Improved bispecific proteins for glycan-dependent immunotherapy with longer half-lives

Abstract

Compositions and methods are provided for treating diseases associated with abnormal glycosylation of cell surface molecules and the expression of tumor-associated carbohydrate antigens (TACAs). Also provided are a fusion protein specific for a tumor-associated carbohydrate antigen (TACA) comprising a half-life extender molecule, a vector encoding the TACA fusion protein, and a recombinant cell comprising the TACA-specific fusion protein.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 351,634, filed on June 13, 2022, which is hereby incorporated by reference in its entirety for all purposes.

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

[0003] This disclosure generally relates to the fields of pharmacology and immunology, and more specifically to bispecific fusion proteins (TACA bispecific fusion proteins) that target tumor - associated carbohydrate antigens and have a long half - life, as well as to the use of immune cells expressing TACA bispecific fusion proteins for treating diseases associated with abnormal glycosylation of cell - surface molecules.

Background Art

[0004] Antigen-targeted cancer immunotherapies, such as bispecific antibodies (e.g., bispecific T cell engagers) or chimeric antigen receptor T cells (e.g., engineered immune cells expressing a chimeric antigen receptor (CAR)), are known as the most powerful immunotherapies. Both cause T cell-mediated killing of cancer cells, and the complete response rate of CAR T cells in relapsed / refractory B cell malignancies reaches as high as about 90%. Both utilize single-chain variable fragments (scFvs) derived from the variable heavy and variable light chains of monoclonal antibodies to target antigens expressed in cancer. In bispecific antibodies, the antigen-specific scFv is fused to a second scFv specific for CD3, while in CARs, the antigen-specific scFv is fused to a transmembrane domain and one or more cytoplasmic signaling domains derived from immune cell receptors. Both types of chimeric molecules are genetically expressed within T cells. Both therapies are currently + approved for the treatment of CD19 B cell malignancies. To apply bispecific proteins and / or CAR T cells to a wide variety of cancer types, it is first necessary to identify cell surface cancer antigens that can be safely targeted. This is a major challenge, especially for solid tumors.

[0005] A potential approach to address all these issues is to target “tumor-associated carbohydrate antigens” (TACAs), which are overexpressed in various cancer types and at even higher levels in metastatic and invasive diseases. Carbohydrates (glycans), like glycoproteins and glycolipids, are major cell surface components. Thus, bispecific proteins and chimeric antigen receptor (CAR) T cells have been generated for immunotherapy to target cells for killing by T cells rather than antibodies, using tumor-associated carbohydrate antigen (TACA)-binding domains derived from lectins. This novel technology is called “glycan-dependent T cell recruiter” or GlyTR (pronounced “glitter”). However, like many useful therapeutic agents and conventional immunotherapies (e.g., bispecific T cell engagers (BiTE®), tandem diabodies (TandAbs) or dual-affinity retargeting proteins (DART®)), GlyTR is rapidly cleared from the body when administered to a subject. This rapid removal requires administration of either high doses or multiple doses of GlyTR to the subject to achieve a desirable therapeutic effect.

[0006] Accordingly, there is a need for an improved immunotherapy approach with improved pharmacokinetic properties for targeting antigens present in multiple common cancers. The present disclosure meets this unmet need. SUMMARY OF THE INVENTION

[0007] Embodiments relate to a novel class of immunotherapy fusion proteins (trispecific fusion proteins) having an extended serum half-life, isolated nucleic acids and vectors encoding TACA fusion proteins, recombinant (i.e., modified or host) cells comprising TACA-specific fusion proteins; compositions and methods comprising fusion proteins for immunotherapy, for treating diseases related to abnormal glycosylation of cell surface molecules and / or expression of tumor-associated carbohydrate antigens (TACAs).

[0008] One aspect of the present disclosure provides an isolated nucleic acid molecule encoding a fusion protein, the 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 that is a polypeptide capable of extending the half-life of the fusion protein.

[0009] 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 albumin, albumin, serum albumin, the Fc domain of an antibody, a polyethylene glycol moiety (PEG), a poly(lactic-co-glycolic acid) (PLGA) polymer, a polymeric hydrogel, a nanoparticle, a fatty acid chain, an acyl group, a myristic acid group, a palmitoyl group, and a sterol group. In one embodiment, the half-life extension domain comprises the Fc domain of an antibody selected from the IgG1, IgG2, IgG3, or IgG4 Fc region. In one embodiment, the half-life extension domain comprises a PEG moiety. In that 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.

[0010] In some embodiments, the half-life extension domain comprises a molecule capable of binding serum albumin. In some embodiments, the half-life extension domain comprises a polypeptide having 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). In those embodiments, Xaa is any amino acid. In some embodiments, the half-life extension domain comprises a fatty acid chain conjugated polypeptide. In those embodiments, 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 conjugate molecule. In some embodiments, the half-life extension domain comprises 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).

[0011] In some embodiments, the half-life extension domain comprises a serum albumin polypeptide. In those embodiments, the serum albumin is human serum albumin. In some embodiments, the half-life extension domain comprises an amino acid sequence of SEQ ID NO: 57.

[0012] In some embodiments, the half-life of the fusion protein 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 fusion protein lacking the half-life extension domain. In some embodiments, the half-life extension is based on the average plasma residence time of the fusion protein.

[0013] In some embodiments, the antigen-binding domain comprises more than one TACA-binding domain. In some embodiments, the antigen-binding domain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 TACA-binding domains. In such embodiments, the TACA-binding domains are operably linked by a linker. In some embodiments, the linker is selected from the group consisting of a peptide linker, a non-peptide linker, a chemical unit, a disulfide cross-linking linker, and a non-disulfide cross-linking linker.

[0014] In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker has a length of at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, or at least about 15 amino acids. 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.

[0015] In some embodiments, the antigen-binding domain comprises a TACA-binding domain derived from a lectin. In some embodiments, the antigen-binding domain is a lectin selected from galectin, siglec, selectin; C-type lectin; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Phaseolus vulgaris leukocyte agglutinin); E-PHA (Phaseolus vulgaris erythroagglutinin); tomato lectin (Lycopersicon esculentum lectin; LEA); peanut lectin (Arachis hypogaea lectin; PNA); potato lectin (Solanum tuberosum lectin), pokeweed mitogen (Phytolacca americana lectin), wheat germ agglutinin (Triticum aestivum germ lectin); Artocarpus polyphyllus lectin (jacalin lectin); hairy vetch lectin (VVA); apple snail lectin (HPA); fucose lectin (WFA); Sambucus nigra lectin (SNA), BC2L-CNt (lectin from the gram-negative bacterium Burkholderia cenocepacia), dog kidney leukocyte agglutinin (MAL), Pleurotus ostreatus (PVL), Sclerotium rolfsii lectin (SRL), Echinops sphaerocephalus lectin (ESA), CLEC17A (prolectin), Hericium erinaceus lectin, Allium sativum lectin (SSA), Glechoma hederacea lectin (Gleheda), Momordica charantia lectin (Morniga G), Orchis mascula lectin, Salvia bogotensis lectin, Salvia horminum lectin, Cuscutaceae lectin, Calceolaria integrifolia lectin, Glyphomia simplificifolia (GsLA4), Vicia faba (acidic WBAI), Vigna angularis lectin, Apios americana lectin, Amaranthus leucocarpus lectin, Relya autumnalis lectin, Paramignya monophylla lectin, Ulex europaeus lectin, Artocarpus lakoocha lectin, Himalayan Vicia faba lectin, Himalayan Vicia faba lectin, soybean lectin and mushroom lectin, and comprises at least two TACA-binding domains derived from the selected lectins.

[0016] In some embodiments, the antigen-binding domain comprises an amino acid sequence set forth 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%, or at least 99% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 33-56. In some embodiments, the antigen-binding comprises an amino acid sequence having at least 90% homology to SEQ ID NOs: 33-56.

[0017] 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 an NK cell.

[0018] In some embodiments, the immune cell recognition domain comprises (i) an antibody Fc domain (optionally the 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).

[0019] 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. In one embodiment, the receptor on the immune effector cell is a 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 an NK cell receptor selected from the group consisting of NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1.

[0020] In some embodiments, the immune cell recognition domain comprises an scFv that selectively binds 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 having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 59, 60, or 61.

[0021] In some embodiments, the encoded fusion protein is an Fc fusion protein comprising an antigen-binding domain that selectively binds a tumor-associated carbohydrate antigen (TACA) and an Fc domain. In that embodiment, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 69, or SEQ ID NOs: 91-94.

[0022] In some embodiments of the present disclosure, the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 1 to 32, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1 to 32. In some embodiments, the encoded fusion protein selectively targets a TACA selected from the group consisting of β1,6-branched, β1,6GlcNAc-branched N-glycan, T antigen, Tn antigen, sialyl-T epitope, Tn epitope, sialyl-Tn antigen or epitope, α2,6-sialylation, sialylation, sialyl-Lewis x / a , disialyl-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-glycan, GalNAc, Tn antigen, GalNAcα-ser, GalNAcα-thr, GalNAc, or GalNAcβ1.

[0023] 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 the amino acid sequence of SEQ ID NOs: 13-32. In some embodiments, the encoded fusion protein binds to β1,6GlcNAc branched type N-glycan expressing tumor cells as compared to a bispecific fusion protein comprising a flexible linker in the antigen binding domain. In some embodiments, the encoded fusion protein binds to Thomsen-nouveau (Tn) antigen expressing tumor cells as compared to a fusion protein comprising a flexible linker in the antigen binding domain. In those embodiments, the flexible linker is a glycine-serine linker, or 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, and is a linker comprising the same.

[0024] In some embodiments, the isolated nucleic acid comprises an expression vector and / or in vitro transcribed RNA.

[0025] 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 the isolated nucleic acid described herein.

[0026] Another aspect of the present disclosure is 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%, or at least 99% sequence identity to the amino acid sequence set forth 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 that is a polypeptide capable of extending the half-life of the fusion protein.

[0027] 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 albumin, albumin, serum albumin, the Fc domain of an antibody, a polyethylene glycol moiety (PEG), a poly(lactic-co-glycolic acid) (PLGA) polymer, a polymeric hydrogel, a nanoparticle, a fatty acid chain, an acyl group, a myristic acid group, a palmitoylation group, and a sterol group.

[0028] In some embodiments, the half-life extension domain comprises the Fc domain of an antibody selected from an IgG1, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the half-life extension domain comprises a PEG moiety. In those embodiments, 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.

[0029] In some embodiments, the half-life extension domain comprises a molecule capable of binding serum albumin. In some embodiments, the half-life extension domain comprises a polypeptide having 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). In those embodiments, Xaa is any amino acid. In some embodiments, the half-life extension domain comprises a fatty acid chain conjugated polypeptide, and 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 that selectively binds serum albumin, optionally a single domain antibody, a CDR of a single domain antibody, or a single chain variable fragment (scFv).

[0030] In some embodiments, the half-life extension domain comprises a serum albumin polypeptide. In those embodiments, 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.

[0031] In some embodiments, the half-life of the fusion protein is 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 extended compared to a fusion protein lacking the half-life extension domain.

[0032] In some embodiments, the half-life extension is based on the average plasma residence time of the fusion protein. In some embodiments, the immune cell recognition domain comprises the Fc domain of an antibody. In one embodiment, the immune cell recognition domain comprises the Fc domain of an IgG molecule. In some embodiments, the immune cell recognition domain comprises 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 the immune cell recognition domain comprises the constant region domains CH2 and / or CH3 of an antibody (preferably CH2 and CH3, optionally with or without a hinge region).

[0033] In some embodiments, the receptor on the immune effector cell is selected from the group consisting of T cell receptor (TCR) 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.

[0034] 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 with an amino acid sequence selected from SEQ ID NOs: 1-32. In some embodiments, the fusion protein selectively targets a TACA selected from the group consisting of β1,6-branched, β1,6GlcNAc-branched N-glycan, T antigen, Tn antigen, sialyl-T epitope, Tn epitope, sialyl-Tn antigen or epitope, α2,6-sialylation, sialylation, sialyl-Lewis x / a , disialyl-Lewis x / a , sialyl 6-sulfo Lexis x , Lewis-y (Le y ), Globo H, GD2, GD3, GM3 and fucosyl GM1.

[0035] In some embodiments, the fusion protein selectively targets the Tn antigen or the β1,6GlcNAc-branched N-glycan. In some embodiments, the fusion protein that selectively targets the 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 with an amino acid sequence selected from SEQ ID NOs: 36-42, 52-56, or 62. In some embodiments, the fusion protein that selectively targets the 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 with an amino acid sequence selected from SEQ ID NOs: 13-32.

[0036] In some embodiments, the fusion protein that selectively targets the β1,6GlcNAc-branched N-glycan 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 with an amino acid sequence selected from SEQ ID NOs: 33-35 or 43-51. In some embodiments, the fusion protein that selectively targets the β1,6GlcNAc-branched N-glycan 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.

[0037] 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%, or at least 99% sequence identity to the amino acid sequence set forth 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. In some embodiments, the half-life extension domain is a polypeptide 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.

[0038] 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%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 33-56; and (ii) the Fc domain of an antibody. In some embodiments, the Fc domain is a half-life extension domain. In some embodiments, the Fc domain is an IgG molecule or the Fc domain comprises the amino acids of SEQ ID NO: 69 or 91-94.

[0039] One aspect of the present disclosure provides an expression construct comprising an 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 the EF-lα promoter, the T cell receptor alpha (TRAC) promoter, the interleukin 2 (IL-2) promoter, or the cytomegalovirus (CMV) promoter, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter or the Rous sarcoma virus promoter. 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.

[0040] One aspect of the present disclosure provides a modified cell comprising an isolated nucleic acid, a fusion protein, or an expression construct described herein. In some embodiments, the modified cell (e.g., a host cell) is selected from the group consisting of a bacterial cell, a fungal cell, an insect cell, or a mammalian cell. 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 a yeast cell, Saccharomyces cerevisiae, or Pichia pastoris. In some embodiments, the modified cell is an insect cell selected from a lepidopteran insect cell or Spodoptera frugiperda. In some embodiments, the modified cell is a mammalian cell selected from Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, monkey kidney cells, HeLa cells, human hepatocellular carcinoma cells, or human fetal kidney 293. In one embodiment, the modified cell is a CHO cell or a HEK293 cell.

[0041] In some embodiments, the modified cells are T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs) or regulatory T cells. In such embodiments, the T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs) or regulatory T cells comprise a chimeric antigen receptor (CAR). In some embodiments, the 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, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, Claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRα) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Rα, IL13Rα2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin 4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP 1-6), NY-ESO-1, p16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof. In one embodiment, 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 a tumor antigen. In some embodiments, the modified cells are CAR T cells that specifically target a tumor-associated carbohydrate antigen (TACA).

[0042] Another aspect of the present disclosure provides a method for generating a modified cell comprising the fusion protein described herein, the method comprising: (a) introducing into the cell an isolated nucleic acid, fusion protein or expression construct described herein; (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.

[0043] Another aspect of the present disclosure provides a composition comprising (a) a fusion protein encoded by an isolated nucleic acid described herein, (b) a fusion protein described herein, (c) a modified cell described herein, or (d) a fusion protein encoded by an expression construct 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 such embodiments, the T cell, natural killer (NK) cell, cytotoxic T lymphocyte (CTL), or regulatory T cell comprises a chimeric antigen receptor (CAR) that targets a tumor antigen. 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 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, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, Claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRα) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin 4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP 1-6), NY-ESO-1, p16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof.

[0044] 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 an immunotherapeutic composition comprising (a) a fusion protein encoded by an isolated nucleic acid described herein, (b) a fusion protein described herein, (c) a modified cell described herein, or (d) a composition 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 such embodiments, the T cell, natural killer (NK) cell, cytotoxic T lymphocyte (CTL), or 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 such embodiments, the T cell, natural killer (NK) cell, cytotoxic T lymphocyte (CTL), or 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 cell is a CAR T cell that specifically targets a tumor antigen.

[0045] 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, CDI 17, CD123, CD133, CD147, CDI 71, CD276, CEA, Claudin 18.2, c-Met, DLL3, DRS, EGFR, EGFRvlll, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate-binding protein (FBP), GD-2, glycolipid F77, glypican-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, nectin 4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP I-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-associated carbohydrate antigen (TACA).

[0046] In some embodiments, the cancer is selected from the group consisting of hematological malignancies, solid tumors, primary or metastatic tumors, leukemia, carcinoma, blastoma, sarcoma, leukemia, malignant lymphoma, melanoma and lymphoma.

[0047] Another aspect of the present disclosure is a method of treating cancer in a subject in need of treating cancer, the method comprising administering to the subject a therapeutically effective composition comprising modified cells comprising a fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), the fusion protein 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 any of the amino acid sequences set forth 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 that is a polypeptide capable of extending the half-life of the fusion protein.

[0048] In some embodiments, the immune cell recognition domain specifically binds CD3. In some embodiments, the immune cell recognition domain is the Fc domain of an antibody. In some embodiments, the immune cell recognition domain is the Fc domain of an antibody and a domain that specifically binds CD3. In some embodiments, the immune cell recognition domain is the Fc domain of an antibody.

[0049] 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 NOs: 1-32.

[0050] Another aspect of the present disclosure is a method of treating cancer in a subject in need of treating cancer, 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), the fusion protein 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 with any of the amino acid sequences set forth 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.

[0051] Another aspect of the present disclosure provides a method of providing anti-tumor immunity to a mammal, the method comprising administering to the mammal a therapeutically effective amount of (a) a fusion protein encoded by an isolated nucleic acid described herein, (b) a fusion protein having an extended serum half-life described herein, (c) a modified cell population described herein, or (d) a composition 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 such embodiments, the T cell, natural killer (NK) cell, cytotoxic T lymphocyte (CTL), or regulatory T cell comprises a chimeric antigen receptor (CAR) that selectively or specifically binds to a tumor antigen. In some embodiments, the tumor antigen is a tumor-associated carbohydrate antigen (TACA), alpha-fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-IX, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, Claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRα) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Rα, IL13Rα2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin 4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP 1-6), NY-ESO-1, p16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof. 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 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 (TACAs).

Brief Description of the Drawings

[0052]

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Mode for Carrying Out the Invention

[0053] I. Summary In the co-filed International Application No. PCT / US2023 / 024898, titled "Bispecific Fusion Proteins and Chimeric Antigen Receptors for Improved Glycan-Dependent Immunotherapy", a new class of bispecific fusion proteins and CARs for immunotherapy that effectively target TACAs for immunotherapy have been developed. Specifically, bispecific fusion proteins and CARs that target TACAs independently of carrier proteins were engineered using antigen-binding domains derived from lectins rather than monoclonal antibodies or fragments thereof. This novel technology is referred to as "Glycan-Dependent T Cell Recruiter" or GlyTR (pronounced "glitter"). One set of GlyTR therapeutics is a TACA bispecific fusion protein comprising a sugar chain recognition domain (e.g., a TACA-binding domain) from a lectin operably linked, conjugated, or fused to an immunocyte recognition domain that specifically binds to a receptor on immune effector cells. Another set of GlyTR therapeutics is a chimeric antigen receptor comprising an antigen-binding domain that includes a TACA-binding domain derived from a lectin. In all cases, the TACA-binding domain specifically binds to TACAs expressed on tumor cells, and the TACA-binding domain includes one or more TACA-binding domains derived from lectins.

[0054] Since GlyTR can target antigens present in multiple common cancers, the GlyTR disclosed in that application represents a significant improvement for immunotherapy. This is because the glycosylation changes that generate TACAs are an almost universal feature of cancer. TACAs provide the most abundant and widespread cell surface cancer antigens known, and their target density is up to about 100 - 1000 times higher than that of common protein antigens. Furthermore, the TACA target density is about 100 - 1000 times higher than that of common protein antigens. GlyTR also has high avidity binding, which is achieved by the combination of high-density target expression on tumor cells and the presence of multiple glycan-binding domains of engineered GlyTR. This combination of high target density and multiple binding sites increases the specificity of GlyTR for high-TACA-expressing cells (e.g., cancer cells) compared to low-expressing cells (such as normal cells).

[0055] However, like many useful therapeutic agents and conventional immunotherapies (e.g., bispecific T cell engagers (BiTE®), tandem diabodies (TandAbs) or dual affinity retargeting proteins (DART®)), GlyTR is rapidly cleared from the body when administered to a subject. Therefore, a TACA-specific bispecific fusion protein with an extended serum half-life is needed to increase the therapeutic potential (such as low-dose pharmaceutical formulations, reduced frequency of administration, and / or novel pharmaceutical compositions).

[0056] In the present disclosure, a novel class of trispecific fusion proteins for immunotherapy with an extended serum half-life that effectively targets TACAs for immunotherapy has been developed. The novel TACA trispecific fusion protein includes a first domain that is an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), a second domain that is an immune cell recognition domain that specifically binds to a receptor (such as CD3) within immune cells, and a third domain that 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 rearranged in various manners as described below. For example, the half-life extension domain can be located at the N-terminus, the middle, or the C-terminus of the trispecific fusion protein.

[0057] A. Summary of Experimental Results The novel trispecific fusion protein disclosed herein has a serum half-life that is at least 5-fold longer than that of the parental bispecific fusion protein, while maintaining substantially the same binding to TACA-positive cancer cells, killing using the same, and T cell activation as compared to the parental bispecific fusion protein. As shown in FIG. 6B, the serum half-life of the dimer GlyTR1 LPHA(2)xCD3 was approximately 2.7 hours (FIG. 6B). Most of GlyTR1 LPHA(2)xCD3 accumulated in the liver, with only much smaller amounts accumulating in the spleen and kidney (FIGS. 6A, 6C-6E). The serum half-life of GlyTR2 CD301(3)xCD3 was substantially the same as that of GlyTR1 LPHA(2)xCD3 (FIG. 19A). GlyTR2 slCD301(4)xCD3 also accumulated in the liver, with only minimal amounts accumulating in the kidney, spleen, lung, and intestine (FIGS. 20A, 20B). However, GlyTR2 slCD301(4)xCD3It did not significantly bind to human hepatocytes or mouse hepatocytes, so liver accumulation did not indicate binding to GalNAc-containing glycans on hepatocytes (Figure 20C, Figure 20D, data not shown). These observations indicate that the GlyTR molecule may be rapidly removed by the liver for degradation, reducing the therapeutic effect of the GlyTR molecule. The observed half-life of the GlyTRs fusion protein is similar to the observed half-life of the FDA-approved therapeutic agent BLINCYTO® (blinatumomab). BLINCYTO® is a BiTE® immunotherapy that binds to CD3 and CD19 and has a short half-life, so it is necessary to continuously intravenously inject a 28-day treatment regimen twice. To avoid this cumbersome regimen and extend the half-life of GlyTR, the inventors generated a novel and improved GlyTR that further includes a half-life extension domain that can extend the half-life of the fusion protein. As shown in Figure 22E, the half-life of the trispecific fusion protein containing GlyTR slCD301(4)xCD3 and the human serum albumin (HSA) domain was 5-fold longer than that of the parental GlyTR slCD301(4)xCD3 2 hours after intravenous injection. Furthermore, the novel trispecific fusion protein HSA-GlyTR slCD301(4)xCD3 showed substantially similar binding and killing to Tn antigen-positive cancer cells compared to the parental GlyTR slCD301(4)xCD3 (Figures 22A - 22C). Despite the additional domain, the novel trispecific fusion protein HSA-GlyTR slCD301(4)xCD3 also showed substantially similar levels of T cell activation compared to the parental GlyTR slCD301(4)xCD3 (Figure 22D).

[0058] B. Typical advantages of the GlyTR fusion protein The GlyTR trispecific fusion protein described herein is designed to be able to specifically and selectively target cells expressing any TACA by mobilizing cytotoxic T cells. The GlyTR trispecific fusion protein binds to receptors of immune effector cells (such as CD3), thereby crosslinking cytotoxic T cells with cells expressing TACA in a very specific manner, and thus can direct the cytotoxic performance of T cells to target cells. The GlyTR trispecific fusion protein described herein involves cytotoxic T cells by binding to surface-expressed immune receptors (for example, the CD3 protein that forms part of the TCR). When some GlyTR trispecific fusion proteins simultaneously bind to CD3 and TACA expressed on the surface of specific cells, it causes T cell activation and subsequently results in the lysis of specific TACA-expressing cells. Therefore, the GlyTR trispecific fusion protein is intended to exhibit potent, specific, and efficient target cell killing.

[0059] A further advantage of the GlyTR trispecific fusion protein described herein over conventional monoclonal antibodies and other smaller bispecific molecules is the extended half-life. Generally, the efficacy of recombinant protein pharmaceuticals largely depends on the intrinsic pharmacokinetics of the protein itself. Here, the main advantage of the GlyTR trispecific fusion protein described herein is that it has an extended pharmacokinetic elimination half-life thanks to a half-life extension domain (for example, a domain specific for HSA).

[0060] The GlyTR triple-specific fusion protein described herein has an extended serum elimination half-life that is 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 longer compared to a fusion protein lacking a half-life extension domain (e.g., a GlyTR dual-specific fusion protein or other molecules for immunotherapy known in the art, examples of which include bispecific T cell engagers (BiTE®), tandem diabodies (TandAbs), or bispecific affinity retargeting proteins (DART®) molecules). BiTE® or DART® molecules have relatively shorter elimination half-lives (e.g., BiTE® has a short elimination half-life (mean ± SD) of 1.25 ± 0.63 hours and is thus rapidly cleared from the circulation). For these reasons, for example, in the case of the BiTE® CD19×CD3 bispecific fusion molecule, continuous administration at a high concentration (15 - 28 μg per day) is required to mobilize and activate a large number of suboptimal T cells to achieve maximal half-maximal target cell lysis. Thus, BiTE® CD19×CD3 is administered as a 4-week continuous intravenous infusion (i.v.) to maintain a sufficient therapeutic serum concentration.

[0061] Accordingly, the longer intrinsic half-life of the GlyTR triple-specific fusion protein described herein solves the problem of short half-lives and can thereby increase the potential for therapy, examples of which include, for example, low-dose pharmaceutical formulations, reduced frequency of administration, and / or novel pharmaceutical compositions.

[0062] In one aspect, the present disclosure provides an isolated nucleic acid molecule encoding a fusion protein (e.g., a trispecific 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 capable of extending 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.

[0063] 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 the isolated nucleic acid molecule disclosed herein. The antigen-binding domain of the fusion protein disclosed herein is selected from the group consisting of 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 sequences set forth in SEQ ID NOs: 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 (e.g., a polypeptide capable of binding albumin, albumin, serum albumin, the Fc domain of an antibody, a polyethylene glycol moiety (PEG), a poly(lactic-co-glycolic acid) (PLGA) polymer, a polymeric hydrogel, a nanoparticle, a fatty acid chain, an acyl group, a myristic acid group, a palmitoylation group, and a sterol group) 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: 110.

[0064] In one aspect, the present disclosure provides modified cells comprising the isolated nucleic acid molecules disclosed herein and the fusion proteins disclosed herein. The cells are, for example, T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. In another aspect, the present disclosure provides an expression construct comprising the isolated nucleic acid and / or promoter disclosed herein. Also provided are compositions comprising the isolated nucleic acid, fusion protein, modified cell, or expression construct disclosed herein. In one aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an immunotherapeutic composition disclosed herein.

[0065] II. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, but the preferred methods and materials are described below. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0066] In the practice of the present disclosure, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the skill of those of ordinary skill in the art are employed. For example, Green and Sambrook eds. (2012) Molecular Cloning: A Laboratory Manual, 4th edition; the series Ausubel et al. eds. (2015) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (2015) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; McPherson et al. (2006) PCR: The Basics (Garland Science); Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Greenfield ed. (2014) Antibodies, A Laboratory Manual; Freshney (2010) Culture of Animal Cells: A Manual of Basic Technique, 6th edition; Gait ed. (1984) Oligonucleotide Synthesis; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Herdewijn ed. (2005) Oligonucleotide Synthesis: Methods and Applications; Hames and Higgins eds. (1984) Transcription and Translation; Buzdin and Lukyanov ed.(2007) Nucleic Acids Hybridization: Modern Applications; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi ed. (2007) In Vitro Transcription and Translation Protocols, 2nd edition; Guisan ed. (2006) Immobilization of Enzymes and Cells; Perbal (1988) A Practical Guide to Molecular Cloning, 2nd edition; Miller and Calos eds., (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Lundblad and Macdonald eds. (2010) Handbook of Biochemistry and Molecular Biology, 4th edition; and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology, 5th edition. See also.

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

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

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

[0070] As used herein, the term "affinity" refers to the strength of the interaction between a bispecific fusion protein described herein and a TACA at a single antigenic site. Within each antigenic site, the TACA-binding domain derived from a lectin interacts with the antigen at multiple sites by weak non-covalent forces, and the greater the number of interactions, the stronger the affinity.

[0071] As used herein, the term "avidity" refers to a useful measure of the overall stability or strength of a bispecific fusion protein-TACA antigen complex. This is controlled by three main factors: namely, the TACA-binding domain derived from lectin affinity, the valency of both TACA and the 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 the correct antigen epitope.

[0072] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in mean survival, or an improvement in various physiological symptoms associated with the cancer pathology. "Anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present disclosure to prevent the occurrence of tumors in the first place.

[0073] As used herein, the term "self" is intended to refer to any material that is derived from the same individual and later reintroduced into that individual.

[0074] 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, the activation of specific immunocompetent cells, or both. One of ordinary skill in the art will understand that virtually any macromolecule, including substantially all proteins or peptides, can serve as an antigen. Additionally, an antigen can be derived from recombinant DNA or genomic DNA. One of ordinary skill in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will, therefore, encode an "antigen" as the term is used herein. Further, one of ordinary skill in the art will understand that an antigen need not necessarily be encoded by the full-length nucleotide sequence of a gene. The present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and it is readily apparent that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Moreover, one of ordinary skill in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated synthetically or produced from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0075] As used herein, the term "allogeneic" refers to a graft that is derived from different animals of the same species.

[0076] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody may be an intact immunoglobulin derived from a natural source or a recombinant source, or may be an immunoreactive portion of an intact immunoglobulin. Antibodies are generally tetramers of immunoglobulin molecules. The antibodies in the present disclosure may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, F(ab)2, and single-chain antibodies (scFv), as well as humanized antibodies. In some embodiments, an antibody refers to an assembly (e.g., an intact antibody molecule, an immunoadhesin, or a variant thereof) having significant known specific immunoreactive activity against a target antigen (e.g., a tumor-associated antigen). Antibodies and immunoglobulins include light and heavy chains, which may or may not have interchain disulfide bonds. The basic immunoglobulin structure in the vertebrate system is relatively well understood.

[0077] As used herein, the term "antibody fragment" refers to a portion of an intact antibody and to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0078] As used herein, the term "antibody heavy chain" refers to the larger of the two polypeptide chains that exist in a conformation naturally occurring in all antibody molecules.

[0079] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains that exist in a conformation naturally occurring in all antibody molecules.

[0080] As used herein, the term "antibody variant" includes synthetic and engineered forms of an antibody that have been altered such that they do not occur naturally, examples of which include antibodies that contain at least two heavy chain portions but do not contain two full-length heavy chains (such as domain-deleted antibodies or minibodies), multispecific forms of antibodies that have been altered to bind two or more different antigens or to bind different epitopes on a single antigen (e.g., bispecific, trispecific, etc.), heavy chain molecules conjugated to scFv molecules, and the like. Further, the term "antibody variant" includes multivalent forms of an antibody (e.g., trivalent, tetravalent, etc., antibodies that bind three, four, or more copies of the same antigen).

[0081] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells may spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

[0082] As used herein, the terms "cancer-related antigen" or "tumor antigen" interchangeably refer to a molecule (typically a protein, carbohydrate, or lipid) that is expressed on the surface of cancer cells, either as a whole or in fragments (e.g., MHC / peptide), and that is useful for the preferential targeting of an agent with pharmacological activity to those cancer cells. In some embodiments, the tumor antigen is a marker expressed by both normal and cancer cells (e.g., a lineage marker such as CD19 on B cells). In some embodiments, the tumor antigen is a cell surface molecule that is overexpressed in cancer cells compared to normal cells (e.g., 1-fold overexpression, 2-fold overexpression, 3-fold or greater overexpression compared to normal cells). In some embodiments, the tumor antigen is a cell surface molecule that is inappropriately synthesized within cancer cells (e.g., a molecule containing deletions, additions, or mutations compared to a molecule expressed on normal cells). In some embodiments, the tumor antigen is expressed exclusively on the cell surface of cancer cells, either as a whole or in fragments (e.g., MHC / peptide), and is not synthesized or expressed on the surface of normal cells. In some embodiments, the CARs of the present disclosure include CARs that include an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide. Peptides derived from endogenous proteins typically fill the pockets of major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets in immunotherapy. TCR-like antibodies targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described. For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage display libraries.

[0083] 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, CDI 17, CD123, CD133, CD147, CDI 71, CD276, CEA, Claudin 18.2, c-Met, DLL3, DRS, EGFR, EGFRvlll, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate-binding protein (FBP), GD-2, glycolipid F77, glypican-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, nectin 4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP I-6), NY-ESO-1, Pl 6, PD-LI, PSCA, PSMA, RORI, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof.

[0084] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell receptor that is expressed on an immune effector cell or its progenitor cell and engineered to specifically bind an antigen. The CAR may be used in adoptive cell therapy by adoptive cell transfer. In some embodiments, adoptive cell transfer (or therapy) includes removing T cells from a patient and modifying the T cells to express a receptor specific for a particular antigen. In some embodiments, the CAR is specific for a selected target, such as a tumor-associated carbohydrate antigen (TACA). The CAR may also include an extracellular domain that includes an antigen-binding region, a transmembrane domain, and an intracellular activation domain.

[0085] As used herein, the term "derived from" refers to the relationship between a first molecule and a second molecule. This defines the structural similarity between the first and second molecules and implies or encompasses no 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 the CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure to have the required function (i.e., the ability to generate a signal under appropriate conditions). This implies or encompasses no limitation on the specific process for producing the intracellular signaling domain. It does not mean that one has to start with the CD3 zeta sequence and delete unnecessary sequences or introduce mutations to arrive at the intracellular signaling domain.

[0086] As used herein, "disease" refers to the health state of an animal in which the animal cannot maintain homeostasis and, if the disease is not improved, the animal's health continues to deteriorate. In contrast, an "ailment" of an animal is a health state in which the animal can maintain homeostasis, but the animal's health state is less favorable than it would be in the absence of the ailment. The animal's health state is not necessarily further reduced by the ailment if left untreated.

[0087] As used herein, the term "disease associated with the expression of a tumor antigen" includes, but is not limited to, a disease associated with the expression of a tumor antigen or a condition associated with a cell expressing the tumor antigen, including, but not limited to, a proliferative disease such as cancer or malignancy, or a pre-cancerous condition such as myelodysplasia, myelodysplastic syndrome or pre-leukemia; or a non-cancer related indication associated with a cell expressing the tumor antigen. In some embodiments, the cancer associated with the expression of the tumor antigen is a blood cancer. In some embodiments, the cancer associated with the expression of the tumor antigen is a solid cancer. Further diseases associated with the expression of the tumor antigen include, but are not limited to, atypical and / or non-classical cancers, malignancies, pre-cancerous conditions, or proliferative diseases associated with the expression of the tumor antigen. Non-cancer related indications associated with the expression of the tumor antigen include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, the tumor antigen-expressing cells express the mRNA encoding the tumor antigen or express it at any point in time. In some embodiments, the tumor antigen-expressing cells produce a tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal or reduced levels. In some embodiments, the tumor antigen-expressing cells produce a detectable level of the tumor antigen protein at one point in time and then substantially no longer produce a detectable tumor antigen protein.

[0088] As used herein, the term "downregulation" refers to a decrease or elimination of gene expression of one or more genes.

[0089] As used herein, "effective amount" or "therapeutically effective amount" means an amount of a compound, formulation, material, pharmaceutical agent, or composition as described herein effective to achieve the desired physiological, therapeutic, or prophylactic result in a subject in need thereof. Such results can include, but are not limited to, an amount that causes a detectable level of an immune response when compared to an immune response detected when no composition of the disclosure is administered to a mammal. The immune response can be readily evaluated by a variety of methods recognized in the art. One of ordinary skill in the art will understand that the amount of the composition administered herein will vary and can be readily determined based on multiple factors such as the disease or condition being treated, the age of the mammal being treated, as well as the health and physical condition, the severity of the disease, the particular compound being administered, etc. The effective amount may vary for each subject depending on the health and physical condition of the subject being treated, the taxonomic group of the subject being treated, the formulation of the composition, the evaluation of the medical condition of the subject, and other relevant factors.

[0090] As used herein, the term "encode" refers to the inherent property of a specific sequence of nucleotides within a polynucleotide such as a gene, cDNA, or mRNA, and serves as a template for the synthesis of other polymers and macromolecules in a biological process having either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, when a protein is produced in a cell or other biological system by transcription and translation of mRNA corresponding to a gene, that gene encodes the protein. Both the nucleotide sequence that is identical to the mRNA sequence, usually the coding strand provided in the sequence listing, and the non-coding strand that is used as a template for transcription of the gene or cDNA can be said to encode the protein or other product of that gene or cDNA.

[0091] As used herein, "endogenous" refers to any substance that is from within or produced within a living organism, cell, tissue, or system.

[0092] As used herein, the term "epitope" is defined as a small chemical moiety on an antigen that can elicit an immune response and induce a B cell response and / or a T cell response. An antigen can have one or more epitopes. Most antigens have multiple epitopes; that is, they are multivalent. Generally, an epitope is approximately on the order of about 10 amino acids and / or sugars in size. In certain exemplary embodiments, an epitope is about 4 - 18 amino acids, about 5 - 16 amino acids, about 6 - 14 amino acids, about 7 - 12 amino acids, or about 8 - 10 amino acids. Generally, the overall three-dimensional structure rather than the specific linear sequence of the molecule is the main criterion for antigen specificity, and thus, those skilled in the art understand that this overall three-dimensional structure distinguishes one epitope from another. Based on the present disclosure, the peptides used in the present disclosure can serve as epitopes. As used herein, the term "exogenous" refers to any substance that is introduced from outside or produced outside a living organism, cell, tissue, or system.

[0093] As used herein, the term "expansion" as used herein refers to an increase in number, such as an increase in the number of immune cells (e.g., T cells). In some embodiments, the number of immune cells (e.g., T cells) expanded ex vivo increases compared to the number originally present in the culture. In another embodiment, the number of immune cells (e.g., T cells) expanded ex vivo increases compared to other cell types in the culture.

[0094] As used herein, the term "exogenous" refers to any substance that is introduced from outside or produced outside a living organism, cell, tissue, or system.

[0095] As used herein, the term "expression" as used herein is defined as the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0096] As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains cis-acting elements sufficient for expression, and other elements for expression can be supplied by the host cell or an in vitro expression system. Expression vectors include all those known in the art, examples of which include cosmids, plasmids (e.g., naked or contained in liposomes), and viruses incorporating recombinant polynucleotides (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0097] As used herein, the term "ex vivo" refers to cells removed from a living body (e.g., a human) and grown outside the living body (e.g., in a culture dish, test tube, or bioreactor).

[0098] As used herein, the term "Fc portion" or "Fc monomer" in the context of the present disclosure means a polypeptide comprising at least one domain having the function of the CH2 domain of an immunoglobulin molecule and at least one domain having the function of the CH3 domain. As is apparent from the term "Fc monomer", the polypeptide containing these CH domains is a "polypeptide monomer". The Fc monomer can be a polypeptide containing at least a fragment of the constant region of an immunoglobulin that excludes the first constant region immunoglobulin domain (CH1) of the heavy chain but maintains a functional portion of at least one CH2 domain and a functional portion of one CH3 domain (the CH2 domain being the amino terminus of the CH3 domain). In a preferred embodiment of this definition, the Fc monomer can be a polypeptide constant region containing the CH2 region and the CH3 region, which are part of the Ig-Fc hinge region, and the hinge region is the amino terminus of the CH2 domain.

[0099] The hinge region of the present disclosure is assumed to promote dimerization. Such Fc polypeptide molecules can be obtained, for example, by papain digestion of the immunoglobulin region (of course, a dimer of two Fc polypeptides is formed). However, this is an example and not a limitation. In another aspect of this definition, the Fc monomer can be a polypeptide region comprising a part of the CH2 region and the CH3 region. Such Fc polypeptide molecules can be obtained by pepsin digestion of immunoglobulin molecules. However, this is an example and not a limitation. In one embodiment, the polypeptide sequence of the Fc monomer is substantially similar to the Fc polypeptide sequences of the IgG1 Fc region, IgG2 Fc region, IgG3 Fc region, IgG4 Fc region, IgM Fc region, IgA Fc region, IgD Fc region, and IgE Fc region. (See, for example, Padlan, Molecular Immunology, 31(3), 169-217 (1993)). Since there are some variations among immunoglobulins, for simplicity, the Fc monomer refers to the last two heavy chain constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three heavy chain constant region immunoglobulin domains of IgE and IgM.

[0100] The Fc monomer can also include a flexible hinge at the N-terminus of these domains. In the case of IgA and IgM, the Fc monomer may include a J chain. In the case of IgG, the Fc portion includes the immunoglobulin domains CH2 and CH3, and the hinge between the first two domains and CH2. Although the boundaries of the Fc portion can vary, an example of the human IgG heavy chain Fc portion containing a functional hinge, CH2, and CH3 domains can be defined to include the amino acid sequences of SEQ ID NOs: 63-68.

[0101] The IgG hinge region can be identified by analogy using Kabat. In one embodiment, the hinge domain / region of the present disclosure comprises amino acid residues corresponding to the IgG1 sequence stretch from D234 to P243 according to Kabat numbering. In some embodiments, the hinge domain / region of the present disclosure comprises or consists of the IgG1 hinge sequence DKTHTCPPCP (SEQ ID NO: 63 or 64).

[0102] In one embodiment, the IgG1 hinge domain / region comprises the amino acid sequence of EPKSCDKTHTCPPCP (SEQ ID NO: 64).

[0103] In a further embodiment of the present disclosure, the hinge domain / region comprises or consists of the IgG2 subtype hinge sequence ERKCCVECPPCP (SEQ ID NO: 65), the IgG3 subtype hinge sequence ELKTPLDTTHTCPRCP (SEQ ID NO: 66) or ELKTPLGDTTHTCPRCP (SEQ ID NO: 67), and / or the 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 sequences disclosed in Table 2 or Table 3.

[0104] In some embodiments, the fusion protein further comprises a third domain comprising two polypeptide monomers, each monomer comprising a hinge, a CH2 domain and a CH3 domain. In one embodiment, the third domain comprises, in amino to carboxyl order, hinge-CH2-CH3-linker-hinge-CH2-CH3. In one embodiment, the CH2 domain comprises an intra-domain cysteine disulfide bridge.

[0105] In another embodiment, two polypeptide monomers are fused to each other via a peptide linker. In yet another embodiment, the first domain and the second domain are fused to the third domain via a peptide linker. In some embodiments, the peptide linker of the fusion protein of the present disclosure comprises the amino acid sequence of GGGGS (e.g., Gly4Ser (SEQ ID NO: 86)), or a polymer thereof (e.g., (Gly4Ser)n, where n is an integer of 5 or more (e.g., 5, 6, 7, 8, etc., or more)). In some embodiments, the peptide linker of the fusion protein of the present disclosure comprises the amino acid sequences of SEQ ID NOs: 74 to 90.

[0106] As used herein, the term "half-life extending molecule" refers to a biological or chemical substance 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 substance (e.g., polyethylene glycol (PEG)), which extend the half-life of the bispecific fusion protein disclosed herein. The half-life extending molecule can extend 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 more, as determined by the assays disclosed herein.

[0107] As used herein, the term "homologous" refers to sequence similarity or identity between two polypeptides or between two nucleic acid molecules. When the positions of both of the two sequences being compared are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences correlates with the value obtained by dividing the number of matching or homologous positions shared by the two sequences by the number of positions compared × 100. For example, if 6 out of 10 positions of two sequences match or are homologous, the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, the comparison is made when the two sequences are aligned to obtain the maximum homology.

[0108] As used herein, the term "identity" refers to subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules. When two amino acid sequences have the same residue at the same position, for example, if each position of two polypeptide molecules is occupied by arginine, then they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position within an alignment is often expressed as a percentage. The identity between two amino acid sequences correlates directly with the number of matching or identical positions. For example, if half of the positions within two sequences (e.g., 5 out of 10 positions in a 10 - amino - acid polymer) are identical, the two sequences are 50% identical, and if 90% of the positions (e.g., 9 out of 10) match or are identical, the two amino acid sequences are 90% identical.

[0109] As used herein, the terms "immunoglobulin" or "Ig" are defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes called BCR (B cell receptor) or antigen receptors. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the major antibody present in body secretions, examples of which include saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and urogenital tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important in defense against bacteria and viruses. IgD is an immunoglobulin whose antibody function is not known but may serve as an antigen receptor. IgE is an immunoglobulin that causes the release of mediators from mast cells and basophils upon exposure to allergens, resulting in immediate hypersensitivity.

[0110] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify an antigen molecule as foreign, induce the formation of antibodies, and / or activate lymphocytes to remove the antigen. As used herein, the term "immunostimulation" is used to refer to an increase in the overall immune response. As used herein, the term "immunosuppression" is used to refer to a decrease in the overall immune response.

[0111] As used herein, the term "immune response" as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify an antigen molecule as foreign, induce the formation of antibodies, and / or activate lymphocytes to remove the antigen.

[0112] As used herein, the term "immune effector cell" refers to a cell that is involved in an immune response (e.g., in promoting an immune effector response). Examples of immune effector cells include T cells (e.g., alpha / beta T cells, and gamma / delta T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0113] As used herein, the term "immune effector function" or "immune effector response" refers to a function or response that enhances or promotes an immune attack on a target cell. In some embodiments, the immune effector function or response refers to properties of T cells or NK cells that promote killing of target cells, or inhibition of growth or proliferation. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0114] As used herein, "explanatory materials" include publications, records, charts, or any other medium of expression that can be used to convey the usefulness of the disclosed compositions and methods. The explanatory materials of the kits of the present disclosure may be affixed, for example, to a container containing the nucleic acids, peptides and / or compositions of the present disclosure, or shipped together with a container containing the nucleic acids, peptides and / or compositions of the present disclosure. Alternatively, the explanatory materials may be shipped separately from the container, with the intention that the recipient will use the explanatory materials in concert with the compound.

[0115] As used herein, the term "isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that naturally occurs in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from the substances that coexist in 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.

[0116] In the context of the present disclosure, the following abbreviations are generally used for the commonly occurring nucleobases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0117] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may include introns if the nucleotide sequence encoding the protein may contain introns in any version.

[0118] As used herein, the term "lectin" or "hemagglutinin" refers to a protein or peptide that binds to a sugar chain structure. One skilled in the art will understand that lectins are proteins or peptides that are highly specific for binding to the sugar moiety. Lectins are sugar chain-binding proteins that are highly specific for sugar chains present on proteins and / or lipids, and cause aggregation of specific cells or precipitation of glycoconjugates and polysaccharides. Lectins play a role in recognition at the cellular and molecular levels and play numerous roles in biological recognition phenomena involving cells, sugar chains, and proteins. Lectins also mediate the attachment and binding of bacteria, viruses, and fungi to their targets. In one embodiment, a "lectin" can be defined as a non-immunoglobulin protein or glycoprotein that can specifically recognize and reversibly bind to the sugar chain portion of a complex glycoconjugate (protein and / or lipid) without altering any covalent structure of the recognized glycosyl ligand. Lectins are glycoproteins having a sugar chain-binding domain with the ability to reversibly bind to glycoproteins or glycolipids, as well as specific sugar moieties within free monosaccharides and glycan structures. Many organisms express lectins or lectin-like biomolecules, but most of the scientifically important lectins identified recently have been purified from plant sources.

[0119] As used herein, the term "tumor-associated carbohydrate antigen" or "TACA" refers to carbohydrate structures found in disorders associated with altered glycosylation (e.g., cancer). Carbohydrate-containing polymers (glycans) are ubiquitous in biological systems and are essential for many biological functions. Carbohydrates can attach to proteins (glycoproteins), lipids (glycolipids), and exist as chains of glycosaminoglycans. Changes in the structure of these carbohydrate-containing polymers (glycosylation) 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 because they display abnormal levels and types of carbohydrate structures on their surface.

[0120] Three general changes in carbohydrate-containing polymers, namely increased expression of truncated or incomplete glycans, increased branching of N-glycans, and increased or altered presence of sialic acid-containing glycans, are associated with cancer. For example, in cancer-associated glycans, the amount of sialic acid is often increased, and this hyper-sialylation enhances the activation of sialic acid-binding receptors such as selectins and siglecs, leading to cancer progression. Another of the most common cancer-associated changes in glycosylation is the cleavage of O-linked carbohydrate chains such as mucins (cleavage of O-glycoproteins). Under normal conditions, N-acetylgalactosamine (GalNAc) sugar residues attach to serine or threonine of glycoproteins (GalNAcα1-O-Ser / Thr, Tn antigen) and are usually elongated by T synthase (core 1 β3-galactosyltransferase) in the Golgi apparatus, which attaches a galactose residue to the Tn antigen (Thomsen-Friedenreich (TF) antigen). In various cancers, this process is altered, and the glycosylation of the Tn antigen or its sialylated form (sialyl-Tn (STn) antigen) is changed, resulting in truncated T, Tn, and STn antigens. In addition, increased branching of N-glycoproteins that stimulate galectin-3 and changes in glycolipids such as gangliosides (GM3, GM2, CD3, and GD2) have also been observed.

[0121] The following TACAs have been observed in various cancers. That is, (i) H / Le y / ILe a in primary non-small cell lung tumors, (ii) sialyl-Le x (SLe x ) and sialyl-Lea (SLea) in various types of cancers, (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), (v) globo-H in breast, ovarian, and prostate cancers, (vi) dialsylgalactosylgloboside in renal cell carcinomas.

[0122] Thus, the term "tumor-associated carbohydrate antigen (TACA)" encompasses all altered carbohydrate chain structures on proteins and / or lipids that are expressed on tumor cells and promote cancer metastasis, cancer progression, cancer and non-cancer immunosuppression, and / or promote autoimmune disorders. A skilled artisan will understand that carbohydrate chain structures are composed of one or more linked sugars or monosaccharides. See, for example, Mantuano et al, J. Immunotherapy Cancer 8(2): 8:e001222 (2020); Hakomori, Si. (2001). Tumor-Associated Carbohydrate Antigens Defining Tumor Malignancy: Basis for Development of Anti-Cancer Vaccines, in The Molecular Immunology of Complex Carbohydrates -2. Advances in Experimental Medicine and Biology, vol 491. Springer, Boston, MA (Wu et al (eds)). A skilled artisan will understand that carbohydrate chain structures can exist independently of, and / or attach to, proteins or lipids known as glycoproteins and glycolipids. A skilled artisan will understand that these carbohydrate chain structures bind to lectins.

[0123] As used herein, the term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells and can deliver large amounts of genetic information into the DNA of host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0124] As used herein, the term "limited toxicity" means that the peptides, polynucleotides, cells, and / or antibodies of the present disclosure do not exert substantially negative biological effects, anti-tumor effects, or substantially negative physiological symptoms on healthy cells, non-tumor cells, non-disease cells, non-target cells, or populations of such cells, either in vitro or in vivo.

[0125] As used herein, the term "flexible polypeptide linker" or "linker" when used in the context of scFv refers to a peptide linker composed of amino acids such as glycine and / or serine residues that are used alone or in combination to link the variable heavy chain region and the variable light chain region together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and includes the amino acid sequence (Gly-Gly-Gly-Gly-Ser)n, where n is a positive integer of 1 or more. For example, n = 1, n = 2, n = 3, n = 4, n = 5, n = 6, n = 7, n = 8, n = 9, n = 10 (SEQ ID NOs: 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).

[0126] As used herein, the term "modification" means an altered state or structure of a molecule or cell of the present disclosure. A molecule may be modified in many ways, including chemical, structural, and functional modifications. A cell may be modified by introduction of nucleic acid.

[0127] As used herein, the term "modulate" means to mediate a detectable increase or decrease in the level of response in a subject as compared to the level of response in the subject in the absence of treatment or compound and / or as compared to the level of response in an otherwise identical untreated subject. This term encompasses disturbing and / or affecting the original signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0128] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may include introns.

[0129] As used herein, the term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence if the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, are in the same reading frame.

[0130] As used herein, the terms "overexpressed tumor antigen" or "overexpression of a tumor antigen" are intended to indicate that the expression level of a tumor antigen in cells of a disease area, such as a solid tumor in a patient's specific tissue or organ, is abnormal compared to the expression level in normal cells of that tissue or organ. A patient having a solid tumor or hematological malignancy characterized by overexpression of a tumor antigen can be determined by standard assays known in the art.

[0131] As used herein, the term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.) injection, intravenous (i.v.) injection, intramuscular (i.m.) injection or intrasternal injection, or infusion techniques.

[0132] As used herein, terms such as "patient", "subject" and "individual" are used interchangeably herein and refer to any animal or its cells suitable for the methods described herein, whether in vitro or in situ. The subject can be a mammal, examples of which include non - primates (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or primates (e.g., monkeys and humans). In certain embodiments, the term "subject" as used herein refers to a vertebrate such as a mammal. Mammals include, but are not limited to, humans, non - human primates, wild animals, feral animals, livestock, sport animals, pets, etc. Any organism capable of eliciting an immune response can also be a subject or patient. In certain exemplary embodiments, the subject is a human.

[0133] As used herein, the term "polynucleotide" as used herein is defined as a chain of nucleotides. Further, a nucleic acid is a polymer of nucleotides. Thus, the nucleic acids and polynucleotides as used herein are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide and can be hydrolyzed into monomeric "nucleotides". The 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 recombinant means, i.e., those using conventional cloning techniques and PCR (registered trademark), etc., and synthetic means, including cloning of nucleic acid sequences from recombinant libraries or cell genomes, but not limited thereto.

[0134] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise the sequence of a protein or peptide. A polypeptide includes any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, generally also referred to in the art as peptides, oligopeptides and oligomers, and long chains, generally referred to in the art as proteins, of which there are many types. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0135] As used herein, the term "promoter" is defined as a DNA sequence recognized by the cellular synthetic machinery, or introduced synthetic machinery, necessary to initiate specific transcription of a polynucleotide sequence.

[0136] As used herein, the term "promoter" or " / regulatory sequence" means a nucleic acid sequence necessary for the expression of a gene product operably linked to the "promoter" or "regulatory sequence". In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include enhancer sequences and other regulatory elements necessary for the expression of the gene product. The promoter / regulatory sequence may, for example, express the gene product in a tissue-specific manner.

[0137] As used herein, a "constitutive promoter" is a nucleotide sequence which, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced intracellularly under most or all physiological conditions of the cell.

[0138] As used herein, an "inducible promoter" is a nucleotide sequence which, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced intracellularly only when an inducer substantially corresponding to the promoter is present intracellularly.

[0139] As used herein, a "tissue-specific promoter" is a nucleotide sequence which, when operably linked to a polynucleotide encoded or specified by a gene, causes the gene product to be produced intracellularly only when the cell is substantially a cell of the tissue type corresponding to the promoter.

[0140] As used herein with respect to an antibody, antigen-binding domain, CAR or bispecific fusion protein, the terms "specifically binds" or "selectively binds" mean an antibody, antigen-binding domain, CAR or bispecific fusion protein that recognizes a specific antigen (e.g., TACA) but does not substantially recognize or bind other molecules in a sample. For example, an antibody, antigen-binding domain, CAR or bispecific fusion protein that specifically binds to a particular antigen (e.g., TACA) may also bind to one or more species of that antigen. However, such cross-reactivity per se does not change the classification of the antibody as specific. In another example, an antibody, antigen-binding domain, CAR or bispecific fusion protein that specifically binds to an antigen (e.g., TACA) may also bind to different allelic forms of that antigen (e.g., TACA). However, such cross-reactivity per se does not change the classification of the antibody, antigen-binding domain, CAR or bispecific fusion protein as specific. In some instances, the terms "specific binding" or "specifically binds" are used with respect to the interaction of an antibody, protein, peptide, antigen-binding domain, CAR or bispecific fusion protein with a second chemical species and can mean that the interaction depends on the presence of a particular structure (e.g., antigen determinant or epitope) with respect to the chemical species. For example, an antibody, antigen-binding domain, CAR or bispecific fusion protein generally recognizes and binds to a specific protein structure (TACA) rather than a protein per se. If an antibody, antigen-binding domain, CAR or bispecific fusion protein is specific for epitope "A", in a reaction containing the label "A" and the antibody, antigen-binding domain, CAR or bispecific fusion protein, the amount of labeled A bound to the antibody may decrease if a molecule containing epitope A (or free, unlabeled A) is present.

[0141] As used herein, the term "single-chain antibody" refers to an antibody formed by recombinant DNA techniques in which immunoglobulin heavy and light chain fragments are linked to the Fv region via a manipulated range of amino acids. A variety of methods for generating single-chain antibodies are known, including those described in U.S. Patent No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Ward et al., Nature 334:544-54 (1989); Skerra et al., Science 242:1038-1041 (1988).

[0142] As used herein, the term "specificity" refers to the ability to specifically bind (e.g., immunoreact with) a given target antigen (e.g., a human target antigen). A chimeric antigen receptor is monospecific and may contain one or more binding sites that specifically bind a target, or a chimeric antigen receptor is multispecific and may contain two or more binding sites that specifically bind the same or different targets. In certain embodiments, the chimeric antigen receptor is specific for two different (e.g., non-overlapping) portions of the same target. In certain embodiments, the chimeric antigen receptor is specific for more than one target.

[0143] As used herein, the terms "specifically binds to" or "selectively binds to" with respect to an antibody mean an antibody or a binding fragment thereof (e.g., an scFv) that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to a certain antigen may also bind to one or more species of that antigen. However, such interspecies reactivity per se does not change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity per se does not change the classification of the antibody as specific.

[0144] As used herein, the terms "specific binding" or "specifically binds" can be used with respect to the interaction of an antibody, protein, chimeric antigen receptor or peptide with a second chemical species, meaning that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) with respect to the chemical species. For example, a chimeric antigen receptor generally recognizes and binds to a specific protein structure rather than a protein. If an antibody is specific for an epitope "A", in a reaction containing the label "A" and the antibody, the amount of labeled A bound to the antibody will decrease if a molecule containing epitope A (or free unlabeled A) is present. In some embodiments, the term "specific binding" between an antibody, protein, chimeric antigen receptor or peptide and a second chemical species is at least about 10 2 M -1 、 at least about 5×10 2 M -1 、 at least about 10 3 M -1 、 at least about 5×10 3 M -1 、 at least about 10 4 M -1 at least about 5×10 4 M -1 、 at least about 10 5 M -1 、 at least about 5×10 5 M -1 、 at least about 10 6 M -1 、 at least about 5×10 6 M -1 、 at least about 10 7 M -1 、 at least about 5×10 7 M -1 、 at least about 10 8 M -1 、 at least about 5×10 8 M -1 、 at least about 10 9 M -1 、 at least about 5×10 9 M -1 、 at least about 10 10 M -1, at least about 5×10 10 M -1 , at least about 10 11 M -1 , at least about 5×10 11 M -1 , at least about 10 12 M -1 , at least about 5×10 12 M -1 , at least about 10 13 M -1 , at least about 5×10 13 M -1 , at least 10 14 M -1 , at least about 5×l0 14 M -1 , at least about 10 15 M -1 , or at least about 5×10 15 M -1 of the equilibrium constant (K A )(k on / k off ), which means a binding having.

[0145] In some embodiments, the terms "specifically binds" or "selectively binds" refer to the binding reaction between a TACA bispecific fusion protein and its cognate antigen (e.g., TACA on tumor cells) that determines the presence of the cognate antigen in a heterogeneous population of proteins and other biologics.

[0146] In addition to the equilibrium constant (K A ) shown above, the TACA bispecific fusion proteins disclosed herein typically have less than 5×10 -2 M, less than 10 -2 M, less than 5×10 -3 M, less than 10 -3 M, 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 less than M, 10 -8 less than M, 5×10 -9 less than M, 10 -9 less than M, 5×10 -1 less than °M, 10 -1 less than °M, 5×10 -11 less than M, 10 -11 less than M, 5×10 -12 less than M, 10 -12 less than M, 5×10 -13 less than M, 10 -13 less than M, 5×10 -14 less than M, 10 -14 less than M, 5×10 -15 less than or 10 -15 less than M, or a dissociation rate constant (K D ) that is lower than that, and binds to TACA with an affinity that is at least 2-fold stronger than the affinity when binding to a non-specific antigen. In one embodiment, the TACA bispecific fusion protein disclosed herein has a dissociation constant (K d ) 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 when evaluated using the methods described herein or methods known to those of skill in the art (e.g., Biacore assay, ELISA, FACS, SET) (Biacore International AB, Uppsala, Sweden).

[0147] 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, and the ratio of K d to K a (i.e., K d / K a ) obtained and expressed as molar concentration (M). The K of the bispecific fusion protein disclosed herein D value can be determined using methods well established in the art. The method for determining the K of an antibody D is to use surface plasmon resonance or a biosensor system such as the Biacore® system.

[0148] As used herein, the terms "percent identical" or "percent identity" refer to two or more sequences or subsequences that are identical in the context of two or more nucleic acid or polypeptide sequences. When two sequences are compared and aligned to obtain the maximum match over a comparison window, or a specified region, and are measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, the two sequences have the amino acid residues or nucleotides that are identical at the specifically recited percentage (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over the specifically recited region or, if not specifically recited, over the entire sequence), the two sequences are "substantially identical". Optionally, the identity exists over a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200, or more amino acids) in length.

[0149] In array comparison, usually one array functions as a reference array against which a test array is compared. When using an array comparison algorithm, the test array and the reference array are input into a computer, and if necessary, partial array coordinates are specified, and array algorithm program parameters are specified. Default program parameters can be used. Or alternative parameters can be specified. Then, the array comparison algorithm calculates the percent sequence identity of the test array to the reference array based on the program parameters.

[0150] As used herein, the term "comparison window" includes reference to any one segment of a number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150, where the two arrays may be compared after optimally aligning this array and a reference array of the same number of contiguous positions. Methods of aligning arrays for comparison are well known in the art. Optimal alignment of arrays for comparison can be performed by the following methods. For example, the local homology algorithm (Smith and Waterman (1970) Adv. Appl. Math. 2:482c), the homology alignment algorithm (Needleman and Wunsch (1970) J. Mol. Biol. 48:443), the similarity search method (Pearson and Lipman, (1988) Proc. Nat’l. Acad. Sci. USA 85:2444), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or manual alignment and visual inspection (see Brent et al., (2003) Current Protocols in Molecular Biology).

[0151] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms. These are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves identifying high-scoring sequence pairs (HSPs) by first identifying short words of length W within the query sequence. These short words either match or satisfy a certain positive threshold score T when aligned with words of the same length within the database sequences. T is referred to as the neighborhood word score threshold (see Altschul et al., supra). These initial neighborhood word hits serve as seeds to initiate a search for longer HSPs that contain them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. The cumulative score is calculated using parameters M (reward score for pairs of matching residues, always >0) and N (penalty score for non-matching residues, always <0) for nucleotide sequences. In the case of amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction stops when the cumulative alignment score drops by amount X from its maximum achieved value, or when the cumulative score becomes zero or less due to the accumulation of one or more negative-score residue alignments, or when the end of either sequence is reached. The sensitivity and speed of the alignment are determined by the BLAST algorithm parameters W, T, and X. The BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands.In the case of amino acid sequences, the BLASTP program, by default, uses a word length of 3, an expectation value (E) of 10, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915), an alignment (B) of 50, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands.

[0152] 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 minimum total probability (P(N)). This provides an indication of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum total probability in a comparison of a test nucleic acid and a reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0153] Also, the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. 4:11-17 (1988)). This is incorporated into the ALIGN program (version 2.0) using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Further, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J.J. Mol, Biol. 48:444-453 (1970)) incorporated into the GAP program of the GCG software package (available at www.gcg.com), using either the Blossom62 matrix or the PAM250 matrix, and using a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0154] In addition to the percentage of sequence identity described above, another indication 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 an antibody raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, the polypeptide is typically substantially identical to the second polypeptide; for example, the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complementary molecules hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the sequences can be amplified using the same primers.

[0155] As used herein, the term "stimulation" means a primary response induced by binding a stimulatory molecule (e.g., TCR / CD3 complex) to its cognate ligand, thereby mediating a signaling event, examples of which include, but are not limited to, signaling through the TCR / CD3 complex. Stimulation can mediate changes in the expression of specific molecules, examples of which include down-regulation of TGF-β, and / or reorganization of the cytoskeletal structure, clonal expansion, and differentiation into different subsets.

[0156] 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.

[0157] As used herein, the term "stimulatory ligand" means a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), specifically binds to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby being able to mediate a primary response by the T cell, which primary response includes, but is not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, inter alia, MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0158] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cell types with which it is normally associated in its native state. In some instances, a population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to cells that have been separated from the cell, which are naturally associated with the cell in its native state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0159] As used herein, a "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.

[0160] As used herein, the terms "T cell receptor" or "TCR" refer to a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. The TCR plays a role in recognizing antigens bound to major histocompatibility complex molecules. The TCR is composed of a heterodimer of an alpha (α) chain and a beta (β) chain coupled to three dimeric modules, CD3δ / CD3ε, CD3γ / CD3ε, and CD3ζ / CD3ζ. In some cells, the TCR is composed of a gamma chain and a delta (γ / δ) chain (CD3γ / CD3ε). The TCR may exist in alpha / beta and gamma / delta forms, which are structurally similar but have different anatomical locations and functions. Each chain is composed of two extracellular domains, namely a variable domain and a constant domain. In some embodiments, the TCR may be modified on any cell containing the TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells.

[0161] As used herein, the term "treatment" as used herein means treatment and / or prevention. A therapeutic effect is obtained by suppression, remission, or eradication of a medical condition.

[0162] The terms "therapeutically effective amount" or "effective amount" refer to the amount of a subject compound that induces a biological or medical response in a tissue, system, or subject sought by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of a compound sufficient to prevent, or to some extent alleviate, the onset of one or more signs or symptoms of a disorder or disease in a subject being treated. The therapeutically effective amount varies depending on the compound, the disease and its severity, the age, weight, etc. of the patient being treated.

[0163] As used herein, the term "treatment" refers to any protocol, method, and / or agent (e.g., CAR-T) that can be used for the prevention, management, treatment, and / or amelioration of a disease or a symptom associated therewith. In some embodiments, the terms "therapy" and "therapies" refer to biological therapies (e.g., adoptive cell therapy), supportive therapies (e.g., lymphodepletion therapy), and / or other therapies useful for the prevention, management, treatment, and / or amelioration of a disease or a symptom associated therewith that are known to those of ordinary skill in the art, such as medical professionals.

[0164] As used herein, the terms "treating," "treatment," and "treatment of" refer to a decrease or amelioration in the progression, severity, frequency, and / or duration of a disease or a symptom associated therewith that results from the administration of one or more treatments (including, but not limited to, CAR-T therapy directed to the treatment of solid tumors). The term "treatment" as used herein can also refer to changing the course of a disease in a subject being treated. The therapeutic effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of a disease, alleviation of symptoms, reduction of direct or indirect pathological consequences of a disease, decrease in the rate of disease progression, improvement of a medical condition or alleviation of pain, and remission or improvement of prognosis. In some embodiments, "treatment" of a disease as used herein means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0165] As used herein, the term "transfected" or "transformed" or "transduced" refers to the process by which exogenous nucleic acid is introduced or transferred into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. This cell includes primary subject cells and their progeny.

[0166] As used herein, the phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct position and orientation with respect to the polynucleotide and controls the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0167] 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 into the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes self-replicating plasmids or viruses. This term should also be construed to include non-plasmid and non-viral compounds that facilitate the entry of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0168] As used herein, the term "heterologous" refers to a graft derived from animals of different species.

[0169] Range: Throughout this disclosure, the various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, a description of a range should be considered to specifically disclose not only the individual numerical values within that range but also all possible sub-ranges. For example, a description of a range such as 1-6 should be considered to specifically disclose sub-ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as the individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0170] III. Fusion Proteins Having an Extended Half-Life One aspect of the present disclosure provides a fusion protein (e.g., a trispecific fusion protein) having an extended half-life, which includes 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 capable of extending the half-life of the entire fusion protein. The three domains of the fusion protein can be made in any order. For example, the half-life extension domain can be located at the N-terminus, the middle, or the C-terminus of the fusion protein.

[0171] The TACA fusion protein (e.g., a trispecific fusion protein) of the present disclosure can include a first domain (I) that is an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), a second domain (II) that is an immune cell recognition domain that specifically binds to a receptor (such as CD3) within an immune cell, and a third domain (III) that is a half-life extension domain. In some embodiments, the TACA fusion protein (e.g., a trispecific fusion protein) can include a first domain (I) that is an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), a second domain (II) that is a half-life extension domain, and a third domain (III) that is an immune cell recognition domain that specifically binds to a receptor (e.g., CD3) within an immune cell. In some embodiments, the TACA trispecific fusion protein includes a first domain (I) that is an immune cell recognition domain that specifically binds to a receptor (e.g., CD3) within an immune cell, a second domain (II) that is an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), and a third domain (III) that is a half-life extension domain.

[0172] In some embodiments, the TACA fusion protein (e.g., a trispecific fusion protein) comprises a first domain (I) that is an immune cell recognition domain that specifically binds to a receptor (e.g., CD3) within an immune cell, a second domain (II) that is a half-life extension domain, and a third domain (III) that is an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA). In some embodiments, the TACA fusion protein (e.g., a trispecific fusion protein) comprises a first domain (I) that is a half-life extension domain, a second domain (II) that is an immune cell recognition domain that specifically binds to a receptor (e.g., CD3) within an immune cell, and a third domain (III) that is an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA). In some embodiments, the TACA fusion protein (e.g., a trispecific fusion protein) comprises a first domain (I) that is a half-life extension domain, a second domain (II) that is an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), and a third domain (III) that is an immune cell recognition domain that specifically binds to a receptor (e.g., CD3) within an immune cell. In some embodiments, the first, second, and third domains are linked by a linker.

[0173] A. Half-life extension domain The half-life extension domain extends the elimination half-life of the protein of interest. The half-life extension domain also alters the pharmacodynamic properties of the protein, including changes in the tissue distribution, penetration, and diffusion of the trispecific antigen-binding protein. In some embodiments, the half-life extension domain provides improved tissue (including tumor) targeting, tissue distribution, tissue penetration, tissue diffusion, and efficacy as compared to a protein without a half-life extension domain. Therapeutic methods comprising a TACA fusion protein (e.g., a trispecific fusion protein) having a half-life extension domain of the present disclosure effectively and efficiently utilize a reduced amount of the GlyTR fusion protein, resulting in reduced side effects, such as a reduction in the cytotoxicity of non-tumor cells. Thus, the half-life extension domain enhances the therapeutic utility of GlyTR.

[0174] Furthermore, the binding affinity of the half-life extension domain can be selected to target the specific elimination half-life in a particular trispecific antigen-binding protein. Thus, 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 still other embodiments, the half-life extension domain has a low or marginal binding affinity. Exemplary binding affinities include KD concentrations of 10 nM or less (high), between 10 nM and 100 nM (moderate), and greater than 100 nM (low). As described above, the binding affinity to a half-life extension domain (e.g., serum albumin) can be determined by known methods such as surface plasmon resonance (SPR).

[0175] 1. Molecules capable of extending serum half-life 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 albumin, albumin, serum albumin, the Fc domain of an antibody, a polyethylene glycol moiety (PEG), a poly(lactic-co-glycolic acid) (PLGA) polymer, a polymeric hydrogel, nanoparticles, a fatty acid chain, an acyl group, a myristic acid group, a palmitoylated group, and a sterol group. In some embodiments, the half-life extension domain binds human serum albumin.

[0176] Generally, a polypeptide that extends the serum half-life in vivo is a polypeptide that occurs naturally in vivo and / or is resistant to degradation or removal by endogenous mechanisms that remove unwanted substances from an organism (e.g., a mammal or a human). For example, a polypeptide that extends the serum half-life in vivo can be selected from a protein derived from the extracellular matrix, a protein present in the blood, a protein present in the blood-brain barrier or neural tissue, a protein localized in the kidney, liver, lung, heart, skin or bone, a stress protein, a disease-specific protein, or a protein involved in Fc transport.

[0177] In some embodiments, suitable polypeptides that include a half-life extension domain that extends the serum half-life in vivo include, for example, a transferrin receptor-specific ligand-neuropharmaceutical fusion protein (see U.S. Patent No. 5,977,307, the teachings of which are incorporated herein by reference), a brain capillary endothelial cell receptor, transferrin, a transferrin receptor (e.g., a soluble transferrin receptor), insulin, an insulin-like growth factor 1 (IGF 1) receptor, an insulin-like growth factor 2 (IGF 2) receptor, an insulin receptor, a blood coagulation factor X, α1-antitrypsin, and HNF 1α. Suitable polypeptides that extend the serum half-life also include α-1 glycoprotein (orosomucoid, AAG), α-1 antichymotrypsin (ACT), α-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), haptoglobin (HPX), lipoprotein(a) (Lp(a)), mannose-binding protein (MBP), myoglobin (Myo), prealbumin (transthyretin, PAL), retinol-binding protein (RBP), and rheumatoid factor (RF).

[0178] In some embodiments, suitable polypeptides comprising a half-life extension domain that extends the in vivo serum half-life include proteins derived from the extracellular matrix, including, for example, collagen, laminin, integrin, and fibronectin. Collagen is a major protein of the extracellular matrix. Currently, about 15 types of collagen molecules are known, which are present in various parts of the body. Examples include type I collagen present in bone, skin, tendon, ligament, cornea, and internal organs (accounting for 90% of the collagen in the body), or type II collagen present in cartilage, intervertebral disc, notochord, and vitreous humor of the eye.

[0179] In some embodiments, suitable polypeptides comprising a half-life extension domain that extends the in vivo serum half-life include blood-derived proteins, examples of which include plasma proteins (such as fibrin, α-2 macroglobulin, serum albumin, fibrinogen (such as fibrinogen A, fibrinogen B), serum amyloid protein A, haptoglobin, profilin, ubiquitin, uteroglobin, and β-2 microglobulin), enzymes and enzyme inhibitors (such as plasminogen, lysozyme, cystatin C, α-1 antitrypsin, and pancreatic trypsin inhibitor), proteins of the immune system, such as immunoglobulin proteins (such as IgA, IgD, IgE, IgG, IgM, immunoglobulin light chain (kappa / lambda)), transport proteins (such as retinol-binding protein, neutrophil defensin (such as α-1 microglobulin), defensin (such as β-defensin 1, neutrophil defensin 1, neutrophil defensin 2, neutrophil defensin 3), etc.). Suitable proteins present in the blood-brain barrier or neural tissue include, for example, melanocortin receptor, myelin, ascorbic acid transporter, etc.

[0180] In some embodiments, suitable polypeptides comprising a half-life extension domain that extends the serum half-life in vivo include proteins localized in the kidney (e.g., polycystin, type IV collagen, organic anion transporter K1, Heymann antigen), proteins localized in the liver (e.g., alcohol dehydrogenase, G250), proteins localized in the lung (e.g., secretory component that binds to IgA), proteins localized in the heart (e.g., HSP 27 associated with dilated cardiomyopathy), proteins localized in the skin (e.g., keratin), bone-specific proteins such as morphogenetic proteins (BMPs), which are a subset of the transforming growth factor-β superfamily of proteins 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, cathepsin (e.g., cathepsin B that may be present in the liver and spleen)).

[0181] In some embodiments, disease-specific proteins that can extend the serum half-life of a protein include, for example, antigens expressed only on activated T cells, such as 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 I (HTLV-I) producing cells, see Immunol. 165(1):263-70 (2000)). Suitable disease-specific proteins also include, for example, metalloproteases (associated with arthritis / cancer) such as CG6512 Drosophila, human parapegyrin, human FtsH, human AFG3L2, mouse ftsH, and angiogenic growth factors such as 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 (TGF α), tumor necrosis factor-α (TNF-α), angiogenin, 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 fractalkine.

[0182] In some embodiments, suitable polypeptides comprising a half-life extension domain that extends the in vivo serum half-life include stress proteins such as heat shock proteins (HSP). HSP are normally present intracellularly. When these are found extracellularly, it is an indicator that the cells have died and released their contents. This unprogrammed cell death (necrosis) occurs as a result of trauma, disease, or injury when extracellular HSP elicits a response from the immune system. Binding to extracellular HSP may result in localization of the compositions of the present disclosure to the disease site.

[0183] 2. Human Serum Albumin In some embodiments, the half-life extension domain comprises human serum albumin or a molecule capable of binding serum albumin. Human serum albumin (HSA) (molecular weight approximately 67 kDa) is the most abundant protein in plasma. The mature form of HSA contains approximately 585 amino acids. HSA functions to maintain plasma pH, contributes to colloid osmotic pressure, serves as a carrier for many metabolites and fatty acids, and acts as the major drug transport protein in plasma. HSA accounts for a significant proportion of the osmotic pressure of serum and also functions as a carrier for endogenous and exogenous ligands. HSA is present in plasma at a concentration of approximately 50 mg / ml (600 μM) and has a half-life of approximately 20 days in humans.

[0184] The role of albumin as a carrier molecule and its inert nature are desirable properties for use as a carrier and transporter of polypeptides in vivo. The use of albumin as a carrier for various proteins as a component of albumin fusion 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, a fragment of HSA can be used in the fusion proteins of the present disclosure (e.g., a trispecific fusion protein). The HSA fragment can be an N-terminal fragment. Thus, by genetically or chemically fusing or conjugating the fusion proteins described herein to human albumin, the serum half-life is stabilized or extended and / or the activity of the molecule is retained in solution over a long period in vitro and / or in vivo.

[0185] 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 set forth in Table 2. In some embodiments, the fusion protein comprises the amino acid sequence set forth in Table 2 or Table 3.

[0186] a. Modified HSA In some embodiments, the HSA is mutated. The mutated HSA may have two amino acid substitutions compared to wild-type HSA. For example, the cysteine at position 34 (“C34S”) may be substituted with serine, 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) can be mutated to any amino acid residue other than cysteine (e.g., serine, threonine or alanine). Similarly, the asparagine residue at position 503 can be mutated to any amino acid residue other than asparagine (e.g., glutamine, serine, histidine or alanine). Specifically, substituting cysteine with serine at amino acid residue 34 can result in a decrease in the oxidation and protein heterogeneity of HSA. In wild-type HSA, the asparagine at amino acid residue 503 is sensitive to deamination, and as a result, the pharmacological half-life is likely to decrease. Substituting asparagine with glutamine at amino acid residue 503 can result in an extension of the pharmacological half-life of HSA, and correspondingly, the pharmacological half-life of the bispecific fusion proteins disclosed herein, as well as their cells, tissues or organs, can be extended.

[0187] In some embodiments, the half-life extension domain comprises a truncated wild-type HSA polypeptide operably linked to the bispecific fusion protein, with or without an attachment 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 deletions of internal residues. The truncation of more than one amino acid residue need not be linear (i.e., continuous).

[0188] The half-life extending domain (e.g., HSA) may be modified by site-specific chemical modification of amino acid residues within HSA, but need not necessarily be altered. In the properly folded tertiary structure of HSA, certain amino acid residues are displayed on the outer surface of the protein. Chemically reactive amino acid residues (e.g., cysteine) can be used in place of these surface-exposed residues to enable site-specific conjugation of other agents.

[0189] Alternatively, or additionally, the half-life extending domain (e.g., HSA) may optionally be modified by addition or removal of asparagine, serine or threonine residues from the HSA sequence to alter the glycosylation of these amino acid residues. Glycosylation sites added to HSA can be exposed on the surface. Glycosyl or other sugar chain moieties introduced into HSA can be directly conjugated to a diagnostic agent, a therapeutic agent or a cytotoxic agent.

[0190] Amino acid residues exposed on the surface of a half-life extended domain (e.g., HSA) may be replaced with cysteine residues to enable chemical conjugates of diagnostic, therapeutic, or cytotoxic agents. Cysteine residues exposed on the surface of HSA (when folded in its native tertiary structure) enable specific conjugation of diagnostic, therapeutic, or cytotoxic agents to thiol-reactive groups such as maleimide or haloacetyl. The nucleophilic reactivity of the thiol functional group of cysteine residues is approximately 1000-fold higher compared to other arbitrary amino acid functional groups within the protein, such as the amino group of lysine residues or the N-terminal amino group. Thiol-specific functional groups in iodoacetyl and maleimide reagents may react with amine groups, but higher pH (>9.0) and longer reaction times are required (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London). The amount of free thiol in the protein may be estimated using a standard Ellman assay. In some instances, 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.

[0191] The site of cysteine substitution can be identified by analysis of the surface accessibility of HSA. The surface accessibility can be represented as the surface area (e.g., square angstroms) that solvent molecules such as water can contact. The space occupied by water is approximated as a sphere with a radius of 1.4 angstroms. Software for calculating the surface accessibility of each amino acid of a protein is available free of charge or can be licensed. For example, the crystallography program CCP4 Suite of the CCP4 Suite (“The CCP4 Suite: Programs for Protein Crystallography” Acta. Cryst. D50:760-763 (1994); ccp4.ac.uk / dist / html / INDEX.html), which employs an algorithm for calculating the surface accessibility of each amino acid of a protein using coordinates obtained from known X-ray crystallographic analysis. Also, the solvent accessibility may be evaluated using the free software DeepView Swiss PDB Viewer downloaded from the Swiss Institute of Bioinformatics. Substituting cysteine at a site exposed on the surface enables the conjugate of a reactive cysteine to a thiol-reactive group linked to a diagnostic agent or a therapeutic agent.

[0192] b. Glycosylated HSA Furthermore, by engineering a glycosylation site into the half-life extension domain (e.g., HSA), alteration of the clearance rate can be achieved. In certain embodiments, HSA can be glycosylated. Glycosylation of a polypeptide is typically either N-linked or O-linked. N-linked refers to the attachment of a sugar moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X represents any amino acid other than proline) are recognition sequences for the enzymatic attachment of a sugar moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences within a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid (most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used).

[0193] Addition or deletion of a glycosylation site to HSA can be readily achieved by altering the amino acid sequence such that one or more of the above-described tripeptide sequences (for N-linked glycosylation sites) are created. Alternatively, the alteration may be made by addition, deletion or substitution of one or more serine or threonine residues to the sequence of HSA (for O-linked glycosylation sites). The sugar chain structure obtained on HSA can also be used for site-specific conjugates of cytotoxic agents, immunomodulatory agents or cell growth inhibitory agents. In some embodiments, human serum albumin is glycosylated.

[0194] c. Generation of HSA fusion proteins Fusion of albumin to another protein may be achieved by genetic manipulation such as DNA encoding HSA or a fragment thereof. A suitable host is then transformed or transfected with a fusion nucleotide sequence that expresses the fusion polypeptide by being placed on a suitable plasmid. Expression can occur, for example, in vitro from prokaryotic or eukaryotic cells or in vivo (e.g., from transgenic organisms). Additional methods related to HSA fusion are described, for example, in WO2001 / 077137 and WO2003 / 06007, which are incorporated herein by reference.

[0195] The elimination half-life of the short-lived protein is extended by non-covalent association between the protein of interest and HSA. For example, the recombinant fusion of HSA to GlyTR of the present disclosure resulted in at least a 5-fold increase in half-life when administered intravenously to mice compared to administration of GlyTR alone. FIGS. 22A-22E. In some embodiments, the half-life of the fusion protein is 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 extended compared to a fusion protein lacking a half-life extension domain. In some embodiments, the half-life extension is based on the average plasma residence time of the fusion protein. The clearance rate of the fusion protein containing HSA can be optimized by testing conjugates containing cleaved wild-type HSA.

[0196] A molecule that can extend the half-life of another molecule, such as HSA, can be incorporated into a fusion protein by direct or indirect linkage to the fusion protein. The term "direct" linkage refers to the fusion protein binding directly to the half-life extension domain, eliminating the 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" linkage refers to the bispecific fusion protein binding not directly to the half-life extension domain, but rather through an amino acid "attachment linker" between the bispecific fusion protein and the half-life extension domain. Examples of amino acid attachment linkers include, but are not limited to, linkers composed entirely of glycine, alanine, serine, glutamine, leucine or valine residue linkers, or any combination of these residues. These amino acid attachment 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 in length, providing a flexible tether between the fusion protein (e.g., a TACA bispecific fusion protein) and the half-life extension domain. An amino acid attachment linker that can attach to the C-terminus or N-terminus of a half-life extension domain such as HSA or mutant HSA (e.g., by a covalent bond (e.g., a peptide bond), an ionic bond or a hydrophobic bond, or through a high-affinity protein-protein binding interaction (e.g., biotin and avidin)).

[0197] d. A molecule capable of binding serum albumin In some embodiments, the half-life extension domain comprises a molecule capable of binding serum albumin. For example, a peptide ligand that binds human serum albumin may comprise the amino acid sequence of D-Xaa-CLP-Xaa-WGCLW (SEQ ID NO: 70), and may be fused with a fusion protein (e.g., bispecific GlyTR) disclosed herein to mobilize HSA when administered to a subject. In some embodiments, Xaa can be any amino acid. A peptide ligand that binds mammalian serum albumin can be identified by its ability to compete for binding of human serum albumin in an in vitro assay with the peptide ligand. In some embodiments, a peptide ligand that binds 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).

[0198] In some embodiments, the half-life extension domain comprises a fatty acid chain conjugated polypeptide. The fatty acid chain may 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 conjugate 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 complementarity determining region (CDR) of a single domain antibody, or a single chain variable fragment (scFv).

[0199] 3 Fc fusion Natural antibody molecules are composed of two identical heavy chains and two identical light chains. The heavy chain constant region includes CH1, the hinge region, CH2, and CH3. Papain digestion of the antibody produces two fragments, Fab and Fc. The Fc fragment is composed of CH2, CH3, and part of the hinge region. The Fc region has been recognized as important for maintaining the serum half-life of antibodies of class IgG (Ward and Ghetie, Ther. Immunol. 2:77-94 (1995)). Studies have shown 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 made it possible to prepare novel chimeric polypeptides with multiple functional domains. The most common among such chimeric polypeptides are immunoglobulin (Ig) fusion proteins. These proteins are composed of the Fc region of an antibody, usually a mouse or human antibody, fused to an unrelated protein or protein fragment.

[0200] As used herein, the term "Fc" refers to a polypeptide comprising at least a portion of CH3, CH2, and the hinge region of the constant domain of an antibody. Optionally, the Fc region may include the CH4 domain present in some antibody classes. Fc may include the entire hinge region of the constant domain of the antibody. In one embodiment, the fusion protein includes the Fc region and the CH1 region of the antibody. In one embodiment, the fusion protein includes the Fc region and the CH3 region of the antibody. In another embodiment, the fusion protein includes Fc, the CH1 region, and the kappa / lambda region from the constant domain of the antibody. 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, etc.

[0201] 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 to 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 with the amino acid sequence set forth in SEQ ID NOs: 33 to 56, and the Fc domain of an antibody. In some embodiments, the Fc domain is a half-life extension domain. In one embodiment, the half-life extension domain is the Fc domain of an IgG antibody. In one embodiment, the half-life extension domain is the Fc domain of an antibody selected from the IgG1, IgG2, IgG3 or IgG4 Fc regions. 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 set forth in SEQ ID NO: 69, or SEQ ID NOs: 91 to 94. In some embodiments, the half-life extension domain is an Fc fusion protein.

[0202] The fusion proteins of the present disclosure that include an Fc region / domain as a half-life extension domain may be produced by standard recombinant DNA techniques or by protein synthesis techniques (e.g., by use of a peptide synthesizer). For example, a nucleic acid molecule encoding an Fc fusion protein can be synthesized by conventional techniques including an automated DNA synthesizer. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers. The anchor primers can generate complementary overhangs between two consecutive gene fragments, which can then be annealed and reamplified to generate a chimeric gene sequence (see, e.g., “Current Protocols in Molecular Biology”, Ausubel et al., eds., John Wiley & Sons, (1992)). Moreover, the nucleic acid encoding the fusion proteins disclosed herein can be cloned into an expression vector containing an Fc domain or a fragment thereof such that the fusion proteins disclosed herein are ligated in-frame to the constant domain or a fragment thereof.

[0203] Methods of fusing or conjugating a polypeptide to the constant region of an antibody are disclosed herein and / or are known in the art. See, for example, U.S. Pat. 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, 5,112,946; European Patent Publications, EP 0 307 434; EP 0 367 166; EP 0 394 827; PCT Publications WO 91 / 06570, WO 96 / 04388, WO 96 / 22024, WO 97 / 34631, 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. Pat. 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 an Fc modified as a half-life extending domain are also within the scope of the present disclosure.

[0204] In some embodiments, suitable polypeptides comprising a half-life extension domain that extends the in vivo serum half-life include proteins involved in Fc transport. These proteins include, for example, the Brambell receptor (also called FcRB). This Fc receptor has two functions, both of which may be useful for delivery. The functions are (1) transport of IgG from mother to child via the placenta, and (2) protecting IgG from degradation, thereby extending the serum half-life. The receptor is thought to recycle IgG from the endosome. (See Holliger et al, Nat Biotechnol 15(7):632-6 (1997).)

[0205] 4. PEGylation In another embodiment, the half-life extension domain comprises polyethylene glycol (PEG). Efforts to reduce the clearance of molecules having a molecular weight of 60 kDa have generally focused on increasing the molecular size of these proteins by protein fusion, glycosylation, or addition of polyethylene glycol polymers (i.e., PEG). To extend the in vivo serum circulation of the bispecific fusion proteins disclosed herein, the bispecific fusion protein (i.e., GlyTR) can be linked to an inert polymer molecule such as high molecular weight PEG. The linkage may or may not have an attachment linker. The linkage can be either by site-specific conjugation of PEG to the N-terminus or C-terminus of the bispecific fusion, or via the epsilon amino groups present in lysine residues. For PEGylation, typically the bispecific fusion is reacted with polyethylene glycol (PEG) (such as a reactive ester or aldehyde derivative of PEG) under conditions such that one or more PEG groups attach to the bispecific fusion. PEGylation can be carried out by an acylation or alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer).

[0206] As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, examples of which include mono (C1-C10) alkoxy or aryloxy polyethylene glycol or polyethylene glycol maleimide. Derivatization of linear or branched polymers that minimizes loss of biological activity can be used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to bispecific fusions, resulting in the production of trispecific fusion proteins. Unreacted PEG can be separated from the trispecific fusion protein by size exclusion or by ion exchange chromatography. The trispecific fusion protein can be tested for binding activity and even in vivo efficacy using methods well known to those of skill in the art (e.g., by the immunological assays described herein). Methods for PEGylating proteins are known in the art (see, e.g., EP 0 154 316 by Nishimura et al. and EP 0401384 by Ishikawa et al.).

[0207] Other improved PEGylation techniques include a reconstituted chemical orthogonal manipulation technique (ReCODE PEG) that incorporates chemically specified side chains into biosynthetic proteins via a reconstitution system containing a tRNA synthetase and tRNA. This technique enables the incorporation of more than 30 new amino acids into biosynthetic proteins in E. coli, yeast, and mammalian cells. The tRNA incorporates unnatural amino acids wherever an amber codon is placed and converts the amber from a stop codon to a codon that signals the incorporation of a chemically specified amino acid.

[0208] In addition, recombinant PEGylation technology (rPEG) can be used for serum half-life extended proteins. This technology involves genetically fusing an unstructured protein tail of 300 to 600 amino acids to a bispecific fusion protein. Since the apparent molecular weight of such an unstructured protein chain is approximately 15 times larger than the actual molecular weight, the serum half-life of the trispecific fusion protein is significantly increased. In contrast to conventional PEGylation that requires chemical conjugation and repurification, the manufacturing process is greatly simplified and the product becomes homogeneous.

[0209] In some embodiments, the half-life extended 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.

[0210] 5. PSA PSA polymers naturally exist in the human body. PSA polymers are adopted by certain bacteria and have evolved over millions of years to cover their walls with PSA polymers. These natural polysialylated bacteria were then able to deceive the body's defense system, thanks to molecular mimicry. PSA, the ultimate stealth technology in nature, can be easily mass-produced from such bacteria with predetermined physical properties. Since bacterial PSA is chemically identical to PSA in the human body, it remains completely non-immunogenic even when coupled to a protein.

[0211] Polysialylation is another technique 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 (sugar). When used for drug delivery of proteins and therapeutic peptides, polysialic acid provides a protective microenvironment during conjugation. This extends the active lifespan of bispecific fusion proteins in circulation and prevents their recognition by the immune system. In some embodiments, the half-life extension domain can include polysialic acid (PSA).

[0212] 6. HESylation HES is a modified natural polymer derived from waxy maize starch and can be metabolized by enzymes in the body. HES solutions are typically administered to replenish depleted blood volume and improve the rheological properties of blood. HESylation of bispecific fusion proteins increases the stability of the molecule and further reduces renal clearance, allowing for an extended circulation half-life and resulting in increased biological activity. By varying various 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 includes hydroxyethyl starch ("HES"). HES derivatives linked to the bispecific fusion proteins disclosed herein can result in trispecific fusion proteins with an extended half-life.

[0213] B. Lectin Malignant transformation of cells is almost universally associated with 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 cancers and human cancers (Hakomori, 2002, PNAS USA, 99:10231-10233), and these altered sugar structures are called tumor-associated carbohydrate antigens (TACAs) (Table 1). Due to this tumor-specific property, cell surface TACAs are excellent target antigens for the production of monoclonal antibodies targeting many common cancers. However, despite decades of effort, specific antibodies with high affinity for TACAs are not yet available because it is difficult to generate antibodies against carbohydrate antigens.

[0214] Therefore, the inventors of the present disclosure have engineered therapeutic bispecific fusion proteins and CARs that selectively target TACAs on tumor cells, relying on lectins rather than antibodies. Lectins and their binding partners are well known in the art. See, for example, functionalglycomics.org / glycomics / publicdata / primaryscreen.jsp. The lectin-binding proteins of the present disclosure have significant advantages over existing technologies (e.g., GlyTR chimeric proteins) and provide opportunities for the development of a new class of therapeutic agents for cancer immunotherapy. Based on the concept of GlyTR and the availability of a number of different lectins specific for a number of different TACAs, multiple GlyTRs can be generated by replacing L-PHA with other lectins. Alternatively, chimeric proteins composed of lectins and scFvs that can recruit other immune effector cells can also be produced. The functionality of the lectin domain can be improved by mutation. For example, GlyTR L-PHA x CD3 can be further improved by exchanging the sugar-binding domain of E-PHA with L-PHA, resulting in a 20- to 30-fold increase in binding (Kaneda et al., 2002, J Biol Chem, 277:16928-16935).

[0215] 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.

[0216] Additional information regarding the GlyTR technology and subsequent improvements is disclosed in International Application Nos. 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 hereby incorporated by reference in their entirety. [Table 1]

[0217] The antigen-binding domain of the bispecific fusion proteins disclosed herein is 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 include an affinity for a target antigen (e.g., a tumor-associated carbohydrate antigen) on a target cell (e.g., a cancer cell). The target antigen may include any type of protein or epitope thereof that associates with the target cell. For example, the bispecific fusion protein may have an affinity for a target antigen on the target cell that indicates a specific state of the target cell.

[0218] In some embodiments, the antigen-binding domain comprises one or more TACA-binding domains. In some embodiments, the antigen-binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more TACA-binding domains. In one embodiment, the antigen-binding domain comprises one TACA-binding domain. In one embodiment, the antigen-binding domain comprises two TACA-binding domains. In one embodiment, the antigen-binding domain comprises three TACA-binding domains. In one embodiment, the antigen-binding domain comprises four TACA-binding domains.

[0219] In some embodiments, the lectin is selected from the group consisting of galectin, siglec, selectin; C-type lectin; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Phaseolus vulgaris leukocyte agglutinin); E-PHA (Phaseolus vulgaris erythroagglutinin); tomato lectin (Lycopersicon esculentum lectin; LEA); peanut lectin (Arachis hypogaea lectin; PNA); potato lectin (Solanum tuberosum lectin), pokeweed mitogen (Phytolacca americana lectin), wheat germ lectin (Triticum aestivum germ lectin); Artocarpus polyphyllus lectin (jacalin lectin); hairy vetch lectin (VVA); apple snail lectin (HPA); fucose lectin (WFA); Sambucus nigra lectin (SNA), BC2L-CNt (lectin from the Gram-negative bacterium Burkholderia cenocepacia), dog spleen leukocyte lectin (MAL), Pleurotus ostreatus (PVL), Sclerotium rolfsii lectin (SRL), Euonymus sieboldianus lectin (ESA), CLEC17A (prolectin), Hypsizygus marmoreus lectin, Allium fistulosum lectin (SSA), Glechoma hederacea lectin (Gleheda), Momordica charantia lectin (Morniga G), Salvia officinalis lectin, Salvia bogotensis lectin, Salvia horminum lectin, Cuscutaceae lectin, Calceolaria integrifolia lectin, Glycine max lectin, Phaseolus coccineus lectin, Amaranthus leucocarpus lectin, Relia autumnalis lectin, Paramignya monophylla lectin, Ipomoea batatas lectin, Himalayan Vicia faba lectin, Himalayan Vicia faba lectin, soybean lectin and mushroom lectin.

[0220] In some embodiments, the lectin can be a galectin selected from the group consisting of galectin-1, galectin-2, galectin-3, galectin-4, galectin-5, galectin-6, galectin-7, galectin-8, galectin-9, galectin-10, galectin-11, galectin-12, galectin-13, galectin-14 and galectin-15. In some embodiments, the lectin can be a siglec selected from the group consisting of siglec-1 (sialoadhesin), siglec-2 (CD22), siglec-3 (CD33), siglec-4 (myelin-associated glycoprotein), siglec-5, siglec-6, siglec-7, siglec-8, siglec-9, siglec-10, siglec-11, siglec-12, siglec-13, siglec-14, siglec-15, siglec-16, siglec-17, siglec-E, siglec-F, siglec-G and siglec-H. In some embodiments, the TACA-binding domain is derived from a selectin or a C-type lectin.

[0221] In some embodiments, the lectin is a polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) that can be selected from the group consisting of ppGalNAc-T1 (GALNT1), ppGalNAc-T2 (GALNT2), ppGalNAc-T3 (GALNT3), ppGalNAc-T4 (GALNT4), ppGalNAc-T5 (GALNT5), ppGalNAc-T6 (GALNT6), ppGalNAc-T7 (GALNT7), ppGalNAc-T8 (GALNT8), ppGalNAc-T9 (GALNT9), ppGalNAc-T10 (GALNT10), ppGalNAc-T12 (GALNT12), ppGalNAc-T13 (GALNT13), ppGalNAc-T14 (GALNT14), ppGalNAc-T15 (GALNT15), ppGalNAc-T16 (GALNT16), ppGalNAc-T17 (GALNT17), ppGalNAc-T18 (GALNT18), ppGalNAc-T Like 5 (GALNTL5) and ppGalNAc-T Like 6 (GALNTL6).

[0222] In some embodiments, the antigen-binding domains of the bispecific fusion proteins described herein are β1,6-branched, β1,6GlcNAc-branched N-glycans, T antigen, Tn antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6-sialylation, sialylation, Lewis-y (Le y )), sialyl-Lewisx / a, disialyl-Lewis x / a , sialyl 6-sulfo Lexisx, Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GM1, and selectively target TACAs selected from the group consisting of.

[0223] C. TACAs The TACA-binding domain may include any peptide, protein, lectin, lectin fragment, antibody, antibody fragment, small molecule, nucleic acid, etc. that can specifically bind to TACAs. 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.

[0224] Exemplary TACAs and their binding partners are listed in Table 1. Exemplary TACAs include, but are not limited to, β1,6-branched, T antigen, sialyl-T epitope, Tn epitope, sialyl-Tn epitope, a2,6-sialylation, sialylation, sialyl-Lewis^, disialyl-Lewis^, sialyl 6-sulfo Lexis x , Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GMl. In some embodiments, the bispecific fusion is β1,6-branched, β1,6GlcNAc-branched N-glycans, T antigen, Tn antigen, sialyl-T epitope, Tn epitope, sialyl-Tn epitope, α2,6-sialylation, sialylation, Lewis-y (Le y ))), sialyl-Lewis x / a, dicyaryllium-Lewis x / a , sialyl 6-sulfo Lexis x It selectively targets TACAs selected from the group consisting of 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.

[0225] In one embodiment, the TACA binding domain binds to N-glycans. In certain embodiments, the TACA binding domain binds to tri- and tetra-branched oligosaccharides. In one embodiment, the TACA binding domain binds to β1,6GlcNAc-branched N-glycans. In one embodiment, the TACA binding domain binds to the Tn epitope.

[0226] In some embodiments, the antigen binding domain comprises the amino acid sequence set forth 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 with the amino acid sequence set forth in SEQ ID NOs: 33-56. In one embodiment, the antigen binding comprises an amino acid sequence having at least 90% homology with SEQ ID NOs: 33-56.

[0227] D. Immunorecognition domain In some embodiments, the bispecific fusion protein comprises an immunocyte recognition domain that selectively binds to a receptor on an immune effector cell. In such embodiments, the immune effector cell can be selected from the group consisting of T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, monocytes, dendritic cells, and neutrophils. In such embodiments, the immune effector cell can be a T cell. In another embodiment, the immune effector cell can be an NK cell.

[0228] The receptor on the immune effector cell can be selected from the group consisting of T cell receptor (TCR) alpha, TCR beta, TCR gamma, TCR delta, 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 an NK cell, and the NK cell receptor may be selected from the group consisting of NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1.

[0229] In some embodiments, the immunocyte 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 the immune effector cell. The immunocyte recognition domain may comprise an scFv that can selectively bind CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1. In some embodiments, the immunocyte recognition domain specifically binds CD3. In some embodiments, the immunocyte recognition domain may comprise the amino acid sequences of SEQ ID NOs: 75-77.

[0230] 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 with the amino acid sequence of SEQ ID NO: 59, 60 or 61.

[0231] Alternatively, the immune cell recognition domain comprises an antibody Fc domain and optionally the 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 Fc domain of an antibody and a domain that specifically binds 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 that embodiment, the Fc domain may comprise the amino acid sequence set forth in SEQ ID NO: 69, or 91-94. In another embodiment, the immune cell recognition domain comprises the constant region domains CH2 and / or CH3 of an antibody, preferably CH2 and CH3. The constant region domains CH2 and / or CH3 of the antibody may or may not include a hinge region.

[0232] E. Fusion Protein 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 capable of extending the half-life of the fusion protein.

[0233] Bispecific fusion proteins contain two different binding specificities and thus bind to two different antigens. In one embodiment, the bispecific fusion protein comprises a first antigen recognition domain that binds to a first antigen (e.g., TACA) and a second antigen recognition domain that binds to a second antigen. In one embodiment, the first antigen recognition domain is a TACA binding domain. Examples of TACA are described elsewhere in this specification, and all of them may be targeted by the bispecific fusion proteins of the present disclosure. In certain embodiments, the second antigen recognition domain binds to immune effector cells. 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.

[0234] In some embodiments, the bispecific fusion protein selectively targets a TACA selected from the group consisting of β1,6-branched, β1,6GlcNAc-branched N-glycans, T antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6-sialylation, sialylation, Lewis-y (Le y )), sialyl-Lewis x / a , disialyl-Lewis x / a , sialyl 6-sulfo Lexis x , Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GM1. In one embodiment, the bispecific fusion protein selectively targets the 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.

[0235] In some embodiments, the fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 1 to 32, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1 to 32. In some embodiments, the fusion protein (e.g., a trispecific fusion protein) comprises an amino acid sequence selected from SEQ ID NOs: 1 to 12. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NOs: 13 to 32. In some embodiments, the fusion protein comprises the amino acid sequence disclosed in Table 2 or Table 3.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

[0236] In some embodiments, the fusion protein showed enhanced binding to Thomsen-nouveau (Tn) antigen-expressing tumor cells or β1,6GlcNAc branched N-glycan-expressing tumor cells as compared to a fusion protein containing a flexible linker in the antigen-binding domain. In some embodiments, the flexible linker is a glycine-serine linker, or an amino acid sequence selected from 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 with an amino acid sequence selected from GGGGS (SEQ ID NO: 86), GGGGSGGGGS (SEQ ID NO: 87), or GGGGSGGGGSGGGGS (SEQ ID NO: 85).

[0237] In some embodiments, the fusion protein selectively targets Tn antigen or β1,6GlcNAc branched N-glycan. In some embodiments, a fusion protein that selectively targets 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 with an amino acid sequence selected from SEQ ID NOs: 36-42, 52-56, or 62. In some embodiments, a fusion protein that selectively targets 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 with an amino acid sequence selected from SEQ ID NOs: 13-32.

[0238] 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 with 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 with an amino acid sequence selected from SEQ ID NOs: 1-12.

[0239] 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 with the amino acid sequence set forth in SEQ ID NOs: 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 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.

[0240] 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 to 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 sequences set forth in SEQ ID NOs: 33 to 56, and the 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 extension domain. In some embodiments, the half-life extension domain is a polypeptide 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 to 94.

[0241] F. Production of Fusion Proteins The fusion proteins or peptides of the present disclosure may be produced using chemical methods. For example, peptides, fusion proteins (bispecific or trispecific fusion proteins) can be synthesized by solid-phase techniques (Roberge J Y et al (1995) Science 269:202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis may be achieved using, for example, an ABI 431A peptide synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

[0242] The peptides and fusion proteins of the present disclosure may be synthesized by conventional techniques. For example, the peptides or fusion proteins may be synthesized by chemical synthesis using solid-phase peptide synthesis methods. In these methods, either a solid-phase synthesis method or a liquid-phase synthesis method is employed (for example, for solid-phase synthesis techniques, see J. M. Stewart, and J. D. Young, Solid Phase Peptide Synthesis, 2 nd Ed., Pierce Chemical Co., Rockford 111. (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254; for classical solution synthesis, see M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, suprs, Vol 1). As an example, the peptides of the present disclosure may be synthesized by directly incorporating phosphothreonine as an N-fluorenylmethoxycarbonyl-O-benzyl-L-phosphothreonine derivative using 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase chemistry.

[0243] An N-terminal fusion protein or C-terminal fusion protein comprising a peptide or fusion protein of the present disclosure conjugated to another molecule may be prepared by recombinant techniques by fusing the N-terminus or C-terminus of the peptide or fusion protein with the sequence of a selected protein or selectable marker having a desired biological function. The resulting fusion protein contains the peptide of the fusion protein of the present disclosure fused to the selected protein or marker protein as described herein. Examples of proteins that may be used in the preparation of the fusion protein include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.

[0244] The peptides or fusion proteins of the present disclosure may be developed using biological expression systems. By using these systems, it is possible to produce large libraries of random peptide sequences and screen these libraries for peptide sequences that bind to specific proteins. Libraries may be produced by cloning synthetic DNA encoding random peptide sequences into 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 may also be constructed by the simultaneous synthesis of overlapping peptides (see U.S. Patent No. 4,708,871).

[0245] 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 peptide or fusion protein that is "substantially homologous" is about 50% homologous to the amino acid sequence of the peptides disclosed herein, more preferably about 70% homologous, even more preferably about 80% homologous, more preferably about 90% homologous, even more preferably about 95% homologous, and even more preferably about 99% homologous.

[0246] 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%, at least 99% sequence identity with the amino acid sequences set forth 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 with the amino acid sequences set forth in SEQ ID NOs: 1-32. The peptide or fusion protein may alternatively be produced by recombinant means or by cleavage from a longer polypeptide.

[0247] The variants of the bispecific fusion proteins and peptides according to the present disclosure are those in which (i) one or more of the amino acid residues are substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and such substituted amino acid residues may or may not be encoded by the genetic code, (ii) those in which one or more modified amino acid residues (e.g., residues modified by the attachment of substituents) are present, (iii) those in which the peptide is an alternative splice variant of the peptide of the present disclosure, (iv) fragments of the peptide, and / or (v) those in which the peptide may be fused to another peptide such as a leader or secretion sequence, or a sequence employed for purification (e.g., His-tag) or detection (e.g., Sv5 epitope tag). The fragments include peptides generated by proteolytic cleavage (including multi-site proteolysis) of the original sequence. The variants may be subject to post-translational or chemical modifications. Such variants are determined to be within the scope of those skilled in the art according to the teachings herein.

[0248] As is known in the art, the "similarity" between two peptides is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions to the sequence of a second polypeptide. Variants are defined as those that include a peptide sequence that differs from the original sequence, i.e., preferably less than 40% of the residues per targeted segment differ from the original sequence, more preferably less than 25% of the residues per targeted segment differ from the original sequence, more preferably less than 10% of the residues per targeted segment differ from the original sequence, and most preferably only a very small number of residues per targeted segment differ from the original protein sequence, while at the same time being homologous enough to maintain the functionality of the original sequence and / or the ability to bind to TACA. The present disclosure includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90% or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods well known to those of skill in the art. The identity between two amino acid sequences is preferably determined using the BLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)].

[0249] The fusion proteins of the present disclosure can be post-translationally modified. For example, post-translational modifications included within the scope of the present disclosure include cleavage of signal peptides, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding, proteolytic processing, and the like. Some modification or processing events require the introduction of additional biological machinery. For example, processing events such as cleavage of signal peptides and core glycosylation are examined by adding dog microsomal membranes or Xenopus laevis oocyte extracts (U.S. Patent No. 6,103,489) to the standard translation reaction.

[0250] In some embodiments, the bispecific fusion proteins of the present disclosure may include non-natural amino acids formed by post-translational modifications or by introducing non-natural amino acids during translation. Various approaches are available for introducing non-natural amino acids during protein translation.

[0251] The peptides or fusion proteins of the present disclosure may be conjugated with other molecules such as proteins to prepare fusion proteins. This can be achieved, for example, by the synthesis of N-terminal or C-terminal fusion proteins. However, it is necessary that the resulting fusion protein retains the functionality of the peptide. The peptides or bispecific fusion proteins of the present disclosure may be phosphorylated using conventional methods, examples of which include the methods described in Reedijk et al. The EMBO Journal 11(4):1365(1992).

[0252] Cyclic derivatives of the peptides or fusion proteins of the present disclosure are also contemplated. Cyclization may enable the peptide to adopt a preferred conformation upon association with other molecules. Cyclization may be achieved using techniques known in the art. For example, a disulfide bond may be formed between two components having free sulfhydryl groups and appropriately spaced apart, or an amide bond may be formed between the amino group of one component and the carboxyl group of another component. Also, cyclization may be achieved using azobenzene-containing amino acids as described in Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The components forming the bond may be side chains of amino acids, non-amino acid components, or a combination of the two. In embodiments of the present disclosure, the cyclic peptide may contain a β-turn in the correct position. A β-turn may be introduced into the peptide of the present disclosure by adding the amino acids Pro-Gly in the correct position. It may be desirable to produce a cyclic peptide that is more flexible than a cyclic peptide containing a peptide bond linkage as described above. A more flexible peptide may be prepared by introducing 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 as not to deform or rotate the β-sheet. As a result of the shorter length of the disulfide linkage and the fewer number of hydrogen bonds in the β-sheet portion, the peptide becomes more flexible. The relative flexibility of the cyclic peptide can be determined by molecular dynamics simulations.

[0253] One aspect of the present disclosure provides a bispecific fusion protein, fusion protein, CAR or peptide fused or integrated with a target protein, and / or a targeting domain capable of directing the bispecific fusion protein, fusion protein, CAR or peptide to a desired cellular component or cell type or tissue. The bispecific fusion protein, fusion protein, CAR or peptide may also contain additional amino acid sequences or domains. The bispecific fusion protein, fusion protein, CAR or peptide is recombinant in the sense that the various components are from various sources and thus are not found together in nature (i.e., are heterologous).

[0254] In one embodiment, the targeting domain can be a transmembrane domain, a membrane-binding domain, or a sequence that directs a protein to associate with, for example, a vesicle or nucleus. In one embodiment, the targeting domain can enable a peptide to reach a particular cell type or tissue. For example, the targeting domain can be a cell surface ligand or antibody against a cell surface antigen of a target tissue (e.g., bone, regenerated bone, degenerated bone, cartilage). The targeting domain may enable the peptides of the present disclosure to reach cellular components.

[0255] IV. Nucleic Acids and Expression Vectors A. Bispecific Fusion Protein 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 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 capable of extending the half-life of the fusion protein. In some embodiments, the half-life extension domain is located at the N-terminus, central or C-terminus of the fusion protein.

[0256] 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.

[0257] 1. Half-life extension In some embodiments, the half-life of the fusion protein 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 fusion protein lacking the half-life extension domain. In some embodiments, the half-life extension is based on the average plasma residence time of the fusion protein.

[0258] In some embodiments, the half-life extension domain comprises a molecule selected from the group consisting of a polypeptide capable of binding albumin, albumin, serum albumin, the Fc domain of an antibody, a polyethylene glycol moiety (PEG), a poly(lactic-co-glycolic acid) (PLGA) polymer, a polymeric hydrogel, a nanoparticle, a fatty acid chain, an acyl group, a myristic acid group, a palmitoylation group, and a sterol group.

[0259] In some embodiments, the half-life extension domain comprises the Fc domain of an antibody selected from the IgG1, IgG2, IgG3, or IgG4 Fc region. In one embodiment, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 69, or SEQ ID NOs: 91-94. In another embodiment, the half-life extension domain comprises a PEG moiety. In one embodiment, the PEG moiety is less than about 0.5k, 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.

[0260] In some embodiments, the half-life extension domain comprises a serum albumin polypeptide (e.g., human serum albumin). In some embodiments, the half-life extension domain comprises the amino acid sequence of SEQ ID NO: 57.

[0261] In some embodiments, the half-life extension domain comprises a molecule capable of binding serum albumin. For example, a peptide ligand that binds human serum albumin may comprise the amino acid sequence of D-Xaa-CLP-Xaa-WGCLW (SEQ ID NO: 70), and may be fused with bispecific GlyTR to mobilize HSA when administered to a subject. Xaa is any amino acid. A peptide ligand that binds mammalian serum albumin can be identified by its ability to compete for binding of human serum albumin in an in vitro assay with the peptide ligand. In some embodiments, the peptide ligand that binds 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).

[0262] In some embodiments, the half-life extension domain comprises a fatty acid chain conjugated polypeptide. The fatty acid chain may 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 conjugate 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 complementarity determining region (CDR) of a single domain antibody, or a single chain variable fragment (scFv).

[0263] 2. Antigen binding domain 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 galectin, siglec, selectin; C-type lectin; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Phaseolus vulgaris leukocyte agglutinin); E-PHA (Phaseolus vulgaris erythroagglutinin); tomato lectin (Lycopersicon esculentum lectin; LEA); peanut lectin (Arachis hypogaea lectin; PNA); potato lectin (Solanum tuberosum lectin), pokeweed mitogen (Phytolacca americana lectin), wheat germ agglutinin (Triticum aestivum germ lectin); Artocarpus polyphyllus lectin (jacalin lectin); hairy vetch lectin (VVA); apple snail lectin (HPA); fucose lectin (WFA); Sambucus nigra lectin (SNA), BC2L-CNt (lectin from the gram-negative bacterium Burkholderia cenocepacia), dog kidney leukocyte agglutinin (MAL), Lentinus edodes (PVL), Sclerotium rolfsii lectin (SRL), Euryale ferox lectin (ESA), CLEC17A (prolectin), Hericium erinaceus lectin, Allium sativum lectin (SSA), Glechoma hederacea lectin (Gleheda), Momordica charantia lectin (Morniga G), Onosma aliciae lectin, Salvia bogotensis lectin, Salvia horminum lectin, Cuscutae semen lectin, Salvia virgata lectin, Glyphomia simplificolia (GsLA4), Vicia faba (acidic WBAI), Vigna angularis lectin, Cirsium japonicum lectin, Amaranthus leucocarpus lectin, Relya autumnalis lectin, Paramignya monophylla lectin, Ipomoea batatas lectin, Altocarpus lakoocha lectin, Himalayan Vicia faba lectin, Himalayan Vicia faba lectin, soybean lectin and mushroom lectin.

[0264] In some embodiments, the lectin can be a galectin selected from the group consisting of galectin-1, galectin-2, galectin-3, galectin-4, galectin-5, galectin-6, galectin-7, galectin-8, galectin-9, galectin-10, galectin-11, galectin-12, galectin-13, galectin-14, and galectin-15. In some embodiments, the lectin can be a siglec selected from the group consisting of siglec-1 (sialoadhesin), siglec-2 (CD22), siglec-3 (CD33), siglec-4 (myelin-associated glycoprotein), siglec-5, siglec-6, siglec-7, siglec-8, siglec-9, siglec-10, siglec-11, siglec-12, siglec-13, siglec-14, siglec-15, siglec-16, siglec-17, siglec-E, siglec-F, siglec-G, and siglec-H.

[0265] In some embodiments, the lectin can be a polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) selected from the group consisting of ppGalNAc-T1 (GALNT1), ppGalNAc-T2 (GALNT2), ppGalNAc-T3 (GALNT3), ppGalNAc-T4 (GALNT4), ppGalNAc-T5 (GALNT5), ppGalNAc-T6 (GALNT6), ppGalNAc-T7 (GALNT7), ppGalNAc-T8 (GALNT8), ppGalNAc-T9 (GALNT9), ppGalNAc-T10 (GALNT10), ppGalNAc-T12 (GALNT12), ppGalNAc-T13 (GALNT13), ppGalNAc-T14 (GALNT14), ppGalNAc-T15 (GALNT15), ppGalNAc-T16 (GALNT16), ppGalNAc-T17 (GALNT17), ppGalNAc-T18 (GALNT18), ppGalNAc-T Like 5 (GALNTL5), and ppGalNAc-T Like 6 (GALNTL6).

[0266] In some embodiments, the antigen-binding domain of the fusion protein described herein selectively targets a TACA selected from the group consisting of β1,6-branched, β1,6GlcNAc-branched N-glycan, T antigen, Tn antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6-sialylation, sialylation, Lewis-y (Le y )), sialyl-Lewisx / a, disialyl-Lewisx / a, sialyl 6-sulfo Lexisx, Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GM1. In some embodiments, the antigen-binding domain selectively targets β1,6GlcNAc-branched N-glycan, Tn epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), GalNAcα-serine, GalNAcα-threonine, GalNAc, or GalNAcβ1.

[0267] In some embodiments, the antigen-binding domain comprises one or more TACA-binding domains. In one embodiment, the antigen-binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 TACA-binding domains. In some embodiments, the TACA-binding domains are operably linked by a linker. The linker can be selected from a peptide linker, a non-peptide linker, a chemical unit, a disulfide-crosslinking linker, or a non-disulfide-crosslinking linker. In one embodiment, the linker is a peptide linker (e.g., a glycine-serine linker). In some embodiments, 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. 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.

[0268] In some embodiments, the antigen-binding domain comprises the amino acid sequence set forth 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 with the amino acid sequence set forth in SEQ ID NOs: 33-56. In one embodiment, the antigen-binding comprises an amino acid sequence having at least 90% homology with SEQ ID NOs: 33-56.

[0269] 3. Immune recognition domain The fusion proteins disclosed herein can bind to a molecule expressed on the surface of an immune effector cell (e.g., an "effector cell protein") and another molecule expressed on the surface of a target cell (e.g., a "target cell protein"). The immune effector cell can be a T cell, NK cell, macrophage or neutrophil. In some embodiments, the effector cell protein is a protein contained in the T cell receptor (TCR)-CD3 complex. The TCR-CD3 complex is a hetero-oligomer comprising a hetero-dimer containing TCRα and TCRβ, or TCRγ and TCRδ plus various CD3 chains (from among the CD3 zeta (CD3ζ) chain, CD3 epsilon (CD3ε) chain, CD3 gamma (CD3γ) chain and CD3 delta (CD3δ) chain). In some embodiments, the effector cell protein can be the human CD3 epsilon (CD3ε) chain, which can be part of a multimeric protein. Alternatively, the effector cell protein can be a human and / or cynomolgus TCRα, TCRβ, TCRδ, TCRγ, CD3 beta (CD3β) chain, CD3 gamma (CD3γ) chain, CD3 delta (CD3δ) chain or CD3 zeta (CD3ζ) chain. In some embodiments, the immune cell recognition domain may comprise CD3 or the amino acid sequences of SEQ ID NOs: 75-77.

[0270] In addition, in some embodiments, the fusion proteins disclosed herein can also bind to CD3ε chains derived from non-human species such as mouse, rat, rabbit, New World monkeys and / or Old World monkey species. Such species include, but are not limited to, the following mammalian species: house mouse, brown rat, Norway rat, cynomolgus monkey, Macaca fascicularis, hamadryas baboon, Papio hamadryas, guinea baboon, Papio papio, olive baboon, Papio anubis, yellow baboon, Papio cynocephalus, chacma baboon, Papio ursinus, common marmoset, cottontop tamarin, and squirrel monkey. Having a therapeutic agent with equivalent activity in humans and species commonly used in preclinical trials (such as mice and monkeys) as is known in the field of protein therapeutic development can simplify and accelerate drug development. Such advantages can be extremely important in the long and costly process of bringing a pharmaceutical product to market.

[0271] In a more specific embodiment, the heterodimeric bispecific antibody can bind to an epitope within the first 27 amino acids of the CD3ε chain, which may be a human CD3ε chain or a CD3ε chain derived from one of various species, particularly one of the mammalian species listed above. The advantages of antibodies that bind such epitopes 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, for example, by alanine scanning as described in U.S. Patent Application Publication No. 2010 / 183615, the relevant portions of which are incorporated herein by reference.

[0272] When the T cell is an immune effector cell, effector cell proteins to which the fusion proteins disclosed herein can bind include, but are not limited to, CD3ε chain, CD3γ, CD3δ chain, CD3ζ chain, TCRα, TCRβ, TCRγ, and TCRδ. When the NK cell or cytotoxic T cell is an immune effector cell, for example, NKG2D, CD352, NKp46, or CD16a can be an effector cell protein. CD8 + When the T cell is an immune effector cell, for example, 4-1BB or NKG2D can be an effector cell protein. Alternatively, the fusion proteins disclosed herein can bind to other effector cell proteins expressed on T cells, NK cells, macrophages, or neutrophils.

[0273] a. Target cell As described above, the fusion proteins disclosed herein can bind to effector cell proteins and target cell proteins. Target cell proteins can be expressed, for example, on the surface of cancer cells, cells infected with a pathogen, or cells that mediate a disease (e.g., inflammatory, autoimmune, and / or fibrotic pathologies). In some embodiments, the target cell protein can be highly expressed on the target cell, although a high level of expression is not necessarily required.

[0274] When the target cell is a cancer cell, the fusion protein can bind to the cancer cell antigen as described herein and as described above. The cancer cell antigen may be a human protein or may be a protein of another species. For example, the fusion protein may bind to target cell proteins from, inter alia, species such as mouse, rat, rabbit, New World monkeys and / or Old World monkeys. Such species include, but are not limited to, the following species: Mus musculus, Rattus norvegicus, Rattus rattus, cynomolgus monkey (Macaca fascicularis), hamadryas baboon (Papio hamadryas), Guinea baboon, Papio papio, olive baboon (Papio anubis), yellow baboon (Papio cynocephalus), chacma baboon (Papio ursinus), common marmoset, cottontop tamarin, squirrel monkey.

[0275] In other embodiments, the target cells can be cells that mediate an autoimmune or inflammatory disease. For example, human eosinophils in asthma can be target cells, and in that case, for example, an EGF-like module containing mucin-like hormone receptor (EMR1) can be a target cell protein. Alternatively, excess human B cells in patients with systemic lupus erythematosus can be target cells, and in that case, for example, CD19 or CD20 can be target cell proteins. In other autoimmune conditions, excess human Th2 T cells can be target cells, and in that case, for example, CCR4 can be a target cell protein. Similarly, the target cells can be fibrotic cells that mediate a disease, examples of which include atherosclerosis, chronic obstructive pulmonary disease (COPD), cirrhosis, scleroderma, renal transplant fibrosis, renal allograft nephropathy, or pulmonary fibrosis (including idiopathic pulmonary fibrosis and / or idiopathic pulmonary hypertension). In such fibrotic conditions, for example, fibroblast activation protein alpha (FAP alpha) can be used as the target cell protein.

[0276] b. Target cell lysis assay An assay to determine whether a fusion protein can induce in vitro cell lysis of target cells by immune effector cells as described herein is set forth in the following examples. In this assay, the immune effector cells are T cells. When the immune effector cells are NK cells, a very similar assay as follows can be used. Target cell lines expressing the target cell protein of interest can be labeled with 2 μM carboxyfluorescein succinimidyl ester (CFSE) at 37° C. for 15 minutes and then washed. Next, an appropriate number of labeled target cells can be incubated at 4° C. for 40 minutes in one or more 96-well flat-bottom culture plates with or without bispecific proteins, control proteins, or in the absence of adding various concentrations of proteins. NK cells isolated from healthy human donors can be isolated using the Miltenyi NK Cell Isolation Kit II (Miltenyi Biotec, Auburn, Calif.) and then added to the target cells at an effector:target ratio of 10:1. The NK cells, which are the immune effector cells of this assay, can be used immediately after isolation or after culturing overnight at 37° C. Plates containing tumor target cells, fusion proteins (e.g., bispecific) and immune effector cells can be cultured at 37° C. with 5% CO2 for 18-24 hours. Also, appropriate control wells can be set up. After an assay period of 18-24 hours, all cells can be removed from the wells. A 7-AAD solution in a volume equal to the volume of the well contents can be added to each sample. Then, as described in the following examples, the samples can be assayed by flow cytometry to determine the ratio of live target cells to dead target cells.

[0277] In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an immunocyte recognition domain that selectively binds to a receptor on an immune effector cell. In such embodiments, the immune effector cell can be selected from the group consisting of T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, monocytes, dendritic cells, and neutrophils. In such embodiments, the immune effector cell can be a T cell. In another embodiment, the immune effector cell can be an NK cell. The receptor on the immune effector cell can be selected from the group consisting of T cell receptor (TCR) alpha, TCR beta, TCR gamma, TCR delta, 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 an NK cell, and the NK cell receptor can be selected from the group consisting of NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1.

[0278] In some embodiments, the immunocyte 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 immunocyte recognition domain may comprise an scFv that can selectively bind CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1. In some embodiments, the immunocyte recognition domain specifically binds CD3. Alternatively, the immunocyte recognition domain may comprise the amino acid sequence of SEQ ID NO: 59, 60, or 61. In some embodiments, the immunocyte recognition domain may comprise an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 59, 60, or 61.

[0279] Alternatively, the immune cell recognition domain comprises the Fc domain of an antibody. In some embodiments, the Fc region of an IgG molecule. In some embodiments, the immune cell recognition domain is a domain that specifically binds an antibody Fc domain and CD3. In one embodiment, the fusion protein is an Fc fusion protein comprising an antigen-binding domain that selectively binds a tumor-associated carbohydrate antigen (TACA). In another embodiment, the immune cell recognition domain comprises the constant region domains CH2 and / or CH3 of an antibody. In one embodiment, the constant region is preferably CH2 and CH3. The constant region domains CH2 and / or CH3 of the antibody may or may not include the hinge region.

[0280] 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 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 extension domain comprises the Fc domain of an antibody selected from the IgG1, IgG2, IgG3 or IgG4 Fc regions.

[0281] B. Nucleic Acids 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, examples of which include, for example, screening a library from cells expressing the gene, deriving the gene from a vector known to contain the gene, or isolating it directly from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced by synthesis rather than cloning.

[0282] The isolated nucleic acid may include any type of nucleic acid, including but not limited to DNA and RNA. For example, in one embodiment, the composition includes an isolated DNA molecule, which may include, for example, an isolated cDNA molecule encoding a peptide of the present disclosure, or a functional fragment thereof. In one embodiment, the composition includes an isolated RNA molecule encoding a peptide of the present disclosure, or a functional fragment thereof.

[0283] The nucleic acid molecules of the present disclosure can be modified to improve their stability in serum or in growth media for cell culture. Modifications can be added to enhance stability, functionality, and / or specificity, and to minimize the immunostimulatory properties of the nucleic acid molecules of the present disclosure. For example, to enhance stability, the 3' residue may be stabilized against degradation, for example, the 3' residue may be selected such that it is composed of a purine nucleotide, particularly adenosine or guanosine nucleotide. Alternatively, substitution with a modified analog of a pyrimidine nucleotide, for example, substitution of uridine with 2'-deoxythymidine, is tolerated and does not affect the function of the molecule. In one embodiment of the present disclosure, the nucleic acid molecule may contain at least one modified nucleotide analog. For example, the termini may be stabilized by incorporating modified nucleotide analogs.

[0284] Non-limiting examples of nucleotide analogs include ribonucleotides that are sugar-modified and / or backbone-modified (i.e., including modifications to the phosphate-sugar backbone). For example, the phosphodiester bond of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In preferred backbone-modified ribonucleotides, the phosphate ester group that binds adjacent ribonucleotides is replaced by a modified group such as a phosphorothioate group. In preferred sugar-modified ribonucleotides, the 2' OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or ON, where R is Ci-Ce alkyl, alkenyl, or alkynyl, and halo is F, CI, Br, or I.

[0285] Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. The base may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include uridine and / or cytidine modified at the 5-position (e.g., 5-(2-amino)propyluridine, 5-bromouridine), adenosine and / or guanosine modified at the 8-position (e.g., 8-bromoguanosine), deazanucleotides (e.g., 7-deazaadenosine), O- and N-alkylated nucleotides (e.g., N6-methyladenosine is preferred), but are not limited thereto. It should be noted that the above modifications may be combined.

[0286] For example, a nucleic acid molecule comprises at least one of the following chemical modifications, i.e., modification of 2'-H, 2'-O-methyl or 2'-OH of one or more nucleotides. In certain embodiments, the nucleic acid molecules of the present disclosure can have enhanced resistance to nucleases. To improve nuclease resistance, the nucleic acid molecule can comprise, for example, 2'-modified ribose units and / or phosphorothioate linkages. For example, the 2'-hydroxyl group (OH) can be modified or replaced with a plurality of different "oxy" substituents or "deoxy" substituents. To improve nuclease resistance, the nucleic acid molecules of the present disclosure can comprise 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. Also, specific nucleobase modifications such as locked nucleic acid (LNA), ethylene nucleic acid (ENA) (e.g., 2'-4'-ethylene-bridged nucleic acid), and 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modification can improve the binding affinity to the target.

[0287] In one embodiment, the nucleic acid molecule comprises 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-methylacetamide (2'-O-MA). In one embodiment, the nucleic acid molecule comprises at least one 2'-O-methyl modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule comprise 2'-O-methyl modification.

[0288] In some embodiments, the nucleic acid molecules of the present disclosure preferably have one or more of the following properties, namely, the nucleic acid agents discussed herein are unmodified RNA and DNA in other respects, and further RNA and DNA modified, for example, to improve efficacy, and polymers of nucleoside analogs.

[0289] Unmodified RNA refers to a molecule in which the components of the nucleic acid (i.e., the sugar, base, and phosphate moieties) are the same as or essentially the same as those found in nature, preferably those naturally occurring in the human body. In the art, rare or abnormal but naturally occurring RNAs are referred to as modified RNAs. See, for example, Limbach et al., Nucleic Acids Res., 1994, 22:2183-2196. Such rare or abnormal RNAs are often referred to as modified RNAs and are generally the result of post-transcriptional modification and are within the scope of the term unmodified RNA as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid (i.e., the sugar, base, and phosphate moieties) are different from those found in nature, preferably different from those present in the human body. Although these are called "modified RNAs", they include molecules that are not strictly RNAs due to the modifications. Nucleoside analogs are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct, which allows the bases to be presented in the correct spatial relationship, resulting in hybridization that is substantially similar to that seen with a ribophosphate backbone (e.g., an uncharged mimic of the ribophosphate backbone). Modifications of the nucleic acids of the present disclosure may be present in one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobase.

[0290] C. Expression Vector 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.

[0291] Expression of a natural or synthetic nucleic acid encoding a peptide of the present disclosure is generally achieved by operably linking a nucleic acid encoding the peptide or a portion thereof to a promoter and incorporating the construct into an expression vector. The vectors used are suitable for replication and optionally integration into eukaryotic cells. Typical vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0292] Also, the vectors of the present disclosure may be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466. These patents are hereby incorporated by reference in their entirety. In another embodiment, the present disclosure provides a gene therapy vector.

[0293] The isolated nucleic acids of the present disclosure can be cloned into a plurality of types of vectors. For example, the nucleic acid can be cloned into a vector, including but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0294] Furthermore, the vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), as well as in other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0295] 1. Virus-based systems In some embodiments, the expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. In some embodiments, the expression construct is a lentiviral vector. In some embodiments, the expression construct is a self-inactivating lentiviral vector. In some embodiments, the expression construct comprises an isolated nucleic acid encoding the bispecific fusion protein described herein.

[0296] Multiple virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector using techniques known in the art and packaged into retroviral particles. The recombinant virus can then be isolated and delivered to the target cells either in vivo or ex vivo. Multiple retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Multiple adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0297] For example, vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow for stable integration of the transgene over time and its propagation in daughter cells. Lentiviral vectors have the additional advantage of being able to transduce non-proliferating cells such as hepatocytes compared to vectors derived from oncogenic retroviruses such as murine leukemia virus. They also have the additional advantage of low immunogenicity. In one embodiment, the composition comprises a vector derived from adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors have multiple characteristics that make them ideally suited for gene therapy, including lack of pathogenicity, minimal immunogenicity, and the ability to transduce post-mitotic cells in a stable and efficient manner. The expression of a specific gene contained within an AAV vector can be targeted specifically to one or more types of cells by selecting an appropriate combination of AAV serotype, promoter, and delivery method.

[0298] 2. Regulatory elements In some embodiments, the vector also includes conventional control elements that are operably linked to the transgene in a manner that enables transcription, translation, and / or expression in cells transfected with the plasmid vector or in cells infected with the virus produced herein. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation sequences, transcription termination sequences, promoter sequences, and enhancer sequences; efficient RNA processing signals such as splicing signals and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, optionally, sequences that enhance the secretion of the encoded product. A number of expression control sequences, including constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be utilized.

[0299] Additional promoter elements (e.g., enhancers) regulate the frequency of transcriptional initiation. In general, these are located in the region 30 to 110 bp upstream of the start site, although recently it has been shown that multiple promoters also contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is maintained even when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased up to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements can function either cooperatively or independently to activate transcription.

[0300] 3. Promoter In some embodiments, the expression construct further comprises a promoter. The promoter may be selected from the EF-lα promoter, the T cell receptor alpha (TRAC) promoter, the interleukin 2 (IL-2) promoter, or the cytomegalovirus (CMV) promoter, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter or the Rous sarcoma virus promoter.

[0301] The immediate early cytomegalovirus (CMV) promoter sequence is an example of a strong constitutive promoter sequence capable of driving high-level expression of any operably linked polynucleotide sequence. Another example of a suitable promoter is elongation growth factor - la (EF - la). However, other constitutive promoter sequences may also be used, including the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein - Barr virus immediate early promoter, Rous sarcoma virus promoter, and furthermore human gene promoters (examples include, but are not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter). Furthermore, the expression constructs disclosed herein should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present disclosure. The use of an inducible promoter provides a molecular switch that can turn on the expression of an operably linked polynucleotide sequence when such expression is desired, or turn off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0302] Enhancer sequences present on the vector also regulate the expression of the genes contained therein. Generally, enhancers bind to protein factors to enhance gene transcription. The enhancer may be placed upstream or downstream of the gene it regulates. Also, the enhancer may be made tissue - specific to enhance transcription in specific cell or tissue types. In one embodiment, the vectors of the present disclosure include one or more enhancers to facilitate the transcription of genes present within the vector.

[0303] 4. Selectable Marker To evaluate peptide expression, the expression vector introduced into the cells may also contain either or both of a selectable marker gene and a reporter gene to facilitate the identification and selection of expressing cells from the population of cells to be transfected or infected by the viral vector. In other embodiments, the selectable marker may be carried on a separate DNA fragment and used in a co-transfection procedure. Appropriate regulatory sequences may be placed on both the selectable marker and the reporter gene to permit expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

[0304] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression appears as some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at a suitable time point after the DNA has been introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, a construct having a minimal 5' flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such a promoter region may be ligated to the reporter gene and used to evaluate agents for their ability to modulate promoter-driven expression.

[0305] V. Modified Cells One aspect of the present disclosure provides a genetically modified (e.g., engineered) cell that contains and stably expresses the fusion protein of the present disclosure. In one aspect, the modified cell contains an isolated nucleic acid molecule encoding a fusion protein that includes 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 capable of extending the half-life of the fusion protein.

[0306] In some embodiments, the modified cell contains the fusion protein described herein. In another embodiment, the modified cell contains an isolated nucleic acid molecule encoding the bispecific fusion protein described herein. In another embodiment, the modified cell contains the expression construct described herein.

[0307] In some embodiments, the modified cell is a genetically modified immune cell (e.g., a T cell) or a progenitor cell thereof that contains a fusion protein having an affinity for a tumor-associated carbohydrate antigen (TACA) as described herein. In some embodiments, the fusion protein includes 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, and the half-life extension domain is a polypeptide capable of extending the half-life of the fusion protein.

[0308] In some embodiments, the genetically modified immune cell (e.g., a T cell) or a progenitor cell thereof of the present disclosure is β1,6-branched, β1,6GlcNAc-branched N-glycan, T antigen, Tn antigen, sialyl-T epitope, Tn epitope, sialyl-Tn epitope, α2,6-sialylation, sialylation, sialyl-Lewis x / a disialyl-Lewis x / a sialyl 6-sulfo Lewis x Lewis-y (Le y) It includes a bispecific fusion protein having affinity for Lewis Y, Globo H, GD2, GD3, GM3, or fucosyl GM1. In some embodiments, the genetically modified immune cells (e.g., T cells) or their progenitor cells of the present disclosure include 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 progenitor cells of the present disclosure include a bispecific fusion protein having affinity for the Tn antigen or sialyl Tn epitope.

[0309] A. Host cell 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 lepidopteran insect cells or Spodoptera frugiperda.

[0310] In some embodiments, the modified cell is a mammalian cell selected from Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, monkey kidney cells, HeLa cells, human hepatocellular carcinoma cells, or human fetal kidney 293. In one embodiment, the modified cell is CHO cells or HEK293 cells.

[0311] B. Modified immune cells In some embodiments, the modified cell is a T cell, CD4 + T cell, CD8 +It is selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. In some embodiments, the modified cell is a T cell. In certain embodiments, the genetically modified cell is a natural killer (NK) cell. In certain embodiments, the genetically modified cell is an NKT cell. In some embodiments, the modified cell is an autologous cell, a heterologous cell, or an allogeneic cell.

[0312] In some embodiments, the genetically modified cell is a genetically engineered T lymphocyte (T cell), a regulatory T cell (Treg), a naive T cell (TN), a memory T cell (e.g., a central memory T cell (TCM), an effector memory cell (TEM)), a natural killer cell (NK cell), a natural killer T cell (NKT cell), and a macrophage capable of generating progeny relevant to treatment. In some embodiments, the cell is selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. In one embodiment, the cell is a T cell. In one embodiment, the modified cell is an autologous cell.

[0313] In some embodiments, the T cell, natural killer (NK) cell, cytotoxic T lymphocyte (CTL), or regulatory T cell comprises the fusion protein disclosed herein and further comprises a chimeric antigen receptor (CAR) that selectively or specifically binds to a tumor-associated carbohydrate antigen (TACA).

[0314] In some embodiments, the CAR comprises an antigen-binding domain selected from the group consisting of SEQ ID NOs: 33 to 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 with the amino acid sequence set forth in SEQ ID NOs: 33 to 56, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

[0315] In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of a molecule selected from the group consisting of T cell receptor (TCR)-alpha, TCR-beta, CD3-zeta, CD3-epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134 (Ox40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some embodiments, the transmembrane domain comprises the CD8 transmembrane domain. In some embodiments, the co-stimulatory domain of the CAR is a co-stimulatory domain of a molecule selected from the group consisting of CD27, CD28, 4-IBB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD8, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, DAP10, DAP12, Lck, Fas, and combinations thereof. In some embodiments, the co-stimulatory domain comprises the 4-1BB co-stimulatory domain, the CD28 co-stimulatory domain, or both the 4-1BB co-stimulatory domain and the CD28 co-stimulatory domain.

[0316] In some embodiments, the intracellular domain of the CAR comprises the intracellular signaling domain of a molecule selected from the group consisting of T cell receptor (TCR) zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, CDS, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain comprises the CD3 zeta signaling domain.

[0317] In one aspect, the present disclosure provides a population of modified immune cells as described herein. In some embodiments, the modified cells are CAR T cells. In those embodiments, the CAR T cells specifically target a tumor-associated carbohydrate antigen (TACA).

[0318] C. Method for generating modified immune cells In one aspect, the present disclosure provides a method for generating a modified cell as disclosed herein, the method comprising introducing into the cell an isolated nucleic acid encoding a fusion protein, or an expression construct of the present disclosure.

[0319] In another aspect, the present disclosure provides a method for generating a modified cell as described herein, the method comprising introducing into the cell an isolated nucleic acid as described herein, a fusion protein as described herein, or an expression construct as described herein, 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.

[0320] The fusion proteins disclosed herein can be produced 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 host cells cultured by various known methods, examples of which include, for example, transformation, transfection, electroporation, and bombardment with nucleic acid-coated microprojectiles. In some embodiments, the nucleic acid encoding the fusion protein can be inserted into a vector suitable for expression in the host cell prior to introduction into the host cell. Typically, such vectors can contain sequence elements that effect expression at the RNA and protein levels of the inserted nucleic acid. Such vectors are well known in the art and many are commercially available. Host cells containing the nucleic acid can be cultured under conditions such that the cells can express the nucleic acid, and the resulting fusion protein can be collected from the cell mass or the culture medium. Alternatively, the fusion protein can be produced in vivo, for example, in the leaves of plants (see, for example, Scheller et al. (2001), Nature Biotechnol. 19: 573-577 and references cited therein), in avian eggs (see, for example, Zhu et al. (2005), Nature Biotechnol. 23: 1159-1169 and references cited therein), or in mammalian milk (see, for example, Laible et al. (2012), Reprod. Fertil. Dev. 25(1): 315).

[0321] Modified cells (e.g., including a target bispecific fusion protein) may be produced by stably transfecting host cells with an expression vector comprising a nucleic acid of the present disclosure. Additional methods of generating modified cells of the present disclosure include chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes, and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer, and / or hydrodynamic delivery), and / or particle-based methods (e.g., impalefection using a gene gun and / or magnetofection), but are not limited thereto. Transfected cells expressing the target fusion protein of the present disclosure may be expanded ex vivo.

[0322] In some embodiments, cells are genetically modified by contacting the cells with an isolated nucleic acid encoding a bispecific fusion protein as described herein. In some embodiments, the nucleic acid sequence is delivered to the cells using a retroviral or lentiviral vector. For example, retroviral vectors and lentiviral vectors expressing the peptides of the present disclosure can be delivered to various types of eukaryotic cells, as well as tissues and whole organisms, using transduced cells as carriers or using cell-free local or systemic delivery of conjugated or naked encapsulated vectors. This method used can be used for any purpose for which stable expression is required or sufficient.

[0323] In other embodiments, the nucleic acid sequence is delivered to the cells using in vitro transcribed mRNA. In vitro transcribed mRNA can be delivered to various types of eukaryotic cells, as well as tissues and whole organisms, using transfected cells as carriers or using cell-free local or systemic delivery of conjugated or naked encapsulated mRNA. This method used can be used for any purpose for which transient expression is required or sufficient.

[0324] In certain embodiments, the cell may be any suitable cell type capable of expressing the desired peptide. In certain embodiments, the modified cell is used in a manner in which the cell is introduced into a recipient. In certain embodiments, the cell is an autologous cell, an allogeneic cell, a syngeneic cell, or a xenogeneic cell with respect to the recipient.

[0325] The disclosed compositions and methods can be applied to the regulation of T cell activity in basic research and treatment in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including the evaluation of the ability of genetically modified T cells to kill target cancer cells.

[0326] Methods for introducing and expressing genes in cells are known in the art. From the perspective of expression vectors, the vector can be easily introduced into host cells by any method in the art, examples of which include, for example, mammalian cells, bacterial cells, yeast cells, or insect cells. For example, the expression vector can be transferred into host cells by physical means, chemical means, or biological means.

[0327] 1. Physical methods Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0328] 2. Biological methods Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA vectors and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors can be derived from, for example, lentivirus, poxvirus, herpes simplex virus I, adenovirus, adeno-associated virus, etc. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0329] In some embodiments, the nucleic acid encoding the fusion protein of interest of the present disclosure is introduced into cells by an expression vector. Expression vectors containing nucleic acids encoding the fusion protein of interest (e.g., TACA triple specificity fusion) are provided herein. Suitable expression vectors include, but are not limited to, lentiviral vectors, gammaretroviral vectors, foamy viral vectors, adeno-associated virus (AAV) vectors, adenoviral vectors, engineered hybrid viruses, naked DNA, and further include transposon-mediated vectors such as sleeping beauty, piggyback, and integrases such as Phi31. Some other suitable expression vectors include herpes simplex virus (HSV) and retroviral expression vectors.

[0330] Adenoviral expression vectors are based on adenoviruses, which have a low ability to integrate into genomic DNA but a high efficiency of transfection into host cells. Adenoviral expression vectors contain (a) sequences that support the packaging of the expression vector and (b) adenoviral sequences sufficient to ultimately express the target fusion protein (e.g., a trispecific fusion protein) in a host cell. In some embodiments, the adenoviral genome is a 36 kb linear double-stranded DNA, and to produce the expression vectors of the present disclosure, a foreign DNA sequence (e.g., a nucleic acid encoding a TACA fusion protein) may be inserted to replace a large piece of the adenoviral DNA. See, e.g., Danthinne and Imperiale, Gene Therapy 7(20):1707-1714(2000).

[0331] Another type of expression vector is based on adeno-associated virus (AAV) and utilizes an adenoviral coupling system. This AAV expression vector has a high frequency of integration into the host genome and is useful for delivering genes to mammalian cells, e.g., in tissue culture or in vivo, because it can infect non-dividing cells. With respect to infectivity, AAV vectors have a broad host range. Details regarding the production and use of AAV vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797,368.

[0332] Retroviral expression vectors integrate into the host genome, deliver large amounts of foreign genetic material, infect a wide range of species and cell types, and can be packaged into specific cell lines. Retroviral vectors are constructed by inserting a nucleic acid (e.g., a nucleic acid encoding a TACA fusion protein) into a specific position in the viral genome to produce a replication-defective virus. Retroviral vectors can infect a variety of cell types, but integration and stable expression of the target fusion protein require division of the host cell.

[0333] Lentiviral vectors are derived from lentiviruses, which are complex retroviruses that contain, in addition to the gag, pol, and env genes, which are common retroviral genes, other genes with regulatory or structural functions. See, for example, U.S. Pat. Nos. 6,013,516 and 5,994,136. Some examples of lentiviruses include human immunodeficiency virus (HTV-1, HTV-2) and simian immunodeficiency virus (SIV). Lentiviral vectors are produced by complex attenuation of the HIV virulence genes, for example, the env, vif, vpr, vpu, and nef genes are deleted, and the vector becomes biologically safe. Lentiviral vectors can infect non-dividing cells and can be used, for example, for gene transfer and expression of nucleic acids encoding a target fusion protein both in vivo and ex vivo.

[0334] The expression vector containing the nucleic acid of the present disclosure can be introduced into a host cell by any means known to those skilled in the art. The expression vector may optionally contain viral sequences for transfection. Alternatively, the expression vector may be introduced by fusion, electroporation, biolistics, transfection, lipofection, etc. The host cell can be grown and expanded in culture prior to introduction of the expression vector, followed by appropriate treatment for vector introduction and integration. The host cell can then be expanded and screened by a marker present in the vector.

[0335] A variety of markers that can be used are known in the art and include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, and the like. As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. In some embodiments, the host cell is an immune cell or a progenitor cell thereof, examples of which include T cells, NK cells, or NKT cells.

[0336] 3. Chemical methods Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems used as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).

[0337] When using non-viral delivery systems, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule associated with both the liposome and the oligonucleotide, incorporated into a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in a micelle or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they may exist as micelles within a bilayer structure or in a "disrupted" structure. They may also simply be dispersed in solution and may form aggregates that are not uniform in size or shape. Lipids are fatty substances and may be naturally occurring lipids or synthetic lipids. For example, lipids include, in addition to lipid droplets that naturally occur within the cytoplasm, a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, examples of which include fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0338] Suitable lipids can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine (“DMPC”) can be obtained from Sigma (St. Louis, Missouri), dicetyl phosphate (“DCP”) can be obtained from K&K Laboratories (Plainview, New York), cholesterol (“Choi”) can be obtained from Calbiochem-Behring, and dimyristoyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Alabama). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Since chloroform evaporates more readily than methanol, it is used as the sole solvent. “Liposome” is a general term encompassing various single and multi-layer lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of aqueous solution, they form spontaneously. The lipid components undergo self-arrangement before forming a closed structure and take up water and dissolved solutes between the lipid bilayers. Ghosh et al., Glycobiology 5:505-10 (1991). However, compositions having structures different from normal vesicular structures in solution are also included. For example, the lipids may adopt a micellar structure or simply exist as a heterogeneous aggregate of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0339] Regardless of the method used to introduce exogenous nucleic acids into a host cell, various assays may be performed to confirm the presence of a recombinant DNA sequence within the host cell. Such assays include, for example, "molecular biology" assays (such as Southern blotting, Northern blotting, RT-PCR, and PCR, which are well known to those skilled in the art), and "biochemical" assays (such as detecting the presence or absence of a specific peptide by immunological means (ELISA and Western blot), or by the assays described herein for identifying agents within the scope of the present disclosure).

[0340] Moreover, the nucleic acid may be introduced by any means, examples of which include causing transduction in expanded T cells, transfecting expanded T cells, and performing electroporation on expanded T cells. One nucleic acid may be introduced into T cells by one method, and another nucleic acid may be introduced into T cells by a different method.

[0341] 4. RNA RNA has several advantages over more traditional plasmid or viral approaches. Gene expression from an RNA source does not require transcription, and protein products are rapidly produced after transfection. Furthermore, RNA only needs to access the cytoplasm rather than the nucleus, and thus extremely high transfection rates occur with common transfection methods. Additionally, plasmid-based approaches require that the promoter driving the expression of the gene of interest be active within the cell under study.

[0342] One advantage of the RNA transfection method of the present disclosure is that RNA transfection is essentially transient and does not require a vector. The RNA transgene can be delivered to lymphocytes as a minimal expression cassette without the need for any additional viral sequences and expressed there after a short period of in vitro cell activation. Under these conditions, the probability that the transgene integrates into the genome of the host cell is low. Due to the efficiency of RNA transfection and the ability to uniformly modify the entire lymphocyte population, cell cloning is not necessary.

[0343] Gene modification of host cells by in vitro transcribed RNA (TVT-RNA) uses two different strategies, both of which are being continuously tested in various animal models. Cells are transfected with in vitro transcribed RNA by means such as lipofection or electroporation. To achieve long-term expression of the transferred IVT-RNA, it is desirable to stabilize the IVT-RNA using various modifications. Several IVT vectors are known in the literature that are utilized in a standardized manner as templates for in vitro transcription and are genetically modified to produce stabilized RNA transcripts.

[0344] Currently, the protocols used in this technical field are based on the following structure, namely, a 5' RNA polymerase promoter that enables RNA transcription, followed by a target gene adjacent to an untranslated region (UTR) either at the 3' and / or 5', and a plasmid vector having a 3' polyadenylation cassette containing 50 - 70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized by a type II restriction enzyme downstream of the polyadenylation cassette (the recognition sequence corresponds to the cleavage site). Thus, the polyadenylation cassette corresponds to the late poly(A) sequence within the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization, extending or masking the poly(A) sequence at the 3' end. Whether this non - physiological overhang affects the amount of protein produced intracellularly from such constructs is not clear.

[0345] In one embodiment, the isolated nucleic acid encoding the bispecific fusion protein of the present disclosure and introduced into the cells of the present disclosure contains RNA. In one embodiment, the RNA is mRNA. In one embodiment, the RNA is in vitro transcribed (IVT) RNA. The RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). DNA of interest from any source can be directly converted to a template for in vitro mRNA synthesis by PCR using appropriate primers...

Claims

1. An isolated nucleic acid molecule encoding a fusion protein, wherein the fusion protein is (i) an antigen-binding domain that selectively binds tumor-associated glycosylation antigens (TACAs), (ii) An immune cell recognition domain that specifically binds to receptors on immune effector cells, (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, and The isolated nucleic acid molecule, including the above.

2. The half-life extension domain is (i) Molecules capable of binding serum albumin; (ii) A polypeptide comprising the amino acid sequence D-Xaa-CLP-Xaa-WGCLW (SEQ ID NO: 70), QGLIGDICLPRWGCLWGDSVK (SEQ ID NO: 71), RLIEDICCLPRWGCLWEDD (SEQ ID NO: 72), or EDICLPRWGCLWED (SEQ ID NO: 73), wherein Xaa is optionally any amino acid; (iii) A fatty acid chain conjugate polypeptide wherein the fatty acid chain is selected from a C-16 fatty acid chain or a C-18 fatty acid chain; (iv) C-16 fatty acid conjugate molecule; (v) Antibody fragments that selectively bind serum albumin, optionally single-domain antibodies, single-domain antibody CDRs, or single-strand variable fragments (scFv); (vi) A molecule selected from the group consisting of polypeptides capable of binding albumin, albumin, serum albumin, the Fc domain of an antibody, polyethylene glycol moiety (PEG), poly(lactic acid-glycolic acid copolymer) (PLGA) polymer, polymer hydrogel, nanoparticles, fatty acid chains, acyl groups, myristic acid groups, palmitoyl groups, and steryl groups; or (vii) Human serum albumin, The isolated nucleic acid molecule according to claim 1, comprising

3. The isolated nucleic acid molecule according to claim 2, wherein the half-life extension domain includes the amino acid sequence of SEQ ID NO:

57.

4. The isolated nucleic acid molecule according to claim 1, wherein the half-life of the fusion protein is extended by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 8 times, at least about 10 times, at least about 15 times, at least about 16 times, at least about 18 times, or at least about 20 times compared to a fusion protein lacking the half-life extension domain.

5. The antigen-binding domain is (a) TACA-binding domain derived from lectin; (b) More than one TACA binding domain; and (c) Two, three, four, five, six, seven, eight, nine, or ten TACA binding domains, An isolated nucleic acid molecule according to claim 1, comprising one or more of the above.

6. The antigen-binding domain comprises one or more TACA-binding domains, (a) The TACA-binding domain is operably linked by a linker; or (b) The TACA-binding domain is operably linked by a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 86, 87, 85, 88, 89, and 90. The isolated nucleic acid molecule according to claim 5.

7. The isolated nucleic acid molecule according to claim 1, wherein the antigen-binding domain comprises an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence described in any one of sequence numbers 33 to 56.

8. The isolated nucleic acid molecule according to claim 1, wherein the immune effector cells are selected from the group consisting of T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, monocytes, dendritic cells, and neutrophils.

9. The aforementioned immune cell recognition domain is (i) scFv that selectively binds proteins selected from the group consisting of CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1; (ii) Any one of the amino acid sequences of SEQ ID NOs. 59, 60, and 61; or (iii) an amino acid sequence having at least 90% sequence identity with any one of the amino acid sequences of sequence numbers 59, 60, and 61, The isolated nucleic acid molecule according to claim 1, comprising

10. The isolated nucleic acid molecule according to claim 1, wherein the encoded fusion protein is an Fc fusion protein comprising an antigen-binding domain for selectively binding tumor-associated glycosylation antigens (TACAs) and an Fc domain, wherein the Fc domain optionally comprises an amino acid sequence described in SEQ ID NO: 69 or SEQ ID NOs: 91-94.

11. The isolated nucleic acid molecule according to claim 1, wherein the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 1 to 32, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1 to 32.

12. The isolated nucleic acid molecule according to claim 1, wherein the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 1 to 12.

13. The isolated nucleic acid molecule according to claim 1, wherein the isolated nucleic acid molecule encodes a fusion protein comprising an amino acid sequence selected from SEQ ID NOs. 13 to 32.

14. The fusion protein is (a) Enhanced binding to β1,6GlcNAc branched N-glycan expressing tumor cells compared to a bispecific fusion protein containing a flexible linker in the antigen-binding domain; or (b) Compared to a fusion protein containing a flexible linker in the antigen-binding domain, enhanced binding to Thomsen-nouveau (Tn) antigen-expressing tumor cells. Show, The flexible linker is a glycine-serine linker, or a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 86, SEQ ID NO: 87, and SEQ ID NO: 85, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 86, SEQ ID NO: 87, and SEQ ID NO:

85. The isolated nucleic acid molecule according to claim 12.

15. A fusion protein that selectively binds tumor-associated glycosylation antigens (TACAs), wherein the fusion protein is encoded by the isolated nucleic acid molecule described in claim 1.

16. A fusion protein, (i) 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs. 33 to 56, or an amino acid sequence described in SEQ ID NOs. 33 to 56, (ii) An immune cell recognition domain that specifically binds CD3 on immune effector cells, (iii) A half-life extension domain which is a polypeptide that can extend the half-life of the fusion protein, Includes, The fusion protein is a fusion protein that selectively binds tumor-associated glycosylation antigens (TACAs).

17. A modified cell comprising the fusion protein described in Claim 15.

18. A composition comprising a fusion protein encoded by the isolated nucleic acid molecule described in Claim 1.

19. An immunotherapy composition for treating cancer in a subject requiring treatment for cancer, comprising the modified cells described in Claim 17.

20. The immunotherapy composition according to claim 19, wherein the cancer is selected from the group consisting of hematological malignancies, solid tumors, primary or metastatic tumors, leukemia, carcinoma, blastoma, sarcoma, lymphoid malignancies, melanoma, and lymphoma.

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