Engineered immune cells

Abstract

To provide an immune cell capable of antibody-dependent cellular cytotoxicity and comprising a nucleic acid sequence encoding a secreted antigen-binding molecule.SOLUTION: Disclosed are immune cells capable of ADCC that are engineered to secrete an antigen binding molecule (e.g., an antibody or antibody-like protein, e.g., scFv-Fc) that targets an antigen expressed in the tumor microenvironment. The present invention also relates to methods of generating immune cells and medical uses of immune cells. Secretion of the antigen-binding molecule can enhance the anti-cancer effects of the immune cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to immune cells capable of antibody-dependent cellular cytotoxicity and comprising a nucleic acid sequence encoding an antigen-binding molecule. The present invention also relates to methods for producing the immune cells and medical uses of the immune cells. [Background technology]

[0002] Alphabeta (ab) T cells expressing chimeric antigen receptors (CARs) form an important part of the immunotherapy toolkit. By expressing CARs in abT cells, the antigen specificity of the abT cells can be altered. In this way, the subject's adaptive immune system can be reprogrammed to target antigens of particular interest in the subject, such as tumor antigens.

[0003] Recently, strategies to improve the therapeutic potential of CAR abT cells have been developed. In particular, "armored" CAR abT cells have been generated. Armored CAR abT cells have the ability to secrete function-enhancing molecules. For example, CAR abT cells can secrete cytokines that enhance their therapeutic efficacy. CAR abT cells can secrete peptides that inhibit protein kinase A (Newick et al., Cancer Immunol Res, June 2016, 4(6):541-551). CAR abT cells can also secrete molecules that overcome immune checkpoints. For example, Rafiq et al. (Nature Biotechnology, August 13, 2018, Vol. 36(9): pp. 847-856) equipped CAR abT cells with the ability to secrete anti-PDL1 antibodies, and Li et al. (Clinical Cancer Research, November 2017, Vol. 23(22): pp. 6982-6992) engineered CAR abT cells to secrete scFv specific for PD-1 on the effector cell surface. Summary of the Invention [Problem to be solved by the invention]

[0004] In contrast to abT cells, gamma delta (gd) T cells are a relatively overlooked innate-like immune cell subset. Unlike abT cells, gdT cells (especially Vδ2+ gdT cells) are capable of potent antibody-dependent cellular cytotoxicity (ADCC) against antibody-labeled tumor cells. Therefore, gdT cell infiltration into tumors correlates with favorable clinical outcomes, and gdT cells have potential in cancer immunotherapy. Myeloid cells are also capable of ADCC and can be used in cancer immunotherapy. To optimize their therapeutic potential, it is desirable to engineer gdT cells and myeloid cells to secrete molecules that enhance their anticancer activity. [Means for solving the problem]

[0005] The present inventors have demonstrated that immune cells capable of ADCC can be engineered to secrete antigen-binding molecules (e.g., antibodies or antibody-like proteins, e.g., scFv-Fc) that target antigens expressed in the tumor microenvironment. Secretion of the antigen-binding molecules can enhance the anti-cancer activity of the immune cells. For example, the secreted antigen-binding molecules can increase target cell killing by ADCC. This exemplary mechanism is shown in Figure 1, in which scFv-Fc secreted by engineered immune cells (e.g., gdT cells) marks target cells expressing the cognate antigen for ADCC. This allows the engineered immune cells to kill the target cells by ADCC. Bystander, non-engineered immune cells can also exert ADCC against target cells. In this way, the antigen-specific cytotoxicity of the engineered bystander immune cells is improved. Therefore, administration of engineered immune cells to a subject provides improved anti-cancer therapy.

[0006] Thus, the present invention provides: - an immune cell capable of ADCC and comprising a nucleic acid sequence encoding an antigen-binding molecule comprising an antigen-binding region; - a method for producing an immune cell of the present invention, comprising introducing a nucleic acid sequence encoding an antigen-binding molecule into an immune cell; - a method of treating a disease in an individual, the method comprising administering to the individual a therapeutically effective number of immune cells of the invention; and - an immune cell of the invention for use in a method of treating a disease in an individual, the method comprising administering to the individual a therapeutically effective number of the immune cell. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows an example of a proposed mechanism by which scFv-Fc secreted by γδ T cells engages Fc receptors on ADCC-competent cells in the tumor microenvironment. [Figure 2] Figure 1 shows the binding of scFv-Fc to cells expressing target antigens as detected by flow cytometry. CEA+ CAPAN-1 cells or CEA- HELA cells were incubated with supernatant from V52 cells or Jurkat cells secreting anti-CEA scFv-Fc fusion protein. Anti-human Fc was used to detect binding of the scFv-Fc fusion protein. Purified scFv fusion protein was used as a positive control. Similar experiments were performed using supernatant from GD2+ / - SupT1 cells and V52 cells secreting anti-GD2 scFv-Fc fusion protein. In this case, clinical-grade dinutuximab (anti-GD2) was used as a positive control. [Figure 3] FIG. 1 shows the experimental set-up showing the conditions used in the cytotoxicity assay to test the direct and indirect cytotoxic benefits of scFv-Fc fusion protein expression. [Figure 4A-C]Figure 1 shows cytotoxicity in cell contact-dependent and -independent settings. All cytotoxicity experiments were performed at a 1:1 effector:target ratio with 18 h co-culture. Target cells were labeled with CellTrace Violet™ and killing was detected by staining with GhostRed fixable viability dye. Target cell death is shown as all values ​​minus background death (in the absence of effectors). A) Killing of CEA+ CAPAN-1 or CEA- HELA cells by V52 cells secreting anti-CEA scFv-Fc or untransduced V52 cells. B) Killing of CEA+ CAPAN-1 or CEA- HELA cells by untransduced V52 cells in the presence or absence of supernatant from V52 cells secreting anti-CEA-scFv-Fc. C) Killing of CEA+ CAPAN-1 cells or CEA- HELA cells by V52 cells secreting anti-CEA scFv-Fc or non-transduced controls sequestered behind a semipermeable membrane. [Figure 4D-F] Figure 1 shows cytotoxicity in cell contact-dependent and -independent settings. D) Killing of GD2+ SupT1 or GD2-wild-type SupT1 cells by non-transduced V52 cells or secreting anti-GD2 scFv-Fc. E) Killing of GD2+ SupT1 or GD2-wild-type SupT1 cells by non-transduced V52 cells in the presence or absence of supernatant from V52 cells secreting anti-GD2-scFv-Fc. F) Killing of GD2+ SupT1 or GD2-wild-type SupT1 cells by non-transduced V52 cells sequestered behind a semi-permeable membrane with V52 cells secreting anti-GD2 scFv-Fc or a non-transduced control. [Figure 5] FIG. 1 shows IFNγ concentrations in the supernatant after 18 hours of co-culture of CEA+ CAPAN-1 or CEA- HELA cells with non-transduced V52 cells, where anti-CEA scFv-Fc-secreting V52 cells or a non-transduced control were sequestered behind a semi-permeable membrane. [Figure 6]Figure 1 shows the binding of anti-GD2 antibody (SEQ ID NO: 17) produced by 293T cells to GD2+ / - target cells as detected by flow cytometry. 293T cells were treated with decreasing volumes of lentivirus encoding the whole anti-GD2 antibody 14G2a. Isogenic SupT1_wt(GD2-) or Sup T1_GD2(GD2+) was incubated with supernatants from transduced 293T cells. Antibody binding was detected using a PE-conjugated anti-human Fc antibody. A pure anti-GD2 antibody (dinutuximab, ch14.18, clone 14G2a) was used as a positive control. Antibodies were detected in the supernatants of transduced 293T cells at levels dependent on the amount of virus applied. [Figure 7] Figure 1 shows binding of antibodies produced by γδ T cells to cells expressing the target antigen. Isogenic SupT1_wt(GD2-) or Sup T1_GD2(GD2+) cells were incubated with supernatant from transduced V52 to express whole anti-GD2 antibody (clone 14G2a). Antibody binding was detected using an anti-human IgG secondary antibody; pure anti-GD2 (dinutuximab, ch14.18, clone 14G2a) was used as a positive control. [Figure 8A] Figure 1 shows cytotoxicity in cell contact-dependent and -independent settings. All cytotoxicity experiments were performed at a 1:1 effector:target ratio with 18 h co-culture. Target cells were labeled with CellTrace Violet™ and death was detected by staining with Live / Dead Blue fixable viability dye (detected on the DAPI channel). Live / Dead Blue staining of target cells is shown and the percentage of dead cells is indicated. A) Experimental setup showing how direct bystander cytotoxicity was determined. [Figure 8B] Cytotoxicity in cell contact-dependent and -independent settings. B) Killing of GD2+ SupT1 or GD2-wild-type SupT1 cells by cells secreting anti-GD2 antibodies or non-transduced V52 cells. [Figure 8C]Cytotoxicity in cell contact-dependent and -independent settings. C) Killing of GD2+ SupT1 or GD2-wildtype SupT1 cells by non-transduced V52 cells in the presence or absence of supernatant from V52 cells secreting anti-GD2 antibodies. DETAILED DESCRIPTION OF THE INVENTION

[0008] Sequence Listing Description SEQ ID NO: 1 is the V of the CEA-specific scFv used in the examples H Provides an array of domains. SEQ ID NO: 2 is the V of the CEA-specific scFv used in the examples L Provides an array of domains. SEQ ID NO: 3 provides the sequence of the CEA-specific scFv-Fc used in the examples. SEQ ID NO: 4 is the V of the GD2-specific scFv used in the examples H Provides an array of domains. SEQ ID NO: 5 is the V of the GD2-specific scFv used in the examples L Provides an array of domains. SEQ ID NO: 6 provides the sequence of the GD2-specific scFv-Fc used in the examples. SEQ ID NO: 7 provides the sequence of the CEA-specific scFv used in the examples. SEQ ID NO: 8 provides the sequence of the GD2-specific scFv used in the examples. SEQ ID NO: 9 is the V of the B7H3-specific scFv H Provides an array of domains. SEQ ID NO: 10 is the V of the B7H3-specific scFv L Provides an array of domains. SEQ ID NO: 11 provides the sequence of the B7H3-specific scFv-Fc. SEQ ID NO: 12 provides the sequence of the B7H3-specific scFv. SEQ ID NO: 13 is the V of the CD20-specific scFv H Provides an array of domains. SEQ ID NO: 14 is the V of the CD20-specific scFv L Provides an array of domains. SEQ ID NO: 15 provides the sequence of the CD20-specific scFv-Fc. SEQ ID NO: 16 provides the sequence of a CD20-specific scFv. SEQ ID NO: 17 provides the sequence of the GD2-specific IgG1 used in the examples, with the cleavage site between the light and heavy chains. SEQ ID NO:18 provides the sequence of the light chain of GD2 IgG1 of SEQ ID NO:17. SEQ ID NO: 19 provides the sequence of the cleavage sequence (Furin-V5-SG-P2A) of GD2 IgG1 of SEQ ID NO: 17. SEQ ID NO:20 provides the sequence of the heavy chain of GD2 IgG1 of SEQ ID NO:17.

[0009] Detailed Description of the Invention It is to be understood that different applications of the disclosed products and methods may be tailored to the particular needs of the art, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0010] Furthermore, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid" includes "nucleic acids," reference to "scFv-Fc" includes two or more such scFv-Fc, reference to "a T cell" includes two or more such T cells, etc.

[0011] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0012] immune cells The present invention provides immune cells capable of ADCC and comprising a nucleic acid sequence encoding an antigen-binding molecule. The immune cells can be any immune cells capable of ADCC. Immune cells capable of ADCC are known in the art.

[0013] ADCC is a well-known mechanism of adaptive cellular immunity. During ADCC, immune effector cells actively lyse target cells whose surface antigens are bound by specific antibodies. Specifically, antibodies bind to their cognate antigens on the surface of target cells. Fc receptors present on the surface of immune effector cells recognize the Fc region of the bound antibody. Cross-linking of Fc receptors induces the formation of a lytic synapse between the immune effector cell and the target cell, causing the immune effector cell to degranulate lytic granules. Thus, apoptosis of the target cell is induced. Immune effector cells capable of ADCC are known to include natural killer (NK) cells, macrophages, neutrophils, and eosinophils. gdT cells are also capable of ADCC.

[0014] The immune cells can be from any species, for example, human, dog, cat, mouse, rat, pig, sheep, cow, goat, or horse. The immune cells are typically human immune cells. The immune cells can be dog, cat, mouse, pig, sheep, goat, cow, or horse immune cells.

[0015] Preferably, the immune cells are not abT cells. abT cells are T cells that have a T cell receptor (TCR) containing an alpha chain and a beta chain. They are usually activated in an MHC-dependent manner. ADCC has not been reported for abT cells. abT cells are often considered "conventional" T cells.

[0016] The immune cells can be gdT cells. gdT cells are T cells that have a gdT cell receptor (TCR) on their surface. That is, gdT cells have a TCR that includes a gamma chain and a delta chain. Thus, gdT cells are structurally distinct from abT cells. gdT cells are also functionally distinct from abT cells. In particular, gdT cells are capable of ADCC. gdT cells are typically activated in an MHC-independent manner. gdT cells are often considered "non-conventional" T cells.

[0017] There are several subsets of gdT cells. For example, gdT cells can be V52+ gdT cells, V51+ gdT cells, or V51- / V52- gdT cells. Preferably, gdT cells are V52+ gdT cells. V52+ gdT cells, V51+ gdT cells, and V51- / V52- T cells all have excellent ADCC capabilities and exhibit good anti-tumor cytotoxicity.

[0018] Methods for expanding gdT cells are known in the art. For example, gdT cells can be expanded by culturing them in the presence of IL-2 and zoledronic acid (Fisher J et al., Effective combination treatment using anti-GD2 ch14.18 / CHO antibody with V52+ γδT cells in Ewing sarcoma and neuroblastoma. Oncoimmunology 2015 Apr. 27; Vol. 5(1): e1025194). Thus, gdT cells are readily available for use in the present invention.

[0019] The immune cells can be bone marrow cells. Bone marrow cells are cells derived from common myeloid precursor cells, such as platelets, erythrocytes, mast cells, macrophages, basophils, neutrophils, and eosinophils. ADCC has been reported for macrophages, basophils, neutrophils, and eosinophils. Therefore, preferably, the bone marrow cells are macrophages, basophils, neutrophils, or eosinophils. Methods for isolating and expanding bone marrow cells are known in the art.

[0020] Natural killer (NK) cells are also capable of ADCC. The immune cells can be NK cells. NK cells are a class of innate immune lymphocytes that play a role in immunity against various diseases. For example, NK cells play a role in detecting and controlling cancer and killing virus-infected cells. Methods for isolating and expanding NK cells are known in the art.

[0021] Preferably, the immune cells do not express a chimeric antigen receptor (CAR), and therefore preferably the immune cells are not CAR T cells.

[0022] Nucleic acid sequence The immune cells of the present invention comprise a nucleic acid sequence encoding an antigen-binding molecule. The nucleic acid sequence may comprise DNA. The nucleic acid sequence may comprise RNA. The nucleic acid sequence may comprise DNA and RNA.

[0023] An immune cell may contain one or more nucleic acid sequences, each encoding an antigen-binding molecule. For example, an immune cell may contain two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, or twenty or more nucleic acid sequences, each encoding an antigen-binding molecule. When an immune cell contains multiple nucleic acid sequences, each encoding an antigen-binding molecule, the antigen-binding molecules encoded by each nucleic acid sequence may be the same or different. Preferably, each of the antigen-binding molecules is different. Preferably, each of the antigen-binding molecules is specific for a different antigen. Preferably, the antigen is a tumor antigen. The antigen may be expressed on or by cancer cells. The antigen may be expressed on or by non-cancer cells in the tumor microenvironment. The antigen may be secreted into the tumor microenvironment.

[0024] Each nucleic acid sequence may encode one or more antigen-binding molecules. For example, the nucleic acid sequence may encode two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, or twenty or more antigen-binding molecules. When a nucleic acid sequence encodes multiple antigen-binding molecules, each of the antigen-binding molecules can be the same or different. Preferably, each of the antigen-binding molecules is different. Preferably, each of the antigen-binding molecules is specific for a different antigen. Preferably, the antigen is a tumor antigen. The antigen may be expressed on or by cancer cells. The antigen may be expressed on or by non-cancer cells in the tumor microenvironment. The antigen may be secreted into the tumor microenvironment.

[0025] The nucleic acid sequence may comprise an exogenous promoter sequence to which the sequence encoding the antigen-binding molecule is operably linked. The exogenous promoter may be an inducible promoter. Alternatively, the nucleic acid sequence may lack an exogenous promoter sequence. In this case, the nucleic acid sequence may be integrated into the genome of an immune cell such that expression of the antigen-binding molecule is controlled by an endogenous promoter in the genome. Activation of the exogenous or endogenous promoter may be controlled by an inducible signaling pathway. For example, the exogenous or endogenous promoter may be activated upon engagement of a synNotch receptor with a cognate antigen.

[0026] The nucleic acid sequence can be integrated into the genome of the immune cell. Alternatively, the nucleic acid sequence cannot be integrated into the genome of the immune cell. If the nucleic acid sequence is not integrated into the genome of the immune cell, it can be contained in a plasmid, vector, or artificial chromosome. The vector can be a viral vector or a non-viral vector. The artificial chromosome can be a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a human artificial chromosome (HAC). Preferably, the artificial chromosome is a HAC.

[0027] antigen binding molecule The immune cells of the present invention comprise a nucleic acid sequence encoding an antigen-binding molecule.

[0028] An antigen-binding molecule comprises an antigen-binding region. An antigen-binding region is a region of an antigen-binding molecule that can specifically bind to one or more antigens. For example, an antigen-binding region may be capable of specifically binding to two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more different antigens. Exemplary antigen-binding regions are known in the art and include at least scFv (single-chain variable fragment), Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, dAb, Fd, dsFv, ds-scFv, scFv2, bispecific T-cell engagers, nanobodies, DARPins, antibody mimetics, diabodies, triabodies, and tetrabodies. Thus, the antibody binding region can comprise an scFv, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, dAb, Fd, dsFv, ds-scFv, scFv2, bispecific T-cell engager, nanobody, DARPin, antibody mimetic, diabody, triabody, or tetrabody, alone or in any combination. The antigen-binding molecule can comprise an scFv, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, dAb, Fd, dsFv, ds-scFv, scFv2, bispecific T-cell engager, nanobody, DARPin, antibody mimetic, diabody, triabody, tetrabody, or polypeptide ligand of a receptor expressed on the surface of a cell targeted by an immune cell, alone or in any combination.

[0029] Preferably, the antigen-binding molecule comprises an antigen-binding region comprising an scFv. The antigen-binding region may comprise two or more, for example, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more scFvs. scFvs are known in the art. An scFv is a molecule comprising a variable region (V) of an antibody light chain. L ) linked to the variable region of the heavy chain (V H ) is a fusion protein containing H and V Lare connected by a linker peptide. The linker peptide can be about 5 to about 30 amino acids in length. For example, the linker peptide is about 6 to about 29, about 7 to about 28, about 8 to about 27, about 9 to about 26, about 10 to about 25, about 11 to about 24, about 12 to about 23, about 13 to about 22, about 14 to about 21, about 15 to about 20, about 16 to about 19, about 17, or about 18 amino acids in length.

[0030] The antigen-binding molecule can bind to an Fc receptor. For example, the antigen-binding molecule can comprise an Fc (fragment crystallizable) region. Fc regions are known in the art. The Fc region is the tail region of an antibody that interacts with Fc receptors and several proteins of the complement system. This property allows the antibody to activate the immune system. The Fc region comprises at least two heavy chain constant (CH) domains. Specifically, in an Fc domain derived from an IgG, IgA, or IgD antibody, the Fc region comprises the CH2 and CH3 domains of the antibody. In an Fc region derived from an IgM or IgE antibody, the Fc region comprises the CH2, CH3, and CH4 domains of the antibody. The Fc region can be a modified Fc region. For example, the Fc region can be an Fc region modified to optimize its ability to bind to FcR. Such optimization is known in the art and is described, for example, in (i) Mossner E et al., Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity. Blood. 2010;115(22):4393-4402; and (ii) Wang et al., 2018, IgG Fc engineering to modulate antibody effector functions. Protein Cell. 2018 January;9(1):63-73. doi:10.1007 / s13238-017-0473-8 (references contained therein).

[0031] An antigen-binding molecule may be capable of binding to an Fc receptor via a region other than the Fc region. That is, an antigen-binding molecule does not need to contain an Fc region to be able to bind to an Fc receptor. For example, an antigen-binding molecule may contain an antigen-binding region capable of binding to an Fc receptor (e.g., scFv, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, dAb, Fd, dsFv, ds-scFv, scFv2, bispecific T cell engager, nanobody, DARPin, antibody mimetic, diabody, triabody, tetrabody, or polypeptide ligand of a receptor expressed on the surface of a cell targeted by an immune cell). An antigen-binding molecule may contain an antigen-binding region capable of binding to an Fc receptor and an antigen-binding region capable of binding to a different antigen, for example, an antigen expressed in the tumor microenvironment. For example, an antigen-binding molecule may contain an scFv capable of binding to an Fc receptor and an scFv capable of binding to a different antigen, for example, an antigen expressed in the tumor microenvironment. The antigen binding molecule can comprise a bispecific T cell engager comprising an scFv capable of binding to CD3 and an scFv capable of binding to a different antigen, for example, an antigen expressed in the tumor microenvironment.

[0032] The antigen-binding molecule may comprise an antigen-binding region and an Fc region. The antigen-binding molecule may be an antibody, scFv-Fc, dAb-Fc, or heavy-chain antibody. Exemplary heavy-chain antibodies include IgNAR and camelid antibodies. The antibody may comprise, for example, (a) a light chain encoded by SEQ ID NO: 18, (b) a heavy chain encoded by SEQ ID NO: 20, and / or (c) a truncated sequence encoded by SEQ ID NO: 19. The antibody molecule may comprise, for example, (a); (b); (c); (a) and (b); (a) and (c); (b) and (c); or (a), (b) and (c). The antibody may be encoded by SEQ ID NO: 17. The light chain encoded by SEQ ID NO: 18 may comprise a V chain encoded by SEQ ID NO: 5. L The heavy chain encoded by SEQ ID NO:20 comprises the VH Includes.

[0033] Preferably, the antigen-binding molecule is an scFv-Fc. An scFv-Fc is a fusion protein comprising an scFv fused to an Fc region. The structures of scFvs and Fc regions are known in the art and are described above. According to these structures, an scFv-Fc is a fusion protein comprising a V H and V L scFv-Fc can comprise a V domain, a CH2 domain, and a CH3 domain, which together form the Fc region. H and V L The scFv may comprise a V domain (together forming an scFv), and a CH2 domain, a CH3 domain, and a CH4 domain (together forming an Fc region). In an scFv-Fc, the scFv is linked to the Fc region. Preferably, the V domain in the scFv L or V H is linked to CH2 in the Fc region to link the scFv to the Fc region. The scFv can be linked directly to the Fc region, i.e., without a linker. The scFv can be linked to the Fc region via a linker. The linker can be (Ser(Gly)4), (Ser(Gly)4)2, (Ser(Gly)4)3, (Ser(Gly)4)4, or (Ser(Gly)4)5. The linker can be an amino acid consisting of about two amino acids or a short oligopeptide. The linker can form a hinge region. The scFv-Fc can be a bivalent scFv-Fc. A bivalent scFv-Fc contains two different scFvs, each linked to an Fc region. Essentially, a bivalent scFv-Fc contains two arms, each containing an scFv linked to an Fc region. scFvs, Fc regions, and linkages have been discussed above. The two arms are preferably linked. The link between the two arms preferably connects the linker between the scFv region and the Fc region in one arm to the linker between the scFv region and the Fc region in the other arm. The two arms are preferably connected at a point between the scFv and Fc regions in each arm.

[0034] Antigen-binding molecules can function to increase the cytotoxicity of target cells. That is, antigen-binding molecules can increase the killing of cells expressing the cognate antigen of the antigen-binding region. For example, antigen-binding molecules can increase the killing of tumor cells, endothelial cells, and / or immune cells. Increased killing can be mediated by engineered immune cells (i.e., immune cells containing a nucleic acid sequence encoding the antigen-binding molecule). Increased killing can be mediated by non-engineered ("bystander") immune cells (i.e., immune cells not containing a nucleic acid sequence encoding the antigen-binding molecule). Increased killing can be mediated by both engineered and non-engineered immune cells. Increased killing can be by any mechanism known in the art. Preferably, increased killing is mediated by increased ADCC. Increased killing can be mediated by increased engagement of AB T cells.

[0035] The antigen-binding molecule is preferably an opsonin. An opsonin is a molecule that binds to an antigen and enhances phagocytosis. Binding of an opsonin to an antigen promotes the interaction of the antigen with a cell surface receptor on an immune cell, thereby promoting the dynamics of phagocytosis. Therefore, an antigen-binding molecule can enhance phagocytosis. In particular, an antigen-binding molecule can enhance the phagocytosis of an antigen for which the antigen-binding region is specific. In other words, an antigen-binding molecule can enhance the phagocytosis of an antigen bound to the antigen-binding region (and therefore the antigen-binding molecule).

[0036] The antigen-binding region (and antigen-binding molecule) may be capable of binding to any antigen. That is, any antigen can be bound to the antigen-binding region (and antigen-binding molecule). Preferably, the antigen-binding region (and antigen-binding molecule) can bind to an antigen expressed on or by cells in the tumor microenvironment. An antigen is expressed in the tumor microenvironment if it is expressed by any type of cell present in the tumor microenvironment. For example, the antigen may be expressed by tumor cells, endothelial cells, or immune cells in the tumor microenvironment. Thus, the antigen-binding region (and antigen-binding molecule) can bind to a tumor antigen, an endothelial antigen, or an immune cell antigen. The immune cell may be, for example, a CD4+ T cell, a CD8+ T cell, a gdT cell, a B cell, a NK cell, a NKT cell, a macrophage, a monocyte, a basophil, an eosinophil, or a neutrophil.

[0037] Antigen-binding regions (and antigen-binding molecules) include TSHR, CD19, CD123, CD22, CD20, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7-H3, KIT, IL-13Ra2, mesothelin, IL-IIRa, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, and bcr-ab. l, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OYTES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut It may be capable of binding to an antigen selected from the group consisting of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, CEA, LINGO1, CD70, IL13Rα2, MUC-16, PSCA, ROR1, and IGLL1.

[0038] The antigen-binding region (and antigen-binding molecule) may be capable of binding to carcinoembryonic antigen (CEA). The CEA may be CEA-CAM5. CEA may be expressed in a number of cancers, including colon and / or rectal cancer, gastric cancer, breast cancer, pancreatic cancer, lung cancer, thyroid cancer, cervical cancer, or ovarian cancer.

[0039] The CEA-specific antigen-binding region (and antigen-binding molecule) is V encoded by SEQ ID NO: 1. H The CEA-specific antigen-binding region (and antigen-binding molecule) may comprise the V domain encoded by SEQ ID NO:2. L The CEA-specific antigen-binding region (and antigen-binding molecule) may comprise the V domain encoded by SEQ ID NO: 1. H Domain and V encoded by SEQ ID NO:2 L The CEA-specific antigen-binding region (and antigen-binding molecule) may comprise the V domain encoded by SEQ ID NO: 1. H Domain and / or V encoded by SEQ ID NO:2 L The CEA-specific antigen-binding region (and antigen-binding molecule) may comprise a complementarity-determining region (CDR) derived from the V domain encoded by SEQ ID NO: 1. H The CEA-specific antigen-binding region (and antigen-binding molecule) may comprise heavy chain CDR1, CDR2 and / or CDR3 from the V domain encoded by SEQ ID NO:2. L The CEA-specific antigen-binding region (and antigen-binding molecule) may comprise a light chain CDR1, CDR2 and / or CDR3 derived from the V domain encoded by SEQ ID NO: 1. H Heavy chain CDR1, CDR2 and / or CDR3 from the domain and V encoded by SEQ ID NO:2 L The CEA-specific antigen-binding molecule may comprise a light chain CDR1, CDR2, and / or CDR3 derived from the domain. The CEA-specific antigen-binding molecule may be an scFv-Fc encoded by SEQ ID NO: 3. The scFv may have the amino acid sequence of SEQ ID NO: 7.

[0040] The antigen-binding region (and antigen-binding molecule) can bind to GD2, which can be expressed by cancers of neuroectodermal origin, such as neuroblastoma and melanoma.

[0041] The GD2-specific antigen-binding region (and antigen-binding molecule) is V encoded by SEQ ID NO:4. H The GD2-specific antigen-binding region (and antigen-binding molecule) may comprise the V domain encoded by SEQ ID NO:5. L The GD2-specific scFv-Fc antigen-binding region (and antigen-binding molecule) may comprise the V domain encoded by SEQ ID NO:4. H Domain and V encoded by SEQ ID NO:5 L The GD2-specific antigen-binding region (and antigen-binding molecule) may comprise the V domain encoded by SEQ ID NO:4. H CDRs from the domain and / or V encoded by SEQ ID NO:5 L That is, the GD2 antigen-binding region (and antigen-binding molecule) may comprise CDRs from the V domain encoded by SEQ ID NO:4. H The GD2-specific antigen-binding region (and antigen-binding molecule) may comprise heavy chain CDR1, CDR2, and / or CDR3 from the V domain encoded by SEQ ID NO:5. L The GD2-specific antigen-binding region (and antigen-binding molecule) may comprise a light chain CDR1, CDR2, and / or CDR3 derived from the V domain encoded by SEQ ID NO:4. H Heavy chain CDR1, CDR2 and / or CDR3 from the domain and V encoded by SEQ ID NO:5 L The GD2-specific antigen-binding molecule may comprise a light chain CDR1, CDR2, and / or CDR3 derived from the GD2 domain. The GD2-specific antigen-binding molecule may be an scFv-Fc encoded by SEQ ID NO: 6. The scFv may have the amino acid sequence of SEQ ID NO: 8.

[0042] A GD2-specific antigen-binding molecule may comprise (a) a light chain encoded by SEQ ID NO: 18, (b) a heavy chain encoded by SEQ ID NO: 20, and / or (c) a cleavage sequence. A GD2-specific antigen-binding molecule may comprise, for example, (a); (b); (c); (a) and (b); (a) and (c); (b) and (c); or (a), (b) and (c). Preferably, a GD2-specific antigen-binding molecule comprises (a), (b), and (c) in that order. In any of the embodiments described herein, the cleavage sequence (c) may be, for example, Furin-V5-SG-P2A. The cleavage sequence may be, for example, encoded by SEQ ID NO: 19. Other cleavage sequences are also known in the art and can be used as the cleavage sequence (c). For example, the cleavage sequence (c) may comprise or consist of P2A, E2A, F2A, or T2A. An IRES or IRES2 sequence can be used in place of the cleavage sequence (c) in any of the embodiments described herein. The GD2-specific antigen-binding molecule can be an antibody encoded by SEQ ID NO: 17.

[0043] The antigen-binding region (and antigen-binding molecule) can bind to B7-H3, which can be expressed by cancers of neuroectodermal origin, such as neuroblastoma and melanoma.

[0044] The B7-H3 specific antigen-binding region (and antigen-binding molecule) is V encoded by SEQ ID NO:9. H The B7-H3-specific antigen-binding region (and antigen-binding molecule) may comprise the V domain encoded by SEQ ID NO: 10. L The B7-H3 specific antigen-binding region (and antigen-binding molecule) may comprise the V domain encoded by SEQ ID NO:9. H Domain and V encoded by SEQ ID NO: 10 L The B7-H3 specific antigen-binding region (and antigen-binding molecule) may comprise the V domain encoded by SEQ ID NO:9. H CDRs from the domain and / or V encoded by SEQ ID NO: 10 LThat is, the B7-H3-specific antigen-binding region (and antigen-binding molecule) may comprise CDRs from the V domain encoded by SEQ ID NO:9. H The B7-H3 specific antigen-binding region (and antigen-binding molecule) may comprise heavy chain CDR1, CDR2 and / or CDR3 from the V domain encoded by SEQ ID NO: 10. L The B7-H3 specific antigen-binding region (and antigen-binding molecule) may comprise a light chain CDR1, CDR2 and / or CDR3 from the V domain encoded by SEQ ID NO:9. H Heavy chain CDR1, CDR2 and / or CDR3 from the domain and V encoded by SEQ ID NO: 10 L The B7-H3-specific antigen-binding molecule may comprise a light chain CDR1, CDR2, and / or CDR3 derived from the domain. The B7-H3-specific antigen-binding molecule may be an scFv-Fc encoded by SEQ ID NO: 11. The scFv may have the amino acid sequence of SEQ ID NO: 12.

[0045] The antigen-binding region (and antigen-binding molecule) may be capable of binding to CD20. CD20 is expressed during B cell development, from memory cells to the late pro-B cell stage (but not early pro-B cells or plasmablasts and plasma cells). CD20 is also expressed in B cell lymphoma, hairy cell leukemia, B cell chronic lymphocytic leukemia, and melanoma cancer stem cells.

[0046] The CD20-specific antigen-binding region (and antigen-binding molecule) is V encoded by SEQ ID NO: 13. H The CD20-specific antigen-binding region (and antigen-binding molecule) may comprise the V domain encoded by SEQ ID NO: 14. L The CD20-specific antigen-binding region (and antigen-binding molecule) may comprise the V domain encoded by SEQ ID NO: 13. H Domain and V encoded by SEQ ID NO: 14 L The CD20-specific antigen-binding region (and antigen-binding molecule) may comprise the V domain encoded by SEQ ID NO: 13. H CDRs from the domain and / or V encoded by SEQ ID NO: 14 LThat is, the CD20-specific antigen-binding region (and antigen-binding molecule) may comprise CDRs from the V domain encoded by SEQ ID NO: 13. H The CD20-specific antigen-binding region (and antigen-binding molecule) may comprise heavy chain CDR1, CDR2 and / or CDR3 from the V domain encoded by SEQ ID NO: 14. L The CD20-specific antigen-binding region (and antigen-binding molecule) may comprise a light chain CDR1, CDR2, and / or CDR3 derived from the V domain encoded by SEQ ID NO: 13. H Heavy chain CDR1, CDR2 and / or CDR3 from the domain and V encoded by SEQ ID NO: 14 L The CD20-specific antigen-binding molecule may comprise a light chain CDR1, CDR2, and / or CDR3 derived from the domain. The CD20-specific antigen-binding molecule may be an scFv-Fc encoded by SEQ ID NO: 15. The scFv may have the amino acid sequence of SEQ ID NO: 16.

[0047] The antigen-binding molecule is preferably expressed by immune cells containing a nucleic acid sequence encoding the antigen-binding molecule. The expression of the antigen-binding molecule can be determined based on the presence of mRNA encoding the antigen-binding molecule in the immune cells. Preferably, the expression of the antigen-binding molecule is determined based on the presence of the antigen-binding molecule itself in the immune cells. Methods for determining the presence of mRNA or protein in cells are well known in the art. For example, the presence of mRNA in cells can be determined using reverse transcriptase PCR or Northern blotting. The presence of protein in cells can be determined using flow cytometry, immunofluorescence imaging, or Western blotting.

[0048] How to generate immune cells The present invention provides a method for producing immune cells of the present invention. The method comprises introducing a nucleic acid sequence encoding an antigen-binding molecule into immune cells. Antigen-binding molecules are described in detail above. The nucleic acid sequence can be introduced into immune cells, for example, by transduction or transfection. For example, gdT cells can be transduced with the nucleic acid sequence using T cell transduction methods known in the art. Prior to such transduction, gdT cells can be expanded, for example, by culturing them in the presence of IL-2 and zoledronic acid for about 5 days (e.g., about 3 days, about 4 days, about 6 days, or about 7 days).

[0049] The term "transduction" can be used to describe viral-mediated nucleic acid transfer. Viral vectors can be used to transduce cells with nucleic acid sequences. Thus, the nucleic acid sequence can be contained in a viral vector. The viral vector can be a retroviral, lentiviral, adenoviral, adeno-associated (AAV) or herpes simplex virus (HSV) vector. Preferably, the viral vector is a retroviral vector. Methods for producing and purifying such vectors are known in the art. Immune cells can be transduced using any method known in the art. Transduction can be in vitro or ex vivo.

[0050] The term "transfection" can be used to describe non-viral-mediated nucleic acid transfer. Immune cells can be transfected using any method known in the art. Transfection can be in vitro or ex vivo. Any vector capable of transfecting immune cells can be used, such as conventional plasmid DNA or RNA transfection. Human artificial chromosomes and / or naked RNA and / or siRNA can be used to transfect cells with nucleic acid sequences. Human artificial chromosomes are described, for example, in Mol. Ther. 19(9):1591-1601 (2011) and Kouprina et al., Expert Opinion on Drug Delivery 11(4):517-535 (2014). Alternative non-viral delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles, such as liposomes. Non-viral methods for nucleic acid delivery include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced uptake of DNA. Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those described in Felgner, WO91 / 17424; WO91 / 16024.The preparation of targeted liposomes, e.g., lipid:nucleic acid complexes, including immunolipid complexes, is well known to those of skill in the art (e.g., Crystal Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0051] Cells can be transfected with nucleic acid sequences or nucleic acid constructs using nanoparticle delivery systems, including but not limited to lipid-based systems, liposomes, micelles, microvesicles, and exosomes. For nanoparticles capable of delivering RNA, see, e.g., Alabi et al., Proc Natl Acad Sci U S A. 2013 Aug. 6; 110(32):12881-6; Zhang et al., Adv Mater. 2013 Sep. 6; 25(33):4641-5; Jiang et al., Nano Lett. 2013 Mar. 13; 13(3):1059-64; Karagiannis et al., ACS Nano. 2012 Oct. 23; 6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug. 28; 6(8):6922-9; and Lee et al., Nat Nanotechnol. 2012 Jun. 3; 7(6):389-93. Lipid nanoparticles, spherical nucleic acid (SNA™) constructs, nanoplexes and other nanoparticles (particularly gold nanoparticles) are also contemplated in the present invention as delivery vehicles for constructs or vectors.

[0052] Uptake of nucleic acid constructs can be enhanced by several known transfection techniques, such as those involving the use of transfection agents. Examples of these agents include cationic agents, such as calcium phosphate and DEAE-dextran, and lipofectants, such as lipofectamine, fugene, and transfectam.

[0053] Immune cells can be transfected under appropriate conditions. Immune cells can be contacted with the drug or vector for, for example, 5 minutes to 10 days, preferably 1 hour to 5 days, more preferably 5 hours to 2 days, and even more preferably 12 hours to 1 day.

[0054] The nucleic acid sequence transduced or transfected into immune cells induces expression of the antigen-binding molecule in the immune cells. The nucleic acid sequence preferably comprises a promoter operably linked to the sequence encoding the antigen-binding molecule. The promoter may be constitutively active in immune cells, or may be inducible in immune cells.

[0055] Agents, Methods and Therapeutic Uses The immune cells of the invention can be used in methods of treatment of the human or animal body. Accordingly, the invention provides a method of treating disease in an individual, the method comprising administering to the individual a therapeutic number of immune cells of the invention. The invention further provides the immune cells of the invention for use in a method of treating disease in an individual, the method comprising administering to the individual a therapeutic amount of immune cells.

[0056] The individual can be of any species. For example, the individual can be a human, dog, cat, mouse, rat, pig, sheep, cow, goat, or horse. Preferably, the individual is human. Preferably, the immune cells are of the same species as the individual. The immune cells can be autologous with respect to the individual. The immune cells can be allogeneic with respect to the individual. The individual can be an infant, young child, or adult. The patient can have, be susceptible to, or be at risk for the disease.

[0057] The present invention relates to administering a therapeutically effective number of immune cells of the present invention to an individual. A therapeutically effective number is a number that ameliorates one or more symptoms of a disease. A therapeutically effective number is preferably a number that treats the disease. Any suitable number of immune cells can be administered to an individual. As a guideline, the number of immune cells administered is typically between 10 and 100 mg / ml. 5 ~10 9 pieces, preferably 10 6 ~10 8 For example, at least or about 0.2 x 10 per kg of individual. 6 , 0.25×10 6 , 0.5×10 6 , 1.5×10 6 , 4.0×10 6 or 5.0 x 10 6 For example, at least or about 10 immune cells can be administered. 5 , 10 6 , 10 7 , 10 8 , or 10 9 At least about 1 x 10 immune cells can be administered. 6 , at least about 2 × 10 6 , at least about 2.5 x 10 6 , at least about 5 × 10 6 , at least about 1 x 10 7 , at least about 2 × 10 7 , at least about 5 × 10 7 , at least about 1 x 10 8 , or at least about 2×10 8 The immune cells can be administered.

[0058] Immune cells can be used in combination with other means and substances for treating disease. For example, immune cells can be used in combination with one or more cancer therapies. For example, immune cells can be used in combination with one or more chemotherapeutic agents. Immune cells can be used in combination with ab T cells expressing one or more CARs. Immune cells can be used in combination with radiation therapy. Immune cells can be used in combination with surgery, for example, surgery to resect or remove a tumor.

[0059] The immune cells can be used in combination with one or more therapies to treat an infectious disease, such as a viral or bacterial infection. For example, the immune cells can be used in combination with one or more antiviral drugs. The immune cells can be used in combination with one or more antibiotics.

[0060] Immune cells may be used in combination with a substance that supports immune cell function. For example, immune cells may be used in combination with an aminobisphosphonate, such as zoledronic acid. For example, when the immune cells are V52+gdT cells, immune cells may be used in combination with an aminobisphosphonate. Immune cells may be used in combination with one or more immune-stimulating cytokines, such as IL-2, GM-CSF, or G-CSF. IL-2, GM-CSF, or G-CSF may each enhance other populations of ADCC-competent cells.

[0061] When immune cells are used in combination with one or more other substances, the immune cells can be administered simultaneously, sequentially, or separately with the other substance(s).The immune cells can be used in combination with existing treatments for treating diseases, for example, simply mixed with such treatments.Therefore, the immune cells can be used to increase the efficacy of existing treatments for diseases.

[0062] Immune cells can be formulated for administration using any suitable method. Formulation of cells using standard pharmaceutically acceptable carriers and / or excipients can be carried out using conventional methods in the pharmaceutical field. The exact nature of the formulation depends on several factors, including the cells to be administered and the desired route of administration. Suitable types of formulations are fully described in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.

[0063] The immune cells can be formulated with a physiologically acceptable carrier or diluent. Typically, such a formulation is prepared as a liquid suspension of cells. The cells can be mixed with an excipient that is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, etc., and combinations thereof. Furthermore, if desired, the pharmaceutical composition of the present invention can contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and / or adjuvants that enhance effectiveness.

[0064] The immune cells may be administered by any route. Suitable routes include, but are not limited to, intravenous, intramuscular, intraperitoneal, or other suitable routes of administration. The immune cells are preferably administered intravenously.

[0065] The disease can be any disease in which a patient can benefit from targeted ADCC. The disease can be any disease in which a patient can benefit from a targeted T cell response. For example, the disease can be a disease in which a subject can benefit from the killing of unwanted cells. The unwanted cells can be, for example, cancer cells. The unwanted cells can be cells infected with bacteria, viruses, fungi, protozoa, or parasites. The unwanted cells can be aberrant immune cells, i.e., immune cells that cause a harmful immune response. For example, the aberrant immune cells can be autoimmune cells. Thus, the disease can be an infectious disease (e.g., a bacterial, viral, fungal, protozoan, or other parasitic infection) or an autoimmune disease. Preferably, the disease is cancer. The cancer can be a cancer of the hematopoietic tissue and / or lymphoid tissue. Preferably, the cancer is a solid tumor.

[0066] The cancer can be a primary cancer or a secondary cancer. The cancer can be anal cancer, bile duct cancer (cholangiocarcinoma), bladder cancer, blood cancer, bone cancer, intestinal cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, endocrine tumors, eye cancer (such as ocular melanoma), fallopian tube cancer, gallbladder cancer, head and / or neck cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymph node cancer, lymphoma, melanoma, mesothelioma, myeloma, neuroendocrine tumors, ovarian cancer, esophageal cancer, pancreatic cancer, penile cancer, primary peritoneal cancer, prostate cancer, skin cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, tracheal cancer, cancer of unknown primary, vaginal cancer, vulvar cancer, or endometrial cancer. The leukemia can be acute lymphoblastic leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia, or chronic myelogenous leukemia. The lymphoma can be Hodgkin's lymphoma or non-Hodgkin's lymphoma. The cancer can be neuroblastoma or melanoma. The cancer can be colon cancer, rectal cancer, gastric cancer, breast cancer, lung cancer, thyroid cancer, or ovarian cancer. The cancer can be carcinoma. Preferably, the cancer is colon cancer or neuroendocrine tumor.

[0067] [Example] Vδ2 + γδ T cells can be transduced to secrete proteins that specifically bind to antigens on target cells Vδ2 + γδ T cells were transduced to secrete scFv fusion peptides targeting the tumor-associated antigens CEA and GD2. Supernatants from transduced cells were collected and analyzed for CEA. + CAPAN-1 cells and CEA-HELA cells, or GD2 + / - The scFv-Fc fusion protein was applied to SupT1 cells. Binding of the scFv-Fc fusion protein was detected using a phycoerythrin-conjugated anti-human Fc antibody. Purified anti-CEA scFv-Fc fusion protein produced in the cell line or purified anti-GD2 antibody was used as a positive control.

[0068] As shown in Figure 2, components of the supernatants from the transduced cell lines and transduced V52 γδ T cells were able to specifically bind to antigen-positive cells, resulting in the detection of human Fc on their surface.

[0069] Cytotoxicity experiments showing the effect of direct and indirect contact with Vδ2 secreting scFv-Fc proteins Because engineered T cells secrete proteins that can affect the cytotoxicity of bystander, unengineered cells, a series of experimental conditions were developed to demonstrate this. The experimental setup is shown in Figure 3. Briefly, in addition to comparing the cytotoxicity of transduced and untransduced V52 when directly cocultured with target cells, two additional conditions were used to demonstrate cell-contact-autonomous effects. First, supernatant from transduced V52 was added to cocultures of untransduced V52 and target cells. Second, untransduced cells were cocultured with targets in the presence of transduced cells sequestered behind a semipermeable membrane that blocks cell passage but not secreted molecules. All cocultures were for 18 hours, with an effector:target ratio of 1:1. To demonstrate the effect of anti-CEA scFv-Fc fusion proteins, CEA+CAPAN-1 and CEA-HELA cells were used. To demonstrate the effect of the anti-GD2 scFv-Fc fusion protein, GD2 or isogenic GD2 -SupT1 engineered to express wild-type SupT1 was used.

[0070] Vδ2 cells transduced to secrete anti-CEA scFv-Fc fusion proteins showed increased CEA expression compared to NT Vδ2s + V52 transduced to express anti-CEA scFv-Fc fusion protein resulted in significantly higher target mortality (p<0.0001, Figure 4A). + Supernatants from cells express CEA by non-transduced Vδ2 + Enhanced target killing (p<0.0001) and CEA - It also slightly increased target killing (p=0.031, Figure 4B). When V52 secreting anti-CEA scFv-Fc fusion proteins were sequestered behind a semipermeable membrane, the non-transduced V52 secreted CEA compared to the isolation of non-transduced cells. + It was also able to enhance the ability to kill the target (p=0.012, Figure 4C).

[0071] Similar data were observed for V52 transduced to secrete an anti-GD2 scFv-Fc fusion protein. Transduced V52 showed better GD2 secretion than non-transduced ones (p=0.03). + Kills the target and produces the same GD2 - Greater GD2 than controls + The supernatant from the anti-GD2-scFv-Fc fusion protein expressing Vδ2 showed cytotoxicity against the target (p=0.0049, Figure 4D). The supernatant from the anti-GD2-scFv-Fc fusion protein expressing Vδ2 showed cytotoxicity against the target (p=0.0049, Figure 4D). - GD2 of non-transduced Vδ2 when compared to targets (p=0.0002, Figure 4E) + Sequestration of V52 secreting anti-GD2 scFv-Fc fusion proteins behind a semipermeable membrane significantly enhanced their ability to kill targets compared with sequestration of non-transduced cells (p=0.0158) or GD2 - Compared to target killing (p=0.0003, Figure 4F), non-transduced Vδ2 GD2 + It could also enhance its ability to kill targets.

[0072] CEA non-transduced Vδ2 ± When targets were co-cultured in the presence of V52 secreting anti-CEA scFv-Fc fusion protein sequestered behind a semipermeable membrane or untransduced controls, a significant increase in IFNγ production was observed only when cells secreting anti-CEA scFv-Fc fusion protein were sequestered (p=0.0014 compared to sequestered NT V52), suggesting that CEA + Targets were used (CEA - p = 0.012 compared to target, Figure 5).

[0073] Vδ2 + γδ T cells can be transduced to secrete whole antibodies that specifically bind to antigens on target cells. HEK293T and Vδ2 + γδ T cells were transduced to secrete IgG1 antibodies targeting the tumor-associated antigen GD2 (SEQ ID NO: 17). Supernatants from HEK293T cells transduced with reduced volumes of virus were collected and GD2 + / - The transduced V52 cells were applied to the scFv-Fc fusion protein. Binding of the scFv-Fc fusion protein was detected using a phycoerythrin-conjugated anti-human Fc antibody (Figure 6). + Supernatants from γδT cells were also collected and analyzed using GD2 + / - SupT1 cells and antibody binding was detected using an anti-human IgG antibody conjugated with AlexaFluor 647 (Figure 7). Purified anti-GD2 antibody produced in a cell line was used as a positive control.

[0074] As shown in Figures 6 and 7, the transduced cell lines and transduced V52 + Components of the γδ T cell-derived supernatant were able to specifically bind to antigen-positive cells, resulting in the detection of human Fc or IgG on their surface.

[0075] Cytotoxicity experiments demonstrating the effects of direct and indirect contact with antibody-secreting Vδ2 To demonstrate cell contact-dependent and cell contact-autonomous effects, two experimental systems were used (Figure 8A). Briefly, untransduced Vδ2 + γδ T cells or Vδ2+ γδ T cells transduced to express anti-GD2 IgG1 were used as GD2 + / - In a second experiment, untransduced Vδ2 cells were co-cultured with SupT1 at a 1:1 effector:target ratio. + Supernatants from γδ T cells or Vδ2+ γδ T cells transduced to express anti-GD2 IgG1 were used to treat non-transduced Vδ2+ γδ T cells and GD2 + / - V52 cells transduced to express anti-GD2 IgG1 were added to a 1:1 co-culture of V52 cells transduced to express anti-GD2 IgG1. Target cell death was determined using flow cytometry, and Figures 8B and 8C show representative data from three replicates. + γδ T cells exhibited enhanced cytotoxicity against SupT1-GD2 but not against SupT1-wt (Fig. 8B). Vδ2 transduced to express anti-GD2 IgG1 + Supernatants from γδ T cells express untransduced Vδ2 against SupT1-GD2 + It enhanced the cytotoxicity of γδT cells against SupT1-wt but not against SupT1-wt ( Fig. 8C ).

[0076] Materials and Methods cell line CAPAN-1, HELA, and SupT1 cell lines were obtained from ATCC. SupT1-GD2 was generated by transducing wild-type SupT1 with a vector encoding GD2 / GD3 synthase and isolating clones of successfully transduced cells.

[0077] Donor selection for PBMCs PBMCs were isolated from the blood of healthy donors aged 20–36 years. Donors were screened for cross-reactivity between the SFP construct and expanded Vδ2 cells prior to inclusion. Donors with evidence of cross-reactivity were not included in the cytotoxicity studies.

[0078] Isolation of fresh PBMCs and pre-stimulation Twenty ml of whole blood was diluted with 10 ml of PBS plus 500 μl of 100 mM EDTA and layered on 20 ml of Percoll. Interface PBMCs (20 min, 300 × g, RT) were washed in PBS, resuspended in 25 ml of T cell medium (X-VIVO 15 (Lonza BioWhittaker, Maryland, USA)) supplemented with penicillin / streptomycin (100 IU / ml penicillin, 100 μg / ml streptomycin (Sigma-Aldrich, Missouri, USA)), and cultured overnight before use.

[0079] Vδ2 + T cell expansion Specific Vδ2 + For γδ T cell expansion, PBMCs were isolated as described above and cultured in RPMI-1640 medium supplemented with L-glutamine (2 mM, Sigma-Aldrich), penicillin / streptomycin (100 IU / ml penicillin, 100 μg / ml streptomycin (Sigma-Aldrich)), and 10% FCS (v / v, Gibco, Massachusetts, USA). + γδ T cell expansion was stimulated with 5 μM zoledronic acid (Actavis, New Jersey, USA) and 100 IU / ml IL-2 (Aldesleukin Novartis, Frimley, UK), which were added to the PBMC suspension after PBMC isolation (day 1). IL-2 was replenished every 2–3 days by removing half of the medium from the wells and replacing it with fresh medium containing 200 IU / ml IL-2.

[0080] Construction of retroviral constructs The gumma retroviral vector used in all constructs was SFG (Riviere et al., 1995), pseudotyped with the RD114 envelope. DNA fragments were amplified using Phusion HT II polymerase according to the manufacturer's instructions (Thermo Scientific, Massachusetts, USA). PCR was performed on a PTC-200 DNA Engine (MJ Research, Massachusetts, USA). PCR products were extracted from 1% agarose gels using a Wizard SV Gel & PCR Clean-Up Kit (Promega, Wisconsin, USA). Sample concentrations were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Massachusetts, USA). Constructs contain one of a series of scFvs against a panel of targets, including human GD2 (clone 14G2A) or human CEA (clone SM3EL), linked to the Fc portion of human IgG1.

[0081] In addition to the scFv-Fc fusion protein construct, we included a marker bearing the CD34 epitope, RQR8 (Philip et al., 2014), separated from the scFv-Fc fusion protein by a cleavable 2A peptide, which allows for the detection of cells expressing the scFv-Fc fusion protein by flow cytometry after staining with the anti-CD34 antibody clone QBend10.

[0082] Construction of lentiviral constructs The lentiviral vector used for all antibody transduction was pCCL (Dull et al., 1998), pseudotyped with the RDPro envelope (Cosset et al., 1995). DNA fragments were amplified using Phusion HT II polymerase according to the manufacturer's instructions (Thermo Scientific, Massachusetts, USA). PCR was performed on a PTC-200 DNA Engine (MJ Research, Massachusetts, USA). PCR products were extracted from 1% agarose gels using the Wizard SV Gel & PCR Cleanup Kit (Promega, Wisconsin, USA). Sample concentrations were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Massachusetts, USA). The construct contained an anti-GD2 IgG1 (clone 14G2A).

[0083] In addition to the IgG1 construct, eGFP was included, separated from the IgG1 protein by a cleavable 2A peptide, which allows for the detection of IgG1-expressing cells by flow cytometry.

[0084] Production of viral particles by transfection 1.5×10 6 293T cells / 100mm 2Dishes (Nucleon Delta Surface, Thermo Fisher Scientific) were seeded in 293T medium (D-MEM, 10% FCS (v / v)) on day 1. γ-Retroviral or lentiviral particles were generated by co-transfecting 293T cells on day 2 using Gene Juice transfection reagent (Novagen / Millipore, Massachusetts, USA) according to the manufacturer's instructions. The supernatant containing the viral particles was collected on day 4, medium was added, and the supernatant was collected on day 5. γ-Retroviral supernatants were pooled, filtered (0.45 μm filter, Millipore), and either used directly for transduction or stored overnight at 4°C before use. Lentiviral supernatants were concentrated by ultracentrifugation and frozen for later use.

[0085] Gamma retroviral transduction of T cells The viral supernatant was preloaded onto a 24-well plate coated with Retronectin (Takara Bio, Tokyo, Japan), and T cells were transduced using 0.5 × 10 cells suspended in 0.5 ml of T cell medium plus 400 IU of IL-2. 6 T cells were mixed with 1.5 ml viral supernatant and centrifuged at 1000 × g for 40 minutes at room temperature. Typically, 12 × 10 cells were collected per donor. 6 T cells were seeded for transduction.

[0086] γδ T cell expansion was stimulated with 5 μM zoledronic acid (Actavis, New Jersey, USA) and 100 IU / ml IL-2 (Aldesleukin) and transduction was performed on day 5. On day 8 of culture (day 3 post-transduction), cells were pooled, washed, and cultured at 2 × 10 cells in T cell medium + 100 IU IL-2 / ml (24-well plate, Nucleon Delta Surface, Thermo Scientific, Massachusetts, USA). 6 Cells were seeded at 1000 x g / ml. Transduction efficiency was determined by flow cytometry on day 10 (day 5 post-transduction).

[0087] Lentiviral transduction of T cells T cell transduction was performed in 96-well plates, with each well containing 0.3 x 10 cells suspended in 0.3 ml of T cell medium. 6 Concentrated lentivirus containing T cells was added and the plates were centrifuged for 40 minutes at 1000 x g at room temperature.

[0088] Expansion of γδ T cells was stimulated with 5 μM zoledronic acid (Actavis, New Jersey, USA) and 100 IU / ml IL-2 (Aldesleukin), and transduction was performed on day 2. On day 5 of culture (day 3 post-transduction), cells were pooled, washed, and transduced into 1 × 10 cells in T cell medium + 100 IU IL-2 / ml. 6 cells / cm 2 Transduction efficiency was determined by flow cytometry on day 7 (day 5 post-transduction).

[0089] Cytotoxicity assay Target cell lines were labeled with CellTrace Violet (Thermo Fisher) and then co-cultured with effector cells at an effector:target ratio of 1:1 in either RPMI1640 + 10% FCS + 1% Pen / Step + 1% L-glutamine supplemented with 100 μg / ml IL-2, or supernatant from V52 cells secreting scFv-Fc fusion proteins cultured in the same medium.

[0090] For assays involving effector cells sequestered behind a semipermeable membrane, cells were separated using 0.4 μm pore size TransWells (Thermo Fisher). Equal numbers of effectors were placed in the transwells and main wells. Target cells were placed in the main wells, and a 1:1 effector:target ratio was calculated for cells in the main well.

[0091] After 18 hours of co-culture, cells were harvested and analyzed by flow cytometry. Cell death was identified by staining with Ghost Red fixable viability dye (Tonbo Biosciences, San Diego, CA) on violet-labeled cells.

[0092] The present invention encompasses, for example, the following embodiments: [Embodiment 1] An immune cell capable of antibody-dependent cellular cytotoxicity (ADCC) and comprising a nucleic acid sequence encoding an antigen-binding molecule comprising an antigen-binding region. [Embodiment 2] The immune cell of embodiment 1, wherein the antigen-binding region comprises an scFv, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, dAb, Fd, dsFv, ds-scFv, scFv2, bispecific T cell engager, nanobody, DARPin, antibody mimetic, diabody, triabody, tetrabody, or polypeptide ligand of a receptor expressed on the surface of a cell targeted by the immune cell. [Embodiment 3] The immune cell of embodiment 1 or 2, wherein the antigen-binding molecule is capable of binding to an Fc receptor. [Embodiment 4] An immune cell according to any one of embodiments 1 to 3, wherein the antigen-binding molecule comprises an Fc region or a modified Fc region. [Embodiment 5] The immune cell according to any one of embodiments 1 to 4, wherein the antigen-binding molecule is an antibody, scFv-Fc, dAb-Fc, heavy chain antibody, IgNAR, or camel antibody. [Embodiment 6] An immune cell according to any one of embodiments 1 to 5, wherein the immune cell is not an alpha beta T cell. [Embodiment 7] An immune cell described in any one of embodiments 1 to 6, wherein the immune cell is a gamma delta T cell or an NK cell. [Embodiment 8] The immune cells of embodiment 7, wherein the gamma delta T cells are Vδ1+ gamma delta T cells, Vδ2+ gamma delta T cells, or Vδ1- / Vδ2- gamma delta T cells. [Embodiment 9] The immune cells of embodiment 8, wherein the gamma delta T cells are Vδ2+ gamma delta T cells. [Embodiment 10] An immune cell according to any one of embodiments 1 to 6, wherein the immune cell is a bone marrow cell. [Embodiment 11] The immune cells described in embodiment 10, wherein the bone marrow cells are macrophages, basophils, eosinophils, or neutrophils. [Embodiment 12] An immune cell described in any one of embodiments 1 to 11, wherein the immune cell does not express a chimeric antigen receptor (CAR). [Embodiment 13] An immune cell described in any one of embodiments 1 to 12, wherein the antigen-binding region is capable of binding to an antigen expressed in the tumor microenvironment. [Embodiment 14] An immune cell described in any one of embodiments 1 to 13, wherein the antigen-binding region is capable of binding to a tumor antigen, an endothelial antigen, or an immune cell antigen. [Embodiment 15] The antigen-binding region is selected from the group consisting of CEA, B7-H3, TSHR, CD3, CD16, CD32, CD64, CD19, CD123, CD22, CD20, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, and Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, EPCAM, KIT, IL-13Ra2, mesothelin, IL-llRa, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosine Enzyme, EphA2, Fucosyl-GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OYTES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut15. The immune cell of any of embodiments 1 to 14, which is capable of binding to an antigen selected from the group consisting of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, LINGO1, CD70, IL13Rα2, MUC-16, PSCA, ROR1, and IGLL1. [Embodiment 16] The immune cell described in embodiment 15, wherein the tumor antigen is CEA, B7-H3, CD20, or GD2. [Embodiment 17] An immune cell described in any one of embodiments 1 to 16, wherein the antigen-binding molecule is an opsonin. [Embodiment 18] An immune cell described in any one of embodiments 1 to 17, wherein the immune cell expresses an antigen-binding molecule. [Embodiment 19] An immune cell described in any one of embodiments 1 to 18, wherein the antigen-binding molecule comprises a VH domain encoded by SEQ ID NO: 1 and / or a VL domain encoded by SEQ ID NO: 2. [Embodiment 20] The immune cell described in embodiment 19, wherein the antigen-binding molecule is encoded by SEQ ID NO: 3. [Embodiment 21] An immune cell described in any one of embodiments 1 to 18, wherein the antigen-binding molecule comprises a VH domain encoded by SEQ ID NO: 4 and / or a VL domain encoded by SEQ ID NO: 5. [Embodiment 22] The immune cell described in embodiment 21, wherein the antigen-binding molecule is encoded by SEQ ID NO: 6. [Embodiment 23] An immune cell described in any one of embodiments 1 to 18 and 21, wherein the antigen-binding molecule comprises a heavy chain encoded by SEQ ID NO: 20 and / or a light chain encoded by SEQ ID NO: 18. [Embodiment 24] The immune cell described in embodiment 23, wherein the antigen-binding molecule is encoded by SEQ ID NO: 17. [Embodiment 25] An immune cell described in any one of embodiments 1 to 24, wherein the nucleic acid sequence encodes two or more different antigen-binding molecules. [Embodiment 26] An immune cell according to any one of embodiments 1 to 25, wherein the immune cell comprises two or more nucleic acid sequences each encoding a different antigen-binding molecule. [Embodiment 27] A method for generating an immune cell according to any one of embodiments 1 to 26, comprising introducing a nucleic acid sequence encoding an antigen-binding molecule into an immune cell. [Embodiment 28] The method of embodiment 27, wherein the nucleic acid sequence is contained in a vector, optionally a viral vector. [Embodiment 29] A method for treating a disease in an individual, comprising administering to the individual a therapeutically effective number of immune cells described in any one of embodiments 1 to 26. [Embodiment 30] An immune cell described in any one of embodiments 1 to 26 for use in a method for treating a disease in an individual, the method comprising administering a therapeutically effective number of the immune cell to the individual. [Embodiment 31] The immune cell for use as described in embodiment 29 or embodiment 30, wherein the disease is cancer. [Embodiment 32] The method or immune cell for use according to embodiment 31, wherein the cancer is a solid tumor. Materials and Methods References I. Riviere, K. Brose, RC Mulligan, Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells, Proc. Natl. Acad. Sci. USA 92, 6733-6737 (1995). B. Philip, E. Kokalaki, L. Mekkaoui, S. Thomas, K. Straathof, B. Flutter, V. Marin, T. Marafioti, R. Chakraverty, D. Linch, S. A. Quezada, K. S. Peggs, M. Pule, A highly compact epitope-based marker / suicide gene for easier and safer T-cell therapy, Blood 124, 1277-1287 (2014). T. Dull, R. Zufferey. M. Kelly, R. J. Mandel, M. Nguyen, D. Trono, L. Naldini. A Third generation lentivirus vector with a conditional packaging system. Journal of Virology, Nov 1998; 72(11):8463-71 F. L. Cosset, Y. Takeuchi, J. L. Battini, R. A. Weiss, M. K. Collins. High-titer packaging cells producing recombinant retroviruses resistant to human serum. Journal of Virology, Dec 1995; 69(12):7430-6

[0093] [Sequence Listing] SEQUENCE LISTING <110> UCL Business Ltd <120> Engineered Immune Cells <130> PA25-401 <150> GB 2000934.6 <151> 2020-01-22 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH domain of a CEA-specific scFv used in the examples <400> 1 Gln Val Lys Leu Glu Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser 20 25 30 Tyr Met His Trp Leu Arg Gln Gly Pro Gly Gln Cys Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Phe Thr Thr Asp Thr Ser Ala Asn Thr Ala Tyr 65 70 75 80 Leu Gly Leu Ser Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> VL domain of a CEA-specific scFv used in the examples <400> 2 Glu Asn Val Leu Thr Gln Ser Pro Ser Ser Met Ser Val Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ala Cys Ser Ala Ser Ser Ser Val Pro Tyr Met 20 25 30 His Trp Leu Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Val Gln Pro Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr 85 90 95 Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> CEA-specific scFv-Fc used in the examples <400> 3 Met Ala Gln Val Lys Leu Glu Gln Ser Gly Ala Glu Val Val Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys 20 25 30 Asp Ser Tyr Met His Trp Leu Arg Gln Gly Pro Gly Gln Cys Leu Glu 35 40 45 Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro 50 55 60 Lys Phe Gln Gly Lys Ala Thr Phe Thr Thr Asp Thr Ser Ala Asn Thr 65 70 75 80 Ala Tyr Leu Gly Leu Ser Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asn 130 135 140 Val Leu Thr Gln Ser Pro Ser Ser Met Ser Val Ser Val Gly Asp Arg 145 150 155 160 Val Thr Ile Ala Cys Ser Ala Ser Ser Ser Val Pro Tyr Met His Trp 165 170 175 Leu Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile Tyr Leu Thr 180 185 190 Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 195 200 205 Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Ala 210 215 220 Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr Phe Gly 225 230 235 240 Cys Gly Thr Lys Leu Glu Ile Lys Arg Ser Ser Arg Ala Ala Ala Ser 245 250 255 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 260 265 270 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 275 280 285 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 290 295 300 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 305 310 315 320 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 325 330 335 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 340 345 350 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 355 360 365 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 370 375 380 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 385 390 395 400 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 405 410 415 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 420 425 430 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 435 440 445 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 450 455 460 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 465 470 475 480 Pro Gly Lys <210> 4 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> VH domain of a GD2-specific scFv (clone 14G2A) used in the examples <400> 4 Glu Val Lys Leu Gln Gln Ser Gly Pro Ser Leu Val Glu Pro Gly Ala 1 5 10 15 Ser Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met His Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 100 105 110 Ser <210> 5 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> VL domain of a GD2-specific scFv (clone 14G2A) used in the examples <400> 5 Asp Ile Leu Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 6 <211> 474 <212> PRT <213> Artificial Sequence <220> <223> GD2-specific scFv-Fc (clone 14G2A) used in the examples <400> 6 Glu Val Lys Leu Gln Gln Ser Gly Pro Ser Leu Val Glu Pro Gly Ala 1 5 10 15 Ser Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met His Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Asp Ile Leu Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 130 135 140 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg 145 150 155 160 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 165 170 175 Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser Gly Val Pro 180 185 190 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 195 200 205 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 210 215 220 Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 225 230 235 240 Lys Arg Ser Ala Ala Ala Ser Asp Lys Thr His Thr Cys Pro Pro Cys 245 250 255 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 290 295 300 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 355 360 365 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 370 375 380 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440 445 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455 460 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 7 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> CEA-specific scFv used in the examples <400> 7 Met Ala Gln Val Lys Leu Glu Gln Ser Gly Ala Glu Val Val Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys 20 25 30 Asp Ser Tyr Met His Trp Leu Arg Gln Gly Pro Gly Gln Cys Leu Glu 35 40 45 Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro 50 55 60 Lys Phe Gln Gly Lys Ala Thr Phe Thr Thr Asp Thr Ser Ala Asn Thr 65 70 75 80 Ala Tyr Leu Gly Leu Ser Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asn 130 135 140 Val Leu Thr Gln Ser Pro Ser Ser Met Ser Val Ser Val Gly Asp Arg 145 150 155 160 Val Thr Ile Ala Cys Ser Ala Ser Ser Ser Val Pro Tyr Met His Trp 165 170 175 Leu Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile Tyr Leu Thr 180 185 190 Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 195 200 205 Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Ala 210 215 220 Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr Phe Gly 225 230 235 240 Cys Gly Thr Lys Leu Glu Ile Lys 245 <210> 8 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> GD2-specific scFv (clone 14G2A) used in the examples <400> 8 Glu Val Lys Leu Gln Gln Ser Gly Pro Ser Leu Val Glu Pro Gly Ala 1 5 10 15 Ser Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met His Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Asp Ile Leu Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 130 135 140 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg 145 150 155 160 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 165 170 175 Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser Gly Val Pro 180 185 190 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 195 200 205 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 210 215 220 Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 225 230 235 240 Lys <210> 9 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> VH domain of a B7H3-specific scFv <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL domain of a B7H3-specific scFv <400> 10 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 477 <212> PRT <213> Artificial Sequence <220> <223> B7H3-specific scFv-Fc <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser 130 135 140 Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 145 150 155 160 Lys Ala Ser Gln Asn Val Asp Thr Asn Val Ala Trp Tyr Gln Gln Lys 165 170 175 Pro Gly Lys Ala Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr 180 185 190 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 210 215 220 Cys Gln Gln Tyr Asn Asn Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys 225 230 235 240 Leu Glu Ile Lys Arg Ser Ala Ala Ala Ser Asp Lys Thr His Thr Cys 245 250 255 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 260 265 270 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 275 280 285 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 290 295 300 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 305 310 315 320 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 325 330 335 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 340 345 350 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 355 360 365 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 370 375 380 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 385 390 395 400 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 405 410 415 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 420 425 430 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 435 440 445 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 450 455 460 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 <210> 12 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> B7H3-specific scFv <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser 130 135 140 Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 145 150 155 160 Lys Ala Ser Gln Asn Val Asp Thr Asn Val Ala Trp Tyr Gln Gln Lys 165 170 175 Pro Gly Lys Ala Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr 180 185 190 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 210 215 220 Cys Gln Gln Tyr Asn Asn Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys 225 230 235 240 Leu Glu Ile Lys <210> 13 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH domain of a CD20-specific scFv <400> 13 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ala 115 120 <210> 14 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> VL domain of a CD20-specific scFv <400> 14 Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 15 <211> 475 <212> PRT <213> Artificial Sequence <220> <223> CD20-specific scFv-Fc <400> 15 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Ser Gln Ser Pro 130 135 140 Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg 145 150 155 160 Ala Ser Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly 165 170 175 Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly 180 185 190 Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu 195 200 205 Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln 210 215 220 Gln Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys Arg Ser Ala Ala Ala Ser Asp Lys Thr His Thr Cys Pro Pro 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 370 375 380 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 <210> 16 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> CD20-specific scFv <400> 16 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Ser Gln Ser Pro 130 135 140 Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg 145 150 155 160 Ala Ser Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly 165 170 175 Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly 180 185 190 Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu 195 200 205 Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln 210 215 220 Gln Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys <210> 17 <211> 744 <212> PRT <213> Artificial Sequence <220> <223> GD2 specific IgG1 (clone 14G2A) used in the examples, having a cleavage site between the light and heavy chains <400> 17 Met Leu Pro Ser Gln Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala 1 5 10 15 Ser Arg Gly Asp Ile Leu Leu Thr Gln Thr Pro Leu Ser Leu Pro Val 20 25 30 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu 35 40 45 Val His Arg Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys 100 105 110 Ser Gln Ser Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys 115 120 125 Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Arg 225 230 235 240 Ala Lys Arg Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser 245 250 255 Thr Ser Gly Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln 260 265 270 Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Asp Trp Thr Trp Arg 275 280 285 Ile Leu Phe Leu Val Ala Ala Ala Thr Gly Ala His Ser Glu Val Lys 290 295 300 Leu Gln Gln Ser Gly Pro Ser Leu Val Glu Pro Gly Ala Ser Val Met 305 310 315 320 Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Gly Tyr Asn Met Asn 325 330 335 Trp Val Arg Gln Asn Ile Gly Lys Ser Leu Glu Trp Ile Gly Ala Ile 340 345 350 Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys Gly Arg 355 360 365 Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met His Leu 370 375 380 Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Val Ser Gly 385 390 395 400 Met Glu Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser 405 410 415 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 420 425 430 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 435 440 445 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 450 455 460 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 465 470 475 480 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 485 490 495 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 500 505 510 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 515 520 525 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 530 535 540 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 545 550 555 560 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 565 570 575 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 580 585 590 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 595 600 605 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 610 615 620 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 625 630 635 640 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 645 650 655 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 660 665 670 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 675 680 685 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 690 695 700 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 705 710 715 720 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 725 730 735 Ser Leu Ser Leu Ser Pro Gly Lys 740 <210> 18 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> light chain of the GD2 IgG1 of SEQ ID NO: 17 <400> 18 Met Leu Pro Ser Gln Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala 1 5 10 15 Ser Arg Gly Asp Ile Leu Leu Thr Gln Thr Pro Leu Ser Leu Pro Val 20 25 30 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu 35 40 45 Val His Arg Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys 100 105 110 Ser Gln Ser Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys 115 120 125 Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 19 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> cleavage sequence (Furin-V5-SG-P2A) of the GD2 IgG1 of SEQ ID NO: 17 <400> 19 Arg Ala Lys Arg Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp 1 5 10 15 Ser Thr Ser Gly Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys 20 25 30 Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 35 40 <210> 20 <211> 462 <212> PRT <213> Artificial Sequence <220> <223> heavy chain of the GD2 IgG1 of SEQ ID NO: 17 <400> 20 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser Glu Val Lys Leu Gln Gln Ser Gly Pro Ser Leu Val Glu 20 25 30 Pro Gly Ala Ser Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe 35 40 45 Thr Gly Tyr Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu 50 55 60 Glu Trp Ile Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met His Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val 115 120 125 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 130 135 140 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 145 150 155 160 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 165 170 175 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 180 185 190 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 195 200 205 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 210 215 220 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 245 250 255 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 275 280 285 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 305 310 315 320 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 325 330 335 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 355 360 365 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 370 375 380 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 405 410 415 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420 425 430 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 435 440 445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460

Claims

1. An immune cell capable of antibody-dependent cellular cytotoxicity (ADCC) and containing a nucleic acid sequence encoding an antigen-binding molecule containing an antigen-binding region.

2. 2. The immune cell of claim 1, wherein the antigen-binding region comprises an scFv, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, dAb, Fd, dsFv, ds-scFv, scFv2, bispecific T cell engager, nanobody, DARPin, antibody mimetic, diabody, triabody, tetrabody, or a polypeptide ligand of a receptor expressed on the surface of a cell targeted by the immune cell.

3. The immune cell of claim 1 or 2, wherein the antigen-binding molecule is capable of binding to an Fc receptor.

4. The immune cell of any one of claims 1 to 3, wherein the antigen-binding molecule comprises an Fc region or a modified Fc region.

5. The immune cell of any one of claims 1 to 4, wherein the antigen-binding molecule is an antibody, scFv-Fc, dAb-Fc, heavy chain antibody, IgNAR, or camel antibody.

6. 6. The immune cell of any one of claims 1 to 5, wherein the immune cell is not an alpha beta T cell.

7. 7. The immune cell of claim 1, wherein the immune cell is a gamma delta T cell or a NK cell.

8. The immune cell of claim 7, wherein the gamma delta T cell is a V51+ gamma delta T cell, a V52+ gamma delta T cell, or a V51- / V52- gamma delta T cell.

9. The immune cell of claim 8, wherein the gamma delta T cell is a V52+ gamma delta T cell.

10. The immune cell of any one of claims 1 to 6, wherein the immune cell is a bone marrow cell.

11. The immune cell of claim 10, wherein the bone marrow cell is a macrophage, basophil, eosinophil, or neutrophil.

12. 12. The immune cell of any one of claims 1 to 11, wherein the immune cell does not express a chimeric antigen receptor (CAR).

13. 13. The immune cell of any one of claims 1 to 12, wherein the antigen-binding region is capable of binding to an antigen expressed in the tumor microenvironment.

14. 14. The immune cell of claim 1, wherein the antigen-binding region is capable of binding to a tumor antigen, an endothelial antigen, or an immune cell antigen.

15. The antigen binding region is CEA, B7-H3, TSHR, CD3, CD16, CD32, CD64, CD19, CD123, CD22, CD20, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, EPCAM, KIT, IL-13Ra2, mesothelin, IL-llRa, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosine Enzyme, EphA2, Fucosyl-GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OYTES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut15. The immune cell of any one of claims 1 to 14, which is capable of binding to an antigen selected from the group consisting of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, LINGO1, CD70, IL13Rα2, MUC-16, PSCA, ROR1, and IGLL1.

16. The immune cell of claim 15, wherein the tumor antigen is CEA, B7-H3, CD20, or GD2.

17. The immune cell of any one of claims 1 to 16, wherein the antigen-binding molecule is an opsonin.

18. 18. The immune cell of any one of claims 1 to 17, wherein the immune cell expresses an antigen-binding molecule.

19. The antigen-binding molecule comprises a V encoded by SEQ ID NO: 1 H Domain and / or V encoded by SEQ ID NO:2 L 19. An immune cell according to any one of claims 1 to 18, comprising a domain.

20. The immune cell of claim 19, wherein the antigen-binding molecule is encoded by SEQ ID NO:

3.

21. The antigen-binding molecule comprises a V encoded by SEQ ID NO: 4 H Domain and / or V encoded by SEQ ID NO:5 L 19. An immune cell according to any one of claims 1 to 18, comprising a domain.

22. The immune cell of claim 21, wherein the antigen-binding molecule is encoded by SEQ ID NO:

6.

23. 22. The immune cell of any one of claims 1 to 18 and 21, wherein the antigen-binding molecule comprises a heavy chain encoded by SEQ ID NO: 20 and / or a light chain encoded by SEQ ID NO:

18.

24. The immune cell of claim 23, wherein the antigen-binding molecule is encoded by SEQ ID NO:

17.

25. 25. The immune cell of any one of claims 1 to 24, wherein the nucleic acid sequence encodes two or more different antigen-binding molecules.

26. 26. The immune cell of any one of claims 1 to 25, wherein the immune cell comprises two or more nucleic acid sequences each encoding a different antigen-binding molecule.

27. 27. A method for generating the immune cell of any one of claims 1 to 26, comprising introducing a nucleic acid sequence encoding the antigen-binding molecule into the immune cell.

28. 28. The method of claim 27, wherein the nucleic acid sequence is comprised in a vector, optionally a viral vector.

29. 27. A method of treating a disease in an individual comprising administering to the individual a therapeutically effective number of immune cells according to any one of claims 1 to 26.

30. 27. An immune cell according to any one of claims 1 to 26 for use in a method of treating a disease in an individual, the method comprising administering to the individual a therapeutically effective number of the immune cell.

31. 31. The method of claim 29 or the immune cell for use according to claim 30, wherein the disease is cancer.

32. 32. The method or immune cell for use according to claim 31 , wherein the cancer is a solid tumor.

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