human serum albumin comprises the amino acid sequence QGLIGDICLPRWGCLWGDSVK (SEQ ID NO: 71), RLIEDICLPRWGCLWEDD, (SEQ ID NO: 72), or EDICLPRWGCLWED (SEQ ID NO: 73).

[0239] In some embodiments, the half-life extension domain comprises a fatty acid chain conjugated polypeptide. The fatty acid chain can be selected from a C-16 fatty acid chain or a C-18 fatty acid chain. In some embodiments, the fatty acid chain is a C-16 fatty acid conjugated molecule. In some embodiments, the half-life extension domain comprises an antibody fragment that selectively binds serum albumin. In one embodiment, the antibody fragment is a single domain antibody, a complementary determining region (CDR) of a single domain antibody, or a single chain variable fragment (scFv).

[0240] 3Fc fusion

[0241] A natural antibody molecule consists of two identical heavy chains and two identical light chains. The heavy chain constant region includes CH1, hinge region, CH2, and CH3. Papain digestion of an antibody produces two fragments, Fab and Fc. The Fc fragment consists of CH2, CH3, and part of the hinge region. It has been recognized that the Fc region is crucial for maintaining the serum half-life of IgG antibodies (Ward and Ghetie, Ther. Immunol. 2:77-94 (1995)). Studies have found that the serum half-life of IgG antibodies is mediated by the binding of Fc to the neonatal Fc receptor (FcRn). FcRn is a heterodimer consisting of a transmembrane α chain and a soluble β chain (β2-microglobulin). Advances in molecular biology techniques have allowed the preparation of novel chimeric polypeptides with multiple functional domains. The most common of these chimeric polypeptides is immunoglobulin (Ig) fusion proteins. These proteins consist of the Fc region of an antibody (usually a mouse or human antibody) fused to an unrelated protein or protein fragment.

[0242] As used herein, the term "Fc" refers to a polypeptide comprising the CH3, CH2, and at least a portion of the hinge region of an antibody constant domain. Optionally, the Fc region may include a CH4 domain present in some antibody classes. Fc may comprise the entire hinge region of an antibody constant domain. In one embodiment, the fusion protein comprises the Fc region and CH1 region of an antibody. In one embodiment, the fusion protein comprises the Fc region and CH3 region of an antibody. In another embodiment, the fusion protein comprises the Fc, CH1, and κ / λ regions from an antibody constant domain. Exemplary modifications include the addition, deletion, or substitution of one or more amino acids in one or more domains. Such changes may be included to optimize effector function, half-life, and the like.

[0243] One aspect of the present disclosure provides a 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 NOs: 33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NOs: 33-56; and an Fc domain of an antibody. In some embodiments, the Fc domain is a half-life extending domain. In one embodiment, the half-life extending domain is the Fc domain of an IgG antibody. In one embodiment, the half-life extension domain is an Fc domain of an antibody selected from the Fc region of IgG1, IgG2, IgG3 or IgG4. In some embodiments, the 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. In some embodiments, the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 69 or 91-94. In some embodiments, the half-life extension domain is an Fc fusion protein.

[0244] The fusion protein of the present disclosure comprising an Fc region / domain as a half-life extension domain can be produced by standard recombinant DNA techniques or by protein synthesis techniques (e.g., by using a peptide synthesizer). For example, nucleic acid molecules encoding Fc fusion proteins can be synthesized by conventional techniques (including automated DNA synthesizers). Alternatively, PCR amplification of gene fragments can be performed using anchor primers that produce complementary overhangs between two consecutive gene fragments, which can then be annealed and re-amplified to generate a chimeric gene sequence (see, e.g., "Current Protocols in Molecular Biology", edited by Ausubel et al., John Wiley & Sons, (1992)). In addition, nucleic acids encoding fusion proteins disclosed herein can be cloned into expression vectors containing Fc domains or fragments thereof so that the fusion proteins disclosed herein are in-frame connected to constant domains or fragments thereof.

[0245] Methods for fusing or conjugating polypeptides to the constant regions of antibodies are disclosed herein and / or known in the art. See, for example, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946; European Patent Publication Nos. EP 0 307 434, EP 0 367 166, EP 0 394 827; PCT Publications Nos. WO 91 / 06570, WO 96 / 04388, WO 96 / 22024, WO 97 / 34631, and WO 99 / 04813; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Traunecker et al., Nature 331:84-86 (1988); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341 (1992), WO 98 / 23289; WO 97 / 34631; U.S. Patent No. 6,277,375; WO 93 / 15199, WO 93 / 15200; WO 2001 / 77137; and EP 413,622, each of which is incorporated herein by reference in its entirety. Trispecific fusion proteins comprising a modified Fc as a half-life extension domain are also within the scope of the present disclosure.

[0246] In some embodiments, suitable polypeptides comprising a half-life extension domain that enhances serum half-life in vivo include proteins involved in Fc transport. These proteins include, for example, the Brambell receptor (also known as FcRB). This Fc receptor has two functions, both of which may be used for delivery. The functions are (1) transporting IgG from the mother to the fetus across the placenta and (2) protecting IgG from degradation, thereby extending its serum half-life. It is believed that the receptor recovers IgG from endosomes. (See, Holliger et al., Nat Biotechnol 15(7):632-6 (1997).)

[0247] 4.PEGylation

[0248] In another embodiment, the half-life extension domain includes polyethylene glycol (PEG). Efforts to reduce the clearance of molecules with a molecular weight of 60kDa are generally focused on increasing the molecular size of these proteins by protein fusion, glycosylation or the addition of polyethylene glycol polymers (i.e., PEG). In order to extend the serum circulation in vivo of the bispecific fusion proteins disclosed herein, the bispecific fusions (i.e., GlyTR) can be connected to an inert polymer molecule, such as a high molecular weight PEG. The connection may have or not have a connection joint. The connection can be by conjugating the PEG site-specifically to the N-terminal or C-terminal of the bispecific fusion, or by carrying out the ε-amino group present on the lysine residue. For pegylation, under the condition that one or more PEG groups are connected to the bispecific fusion body, the bispecific fusion body is usually reacted with polyethylene glycol (PEG) (such as the reactive ester or aldehyde derivative of PEG). Pegylation can be carried out by carrying out acylation reaction or alkylation reaction with reactive PEG molecules (or similar reactive water-soluble polymers).

[0249] As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG for derivatizing other proteins, such as single (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. Straight or branched polymer derivatization that results in minimal loss of biological activity can be used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure that the PEG molecule is correctly conjugated to the bispecific fusion body, thereby producing a trispecific fusion protein. Unreacted PEG can be separated from the trispecific fusion protein by size exclusion or by ion exchange chromatography. Methods well known to those skilled in the art, such as by immunoassays as described herein, can be used to test the binding activity and in vivo efficacy of the trispecific fusion protein. Methods for pegylated proteins are known in the art (see, for example, EP 0154 316 by Nishimura et al. and EP 0401384 by Ishikawa et al.).

[0250] Other modified PEGylation technologies include reconstructed chemical orthogonal directed engineering (ReCODE PEG), which incorporates chemically specified side chains into biosynthetic proteins via a reconstructed system including tRNA synthetase and tRNA. This technology is capable of incorporating more than 30 new amino acids into biosynthetic proteins in E. coli, yeast, and mammalian cells. tRNA will incorporate unnatural amino acids at any position where an amber codon is located, converting the amber codon from a stop codon to a codon indicating the incorporation of a chemically specified amino acid.

[0251] Recombinant PEGylation (rPEG) can also be used to extend the serum half-life of proteins. This technology involves genetically fusing a 300-600 amino acid unstructured protein tail to a bispecific fusion protein. Because the apparent molecular weight of this unstructured protein chain is approximately 15 times greater than its actual molecular weight, the serum half-life of the trispecific fusion protein will be greatly increased. Compared to traditional PEGylation, which requires chemical conjugation and repurification, the manufacturing process is greatly simplified, and the product is homogeneous.

[0252] In some embodiments, the half-life extending domain comprises a PEG moiety of less than about 0.5k, less than about 1.0k, less than about 2.0k, less than about 3.0k, less than about 4.0k, less than about 5.0k, less than about 6.0k, less than about 7.0k, less than about 6.0k, less than about 7.0k, less than about 8.0k, less than about 10.0k, less than about 12.0k, less than about 14.0k, less than about 16.0k, less than about 18.0k, or less than about 20.0k.

[0253] 5.PSA

[0254] PSA polymers occur naturally in the human body. Certain bacteria, over millions of years of evolution, use these polymers to coat their cell walls. These naturally polysialylated bacteria are then able to subvert the body's defenses through molecular mimicry. PSA, nature's ultimate stealth technology, can be easily produced in large quantities from these bacteria and possesses predetermined physical characteristics. Bacterial PSA is completely non-immunogenic, even when conjugated to proteins, because it is chemically identical to PSA found in the human body.

[0255] Polysialylation is another technology that uses natural polymers to extend the active lifespan and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (a sugar). When used for protein and therapeutic peptide drug delivery, polysialic acid provides a protective microenvironment when conjugated. This increases the active lifespan of the bispecific fusion protein in the circulation and prevents it from being recognized by the immune system. In some embodiments, the half-life extension domain may comprise polysialic acid (PSA).

[0256] 6. HES

[0257] HES is a modified natural polymer derived from waxy corn starch and can be metabolized by human enzymes. HES solution is usually used to replace insufficient blood volume and improve the rheological properties of blood. The hydroxyethylation of bispecific fusion protein can extend the circulation half-life by increasing the stability of the molecule and by reducing the renal clearance rate, thereby leading to improved biological activity. By changing different parameters, such as the molecular weight of HES, a wide range of HES trispecific fusion proteins can be customized. In another embodiment, the half-life extension domain comprises hydroxyethyl starch (" HES "). The HES derivatives connected with the bispecific fusion protein disclosed herein can produce trispecific fusion proteins with extended half-life.

[0258] B. Lectins

[0259] 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 (TACAs) (Table 1). This tumor-specific property makes cell surface TACAs an excellent target antigen for the production of monoclonal antibodies targeting many common cancers. However, despite decades of effort, due to the difficulty in generating antibodies against carbohydrate antigens, specific antibodies with high affinity for TACAs have not yet been obtained.

[0260] 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 proteins disclosed herein provide an opportunity for developing new therapeutic drug categories for cancer immunotherapy, which have significant advantages over prior art (e.g., GlyTR chimeric proteins). Based on the GlyTR concept and the availability of a variety of different lectins that are specific for 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 that 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).

[0261] 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. In some embodiments, the antigen binding domain comprises a TACA binding domain derived from a lectin. In some embodiments, the antigen binding domain comprises one or more TACA binding domains.

[0262] Additional information regarding GlyTR technology and subsequent improvements is disclosed in International Application No. PCT / US2016 / 030113 and co-pending International Application No. PCT / US2023 / 024898 (entitled “Improved Glycan-Dependent Immunotherapeutic Bi-Specific Fusion Proteins and Chimeric Antigen Receptors”), which are incorporated herein by reference in their entireties.

[0263]

[0264]

[0265] The antigen binding domains of the bispecific fusion proteins disclosed herein are designed to specifically target glycoproteins and / or glycolipids (i.e., carbohydrate-containing macromolecules) on tumor cells. In some embodiments, the bispecific fusion proteins of the present disclosure comprise 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 epitope thereof that associates with the target cell. For example, the bispecific fusion protein may comprise affinity for a target antigen on a target cell that indicates a particular state of the target cell.

[0266] In some embodiments, the antigen binding domain comprises one or more TACA binding domains. In some embodiments, the antigen binding domain comprises one, two, three, four, five, six, seven, eight, nine, ten or more TACA binding domains. In one embodiment, the antigen binding domain comprises one TACA binding domain. In one embodiment, the antigen binding domain comprises two TACA binding domains. In one embodiment, the antigen binding domain comprises three TACA binding domains. In one embodiment, the antigen binding domain comprises four TACA binding domains.

[0267] In some embodiments, the lectin is selected from the group consisting of: a galectin, a sialic acid-binding immunoglobulin-like lectin, a selectin; a C-type lectin; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Phaseolus vulgaris leukoagglutinin); 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 (Phytolacca Americana lectin), wheat germ agglutinin (Wheat germ agglutinin), and lectin. agglutinin (wheat germ agglutinin); Artocarpus polyphemus lectin (jacalin letin); Vicia fragrans agglutinin (VVA); Snail agglutinin (HPA); Wisteria multiflora agglutinin (WFA); Sambucus nigra agglutinin (SNA); BC2L-CNt (lectin from the Gram-negative bacterium Burkholderia cenocepacia); Mulberry leukocyte agglutinin (MAL); Pleurotus eryngii agglutinin (PVL); Sclerotium sclerotiorum agglutinin (SRL); Eucheuma serrata agglutinin (ESA); CLEC17A (protectin); Dictyocystis aurantiacus agglutinin; Sessile elderberry agglutinin (SSA); Gleheda lectin; Morniga G agglutinin; Salvia miltiorrhiza agglutinin; bogotensis agglutinin, Salvia officinalis agglutinin, Dichroa haizhouensis agglutinin, Concha striata agglutinin, Griffonia suffruticosa (GsLA4), Quadrangularis bean (acidic WBAI), Abrus precatorius lectin, Amaranthus caudatus lectin, Ginkgo biloba lectin, Orchidaceae agglutinin, Jackfruit agglutinin, Citrus aurantium agglutinin, Jackfruit agglutinin, Dolichos biflorus agglutinin, Dolichos biflorus lectin, Soybean agglutinin, and Agaricus bisporus agglutinin.

[0268] 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 In some embodiments, the TACA binding domain is derived from a selectin or a C-type lectin.

[0269] 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), ppGalNAc-T10 (GALNT11), ppGalNAc-T12 (GALNT13), ppGalNAc-T14 (GALNT15), ppGalNAc-T15 (GALNT16), ppGalNAc-T16 (GALNT17), ppGalNAc-T17 (GALNT18), ppGalNAc-T19 (GALNT19), ppGalNAc-T10 (GALNT11), ppGalNAc-T11 (GALNT12), ppGalNAc-T12 (GALNT13), ppGalNAc-T13 (GALNT14), 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).

[0270] In some embodiments, the antigen binding domain of the bispecific fusion protein described herein 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, Lewis-y (Lewis antigen), y ), Sialyl-Lewisx / a, Disialo-Lewis x / a , sialyl 6-sulfoLexisx, Lewis Y, Globo H, GD2, GD3, GM3 and fucosyl GM1.

[0271] C.TACA

[0272] The TACA binding domain can 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.

[0273] 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, Globo H, GD2, GD3, GM3, and Fucosyl GM1. In some embodiments, the bispecific fusion 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, Lewis-y (Le y ), sialic acid group – Lewis x / a , disialo-Lewis x / a , sialyl-6-sulfo Lexis x , Globo H, GD2, GD3, GM3 and fucosyl GM1. In some embodiments, the fusion protein selectively targets β1,6GlcNAc branched N-glycans, GalNAc, Tn antigen, GalNAcα-ser, GalNAc or GalNAcβ1.

[0274] 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.

[0275] In some embodiments, the antigen binding domain comprises the amino acid sequence shown in SEQ ID NOs: 33-56, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NOs: 33-56. In one embodiment, the antigen binding domain comprises an amino acid sequence having at least 90% homology to SEQ ID NOs: 33-56.

[0276] D. Immune recognition domain

[0277] In some embodiments, bispecific fusion protein comprises the immune cell recognition domain that selectively binds to 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.

[0278] The receptor on the immune effector cell can be 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. Alternatively, the receptor on the immune effector cell is a 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 can be selected from the group consisting of: NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA and CEACAM1.

[0279] In some embodiments, the immune cell recognition domain of the bispecific fusion protein comprises a peptide, protein, antibody, single domain antibody, nano antibody, 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. In some embodiments, the immune cell recognition domain may comprise the amino acid sequence of SEQ ID NO:75-77.

[0280] Alternatively, the immune cell recognition domain may comprise the amino acid sequence of SEQ ID NO: 59, 60, or 61. In some embodiments, the immune cell recognition domain may comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 59, 60, or 61.

[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 some embodiments, the 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. In this embodiment, the Fc domain may comprise the amino acid sequence shown in SEQ ID NO:69 or 91-94. 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 comprise a hinge region.

[0282] E. Fusion protein

[0283] One aspect of the present disclosure provides a fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising (i) an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA); (ii) an immune cell recognition domain that specifically binds to a receptor on an immune effector cell; and (iii) a half-life extension domain. In some embodiments, the half-life extension domain is a polypeptide that can extend the half-life of the fusion protein.

[0284] 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 bound to a first antigen (e.g., TACA) and a second antigen recognition domain bound to 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 disclosure. In certain embodiments, the second antigen recognition domain is bound to 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.

[0285] In some embodiments, the bispecific fusion protein selectively targets a TACA selected from the group consisting of: β1,6 branching, β1,6GlcNAc branched N-glycan, T antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6 sialylation, sialylation, Lewis-y (Lewis y ), sialic acid group – Lewis x / a , disialo-Lewis x / a , sialyl 6-sulfo Lexis x , Lewis Y, GloboH, GD2, GD3, GM3 and fucosyl GM1. In one embodiment, the bispecific fusion protein selectively targets Tn antigen or β1,6GlcNAc branched N-glycan. In some embodiments, the bispecific fusion protein that selectively targets β1,6GlcNAc branched N-glycan comprises an antigen binding domain having an amino acid sequence selected from the group consisting of

[0286] In some embodiments, the fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 1-32; or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-32. In some embodiments, the fusion protein (e.g., a trispecific fusion protein) comprises an amino acid sequence selected from SEQ ID NOs: 1-12. In some embodiments, the fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 13-32. In some embodiments, the fusion protein comprises an amino acid sequence disclosed in Table 2 or Table 3.

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316] In some embodiments, the fusion protein exhibits enhanced binding to tumor cells expressing the Thomsen-nouveau (Tn) antigen or tumor cells expressing β1,6GlcNAc branched N-glycans compared to a 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 the group consisting of GGGGS (SEQ ID NO: 86), GGGGSGGGGS (SEQ ID NO: 87), or GGGGSGGGGSGGGGS (SEQ ID NO: 85); or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of GGGGS (SEQ ID NO: 86), GGGGSGGGGS (SEQ ID NO: 87), or GGGGSGGGGSGGGGS (SEQ ID NO: 85).

[0317] In some embodiments, the bispecific fusion protein selectively targets a Tn antigen or a β1,6GlcNAc branched N-glycan. In some embodiments, the fusion protein that selectively targets a Tn antigen comprises an antigen binding domain having an amino acid sequence selected from SEQ ID NOs: 36-42, 52-56, or 62; or an amino acid sequence that has at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 36-42, 52-56, or 62. In some embodiments, the fusion protein that selectively targets a Tn antigen comprises an amino acid sequence selected from SEQ ID NOs: 13-32; or an amino acid sequence that has at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 13-32.

[0318] In some embodiments, the 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: 33-35 or 43-51, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 33-35 or 43-51. In some embodiments, the fusion protein that selectively targets 1,6GlcNAc branched N-glycans comprises an amino acid sequence selected from SEQ ID NOs: 1-12, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-12.

[0319] One aspect of the present disclosure provides a 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: 33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 33-56; an immune cell recognition domain that specifically binds to CD3 on immune effector cells; and a half-life extension domain. In some embodiments, the half-life extension domain is a polypeptide that is capable of extending the half-life of the fusion protein. In some embodiments, the half-life extension domain comprises human serum albumin or the amino acid sequence of SEQ ID NO: 57.

[0320] Another aspect of the present disclosure provides a 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 NOs: 33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NOs: 33-56; an Fc domain of an antibody. In some embodiments, the Fc domain is the Fc domain of an IgG molecule. In some embodiments, the Fc domain is a half-life extending domain. In some embodiments, the half-life extending domain is a polypeptide that is capable of extending the half-life of the fusion protein. In some embodiments, the Fc domain is an IgG molecule.In some embodiments, the Fc domain comprises the amino acids of SEQ ID NO: 69 or 91-94.

[0321] F. Generation of Fusion Proteins

[0322] Fusion proteins or peptides disclosed herein can be prepared using chemical methods. For example, peptides, fusion proteins (bispecific fusion proteins or trispecific fusion proteins) can be synthesized by solid phase techniques (Roberge JY et al. (1995) Science 269: 202-204), cut from resin, and purified by preparative high performance liquid chromatography. For example, automatic synthesis can be achieved using an ABI 431A peptide synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

[0323] Peptide of the present disclosure, fusion protein can be synthesized by conventional techniques.For example, solid phase peptide synthesis can be used to synthesize peptide or fusion protein by chemical synthesis.These methods adopt solid phase or solution phase synthesis method (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 RBMerrifield, The Peptides:Analysis Synthesis, Biology editors E.Gross and J.Meienhofer 2nd volume Academic Press, New York, 1980, 3-254 pages; And about classical solution synthesis, M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E.Gross and J.Meienhofer edit, The Peptides:Analysis, Synthesis, Biology, suprs, 1st volume). For example, the peptides of the present disclosure can be synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid phase chemistry by direct incorporation of phosphothreonine as N-fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivatives.

[0324] The N-terminal or C-terminal fusion protein comprising the peptide or fusion protein of the present invention conjugated to other molecules can be prepared by recombinant technology by fusing the N-terminal or C-terminal of the peptide or fusion protein to the sequence of the selected protein or selectable marker with the desired biological function. The resulting fusion protein contains the peptide of the fusion protein of the present invention fused to the 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.

[0325] The peptides or 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 from these libraries, peptide sequences that bind to specific proteins can be screened. Libraries can be produced 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).

[0326] In one aspect, the present disclosure provides any form of peptide or fusion protein having substantial homology to the peptides or fusion proteins disclosed herein. Preferably, a "substantially homologous" peptide or fusion protein has about 50% homology to the amino acid sequence of the peptide 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.

[0327] In some embodiments, the peptide or fusion protein comprises an antigen binding domain comprising 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 NOs:33-56. In some embodiments, the peptide or fusion protein comprises 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 shown in SEQ ID NO: 1-32. The peptide or fusion protein can alternatively be prepared recombinantly or by cleavage from a longer polypeptide.

[0328] Variants of peptides and bispecific fusion proteins according to the present disclosure may be (i) variants 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 amino acid residues replaced may or may not be amino acid residues encoded by the genetic code, (ii) variants in which there are one or more modified amino acid residues (e.g., residues modified by the connection of substituents), (iii) variants in which the peptide is an alternative splice variant of a peptide of the present disclosure, (iv) fragments of the peptide, and / or (v) variants in which the peptide is fused to another peptide, such as a leader sequence or secretory sequence or a sequence for purification (e.g., a His tag) or for detection (e.g., an Sv5 epitope tag). Fragments include peptides generated by proteolytic cleavage (including multisite 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.

[0329] 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 to the sequence of a second polypeptide. Variants are defined as comprising peptide sequences that differ from the original sequence, preferably, each fragment of interest differs from the original sequence by less than 40% of the residues, more preferably, each fragment of interest differs from the original sequence by less than 25% of the residues, more preferably, each fragment of interest differs from the original sequence by less than 10% of the 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 disclosure 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 [BLASTP algorithm, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S. et al., J. Mol. Biol. 215: 403-410 (1990)].

[0330] Fusion proteins of the present disclosure can be post-translationally modified. For example, post-translational modifications falling within the scope of the present disclosure 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 can be examined by adding canine microsomal membranes or African clawed frog egg extract (U.S. Patent number 6,103,489) to standard translation reactions.

[0331] In some embodiments, the bispecific fusion proteins of the present disclosure may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation.A variety of methods can be used to introduce unnatural amino acids during protein translation.

[0332] The peptides or fusion proteins of the present disclosure can be conjugated to 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 peptides or bispecific fusion proteins of the present disclosure can be phosphorylated using conventional methods (such as those described in Reedijk et al. The EMBO Journal 11(4):1365 (1992)).

[0333] Cyclic derivatives of the peptides or fusion proteins of the present disclosure are also contemplated. Cyclization can allow the peptide to assume a more favorable conformation so as 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 may contain a β-turn at the right position. The β-turn can be introduced into the peptide of the present disclosure 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 peptides containing peptide bonds connected as described above. More flexible peptides 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 cyclic peptides can be determined by molecular dynamics simulations.

[0334] 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 sequence or domain. In the sense that various components are from different sources, bispecific fusion protein, fusion protein, CAR or peptide are recombinant, therefore will not be found together in nature (i.e., heterologous).

[0335] 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 disclosure to a cellular component.

[0336] IV. Nucleic Acids and Expression Vectors

[0337] A. Bispecific Fusion Proteins

[0338] One aspect of the present disclosure provides an isolated nucleic acid molecule encoding a bispecific fusion disclosed herein. Another aspect of the present disclosure provides an isolated nucleic acid molecule encoding a fusion protein, the fusion protein comprising an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA); an immune cell recognition domain that specifically binds to a receptor on an immune effector cell; and a half-life extension domain. In some embodiments, the half-life extension domain is a polypeptide that can extend the half-life of the fusion protein. In some embodiments, the half-life extension domain is located at the N-terminus, middle, or C-terminus of the fusion protein.

[0339] In some embodiments, the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 1-32, or an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-32. In some embodiments, the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-32.

[0340] 1. Extended half-life

[0341] In some embodiments, the half-life of the fusion protein is enhanced by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 16-fold, at least about 18-fold, or at least about 20-fold compared to a fusion protein lacking the half-life extension domain. In some embodiments, the half-life extension is based on the mean plasma residence time of the fusion protein.

[0342] In some embodiments, the half-life extending domain comprises a molecule selected from the group consisting of a polypeptide capable of binding to albumin, albumin, serum albumin, an Fc domain of an antibody, a polyethylene glycol moiety (PEG), a poly(lactic-co-glycolic acid) (PLGA) copolymer, a polymer hydrogel, a nanoparticle, a fatty acid chain, an acyl group, a myristic acid group, a palmitoylated group, and a steryl group.

[0343] In some embodiments, the half-life extension domain comprises an Fc domain of an antibody selected from the group consisting of IgG1, IgG2, IgG3, or IgG4 Fc regions. In one embodiment, the Fc domain comprises the amino acid sequence set forth in SEQ ID NOs: 69 or 91-94. In another embodiment, the half-life extension domain comprises a PEG moiety. In one embodiment, the PEG moiety is less than about 0.5 kJ, less than about 1.0 kJ, less than about 2.0 kJ, less than about 3.0 kJ, less than about 4.0 kJ, less than about 5.0 kJ, less than about 6.0 kJ, less than about 7.0 kJ, less than about 6.0 kJ, less than about 7.0 kJ, less than about 8.0 kJ, less than about 10.0 kJ, less than about 12.0 kJ, less than about 14.0 kJ, less than about 16.0 kJ, less than about 18.0 kJ, or less than about 20.0 kJ.

[0344] In some embodiments, the half-life extension domain comprises a serum albumin polypeptide, such as human serum albumin. In some embodiments, the half-life extension domain comprises the amino acid sequence of SEQ ID NO: 57.

[0345] In some embodiments, the half-life extension domain comprises a molecule capable of binding to serum albumin. For example, a peptide ligand that binds to human serum albumin may comprise the amino acid sequence of D-Xaa-CLP-Xaa-WGCLW (SEQ ID NO: 70) and, when administered to a subject, may be fused to a bispecific GlyTR to recruit HSA. Xaa is any amino acid. Peptide ligands that bind to mammalian serum albumin can be identified by their ability to compete with the peptide ligand for binding to human serum albumin in an in vitro assay. In some embodiments, the peptide ligand that binds to human serum albumin comprises the amino acid sequence of QGLIGDICLPRWGCLWGDSVK (SEQ ID NO: 71), RLIEDICLPRWGCLWEDD, (SEQ ID NO: 72), or EDICLPRWGCLWED (SEQ ID NO: 73).

[0346] In some embodiments, the half-life extension domain comprises a fatty acid chain conjugated polypeptide. The fatty acid chain can be selected from a C-16 fatty acid chain or a C-18 fatty acid chain. In some embodiments, the fatty acid chain is a C-16 fatty acid conjugated molecule. In some embodiments, the half-life extension domain comprises an antibody fragment that selectively binds serum albumin. In one embodiment, the antibody fragment is a single domain antibody, a complementary determining region (CDR) of a single domain antibody, or a single chain variable fragment (scFv).

[0347] 2. Antigen binding domain

[0348] In some embodiments, the antigen binding domain comprises a TACA binding domain derived from a lectin. In some embodiments, the lectin is selected from the group consisting of: a galectin, a sialic acid-binding immunoglobulin-like lectin, a selectin; a C-type lectin; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Phaseolus vulgaris leukoagglutinin); 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 (Phytolacca Americana lectin), wheat germ agglutinin (Wheat germ agglutinin), and lectin. agglutinin (wheat germ agglutinin); Artocarpus polyphemus lectin (jacalin letin); Vicia fragrans agglutinin (VVA); Snail agglutinin (HPA); Wisteria multiflora agglutinin (WFA); Sambucus nigra agglutinin (SNA); BC2L-CNt (lectin from the Gram-negative bacterium Burkholderia cenocepacia); Mulberry leukocyte agglutinin (MAL); Pleurotus eryngii agglutinin (PVL); Sclerotium sclerotiorum agglutinin (SRL); Eucheuma serrata agglutinin (ESA); CLEC17A (protectin); Dictyocystis aurantiacus agglutinin; Sessile elderberry agglutinin (SSA); Gleheda lectin; Morniga G agglutinin; Salvia miltiorrhiza agglutinin; bogotensis agglutinin, Salvia officinalis agglutinin, Dichroa haizhouensis agglutinin, Concha striata agglutinin, Griffonia suffruticosa (GsLA4), Quadrangularis bean (acidic WBAI), Abrus precatorius lectin, Amaranthus caudatus lectin, Ginkgo biloba lectin, Orchidaceae agglutinin, Jackfruit agglutinin, Citrus aurantium agglutinin, Jackfruit agglutinin, Dolichos biflorus agglutinin, Dolichos biflorus lectin, Soybean agglutinin, and Agaricus bisporus agglutinin.

[0349] 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 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.

[0350] 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), ppGalNAc-T10 (GALNT11), ppGalNAc-T12 (GALNT13), ppGalNAc-T14 (GALNT15), ppGalNAc-T15 (GALNT16), ppGalNAc-T16 (GALNT17), ppGalNAc-T17 (GALNT18), ppGalNAc-T19 (GALNT19), ppGalNAc-T10 (GALNT11), ppGalNAc-T11 (GALNT12), ppGalNAc-T12 (GALNT13), ppGalNAc-T13 (GALNT14), 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).

[0351] In some embodiments, the antigen binding domain of the fusion protein described herein selectively targets 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, Lewis-y (Lewis antigen), y ), sialyl-Lewisx / a, disialyl-Lewisx / a, sialyl 6-sulfoLexisx, 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 epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), GalNAcα-serine, GalNAcα-threonine, GalNAc, or GalNAcβ1.

[0352] In some embodiments, the antigen binding domain comprises one or more TACA binding domains. In one embodiment, the antigen binding domain comprises one, two, three, four, five, six, seven, eight, nine or ten TACA binding domains. In some embodiments, the TACA binding domains are operably connected via a linker. The linker may be a peptide linker, a non-peptide linker, a chemical unit, a hindered cross-linking agent, or a non-hindered cross-linking agent. In one embodiment, the linker is a peptide linker, such as a glycine serine 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 linker comprises an amino acid sequence selected from the group consisting of: GGGGS (SEQ ID NO: 86), GGGGSGGGGS (SEQ ID NO: 87), GGGGSGGGGSGGGGS (SEQ ID NO: 85), AEAAAKA (SEQ ID NO: 88), AEAAAKAAEAAAKA (SEQ ID NO: 89), and AEAAAKAAEAAAKAAEAAAKA (SEQ ID NO: 90). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 85 or 89.

[0353] In some embodiments, the antigen binding domain comprises the amino acid sequence shown in SEQ ID NOs: 33-56, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NOs: 33-56. In one embodiment, the antigen binding domain comprises an amino acid sequence having at least 90% homology to SEQ ID NOs: 33-56.

[0354] 3. Immune recognition domain

[0355] The fusion proteins disclosed herein can be bound to molecules expressed on the surface of immune effector cells (e.g., "effector cell proteins") and to another molecule expressed on the surface of target cells (e.g., "target cell proteins"). Immune effector cells can be T cells, NK cells, macrophages, or neutrophils. In some embodiments, the effector cell protein is a protein contained in a T cell receptor (TCR)-CD3 complex. The TCR-CD3 complex is a heteromultimer comprising a heterodimer comprising TCRα and TCRβ or TCRγ and TCRδ plus various CD3 chains from CD3 zeta (CD3ζ) chain, CD3epsilon (CD3ε) chain, CD3 gamma (CD3γ) chain, and CD3 delta (CD3δ) chain. In some embodiments, the effector cell protein can be a human CD3 epsilon (CD3ε) chain, which can be part of a multimeric protein. Alternatively, the effector cell protein can be human and / or cynomolgus monkey TCRα, TCRβ, TCRδ, TCRγ, CD3 beta (CD3β) chain, CD3gamma (CD3γ) chain, CD3 delta (CD3δ) chain or CD3 zeta (CD3ζ) chain. In some embodiments, the immune cell recognition domain can comprise the amino acid sequence of CD3 or SEQ ID NO: 75-77.

[0356] Furthermore, in some embodiments, the fusion proteins disclosed herein may also bind to CD3 epsilon chains from non-human species, such as mouse, rat, rabbit, New World monkey, and / or Old World monkey species. Such species include, but are not limited to, the following mammalian species: Mus musculus, Rattus rattus, Rattus norvegicus, cynomolgus monkey, Macaca fascicularis, hamadryas baboon, Papio hamadryas, Guinea baboon, Papio papio, olive baboon, Papio anubis, yellow baboon, Papio cynocephalus, Chacma baboon, Papio ursinus, Callithrix jacchus, Saguinus oedipus, and squirrel monkey. As is known in the field of protein therapeutic development, having a therapeutic agent with comparable activity in humans and species commonly used for preclinical testing (such as mice and monkeys) can simplify and speed up drug development. Such advantages are crucial in the long and expensive process of bringing a drug to market.

[0357] In a more specific embodiment, the heterodimeric bispecific antibody can bind to an epitope within the first 27 amino acids of the CD3 epsilon chain, which can be human CD3 epsilon or a CD3 epsilon chain from a different species, particularly one of the mammalian species listed above. The advantages of antibodies that bind to this epitope are described in detail in U.S. Patent Application Publication No. 2010 / 183615, the relevant portions of which are incorporated herein by reference. The epitope to which the antibody binds can be determined by alanine scanning, as described, for example, in U.S. Patent Application Publication No. 2010 / 183615, the relevant portions of which are incorporated herein by reference.

[0358] When T cells are immune effector cells, the effector cell proteins that the fusion proteins disclosed herein can bind to include, but are not limited to, CD3ε chain, CD3γ, CD3δ chain, CD3ζ chain, TCRα, TCRβ, TCRγ, and TCRδ. When NK cells or cytotoxic T cells are immune effector cells, NKG2D, CD352, NKp46, or CD16a can, for example, be effector cell proteins. When CD8 +When T cells are immune effector cells, 4-1BB or NKG2D, for example, can be effector cell proteins. Alternatively, the fusion proteins disclosed herein can bind to other effector cell proteins expressed on T cells, NK cells, macrophages, or neutrophils.

[0359] a. Target cells

[0360] As described above, the fusion proteins disclosed herein can bind to effector cell proteins and target cell proteins. For example, target cell proteins can be expressed on the surface of cancer cells, cells infected by pathogens, or cells that mediate diseases (e.g., inflammatory diseases, autoimmune diseases, and / or fibrotic diseases). In some embodiments, target cell proteins can be highly expressed on target cells, although high levels of expression are not necessarily required.

[0361] When the target cell is a cancer cell, the fusion protein as described herein can be bound to a cancer cell antigen as described above. The cancer cell antigen can be a human protein or a protein from another species. For example, the fusion protein can be bound to a target cell protein from a mouse, rat, rabbit, New World monkey and / or Old World monkey species. Such species include, but are not limited to, the following species: house mouse, black mouse, brown rat, cynomolgus monkey, Macaca fascicularis, Hamadryas baboon, Papio hamadryas, Guinea baboon, Papio papio, olive baboon, East African baboon, yellow baboon, grassland baboon, Chakma baboon, pig-tailed baboon, common marmoset, cashmere-top tamarin; and squirrel monkey.

[0362] In other aspects, the target cell can be a cell that mediates an autoimmune disease or an inflammatory disease. For example, human eosinophils in asthma can be target cells, in which case, for example, a mucin-like hormone receptor (EMR1) containing an EGF-like module can be a target cell protein. Alternatively, excessive human B cells in systemic lupus erythematosus patients can be target cells, in which case, for example, CD19 or CD20 can be target cell proteins. In other autoimmune diseases, excessive human Th2T cells can be target cells, in which case, for example, CCR4 can be a target cell protein. Similarly, the target cell can be a fibrotic cell that mediates a disease such as atherosclerosis, chronic obstructive pulmonary disease (COPD), cirrhosis, scleroderma, renal transplant fibrosis, renal transplant nephropathy or pulmonary fibrosis, including idiopathic pulmonary fibrosis and / or idiotype pulmonary hypertension. For such fibrotic diseases, for example, fibroblast activation protein alpha (FAPα) can be a target cell protein.

[0363] b. Target Cell Cytolysis Assay

[0364] In the following examples, a method for determining whether a fusion protein as described herein can induce immune effector cells to dissolve target cells in vitro is described. In this assay, immune effector cells are T cells. When immune effector cells are NK cells, the following very similar assays can be used. Target cell lines expressing target cell proteins of interest can be labeled with 2 μM carboxyfluorescein succinimidyl ester (CFSE) at 37 ° C for 15 minutes and then washed. The target cells of an appropriate number of markers can then be incubated at 4 ° C for 40 minutes in one or more 96-well flat-bottomed culture plates, containing or not containing bispecific proteins, control proteins, or without adding different concentrations of protein. Miltenyi NK cell separation kit II (Miltenyi Biotec, Auburn, Calif.) can be used to separate NK cells isolated from healthy human donors, and then with an effector cell of 10: 1: target cell ratio is added to the target cells. NK cells are immune effector cells in this assay and can be used immediately after separation or after overnight culture at 37 ° C. Plates containing tumor target cells, fusion proteins (e.g., bispecifics), and immune effector cells can be cultured at 37°C under 5% CO2 for 18-24 hours. Appropriate control wells can also be set up. After the 18-24 hour detection period, all cells can be removed from the wells. A volume of 7-AAD solution equal to the volume of the well contents can be added to each sample. The samples can then be assayed to determine the percentage of live and dead target cells by flow cytometry, as described in the Examples below.

[0365] 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, the 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.

[0366] 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 the amino acid sequence of SEQ ID NO: 59, 60 or 61. In some embodiments, the immune cell recognition domain may comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 59, 60 or 61.

[0367] Alternatively, the immune cell recognition domain comprises an antibody Fc domain. In some embodiments, the 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 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 the constant region domain CH2 and / or CH3 of an antibody. In one embodiment, the constant region is preferably CH2 and CH3. The constant region domain CH2 and / or CH3 of an antibody may or may not include a hinge region.

[0368] In some embodiments of the nucleic acids disclosed herein, the encoded 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. In one embodiment, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 69 or 91-94. In one embodiment, the half-life extending domain comprises an Fc domain of an antibody selected from the group consisting of IgG1, IgG2, IgG3, or IgG4 Fc regions.

[0369] B. Nucleic Acids

[0370] The isolated nucleic acid sequence encoding the bispecific fusion protein of the present disclosure can be obtained using any of a number of 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.

[0371] 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.

[0372] The nucleic acid molecules of the present invention can be modified to improve their stability in serum or in the 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 invention. For example, in order to enhance stability, 3'-residues can be stabilized to prevent degradation, for example, they can be selected so that they are composed of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, it is tolerable to replace pyrimidine nucleotides with modified analogs (e.g., replacing uridine with 2'-deoxythymidine) and do not affect the function of the molecule. In one embodiment of the present disclosure, the nucleic acid molecules can contain at least one modified nucleotide analog. For example, the end can be stabilized by incorporating modified nucleotide analogs.

[0373] 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 selected from H, OR, R, halo, SH, SR, NH , NHR, NR , or ON group replacement, wherein R is C -C alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

[0374] Examples of other modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides containing at least one non-naturally occurring nucleobase in place of a naturally occurring nucleobase. The base can 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 can be combined.

[0375] For example, nucleic acid molecules include at least one of the following chemical modifications: 2'-H, 2'-O-methyl or 2'-OH modifications of one or more nucleotides. In certain embodiments, nucleic acid molecules of the present disclosure may have enhanced nuclease resistance. In order to increase nuclease resistance, 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, 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) (for example, 2'-4'-ethylene-bridged nucleic acid) and certain core base modifications (such as 2-amino-A, 2-thio (for example, 2-thio-U), G-clamp modifications) can also increase the binding affinity to the target.

[0376] In one embodiment, the nucleic acid molecule includes 2'-modified nucleotides, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-MA). In one embodiment, the nucleic acid molecule includes at least one 2'-O-methyl modified nucleotide, and in some embodiments, all nucleotides of the nucleic acid molecule include a 2'-O-methyl modification.

[0377] 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 polymers of RNA and DNA that have been modified (e.g., to improve efficacy) and nucleoside surrogates.

[0378] 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 nucleic acid components naturally occurring in humans. Rare or uncommon but naturally occurring RNAs are referred to in the art as modified RNAs, see, for example, 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 that 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 nucleic acids disclosed herein can be present at one or more of a phosphate group, a sugar group, a backbone, an N-terminal, a C-terminal or a core base.

[0379] C. Expression vector

[0380] In one aspect, the present disclosure provides an expression construct comprising an isolated nucleic acid encoding a fusion protein disclosed herein. In some embodiments of the present disclosure, the isolated nucleic acid described herein comprises an expression vector. In another embodiment, the isolated nucleic acid comprises in vitro transcribed RNA. In another embodiment, the expression construct comprises an isolated nucleic acid encoding a fusion protein described herein.

[0381] The expression of natural or synthetic nucleic acids encoding the peptides of the present invention is generally achieved by operably linking the nucleic acid encoding the peptide or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector to be used is suitable for replication and optionally integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating expression of the desired nucleic acid sequence.

[0382] 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, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present disclosure provides a gene therapy vector.

[0383] 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.

[0384] 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 a replication start point, a promoter sequence, a convenient restriction endonuclease site 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.

[0385] 1. Virus-based systems

[0386] 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.

[0387] 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 gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of a subject in vivo or ex vivo. 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.

[0388] 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 the 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, compositions includes the 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, minimum 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.

[0389] 2. Control elements

[0390] In some embodiments, the vector further comprises conventional control elements that are operably linked 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 herein. As used herein, an "operably linked" sequence includes 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.

[0391] The frequency of transcription initiation is regulated by additional promoter elements (e.g., enhancers). Typically, they are located in the region 30bp-110bp upstream of the start site, but recently multiple promoters have been shown to contain functional elements downstream of the start site. The interval between promoter elements is often flexible, and therefore still retains promoter function when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, before activity begins to decline, the interval between promoter elements can be increased to 50bp. Depending on the promoter, each element may act collaboratively or independently to activate transcription.

[0392] 3. Promoter

[0393] In some embodiments, the expression construct further comprises a promoter. The promoter can be selected from: 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, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter or Rous sarcoma virus promoter.

[0394] 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 can 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, 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, expression construct disclosed herein should not limit the use of constitutive promoters. Inducible promoters are also considered as a part of the present disclosure. 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 turn off expression when not expecting this expression. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0395] Enhancer sequences found on vectors also regulate the expression of genes contained in the vector. Typically, enhancers bind to protein factors to enhance the transcription of genes. Enhancers may be located upstream or downstream of the gene they regulate. Enhancers may also be tissue-specific to enhance transcription in specific cell or tissue types. In one embodiment, the vectors of the present disclosure contain one or more enhancers to enhance the transcription of genes present in the vector.

[0396] 4. Selectable markers

[0397] In order to assess 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 may be flanked by appropriate regulatory sequences to achieve expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo etc.

[0398] Reporter gene is used to identify the cells of potential transfection and to evaluate the function of regulatory sequences. In general, reporter gene is a gene that is not present in a receptor organism or tissue or is not expressed by a receptor organism or tissue, and the expression of the polypeptide encoded by the reporter gene is expressed as some easily detected 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 can include a gene encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase (chloramphenicol acetyl transferase), secretory alkaline phosphatase or a green fluorescent protein gene (for example, Ui-Tei et al., 2000FEBS Letters 479:79-82). Suitable expression system is well known, and known technology can be used to prepare or commercially available. In general, the construct with minimum 5' flanking region showing the highest level of reporter gene expression is identified as a promoter. This type of promoter region can be connected with a reporter gene and used to assess the ability of the promoter driven by the agent.

[0399] V. Modified cells

[0400] One aspect of the present disclosure provides genetically modified (e.g., engineered) cells comprising and stably expressing the subject fusion protein of the present disclosure. In one aspect, the modified cells comprise an isolated nucleic acid encoding a fusion protein comprising an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), an immune cell recognition domain that specifically binds to a receptor on an immune effector cell, and a half-life extension domain. In some embodiments, the half-life extension domain is a polypeptide that is capable of extending the half-life of the fusion protein.

[0401] In some embodiments, the modified cell comprises a 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.

[0402] In some embodiments, the modified cell is a genetically modified immune cell (e.g., T cell) or a precursor thereof comprising a fusion protein with affinity for a tumor-associated carbohydrate antigen (TACA) as described herein. In some embodiments, the fusion protein comprises an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA); an immune cell recognition domain that specifically binds to a receptor on an immune effector cell; and a half-life extension domain, wherein the half-life extension domain is a polypeptide that is capable of extending the half-life of the fusion protein.

[0403] In some embodiments, the genetically modified immune cells (e.g., T cells) or precursor cells thereof of the present disclosure comprise a bispecific fusion protein having affinity for: β1,6 branching, β1,6GlcNAc branched N-glycans, 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-sulfoLewis 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 disclosure comprise a bispecific fusion protein having affinity for β1,6 branched 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 disclosure comprise a bispecific fusion protein having affinity for Tn antigen or sialyl-Tn epitope.

[0404] A. Host cells

[0405] 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 insect cells.

[0406] 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.

[0407] B. Modified immune cells

[0408] 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, natural killer (NK) cells, cytotoxic T lymphocytes (CTL) and regulatory T cells. In some embodiments, the modified cells are T cells. In certain embodiments, the genetically modified cells are natural killer (NK) cells. In certain embodiments, the genetically modified cells are NKT cells. In some embodiments, the modified cells are autologous cells, xenogeneic cells or allogeneic cells.

[0409] 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.

[0410] In some embodiments, a T cell, natural killer (NK) cell, cytotoxic T lymphocyte (CTL), or regulatory T cell comprises a fusion protein disclosed herein and further comprises a chimeric antigen receptor (CAR) that selectively or specifically binds a tumor-associated carbohydrate antigen (TACA).

[0411] In some embodiments, the CAR comprises: an antigen binding domain selected from the group consisting of SEQ ID NOs: 33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NOs: 33-56; a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0412] In some embodiments, the transmembrane domain of CAR includes the transmembrane region of the 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 the CD8 transmembrane domain. In some embodiments, 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 combinations thereof. In some embodiments, the costimulatory domain includes a 4-1BB costimulatory domain, a CD28 costimulatory domain; or a 4-1BB costimulatory domain and a CD28 costimulatory domain.

[0413] In some embodiments, 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, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain comprises a CD3 ζ signaling domain.

[0414] In one aspect, the present disclosure provides a modified immune cell population as described herein. In some embodiments, the modified cell is a CAR T cell. In this embodiment, the CAR T cell specifically targets tumor-associated carbohydrate antigens (TACA).

[0415] C. Methods for Generating Modified Immune Cells

[0416] In one aspect, the present disclosure provides a method for producing a modified cell disclosed herein, the method comprising introducing into a cell an isolated nucleic acid encoding a fusion protein or an expression construct disclosed herein.

[0417] In another aspect, the present disclosure provides a method for producing a modified cell as described herein, the method comprising: introducing an isolated nucleic acid as described herein, a fusion protein as described herein; or an expression construct as described herein into a cell; culturing the cell in a culture medium under conditions that induce expression of the fusion protein encoded by the nucleic acid or expression construct; and recovering the fusion protein from the cell mass or the culture medium.

[0418] Fusion proteins disclosed herein can be prepared using methods well known in the art. For example, nucleic acids encoding one or two polypeptide chains of the fusion protein can be introduced into cultured host cells by a variety of known methods, such as, for example, transformation, transfection, electroporation, bombardment with nucleic acid-coated microparticles. In some embodiments, the nucleic acid encoding the fusion protein can be inserted into a vector suitable for expression in the host cell before being introduced into the host cell. Typically, such vectors can contain sequence elements that allow the inserted nucleic acid to be expressed at the RNA and protein levels. Such vectors are well known in the art, and many are commercially available. Host cells containing nucleic acids can be cultured under conditions that allow the cells to express the nucleic acid, and the resulting fusion protein can be collected from cell mass or culture medium. Alternatively, the fusion protein can be produced in vivo, for example, in plant leaves (see, e.g., Scheller et al. (2001), Nature Biotechnol. 19:573-577 and references cited therein), bird eggs (see, e.g., Zhu et al. (2005), Nature Biotechnol. 23:1159-1169 and references cited therein), or mammalian milk (see, e.g., Laible et al. (2012), Reprod. Fertil. Dev. 25(1):315).

[0419] Modified cells (e.g., cells comprising a subject bispecific fusion protein) can be generated by stably transfecting a host cell with an expression vector comprising 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, dendrimers, liposomes, and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer, and / or hydrodynamic delivery), and / or particle-based methods (e.g., puncture transfection, using a gene gun, and / or magnetic transfection). Transfected cells expressing the subject fusion protein of the present disclosure can be expanded ex vivo.

[0420] In some embodiments, cells are genetically modified by contacting them with isolated nucleic acids encoding 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 methods used can be for any purpose requiring stable expression or sufficient stable expression.

[0421] 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 a carrier or using cell-free local or systemic delivery of encapsulated, bound or naked mRNA. The method used can be for any purpose requiring transient expression or for which transient expression is sufficient.

[0422] 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, about the receptor, the cell is autologous, allogeneic, isogenic or xenogeneic.

[0423] The disclosed compositions and methods can be applied to modulation of T 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 T cells to kill target cancer cells.

[0424] 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 readily 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.

[0425] 1. Physical methods

[0426] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for generating 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.

[0427] 2. Biological methods

[0428] 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 lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0429] In some embodiments, nucleic acid encoding the subject fusion protein of the present disclosure is introduced into cells by expression vectors. Provided herein are expression vectors comprising nucleic acids encoding subject fusion proteins (e.g., TACA tri-specific fusions). Suitable expression vectors include lentiviral vectors, gamma retroviral vectors, foamy viral 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 integrases, such as Phi31. Some other suitable expression vectors include herpes simplex virus (HSV) and retroviral expression vectors.

[0430] Adenoviral expression vectors are based on adenovirus, which has a low ability to integrate into genomic DNA but a high efficiency in transfecting host cells. The adenoviral expression vector contains sufficient adenoviral sequences to: (a) support the packaging of the expression vector, and (b) ultimately express the subject fusion protein (e.g., a trispecific fusion protein) in the host cell. In some embodiments, the adenoviral genome is 36 kb, linear, double-stranded DNA, into which foreign DNA sequences (e.g., nucleic acids encoding TACA fusion proteins) can be inserted to replace large fragments of adenoviral DNA in order to prepare the expression vectors of the present disclosure. See, Danthinne and Imperiale, Gene Therapy 7(20):1707-1714 (2000).

[0431] 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 that 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.

[0432] Retroviral expression vectors can be integrated into the host genome, delivering large amounts of foreign genetic material, infecting a wide range of species and cell types, and being packaged in special cell lines. Retroviral vectors are constructed by inserting nucleic acids (e.g., nucleic acids encoding TACA fusion proteins) into certain positions of the viral genome to produce replication-defective viruses. Although retroviral vectors can infect a wide variety of cell types, the integration and stable expression of the subject fusion protein require the division of host cells.

[0433] Lentiviral vectors are derived from lentiviruses, which are complex retroviruses that, in addition to the common retroviral genes gag, pol and env, also contain other genes with regulatory or structural functions. 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 of HIV virulence genes, for example, by deleting genes env, vif, vpr, vpu and nef, making the vector biologically safe. Lentiviral vectors are able to infect non-dividing cells and can be used for, for example, in vivo and ex vivo gene transfer and expression of nucleic acids encoding subject fusion proteins.

[0434] Expression vectors comprising the 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, or the like. Prior to introduction of the expression vector, the host cells can be grown and expanded in culture, followed by appropriate treatment to introduce and integrate the vector. The host cells can then be expanded and screened for markers present in the vector.

[0435] 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 an NKT cell.

[0436] 3. Chemical methods

[0437] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres (microspheres), beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0438] In the case of utilizing non-viral delivery system, exemplary delivery vehicle is liposome. Consider using lipid formulations for nucleic acid to be introduced into host cell (in vitro, in vitro or in vivo). In another aspect, nucleic acid can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of liposome, dispersed in the lipid bilayer of liposome, connected to liposome via the linker molecule that all associates with liposome and oligonucleotide, be wrapped in liposome, with liposome compound, be dispersed in the solution containing lipid, mix with lipid, combine with lipid, be included in lipid as suspension, be included in micelle or with micelle compound or otherwise associate with lipid. The composition that lipid, lipid / DNA or lipid / expression vector are relevant is not limited to any specific structure in solution. For example, they can exist with double-layer structure, micelle or " folding " structure. They also can only be dispersed in solution, may form aggregates of size or shape inhomogeneity. Lipid is a fatty substance, and it can be naturally occurring lipid or synthetic lipid. For example, lipids include the 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.

[0439] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoylphosphatidylcholine ("DMPC") is available from Sigma, St. Louis, MO; dihexadecyl phosphate ("DCP") is available from K&K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other lipids are available 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 sole solvent because it evaporates more easily than methanol. "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar 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. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of aqueous solution, they form spontaneously. The lipid components undergo self-rearrangement before forming a closed structure and 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.

[0440] Regardless of the method used to introduce exogenous nucleic acid into the host cell, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. 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 disclosure.

[0441] 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.

[0442] 4. RNA

[0443] RNA offers several advantages over more traditional plasmid or viral approaches. Gene expression from RNA sources does not require transcription, and protein product is rapidly generated following transfection. Furthermore, because the RNA only needs to enter the cytoplasm rather than the nucleus, typical transfection methods result in extremely high transfection rates. Furthermore, plasmid-based approaches require that the promoter driving expression of the gene of interest be active in the cells being studied.

[0444] One advantage of the RNA transfection method disclosed herein is that RNA transfection is essentially instantaneous and does not require a vector. RNA transgenes can be delivered to lymphocytes after brief in vitro cell activation and expressed therein as minimal expression cassettes without the need for any additional viral sequences. Under these conditions, transgene integration into the host cell genome is unlikely. Due to the transfection efficiency of RNA and its ability to uniformly modify an entire lymphocyte population, cell cloning is not necessary.

[0445] Genetic modification of host cells with in vitro transcribed RNA (TVT-RNA) utilizes two distinct strategies, both of which have been tested in various animal models. Cells are transfected with in vitro transcribed RNA by lipofection or electroporation. Various modifications are contemplated to stabilize IVT-RNA in order to achieve prolonged expression of the transferred IVT-RNA. Several IVT vectors are known in the literature that serve as templates for in vitro transcription in a standardized manner and that have been genetically modified to produce stable RNA transcripts.

[0446] The scheme 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 result of the structure of this procedure, some nucleotides remain as part of the restriction enzyme cleavage site after linearization and extend or mask the poly (A) sequence at the 3' end. It is also unclear whether this non-physiological overhang will affect the amount of protein produced by this construct in the cell.

[0447] In one embodiment, the isolated nucleic acid encoding the bispecific fusion protein of the present invention and introduced into the cell of the present invention comprises RNA. In one embodiment, the RNA is mRNA. In one embodiment, the 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 the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other appropriate source of DNA.

[0448] 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. Exemplary artificial DNA sequences are sequences that contain gene portions that are 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.

[0449] Genes that can be used as DNA sources for PCR include genes that encode polypeptides that provide therapeutic or preventive effects to organisms or can be used to diagnose diseases or conditions of organisms. Preferred genes are genes that can be used for short-term treatments, or genes that have safety issues with the dosage or expression of the gene. For example, for the treatment of cancer, autoimmune disorders, parasites, viruses, bacteria, fungi or other infections, the transgenic gene to be expressed can encode a polypeptide that acts as a ligand or receptor for immune system cells, or can stimulate or suppress the immune system of the organism. In some embodiments, it is not desirable to perform prolonged continuous stimulation of the immune system, nor is it desirable to produce continuous changes after successful treatment, as this may cause new problems. For the treatment of autoimmune disorders, it may be desirable to suppress or block the immune system during an attack, but it cannot be suppressed for a long time, as long-term suppression may cause the patient to be overly sensitive to infection.

[0450] Use PCR to generate a template for in vitro mRNA transcription for transfection. The method for performing PCR is well known in the art. The primers used for PCR are designed to have a region that is 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' UTRs. 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' UTRs. Primers that can be used for PCR are generated by synthetic methods well known in the art.

[0451] A "forward primer" is a primer that contains a nucleotide region that is substantially complementary to nucleotides on a DNA template upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to a position 5' of the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that contains a nucleotide region that is substantially complementary to a double-stranded DNA template downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to a position 3' of the DNA sequence to be amplified relative to the coding strand.

[0452] Any DNA polymerase that can be used for PCR can be used in the methods disclosed herein. Reagents and polymerases are commercially available from a variety of sources. Chemical structures that have the ability to promote stability and / or translation efficiency can also be used. The 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 varied by various methods, including but not limited to designing PCR primers that anneal to different UTR regions. Using this method, one of ordinary skill 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 the naturally occurring endogenous 5' and 3' UTRs of the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by making any other modifications to the template. Using UTR sequences that are not endogenous to the gene of interest can be used to modify the stability and / or translation efficiency of the RNA. For example, it is known that AU-rich elements in the 3' UTR sequence can reduce the stability of the mRNA. Therefore, 3'UTRs can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs well known in the art.

[0453] In one embodiment, the 5'UTR may contain a 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 a 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 RNAs 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 the 3' or 5'UTR to prevent exonucleases from degrading the mRNA.

[0454] In order 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 of the promoter for 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.

[0455] 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 the 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 beyond the last base of the template. Schenborn and Mierendorf, Nuc Acids Res., 13: 6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003).

[0456] 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 cloning procedures not only laborious and time-consuming, but also often unreliable. Therefore, 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.

[0457] 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 (which can range in size from 50T to 5000T), or can be generated after PCR by any other method, including but not limited to DNA ligation or in vitro recombination. The poly(A) tail also provides stability to 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.

[0458] 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 can be attached to the 3' end to increase the stability of the mRNA. This connection can contain modified / artificial nucleotides, aptamers, and other compounds. For example, poly (A) polymerase can be used to incorporate ATP analogs into the poly (A) tail. ATP analogs can further increase the stability of the RNA. The 5' cap also provides stability for 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)).

[0459] 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 of the mRNA and promotes the initiation of translation. Any solute suitable for cell electroporation may be included, which may contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0460] 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.), a 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).

[0461] In some embodiments, RNA is electroporated into the cell, such as in vitro transcribed RNA. The preparation and methodology of nucleic acid construct electroporation into mammalian cells are taught in, for example, US2004 / 0014645, US2005 / 0052630A1, US2005 / 0070841 A1, US 2004 / 0059285A1, US2004 / 0092907A1. The various parameters (including electric field strength) required for the electroporation of any known cell type are typically known in the numerous patents and applications of the relevant research literature and the field. See, for example, U.S. Patent number 6,678,556, U.S. Patent number 7,171,264 and U.S. Patent number 7,173,116. Devices for the therapeutic application of electroporation are commercially available, such as the MedPulser TMDNA electroporation therapy system (Inovio / Genetronics, San Diego, Calif.), and described in patents such as U.S. Pat. No. 6,567,694; U.S. Pat. No. 6,516,223, U.S. Pat. No. 5,993,434, U.S. Pat. No. 6,181,964, U.S. Pat. No. 6,241,701, and U.S. Pat. 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 into cells in vitro.

[0462] 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 skilled in the art, presents an exciting new way to deliver RNA of interest to target cells.

[0463] 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 varying, for example, the promoter or the amount of input RNA, allowing expression levels to be individually modulated. In addition, PCR-based mRNA production technology greatly facilitates the design of mRNAs with different structures and their domain combinations.

[0464] C. Sources of Immune Cells

[0465] Prior to amplification, the immune cell source obtained from the subject is used for ex vivo manipulation. The target cell source for ex vivo manipulation may also include, for example, autologous or allogeneic donor blood, umbilical cord blood, or bone marrow. For example, the source of the immune cells may be from a subject to be treated with the modified immune cells of the present disclosure, such as the subject's blood, the subject's umbilical cord blood, or the subject's bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. In certain exemplary embodiments, the subject is a human.

[0466] 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, for example myeloid cells or l...

Claims

1. An isolated nucleic acid molecule encoding a fusion protein comprising: (i) an antigen binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA); (ii) an immune cell recognition domain that specifically binds to a receptor on an immune effector cell; and (iii) a half-life extension domain, wherein the half-life extension domain is a polypeptide capable of extending the half-life of the fusion protein.

2. The isolated nucleic acid molecule of claim 1, wherein the half-life extension domain is located at the N-terminus or the C-terminus of the fusion protein.

3. The isolated nucleic acid molecule of claim 1 or 2, wherein the half-life extension domain comprises a molecule selected from the group consisting of: a polypeptide capable of binding to albumin, albumin, serum albumin, an Fc domain of an antibody, a polyethylene glycol moiety (PEG), a poly(lactic-co-glycolic acid) (PLGA) copolymer, a polymer hydrogel, a nanoparticle, a fatty acid chain, an acyl group, a myristic acid group, a palmitoylated group, and a steryl group.

4. The isolated nucleic acid molecule of any one of claims 1 to 3, wherein the half-life extension domain comprises: (i) an Fc domain of an antibody selected from the group consisting of an IgG1, IgG2, IgG3 or IgG4 Fc region; or (ii) a PEG moiety, wherein the PEG moiety is less than about 0.5k, less than about 1.0k, less than about 2.0k, less than about 3.0k, less than about 4.0k, less than about 5.0k, less than about 6.0k, less than about 7.0k, less than about 6.0k, less than about 7.0k, less than about 8.0k, less than about 10.0k, less than about 12.0k, less than about 14.0k, less than about 16.0k, less than about 18.0k, or less than about 20.0k.

5. The isolated nucleic acid molecule of any one of claims 1 to 3, wherein the half-life extension domain comprises: (i) a molecule capable of binding to serum albumin; (ii) a polypeptide comprising an amino acid sequence of D-Xaa-CLP-Xaa-WGCLW (SEQ ID NO: 70), QGLIGDICLPRWGCLWGDSVK (SEQ ID NO: 71), RLIEDICLPRWGCLWEDD, (SEQ ID NO: 72) or EDICLPRWGCLWED (SEQ ID NO: 73), optionally wherein Xaa is any amino acid; (iii) a fatty acid chain-conjugated polypeptide, wherein the fatty acid chain is selected from a C-16 fatty acid chain or a C-18 fatty acid chain; (iv) a C-16 fatty acid conjugated molecule; or (v) an antibody fragment that selectively binds serum albumin, optionally a single domain antibody, a CDR of a single domain antibody, or a single chain variable fragment (scFv).

6. The isolated nucleic acid molecule of any one of claims 1 to 3, wherein the half-life extension domain comprises a serum albumin polypeptide.

7. The isolated nucleic acid molecule of claim 6, wherein the serum albumin is human serum albumin.

8. The isolated nucleic acid molecule of claim 6 or 7, wherein the half-life extension domain comprises the amino acid sequence of SEQ ID NO:

57.

9. The isolated nucleic acid molecule of any one of claims 1 to 8, wherein the half-life of the fusion protein is enhanced by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 16-fold, at least about 18-fold, or at least about 20-fold compared to a fusion protein lacking the half-life extension domain.

10. The isolated nucleic acid molecule of any one of claims 1 to 9, wherein the half-life extension is based on the mean plasma residence time of the fusion protein.

11. The isolated nucleic acid molecule of any one of claims 1 to 10, wherein the antigen binding domain comprises a TACA binding domain derived from a lectin.

12. The isolated nucleic acid molecule of any one of claims 1 to 11, wherein the antigen binding domain comprises more than one TACA binding domain.

13. The isolated nucleic acid molecule of any one of claims 1 to 12, wherein the antigen binding domain comprises two, three, four, five, six, seven, eight, nine or ten TACA binding domains.

14. The isolated nucleic acid molecule of claim 12 or 13, wherein the TACA binding domain is operably linked via a linker.

15. The isolated nucleic acid molecule of claim 14, 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.

16. The isolated nucleic acid molecule of claim 15, wherein the linker is a peptide linker.

17. The isolated nucleic acid molecule of claim 15 or 16, 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.

18. The isolated nucleic acid molecule of claim 16 or 17, wherein the peptide linker is a glycine-serine linker.

19. The isolated nucleic acid molecule of any one of claims 15 to 18, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:

90.

20. The isolated nucleic acid molecule of any one of claims 15 to 19, wherein the linker comprises the amino acid sequence of SEQ ID NO:

85.

21. The isolated nucleic acid molecule of any one of claims 15 to 20, wherein the linker comprises the amino acid sequence of SEQ ID NO:

89.

22. The isolated nucleic acid molecule of any one of claims 1 to 21, wherein the antigen binding domain comprises at least two TACA binding domains from a lectin 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 (Phaseolus vulgaris leukocyte agglutinin); 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 agglutinin), wheat germ agglutinin (Wheat germ agglutinin). agglutinin (Triticum Vulgarislectin); Artocarpus polyphemus lectin (Jacalinletin); 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 lectin, Salvia salvia officinalis lectin, Changshan haizhouensis lectin, Concha floridula agglutinin, Griffonia suffruticosa lectin, Griffonia suffruticosa lectin (GsLA4), Quaternary bean (acidic WBAI), Abrus preparata lectin, Amaranthus caudatus lectin, Amaranthus gingkoi lectin, Lactobacillus lanceolata lectin, Artemisia jasminoides lectin, Citrus aurantium lectin, Jackfruit lectin, Dolichos biflorus agglutinin, Dolichos biflorus lectin, Soybean lectin, and Agaricus bisporus lectin.

23. The isolated nucleic acid molecule of any one of claims 1 to 22, wherein the antigen binding domain comprises the amino acid sequence shown in SEQ ID NOs:33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NOs:33-56.

24. The isolated nucleic acid molecule of any one of claims 1 to 22, wherein the antigen binding comprises an amino acid sequence having at least 90% homology to SEQ ID NOs: 33-56.

25. The isolated nucleic acid molecule of any one of claims 1 to 24, wherein the immune effector cell is 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 to 25, wherein the immune effector cell is a T cell.

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

28. The isolated nucleic acid molecule of any one of claims 1 to 27, wherein the immune cell recognition domain comprises: (i) an antibody Fc domain, optionally an Fc domain of an IgG molecule; (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 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 to 28, wherein the receptor on the immune effector cell is selected from the group consisting of: T cell receptor (TCR) α, TCR β, CD3, TCR γ, TCR δ, the invariant TCR from NKT cells, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1.

30. The isolated nucleic acid molecule of any one of claims 1 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 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: 59, 60 or 61; or (iii) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 59, 60 or 61.

32. The isolated nucleic acid molecule of any one of claims 1 to 31, wherein the encoded 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, optionally wherein the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 69 or 91-94.

33. The isolated nucleic acid molecule of any one of claims 1 to 32, wherein the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-32; or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-32.

34. The isolated nucleic acid molecule of any one of claims 1 to 33, wherein the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12.

35. The isolated nucleic acid molecule of any one of claims 1 to 34, wherein the isolated nucleic acid molecule encodes a fusion protein comprising the amino acid sequence of SEQ ID NO: 13-32.

36. The isolated nucleic acid molecule of claim 34 or 35, wherein: (a) the encoded fusion protein binds to tumor cells expressing β1,6GlcNAc branched N-glycans compared to a bispecific fusion protein comprising a flexible linker in the antigen binding domain; or (b) the encoded fusion protein binds to tumor cells expressing Thomsen-nouveau (Tn) antigen compared to a fusion protein comprising a flexible linker in the antigen binding domain.

37. An isolated nucleic acid molecule as described in claim 36, wherein the flexible linker is a glycine-serine linker or a linker comprising an amino acid sequence selected from SEQ ID NO:86, SEQ ID NO:87 or SEQ ID NO:85; or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:86, SEQ ID NO:87 or SEQ ID NO:

85.

38. The isolated nucleic acid molecule of any one of claims 1 to 37, wherein the encoded 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 antigen or 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.

39. The isolated nucleic acid molecule of any one of claims 1 to 39, wherein the encoded fusion protein selectively targets β1,6GlcNAc branched N-glycan, GalNAc, Tn antigen, GalNAcα-ser, GalNAcα-thr, GalNAc or GalNAcβ1.

40. The isolated nucleic acid molecule of any one of claims 1 to 37, wherein the isolated nucleic acid comprises an expression vector; and / or in vitro transcribed RNA.

41. A fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), wherein the fusion protein is encoded by the isolated nucleic acid of any one of claims 1 to 40.

42. A 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 NOs: 33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NOs: 33-56; (ii) an immune cell recognition domain that specifically binds to a receptor on an immune effector cell; and (iii) a half-life extension domain, wherein the half-life extension domain is a polypeptide capable of extending the half-life of the fusion protein.

43. The fusion protein of claim 42, wherein the half-life extension domain is located at the N-terminus or the C-terminus of the fusion protein.

44. The fusion protein of claim 42 or 43, wherein the half-life extension domain comprises a molecule selected from the group consisting of a polypeptide capable of binding to albumin, albumin, serum albumin, an Fc domain of an antibody, a polyethylene glycol moiety (PEG), a poly(lactic-co-glycolic acid) (PLGA) copolymer, a polymer hydrogel, a nanoparticle, a fatty acid chain, an acyl group, a myristic acid group, a palmitoylated group, and a steryl group.

45. The fusion protein of any one of claims 42 to 44, wherein the half-life extension domain comprises: (i) an Fc domain of an antibody selected from an IgG1, IgG2, IgG3 or IgG4 Fc domain; or (ii) a PEG moiety, wherein the PEG moiety is less than about 0.5k, less than about 1.0k, less than about 2.0k, less than about 3.0k, less than about 4.0k, less than about 5.0k, less than about 6.0k, less than about 7.0k, less than about 6.0k, less than about 7.0k, less than about 8.0k, less than about 10.0k, less than about 12.0k, less than about 14.0k, less than about 16.0k, less than about 18.0k, or less than about 20.0k.

46. ​​The fusion protein of any one of claims 42 to 44, wherein the half-life extension domain comprises: (i) a molecule capable of binding to serum albumin; (ii) a polypeptide comprising an amino acid sequence of D-Xaa-CLP-Xaa-WGCLW (SEQ ID NO: 70), QGLIGDICLPRWGCLWGDSVK (SEQ ID NO: 71), RLIEDICLPRWGCLWEDD, (SEQ ID NO: 72) or EDICLPRWGCLWED (SEQ ID NO: 73), optionally wherein Xaa is any amino acid; (iii) a fatty acid chain-conjugated polypeptide, wherein the fatty acid chain is selected from a C-16 fatty acid chain or a C-18 fatty acid chain; (iv) a C-16 fatty acid conjugated molecule; or (v) an antibody fragment that selectively binds serum albumin, optionally a single domain antibody, a CDR of a single domain antibody, or a single chain variable fragment (scFv).

47. The fusion protein of any one of claims 42 to 44, wherein the half-life extension domain comprises a serum albumin polypeptide.

48. The fusion protein of claim 47, wherein the serum albumin is human serum albumin.

49. The fusion protein of claim 47 or 48, wherein the half-life extension domain comprises the amino acid sequence of SEQ ID NO:

57.

50. The fusion protein of any one of claims 42 to 49, wherein the half-life of the fusion protein is enhanced by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 16-fold, at least about 18-fold, or at least about 20-fold compared to a fusion protein lacking the half-life extension domain.

51. The fusion protein of any one of claims 42 to 50, wherein the half-life extension is based on the mean plasma residence time of the fusion protein.

52. The fusion protein of any one of claims 42 to 51, wherein the immune cell recognition domain comprises: (i) an antibody Fc domain, optionally an Fc domain of an IgG molecule; (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 constant region domains CH2 and / or CH3, preferably CH2 and CH3, of an antibody, optionally with or without a hinge region.

53. The fusion protein of any one of claims 42 to 52, wherein the receptor on the immune effector cell is selected from the group consisting of: T cell receptor (TCR) α, TCR β, CD3, TCR γ, TCR δ, the invariant TCR from NKT cells, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1.

54. The fusion protein of any one of claims 42 to 53, wherein the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-32; 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: 1-32.

55. The fusion protein of any one of claims 42 to 54, wherein the 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 antigen or 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.

56. The fusion protein of any one of claims 42 to 55, wherein the fusion protein selectively targets Tn antigen or β1,6GlcNAc branched N-glycans.

57. The fusion protein of any one of claims 42 to 56, wherein the fusion protein that selectively targets a Tn antigen comprises an antigen binding domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-42, 52-56, or 62; 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: 36-42, 52-56, or 62.

58. The fusion protein of any one of claims 42 to 56, wherein the fusion protein that selectively targets a Tn antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-32; 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: 13-32.

59. The fusion protein of any one of claims 42 to 55, wherein the fusion protein that selectively targets β1,6GlcNAc branched N-glycans comprises an antigen binding domain having an amino acid sequence selected from SEQ ID NO: 33-35 or 43-51; or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 33-35 or 43-51.

60. The fusion protein of any one of claims 42 to 55, wherein the fusion protein that selectively targets β1,6GlcNAc branched N-glycans comprises an amino acid sequence selected from SEQ ID NOs: 1-12; or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1-12.

61. A 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 NOs: 33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NOs: 33-56; (ii) an immune cell recognition domain that specifically binds to CD3 on immune effector cells; and (iii) a half-life extension domain, wherein the half-life extension domain is a polypeptide capable of extending the half-life of the fusion protein.

62. A 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 NOs:33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NOs:33-56; and (ii) an Fc domain of an antibody, optionally wherein said Fc domain is said half-life extending domain.

63. The fusion protein of claim 61 or 63, wherein: (a) the half-life extension domain comprises the amino acid sequence of human serum albumin or SEQ ID NO: 57; (b) the Fc domain is an IgG molecule; or (c) the Fc domain comprises the amino acids of SEQ ID NO: 69 or 91-94.

64. An expression construct comprising the isolated nucleic acid of any one of claims 1 to 40.

65. The expression construct of claim 64, wherein the expression construct further comprises: (a) a promoter; or (b) a promoter 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, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter or Rous sarcoma virus promoter.

66. The expression construct of claim 64 or 65, 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.

67. The expression construct of any one of claims 64 to 66, wherein the expression construct is a lentiviral vector.

68. The expression construct of any one of claims 64 to 67, wherein the expression construct is a self-inactivating lentiviral vector.

69. A modified cell comprising the isolated nucleic acid of any one of claims 1 to 40, the fusion protein of any one of claims 41 to 63, or the expression construct of any one of claims 64 to 68.

70. The modified cell of claim 69, wherein: (a) the modified cell is selected from the group consisting of bacterial cells, fungal cells, insect cells or mammalian cells; (b) the modified cell is a bacterial cell selected from Escherichia coli or Bacillus stearothermophilus, (c) the modified cell is a fungal cell selected from yeast cells, Saccharomyces cerevisiae or Pichia pastoris; (d) the modified cell is an insect cell selected from Lepidoptera insect cells or Spodoptera frugiperda insect cells; or (e) The modified cells are mammalian cells 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.

71. The modified cell of claim 69 or 70, wherein the modified cell is a CHO cell or a HEK 293 cell.

72. The modified cell of claim 69, wherein the modified cell is: (a) a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL) or a regulatory T cell, optionally wherein the T cell, the natural killer (NK) cell, the cytotoxic T lymphocyte (CTL) or the regulatory T cell comprises a chimeric antigen receptor (CAR) that selectively or specifically targets a tumor antigen; or (b) CAR T cells that selectively or specifically target tumor antigens, Optionally, wherein 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, ICAMI, IL3Ra, IL13Ra2, LAGE-I, Lewis Y, LMPI (EBV), MAGE-Al, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUCI, Nectin4 / FAP, NKG2D-ligands (MIC-A, MIC-B and ULBPI to 6), NY-ESO-1, Pl 6. PD-LI, PSCA, PSMA, RORI, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2 and any combination thereof.

73. A method for generating a modified cell as claimed in any one of claims 64 to 71, the method comprising: (a) the isolated nucleic acid according to any one of claims 1 to 40; the fusion protein according to any one of claims 41 to 63; or an expression construct as described in any one of claims 64 to 69 is introduced into a cell; (b) culturing the cell in a culture medium under conditions that induce expression of the fusion protein encoded by the nucleic acid or the expression construct; as well as (c) recovering the fusion protein from the cell mass or the culture medium.

74. A composition comprising: (a) a fusion protein encoded by the isolated nucleic acid of any one of claims 1 to 40; (b) the fusion protein of any one of claims 41 to 63; (c) the modified cell of claim 72; or (d) A fusion protein encoded by the expression construct of any one of claims 64 to 69.

75. The composition of claim 74, further comprising a pharmaceutically acceptable carrier.

76. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an immunotherapeutic composition comprising: (a) a fusion protein encoded by the isolated nucleic acid of any one of claims 1 to 40; (b) the fusion protein of any one of claims 41 to 63; (c) the modified cell of claim 72; or (d) The composition of any one of claims 74 to 75.

77. The method of claim 76, 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.

78. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective composition comprising a modified cell comprising a fusion protein that selectively binds a tumor-associated carbohydrate antigen (TACA), wherein the fusion protein comprises: (i) an antigen binding domain selected from the group consisting of SEQ ID NOs: 33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 33-56; (ii) an immune cell recognition domain that specifically binds to a receptor on an immune effector cell; and (iii) a half-life extension domain, wherein the half-life extension domain is a polypeptide capable of extending the half-life of the fusion protein.

79. The method of claim 78, wherein the immune cell recognition domain specifically binds CD3.

80. The method of claim 78, wherein the immune cell recognition domain is: (a) an antibody Fc domain; or (b) Antibody Fc domain and a domain that specifically binds to CD3.

81. The method of any one of claims 78 to 80, wherein: (a) the half-life extension domain comprises the amino acid sequence of human serum albumin or SEQ ID NO: 57; or (b) the half-life extension domain comprises the Fc domain of an IgG molecule; or the amino acid sequence of SEQ ID NO: 69 or 91-94.

82. The method of any one of claims 78 to 81, wherein the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-32.

83. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective composition comprising a modified cell comprising a fusion protein that selectively binds a tumor-associated carbohydrate antigen (TACA), wherein the fusion protein comprises: (i) an antigen binding domain selected from the group consisting of SEQ ID NOs: 33-56; or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 33-56; and (ii) an Fc domain of an antibody, optionally wherein said Fc domain is said half-life extending domain.

84. The method of claim 83, wherein: (a) the Fc domain is an IgG molecule; or (b) the Fc domain comprises the amino acid sequence of SEQ ID NO: 69 or 91-94.

85. A method of providing anti-tumor immunity in a mammal, comprising administering to the mammal a therapeutically effective amount of: (a) a fusion protein encoded by the isolated nucleic acid of any one of claims 1 to 40 or the expression vector of any one of claims 64 to 69; (b) the fusion protein of any one of claims 41 to 63; (c) the modified cell population of claim 72; or (d) The composition of any one of claims 74 to 75.

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