Bispecific fusion proteins and chimeric antigen receptors for improved glycan-dependent immunotherapy

Abstract

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

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 351,596, filed on June 13, 2022, which is hereby incorporated by reference in its entirety for all purposes.

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

[0003] This disclosure generally relates to the fields of pharmacology and immunology, and more specifically, to bispecific fusion proteins and chimeric antigen receptors that target tumor - associated carbohydrate antigens (TACA bispecific fusion proteins; TACA - CAR), and to the use of immune cells expressing TACA bispecific fusion proteins and TACA - CAR for treating diseases associated with abnormal glycosylation of cell - surface molecules.

Background Art

[0004] Antigen-targeted 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 potent immunotherapies. Both cause T cell-mediated killing of cancer cells, and the complete response rate of CAR T cells in relapsed / refractory B cell malignancies reaches a high rate of about 90%. Both utilize single-chain variable fragments (scFvs) derived from the variable heavy and variable light chains of monoclonal antibodies to target antigens expressed in cancer. In bispecific antibodies, the antigen-specific scFv is fused to a second scFv specific for CD3, while in CARs, the antigen-specific scFv is fused to a transmembrane domain and one or more cytoplasmic signaling domains derived from immune cell receptors. Both types of chimeric molecules are genetically expressed within T cells. Both therapies are currently + approved for the treatment of CD19

[0005] Summary of the Invention

[0006] ​The present disclosure provides a novel class of bispecific fusion proteins and chimeric antigen receptors for immunotherapy that effectively target tumor-associated carbohydrate antigens (TACAs) for immunotherapy. 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.

[0007] Accordingly, one aspect of the present disclosure provides an isolated nucleic acid molecule encoding a bispecific fusion protein comprising (a) an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), and (b) an immune cell recognition domain that specifically binds to a receptor on an immune effector cell. In some embodiments, the antigen-binding domain comprises a TACA-binding domain derived from a lectin, and the antigen-binding domain comprises more than one TACA-binding domain.

[0008] Another aspect of the present disclosure provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising (a) an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), (b) a transmembrane domain, (c) a co-stimulatory signaling region, and (d) an intracellular signaling domain. In some embodiments, the antigen-binding domain comprises a TACA-binding domain derived from a lectin, and the antigen-binding domain comprises more than one TACA-binding domain.

[0009] In some embodiments, the antigen-binding domain of the bispecific fusion protein or CAR disclosed herein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more TACA-binding domains.

[0010] In some embodiments, more than one TACA binding domain is 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. In some embodiments, the linker is a peptide 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 peptide linker is a glycine-serine linker. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 127. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 131.

[0011] In some embodiments, the antigen-binding domain of the bispecific fusion protein or CAR disclosed herein comprises a TACA-binding domain derived from a lectin selected from galectin, siglec, selectin; C-type lectin; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Phaseolus limensis leukocyte agglutinin); E-PHA (Phaseolus limensis erythrocyte agglutinin); tomato lectin (Lycopersicon esculentum lectin; LEA); peanut lectin (Arachis hypogaea lectin; PNA); potato lectin (Solanum tuberosum lectin), pokeweed mitogen (Phytolacca americana lectin), wheat germ agglutinin (Triticum aestivum germ lectin); Artocarpus polyphyllus lectin (jacalin lectin); hairy vetch 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), Euryale ferox lectin (ESA), CLEC17A (prolectin), Grifola frondosa lectin, Schizophyllum commune lectin (SSA), Glechoma hederacea lectin (Gleheda), Momordica charantia lectin (Morniga G), Ononis spinosa lectin, Salvia bogotensis lectin, Salvia horminum lectin, Cuscutae semen lectin, Calystegia soldanella lectin, Glyphomia simplificifolia (GsLA4), Vicia sativa (acidic WBAI), Vigna angularis lectin, Apios americana lectin, Amaranthus leucocarpus lectin, Relia autumnalis lectin, Paramicia lectin, Utricularia americana lectin, Artocarpus lakoocha lectin, Himalayan Phaseolus lunatus lectin, Himalayan Phaseolus lunatus lectin, soybean lectin and mushroom lectin.

[0012] In some embodiments, the galectin is selected from the group consisting of galectin-1, galectin-2, galectin-3, galectin-4, galectin-5, galectin-6, galectin-7, galectin-8, galectin-9, galectin-10, galectin-11, galectin-12, galectin-13, galectin-14 and galectin-15. In some embodiments, the siglec is 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.

[0013] In some embodiments, the polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) is selected from the group consisting of ppGalNAc-T1 (GALNT1), ppGalNAc-T2 (GALNT2), ppGalNAc-T3 (GALNT3), ppGalNAc-T4 (GALNT4), ppGalNAc-T5 (GALNT5), ppGalNAc-T6 (GALNT6), ppGalNAc-T7 (GALNT7), ppGalNAc-T8 (GALNT8), ppGalNAc-T9 (GALNT9), ppGalNAc-T10 (GALNT10), ppGalNAc-T12 (GALNT12), ppGalNAc-T13 (GALNT13), ppGalNAc-T14 (GALNT14), ppGalNAc-T15 (GALNT15), ppGalNAc-T16 (GALNT16), ppGalNAc-T17 (GALNT17), ppGalNAc-T18 (GALNT18), ppGalNAc-T Like 5 (GALNTL5) and ppGalNAc-T Like 6 (GALNTL6).

[0014] In some embodiments, the antigen-binding domains of the bispecific fusion proteins or CARs disclosed herein are β1,6-branched, β1,6GlcNAc-branched N-glycans, T antigen, Tn antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6-sialylation, sialylation, sialyl-Lewis x / a , disialyl-Lewis x / a , sialyl 6-sulfo Lexis x , Lewis-y (Le y ), Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GM1. In some embodiments, the antigen-binding domain selectively targets a TACA selected from the group consisting of β1,6GlcNAc-branched N-glycans, Tn epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), GalNAcα-serine, GalNAcα-threonine, GalNAc, or GalNAcβ1.

[0015] In some embodiments, the antigen-binding domain of the bispecific fusion protein or CAR disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152, or 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 NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152.

[0016] In some embodiments, the antigen-binding domain comprises an amino acid sequence having at least 90% homology to SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152.

[0017] In some embodiments, the isolated nucleic acid comprises an expression vector and / or in vitro transcribed RNA.

[0018] In some embodiments of the isolated nucleic acid molecules disclosed herein, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, and 63-66, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, and 63-66. In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66. In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising the amino acid sequence of SEQ ID NOs: 32-34, 40-42, 48-50, 56-58, or 64.

[0019] In some embodiments of the isolated nucleic acid molecules disclosed herein, (A) the bispecific fusion protein exhibits enhanced binding to Thomsen-nouveau (Tn) antigen-expressing tumor cells as compared to a bispecific fusion protein comprising a flexible linker in the antigen-binding domain. In some embodiments, the bispecific fusion protein exhibits enhanced binding to 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 these embodiments, the flexible linker is a glycine-serine linker, or an amino acid sequence selected from SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127.

[0020] In some embodiments of the isolated nucleic acid molecules disclosed herein, the immune effector cells targeted by the bispecific fusion protein are selected from the group consisting of T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, monocytes, dendritic cells, and neutrophils. In one embodiment, the immune effector cell is a T cell. In one embodiment, the immune effector cell is an NK cell. In some embodiments, the receptor on the immune effector cell is 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. In some embodiments, the receptor on the immune effector cell is (i) a T cell receptor selected from the group consisting of CD3, CD2, CD28, and CD25, or (ii) an NK cell receptor selected from the group consisting of NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1.

[0021] In some embodiments, the immune cell recognition domain of the bispecific fusion protein disclosed herein comprises (i) a peptide, protein, antibody, single domain antibody, nanobody, antibody fragment, or single-chain variable fragment (scFv) that selectively binds to a receptor on an immune effector cell, (ii) an antibody Fc domain, optionally the Fc region of an IgG molecule, or (iii) the constant region domains CH2 and / or CH3 of an antibody (preferably CH2 and CH3, optionally with or without a hinge region).

[0022] In some embodiments, the immune cell recognition domain comprises (i) an scFv that selectively binds CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1, (ii) the amino acid sequence of SEQ ID NO: 149, 150, or 151, or (iii) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 149, 150, or 151.

[0023] In some embodiments, the bispecific fusion protein is an Fc fusion protein comprising an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA) and an Fc domain.

[0024] In some embodiments of the isolated nucleic acid molecules disclosed herein, 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 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 148.

[0025] In some embodiments of the isolated nucleic acid molecules disclosed herein, 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 (i) a 4-1BB co-stimulatory domain, (ii) the amino acid sequence of SEQ ID NO: 114, (iii) a CD28 co-stimulatory domain, (iv) the amino acid sequence of SEQ ID NO: 113, or (v) 4-1BB and CD28 co-stimulatory domains.

[0026] In some embodiments of the isolated nucleic acid molecules disclosed herein, 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 or the amino acid sequence of SEQ ID NO: 115.

[0027] In some embodiments of the isolated nucleic acid molecules disclosed herein, the CAR further comprises a hinge domain. In some embodiments, the hinge domain is a protein selected from the group consisting of CD8α, the Fc fragment of an antibody, the hinge region of an antibody, the CH2 region of an antibody, the CH3 region of an antibody, and an artificial spacer sequence. In some embodiments, the hinge domain is the CD8α hinge domain or the hinge domain comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the hinge domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 124, 127, 128, 129, 130, 131, 132, and 147.

[0028] One aspect of the present disclosure provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR encoded by the isolated nucleic acid molecule comprises (i) an amino acid sequence set forth in SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, or (ii) an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99.

[0029] One aspect of the present disclosure provides a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA) disclosed herein. In some embodiments, the bispecific fusion protein is encoded by an isolated nucleic acid described herein.

[0030] One aspect of the present disclosure provides a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising: (I) an antigen-binding domain selected from the group consisting of SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152, or 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 NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152; and (ii) an immune cell recognition domain that specifically binds to a receptor on an immune effector cell.

[0031] In some embodiments, the immune cell recognition domain comprises: (i) an antibody Fc domain; (ii) the Fc region of an IgG molecule; (iii) a peptide, protein, antibody, single-domain antibody, antibody fragment, or single-chain variable fragment (scFv) that selectively binds to a receptor on an immune effector cell; or (iv) the constant region domains CH2 and / or CH3 of an antibody (preferably CH2 and CH3, with or without an optional hinge region).

[0032] In some embodiments, the receptor on the immune effector cell is selected from the group consisting of T cell receptor (TCR) alpha, TCR beta, TCR gamma, TCR delta, invariant TCR from NKT cells, CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA, and CEACAM1.

[0033] In some embodiments, the bispecific fusion protein comprises (a) an amino acid sequence selected from SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, and 63-66, or (b) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, and 63-66, or (c) an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66, or (d) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66.

[0034] In some embodiments, the bispecific fusion protein selectively targets a TACA selected from the group consisting of β1,6-branched, β1,6GlcNAc-branched N-glycan, T antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6-sialylation, sialylation, sialyl-Lewis x / a , disialyl-Lewis x / a , sialyl 6-sulfo Lexis x , Lewis-y (Le y ), Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GM1. In some embodiments, the bispecific fusion protein selectively targets the Tn antigen or β1,6GlcNAc-branched N-glycan.

[0035] In some embodiments, the bispecific fusion protein that selectively targets the Tn antigen comprises an antigen-binding domain having an amino acid sequence selected from SEQ ID NOs: 103-109, 142-146, or 152, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 103-109, 142-146, or 152. In some embodiments, the bispecific fusion protein that selectively targets the Tn antigen comprises (a) an amino acid sequence selected from SEQ ID NOs: 26-34, 39-42, 47-50, 55-58, or 63-66, or (b) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 26-34, 39-42, 47-50, 55-58, or 63-66, (c) an amino acid sequence selected from SEQ ID NOs: 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66, or (d) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66.

[0036] In some embodiments, the bispecific 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: 100-102 or 133-141, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 100-102 or 133-141.

[0037] In some embodiments, the bispecific fusion protein that selectively targets the β1,6GlcNAc-branched N-glycan comprises (a) an amino acid sequence selected from SEQ ID NOs: 1-5 and 10-25, or (b) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1-5 and 10-25, (c) an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, or 19-21, or (d) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, or 19-21.

[0038] One aspect of the present disclosure provides a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising: (i) an antigen-binding domain selected from the group consisting of SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152, or 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 NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152; and (ii) an immune cell recognition domain that specifically binds to CD3 on immune effector cells.

[0039] Another aspect of the present disclosure is a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising: (I) a TACA-binding domain selected from the group consisting of SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152; (ii) a peptide, protein, antibody, single-domain antibody, antibody fragment or single-chain variable fragment (scFv) that selectively binds to a receptor on an immune effector cell; and / or (iii) the Fc domain of an antibody, optionally the Fc region of an IgG molecule; or (iv) the constant region domains CH2 and / or CH3 of an antibody (preferably CH2 and CH3, optionally with or without a hinge region).

[0040] One aspect of the present disclosure provides a chimeric antigen receptor that selectively binds to a tumor-associated carbohydrate antigen (TACA). In some embodiments, the CAR is encoded by an isolated nucleic acid disclosed herein.

[0041] One aspect of the present disclosure provides a chimeric antigen receptor that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising: (i) an antigen-binding domain selected from the group consisting of SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152; (ii) a CD8α hinge domain; (iii) a CD8 transmembrane domain; (iv) a CD28 co-stimulatory domain and / or a 4-1BB co-stimulatory domain; and (v) a CD3ζ intracellular signaling domain.

[0042] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 72, 88, 89, 91, 92 or 93. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 93, 94, 95, 96, 97, 98 or 99.

[0043] One aspect of the present disclosure provides an expression construct comprising the isolated nucleic acid described herein.

[0044] In some embodiments, the expression construct further comprises a promoter. In such embodiments, the promoter is selected from an EF-1α promoter, a T cell receptor alpha (TRAC) promoter, an interleukin-2 (IL-2) promoter, or a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a Moloney murine leukemia virus (MoMuLV) promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, or a Rous sarcoma virus promoter.

[0045] In some embodiments, the expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. In one embodiment, the expression construct is a lentiviral vector. In one embodiment, the expression construct is a self-inactivating lentiviral vector.

[0046] In some embodiments, the expression construct comprises an isolated nucleic acid molecule encoding the bispecific fusion protein described herein and an isolated nucleic acid molecule encoding the CAR described herein. In some embodiments, the isolated nucleic acid molecule encoding the bispecific fusion protein described herein and the isolated nucleic acid molecule encoding the CAR described herein are operably linked by a nucleic acid molecule encoding a self-cleaving 2A peptide selected from P2A, T2A, E2A, or F2A.

[0047] One aspect of the disclosure provides a modified cell comprising the isolated nucleic acid, the bispecific fusion protein, the CAR, or the expression vector described herein. In some embodiments, the 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.

[0048] In some embodiments, the modified cell is a T cell. In some embodiments, the modified cell is an autologous cell, a heterologous cell, or an allogeneic cell.

[0049] In some embodiments, the modified cell comprises (A) a bispecific fusion protein disclosed herein and a CAR disclosed herein, or a CAR targeting a tumor antigen, or (b) an isolated nucleic acid molecule encoding a bispecific fusion protein disclosed herein and an isolated nucleic acid molecule encoding a CAR disclosed herein, or a CAR targeting a tumor antigen, or (c) an expression construct disclosed herein.

[0050] One aspect of the present disclosure provides a composition comprising (i) an isolated nucleic acid disclosed herein, (ii) a bispecific fusion protein disclosed herein, (iii) a CAR disclosed herein, (iv) an expression vector disclosed herein, or (v) a modified cell disclosed herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0051] One aspect of the present disclosure provides a method for generating a modified cell disclosed herein, the method comprising introducing into a cell an isolated nucleic acid for generating a modified cell, a bispecific fusion protein for generating a modified cell, a CAR for generating a modified cell, or an expression vector for generating a modified cell.

[0052] One aspect of the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a composition described herein.

[0053] In some embodiments, the composition comprises (a) an isolated nucleic acid disclosed herein, (b) a bispecific fusion protein disclosed herein, (c) a CAR disclosed herein, (d) a bispecific fusion protein disclosed herein and a CAR disclosed herein, (e) an expression vector disclosed herein, or (f) a modified cell disclosed herein.

[0054] In some embodiments, the cancer is selected from the group consisting of hematological malignancies, solid tumors, primary or metastatic tumors, leukemias, carcinomas, blastomas, sarcomas, leukemias, malignant lymphomas, melanomas, and lymphomas.

[0055] One aspect of the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective composition comprising a modified cell, the modified cell comprising a bispecific fusion protein and / or a CAR that selectively binds to a tumor-associated carbohydrate antigen (TACA), the bispecific fusion protein or CAR comprising an antigen-binding domain selected from the group consisting of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, and 152, or 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: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146, or 152.

[0056] In some embodiments, the bispecific fusion protein further comprises an immunocyte recognition domain that specifically binds to a receptor on an immune effector cell. In some embodiments, the immunocyte recognition domain specifically binds to CD3. In some embodiments, the immunocyte recognition domain is an antibody Fc domain and a domain that specifically binds to CD3.

[0057] In some embodiments of the methods of treating cancer in a subject in need thereof described herein, the bispecific fusion protein comprises (a) an amino acid sequence selected from SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, and 63-66, or (b) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, and 63-66, (c) an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66, or (d) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66.

[0058] In some embodiments of the methods of treating cancer in a subject in need thereof described herein, the CAR further comprises a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. In some embodiments, the CAR comprises a CD8 transmembrane domain, a CD28 co-stimulatory domain and / or a 4-1BB co-stimulatory domain, and a CD3 zeta intracellular signaling domain. In some embodiments, the CAR further comprises a hinge domain.

[0059] One aspect of the present disclosure provides a method of providing anti-tumor immunity to a mammal, the method comprising administering to the mammal an effective amount of a population of modified cells disclosed herein.

[0060] The foregoing summary, as well as the following description of the drawings and detailed description, are all illustrative and explanatory. They are intended to provide further details of the disclosure but should not be construed as limiting. Other objectives, advantages, and novel features will become readily apparent to those skilled in the art from the following detailed description of the present disclosure.

Brief Description of the Drawings

[0061]

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Mode for Carrying Out the Invention

[0062] I. Overview Current bispecific antibodies such as Blincyto and AFM11 (CD19 / CD3 TandAb) induce activation of polyclonal T cells in vitro and in vivo in the presence of PBMC. In CAR T immunotherapy, CAR T cells are activated in vitro before being infused into patients. This suggests that peripheral activation of T cells in vivo is important for efficient cancer killing. Consistent with this, animal models of other types of immunotherapy show that activation of peripheral T cells is required for tumor eradication. However, as reported for standard CAR T cells in animal models, overactivation can also lead to a decrease in activity due to T cell exhaustion. Moreover, overactivation of polyclonal T cells can cause life-threatening complications to varying degrees in both CAR T therapy and bispecific therapy, due to the release of excessive pro-inflammatory cytokines (e.g., IL-6), i.e., "cytokine release syndrome".

[0063] To apply bispecific fusion 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. Due to the lack of safe cell surface protein antigens available for targeting, the vast majority of cancer patients are left without the possibility of these new immunotherapies. Moreover, even if new safe cell surface antigens are identified, there is a high likelihood of the need to develop different bispecific T cells and / or CAR T cells for different antigens / cancers. This significantly increases the development time and cost.

[0064] A potential approach to address all of these problems is to target "tumor-associated carbohydrate antigens" (TACAs), which are overexpressed in a wide variety of cancer types and at even higher levels in metastatic and invasive diseases. Carbohydrates (glycans), like glycoproteins and glycolipids, are major cell surface components. Virtually all cell surface proteins are glycosylated, and each protein has multiple glycans. Aberrant expression of glycosyltransferases, glycosidases, and transporters, as well as differences in the abundance of glycan building blocks, result in changes in cellular glycosylation that are common in cancer. These glycosylation changes give rise to unique antigenic glycans known as tumor-associated carbohydrate antigens (TACAs). 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. TACAs are overexpressed in a diverse range of cancer types and are even higher in metastatic and invasive diseases.

[0065] The glycosylation changes that give rise to TACAs are an almost universal feature of cancer. Since virtually all cell surface proteins are glycosylated and each protein has multiple glycans, 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.

[0066] TACA is not only simply a cancer marker but also often serves as an essential driving force for tumor growth and metastasis. For example, abnormal overexpression of β1,6GlcNAc-branched N-glycans in carcinomas drives tumor growth, motility, invasion, and metastasis. See, e.g., Fernandes et al., Cancer research 51: 718-723 (1991); Litynska et al., Melanoma research 11: 205-212 (2001); Lau & Dennis, Glycobiology 18: 750-760 (2008); Dennis et al., Science 236: 582-585 (1987); and Demetrious et al., J. Cell Biology 130: 383-392 (1995). Another related TACA found in tumor cells is the Tn antigen. The Tn antigen is not present on the cell surface of normal human tissues but is expressed in approximately 90% of human carcinomas and many hematopoietic cancers. Indeed, the Tn antigen is one of the most specific human cancer-associated structures known and promotes cell motility, invasiveness, and metastasis. The Tn antigen is a single N-acetyl-galactosamine (GalNAc) α-O-linked to serine / threonine in mucin-like proteins. Tn is the biosynthetic precursor of O-glycans that are usually elongated with α1,3-linked galactose. The chaperone protein COSMC is required by T-synthase to add galactose to GalNAc and is frequently altered in cancer. Mis-localization of enzymes within the ER / Golgi can also lead to abnormal Tn antigen expression in human cancers. The Tn antigen can be abnormally elongated with sialic acid to create the sTn antigen. This is also generally not expressed in normal tissues. Therefore, targeting TACA epitopes may be important in managing various human cancers.

[0067] Bispecific proteins and / or CAR T cells targeting TACA have great therapeutic potential, but the absence of high-affinity and / or highly specific antibodies against glycan targets represents a very important constraint in using glycans as therapeutic targets. It is extremely difficult to generate antibodies against glycans. It has been found that it is difficult to generate monoclonal antibodies with high affinity for complex glycans such as TACA (e.g., Tn antigen or β1,6GlcNAc-branched N-glycan), which hinders the widespread use of TACA as a cancer-specific antigen. Furthermore, anti-glycan antibodies generally have an affinity 1000 - 100000 times lower than that of antibodies against peptide antigens. Also, anti-glycan antibodies generally require additional peptide / lipid epitopes for high-affinity binding. Moreover, antibodies against glycans have low affinity and specificity. Additionally, the recognition of glycan antigens by antibodies depends on the density, valency, presentation, and flexibility of the glycans.

[0068] Therefore, there is a need for TACA-specific bispecific fusion proteins and / or TACA-specific CARs for treating diseases associated with abnormal glycosylation of cell surface molecules not based on the scFv of antigen-specific monoclonal antibodies.

[0069] In the present disclosure, a novel class of bispecific fusion proteins and CARs for immunotherapy that effectively target TACAs for immunotherapy have been developed. Specifically, antigen-binding domains derived from lectins rather than monoclonal antibodies or fragments thereof were used to engineer bispecific fusion proteins and CARs that target TACAs independent of carrier proteins. This novel technology is referred to as a "glycan-dependent T cell recruiter" or GlyTR (pronounced "glitter"). A set of GlyTR therapeutics is a TACA bispecific fusion protein comprising a TACA-binding domain (e.g., a sugar chain recognition domain) from a lectin operably linked, conjugated, or fused to an immune cell recognition domain that specifically binds to a receptor on an immune effector cell. 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 a plurality (e.g., more than one, or at least two) of TACA-binding domains derived from lectins.

[0070] A. Summary of Experimental Results The present disclosure further provides GlyTR therapeutics having enhanced GlyTR binding avidity (FIGS. 1-2, and FIGS. 14-15), killing activity (FIGS. 3-5, FIG. 16, FIGS. 17-18, and FIG. 23), and safety (FIGS. 12-13, and FIGS. 20-21).

[0071] To enhance the binding avidity to high-density TACAs present within cancer cells, GlyTR therapeutics having multiple sugar chain-binding domains derived from lectins were generated. Specifically, GlyTR1 having two L-PHA domains tandemly linked by three flexible linkers (i.e., (GGGGS)3) LPHA(2)xCD3 (GlyTR1 LPHAxLPHAxCD3 ; two TACA-binding domains) was generated (FIG. 1A). By SEC, GlyTR1 LPHA(2)xCD3It was revealed that about 50 - 70% is dimer, and the rest is monomer (about 30 - 40%) or larger multimer (about 10 - 20%) (Figure 2A). GlyTR1 LPHA(2)xCD3 When directly comparing the monomer (two L-PHA domains) fraction and dimer (four L-PHA domains) fraction of GlyTR1 LPHA(2)xCD3 , it was revealed that the binding to β1,6GlcNAc-branched N-glycan was significantly higher in the latter (Figure 2B), and it was further confirmed that increasing the number of TACA-binding domains in the GlyTR1 protein increased the potency. GlyTR1 LPHA(2)xCD3 When directly comparing the monomer (two L-PHA domains) fraction and dimer (four L-PHA domains) fraction of GlyTR1 LPHA(2)xCD3 , it was revealed that the binding to β1,6GlcNAc-branched N-glycan was significantly higher in the latter (Figure 2B), and it was further confirmed that increasing the number of TACA-binding domains in the GlyTR1 protein increased the potency. In fact, dimeric GlyTR1 LPHA(2)xCD3 (four L-PHA domains) binds significantly better to target cancer cells than the original dimeric GlyTR1 LPHAxCD3 (two L-PHA domains), leading to more than a 3000-fold increase in cancer cell killing activity (Figure 2C, Figure 2D). Moreover, dimeric GlyTR1 LPHA(2)xCD3 mediates human T cell-dependent killing against various types of liquid and solid cancers with a low EC of less than 100 femtomolar 50It was strongly induced therein, including multiple myeloma, T-cell leukemia, acute myeloid leukemia (AML), pancreatic cancer, colon cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, and breast cancer (Figures 3A-3I). These improved GlyTR1 binding domains are safe (Figures 12-13 and Figures 20-21), stable (Figures 6 and 19), and selectively kill cancer cells (Figures 3-5, Figure 16, Figures 17-18, and Figure 23). Furthermore, engineered cells containing novel and improved TACA bispecific fusion proteins and / or TACA-CAR did not exhibit any T cell-dependent "on-target / off-cancer" toxicity compared to control cells (Figures 7-11). The high target density and combination of multiple binding sites caused significant specificity for highly expressed cancer cells over lowly expressed normal cells, strongly inducing T cell-mediated killing of highly expressed cancer cells but not of lowly expressed normal cells.

[0072] B. Typical advantages of the GlyTR fusion protein One of the advantages of targeting TACA with immunotherapy is that virtually all cell surface proteins are glycosylated. The TACA target density is about 100-1000 times higher than that of common protein antigens. Therefore, increasing the number of TACA-binding domains in GlyTR may enhance binding avidity and increase cancer cell specificity. This is in contrast to antibodies that use high affinity to achieve specificity. In the present disclosure, high avidity binding was achieved by combining high-density target expression on tumor cells with the presence of multiple glycan-binding domains of engineered GlyTR. The combination of this high target density and multiple binding sites enhanced the specificity of the improved GlyTR for high TACA-expressing cells (e.g., cancer cells) compared to low-expressing cells (e.g., normal cells). Therefore, the specificity of the novel multivalent GlyTR protein (TACA bispecific fusion protein, or TACA CAR) for TACA will be determined by the threshold density of target expression specifically detected by GlyTR having multiple TACA-binding domains, rather than the presence or absence of the target antigen. The multivalent GlyTR proteins of the present disclosure have been shown to improve specificity for high target expression cancer cells and do not harm low expression normal tissues. As shown in the examples described herein, the GlyTR-based immunotherapy of the present disclosure induced sufficient T cell activation to maximize cancer killing in vitro and in vivo, but was insufficient to induce T cell exhaustion or cytokine release syndrome.

[0073] Accordingly, the present disclosure provides a bispecific fusion protein and a chimeric antigen receptor (CAR) that selectively bind TACA on target cells, an isolated nucleic acid molecule encoding the bispecific fusion protein and the CAR, an expression vector comprising the isolated nucleic acid molecule encoding the bispecific fusion protein and the CAR, a modified cell comprising the isolated nucleic acid molecule and / or the expression vector encoding the bispecific fusion protein and the CAR, a composition comprising the modified cell, and a method for treating a condition or disease associated with a tumor-associated carbohydrate antigen (TACA).

[0074] In one aspect, the present disclosure provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), a transmembrane domain, a co-stimulatory signaling region, an intracellular signaling domain, and optionally a hinge domain.

[0075] In another aspect, the present disclosure provides an isolated nucleic acid molecule encoding a bispecific fusion protein comprising an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA) and an immune cell recognition domain that specifically binds to a receptor on an immune effector cell. In some embodiments, the antigen-binding domain of the CAR or bispecific fusion protein comprises a plurality (more than one, or at least two, three, four, or more) of TACA-binding domains derived from a lectin.

[0076] In another aspect, the present disclosure provides a chimeric antigen receptor or bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA), comprising an antigen-binding domain selected from the group consisting of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146.

[0077] The chimeric antigen receptor (CAR) or bispecific fusion protein of the present disclosure includes a tumor-associated carbohydrate antigen (TACA)-binding domain derived from a lectin that targets TACA-expressing cells to kill tumor cells, rather than an antibody or antibody fragment. The CAR (TACA-CAR) or bispecific fusion protein of the present disclosure is improved over the prior art because it includes structural modifications that enhance the specificity of the TACA-CAR or bispecific fusion protein. The first structural modification includes altering the structure of the antigen-binding domain by changing the number, linkage, and sequence of the lectin-derived TACA-binding domains. For example, the antigen-binding domain of the TACA-CAR or bispecific fusion protein includes one or more TACA-binding domains derived from a lectin. Additionally, the linker domain between more than one TACA-binding domain is modified.

[0078] In another aspect, the present disclosure provides a composition comprising a TACA-CAR or bispecific fusion protein disclosed herein, or a modified cell comprising a TACA-CAR or bispecific fusion protein disclosed herein. The compositions of the present disclosure also include an additional peptide comprising a plurality of TACA-binding domains (at least two), a nucleic acid molecule encoding a peptide comprising a TACA-binding domain, a cell modified to express a peptide comprising a plurality of TACA-binding domains, and a substrate comprising a peptide, nucleic acid, cell, or combination thereof.

[0079] In another aspect, the present disclosure provides a method of treating cancer in a subject in need of treating cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising modified cells comprising a chimeric antigen receptor or bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA) of the present disclosure. Diseases or conditions that can be treated using the TACA-CAR or bispecific fusion protein of the present disclosure are, for example, cancer or any condition associated with changes in protein glycosylation. The cancer may be a hematological malignancy, a solid tumor, a primary tumor or a metastatic tumor. In another aspect, the present disclosure provides a method of providing anti-tumor immunity to a mammal, the method comprising administering to the mammal an effective amount of a population of modified cells comprising a chimeric antigen receptor or bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA) of the present disclosure.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] As used herein, the term "about", when referring to a measurable value such as an amount, a temporal duration, and the like, means including a variation of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, such variations being appropriate to practice the disclosed method.

[0084] 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 undergoing cell division.

[0085] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in mean survival, or an improvement in various physiological symptoms associated with the cancer condition. The "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present disclosure in the prevention of tumor development per se.

[0086] As used herein, the term "autologous" is intended to refer to any material that is derived from the same individual and is later reintroduced into that individual.

[0087] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response, which may involve the production of other antibodies, activation of specific immunocompetent cells, or both. One skilled in the art will understand that virtually any macromolecule, including substantially all proteins or peptides, can serve as an antigen. Further, an antigen can be derived from recombinant DNA or genomic DNA. One skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response will thus "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 will be readily apparent that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Moreover, one skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It will be readily apparent that an antigen can be produced synthetically or generated from a biological sample, which can include, but is not limited to, a tissue sample, a tumor sample, a cell, or a biological fluid.

[0088] As used herein, the term "allogeneic" refers to a graft derived from different animals of the same species.

[0089] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from a natural source or a recombinant source, or it can be the 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 contain light and heavy chains, which may or may not have interchain disulfide bonds between them. The basic immunoglobulin structure in the vertebrate system is relatively well understood.

[0090] The term "antibody fragment" refers to a part of an intact antibody and also refers to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0091] As used herein, the term "antibody heavy chain" refers to the larger of the two polypeptide chains that exist in the native conformation in all antibody molecules.

[0092] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains that exist in the native conformation in all antibody molecules.

[0093] As used herein, the term "antibody variant" includes synthetic and engineered forms of an antibody that have been altered so as not to occur naturally, examples of which include antibodies that contain at least two heavy chain portions but do not contain two complete heavy chains (such as domain-deleted antibodies or minibodies), multispecific forms of antibodies that have been altered to bind two or more different antigens or to bind different epitopes on a single antigen (e.g., bispecific, trispecific, etc.), heavy chain molecules conjugated to scFv molecules, and the like. Further, the term "antibody variant" includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind three, four, or more copies of the same antigen).

[0094] 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.

[0095] 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 is useful for the preferential targeting of an agent with pharmacological activity to those cancer cells. In some embodiments, the tumor antigen is a marker expressed by both normal and cancer cells (e.g., a lineage marker such as CD19 on B cells). In some embodiments, the tumor antigen is a cell surface molecule that is overexpressed in cancer cells as compared to normal cells (e.g., 1-fold overexpression, 2-fold overexpression, 3-fold or more overexpression as 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 as 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.

[0096] 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.

[0097] 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.

[0098] As used herein, the term "costimulatory ligand" includes molecules on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.), which specifically bind to cognate costimulatory molecules on T cells and thereby provide a signal that mediates T cell responses including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by the binding of, for example, a peptide-loaded MHC molecule to the TCR / CD3 complex. Costimulatory ligands can include, but are not limited to, CD2, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also include, inter alia, antibodies that specifically bind to costimulatory molecules present on T cells, examples of which include, but are not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0099] As used herein, "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by that T cell, examples of which include, but are not limited to, proliferation. A costimulatory molecule is a cell surface molecule that is neither an antigen receptor nor its ligand and contributes to an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, Toll ligand receptor, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS (CD278), NKG2C, B7-H3 (CD276), and the intracellular domain derived from killer immunoglobulin-like receptor (KIR). In some embodiments, the costimulatory molecule includes OX40, CD27, CD2, CD28, ICOS (CD278), and 4-1BB (CD137).Further examples of such co-stimulatory molecules include ligands that specifically bind to CD8, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0100] As used herein, the term "costimulatory signal" refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules. A costimulatory intracellular signaling domain may be the intracellular portion of a costimulatory molecule. Costimulatory molecules can be represented by the following protein families. They are TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD8, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and ligands that specifically bind to CD83, among others.

[0101] 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 includes 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 includes 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, delete unnecessary sequences, or introduce mutations to arrive at the intracellular signaling domain in order to provide it.

[0102] As used herein, "disease" refers to the health state of an animal where 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 where the animal can maintain homeostasis, but the animal's health state is less favorable than it would be in the absence of the ailment. Even if left untreated, an ailment does not necessarily further reduce the animal's health state.

[0103] As used herein, "disease associated with the expression of a tumor antigen" includes, but is not limited to, diseases associated with the expression of a tumor antigen, or conditions associated with cells that express a tumor antigen, including, but not limited to, proliferative diseases such as cancer or malignancy, or pre-cancerous conditions such as myelodysplasia, myelodysplastic syndrome or pre-leukemia; or non-cancer related indications associated with cells that express a tumor antigen. In some embodiments, the cancer associated with the expression of a tumor antigen is a blood cancer. In some embodiments, the cancer associated with the expression of a tumor antigen is a solid cancer. Further diseases associated with the expression of a tumor antigen include, but are not limited to, non-conventional and / or atypical cancers, malignancies, pre-cancerous conditions, or proliferative diseases associated with the expression of a tumor antigen. Non-cancer related indications associated with the expression of a 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.

[0104] As used herein, the term "downregulation" refers to a decrease or elimination of gene expression of one or more genes.

[0105] 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 administered to a mammal, as compared to an immune response detected in the absence of the composition of the present disclosure. 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, and the like. 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.

[0106] 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, typically the coding strand provided in the sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA can be said to encode the protein or other product of that gene or cDNA.

[0107] As used herein, "endogenous" refers to any substance that is from or produced within a living organism, cell, tissue, or system.

[0108] As used herein, the term "epitope" is defined as a small chemical molecule on an antigen that can induce an immune response and can 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 to 18 amino acids, about 5 to 16 amino acids, about 6 to 14 amino acids, about 7 to 12 amino acids, or about 8 to 10 amino acids. Generally, it is the overall three-dimensional structure rather than the specific linear sequence of the molecule that 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 an organism, cell, tissue, or system or is produced outside of them.

[0109] 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.

[0110] As used herein, the term "exogenous" refers to any substance that is introduced from outside an organism, cell, tissue, or system or is produced outside of them.

[0111] 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.

[0112] 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).

[0113] 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).

[0114] As used herein, the term "Fc portion" or "Fc monomer" means, in the context of the present disclosure, a polypeptide comprising at least one domain having the function of the CH2 domain of an immunoglobulin molecule and at least one domain having the function of the CH3 domain. As is apparent from the term "Fc monomer", the polypeptide comprising these CH domains is a "polypeptide monomer". The Fc monomer can be a polypeptide comprising at least a fragment of the constant region of an immunoglobulin that excludes the first constant region immunoglobulin domain (CH1) of the heavy chain 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 comprising the CH2 region and the CH3 region that are part of the Ig-Fc hinge region, the hinge region being the amino terminus of the CH2 domain.

[0115] 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 including 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.

[0116] 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 including a functional hinge, CH2, and CH3 domains can be defined, for example, to include residue sequence numbers 153-158.

[0117] 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: 153 or 154).

[0118] In one embodiment, the IgG1 hinge domain / region comprises the amino acid sequence of EPKSCDKTHTCPPCP (SEQ ID NO: 154).

[0119] In a further embodiment of the present disclosure, the hinge domain / region comprises or consists of the IgG2 subtype hinge sequence ERKCCVECPPCP (SEQ ID NO: 155), the IgG3 subtype hinge sequence ELKTPLDTTHTCPRCP (SEQ ID NO: 156) or ELKTPLGDTTHTCPRCP (SEQ ID NO: 157), and / or the IgG4 subtype hinge sequence ESKYGPPCPSCP (SEQ ID NO: 158).

[0120] 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 intradomain cysteine disulfide bridge.

[0121] In another embodiment, the 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 has the amino acid sequence of GGGS (e.g., Gly4Ser (SEQ ID NO: 128)), or a polymer thereof (e.g., (Gly4Ser) nincluding where n is an integer of 5 or more (for example, 5, 6, 7, 8, etc., or more).

[0122] 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.

[0123] 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 in 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 (for example, 5 positions within a polymer of 10 amino acids in length) are identical, the two sequences are 50% identical, and if 90% of the positions (for example, 9 out of 10) match or are identical, the two amino acid sequences are 90% identical.

[0124] As used herein, the terms "immunoglobulin" or "Ig" are defined as a class of proteins that function as antibodies. Antibodies expressed by B cells may also be referred to as 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 tract 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.

[0125] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify an antigen molecule as foreign, induce the formation of antibodies, and / or activate lymphocytes to eliminate the antigen. As used herein, the term "immunostimulation" is used to refer to an increase in the overall immune response. As used herein, the term "immunosuppression" is used to refer to a decrease in the overall immune response.

[0126] 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 eliminate the antigen.

[0127] 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.

[0128] 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.

[0129] 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, for example, affixed to a container containing the nucleic acids, peptides, and / or compositions of the present disclosure, or shipped together with a container containing the nucleic acids, peptides, and / or compositions of the present disclosure. Alternatively, the explanatory materials may be shipped separately from the container, with the intention that the recipient will use the explanatory materials in concert with the compound.

[0130] As used herein, the term "isolated" means changed or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from the substances that coexist in the natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-natural environment such as, for example, a host cell.

[0131] 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.

[0132] 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 (s) in any version.

[0133] As used herein, the terms "lectin" or "hemagglutinin" refer to proteins or peptides that bind to sugar chain structures. One skilled in the art will understand that lectins are proteins or peptides that are highly specific for binding to sugar moieties. Lectins are sugar chain-binding proteins that are highly specific for sugar chains present on proteins and / or lipids, and cause aggregation of specific cells or precipitation of glycoconjugates and polysaccharides. Lectins play a role in recognition at the cellular and molecular levels and play numerous roles in biological recognition phenomena involving cells, sugar chains, and proteins. Lectins also mediate the attachment and binding of bacteria, viruses, and fungi to their targets. In one embodiment, a "lectin" can be defined as a non-immunoglobulin protein or glycoprotein that can specifically recognize and reversibly bind to the sugar chain portion of a complex glycoconjugate (protein and / or lipid) without altering any covalent structure of the recognized glycosyl ligand. Lectins are glycoproteins having a sugar chain-binding domain with the ability to reversibly bind to glycoproteins or glycolipids, as well as to specific sugar moieties within free monosaccharides and glycan structures. Many organisms express lectins or lectin-like biomolecules, but most of the scientifically important lectins identified recently have been purified from plant sources.

[0134] 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 macromolecules (glycans) are ubiquitous in biological systems and are essential for a number of biological functions. Carbohydrates can attach to proteins (glycoproteins), lipids (glycolipids), and exist as chains of carbohydrates (glycosaminoglycans). Changes in the structure of these carbohydrate-containing macromolecules (glycosylation) have a major 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 often can be distinguished from normal cells because they display abnormal levels and types of carbohydrate structures on their surfaces.

[0135] Three general changes in carbohydrate-containing macromolecules, namely increased expression of truncated or incomplete glycans, increased branching of N-glycans, and increased presence or change in 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 that attaches galactose residues to the Tn antigen (Thomsen-Friedenreich (TF) antigen). In various cancers, this process is altered, the glycosylation of the Tn antigen or its sialylated form (sialyl-Tn (STn) antigen) is changed, and truncated T, Tn, and STn antigens are produced. Furthermore, an increase in the branching of N-glycoproteins that stimulate galectin-3, and changes in glycolipids such as gangliosides (GM3, GM2, CD3, and GD2) have also been observed.

[0136] The following TACAs have been observed in various cancers. Namely, (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 cancer, lung cancer, breast cancer, and many other cancers, (iv) GM2, GD2, and GD3 gangliosides in neuroectodermal tumors (melanoma and neuroblastoma), (v) globo-H in breast cancer, ovarian cancer, and prostate cancer, (vi) dialsylgalactosylgloboside in renal cell carcinoma.

[0137] Thus, the term "tumor-associated carbohydrate antigen (TACA)" encompasses all altered sugar 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 a sugar chain structure is composed of one or more linked sugars or monosaccharides. See, for example, Mantuano et al, J. Immunotherapy Cancer 8(2): 8:e001222 (2020); Hakomori, Si. (2001). Tumor-Associated Carbohydrate Antigens Defining Tumor Malignancy: Basis for Development of Anti-Cancer Vaccines, in The Molecular Immunology of Complex Carbohydrates -2. Advances in Experimental Medicine and Biology, vol 491. Springer, Boston, MA (Wu et al (eds)). A skilled artisan will understand that a sugar chain structure can exist independently of and / or attach to proteins or lipids known as glycoproteins and glycolipids. A skilled artisan will understand that these sugar chain structures bind to lectins.

[0138] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells and can deliver large amounts of genetic information into the DNA of host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0139] As used herein, the term "limited toxicity" means that the peptides, polynucleotides, cells, and / or antibodies of the present disclosure do not exert a substantially negative biological effect, anti-tumor effect, or substantially negative physiological symptoms on healthy cells, non-tumor cells, non-disease cells, non-target cells, or populations of such cells, either in vitro or in vivo.

[0140] 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-Ser)n, where n is a positive integer of 1 or more. For example, n = 1, n = 2, n = 3, n = 4, n = 5, n = 6, n = 7, n = 8, n = 9, n = 10 (SEQ ID NO: 6592). In one embodiment, the flexible polypeptide linker includes, but is not limited to, (Gly4Ser)4 or (Gly4Ser)3. In another embodiment, the linker includes multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser).

[0141] 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.

[0142] 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. The term encompasses disturbing and / or affecting the original signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0143] 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.

[0144] 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, in the same reading frame.

[0145] 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. Patients having a solid tumor or a hematological malignancy characterized by overexpression of a tumor antigen can be determined by standard assays known in the art.

[0146] 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.

[0147] 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.

[0148] 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 by recombinant means, i.e., using conventional cloning techniques and PCR (registered trademark), etc., and by synthetic means, including cloning of nucleic acid sequences from recombinant libraries or cell genomes, but are not limited thereto.

[0149] 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 containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which are generally also referred to in the art as peptides, oligopeptides and oligomers, and long chains, which are generally referred to in the art as proteins and which come in many types. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0150] 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.

[0151] 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.

[0152] As used herein, a "constitutive promoter" is a nucleotide sequence which, when operably linked to a polynucleotide encoding or specifying a gene product, produces the gene product in a cell under most or all physiological conditions of the cell.

[0153] As used herein, an "inducible promoter" is a nucleotide sequence which, when operably linked to a polynucleotide encoding or specifying a gene product, produces the gene product in a cell only when an inducer substantially corresponding to the promoter is present in the cell.

[0154] As used herein, a "tissue-specific promoter" is a nucleotide sequence which, when operably linked to a polynucleotide encoded or specified by a gene, produces the gene product in a cell only when the cell is substantially a cell of the tissue type corresponding to the promoter.

[0155] 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.

[0156] As used herein, the term "single-chain antibody" refers to an antibody formed by recombinant DNA techniques in which immunoglobulin heavy and light chain fragments are linked to the Fv region via an engineered range of amino acids. A variety of methods for generating single-chain antibodies are known, including those described in U.S. Patent No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Ward et al., Nature 334:544-54 (1989); Skerra et al., Science 242:1038-1041 (1988).

[0157] 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 may be monospecific, containing one or more binding sites that specifically bind a target, or a chimeric antigen receptor may be multispecific, containing two or more binding sites that specifically bind the same or different targets. In certain embodiments, the chimeric antigen receptor is specific for two different (e.g., non-overlapping) portions of the same target. In certain embodiments, the chimeric antigen receptor is specific for more than one target.

[0158] As used herein, the term "specifically binds" with respect to an antibody means an antibody or a binding fragment thereof (e.g., scFv) that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to a certain antigen may also bind to 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 that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity per se does not change the classification of the antibody as specific. In some instances, the terms "specific binding" or "specifically binds" are used with respect to the interaction between an antibody, protein, chimeric antigen receptor or peptide and a second chemical species, and can mean 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 a specific protein structure rather than a protein. If an antibody is specific for epitope "A", in a reaction containing label "A" and the antibody, the amount of label A bound to the antibody will decrease if a molecule containing epitope A (or free, unlabeled A) is present.

[0159] As used herein, the term "stimulate" means a primary response induced by binding a stimulatory molecule (e.g., TCR / CD3 complex) to its cognate ligand, thereby mediating a signaling event, examples of which include, but are not limited to, signaling through the TCR / CD3 complex. Stimulation can mediate a change in the expression of specific molecules, examples of which include downregulation of TGF-β, and / or rearrangement of the cytoskeletal structure, clonal expansion, and differentiation into different subsets.

[0160] 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.

[0161] 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 and thereby can 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.

[0162] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cell types with which it is normally associated in its natural state. In some instances, a population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to cells that have been separated from the cell with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0163] 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 can specifically bind under conditions sufficient for binding to occur.

[0164] 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 gamma and delta (γ / δ) chains (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 that contains 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.

[0165] 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.

[0166] The term "therapeutically effective amount" or "effective amount" refers to the amount of a subject compound that induces a biological or medical response in a tissue, system or subject sought by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of a compound sufficient to prevent, or to alleviate to some extent, the onset of one or more 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.

[0167] 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 personnel.

[0168] As used herein, the terms "treating," "treatment," and "treatment thereof" 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 effect of a treatment includes, but is not limited to, prevention of the occurrence or recurrence of a disease, alleviation of symptoms, reduction of the direct or indirect pathological consequences of a disease, decrease in the rate of progression of a disease, improvement of a medical condition or alleviation of pain, and remission or improvement of a prognosis. In some embodiments, "treatment" of a disease as the term is used herein means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0169] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is introduced or transferred into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. Such cells include primary subject cells and their progeny.

[0170] 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.

[0171] 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. A number of vectors are known in the art and include, but are 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.

[0172] As used herein, the term "heterologous" refers to a graft derived from animals of different species.

[0173] Range: Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, a description of a range should be considered to specifically disclose not only the individual numerical values within that range but also all possible sub-ranges. For example, a description of a range such as 1-6 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.

[0174] III. TACA Antigen-Binding Domain One aspect of the present disclosure provides a bispecific fusion protein or chimeric antigen receptor comprising an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA). In some embodiments, the antigen-binding domain comprises a TACA-binding domain derived from a lectin. In some embodiments, the antigen-binding domain comprises more than one (e.g., plural) TACA-binding domains.

[0175] Malignant transformation of cells is almost invariably associated with abnormal glycosylation of cell surface proteins or lipids (Kim and Varki, 1997, Glycoconj J, 14:569-576). Indeed, changes in cell surface glycosylation have been observed in all types of experimental and human cancers (Hakomori, 2002, PNAS USA, 99:10231-10233), and these altered sugar structures are called tumor-associated carbohydrate antigens (TACAs) (Table 1). Due to this tumor-specific property, cell surface TACAs are excellent target antigens for the production of monoclonal antibodies targeting many common cancers. However, despite decades of effort, specific antibodies with high affinity for TACAs are not yet available because it is difficult to generate antibodies against carbohydrate antigens.

[0176] Accordingly, the inventors of the present disclosure have engineered bispecific fusion proteins and CARs for therapy that selectively target TACAs on tumor cells, relying on lectins rather than antibodies. Lectins and their binding partners are well known in the art. See, for example, functionalglycomics.org / glycomics / publicdata / primaryscreen.jsp. The lectin-binding proteins of the present disclosure have significant advantages over existing technologies (e.g., GlyTR chimeric proteins) and provide opportunities for the development of a new class of therapeutics for cancer immunotherapy. Based on the concept of GlyTR and the availability of multiple different lectins specific for multiple 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).

Table 1

[0177] The antigen-binding domains of the bispecific fusion proteins or CARs disclosed herein are designed to specifically target glycoproteins and / or glycolipids (i.e., carbohydrate-containing macromolecules) on tumor cells. In some embodiments, the bispecific fusion proteins or CARs 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 CAR or bispecific fusion protein may have an affinity for a target antigen on a target cell that indicates a particular state of the target cell.

[0178] In some embodiments, the antigen-binding domain comprises a plurality (e.g., more than one) of TACA-binding domains. In some embodiments, the antigen-binding domain comprises two, three, four, five, six, seven, eight, nine, ten, or more TACA-binding domains. In one embodiment, the antigen-binding domain comprises two TACA-binding domains. In one embodiment, the antigen-binding domain comprises three TACA-binding domains. In one embodiment, the antigen-binding domain comprises four TACA-binding domains.

[0179] A.TACA The TACA-binding domain may include any peptide, protein, lectin, lectin fragment, antibody, antibody fragment, small molecule, nucleic acid, etc. that can specifically bind to TACA. In some embodiments, the antigen-binding domain selectively targets β1,6GlcNAc-branched N-glycan, Tn epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), GalNAcα-serine, GalNAcα-threonine, GalNAc or GalNAcβ1.

[0180] Exemplary TACAs and their binding partners are listed in Table 1. Exemplary TACAs include, but are not limited to, β1,6 branching, T antigen, sialyl-T epitope, Tn epitope, sialyl-Tn epitope, a2,6 sialylation, sialylation, sialyl-Lewis^, disialyl-Lewis^, sialyl 6-sulfo Lexis x 、Lewis-y(Le y )、Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GMl. In some embodiments, the CAR or bispecific fusion comprises β1,6 branching, β1,6GlcNAc-branched N-glycan, T antigen, Tn antigen, sialyl-T epitope, Tn epitope, sialyl-Tn epitope, α2,6 sialylation, sialylation, sialyl-Lewis x / a 、disialyl-Lewis x / a 、sialyl 6-sulfo Lexis x, Lewis-y (Le y ) selectively targets a TACA selected from the group consisting of Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GM1. In some embodiments, the CAR selectively targets a β1,6GlcNAc-branched N-glycan, GalNAc, Tn antigen, GalNAcα-ser, GalNAc, or GalNAcβ1.

[0181] In one embodiment, the TACA binding domain binds to an N-glycan. In certain embodiments, the TACA binding domain binds to tri- and tetra-branched oligosaccharides. In one embodiment, the TACA binding domain binds to a β1,6GlcNAc-branched N-glycan. In one embodiment, the TACA binding domain binds to the Tn epitope.

[0182] B. Lectin In certain embodiments, the TACA binding domain is a peptide sequence derived from a lectin protein. In certain embodiments, the lectin is selected from the group consisting of mammalian lectins, human lectins, plant lectins, bacterial lectins, viral lectins, fungal lectins, and protozoan lectins. In some embodiments, the antigen binding domain comprises a TACA binding domain derived from a lectin. In some embodiments, the antigen binding domain comprises at least two TACA binding domains from a lectin.

[0183] 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); snowdrop lectin (SNA), BC2L-CNt (lectin from the gram-negative bacterium Burkholderia cenocepacia), dogwood leukocyte agglutinin (MAL), Pleurotus ostreatus (PVL), Sclerotium rolfsii lectin (SRL), Euryale ferox lectin (ESA), CLEC17A (prolectin), Grifola frondosa lectin, Allium sativum lectin (SSA), Glechoma hederacea lectin (Gleheda), Momordica charantia lectin (Morniga G), Origanum vulgare lectin, Salvia bogotensis lectin, Salvia horminum lectin, Cuscutaceae lectin, Calceolaria crenatiflora lectin, Glyphomia simplificifolia (GsLA4), Vicia cracca (acidic WBAI), Vigna angularis lectin, Adenophora triphylla lectin, Amaranthus leucocarpus lectin, Relya autumnalis lectin, Paramignya monophylla lectin, Ipomoea batatas lectin, Altocarpus lakoocha lectin, Himalayan Vicia unijuga lectin, Himalayan Vicia unijuga lectin, soybean lectin and mushroom lectin.

[0184] 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.

[0185] 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).

[0186] In some embodiments, the antigen-binding domain of the CAR or bispecific fusion protein described herein is 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, sialyl-Lewisx / a, disialyl-Lewisx / a, sialyl 6-sulfo Lexis x , Lewis-y (Le y ), Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GM1, and selectively targets a TACA selected from the group consisting of

[0187] C. Chimeric Antigen Receptor One aspect of the present disclosure provides compositions and methods for modified immune cells or their progenitor cells (e.g., modified T cells) comprising a chimeric antigen receptor (CAR) having affinity for a tumor-associated carbohydrate antigen (TACA). The CARs of interest in the present disclosure include an antigen-binding domain (e.g., a tumor-associated carbohydrate antigen (TACA)), a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain. The CARs of interest in the present disclosure may optionally include a hinge domain. In some embodiments, each domain of the CAR of interest is separated by a linker. In one aspect, a chimeric antigen receptor that selectively binds a tumor-associated carbohydrate antigen (TACA) is encoded by an isolated nucleic acid disclosed herein.

[0188] One aspect of the present disclosure provides a chimeric antigen receptor (CAR) comprising the amino acid sequence set forth in SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. In some embodiments, the CAR comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

[0189] In another aspect of the present disclosure, a chimeric antigen receptor that selectively binds a tumor-associated carbohydrate antigen (TACA) comprises an antigen-binding domain selected from the group consisting of SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146, or 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 NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146, a CD8 a hinge domain, a CD8 transmembrane domain, a CD28 co-stimulation, and a CD3 zeta intracellular signaling domain.

[0190] In another aspect, a chimeric antigen receptor that selectively binds to a tumor-associated carbohydrate antigen (TACA) comprises an antigen-binding domain selected from the group consisting of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145 or 146, or 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: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146, a CD8 a hinge domain, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta intracellular signaling domain.

[0191] In another aspect, a chimeric antigen receptor that selectively binds to a tumor-associated carbohydrate antigen (TACA) comprises an antigen-binding domain selected from the group consisting of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, or 146, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, or 146, a CD8 a hinge domain, a CD8 transmembrane domain, a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain, and a CD3 zeta intracellular signaling domain.

[0192] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NOs: 72, 88, 89, 91, 92, or 93. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NOs: 93, 94, 95, 96, 97, 98, or 99.

[0193] In some embodiments, the chimeric antigen receptor (CAR) is a 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 diasialyl-Lewis x / a sialyl 6-sulfo Lexis x Lewis-y (Le y) has an affinity for Lewis Y, Globo H, GD2, GD3, GM3, or fucosyl GM1. In some embodiments, the chimeric antigen receptor (CAR) has an affinity for β1,6-branched or β1,6GlcNAc-branched N-glycans. In some embodiments, the chimeric antigen receptor (CAR) has an affinity for the Tn antigen or sialyl-Tn epitope.

[0194] 1. Extracellular domain The antigen-binding domain of the CAR is the extracellular region of the CAR for binding to specific target antigens including proteins, glycans, and glycolipids. In some embodiments, the CAR includes an affinity for a target antigen (e.g., a tumor-associated antigen) on a target cell (e.g., a cancer cell). The target antigen may include any type of protein or an epitope thereof that associates with the target cell. For example, the CAR may include an affinity for a target antigen on a target cell that indicates a specific state of the target cell.

[0195] In some embodiments, the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152. Alternatively, the antigen-binding domain comprises an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152. In some embodiments, the antigen-binding domain comprises the amino acid sequence disclosed in Table 2 or Table 3.

[0196] In some embodiments, the antigen-binding domain comprises an amino acid sequence having at least 90% homology with SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146.

[0197] The antigen-binding domain may be operably linked to another domain of the CAR, such as a transmembrane domain, a co-stimulatory signaling domain or an intracellular signaling domain (each described elsewhere herein), for expression within a cell. The antigen-binding domains described herein can be combined with any of the transmembrane domains, any of the co-stimulatory signaling domains, any of the intracellular signaling domains, or any of the other domains described herein and included in the CARs of the present disclosure.

[0198] 2. Linker In this specification, the terms "linker" and "spacer" are used interchangeably. Linkers generally contain a rich amount of glycine to enhance flexibility and also a rich amount of serine or threonine to enhance solubility. Multiple linkers may be used to connect more than one TACA binding domain. In some embodiments, for example, more than one TACA binding domain can be operably linked by a linker such as a linker that can be selected from the group consisting of a peptide linker, a non-peptide linker, a chemical unit, a disulfide-crosslinked linker, and a non-disulfide-crosslinked linker. In one embodiment, the linker is a peptide linker. The peptide linker can be a glycine-serine linker. The peptide linker can have a length of at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, or at least about 15 amino acids.

[0199] Various linker sequences are known in the art, and non-limiting examples of linkers are disclosed in Shen et ai., Anal. Chem. 80(6):1910-1917(2008) and WO2014 / 087010, the contents of which are hereby incorporated by reference in their entirety. One of ordinary skill in the art would be able to select an appropriate linker sequence for use in the bispecific fusion proteins and / or CARs of the present disclosure.

[0200] In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 127, or the amino acid sequence of SEQ ID NO: 131.

[0201] 3. Transmembrane domain Regarding the transmembrane domain, the CARs of the present disclosure (e.g., TACA CAR) can be designed to include a transmembrane domain that connects the antigen-binding domain of the CAR to the intracellular domain. The transmembrane domain of the target CAR is a region capable of spanning the cell membrane of a cell (e.g., an immune cell or its precursor). The transmembrane domain is intended for insertion into the cell membrane (e.g., a eukaryotic cell membrane). In some embodiments, the transmembrane domain intervenes between the antigen-binding domain and the intracellular domain of the CAR.

[0202] In one embodiment, the transmembrane domain is naturally associated with one or more of the domains within the CAR. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid the binding of such a domain to the transmembrane domain of the same or different surface membrane proteins and minimize its interaction with other members of the receptor complex.

[0203] The transmembrane domain can be derived from either a natural source or a synthetic source. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein (e.g., a type I transmembrane protein). If the source is synthetic, the transmembrane domain can be any artificial sequence that facilitates the insertion of the CAR into the cell membrane, such as an artificial hydrophobic sequence.

[0204] In some embodiments, the chimeric antigen receptor (CAR) comprises a transmembrane domain, which transmembrane domain may comprise the transmembrane region of a molecule selected from the group consisting of T cell receptor (TCR)-alpha, TCR-beta, CD3-zeta, CD3-epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134 (Ox40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In one embodiment, the transmembrane domain comprises the CD8 transmembrane domain. Alternatively, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 148.

[0205] In some embodiments, the transmembrane domain may be a synthetic compound, in which case the transmembrane domain will primarily comprise hydrophobic residues such as leucine and valine. In certain exemplary embodiments, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain.

[0206] The transmembrane domains described herein can be combined with any of the antigen-binding domains described herein, any of the co-stimulatory signaling domains described herein, any of the intracellular signaling domains described herein, or any of the other domains described herein and that may be included in the subject CAR.

[0207] 4. Intracellular Domain The subject CARs of the disclosure also include an intracellular domain. The intracellular domain of the CAR is responsible for activating at least one of the effector functions of the cell in which the CAR is expressed (e.g., an immune cell). The intracellular domain causes transduction of an effector function signal, leading the cell (e.g., an immune cell) to perform specialized functions such as damaging and / or destroying target cells.

[0208] The intracellular domain or, alternatively, the cytoplasmic domain of the CAR is responsible for the activation of the cells in which the CAR is expressed. Examples of intracellular domains used in the present invention include, but are not limited to, the cytoplasmic portion of a surface receptor, a costimulatory molecule, and any molecule that acts in concert to initiate signal transduction within a T cell, as well as any derivative or variant of these elements, and any synthetic sequence having the same functional capacity. In certain embodiments, the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain.

[0209] Examples of intracellular signaling domains include, but are not limited to, either the zeta chain of the T cell receptor complex or a homolog thereof, syk family tyrosine kinases (such as Syk, ZAP 70, etc.), src family tyrosine kinases (such as Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction (such as CD2, CD3, and CD28). In one embodiment, the intracellular signaling domain may be the human CD3 zeta chain, FcγRin, FcεRI, the cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) having a cytoplasmic receptor, and combinations thereof.

[0210] Other examples of intracellular domains include fragments or domains from one or more molecules or receptors, which molecules or receptors specifically bind to TCR, CDS zeta, CDS gamma, CDS delta, CDS epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma R1 la, DAP10, DAP12, T cell receptor (TCR), CDS, CD27, CD28, 4-1BB (CD137), 0X9, 0X40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKpSO (KLRF1), CD127, CD160, CD19, CD4, CDSalpha, CDSbeta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlId, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CD lib, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylOS), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKpSO, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other stimulatory molecules described herein, any derivatives, variants, or fragments thereof, any synthetic sequences of stimulatory molecules having the same functional capacity, and any combinations thereof, including but not limited to these.

[0211] Further examples of intracellular domains include intracellular signaling domains of several types of various other immune signaling receptors, which include first, second, and third generation T cell signaling proteins including CDS, B7 family costimulatory factors, and tumor necrosis factor receptor (TNFR) superfamily receptors, but are not limited thereto (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6):651-653). Further, the intracellular signaling domain may include signaling domains used by NK cells and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6:195), examples of which include NKpSO (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5):2290-2299), and DAP12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7):3201-3212), the signaling domains of NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0212] Intracellular signaling domains suitable for use in the CARs of the present disclosure include any desired signaling domain that provides a distinct and detectable signal (e.g., an increase in the production of one or more cytokines by the cell, a change in the transcription of a target gene, a change in the activity of a protein, a change in cell behavior (e.g., cell death), cell proliferation, cell differentiation, cell survival, regulation of a cell signaling response, etc.) in response to activation of the CAR (i.e., activation by an antigen and a dimerization agent). In some embodiments, the intracellular signaling domain includes at least one (e.g., 1, 2, 3, 4, 5, 6, etc.) GGAM motif, as described below. In some embodiments, the intracellular signaling domain includes a DAP10 / CD28 type signaling chain. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane-bound CAR and instead diffuses within the cytoplasm.

[0213] Intracellular signaling domains suitable for use in the CARs of the present invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, the ITAM motif is repeated twice within the intracellular signaling domain, and the first and second instances of the ITAM motif are separated from each other by 6-8 amino acids. In one embodiment, the intracellular signaling domain of the CAR of interest contains three ITAM motifs. In some embodiments, the intracellular signaling domain includes the signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine-based activation motif (ITAM), examples of which include, but are not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma IIIA, FcRL5 (see, for example, Gillis et al., Front (2014) Immunol. 5:254).

[0214] A suitable intracellular signaling domain can be an ITAM motif-containing portion derived from a polypeptide containing a GGAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3δ (CD3 delta), CD3ε (CDS epsilon), CD3γ (CDS gamma), CD3ζ (CDS zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0215] In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP, TYRO protein tyrosine kinase-binding protein, KARAP, PLOSL, DNAX activation protein 12, KAR-associated protein, TYRO protein tyrosine kinase-binding protein, killer activation receptor-associated protein, killer activation receptor-associated protein, etc.). In one embodiment, the intracellular signaling domain is derived from FCεR1G (also known as FCRG, Fc epsilon receptor I gamma chain, Fc receptor gamma chain, fc-epsilonRI-gamma, fcR gamma, fceRl gamma, high-affinity immunoglobulin epsilon receptor subunit gamma, immunoglobulin E receptor, high-affinity, gamma chain, etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3δ, CD3-DELTA, T3D, CD8 antigen, delta subunit, CD3d antigen, delta polypeptide (TiT3 complex), OKT3, delta chain, T cell receptor T3 delta chain, T cell surface glycoprotein CD8 delta chain, etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3ε, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD8, CD8epsilon, T3e, etc.).

[0216] In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3γ, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CDS zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signaling domain is derived from CD79A (also known as B cell antigen receptor complex-associated protein alpha chain, CD79a antigen (immunoglobulin-related alpha), MB-1 membrane glycoprotein, Ig-α, membrane-bound immunoglobulin-related protein, surface IgM-related protein, etc.). In one embodiment, the intracellular signaling domain suitable for use in the CARs of the present disclosure comprises a DAP10 / CD28 type signaling chain. In one embodiment, the intracellular signaling domain suitable for use in the CARs of the present disclosure comprises a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CDS epsilon, CD3, CD22, CD79a, CD79b or CD66d. In one embodiment, the intracellular signaling domain within the CAR comprises the cytoplasmic signaling domain of human CDS zeta.

[0217] a. Costimulatory domain In certain embodiments, the intracellular domain comprises a costimulatory signaling domain. In some embodiments, the chimeric antigen receptor (CAR) comprises a costimulatory domain, and this costimulatory domain is a costimulatory domain of a molecule selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD8, LIGHT, NKG2C, B7-H3, the ligand that specifically binds to CD83, DAP10, DAP12, Lck, Fas, and combinations thereof.

[0218] In one embodiment, the co-stimulatory domain comprises a 4-1BB co-stimulatory domain or a CD28 co-stimulatory domain. In one embodiment, the co-stimulatory domain comprises a 4-1BB co-stimulatory domain and a CD28 co-stimulatory domain. In one embodiment, the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 114 or SEQ ID NO: 113. In one embodiment, the co-stimulatory domain comprises the amino acid sequences of SEQ ID NO: 114 and SEQ ID NO: 113.

[0219] Tolerable variations of the intracellular domain while maintaining specific activity are known to those skilled in the art. For example, in some embodiments, the intracellular domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any of the amino acid sequences set forth in SEQ ID NO: 113 or 114.

[0220] b. Intracellular signaling domain In certain embodiments, the intracellular domain comprises an intracellular signaling domain. In some embodiments, the isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprises an intracellular domain that is from the intracellular signaling domain of a molecule selected from the group consisting of T cell receptor (TCR) zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, CD3, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the intracellular signaling domain comprises a CD3 zeta signaling domain, or the amino acid sequence of SEQ ID NO: 115.

[0221] Tolerable variations of the intracellular domain while maintaining specific activity are known to those skilled in the art. For example, in some embodiments, the intracellular domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any of the amino acid sequences set forth in SEQ ID NO: 115.

[0222] 5. Hinge The hinge region of the CAR is a hydrophilic region located between the antigen-binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding of the CAR. The hinge region is an optional component of the CAR. In some embodiments, the chimeric antigen receptor (CAR) may further comprise a hinge domain.

[0223] In some embodiments, the hinge domain is a protein selected from the group consisting of CD8α, the Fc fragment of an antibody, the hinge region of an antibody, the CH2 region of an antibody, the CH3 region of an antibody, and an artificial spacer sequence. In one embodiment, the hinge domain is the CD8α hinge domain. In one embodiment, the hinge domain comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the hinge domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 119, SEQ ID NO: 124, 127, 128, 129, 130, 131, 132, and 147. In some embodiments, the CAR of the present disclosure includes a hinge region that connects the antigen-binding domain to the transmembrane domain, which in turn connects to the intracellular domain. The hinge region preferably enables the antigen-binding domain to recognize and bind to a target antigen on the target cell (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2):125-135). In some embodiments, the hinge region is a flexible domain. Thus, the antigen-binding domain can have a structure that optimally recognizes the specific structure and density of the target antigen on cells such as tumor cells. Due to the flexibility of the hinge region, the hinge region can adopt a number of different conformations. In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a hinge region derived from CD8).

[0224] The hinge region can have a length of from about 4 amino acids to about 50 amino acids, such as from about 4 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 40 amino acids, or from about 40 amino acids to about 50 amino acids.

[0225] Suitable hinge regions can be readily selected and can be of any of a plurality of suitable lengths, examples of which include from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids (including from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids), and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0226] D. Bispecific fusion proteins One aspect of the present disclosure provides a bispecific fusion protein comprising an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA) and an immune cell recognition domain that specifically binds to a receptor on an immune effector cell.

[0227] The bispecific fusion protein comprises two different binding specificities and thus binds 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 TACAs are described elsewhere in this specification and all of them may be targeted by the bispecific fusion protein of the present invention. In certain embodiments, the second antigen recognition domain binds to an immune effector cell. In some embodiments, the antigen-binding domain comprises a TACA-binding domain derived from a lectin, and the antigen-binding domain comprises more than one TACA-binding domain as described herein.

[0228] In some embodiments, the bispecific fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, or 63-66. Alternatively, the bispecific fusion protein comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, or 63-66. In some embodiments, the amino acid sequence is selected from SEQ ID NOs: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66. In some embodiments, the bispecific fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 3-5, 10-13, 18-21, 26-34, 39-42, 47-50, 55-58, or 63-66. In some embodiments, the bispecific fusion protein comprises an amino acid sequence disclosed in Table 2 or Table 3. In some embodiments, the bispecific fusion protein comprises the amino acid sequence of SEQ ID NOs: 31-34, 39-42, 47-50, 55-58, 63, or 64.

[0229] In some embodiments, the bispecific fusion protein exhibits enhanced binding to Thomsen - nouveau (Tn) antigen (Tn antigen) - expressing tumor cells as compared to a bispecific fusion protein that includes a flexible linker in the antigen - binding domain. In this embodiment, the antigen - binding domain is derived from CD301 (CLEC10A). In that embodiment, the flexible linker is a glycine - serine linker or a linker comprising an amino acid sequence selected from SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127. Alternatively, the flexible linker is a glycine - serine linker or a linker comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127.

[0230] In some embodiments, the bispecific fusion protein includes an immune cell recognition domain that selectively binds to a receptor on an immune effector cell. In that embodiment, 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 that embodiment, 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.

[0231] 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: 149, 150 or 151. 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: 149, 150 or 151.

[0232] Alternatively, the immunocyte recognition domain comprises an antibody Fc domain and optionally an Fc region of an IgG molecule. In one embodiment, the bispecific fusion protein is an Fc fusion protein comprising an antigen-binding domain that selectively binds a tumor-associated carbohydrate antigen (TACA). In some embodiments, the immunocyte recognition domain comprises an antibody Fc domain and a domain that specifically binds CD3. In another embodiment, the immunocyte recognition domain comprises the constant region domains CH2 and / or CH3 of an antibody, preferably including CH2 and CH3. The constant region domains CH2 and / or CH3 of the antibody may or may not include a hinge region.

[0233] In some embodiments, the bispecific fusion protein is β1,6-branched, β1,6GlcNAc-branched N-glycan, T antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6-sialylation, sialylation, sialyl-Lewis x / a diasialyl-Lewis x / a sialyl 6-sulfo Lexis x Lewis-y (Le y) It selectively targets a TACA selected from the group consisting of Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GM1.

[0234] In one embodiment, the bispecific fusion protein selectively targets the Tn antigen or β1,6GlcNAc-branched N-glycans. In some embodiments, the bispecific fusion protein that selectively targets β1,6GlcNAc-branched N-glycans comprises an antigen-binding domain having an amino acid sequence selected from SEQ ID NOs: 100-102 or 133-141, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 100-102 or 133-141. In another embodiment, the bispecific fusion protein that selectively targets β1,6GlcNAc-branched N-glycans comprises an amino acid sequence selected from SEQ ID NOs: 1-5 and 10-25, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1-25. In some embodiments, the bispecific fusion protein that selectively targets β1,6GlcNAc-branched N-glycans comprises an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, or 19-21, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, or 19-21. In some embodiments, the bispecific fusion protein exhibits enhanced binding to β1,6GlcNAc-branched N-glycan-expressing tumor cells as compared to a bispecific fusion protein that includes 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 SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127, or a linker comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127.

[0235] Another aspect of the present disclosure is an antigen-binding domain selected from the group consisting of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, and 146, or 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: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, or 146, and (ii) an immunocyte recognition domain that specifically binds to a receptor on an immune effector cell, and provides a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA).

[0236] One aspect of the embodiments of the present disclosure is an antigen-binding domain selected from the group consisting of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, and 146, or 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: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, or 146, and an immune cell recognition domain that specifically binds to CD3 on immune effector cells, and provides a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA).

[0237] Another aspect of the present disclosure is a TACA-binding domain selected from the group consisting of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, and 146, or 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: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, or 146, and 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 provides a bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA).

[0238] In some embodiments, the bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA) comprises a TACA binding domain selected from the group consisting of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, and 146, or an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, or 146, and an Fc domain of an antibody. In one embodiment, the domain is the Fc region of an IgG molecule.

[0239] In some embodiments, the bispecific fusion protein that selectively binds to a tumor-associated carbohydrate antigen (TACA) comprises a TACA-binding domain selected from the group consisting of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, and 146, or 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: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152, or 146, and the constant region domains CH2 and / or CH3 of an antibody. In one embodiment, the constant region may be CH2 and CH3. In one embodiment, the CH2 domain and / or CH3 domain comprises a hinge region. In one embodiment, the CH2 domain and / or CH3 domain does not comprise a hinge region. Another aspect of the disclosure provides a bispecific fusion protein encoded by an isolated nucleic acid disclosed herein.

[0240] Generation of E.CAR or Bispecific Fusion Protein The bispecific fusion proteins, CARs or peptides of the present disclosure may be produced using chemical methods. For example, peptides, bispecific fusion proteins or CARs 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 431 A peptide synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

[0241] The peptides, bispecific fusion proteins or CARs of the present disclosure may be synthesized by conventional techniques. For example, peptides or chimeric proteins may be synthesized by chemical synthesis using solid-phase peptide synthesis methods. In these methods, either solid-phase synthesis or liquid-phase synthesis is employed (for example, for solid-phase synthesis techniques, see J. M. Stewart, and J. D. Young, Solid Phase Peptide Synthesis, 2 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 invention may be synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase chemistry by directly incorporating phosphothreonine as an N-fluorenylmethoxycarbonyl-O-benzyl-L-phosphothreonine derivative.

[0242] An N-terminal fusion protein or C-terminal fusion protein comprising a peptide, bispecific fusion protein or CAR of the present disclosure conjugated to another molecule may be prepared by recombinant techniques by fusing the N-terminus or C-terminus of the peptide or chimeric 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 present invention fused to the selected protein or marker protein as described herein. Examples of proteins that can be used in the preparation of the fusion protein include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.

[0243] The peptides or bispecific 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 simultaneous synthesis of overlapping peptides (see U.S. Patent No. 4,708,871).

[0244] In one aspect, the present disclosure provides any form of peptide, bispecific fusion protein or CAR having substantial homology to the peptides, bispecific fusion proteins or CARs disclosed herein. Preferably, a peptide, bispecific fusion protein or CAR that is "substantially homologous" is about 50% homologous to the amino acid sequence of the peptides disclosed herein, more preferably about 70% homologous, even more preferably about 80% homologous, still more preferably about 90% homologous, even more preferably about 95% homologous, and even more preferably about 99% homologous. The peptide, bispecific fusion protein or CAR may alternatively be produced by recombinant means or by cleavage from a longer polypeptide.

[0245] Variants of the peptides, bispecific fusion proteins or CARs according to the present disclosure include (i) those in which one or more of the amino acid residues are replaced with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), and such replaced amino acid residues may or may not be encoded by the genetic code, (ii) those in which one or more modified amino acid residues (e.g., residues modified by attachment of a substituent) 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 is fused to another peptide such as a leader or secretion sequence, or a sequence employed for purification (e.g., His tag) or detection (e.g., Sv5 epitope tag). Fragments include peptides generated by proteolytic cleavage (including multi-site proteolysis) of the original sequence. The variant may be subject to post-translational or chemical modification. Such variants are determined to be within the scope of those skilled in the art according to the teachings herein.

[0246] 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 with the sequence of a second polypeptide. A variant is defined as including a peptide sequence that is different from the original sequence, i.e., preferably less than 40% of the residues per targeted segment are different from the original sequence, more preferably less than 25% of the residues per targeted segment are different from the original sequence, more preferably less than 10% of the residues per targeted segment are different from the original sequence, and most preferably only a very few residues per targeted segment are different 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 invention 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)].

[0247] The bispecific fusion proteins or CARs of the present disclosure can be post-translationally modified. For example, post-translational modifications included within the scope of the present invention include cleavage of 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.

[0248] In some embodiments, the bispecific fusion protein or CAR of the present disclosure may include non-natural amino acids formed by post-translational modification or by introducing non-natural amino acids during translation. Various approaches are available for introducing non-natural amino acids during protein translation.

[0249] The peptides, CARs or bispecific 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, CARs 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).

[0250] Cyclic derivatives of the peptides or bispecific 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 invention by adding the amino acids Pro-Gly in the correct position. It may be desirable to produce a cyclic peptide that is more flexible than a cyclic peptide containing a peptide bond linkage as described above. A more flexible peptide may be prepared by introducing cysteine at the right and left positions of the peptide and forming a disulfide bridge between the two cysteines. The two cysteines are arranged so as not to 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.

[0251] 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 are thus not found together in nature (i.e., are heterologous).

[0252] 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 specific 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 peptide of the present invention to reach a cellular component.

[0253] III. 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 protein comprising an antigen-binding domain that selectively binds a tumor-associated carbohydrate antigen (TACA) and an immune cell recognition domain that specifically binds a receptor on an immune effector cell, wherein the antigen-binding domain comprises a TACA-binding domain derived from a lectin, and the antigen-binding domain comprises more than one (e.g., plural) TACA-binding domains.

[0254] In some embodiments, the antigen-binding domain comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more TACA-binding domains. For example, more than one (e.g., multiple) TACA-binding domains can be operably linked by a linker such as a linker 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. In one embodiment, the linker is a peptide linker. The peptide linker can be a glycine-serine linker. The peptide linker can have a length of at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, or at least about 15 amino acids.

[0255] In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 127, or the amino acid sequence of SEQ ID NO: 131.

[0256] In some embodiments, the lectin is selected from the group consisting of galectin, siglec, selectin; C-type lectin; CD301, polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T), L-PHA (Phaseolus vulgaris leukocyte agglutinin); E-PHA (Phaseolus vulgaris erythroagglutinin); tomato lectin (Lycopersicon esculentum lectin; LEA); peanut lectin (Arachis hypogaea lectin; PNA); potato lectin (Solanum tuberosum lectin), pokeweed mitogen (Phytolacca americana lectin), wheat germ agglutinin (Triticum aestivum germ lectin); Artocarpus polyphyllus lectin (jacalin lectin); hairy bittercress lectin (VVA); apple snail lectin (HPA); fucose lectin (WFA); Sambucus nigra lectin (SNA), BC2L-CNt (lectin from the Gram-negative bacterium Burkholderia cenocepacia), dogwood leukocyte agglutinin (MAL), Pleurotus ostreatus (PVL), Sclerotium rolfsii lectin (SRL), Echinops sphaerocephalus lectin (ESA), CLEC17A (prolectin), Hericium erinaceus lectin, Allium sativum lectin (SSA), Glechoma hederacea lectin (Gleheda), Momordica charantia lectin (Morniga G), Salvia officinalis lectin, Salvia bogotensis lectin, Salvia horminum lectin, Cuscutaceae lectin, Calceolaria integrifolia lectin, Glycine max lectin, Phaseolus angularis lectin, Vicia faba lectin, Amaranthus leucocarpus lectin, Rhamnus autumnalis lectin, Paramignya monophylla lectin, Ipomoea batatas lectin, Himalayan Vicia faba agglutinin, Himalayan Vicia faba lectin, soybean lectin and mushroom lectin.

[0257] 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.

[0258] 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).

[0259] In some embodiments, the antigen-binding domains of the bispecific fusion proteins described herein selectively target TACAs selected from the group consisting of β1,6-branched, β1,6GlcNAc-branched N-glycans, T antigen, Tn antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6-sialylation, sialylation, sialyl-Lewisx / a, disialyl-Lewisx / a, sialyl 6-sulfo Lexis x , Lewis-y (Le y ), Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GM1.

[0260] 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.

[0261] In some embodiments, the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152. Alternatively, the antigen-binding domain comprises an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152. In some embodiments, the antigen-binding domain comprises the amino acid sequence disclosed in Table 2 or Table 3. In some embodiments of the nucleic acid molecules disclosed herein, the antigen-binding domain comprises an amino acid sequence having at least 90% homology with SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146.

[0262] In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, and 63-66. Alternatively, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58, and 63-66. In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 3-5, 10-13, 18-21, 26-34, 39-42, 47-50, 55-58, or 63-66. In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66. In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58, or 64-66. In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein disclosed in Table 2 or Table 3. In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein comprising the amino acid sequence of SEQ ID NOs: 31-34, 39-42, 47-50, 55-58, 63, or 64.

[0263] In some embodiments, the bispecific fusion protein exhibits enhanced binding to Thomsen - nouveau (Tn) antigen (Tn antigen) expressing tumor cells as compared to a bispecific fusion protein that includes a flexible linker in the antigen - binding domain. In those embodiments, the flexible linker is a glycine - serine linker or a linker comprising an amino acid sequence selected from SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127. Alternatively, the flexible linker is a glycine - serine linker or a linker comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127.

[0264] In some embodiments, the isolated nucleic acid molecule encodes a bispecific fusion protein that includes an immunocyte recognition domain that selectively binds to a receptor on an immune effector cell. In those 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 those 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.

[0265] In some embodiments, the immune cell recognition domain of the bispecific fusion protein comprises a peptide, protein, antibody, single domain antibody, nanobody, antibody fragment, or single-chain variable fragment (scFv) that selectively binds to a receptor on immune effector cells. The immune cell 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 immune cell recognition domain specifically binds CD3. Alternatively, the immune cell recognition domain may comprise the amino acid sequence of SEQ ID NO: 149, 150, or 151. In some embodiments, the immune cell recognition domain may comprise an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 149, 150, or 151.

[0266] Alternatively, the immune cell recognition domain comprises an antibody Fc domain and optionally the Fc region of an IgG molecule. In some embodiments, the immune cell recognition domain is an antibody Fc domain and a domain that specifically binds CD3. In one embodiment, the bispecific 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, preferably including CH2 and CH3. The constant region domains CH2 and / or CH3 of the antibody may or may not include a hinge region.

[0267] B. Chimeric Antigen Receptor One aspect of the present disclosure provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), wherein the antigen-binding domain comprises a TACA-binding domain derived from a lectin, and the antigen-binding domain comprises more than one TACA-binding domain, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain. In one embodiment, a first nucleic acid sequence encoding the antigen-binding domain is operably linked to a second nucleic acid sequence encoding the transmembrane domain, and further operably linked to a third nucleic acid sequence encoding the co-stimulatory signaling domain and / or the intracellular signaling domain.

[0268] In some embodiments, the antigen-binding domain comprises two, three, four, five, six, seven, eight, nine, ten, or more TACA-binding domains. For example, more than one (e.g., multiple) TACA-binding domains can be operably linked by a linker such as a linker 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. In one embodiment, the linker is a peptide linker. The peptide linker can be a glycine-serine linker. The peptide linker can have a length of at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, or at least about 15 amino acids.

[0269] In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 127, or the amino acid sequence of SEQ ID NO: 131.

[0270] 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 agglutinin); Artocarpus polyphyllus lectin (jacalin lectin); hairy betel lectin (VVA); apple snail lectin (HPA); fucose lectin (WFA); snowdrop lectin (SNA), BC2L-CNt (lectin from the Gram-negative bacterium Burkholderia cenocepacia), dog kidney leukocyte agglutinin (MAL), oyster mushroom (PVL), Sclerotium rolfsii lectin (SRL), stinging nettle lectin (ESA), CLEC17A (prolectin), shiitake mushroom lectin, Japanese snowdrop lectin (SSA), Glechoma hederacea lectin (Gleheda), Momordica charantia lectin (Morniga G), Salvia officinalis lectin, Salvia bogotensis lectin, Salvia forminaum lectin, heron lectin, Calamintha nepeta lectin, Glyphomia simplificifolia (GsLA4), Vicia angustifolia (acidic WBAI), adzuki bean lectin, honeysuckle lectin, Amaranthus leucocarpus lectin, Relia autumnalis lectin, paramitz lectin, American ivy lectin, Artocarpus lakoocha lectin, Himalayan fava bean lectin, Himalayan fava bean lectin, soybean lectin and mushroom lectin.

[0271] 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.

[0272] 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).

[0273] In some embodiments, the antigen-binding domain of the CARs described herein selectively targets TACAs selected from the group consisting of β1,6-branched, β1,6GlcNAc-branched N-glycans, T antigen, Tn antigen, sialyl-T epitope, Thomsen-nouveau (Tn) epitope (Tn antigen), sialyl-Tn epitope (sialyl-Tn antigen), α2,6-sialylation, sialylation, sialyl-Lewisx / a, disialyl-Lewisx / a, sialyl 6-sulfo Lexis x , Lewis-y (Le y ), Lewis Y, Globo H, GD2, GD3, GM3, and fucosyl GM1.

[0274] 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.

[0275] In some embodiments, the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152. Alternatively, the antigen-binding domain comprises an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152. In some embodiments, the antigen-binding domain comprises the amino acid sequence disclosed in Table 2 or Table 3.

[0276] In some embodiments of the nucleic acid molecules disclosed herein, the antigen-binding domain comprises an amino acid sequence having at least 90% homology with SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 136, 137, 139, 140, 141, 142, 143, 144, 145, 152 or 146.

[0277] In some embodiments, the isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprises a transmembrane domain, which transmembrane domain may comprise the transmembrane region of a molecule selected from the group consisting of T cell receptor (TCR)-alpha, TCR-beta, CD3-zeta, CD3-epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134 (Ox40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In one embodiment, the transmembrane domain comprises the CD8 transmembrane domain. Alternatively, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 148.

[0278] In some embodiments, the isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprises a co-stimulatory domain, which co-stimulatory domain is a co-stimulatory domain of a molecule selected from the group consisting of CD27, CD28, 4-IBB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD8, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, DAP10, DAP12, Lck, Fas, and combinations thereof. In one embodiment, the co-stimulatory domain comprises the 4-1BB co-stimulatory domain or the CD28 co-stimulatory domain. In one embodiment, the co-stimulatory domain comprises the 4-1BB co-stimulatory domain and the CD28 co-stimulatory domain. In one embodiment, the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 114 or SEQ ID NO: 113. In one embodiment, the co-stimulatory domain comprises the amino acid sequences of SEQ ID NO: 114 and SEQ ID NO: 113.

[0279] In some embodiments, the isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprises an intracellular domain, which may be from the intracellular signaling domain of a molecule selected from the group consisting of T cell receptor (TCR) zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, CDS, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the intracellular signaling domain comprises the CD3 zeta signaling domain, or the amino acid sequence of SEQ ID NO: 115.

[0280] In some embodiments, the isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) further comprises a hinge domain. In some embodiments, the hinge domain is a protein selected from the group consisting of CD8α, the Fc fragment of an antibody, the hinge region of an antibody, the CH2 region of an antibody, the CH3 region of an antibody, and an artificial spacer sequence. In one embodiment, the hinge domain is the CD8α hinge domain. In one embodiment, the hinge domain comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the hinge domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 119, SEQ ID NO: 124, 127, 128, 129, 130, 131, 132, and 147.

[0281] One aspect of the present disclosure includes an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising an amino acid sequence set forth in SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. In some embodiments, the CAR comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

[0282] C. Nucleic Acid The isolated nucleic acid sequences encoding the bispecific fusion proteins or chimeric antigen receptors of the present disclosure can be obtained using any of a number of recombinant methods known in the art, examples of which include, for example, screening a library from cells 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.

[0283] The isolated nucleic acid can 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 includes, 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.

[0284] The nucleic acid molecules of the present disclosure can be modified to improve their stability in serum or in growth media for cell culture. Modifications can be added to enhance stability, functionality, and / or specificity, and to minimize the immunostimulatory properties of the nucleic acid molecules of the present disclosure. For example, to enhance stability, the 3' residue may be stabilized against degradation, for example, the 3' residue may be selected such that it is composed of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution with modified analogs of pyrimidine nucleotides, for example, substitution of uridine with 2'-deoxythymidine, is tolerated and does not affect the function of the molecule. In one embodiment of the present disclosure, the nucleic acid molecule may contain at least one modified nucleotide analog. For example, the termini may be stabilized by incorporating modified nucleotide analogs.

[0285] Non-limiting examples of nucleotide analogs include ribonucleotides that are sugar-modified and / or backbone-modified (i.e., including modifications to the phosphate-sugar backbone). For example, the phosphodiester bond of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In preferred backbone-modified ribonucleotides, the phosphate ester group that binds to adjacent ribonucleotides is replaced by a modified group such as a phosphorothioate group. In preferred sugar-modified ribonucleotides, the 2' OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or ON, where R is Ci-Ce alkyl, alkenyl, or alkynyl, and halo is F, CI, Br, or I.

[0286] 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.

[0287] For example, a nucleic acid molecule includes 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 include, for example, 2'-modified ribose units and / or phosphorothioate linkages. For example, the 2'-hydroxyl group (OH) can be modified or replaced with a plurality of different "oxy" substituents or "deoxy" substituents. To improve nuclease resistance, the nucleic acid molecules of the present disclosure can include 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. Also, specific nucleobase modifications such as locked nucleic acid (LNA), ethylene nucleic acid (ENA) (e.g., 2'-4'-ethylene-bridged nucleic acid), and 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modification can be included to improve the binding affinity to the target.

[0288] In one embodiment, the nucleic acid molecule comprises a 2'-modified nucleotide, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamide (2'-O-MA). In one embodiment, the nucleic acid molecule comprises at least one 2'-O-methyl modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule comprise a 2'-O-methyl modification.

[0289] In some embodiments, the nucleic acid molecules of the present disclosure preferably have one or more of the following properties, namely that the nucleic acid agents discussed herein are otherwise unmodified RNA and DNA, as well as RNA and DNA modified, for example, to improve efficacy, and polymers of nucleoside analogs.

[0290] 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, due to the modifications, are not strictly speaking RNAs. Nucleoside analogs are molecules in which the ribophosphate backbone is replaced by a non-ribophosphate construct, which allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to that seen with a ribophosphate backbone (e.g., an uncharged mimic of a ribophosphate backbone). Modifications to the nucleic acids of the present disclosure may be present in one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobase.

[0291] D. Expression Vector In one aspect, the present disclosure provides an expression construct comprising an isolated nucleic acid encoding a bispecific fusion protein and / or CAR disclosed herein. In some embodiments of the present disclosure, the isolated nucleic acid described herein comprises an expression vector and / or in vitro transcribed RNA. In another embodiment, the expression construct comprises an isolated nucleic acid encoding a CAR described herein.

[0292] The expression of natural or synthetic nucleic acids encoding the peptides of the present invention is generally achieved by operably linking a nucleic acid encoding the peptide or a part thereof to a promoter and incorporating the construct into an expression vector. The vectors used are suitable for replication and, optionally, integration into eukaryotic cells. Typical vectors contain transcription terminators and translation terminators, initiation sequences and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0293] 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. Pat. Nos. 5,399,346, 5,580,859, 5,589,466. These patents are hereby incorporated by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0294] The isolated nucleic acids of the present disclosure can be cloned into multiple 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.

[0295] 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).

[0296] 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.

[0297] 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.

[0298] For example, vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow for long-term stable integration of the transgene 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 several 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 directed specifically to one or more types of cells by selecting an appropriate combination of AAV serotype, promoter, and delivery method.

[0299] 2. Regulatory Elements In some embodiments, the vector also includes conventional control elements, which are operably linked to the transgene in a manner that allows for transcription, translation and / or expression in cells transfected with the plasmid vector or in cells infected with the virus produced by the present invention. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation sequences, transcription termination sequences, promoter sequences and enhancer sequences; efficient RNA processing signals such as splicing signals and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, optionally, sequences that enhance the secretion of the encoded product. A number of expression control sequences, including natural, constitutive, inducible and / or tissue-specific promoters, are known in the art and can be utilized.

[0300] Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. Generally, 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 the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased up to 50 bp apart before activity begins to decline. Depending on the promoter, the individual elements appear to be able to function either cooperatively or independently to activate transcription.

[0301] 3. Promoter In some embodiments, the expression construct further comprises a promoter. The promoter may be selected from an EF-lα promoter, a T cell receptor alpha (TRAC) promoter, an interleukin 2 (IL-2) promoter, or a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a Moloney murine leukemia virus (MoMuLV) promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, or a Rous sarcoma virus promoter.

[0302] 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, Moloney murine leukemia virus (MoMuLV) promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and even human gene promoters (examples include, but are not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter). Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of an operably linked polynucleotide sequence when such expression is desired or turn off the expression when it is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0303] 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 located upstream or downstream of the gene it regulates. Also, the enhancer may be made tissue-specific to enhance transcription in specific cell or tissue types. In one embodiment, the vector of the present invention contains one or more enhancers to promote the transcription of genes present within the vector.

[0304] 4. Selectable Marker To evaluate peptide expression, the expression vector introduced into cells may also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the population of cells to be transfected or infected by the viral vector. In other embodiments, the selectable marker may be carried on a separate DNA fragment and used in a co-transfection procedure. Appropriate regulatory sequences may be placed on both the selectable marker and the reporter gene to allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

[0305] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and encodes a polypeptide whose expression appears as some easily detectable property, such as enzyme activity. Expression of the reporter gene is assayed at a suitable time point after the DNA has been introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein (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 a promoter. Such a promoter region may be ligated to the reporter gene and used to evaluate an agent for its ability to regulate promoter-driven expression.

[0306] 5. Combination of CAR and bispecific fusion protein In some embodiments, the expression construct comprises an isolated nucleic acid encoding a CAR polypeptide and a bispecific fusion protein polypeptide. In some embodiments, the isolated nucleic acid molecule encoding the bispecific fusion protein and the isolated nucleic acid molecule encoding the CAR are operably linked by a nucleic acid molecule encoding a self-cleaving 2A peptide. As used herein, "self-cleaving peptide" or "2A peptide" refers to an oligopeptide that enables multiple proteins to be encoded as a polyprotein, which dissociates into constituent proteins during translation. The use of the term "self-cleaving" is not intended to mean a proteolytic cleavage reaction. A variety of self-cleaving peptides or 2A peptides are known to those of skill in the art, including but not limited to peptides found in members of the Picornaviridae family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Theiler's murine encephalomyelitis virus (TaV), and porcine teschovirus-1 (PTV-1), as well as members of the Carioviruses family such as Theiler's virus and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A", "E2A", "P2A", and "T2A", respectively. One of skill in the art will be able to select an appropriate self-cleaving peptide for use in the present invention. In one embodiment, the self-cleaving 2A peptide is selected from P2A, T2A, E2A, or F2A.

[0307] In some embodiments, an isolated nucleic acid molecule encoding a bispecific fusion protein and an isolated nucleic acid molecule encoding a CAR are operably linked by a nucleic acid molecule encoding a linker. The linker used in the present disclosure (e.g., in the context of linking a CAR coding sequence and a bispecific fusion protein coding sequence) enables multiple proteins to be encoded by the same nucleic acid sequence (e.g., a polycistronic or bicistronic sequence), and these multiple proteins are translated as a polyprotein that dissociates into separate protein components. For example, the linker used in the nucleic acids of the present disclosure that include a CAR coding sequence and a bispecific fusion protein coding sequence enables the CAR and the bispecific fusion protein to be translated as a polyprotein that dissociates into separate CAR and bispecific fusion protein components.

[0308] In some embodiments, the linker includes a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, "internal ribosome entry site" or "IRES" refers to an element that leads to cap-independent translation of a gene by facilitating direct internal ribosome entry to the start codon (such as ATG) of a protein coding region. Various internal ribosome entry sites are known to those skilled in the art, including IRESs that can be obtained from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRESs that can be obtained from, for example, cardioviruses, rhinoviruses, aphthoviruses, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV), but are not limited thereto. Those skilled in the art will be able to select an appropriate IRES for use in the present invention.

[0309] IV. Modified Cells One aspect of the present disclosure provides a genetically modified (e.g., engineered) cell that contains and stably expresses the CAR or bispecific fusion protein of the present disclosure. In one aspect, the modified cell contains an isolated nucleic acid encoding a chimeric antigen receptor (CAR), the CAR comprising an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA), a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain. In some embodiments, the modified cell contains a chimeric antigen receptor that selectively binds to a tumor-associated carbohydrate antigen (TACA). In one aspect, the modified cell contains an isolated nucleic acid molecule encoding a bispecific fusion protein comprising an antigen-binding domain that selectively binds to a tumor-associated carbohydrate antigen (TACA) and an immunocyte recognition domain that specifically binds to a receptor on an immune effector cell.

[0310] In some embodiments, the modified cell contains the bispecific fusion protein and / or the CAR described herein. In another embodiment, the modified cell contains an isolated nucleic acid molecule encoding the bispecific fusion protein described herein and / or an isolated nucleic acid molecule encoding the CAR described herein. In another embodiment, the modified cell contains the expression construct described herein.

[0311] In some embodiments, the modified cell contains the bispecific 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. In some embodiments, the modified cell contains the CAR described herein. In another embodiment, the modified cell contains an isolated nucleic acid molecule encoding the CAR described herein. In another embodiment, the modified cell contains the expression construct described herein.

[0312] 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 chimeric antigen receptor (CAR) or a bispecific fusion protein having an affinity for a tumor-associated carbohydrate antigen (TACA). In some embodiments, the genetically modified immune cell (e.g., a T cell) or a progenitor cell thereof of the present invention is a β1,6-branched, β1,6GlcNAc-branched N-glycan, T antigen, Tn antigen, sialyl-T epitope, Tn epitope, sialyl-Tn epitope, α2,6-sialylation, sialylation, sialyl-Lewis x / a , disialyl-Lewis x / a , sialyl 6-sulfo Lexis x , Lewis-y (Le y ), Lewis Y, Globo H, GD2, GD3, GM3 or fucosyl GM1. In some embodiments, the genetically modified immune cell (e.g., a T cell) or a progenitor cell thereof of the present invention contains a CAR or a bispecific fusion protein having an affinity for a β1,6-branched or β1,6GlcNAc-branched N-glycan. In some embodiments, the genetically modified immune cell (e.g., a T cell) or a progenitor cell thereof of the present invention contains a CAR or a bispecific fusion protein having an affinity for a Tn antigen or a sialyl Tn epitope.

[0313] 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 a Chinese hamster ovary (CHO) cell, a baby hamster kidney (BHK) cell, a monkey kidney cell, a HeLa cell, a human hepatocellular carcinoma cell, or a human fetal kidney 293. In one embodiment, the modified cell is a CHO cell or a HEK293 cell.

[0314] A. Modified cell In some embodiments, the modified cell is a T cell, CD4 + T cell, CD8 + T cell, natural killer (NK) cell, cytotoxic T lymphocyte (CTL), and regulatory T cell. 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, an allogeneic cell, or a xenogeneic cell.

[0315] In some embodiments, the modified cells described herein comprise the bispecific fusion protein described herein and the CAR described herein, or a CAR targeting a tumor antigen. In some embodiments, the modified cells described herein comprise an isolated nucleic acid molecule encoding the bispecific fusion protein described herein and an isolated nucleic acid molecule encoding the CAR described herein, or a CAR targeting a tumor antigen. In some embodiments, the modified cells described herein comprise the expression construct described herein and an expression construct comprising a CAR targeting a tumor antigen.

[0316] 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 antigen is TACA.

[0317] In some embodiments, the genetically modified cells are genetically engineered T lymphocytes (T cells), regulatory T cells (Tregs), naive T cells (TN), memory T cells (e.g., central memory T cells (TCM), effector memory cells (TEM)), natural killer cells (NK cells), natural killer T cells (NKT cells), and macrophages capable of giving rise to progeny relevant to the treatment. In some embodiments, the cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. In one embodiment, the cells are T cells. In one embodiment, the modified cells are autologous cells.

[0318] 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, 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 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).

[0319] In one aspect, the present disclosure provides a population of modified immune cells as described herein.

[0320] B. Method for generating modified 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 CAR or bispecific fusion protein, or an expression construct of the present disclosure.

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

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

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

[0324] In certain embodiments, the cell can be any suitable cell type capable of expressing the desired peptide. In certain embodiments, the modified cells are used in the manner in which the cells are introduced into the recipient. In certain embodiments, the cells are autologous, allogeneic, syngeneic or xenogeneic cells to the recipient.

[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 into cells are known in the art. In the context of expression vectors, the vectors can be readily 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] 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.

[0328] 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.

[0329] 1. Physical methods Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. 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.

[0330] 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 lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0331] In some embodiments, the nucleic acid encoding the subject CAR or bispecific fusion protein is introduced into cells by an expression vector. Expression vectors comprising a nucleic acid encoding a subject CAR (e.g., a TACA CAR) or bispecific fusion protein 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.

[0332] The adenovirus expression vector is based on adenovirus, which has a low ability to integrate into genomic DNA but a high efficiency of transfection into host cells. The adenovirus expression vector contains (a) sequences that support the packaging of the expression vector and (b) adenovirus sequences sufficient to ultimately express the target CAR in the host cell. In some embodiments, the adenovirus genome is a 36 kb linear double-stranded DNA, and to produce the expression vector of the present invention, a foreign DNA sequence (e.g., a nucleic acid encoding TACA-CAR or a bispecific fusion protein) may be inserted to replace a large piece of the adenovirus DNA. See, for example, Danthinne and Imperiale, Gene Therapy 7(20):1707-1714(2000).

[0333] Another expression vector is based on adeno-associated virus (AAV) that utilizes an adenovirus coupling system. This AAV expression vector has a high frequency of integration into the host genome. Since it can also infect non-dividing cells, it is useful for delivering genes to mammalian cells, for example, in tissue culture or in vivo. With respect to infectivity, the AAV vector has a broad host range. Details regarding the production and use of AAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0334] Retrovirus expression vectors can be integrated into the host genome, deliver large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged into specific cell lines. Retroviral vectors are constructed by inserting a nucleic acid (e.g., a nucleic acid encoding TACA-CAR or a bispecific fusion protein) at a specific position in the viral genome to produce a replication-defective virus. Retroviral vectors can infect a variety of cell types, but the integration and stable expression of the target CAR or bispecific fusion protein require the division of the host cell.

[0335] Lentiviral vectors are derived from lentiviruses, which are complex retroviruses that contain, in addition to the gag, pol, and env genes that 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 gene transfer and expression, both in vivo and ex vivo, of nucleic acids encoding a subject CAR or bispecific fusion protein (see, for example, U.S. Pat. No. 5,994,136).

[0336] An expression vector containing the nucleic acids of the present disclosure can be introduced into a host cell by any means known to those skilled in the art. The expression vector may optionally contain viral sequences for transfection. Alternatively, the expression vector may be introduced by fusion, electroporation, biolistics, transfection, lipofection, etc. The host cell can be grown and expanded in culture prior to introduction of the expression vector, followed by appropriate treatment for vector introduction and integration. The host cell can then be expanded and screened by a marker present in the vector.

[0337] A variety of markers that can be used are known in the art and include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc. 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.

[0338] 3. Chemical methods Chemical means for introducing polynucleotides into host cells include colloidal dispersions such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems used as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).

[0339] When using non-viral delivery systems, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule associated with both the liposome and the oligonucleotide, incorporated into a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in a micelle or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they may exist as micelles within a bilayer structure or in a "disrupted" structure. They may also simply be dispersed in solution and may form aggregates that are not uniform in size or shape. Lipids are fatty substances and may be naturally occurring lipids or synthetic lipids. For example, lipids include, in addition to lipid droplets that naturally occur within the cytoplasm, a class of compounds that contains long-chain aliphatic hydrocarbons and their derivatives, examples of which include fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0340] 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. “Liposomes” is a general term encompassing various single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of aqueous solution, they form spontaneously. The lipid components undergo self-rearrangement before forming a closed structure and take up water and dissolved solutes between the lipid bilayers. Ghosh et al., Glycobiology 5:505-10 (1991). However, compositions having structures different from 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.

[0341] 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 invention).

[0342] 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.

[0343] 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.

[0344] One of the advantages of the RNA transfection method of the present invention is that RNA transfection is essentially transient and does not require a vector. The RNA transgene can be delivered to lymphocytes as a minimal expression cassette without any additional viral sequences and expressed there after short-term in vitro cell activation. Under these conditions, the probability of the transgene integrating 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.

[0345] 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...

Claims

1. An isolated nucleic acid molecule, (i) A bispecific fusion protein comprising (a) an antigen-binding domain that selectively binds tumor-associated glycosylation antigens (TACAs), and (b) an immune cell recognition domain that specifically binds to receptors on immune effector cells, or (ii) A chimeric antigen receptor (CAR) comprising (a) an antigen-binding domain that selectively binds tumor-associated glycosylation antigens (TACAs), (b) a transmembrane domain, (c) a costimulatory signaling domain, and (d) an intracellular signaling domain. Encode it, The antigen-binding domain includes a lectin-derived TACA-binding domain, and the antigen-binding domain includes more than one TACA-binding domain. The isolated nucleic acid molecule.

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

3. The isolated nucleic acid molecule according to claim 1, wherein the TACA-binding domain is operably linked by a linker.

4. (a) The linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132, or (b) The linker is a peptide linker, or (c) The linker is a glycine-serine linker. The isolated nucleic acid molecule according to claim 3.

5. The antigen-binding domain is an amino acid sequence described in any one of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152, or SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or The 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 any one of the amino acid sequences described in 152, The isolated nucleic acid molecule according to claim 1.

6. The isolated nucleic acid molecule encodes a bispecific fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58 and 63-66, or a bispecific fusion protein comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58 and 63-66. The isolated nucleic acid molecule according to claim 1.

7. The bispecific fusion protein is (a) an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66, or (b) Including the amino acid sequence of SEQ ID NOs. 32-34, 40-42, 48-50, 56-58 or 64, The isolated nucleic acid molecule according to claim 6.

8. The isolated nucleic acid molecule encodes a bispecific fusion protein, (a) The bispecific fusion protein exhibits enhanced binding to Thomsen-nouveau (Tn) antigen-expressing tumor cells compared to a bispecific fusion protein containing a flexible linker in the antigen-binding domain, and / or (b) Compared with a bispecific fusion protein that includes a flexible linker in the antigen-binding domain, the bispecific fusion protein exhibits enhanced binding to β1,6GlcNAc branched N-glycan-expressing tumor cells. The flexible linker is a glycine-serine linker, or a linker comprising an amino acid sequence selected from SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 127, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO:

127. The isolated nucleic acid molecule according to claim 1.

9. The isolated nucleic acid molecule encodes a bispecific fusion protein, and 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. The isolated nucleic acid molecule according to claim 1.

10. The isolated nucleic acid molecule encodes a bispecific fusion protein, (a) The immune cell recognition domain is (i) scFv selectively combines CD3, CD2, CD28, CD25, CD16, NKG2D, NKG2A, CD138, KIR3DL, NKp46, MICA and CEACAM1. (ii) an amino acid sequence selected from sequence numbers 149, 150, or 151, or (iii) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from sequence numbers 149, 150, or 151, or (b) The bispecific fusion protein is an Fc fusion protein comprising an antigen-binding domain that selectively binds tumor-associated glycosylated antigens (TACAs) and an Fc domain. The isolated nucleic acid molecule according to claim 1.

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

12. A bispecific fusion protein that selectively binds tumor-associated glycosylation antigens (TACAs), (i) An amino acid sequence described in any one of sequence numbers 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 and 152, or any one of sequence numbers 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 144, 145, 146 or 152 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 the amino acid sequence described above, (ii) an immune cell recognition domain that specifically binds to a receptor on an immune effector cell, The aforementioned bispecific fusion protein.

13. The bispecific fusion protein is (a) an amino acid sequence selected from SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58 and 63-66, or (b) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1-5, 10-34, 39-42, 47-50, 55-58 and 63-66, or (c) an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66, or (d) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 3-5, 11-13, 19-21, 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66, A bispecific fusion protein according to claim 12, comprising:

14. The bispecific fusion protein is (a) an antigen-binding domain that selectively targets the Tn antigen and has an amino acid sequence selected from SEQ ID NOs: 103-109, 142-146, or 152, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 103-109, 142-146, or 152, or (b) an antigen-binding domain that selectively targets β1,6GlcNAc branched N-glycan and has an amino acid sequence selected from SEQ ID NOs. 100-102 or 133-141, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs. 100-102 or 133-141, The bispecific fusion protein according to claim 12.

15. The bispecific fusion protein that selectively targets the Tn antigen is (a) an amino acid sequence selected from SEQ ID NOs: 26-34, 39-42, 47-50, 55-58 or 63-66, or (b) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 26-34, 39-42, 47-50, 55-58 or 63-66, or (c) an amino acid sequence selected from SEQ ID NOs: 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66, or (d) an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 28-30, 32-34, 40-42, 48-50, 56-58 or 64-66, A bispecific fusion protein according to claim 12, comprising:

16. Modified cells comprising isolated nucleic acids according to any one of claims 1 to 10.

17. The modified cells according to claim 16, wherein the cells are selected from the group consisting of T cells, CD4+ T cells, CD8+ T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells.

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

19. A composition for treating cancer in a subject that needs to be treated for cancer, comprising the modified cells described in claim 16.

20. The 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, or lymphoma.