Improved antigen-binding receptors

Abstract

To provide means having improved safety and effectiveness in adoptive T-cell therapy.SOLUTION: Provided are an antigen-binding receptor that can be specifically bound with a variant Fc domain with reduced Fc receptor bond, and a T-cell expressing the antigen-binding receptors. More specifically, the invention deals with an engineered Fc receptor consisting of an intracellular domain of CD3 bound with an inner domain and a transmembrane domain of CD28. An extracellular part preferably consists of an anti-Pro329Gly antibody variable domain. The receptor is used in treatment and diagnosis of cancer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention generally relates to antigen-binding receptors capable of specifically binding to mutant Fc domains with reduced Fc receptor binding, and T cells expressing these antigen-binding receptors. More specifically, the present invention relates to T cells transfected / transduced with antigen-binding receptors that are recruited by specifically binding / interacting with the mutant Fc domain of a therapeutic antibody. Furthermore, the present invention relates to kits comprising nucleic acid molecules, vectors expressing the T cells of the present invention and / or the antigen-binding receptors of the present invention, and one or more tumor-targeting antibodies comprising a mutant Fc domain. The present invention provides the production and use of T cells in methods for treating specific diseases in combination with tumor-specific antibodies, as well as pharmaceutical compositions / medicines comprising the T cells and / or therapeutic antibodies, wherein the T cells are administered in combination with one or more therapeutic tumor-targeting antibodies comprising a mutant Fc domain with reduced Fc receptor binding. [Background technology]

[0002] background Adoptive T cell therapy (ACT) is a powerful treatment approach that uses cancer-specific T cells (Rosenberg and Restifo, Science 348(6230)(2015), 62-68). ACT can use either naturally occurring tumor-specific cells or genetically engineered T cells with T cell receptors or chimeric antigen receptors (Rosenberg and Restifo, Science 348(6230)(2015), 62-68). ACT can successfully treat and induce remission even in patients with otherwise refractory advanced disease, such as acute lymphoblastic leukemia, non-Hodgkin's lymphoma, or melanoma (Dudley et al., J Clin Oncol 26(32)(2008), 5233-5239 (Non-Patent Document 2); Grupp et al., N Engl J Med 368(16)(2013), 1509-1518 (Non-Patent Document 3); Kochenderfer et al., J Clin Oncol.(2015)33(6):540-549, doi:10.1200 / JCO.2014.56.2025.Epub 2014 Aug 25 (Non-Patent Document 4)).

[0003] However, despite its remarkable clinical efficacy, ACT is limited by treatment-related toxicity. The specificity of engineered T cells used in ACT, and the resulting on-target and off-target effects, are primarily driven by the tumor-targeting antigen-binding moiety implemented in chimeric antigen receptors (CARs). Non-exclusive expression of tumor antigens or temporal differences in expression levels can result in severe side effects, and ACT may even be discontinued due to unacceptable treatment toxicity.

[0004] Additionally, the availability of tumor-specific T cells for efficient tumor cell lysis depends on the long-term survival and proliferation of engineered T cells in vivo, which can lead to unwanted long-term effects due to the persistence of uncontrolled CAR-T responses (Grupp et al. 2013 N Engl J Med 368(16):1509-18 (Non-Patent Document 3), Maude et al. 2014 2014 N Engl J Med 371(16):1507-17 (Non-Patent Document 5)).

[0005] One approach to limiting severe treatment-related toxicity and improving the safety of ACT is to impose constraints on CAR-T cell activation and proliferation by introducing adapter molecules into the immune synapse. Such adapter molecules include small molecule bimodular switches, such as the recently described folate-FITC switch (Kim et al. J Am Chem Soc 2015;137:2832-2835). Another approach involved artificially modified antibodies containing tags that guide and direct the specificity of CAR-T cells to target tumor cells (Ma et al. PNAS 2016;113(4):E450-458 (Non-Patent Document 7), Cao et al. Angew Chem 2016;128:1-6 (Non-Patent Document 8), Rogers et al. PNAS 2016;113(4):E459-468 (Non-Patent Document 9), Tamada et al. Clin Cancer Res 2012;18(23):6436-6445 (Non-Patent Document 10)).

[0006] However, existing approaches have several limitations. The immune synapse, which relies on molecular switches, requires the introduction of additional components, which may induce immune responses or cause nonspecific off-target effects. Furthermore, the complexity of such multicomponent systems can limit the efficacy and tolerability of treatments. Meanwhile, the introduction of tag structures into existing therapeutic monoclonal antibodies can affect the efficacy and safety profile of these constructs.

[0007] Therefore, targeted tumor therapy, especially adoptive T cell therapy, needs to be improved to meet the needs of cancer patients. Therefore, there is still a need to provide improved means that have the potential to improve the safety and efficacy of ACT and overcome the above-mentioned drawbacks. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Rosenberg and Restifo, Science 348(6230)(2015),62-68 [Non-patent document 2] Dudley et al.,J Clin Oncol 26(32)(2008),5233-5239 [Non-patent document 3] Grupp et al.,N Engl J Med 368(16)(2013),1509-1518 [Non-patent document 4] Kochenderfer et al.,J Clin Oncol.(2015)33(6):540-549,doi:10.1200 / JCO.2014.56.2025.Epub 2014 Aug 25 [Non-patent document 5] Maude et al.2014 2014 N Engl J Med 371(16):1507-17 [Non-patent document 6] Kim et al. J Am Chem Soc 2015;137:2832-2835 [Non-Patent Document 7] Ma et al.PNAS 2016;113(4):E450-458 [Non-patent document 8] Cao et al.Angew Chem 2016;128:1-6 [Non-Patent Document 9] Rogers et al.PNAS 2016;113(4):E459-468 [Non-Patent Document 10] Tamada et al. Clin Cancer Res 2012;18(23):6436-6445 Summary of the Invention

[0009] The present invention relates generally to antigen-binding receptors capable of specifically binding to variant Fc domains that have reduced Fc receptor binding, and to T cells that express these antigen-binding receptors.

[0010] In one aspect, the present invention relates to an antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain comprising an antigen-binding portion, wherein the antigen-binding portion is capable of specifically binding to a variant fragment crystallizable (Fc) domain but is incapable of specifically binding to a non-variant parent Fc domain, and the variant Fc domain comprises at least one amino acid substitution compared to the non-variant parent Fc domain.

[0011] In one embodiment, the Fc receptor binding of the variant Fc domain is reduced compared to Fc receptor binding of the non-variant parent Fc domain, particularly the Fc receptor is an Fcγ receptor or neonatal Fc receptor (FcRn). In one embodiment, Fc receptor binding is measured by surface plasmon resonance (SPR) at 25°C.

[0012] In one embodiment, the antigen-binding portion is an scFv, Fab, crossFab, or scFab. In a preferred embodiment, the antigen-binding portion is an scFv. In another preferred embodiment, the antigen-binding portion is a Fab or crossFab.

[0013] In one embodiment, the anchoring transmembrane domain is a transmembrane domain selected from the group consisting of CD8, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10, or DAP12 transmembrane domain, or a fragment thereof.

[0014] In one embodiment, the anchoring transmembrane domain is a CD28 transmembrane domain, and in particular, the anchoring transmembrane domain comprises the amino acid sequence of SEQ ID NO:11.

[0015] In one embodiment, the antigen-binding receptor further comprises at least one stimulatory signaling domain and / or at least one costimulatory signaling domain. In one embodiment, the at least one stimulatory signaling domain is individually selected from the group consisting of the intracellular domain of CD3z, the intracellular domain of FCGR3A, and the intracellular domain of NKG2D, or fragments thereof. In one embodiment, the at least one stimulatory signaling domain is a fragment of the intracellular domain of CD3z, and in particular, the at least one stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:13. In one embodiment, the at least one costimulatory signaling domain is individually selected from the group consisting of the intracellular domain of CD27, the intracellular domain of CD28, the intracellular domain of CD137, the intracellular domain of OX40, the intracellular domain of ICOS, the intracellular domain of DAP10, and the intracellular domain of DAP12, or fragments thereof. In one embodiment, the at least one costimulatory signaling domain is a fragment of the intracellular domain of CD28. In one embodiment, the antigen binding receptor comprises one stimulatory signaling domain comprising the intracellular domain of CD3z or a fragment thereof, and the antigen binding receptor comprises one costimulatory signaling domain comprising the intracellular domain of CD28 or a fragment thereof. In one embodiment, the stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:13, and the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:12.

[0016] In one embodiment, the extracellular domain is linked to the anchoring transmembrane domain, optionally via a peptide linker. In one embodiment, the peptide linker comprises the amino acid sequence GGGGS (SEQ ID NO: 17). In one embodiment, the anchoring transmembrane domain is linked to the co-signaling domain or the signaling domain, optionally via a peptide linker. In one embodiment, the signaling domain and / or the co-signaling domain are optionally linked via at least one peptide linker.

[0017] In one embodiment, the antigen-binding portion is an scFv fragment, which is linked at its C-terminus to the N-terminus of the anchoring transmembrane domain, optionally via a peptide linker.

[0018] In one embodiment, the antigen-binding portion is a Fab fragment or a crossFab fragment, which is linked at the C-terminus of the heavy chain to the N-terminus of the anchoring transmembrane domain, optionally via a peptide linker.

[0019] In one embodiment, the antigen-binding receptor comprises one co-signaling domain, which is linked at its N-terminus to the C-terminus of the anchoring transmembrane domain. In one embodiment, the antigen-binding receptor comprises one stimulatory signaling domain, which is linked at its N-terminus to the C-terminus of the costimulatory signaling domain.

[0020] In one embodiment, the non-mutated parent Fc domain is an IgG1 Fc domain or an IgG4 Fc domain, particularly a human IgG1 Fc domain. In one embodiment, the mutant Fc domain comprises at least one amino acid mutation at a position selected from the group consisting of L234, L235, 1253, H310, P331, P329, and H435 according to EU numbering, particularly the amino acid mutations are L234A, L235A, 1253A, N297A, H310A, P329G, and / or H435A.

[0021] In one embodiment, the variant Fc domain comprises at least one amino acid mutation at a position selected from the group consisting of L234, L235, and P329 according to EU numbering, in particular the amino acid mutations L234A, L235A, and P329G ("PGLALA").

[0022] In one embodiment, the variant Fc domain comprises an amino acid mutation of P329G according to EU numbering, and Fcγ receptor binding of the variant Fc domain is reduced compared to Fcγ receptor binding of the non-variant parent Fc domain, particularly the Fcγ receptor is human FcγRIIIa and / or FcγRIIa.

[0023] In one embodiment, the variant Fc domain comprises at least one amino acid mutation at a position selected from the group consisting of I253, H310, and H435 according to EU numbering, in particular the amino acid mutations I253A, H310A, and H435A ("AAA"), and the FcRn binding of the variant Fc domain is reduced compared to the FcRn binding of the non-variant parent Fc domain.

[0024] In one embodiment, at least one antigen binding moiety is capable of specifically binding to a variant Fc domain comprising a P329G mutation but is unable to specifically bind to a non-variant parent Fc domain, and the antigen binding moiety comprises: (i) (a) heavy chain complementarity determining region (CDR H) 1 amino acid sequence RYWMN (SEQ ID NO:1); (b) CDR H2 amino acid sequence TIFF0007642010000001.tif4128, and (c) CDR H3 amino acid sequence a heavy chain variable region (VH) comprising TIFF0007642010000002.tif4128; (ii)(d) light chain complementarity-determining region (CDR L) 1 amino acid sequence TIFF0007642010000003.tif4128(e) CDR L2 amino acid sequence GTNKRAP (SEQ ID NO:5), and (f) CDR L3 amino acid sequence TIFF0007642010000004.tif4128 containing the light chain variable region (VL) Includes.

[0025] In one embodiment, at least one antigen-binding portion is capable of specifically binding to a variant Fc domain comprising a P329G mutation but is incapable of specifically binding to a non-variant parent Fc domain, and the antigen-binding portion comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:32, and a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:33.

[0026] In one embodiment, at least one antigen-binding portion comprises a heavy chain variable region (VH) of SEQ ID NO:8 and a light chain variable region (VL) of SEQ ID NO:9.

[0027] In one embodiment, at least one antigen-binding portion is an scFv that can specifically bind to the mutated Fc domain comprising the P329G mutation but cannot specifically bind to the non-mutated parent Fc domain, and the antigen-binding receptor comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:31. In one embodiment, the antigen-binding receptor comprises the amino acid sequence of SEQ ID NO:7.

[0028] In one embodiment, at least one antigen-binding portion is a Fab fragment capable of specifically binding to the mutant Fc domain comprising the P329G mutation but incapable of specifically binding to the non-mutated parent Fc domain, and the antigen-binding receptor is a) a heavy chain fusion polypeptide that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:39 and SEQ ID NO:48; b) a light chain polypeptide that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:41 and SEQ ID NO:50; Includes.

[0029] In one embodiment, the antigen binding receptor is a) a heavy chain fusion polypeptide of SEQ ID NO:39; b) the light chain polypeptide of SEQ ID NO:41; Includes.

[0030] In one embodiment, at least one antigen binding moiety is capable of specifically binding to a variant Fc domain comprising I253A, H310A, and H435A ("AAA") mutations, but is unable to specifically bind to a non-variant parent Fc domain, and the antigen binding moiety comprises: (i) (a) heavy chain complementarity determining region (CDR H) 1 amino acid sequence SYGMS (SEQ ID NO:53); (b) the CDR H2 amino acid sequence SSGGSY (SEQ ID NO:54), and (c) CDR H3 amino acid sequence a heavy chain variable region (VH) comprising TIFF0007642010000005.tif4128; (ii)(d) light chain complementarity-determining region (CDR L) 1 amino acid sequence TIFF0007642010000006.tif4128(e) CDR L2 amino acid sequence KVSNRFS (SEQ ID NO:57), and (f) CDR L3 amino acid sequence TIFF0007642010000007.tif4128 containing the light chain variable region (VL) Includes.

[0031] In one embodiment, at least one antigen-binding portion is capable of specifically binding to a variant Fc domain comprising the I253A, H310A, and H435A ("AAA") mutations but is unable to specifically bind to the non-variant parent Fc domain, and the antigen-binding portion comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:61, and a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:62.

[0032] In one embodiment, at least one antigen-binding moiety a) the heavy chain variable region (VH) of SEQ ID NO:61; b) the light chain variable region (VL) of SEQ ID NO:62; Includes.

[0033] In one embodiment, at least one antigen-binding portion is an scFv that can specifically bind to a variant Fc domain comprising I253A, H310A, and H435A ("AAA") mutations but cannot specifically bind to the non-variant parent Fc domain, and the antigen-binding receptor comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 59. In one embodiment, the antigen-binding receptor comprises the amino acid sequence of SEQ ID NO: 59.

[0034] In one embodiment, at least one antigen-binding portion is a Fab fragment capable of specifically binding to the mutated Fc domain comprising the P329G mutation but incapable of specifically binding to the non-mutated parent Fc domain, and the antigen-binding receptor is a) a heavy chain fusion polypeptide that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:39; b) a light chain polypeptide that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:41; Includes.

[0035] In one embodiment, the antigen binding receptor is a) a heavy chain fusion polypeptide of SEQ ID NO:39; b) the light chain polypeptide of SEQ ID NO:41; Includes.

[0036] In one aspect, an isolated polynucleotide encoding an antigen-binding receptor described herein is provided. In one aspect, an isolated polynucleotide encoding a heavy chain fusion polypeptide or a light chain polypeptide of an antigen-binding receptor described herein is provided. In one aspect, a composition encoding an antigen-binding receptor described herein is provided, comprising a first isolated polynucleotide encoding a heavy chain fusion polypeptide and a second isolated polynucleotide encoding a light chain polypeptide.

[0037] In one aspect, there is provided a polypeptide encoded by a polynucleotide described herein or a polypeptide encoded by a composition described herein.

[0038] In one aspect, there are provided vectors, particularly expression vectors, comprising the polynucleotides described herein.

[0039] In one aspect, a transduced T cell is provided that comprises a polynucleotide described herein or a vector described herein. In one aspect, a transduced T cell is provided that is capable of expressing an antigen-binding receptor described herein. In one aspect, a transduced T cell described herein is provided that is co-transduced with a T cell receptor (TCR) that can specifically bind to a target antigen.

[0040] In one aspect, (A) a transduced T cell capable of expressing an antigen-binding receptor described herein; and (B) Antibodies containing variant Fc domains A kit comprising: the antigen-binding receptor is capable of specifically binding to the variant Fc domain but is incapable of specifically binding to the non-variant parent Fc domain; A kit is provided.

[0041] In one aspect, (A) an isolated polynucleotide encoding an antigen-binding receptor described herein; and (B) Antibodies containing variant Fc domains A kit comprising: the antigen-binding receptor is capable of specifically binding to the variant Fc domain but is incapable of specifically binding to the non-variant parent Fc domain; A kit is provided.

[0042] In one aspect, (A) a composition or vector described herein encoding an antigen-binding receptor described herein; and (B) Antibodies containing variant Fc domains A kit comprising: the antigen-binding receptor is capable of specifically binding to the variant Fc domain but is incapable of specifically binding to the non-variant parent Fc domain; A kit is provided.

[0043] In one embodiment, the non-mutated parent Fc domain is an IgG1 Fc domain or an IgG4 Fc domain, particularly a human IgG1 Fc domain. In one embodiment, a mutant Fc domain is provided comprising at least one amino acid mutation at a position selected from the group consisting of L234, L235, 1253, H310, P331, P329, and H435 according to EU numbering, particularly the amino acid mutations are L234A, L235A, 1253A, N297A, H310A, P329G, and / or H435A. In one embodiment, the mutant Fc domain comprises at least one amino acid mutation at a position selected from the group consisting of L234, L235, and P329 according to EU numbering, particularly the amino acid mutations L234A, L235A, and P329G ("PGLALA"). In one embodiment, the mutant Fc domain comprises the amino acid mutation P329G according to EU numbering. In one embodiment, the variant Fc domain comprises at least one amino acid mutation at a position selected from the group consisting of I253, H310, and H435 according to EU numbering, in particular the amino acid mutations I253A, H310A, and H435A ("AAA").

[0044] In one embodiment, an antibody comprising a variant Fc domain can specifically bind to an antigen on the surface of a tumor cell, particularly an antigen selected from the group consisting of FAP, CEA, p95, BCMA, EpCAM, MSLN, MCSP, HER-1, HER-2, HER-3, CD19, CD20, CD22, CD33, CD38, CD52Flt3, FOLR1, Trop-2, CA-12-5, HLA-DR, MUC-1 (mucin), A33 antigen, PSMA, PSCA, transferrin receptor, TNC (tenascin), and CA-IX, and / or to a peptide bound to a molecule of the human major histocompatibility complex (MHC). In one embodiment, an antibody comprising a variant Fc domain can specifically bind to an antigen selected from the group consisting of fibroblast activation protein (FAP), carcinoembryonic antigen (CEA), mesothelin (MSLN), CD20, folate receptor 1 (FOLR1), and tenascin (TNC).

[0045] In one aspect, there is provided a kit as described herein for use as a pharmaceutical.

[0046] In one aspect, there is provided an antigen binding receptor or transduced T cell as described herein for use as a medicament, wherein the transduced T cell expressing the antigen binding receptor is administered before, simultaneously with, or after administration of an antibody comprising a variant Fc domain, and the antigen binding receptor is capable of specific binding to the variant Fc domain but not to the non-variant parent Fc domain.

[0047] In one aspect, there is provided a kit as described herein for use in the treatment of a malignant disease. In one aspect, there is provided an antigen-binding receptor or transduced T cell as described herein for use in the treatment of a malignant disease, wherein the treatment comprises administration of a transduced T cell expressing an antigen-binding receptor prior to, concurrently with, or after administration of an antibody comprising a variant Fc domain, wherein the antigen-binding receptor is capable of specific binding to the variant Fc domain but incapable of specific binding to the non-variant parent Fc domain.

[0048] In one embodiment, the malignancy is selected from cancers of epithelial, endothelial, or mesothelial origin and hematological cancers.

[0049] In one embodiment, the transduced T cells are derived from cells isolated from the subject to be treated. In one embodiment, the transduced T cells are not derived from cells isolated from the subject to be treated.

[0050] In one embodiment, a method of treating a disease in a subject is provided, comprising administering to the subject transduced T cells capable of expressing an antigen-binding receptor described herein; and administering a therapeutically effective amount of an antibody comprising a variant Fc domain before, simultaneously with, or after administration of the transduced T cells, wherein the antigen-binding receptor is capable of specifically binding to the variant Fc domain but not to the non-variant parent Fc domain. In one embodiment, further comprising isolating T cells from the subject, and generating the transduced T cells by transducing the isolated T cells with a polynucleotide, composition, or vector described herein. In one embodiment, the T cells are transduced with a retroviral or lentiviral vector construct, or a non-viral vector construct. In one embodiment, the non-viral vector construct is a Sleeping Beauty minicircle vector.

[0051] In one embodiment, the transduced T cells are administered to a subject by intravenous infusion. In one embodiment, the transduced T cells are contacted with an anti-CD3 antibody and / or an anti-CD28 antibody prior to administration to a subject. In one embodiment, the transduced T cells are contacted with at least one cytokine, preferably interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-15 (IL-15), and / or interleukin-21, or variants thereof, prior to administration to a subject.

[0052] In one embodiment, the disease is a malignant disease. In one embodiment, the malignant disease is selected from cancers of epithelial, endothelial, or mesothelial origin and hematological cancers.

[0053] In one aspect, a method is provided for inducing lysis of a target cell, comprising contacting the target cell with a transduced T cell capable of expressing an antigen binding receptor described herein in the presence of an antibody comprising a variant Fc domain, wherein the antigen binding receptor is capable of specific binding to the variant Fc domain but not to the non-variant parent Fc domain.

[0054] In one embodiment, the target cell is a cancer cell. In one embodiment, the target cell expresses an antigen selected from the group consisting of FAP, CEA, p95, BCMA, EpCAM, MSLN, MCSP, HER-1, HER-2, HER-3, CD19, CD20, CD22, CD33, CD38, CD52Flt3, FOLR1, Trop-2, CA-12-5, HLA-DR, MUC-1 (mucin), A33 antigen, PSMA, PSCA, transferrin receptor, TNC (tenascin), and CA-IX. In one embodiment, the target cell expresses an antigen selected from the group consisting of carcinoembryonic antigen (CEA), mesothelin (MSLN), CD20, folate receptor 1 (FOLR1), and tenascin (TNC).

[0055] In one embodiment, the polynucleotides or transduced T cells described herein are used for the manufacture of a medicament, hi one embodiment, the medicament is for the treatment of a malignant disease. [The present invention 1001] 1. An antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain comprising an antigen-binding portion, wherein the antigen-binding portion is capable of specifically binding to a mutated fragment crystallizable (Fc) domain but is incapable of specifically binding to a non-mutated parent Fc domain, and the mutated Fc domain comprises at least one amino acid substitution compared to the non-mutated parent Fc domain. [The present invention 1002] 1001. An antigen-binding receptor of the present invention, wherein the Fc receptor binding of the mutated Fc domain is reduced compared to the Fc receptor binding of the non-mutated parent Fc domain, particularly wherein the Fc receptor is an Fcγ receptor or a neonatal Fc receptor (FcRn). [The present invention 1003] 1003. The antigen-binding receptor of either of invention 1001 or invention 1002, wherein the antigen-binding portion is an scFv, Fab, crossFab, or scFab. [The present invention 1004] An antigen-binding receptor of any of claims 1001 to 1003, wherein the anchoring transmembrane domain is a transmembrane domain selected from the group consisting of CD8, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10, or DAP12 transmembrane domains, or fragments thereof, and in particular, the anchoring transmembrane domain is a CD28 transmembrane domain or a fragment thereof. [The present invention 1005] The antigen-binding receptor of any of claims 1001 to 1004, further comprising at least one stimulatory signaling domain and / or at least one costimulatory signaling domain. [The present invention 1006] An antigen-binding receptor of any of claims 1001 to 1005, wherein at least one stimulatory signaling domain is individually selected from the group consisting of the intracellular domain of CD3z, the intracellular domain of FCGR3A, and the intracellular domain of NKG2D, or fragments thereof, and in particular, at least one stimulatory signaling domain is the intracellular domain of CD3z or a fragment thereof. [The present invention 1007] An antigen-binding receptor of any of claims 1001 to 1006, wherein at least one costimulatory signaling domain is individually selected from the group consisting of the intracellular domain of CD27, the intracellular domain of CD28, the intracellular domain of CD137, the intracellular domain of OX40, the intracellular domain of ICOS, the intracellular domain of DAP10, and the intracellular domain of DAP12, or fragments thereof, and in particular, wherein at least one costimulatory signaling domain is the intracellular domain of CD28 or a fragment thereof. [The present invention 1008] An antigen-binding receptor of any of claims 1001 to 1007, comprising one stimulatory signaling domain comprising the intracellular domain of CD28 or a fragment thereof, and one costimulatory signaling domain comprising the intracellular domain of CD3z or a fragment thereof. [The present invention 1009] The antigen-binding receptor of any of claims 1001 to 1008, wherein the antigen-binding portion is an scFv fragment, and the scFv fragment is linked at its C-terminus to the N-terminus of the anchoring transmembrane domain, optionally via a peptide linker. [The present invention 1010] The antigen-binding receptor of any of claims 1001 to 1009, wherein the antigen-binding portion is a Fab fragment or a crossFab fragment, and the Fab fragment or crossFab fragment is linked at the C-terminus of the heavy chain to the N-terminus of the anchoring transmembrane domain, optionally via a peptide linker. [The present invention 1011] The antigen-binding receptor of any of claims 1001 to 1010, wherein the mutant Fc domain comprises at least one amino acid mutation at a position selected from the group consisting of L234, L235, I253, H310, P331, P329, and H435 according to EU numbering, and in particular the amino acid mutation is L234A, L235A, I253A, N297A, H310A, P329G, and / or H435A. [The present invention 1012] The antigen-binding receptor of any of claims 1001 to 1011, wherein the mutant Fc domain comprises an amino acid mutation of P329G according to EU numbering, and the Fcγ receptor binding of the mutant Fc domain is reduced compared to the Fcγ receptor binding of the non-mutated parent Fc domain. [The present invention 1013] An antigen-binding receptor of any of claims 1001 to 1011, wherein the variant Fc domain comprises at least one amino acid mutation at a position selected from the group consisting of I253, H310, and H435 according to EU numbering, in particular the amino acid mutations I253A, H310A, and H435A ("AAA"), and wherein the FcRn binding of the variant Fc domain is reduced compared to the FcRn binding of the non-variant parent Fc domain. [The present invention 1014] at least one antigen-binding moiety capable of specifically binding to the variant Fc domain comprising the P329G mutation but not capable of specifically binding to the non-variant parent Fc domain, said antigen-binding moiety comprising: (i) (a) heavy chain complementarity determining region (CDR H) 1 amino acid sequence RYWMN (SEQ ID NO:1); (b) CDR H2 amino acid sequence TIFF0007642010000008.tif4128, and (c) CDR H3 amino acid sequence a heavy chain variable region (VH) comprising TIFF0007642010000009.tif4128; (ii)(d) light chain complementarity-determining region (CDR L) 1 amino acid sequence TIFF0007642010000010.tif4128(e) CDR L2 amino acid sequence GTNKRAP (SEQ ID NO:5), and (f) CDR L3 amino acid sequence TIFF0007642010000011.tif4128 containing the light chain variable region (VL) The antigen-binding receptor of any one of 1001 to 1012 of the present invention, comprising: [The present invention 1015] at least one antigen-binding moiety capable of specifically binding to a variant Fc domain comprising I253A, H310A, and H435A ("AAA") mutations but not to a non-variant parent Fc domain, said antigen-binding moiety comprising: (i) (a) heavy chain complementarity determining region (CDR H) 1 amino acid sequence SYGMS (SEQ ID NO:53); (b) the CDR H2 amino acid sequence SSGGSY (SEQ ID NO:54), and (c) CDR H3 amino acid sequence a heavy chain variable region (VH) comprising TIFF0007642010000012.tif4128; (ii)(d) light chain complementarity-determining region (CDR L) 1 amino acid sequence TIFF0007642010000013.tif4128(e) CDR L2 amino acid sequence KVSNRFS (SEQ ID NO:57), and (f) CDR L3 amino acid sequence TIFF0007642010000014.tif4128 containing the light chain variable region (VL) The antigen-binding receptor of any one of 1001 to 1011 or 1013 of the present invention, comprising: [The present invention 1016] A transduced T cell capable of expressing any one of the antigen-binding receptors of the present invention 1001 to 1015. [The present invention 1017] An isolated polynucleotide encoding any one of the antigen-binding receptors of the present invention 1001 to 1015. [The present invention 1018] A vector, particularly an expression vector, comprising a polynucleotide of the present invention. [The present invention 1019] A transduced T cell capable of expressing any one of the antigen-binding receptors of the present invention 1001 to 1015. [The present invention 1020] (A) a transduced T cell capable of expressing any one of antigen-binding receptors 1001 to 1015 of the present invention; (B) Antibodies containing variant Fc domains A kit comprising: the antigen-binding receptor is capable of specifically binding to the variant Fc domain but is incapable of specifically binding to the non-variant parent Fc domain; kit. [The present invention 1021] (A) an isolated polynucleotide encoding any one of antigen-binding receptors 1001 to 1015 of the present invention; (B) Antibodies containing variant Fc domains A kit comprising: the antigen-binding receptor is capable of specifically binding to the variant Fc domain but is incapable of specifically binding to the non-variant parent Fc domain; kit. [The present invention 1022] The kit of any of the present invention 1020 or 1021, wherein the antibody comprising the variant Fc domain is capable of specifically binding to an antigen selected from the group consisting of fibroblast activation protein (FAP), carcinoembryonic antigen (CEA), mesothelin (MSLN), CD20, folate receptor 1 (FOLR1), and tenascin (TNC). [The present invention 1023] The kit of any one of 1020 to 1022 of the present invention for use as a medicine. [The present invention 1024] The kit of any of claims 1020 to 1022 for use in the treatment of a disease, particularly for use in the treatment of a malignant disease. [The present invention 1025] before, simultaneously with, or after administration of an antibody comprising a variant Fc domain, Antigen-binding receptors capable of specifically binding to the mutated Fc domain but not to the non-mutated parent Fc domain transduced T cells expressing An antigen-binding receptor of any one of claims 1001 to 1015 or a transduced T cell of claim 1019 for use as a pharmaceutical. [The present invention 1026] before, simultaneously with, or after administration of an antibody comprising a variant Fc domain; Antigen-binding receptors capable of specifically binding to the mutated Fc domain but not to the non-mutated parent Fc domain Treatment of malignant diseases comprising administration of transduced T cells expressing 10. The antigen-binding receptor of any one of claims 1001 to 1015 or the transduced T cell of claim 1019 for use in [The present invention 1027] administering to a subject transduced T cells capable of expressing any one of the antigen-binding receptors of the present invention; administering a therapeutically effective amount of an antibody comprising a variant Fc domain prior to, concurrently with, or after administration of the transduced T cells; 1. A method of treating a disease in a subject, comprising: the antigen-binding receptor is capable of specifically binding to the variant Fc domain but is incapable of specifically binding to the non-variant parent Fc domain; method. [The present invention 1028] A method for inducing lysis of a target cell, comprising the step of contacting the target cell with a transduced T cell capable of expressing an antigen-binding receptor according to any one of claims 1001 to 1015 of the present invention, in the presence of an antibody comprising a variant Fc domain, the antigen-binding receptor is capable of specifically binding to the variant Fc domain but is incapable of specifically binding to the non-variant parent Fc domain; method. [The present invention 1029] For the manufacture of a medicament for the treatment of malignant diseases, An antigen-binding receptor of any one of 1001 to 1015 of the present invention, a polynucleotide of 1017 of the present invention, or a transduced T cell of 1019 of the present invention. Use of. [The present invention 1030] 10. An antigen binding receptor substantially as hereinbefore described with reference to any one of the Examples or any one of the accompanying drawings. [Brief explanation of the drawings]

[0056] [Figure 1] Figure 1 shows the architecture of exemplary antigen-binding receptors of the present invention. Figure 1A depicts the architectures of the anti-P329G-scFv-CD28ATD-CD28CSD-CD3zSSD format and the anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD format. The extracellular domain containing an antigen-binding portion capable of specifically binding to a mutant Fc domain containing the P329G mutation is shown. The antigen-binding portion consists of a variable heavy chain and a variable light chain, which are linked by a (Gly4Ser)4 linker. The Gly4Ser linker attached to the variable light chain connects the antigen recognition domain to the CD28 transmembrane domain (TM) fused to the intracellular costimulatory signaling domain (CSD) of CD28, which is in turn fused to the stimulatory signaling domain (SSD) of CD3z. Figure 1B shows the architecture of the anti-P329G-Fab-CD28ATD-CD28CSD-CD3zSSD and anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD formats. The extracellular domains are shown containing an antigen-binding portion capable of specifically binding to a mutant Fc domain containing the P329G mutation. The antigen-binding portion consists of an Ig heavy chain and an Ig light chain. A Gly4Ser linker attached to the heavy chain connects the antigen recognition domain to a CD28 transmembrane domain fused to the intracellular costimulatory signaling domain of CD28, which is in turn fused to the stimulatory signaling domain of CD3z. [Figure 2] Figure 2 is a schematic diagram illustrating the modular composition of an exemplary expression construct encoding an antigen-binding receptor of the invention. Figure 2A shows the P392G-targeted scFv format. Figure 2B shows the P392G-targeted Fab format. [Figure 3]FIG. 3 shows an exemplary IgG1 molecule with a P329G mutation in the Fc domain that is recognized by the anti-P329G antigen-binding receptor of the present invention. [Figure 4] Figure 4 is a schematic diagram of an IgG with the P329G mutation bound to a tumor-associated antigen (TAA), which can then be recognized by anti-P329G antigen-binding receptor-expressing T cells, thereby activating the T cells. [Figure 5] Figure 5 shows a schematic diagram of the Jurkat NFAT T cell reporter assay. IgG with the P329G mutation bound to a TAA can be recognized by Jurkat NFAT T cells expressing anti-P329G antigen-binding receptors. This recognition leads to cell activation, which can be detected by measuring luminescence (cps). [Figure 6] Figure 6 shows a Jurkat NFAT T cell reporter assay using CD20-expressing SUDHDL4 tumor cells as target cells. An anti-CD20 IgG antibody (GA101) with a P329G mutation was used. This antibody recognizes a tumor-associated antigen on the one hand and is recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention on the other hand. In Figure 6A, a sorted pool of Jurkat NFAT T cells expressing anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD was used as effector cells. In Figure 6B, a sorted pool of Jurkat NFAT T cells expressing anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD was used as effector cells. [Figure 7]Figure 7 shows a Jurkat NFAT T cell reporter assay using CD20 tumor cells as target cells. An anti-CD20 IgG antibody (GA101) with a P329G mutation was used. This antibody recognizes a tumor-associated antigen and is recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. In Figure 7A, single clone 5 of anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing Jurkat NFAT T cells was used as effector cells, and WSUDLCL2 cells were used as tumor cells. In Figure 7B, single clone 2 of anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing Jurkat NFAT T cells was used as effector cells, and WSUDLCL2 cells were used as tumor cells. In Figure 7C, single clone 5 of anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing Jurkat NFAT T cells was used as effector cells, and SUDHL4 cells were used as tumor cells. In Figure 7D, single clone 2 of anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing Jurkat NFAT T cells was used as effector cells, and SUDHL4 cells were used as tumor cells. [Figure 8A] Figure 8 shows a Jurkat NFAT T cell reporter assay performed using adherent FAP-expressing NIH / 3T3-huFAP cl 19 tumor cells as target cells. The anti-FAP IgG antibody clone 4B9 with the P329G mutation was used. This antibody recognizes a tumor-associated antigen and is recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. IgG DP47 / vk3 with the P329G mutation was included as an isotype control. In Figure 8A, a sorted pool of anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing Jurkat NFAT T cells was used as effector cells. [Figure 8B]Figure 8 shows a Jurkat NFAT T cell reporter assay performed using adherent FAP-expressing NIH / 3T3-huFAP cl 19 tumor cells as target cells. The anti-FAP IgG antibody clone 4B9 with the P329G mutation was used. This antibody recognizes a tumor-associated antigen and is recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. IgG DP47 / vk3 with the P329G mutation was included as an isotype control. In Figure 8B, a sorted pool of anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD-expressing Jurkat NFAT T cells was used as effector cells. [Figure 8C] Figure 8 shows a Jurkat NFAT T cell reporter assay performed using adherent FAP-expressing NIH / 3T3-huFAP cl 19 tumor cells as target cells. The anti-FAP IgG antibody clone 4B9 with the P329G mutation was used. This antibody recognizes a tumor-associated antigen and is recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. IgG DP47 / vk3 with the P329G mutation was included as an isotype control. In Figure 8C, a sorted pool of anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing Jurkat NFAT T cells was used as effector cells. [Figure 8D] Figure 8 shows a Jurkat NFAT T cell reporter assay performed using adherent FAP-expressing NIH / 3T3-huFAP cl 19 tumor cells as target cells. The anti-FAP IgG antibody clone 4B9 with the P329G mutation was used. This antibody recognizes a tumor-associated antigen and is recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. IgG DP47 / vk3 with the P329G mutation was included as an isotype control. In Figure 8D, a sorted pool of anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD-expressing Jurkat NFAT T cells was used as effector cells. [Figure 9A]Figure 9 shows a Jurkat NFAT T cell reporter assay using adherent CEA-expressing MKN45 tumor cells as target cells. Either the anti-CEA IgG clone A5B7 or the anti-CEA IgG clone T84 LCHA, both of which contain the P329G mutation, was used. These antibodies recognize tumor-associated antigens and are recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. Additionally, the IgG DP47 / vk3, which contains the P329G mutation, was included as an isotype control. In Figures 9A and 9B, a sorted pool of anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing NFAT T cells was used as effector cells. [Figure 9B] Figure 9 shows a Jurkat NFAT T cell reporter assay using adherent CEA-expressing MKN45 tumor cells as target cells. Either the anti-CEA IgG clone A5B7 or the anti-CEA IgG clone T84 LCHA, both of which contain the P329G mutation, was used. These antibodies recognize tumor-associated antigens and are recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. Additionally, the IgG DP47 / vk3, which contains the P329G mutation, was included as an isotype control. In Figures 9A and 9B, a sorted pool of anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing NFAT T cells was used as effector cells. [Figure 9C]Figure 9 shows a Jurkat NFAT T cell reporter assay using adherent CEA-expressing MKN45 tumor cells as target cells. Either the anti-CEA IgG clone A5B7 or the anti-CEA IgG clone T84 LCHA, both of which contain the P329G mutation, was used. These antibodies recognize tumor-associated antigens and are recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. Additionally, the IgG DP47 / vk3, which contains the P329G mutation, was included as an isotype control. In Figures 9C and 9D, a sorted pool of anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD-expressing NFAT T cells was used as effector cells. [Figure 9D] Figure 9 shows a Jurkat NFAT T cell reporter assay using adherent CEA-expressing MKN45 tumor cells as target cells. Either the anti-CEA IgG clone A5B7 or the anti-CEA IgG clone T84 LCHA, both of which contain the P329G mutation, was used. These antibodies recognize tumor-associated antigens and are recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. Additionally, the IgG DP47 / vk3, which contains the P329G mutation, was included as an isotype control. In Figures 9C and 9D, a sorted pool of anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD-expressing NFAT T cells was used as effector cells. [Figure 10A]Figure 10 shows a Jurkat NFAT T cell reporter assay using adherent CEA-expressing MKN45 tumor cells as target cells. Either the anti-CEA clone CH1A1A 98 99 or the anti-CEA IgG clone hMN14 IgG, both of which contain the P329G mutation, was used. These antibodies recognize tumor-associated antigens and are recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. Additionally, the IgG DP47 / vk3, which contains the P329G mutation, was included as an isotype control. In Figures 10A and 10B, a sorted pool of anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD-expressing NFAT T cells was used as effector cells. [Figure 10B] Figure 10 shows a Jurkat NFAT T cell reporter assay using adherent CEA-expressing MKN45 tumor cells as target cells. Either the anti-CEA clone CH1A1A 98 99 or the anti-CEA IgG clone hMN14 IgG, both of which contain the P329G mutation, was used. These antibodies recognize tumor-associated antigens and are recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. Additionally, the IgG DP47 / vk3, which contains the P329G mutation, was included as an isotype control. In Figures 10A and 10B, a sorted pool of anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD-expressing NFAT T cells was used as effector cells. [Figure 10C]Figure 10 shows a Jurkat NFAT T cell reporter assay using adherent CEA-expressing MKN45 tumor cells as target cells. Either the anti-CEA clone CH1A1A 98 99 or the anti-CEA IgG clone hMN14 IgG, both of which contain the P329G mutation, was used. These antibodies recognize tumor-associated antigens and are recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. Additionally, the IgG DP47 / vk3, which contains the P329G mutation, was included as an isotype control. In Figures 10C and 10D, a sorted pool of anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing NFAT T cells was used as effector cells. [Figure 10D] Figure 10 shows a Jurkat NFAT T cell reporter assay using adherent CEA-expressing MKN45 tumor cells as target cells. Either the anti-CEA clone CH1A1A 98 99 or the anti-CEA IgG clone hMN14 IgG, both of which contain the P329G mutation, was used. These antibodies recognize tumor-associated antigens and are recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. Additionally, the IgG DP47 / vk3, which contains the P329G mutation, was included as an isotype control. In Figures 10C and 10D, a sorted pool of anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing NFAT T cells was used as effector cells. [Figure 11A]Figure 11 shows a Jurkat NFAT T cell reporter assay using adherent TNC-expressing CT26TNC cl 19 tumor cells as target cells. The anti-TNC IgG clone A2B10 with the P329G mutation was used as the IgG antibody. This antibody recognizes tumor-associated antigens and is recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. Additionally, the IgG DP47 / vk3 with the P329G mutation was included as an isotype control. In Figures 11A and 11B, a sorted pool of anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD-expressing NFAT T cells was used as effector cells. [Figure 11B] Figure 11 shows a Jurkat NFAT T cell reporter assay using adherent TNC-expressing CT26TNC cl 19 tumor cells as target cells. The anti-TNC IgG clone A2B10 with the P329G mutation was used as the IgG antibody. This antibody recognizes tumor-associated antigens and is recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. Additionally, the IgG DP47 / vk3 with the P329G mutation was included as an isotype control. In Figures 11A and 11B, a sorted pool of anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD-expressing NFAT T cells was used as effector cells. [Figure 11C] Figure 11 shows a Jurkat NFAT T cell reporter assay using adherent TNC-expressing CT26TNC cl 19 tumor cells as target cells. The anti-TNC IgG clone A2B10 with the P329G mutation was used as the IgG antibody. This antibody recognizes tumor-associated antigens and is recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. Additionally, the IgG DP47 / vk3 with the P329G mutation was included as an isotype control. In Figures 11C and 11D, a sorted pool of anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing NFAT T cells was used as effector cells. [Figure 11D]Figure 11 shows a Jurkat NFAT T cell reporter assay using adherent TNC-expressing CT26TNC cl 19 tumor cells as target cells. The anti-TNC IgG clone A2B10 with the P329G mutation was used as the IgG antibody. This antibody recognizes tumor-associated antigens and is recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. Additionally, the IgG DP47 / vk3 with the P329G mutation was included as an isotype control. In Figures 11C and 11D, a sorted pool of anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing NFAT T cells was used as effector cells. [Figure 12A] Figures 12A and 12B show Jurkat NFAT T cell reporter assays using adherent TNC-expressing CT26TNC cl 19 tumor cells as target cells. The anti-TNC IgG clone A2B10 with the P329G mutation was used. This antibody recognizes a tumor-associated antigen and is recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. Additionally, IgG DP47 / vk3 with the P329G mutation was included as an isotype control. A sorted pool of anti-P329G-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing Jurkat NFAT T cells was used as effector cells. [Figure 12B] Figures 12A and 12B show Jurkat NFAT T cell reporter assays using adherent TNC-expressing CT26TNC cl 19 tumor cells as target cells. The anti-TNC IgG clone A2B10 with the P329G mutation was used. This antibody recognizes a tumor-associated antigen and is recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. Additionally, IgG DP47 / vk3 with the P329G mutation was included as an isotype control. A sorted pool of anti-P329G-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing Jurkat NFAT T cells was used as effector cells. [Figure 13A]Figure 13 shows a Jurkat NFAT T cell reporter assay using CD20 tumor cells as target cells. Anti-CD20 IgG antibodies (GA101) containing either the P329G and LALA mutations, the P329G and D265A mutations, or only the LALA mutation, or no mutations, were used to detect tumor-associated antigens, which were recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. In Figure 13A, a cell pool of anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD-expressing Jurkat NFAT T cells was used as effector cells, and SUDHL4 cells were used as tumor cells. [Figure 13B] Figure 13 shows a Jurkat NFAT T cell reporter assay using CD20 tumor cells as target cells. Anti-CD20 IgG antibodies (GA101) containing either the P329G and LALA mutations, the P329G and D265A mutations, or only the LALA mutation, or no mutations, were used to detect tumor-associated antigens, which were recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. In Figure 13B, a pool of Jurkat NFAT T cells expressing anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD was used as effector cells, and SUDHL4 cells were used as tumor cells. [Figure 14A] Figure 14 shows a Jurkat NFAT T cell reporter assay using CD20 tumor cells as target cells. Anti-CD20 IgG antibodies (GA101) with P329G and LALA mutations, P329G mutation only, LALA mutation only, or no mutations were used to detect tumor-associated antigens, which were recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. In Figure 14A, a cell pool of anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD-expressing Jurkat NFAT T cells was used as effector cells, and SUDHL4 cells were used as tumor cells. [Figure 14B] Figure 14 shows a Jurkat NFAT T cell reporter assay using CD20 tumor cells as target cells. Anti-CD20 IgG antibodies (GA101) with P329G and LALA mutations, P329G mutation only, LALA mutation only, or no mutations were used to detect tumor-associated antigens, which were recognized by Jurkat NFAT T cells expressing the antigen-binding receptor of the present invention. In Figure 14B, a cell pool of anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD-expressing Jurkat NFAT T cells was used as effector cells, and SUDHL4 cells were used as tumor cells. DETAILED DESCRIPTION OF THE INVENTION

[0057] Detailed Description definition As used herein, terms are used as commonly used in the art unless otherwise defined below.

[0058] An "activating Fc receptor" is an Fc receptor that, following binding by the Fc domain of an antibody, triggers signaling events that stimulate the receptor-bearing cell to carry out effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0059] Antibody-dependent cell-mediated cytotoxicity ("ADCC") is an immune mechanism that leads to the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which an Fc region-containing antibody or its derivative binds, typically via a protein portion N-terminal to the Fc region. As used herein, the term "reduced ADCC" is defined as a reduction in the number of target cells lysed by the ADCC mechanism defined above in a given time period at a given antibody concentration in the medium surrounding the target cells, and / or an increase in the antibody concentration in the medium surrounding the target cells required to achieve lysis of a given number of targets by the ADCC mechanism in a given time period. The reduced ADCC is compared to the ADCC mediated by the same antibody, but without the introduced mutation, produced by the same type of host cell using the same standard production, purification, formulation, and storage methods (known to those skilled in the art). For example, the reduction in ADCC mediated by an antibody containing an amino acid mutation in its Fc domain that reduces ADCC is compared to the ADCC mediated by the same antibody without this amino acid mutation in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, e.g., PCT Publication No. WO 2006 / 082515 or PCT Publication No. WO 2012 / 130831).

[0060] An "effective amount" of an agent (eg, an antibody) refers to the amount necessary to effect a physiological change in a cell or tissue to which it is administered.

[0061] "Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, "binding affinity," as used herein, refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding moiety and an antigen and / or a receptor and its ligand). The affinity of a molecule X for its partner Y is generally determined by the dissociation and association rate constants (k off and k on) is the dissociation constant (K D ) Thus, equivalent affinities can involve different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by established methods known in the art, including those described herein. A preferred method for measuring affinity is surface plasmon resonance (SPR), with a preferred measurement temperature of 25°C.

[0062] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure different from the general chemical structure of an amino acid but that function similarly to a naturally occurring amino acid. Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0063] As used herein, the term "amino acid mutation" encompasses amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be used to arrive at a final construct, as long as the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor. Deletions and insertions in amino acid sequences include amino- and / or carboxy-terminal deletions and amino acid insertions. A particular amino acid mutation is an amino acid substitution. For purposes such as modifying the binding characteristics of an Fc region, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid that has different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include substitutions with unnatural amino acids or substitutions with natural amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods include site-directed mutagenesis, PCR, gene synthesis, and the like. Altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful. Various names may be used herein to indicate the same amino acid mutation. For example, a proline to glycine substitution at position 329 of the Fc domain is referred to as 329G, G329, G 329 , P329G or Pro329Gly.

[0064] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity. Thus, in the context of the present invention, the term antibody relates to both complete immunoglobulin molecules and parts of such immunoglobulin molecules. Furthermore, the term also relates to modified and / or altered antibody molecules, particularly mutant antibody molecules, as discussed herein. The term also relates to recombinantly or synthetically produced / synthesized antibodies. In the context of the present invention, the term antibody is used interchangeably with the term immunoglobulin.

[0065] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). See also WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. Diabodies are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific. See, for example, EP 404,097, WO 1993 / 01161, Hudson et al., Nat Med 9, 129-134 (2003), and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody (Domantis, Inc., Waltham, Massachusetts; see, for example, U.S. Patent No. 6,248,516 (B1)). Antibody fragments can be produced by a variety of techniques, including but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0066] As used herein, the term "antigen-binding molecule" refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are immunoglobulins and their derivatives, such as fragments thereof, and antigen-binding receptors and their derivatives.

[0067] As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding moiety can target an entity to which it is attached (e.g., an immunoglobulin or antigen-binding receptor) to a target site, such as a specific type of tumor cell or tumor stroma bearing the antigenic determinant, or to an immunoglobulin that binds to the antigenic determinant on a tumor cell. In another embodiment, an antigen-binding moiety can activate signaling by its target antigen. For example, signaling is activated when an antigenic determinant binds to an antigen-binding receptor on a T cell. In the context of the present invention, an antigen-binding moiety can be included in an antibody and fragments thereof, as well as an antigen-binding receptor and fragments thereof, as defined further herein. An antigen-binding moiety comprises an antigen-binding domain comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In certain embodiments, an antigen-binding moiety can comprise an immunoglobulin constant region known in the art, as defined further herein. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include either of two isotypes: kappa and lambda.

[0068] In the context of the present invention, the term "antigen-binding receptor" refers to an antigen-binding molecule comprising an anchoring transmembrane domain and an extracellular domain comprising at least one antigen-binding moiety. An antigen-binding receptor can be composed of polypeptide parts from different sources. Therefore, it can also be understood as a "fusion protein" and / or a "chimeric protein." Typically, a fusion protein is a protein created by joining two or more genes (or preferably cDNAs) that originally encoded separate proteins. Translation of this fusion gene (or fusion cDNA) preferably results in a single polypeptide that possesses functional properties derived from each of the original proteins. Recombinant fusion proteins are artificially created by recombinant DNA technology for use in biological research or therapy. The antigen-binding receptor of the present invention will be described in further detail below. In the context of the present invention, a CAR (chimeric antigen receptor) is understood to be an antigen-binding receptor comprising an extracellular portion comprising an antigen-binding moiety fused to an anchoring transmembrane domain via a spacer sequence, the anchoring transmembrane domain itself being fused to the intracellular signaling domains of CD3z and CD28.

[0069] The term "antigen-binding site" refers to the site, i.e., one or more amino acid residues, of an antigen-binding molecule that interacts with an antigen. For example, the antigen-binding site of an antibody or antigen-binding receptor comprises amino acid residues from the complementarity-determining region (CDR). A native immunoglobulin molecule typically has two antigen-binding sites, while a Fab or scFv molecule typically has one antigen-binding site.

[0070] The term "antigen-binding domain" refers to a part of an antibody or antigen-binding receptor that comprises an area that specifically binds to and is complementary to part or all of an antigen. An antigen-binding domain can be provided, for example, by one or more immunoglobulin variable domains (also referred to as variable regions). In particular, an antigen-binding domain comprises an immunoglobulin light chain variable region (VL) and an immunoglobulin heavy chain variable region (VH).

[0071] The term "variable region" or "variable domain" refers to the domain of an immunoglobulin heavy or light chain that is involved in antigen binding. The variable domains of the heavy and light chains (VH and VL, respectively) of native antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, e.g., Kindt et al., Kuby Immunology, 6 th ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain is typically sufficient to confer antigen-binding specificity.

[0072] As used herein, the term "ATD" refers to an "anchoring transmembrane domain" that defines a polypeptide stretch that can integrate with the cell membrane of a cell. ATM can be fused to additional extracellular and / or intracellular polypeptide domains, in which case these extracellular and / or intracellular polypeptide domains will also be bound to the cell membrane. In the context of the antigen-binding receptor of the present invention, ATM provides membrane attachment and membrane binding of the antigen-binding receptor of the present invention. The antigen-binding receptor of the present invention comprises at least one ATM and an extracellular domain comprising an antigen-binding portion. In addition, ATM may be fused to an additional intracellular signaling domain.

[0073] The term "bind to" as used in the context of the antigen-binding receptor of the present invention defines the mutual binding (interaction) between the "antigen-interaction site" and an antigen. In the antigen-binding receptor of the present invention, the term "antigen-interaction site" defines a polypeptide motif that exhibits the ability to specifically interact with a specific antigen or a specific group of antigens (i.e., a variant Fc domain). Such binding / interaction is also understood to define "specific recognition". The term "specifically recognize" means, in the present invention, that the antigen-binding receptor can specifically interact with and / or bind to a modified molecule as defined herein, while not recognizing an unmodified molecule. The antigen-binding portions of the antigen-binding receptor can recognize, interact with, and / or bind to different epitopes on the same molecule. This term relates to the specificity of the antigen-binding receptor, i.e., its ability to distinguish between specific regions of modified molecules, i.e., a variant Fc domain, as defined herein. The specific interaction of an antigen-interaction site with its specific antigen can result in the initiation of a signal, for example, by inducing conformational changes in the polypeptide containing the antigen, oligomerization of the polypeptide containing the antigen, or oligomerization of the antigen-binding receptor. Thus, the specific motif in the amino acid sequence of the antigen-interaction site and the antigen bind to each other as a result of their primary, secondary, or tertiary structure, and also as a result of secondary modifications of the structure. Therefore, the term "binding to" not only relates to a linear epitope, but also to a conformational epitope, a structural epitope, or a discontinuous epitope consisting of two regions of a target molecule or part thereof. In the context of the present invention, a conformational epitope is defined by two or more discontinuous amino acid sequences that are separate in the primary sequence but assemble on the surface of the molecule when the polypeptide is folded into a native protein (Sela, Science 166 (1969), 1365 and Laver, Cell 61 (1990), 553-536). Furthermore, the term "bind to" is used interchangeably with the term "interact with" in the context of the present invention.The ability of an antigen-binding receptor or an antigen-binding portion of an antibody (e.g., a Fab or scFv domain) to bind to a specific target antigenic determinant can be measured by either enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analyzed with a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of the antigen-binding portion to an unrelated protein is less than about 10% of the binding of the antigen-binding portion to the target antigen, particularly as measured by SPR. In certain embodiments, the antigen-binding portion binds to the target antigen with a binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10. -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 Dissociation constant (K D). As used herein, the term "specific binding" means that the molecules of the present invention do not cross-react or essentially do not cross-react with (poly)peptides of similar structure, i.e., with the non-mutated parent Fc domain (to which the antigen-binding receptor of the present invention can specifically bind). Thus, the antigen-binding receptor of the present invention specifically binds / interacts with the mutant Fc domain. The cross-reactivity of a group of constructs under investigation can be tested, for example, by assessing the binding of a group of antigen-binding moieties to the mutant Fc domain of interest and the non-mutated parent Fc domain under conventional conditions (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988) and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1999)). In the methods provided herein, only constructs (i.e., Fab fragments, scFvs, etc.) that bind to the variant Fc domain of interest but do not or essentially do not bind to the non-variant parent Fc domain are considered specific for the variant Fc domain of interest and are selected for further studies. These methods may include, inter alia, binding studies, blocking, and competition studies with structurally and / or functionally closely related Fc domains. Binding studies may also include FACS analysis, surface plasmon resonance (SPR, e.g., by BIAcore®), analytical ultracentrifugation, isothermal titration calorimetry, fluorescence anisotropy, fluorescence spectroscopy, or radiolabeled ligand binding assays.

[0074] The term "CDR" used herein relates to the "complementarity determining region" as known in the art. CDR is the part of an immunoglobulin or antigen-binding receptor that determines the specificity of the molecule and contacts with specific ligands. CDR is the most variable part of the molecule and contributes to the antigen-binding diversity of these molecules. Each V domain has three CDR regions: CDR1, CDR2 and CDR3. CDR-H refers to the CDR region of the variable heavy chain, and CDR-L relates to the CDR region of the variable light chain. VH refers to the variable heavy chain, and VL refers to the variable light chain. The CDR regions of Ig-derived regions can be determined as described in "Kabat" ("Sequences of Proteins of Immunological Interest", 5th ed. NIH Publication no. 91-3242 USDepartment of Health and Human Services (1991); Chothia J. Mol. Biol. 196 (1987), 901-917) or "Chothia" (Nature 342 (1989), 877-883).

[0075] The term "CD3z" refers to the T-cell surface glycoprotein CD3 zeta chain, also known as the "T-cell receptor T3 zeta chain" and "CD247."

[0076] The term "chimeric antigen receptor" or "chimeric receptor" or "CAR" refers to an antigen-binding receptor composed of an extracellular portion of an antigen-binding moiety (e.g., a single-chain antibody domain) fused to the intracellular signaling domains of CD3z and CD28 by spacer sequences. The present invention also provides antigen-binding receptors in which the antigen-binding moiety is a Fab or crossFab fragment. The term "CAR" in its broadest form is understood to include antigen-binding receptors composed of an extracellular portion comprising an antigen-binding moiety fused to CD3z and fragments thereof and CD28 and fragments thereof, optionally via one or several peptide linkers.

[0077] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0078] A "crossover Fab molecule" (also referred to as "crossFab" or "crossover Fab fragment") refers to a Fab molecule in which the variable or constant regions of the Fab heavy chain and the Fab light chain have been exchanged. That is, a crossFab fragment contains a peptide chain consisting of a light chain variable region and a heavy chain constant region, and a peptide chain consisting of a heavy chain variable region and a light chain constant region. For clarity, the peptide chain containing the heavy chain constant region in a crossFab fragment in which the variable regions of the Fab light chain and the Fab heavy chain have been exchanged is referred to herein as the heavy chain of the crossover Fab molecule. Conversely, the peptide chain containing the heavy chain variable region in a crossFab fragment in which the constant regions of the Fab light chain and the Fab heavy chain have been exchanged is referred to herein as the heavy chain of the crossFab fragment. Thus, a crossFab fragment contains a heavy or light chain (VH-CL) consisting of a heavy chain variable region and a light chain constant region, and a heavy or light chain (VL-CH1) consisting of a light chain variable region and a heavy chain constant region. In contrast, a "conventional Fab" molecule refers to a Fab molecule in its native format, i.e., a Fab molecule comprising a heavy chain (VH-CH1) composed of a heavy chain variable region and a heavy chain constant region, and a light chain (VL-CL) composed of a light chain variable region and a light chain constant region.

[0079] As used herein, the term "CSD" refers to a costimulatory signaling domain.

[0080] The term "effector function" refers to biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0081] As used herein, the terms "engineer," "engineered," and "engineering" are intended to encompass any manipulation or post-translational modification of the peptide backbone of a natural or recombinant polypeptide or fragment thereof. Engineering includes modification of the amino acid sequence, modification of the glycosylation pattern, or modification of the side groups of individual amino acids, as well as combinations of these approaches.

[0082] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide having a series of designated nucleic acid elements that enable transcription of a specific nucleic acid in a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector comprises a nucleic acid sequence to be transcribed and a promoter and other sequences. In certain embodiments, the expression cassettes of the present invention comprise a polynucleotide sequence encoding an antigen-binding molecule of the present invention or a fragment thereof.

[0083] "Fab molecule" refers to a protein consisting of the VH and CH1 domains of the heavy chain ("Fab heavy chain") and the VL and CL domains of the light chain ("Fab light chain") of an antigen-binding molecule.

[0084] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term encompasses native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the "EU numbering" system, also known as the EU index, as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, a subunit of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain that can stably self-associate. For example, a subunit of an IgG Fc domain comprises the IgG CH2 constant domain and the IgG CH3 constant domain.

[0085] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains, FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0086] The term "full-length antibody" refers to an antibody consisting of two "full-length antibody heavy chains" and two "full-length antibody light chains." A "full-length antibody heavy chain" is a polypeptide consisting of, from N- to C-terminus, an antibody heavy chain variable domain (VH), antibody heavy chain constant domain 1 (CH1), antibody hinge region (HR), antibody heavy chain constant domain 2 (CH2), and antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3, and optionally, in the case of antibodies of the subclass IgE, antibody heavy chain constant domain 4 (CH4). Preferably, a "full-length antibody heavy chain" is a polypeptide consisting of, from N- to C-terminus, VH, CH1, HR, CH2, and CH3. A "full-length antibody light chain" is a polypeptide consisting, from N- to C-terminus, of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), abbreviated as VL-CL. The antibody light chain constant domain (CL) can be κ (kappa) or λ (lambda). The two full-length antibody chains are linked together by interpolypeptide disulfide bonds between the CL domain and the CH1 domain and between the hinge regions of the full-length antibody heavy chain. Typical examples of full-length antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE). The full-length antibodies used in accordance with the present invention may be derived from a single species, e.g., human, or may be chimerized or humanized. In some embodiments, the full-length antibodies used in accordance with the present invention, i.e., therapeutic antibodies comprising a variant Fc domain, comprise two antigen-binding sites, each formed by a VH and VL pair, that specifically bind to the same antigen. In further embodiments, the full-length antibodies used in accordance with the present invention comprise two antigen-binding sites, each formed by a VH and VL pair, where the two antigen-binding sites bind to different antigens, e.g., the antibody is bispecific. The C-terminus of the heavy or light chain of the full-length antibody refers to the last C-terminal amino acid of the heavy or light chain.

[0087] By "fused" is meant that the components (eg, the Fab and transmembrane domains) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0088] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells encompass "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. Host cells are any type of cell line that can be used to generate the antibodies used in accordance with the present invention. Host cells include cultured mammalian cells such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name but a few, and also include cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissue.

[0089] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally distinct loops ("hypervariable loops"). Generally, a native four-chain antibody contains six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs generally contain amino acid residues from the hypervariable loops and / or from the complementarity-determining regions (CDRs), the latter of which are most highly sequence variable and / or involved in antigen recognition. With the exception of CDR1 in VH, CDRs generally contain amino acid residues that form the hypervariable loops. Hypervariable regions (HVRs) are also referred to as complementarity-determining regions (CDRs), and these terms are used interchangeably herein to refer to the portions of the variable regions that form the antigen-binding regions. This specific region is described in Kabat et al., US Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J Mol Biol 196:901-917 (1987), and these definitions, when compared with each other, include overlapping or subsets of amino acid residues. Nevertheless, application of either definition to refer to the CDRs of an antibody and / or antigen-binding receptor or variant thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues that encompass the CDRs defined by each of the above-mentioned references are set forth in Table 1 below for comparison. The exact number of residues that encompass a particular CDR will vary depending on the sequence and size of the CDR. One skilled in the art can routinely determine which residues constitute a particular CDR, given the amino acid sequence of the variable region of an antibody.

[0090] (Table 1) CDR definition 1 TIFF0007642010000015.tif33128 1 The numbering of all CDR definitions in Table 1 follows the numbering scheme established by Kabat et al. (see below). 2 As used in Table 1, "AbM" with a lower case "b" refers to the CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0091] Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. One skilled in the art can unambiguously assign this Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth in Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in the antigen-binding portion variable region are in accordance with the Kabat numbering system. The polypeptide sequences in the sequence listing are not numbered according to the Kabat numbering system. However, converting the numbering of the sequences in the sequence listing to Kabat numbering is considered well within the ordinary skill of one of ordinary skill in the art.

[0092] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0093] An "isolated nucleic acid" molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, that has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides encompass polynucleotide molecules contained in cells that normally contain the polynucleotide molecule, but that exist extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive-strand, negative-strand, and double-stranded forms. Isolated polynucleotides or nucleic acids of the present invention further include such molecules produced synthetically. Polynucleotides or nucleic acids may or may not contain regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0094] A nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with other nucleotides, or up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These modifications of the reference sequence can be found at the 5'-terminal or 3'-terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, scattered among the residues in the reference sequence, or scattered in one or more contiguous groups within the reference sequence. In practice, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the invention can be determined conventionally using known computer programs, such as those described below with respect to polypeptides (e.g., ALIGN-2).

[0095] An "isolated polypeptide" or variant or derivative thereof refers to a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated in the present invention, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0096] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after aligning the sequences to maximize percent sequence identity and introducing gaps, if necessary, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, the sequence comparison computer program ALIGN-2 is used to generate % amino acid sequence identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, CA) or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, such as digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. When ALIGN-2 is used for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, or with, or relative to a given amino acid sequence B (which can also be translated as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, or with, or relative to a given amino acid sequence B) is calculated as follows: Fraction X / Y×100 where X is the number of amino acid residues that the sequence alignment program ALIGN-2 reports as an identical match in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not match the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program, as described in the immediately preceding paragraph.

[0097] The term "nucleic acid molecule" refers to the sequence of bases, including purine and pyrimidine bases, contained in a polynucleotide, which bases represent the primary structure of the nucleic acid molecule. As used herein, the term nucleic acid molecule includes DNA, cDNA, genomic DNA, RNA, synthetic DNA, and mixed polymers containing two or more of these molecules. The term nucleic acid molecule also includes both sense and antisense strands. Furthermore, the nucleic acid molecules described herein may contain non-natural or derivatized nucleotide bases, as would be readily understood by one of skill in the art.

[0098] The term "package insert" is used to refer to the instructions customarily included in commercial packaging of a therapeutic product that contain information regarding the indications, usage, dosage, administration, concomitant treatments, contraindications and / or precautions for use of that therapeutic product.

[0099] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional components that are unacceptably toxic to the subject to whom the formulation is to be administered. Pharmaceutical compositions typically include one or more pharmaceutically acceptable carriers.

[0100] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0101] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term polypeptide refers to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, protein, amino acid chain, or any other term used to refer to a chain of two or more amino acids are encompassed within the definition of polypeptide, and the term polypeptide can be used in place of or interchangeably with any of these terms. The term polypeptide also refers to products of post-expression modifications of the polypeptide, such as, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with unnatural amino acids. A polypeptide can be obtained from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated by any method, including chemical synthesis. Polypeptides of the invention can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids in size. Polypeptides can have a defined three-dimensional structure, but do not necessarily have such a structure. Polypeptides that have a defined three-dimensional structure are said to be folded, while polypeptides that do not have a defined three-dimensional structure and can adopt many different conformations are said to be unfolded.

[0102] The term "polynucleotide" refers to an isolated nucleic acid molecule or nucleic acid construct, such as messenger RNA (mRNA), viral-derived RNA, or plasmid DNA (pDNA). A polynucleotide can contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term nucleic acid molecule refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0103] "Reduced binding," e.g., reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, e.g., as measured by SPR. For clarity, the term also encompasses a reduction in affinity to zero (or below the detection limit of the analytical method), i.e., a complete loss of interaction. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0104] The term "regulatory sequences" refers to DNA sequences necessary to achieve expression of coding sequences to which they are ligated. The nature of such control sequences varies depending on the host cell. In prokaryotes, control sequences generally include a promoter, ribosomal binding site, and terminator. In eukaryotes, control sequences generally include a promoter, terminator, and optionally an enhancer, transactivator, or transcription factor. The term "control sequences" is intended to include, at a minimum, all components the presence of which are necessary for expression, and may also include additional advantageous components.

[0105] As used herein, the term "single chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen-binding moieties is an scFv fragment, i.e., a VH domain and a VL domain linked by a peptide linker. In certain embodiments, one of the antigen-binding moieties is a single-chain Fab molecule, i.e., a Fab molecule in which the Fab light chain and the Fab heavy chain are linked by a peptide linker to form a single peptide chain. In certain such embodiments, the C-terminus of the Fab light chain is linked to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

[0106] As used herein, the term "SSD" refers to a stimulatory signaling domain.

[0107] As used herein, "treatment" (and grammatical variants thereof, such as "treat" or "treating") refers to a clinical intervention that seeks to alter the natural course of disease in the individual being treated, and can be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and achieving remission or improved prognosis. In some embodiments, cells expressing the antigen-binding receptor of the present invention are used together with antibodies comprising a variant Fc domain to delay the development of disease or slow the progression of disease.

[0108] As used herein, the term "target antigenic determinant" is synonymous with "target antigen," "target epitope," and "target cell antigen" and refers to a site on a polypeptide macromolecule (e.g., a contiguous stretch of amino acids or a conformational arrangement composed of distinct regions of noncontiguous amino acids) to which an antibody binds to form an antigen-binding moiety-antigen complex. Useful antigenic determinants are found, for example, on the surface of tumor cells, virus-infected cells, other diseased cells, immune cells, or found free in serum and / or the extracellular matrix (ECM). Proteins referred to herein as antigens (e.g., CD20, CEA, FAP, TNC) can be any native form of the protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. In certain embodiments, the target antigen is a human protein. When referring to a specific target protein herein, the term encompasses both unprocessed "full-length" target proteins and any form of target protein resulting from processing in target cells. The term also encompasses natural variants of target proteins, such as splice variants or allelic variants. Exemplary human target proteins useful as antigens include, but are not limited to, CD20, CEA, FAP, TNC, MSLN, FolR1, HER1, and HER2. The ability of an antibody to bind to a specific target antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analyzed by a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of the antibody to the unrelated protein is less than about 10% of the binding of the antibody to the target antigen, as measured, for example, by SPR.In certain embodiments, the antibody has a cytotoxicity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10. -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 Affinity dissociation constant (K D ) binds to the target antigen.

[0109] "Antibodies comprising a variant Fc domain" according to the invention, i.e., therapeutic antibodies, may have one, two, three, or more binding domains and may be monospecific, bispecific, or multispecific. Antibodies may be full-length from a single species or may be chimerized or humanized. In the case of antibodies with more than two antigen-binding domains, some of the binding domains may be identical and / or have the same specificity.

[0110] As used herein, " T cell activation " refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity and expression of activation markers.The antigen-binding receptor of the present invention can induce T cell activation.Suitable assays for measuring T cell activation are known in the art and are described herein.

[0111] In the present invention, the term "T cell receptor" or "TCR" is generally known in the art. In particular, the term "T cell receptor" herein refers to any T cell receptor as long as it meets the following three criteria: (i) tumor specificity, (ii) recognition of (most) tumor cells, which means that the antigen or target must be expressed in (most) tumor cells, and (iii) the TCR matches the HLA type of the subject to be treated. In this regard, suitable T cell receptors that meet the above-mentioned three criteria are known in the art, such as receptors that recognize NY-ESO-1 (see, for example, PCT / GB2005 / 001924 for sequence information) and / or HER2neu (see, for example, WO-A1 2011 / 0280894 for sequence information).

[0112] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the harmful effects of a disease.

[0113] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce a specific gene with which it is operatively associated into a target cell and direct its expression in the target cell. The term encompasses both vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which it is introduced. The expression vectors of the present invention comprise an expression cassette. The expression vector enables the transcription of large amounts of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding an antigen-binding receptor of the present invention or a fragment thereof.

[0114] Antigen-binding receptor capable of specifically binding to a variant Fc domain The present invention relates to an antigen-binding receptor capable of specifically binding to a mutant Fc domain of an antibody, i.e., a therapeutic antibody that targets cancer cells. In particular, the present invention relates to an antigen-binding receptor comprising an extracellular domain that comprises at least one antigen-binding moiety that is capable of specifically binding to the mutant Fc domain but is incapable of specifically binding to the parent, non-mutated Fc domain. In a preferred embodiment, the mutant Fc domain comprises at least one amino acid substitution compared to the non-mutated parent Fc domain, and Fc receptor binding by the mutant Fc domain is reduced compared to Fc receptor binding by the non-mutated Fc domain. In a specific embodiment, the present invention relates to an antigen-binding receptor comprising an extracellular domain comprising at least one antigen-binding portion capable of specifically binding to a variant Fc domain, wherein the at least one antigen-binding portion is incapable of specifically binding to a parent, non-variant Fc domain, and the variant Fc domain comprises at least one amino acid substitution selected from the group consisting of L234, L235, I253, H310, P331, P329, and H435, particularly the amino acid mutations are L234A, L235A, I253A, N297A, H310A, P329G, and / or H435A compared to the non-variant parent Fc domain, and Fc receptor binding by the variant Fc domain is reduced compared to Fc receptor binding by the non-variant Fc domain. In a preferred embodiment, the amino acid mutation is P329G, and Fcγ receptor binding is reduced as measured by SPR at 25°C. In a further preferred embodiment, the amino acid mutations are I253A, H310A, and H435A and binding to neonatal Fc receptor (FcRn) is reduced as measured by SPR at 25°C.

[0115] The present invention further relates to the transduction of T cells, such as CD8+ T cells, CD4+ T cells, CD3+ T cells, γδ T cells, or natural killer (NK) T cells, preferably CD8+ T cells, with the antigen-binding receptors described herein, and the targeted recruitment of these transduced T cells, e.g., to tumors, by antibody molecules, e.g., therapeutic antibodies, comprising a variant Fc domain. In one embodiment, the antibody is capable of specifically binding to a tumor-specific antigen naturally present on the surface of tumor cells.

[0116] As shown in the accompanying examples, as a proof of concept of the present invention, an antigen-binding receptor of the present invention, pETR17096 (SEQ ID NO:7, encoded by the DNA sequence shown in SEQ ID NO:19), comprising an anchoring transmembrane domain and an extracellular domain, was constructed, which is capable of specifically binding to a therapeutic antibody comprising a P329G mutation (represented by an anti-CD20 antibody comprising a heavy chain of SEQ ID NO:112 and a light chain of SEQ ID NO:113). Transduced T cells (Jurkat NFAT T cells) expressing the anti-P329G-scFv-CD28ATD-CD28CSD-CD3zSSD protein (SEQ ID NO:7, encoded by the DNA sequence shown in SEQ ID NO:19) could be strongly activated by co-incubation with an anti-CD20 antibody comprising a P329G mutation in the Fc domain and CD20-positive tumor cells. To support the proof-of-concept of the present invention, the inventors have further prepared several antigen-binding receptor formats that are capable of specifically binding to the variant Fc domain but not to the non-variant parent Fc domain.

[0117] Treatment of tumor cells with a combination of an antibody against a tumor antigen containing the P329G mutation and transduced T cells expressing the anti-P329G-Fab-ds-CD28ATD-CD28CSD-CD3zSSD protein (SEQ ID NO: 44 (DNA) and SEQ ID NOs: 39, 41 (protein)) surprisingly led to stronger activation of the transduced T cells compared to transduced T cells expressing the anti-P329G-scFv-CD28ATD-CD28CSD-CD3zSSD (SEQ ID NO: 19 (DNA) and SEQ ID NO: 7 (protein)) fusion protein (see, e.g., Figures 6 and 8-11).

[0118] Thus, it has been surprisingly and unexpectedly found that T cells, preferably CD8+ T cells, transduced with the antigen-binding receptor of the present invention can be specifically stimulated and recruited by the tumor-specific antibody as a linking element to tumor cells using a tumor-specific antibody comprising a variant Fc domain. Thus, it has been surprisingly and unexpectedly shown in the present invention that pairing a tumor-specific antibody, i.e., a therapeutic antibody, comprising a variant Fc domain with T cells transduced with an antigen-binding receptor comprising / consisting of an extracellular domain comprising an antigen-binding portion capable of specifically binding to the variant Fc domain results in specific activation of the T cells and subsequent lysis of tumor cells. This approach offers significant safety advantages over conventional T cell-based approaches, since T cells are thought to be inactive in the absence of an antibody comprising a variant Fc domain, and their availability can be controlled by the chosen antibody molecule format (i.e., small molecules for short half-lives, large molecules for long half-lives). Thus, the present invention provides a versatile therapeutic platform that can use IgG-type antibodies to mark or label tumor cells as a targeting device for T cells, conferring specificity for the mutant Fc domain of the IgG-type antibody and specifically directing the transduced T cells to host cells. After binding to the mutant Fc domain of the antibody on the surface of tumor cells, the transduced T cells described herein become activated, which will subsequently lyse the tumor cells. This platform is versatile and specific by allowing the use of diverse targeting antibodies (existing or newly developed) or the simultaneous application of multiple antibodies with different antigen specificities but containing the same mutations in their Fc domains. The degree of T cell activation can be further tuned by adjusting the dosage of the co-applied therapeutic antibody or by switching to a different antibody specificity or format. Because the Fc domain mutations described herein are not present in native immunoglobulins, i.e., non-mutated immunoglobulins, the transduced T cells of the present invention are inactive without the simultaneous application of a targeting antibody containing a mutant Fc domain.Thus, in one embodiment, the variant Fc domain does not naturally occur in a wild-type immunoglobulin.

[0119] Thus, the present invention relates to an antigen-binding receptor comprising an extracellular domain comprising at least one antigen-binding moiety capable of specifically binding to a variant Fc domain, wherein said at least one antigen-binding moiety is incapable of specifically binding to a parent, non-variant Fc domain, and wherein the variant Fc domain comprises at least one amino acid mutation compared to the parent, non-variant Fc domain, and wherein Fc receptor binding by the variant Fc domain and / or effector function induced by the variant Fc domain is reduced compared to Fc receptor binding by the non-variant Fc domain and / or effector function induced by the non-variant Fc domain. Since effector function can lead to serious side effects of antibody-based tumor therapy, as further described herein, using therapeutic antibodies with reduced effector function may be particularly desirable in cancer therapy.

[0120] In the context of the present invention, an antigen-binding receptor comprises an extracellular domain that is not naturally found in or on T cells. Thus, the antigen-binding receptor can confer tailored binding specificity to cells expressing the antigen-binding receptor of the present invention. Cells, e.g., T cells, transduced with an antigen-binding receptor of the present invention become capable of specifically binding to a variant Fc domain but are unable to specifically bind to a non-variant parent Fc domain. Specificity is conferred by the antigen-binding portion of the extracellular domain of the antigen-binding receptor, and such antigen-binding portion is considered to be specific for the variant Fc domain defined herein. In the context of the present invention and as described herein, an antigen-binding portion capable of specifically binding to a variant Fc domain binds to / interacts with the variant Fc domain but does not bind to / interact with the non-variant parent Fc domain.

[0121] antigen binding part In an exemplary embodiment of the present invention, as proof of concept, there is provided an antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain comprising at least one antigen-binding moiety, wherein said at least one antigen-binding moiety is capable of specifically binding to a variant Fc domain but is incapable of specifically binding to a non-variant parent Fc domain, wherein the variant Fc domain comprises at least one amino acid substitution compared to the non-variant parent Fc.

[0122] In certain embodiments, at least one of the antigen-binding portions is a conventional Fab fragment, i.e., a Fab molecule consisting of a Fab light chain and a Fab heavy chain. In certain embodiments, at least one of the antigen-binding portions is a crossFab fragment, i.e., a Fab molecule consisting of a Fab light chain and a Fab heavy chain, in which either the variable or constant regions of the Fab heavy and light chains have been exchanged. In certain embodiments, at least one of the antigen-binding portions is an scFv fragment. In certain such embodiments, in the scFv molecule, the C-terminus of the variable heavy chain (VH) is linked to the N-terminus of the variable light chain (VL), optionally via a peptide linker. In certain such embodiments, at least one of the antigen-binding portions is a single-chain Fab molecule, i.e., a Fab molecule in which the Fab light chain and the Fab heavy chain are linked by a peptide linker to form a single peptide chain. In certain such embodiments, in the single-chain Fab molecule, the C-terminus of the Fab light chain is linked to the N-terminus of the Fab heavy chain, optionally via a peptide linker.

[0123] Thus, in the context of the present invention, an antigen-binding portion is capable of specifically binding to a variant Fc domain but is incapable of specifically binding to a non-variant parent Fc domain, wherein the variant Fc domain comprises at least one amino acid substitution compared to the non-variant parent Fc domain.

[0124] Antigen-binding moieties capable of specifically binding to a variant Fc domain can be generated, for example, by immunization with a mammalian immune system. Such methods are known in the art and are described, for example, by Burns in Methods in Molecular Biology 295:1-12 (2005). Alternatively, antigen-binding moieties of the present invention can be isolated by screening combinatorial libraries for antibodies with one or more desired activities. Methods for screening combinatorial libraries are reviewed, for example, by Lerner et al. in Nature Reviews 16:498-508 (2016). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antigen-binding moieties with desired binding characteristics.Such methods are reviewed, for example, by Frenzel et al. in mAbs 8:1177-1194 (2016), by Bazan et al. in Human Vaccines and Immunotherapeutics 8:1817-1828 (2012), by Zhao et al. in Critical Reviews in Biotechnology 36:276-289 (2016), and by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), and also by McCafferty et al., Nature 348:552-554, Clackson et al., Nature 352:624-628 (1991), Marks et al. al., J. Mol. Biol. 222:581-597 (1992), Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003), Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004), Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004), Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004), and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004). In certain phage display methods, repertoires of VH and VL genes can be individually cloned by polymerase chain reaction (PCR), randomly recombined into phage libraries, and then screened for antigen-binding phage, as described by Winter et al., Annual Review of Immunology 12:433-455 (1994). The phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antigen-binding moieties for immunogens without the need for hybridoma construction. Alternatively, naive repertoires (e.g., from humans) can be cloned without any immunization, providing a single source of antigen-binding moieties for a wide range of non-self antigens and also for self antigens, as described by Griffiths et al. in EMBO Journal 12:725-734 (1993). Finally, naive libraries can be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and to perform in vitro rearrangement, as described by Hoogenboom and Winter in Journal of Molecular Biology 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent Nos. 5,750,373, 7,985,840, 7,785,903, and 8,679,490, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2007 / 0117126, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360. Further examples of methods known in the art for screening combinatorial libraries for antibodies with one or more desired activities include ribosome display and mRNA display, as well as methods for antibody display and selection on bacteria, mammalian cells, insect cells, or yeast cells. Methods for yeast surface display are reviewed, for example, by Scholler et al. in Methods in Molecular Biology 503:135-56 (2012), by Cherf et al. in Methods in Molecular Biology 1319:155-175 (2015), and by Zhao et al. in Methods in Molecular Biology 889:73-84 (2012).Methods for ribosome display are described, for example, by He et al. in Nucleic Acids Research 25:5132-5134 (1997) and by Hanes et al. in PNAS 94:4937-4942 (1997).

[0125] In the context of the present invention, an antigen-binding receptor comprising at least one antigen-binding portion capable of specifically binding to a variant Fc domain is provided herein. Thus, transduced cells, i.e., T cells, expressing the antigen-binding receptor of the present invention can specifically bind to the variant Fc domain of an antibody, i.e., a therapeutic antibody. The Fc domain confers advantageous pharmacokinetic properties, including a long serum half-life and a favorable tissue-blood distribution ratio, to antibodies, i.e., therapeutic antibodies, contributing to favorable accumulation in target tissues. However, it can also lead to undesirable targeting of therapeutic antibodies to Fc receptor-expressing cells that are not preferred antigen-bearing cells. Furthermore, simultaneous activation of the Fc receptor signaling pathway can lead to cytokine release, which, upon systemic administration of a therapeutic antibody, results in excessive activation of cytokine receptors and severe side effects. Activation of immune cells other than T cells (Fc receptor-bearing) may even reduce the efficacy of a therapeutic antibody due to potential destruction of the immune cells. Thus, therapeutic antibodies known in the art can be engineered or mutated to exhibit reduced binding affinity for Fc receptors and / or reduced effector function, e.g., compared to a native IgG1 Fc domain. The antigen-binding receptors of the present invention can be used to target effector cells, e.g., T cells, expressing the antigen-binding receptor of the present invention, to target cells, i.e., tumor cells, in vitro and / or in vivo, that are capable of specifically binding to the target cells and that have been labeled with an antibody comprising an altered and / or variant Fc domain as described herein.

[0126] In an exemplary embodiment of the present invention, as proof of concept, an antigen-binding receptor capable of specifically binding to a mutant Fc domain comprising the amino acid mutation P329G, and an effector cell expressing the antigen-binding receptor are provided. The P329G mutation reduces binding to Fcγ receptors and the associated effector function. Thus, a mutant Fc domain comprising the P329G mutation binds to Fcγ receptors with reduced or eliminated affinity compared to a non-mutated Fc domain. In an alternative exemplary embodiment of the present invention, as proof of concept, an antigen-binding receptor capable of specifically binding to a mutant Fc domain comprising the amino acid mutations I253A, H310A, and H435A ("AAA") is provided. The AAA mutation essentially eliminates binding to FcRn.

[0127] However, antibodies comprising a variant Fc domain and having reduced, improved, or diminished Fc receptor (FcR) binding and / or effector function are widely used in the art. Accordingly, provided herein is an antigen-binding receptor capable of specifically binding to an antibody comprising a variant Fc domain; such an antibody is also referred to herein as a targeted antibody. Thus, in one embodiment, the antigen-binding receptor of the present invention can specifically bind to a targeted antibody comprising a variant Fc domain with reduced binding affinity to an Fc receptor and / or reduced effector function. Targeted antibodies with reduced effector function include those with mutations at one or more of residues 238, 265, 269, 270, 297, 327, and 329 of the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with mutations at two or more of amino acids 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with mutations at residues 265 and 297 to alanine (U.S. Patent No. 7,332,581). Several antibody variants with improved or reduced binding to FcR have been described. (See, e.g., U.S. Patent No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)). In certain embodiments, antigen-binding receptors are provided that can specifically bind to antibody variants comprising an Fc region with one or more amino acid mutations that improve ADCC, e.g., an Fc region with mutations at residues 298, 333, and / or 334 (EU numbering) of the Fc region. In certain embodiments, the targeted antibody variant comprises an Fc region with one or more amino acid mutations that reduce or decrease FcRn binding, e.g., mutations at positions 253 and / or 310 and / or 435 (EU residue numbering) of the Fc region. In certain embodiments, the targeted antibody variant comprises an Fc region with amino acid mutations at positions 253, 310, and 435. In one embodiment, the mutations are I253A, H310A, and H435A in the Fc region derived from a human IgG1 Fc region.See, for example, Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

[0128] In certain embodiments, an antigen-binding receptor is provided that can specifically bind to an antibody mutant comprising an Fc region with one or more amino acid mutations that reduce or decrease FcRn binding, e.g., a mutation at one of positions 310, 433, and / or 436 (EU residue numbering) of the Fc region. In certain embodiments, the target antibody mutant comprises an Fc region with amino acid mutations at positions 310, 433, and 436. In one embodiment, the mutations are H310A, H433A, and Y436A in the Fc region derived from a human IgG1 Fc region. In certain embodiments, the target antibody mutant comprises an Fc region with one or more amino acid mutations that increase FcRn binding, e.g., a mutation at positions 252, 254, and / or 256 (EU residue numbering) of the Fc region. In certain embodiments, the target antibody mutant comprises an Fc region with amino acid mutations at positions 252, 254, and 256. In one embodiment, the mutations are M252Y, S254T, and T256E in the Fc region derived from a human IgG1 Fc region. In certain embodiments, an antigen-binding receptor is provided that can specifically bind to an antibody mutant comprising an Fc region with amino acid mutations that reduce FcγR binding, for example, mutations at positions 234, 235, and 329 (EU residue numbering) in the Fc region. In one embodiment, the mutations are L234A and L235A (LALA). In certain embodiments, the target antibody mutant further comprises D265A and / or P329G in the Fc region derived from a human IgG1 Fc region. In one embodiment, the mutation is P329G ("PG") in the Fc region derived from a human IgG1 Fc region. In another embodiment, the mutations are I253A, H310A, and H435A ("AAA") in the Fc region derived from a human IgG1 Fc region.

[0129] In one embodiment, the antigen-binding portion can specifically bind to a variant Fc domain composed of a first subunit and a second subunit that can stably associate. In one embodiment, the Fc domain is an IgG Fc domain, specifically an IgG1 or IgG4 Fc domain. In one embodiment, the Fc domain is a human Fc domain. In one embodiment, the variant Fc domain exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain. In one embodiment, the Fc domain contains one or more amino acid mutations that reduce binding to Fc receptors and / or effector function.

[0130] In a preferred embodiment, the one or more amino acid mutations are at one or more positions selected from the group consisting of L234, L235, and P329 (Kabat numbering). In a specific embodiment, each subunit of the Fc domain contains three amino acid mutations that reduce binding to an activating Fc receptor and / or effector function, the amino acid mutations being L234A, L235A, and P329G. In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0131] In a particular embodiment, the variant Fc domain comprises a P329G mutation. Thus, the variant Fc domain comprising the P329G mutation binds to an Fcγ receptor with reduced or eliminated affinity compared to the non-variant Fc domain. In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to the Fc domain comprising the P329G mutation, said antigen-binding portion comprising: (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of RYWMN (SEQ ID NO: 1); (b) the CDR H2 amino acid sequence of TIFF0007642010000016.tif4128, and (c) CDR H3 amino acid sequence of TIFF0007642010000017.tif4128 The heavy chain variable region comprises at least one of:

[0132] In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising a P329G mutation, said antigen-binding portion comprising: (d) Light chain (CDR L) 1 amino acid sequence of TIFF0007642010000018.tif4128; (e) the CDR L2 amino acid sequence of GTNKRAP (SEQ ID NO:5), and (f) CDR L3 amino acid sequence of TIFF0007642010000019.tif4128 The light chain variable region comprises at least one of:

[0133] In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising a P329G mutation, wherein the antigen-binding portion comprises at least one heavy chain complementarity-determining region (CDR) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and at least one light chain CDR selected from the group of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0134] In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising a P329G mutation, wherein the antigen-binding portion comprises the heavy chain complementarity-determining regions (CDRs) of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 and the light chain CDRs of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0135] In a preferred embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising a P329G mutation, said antigen-binding portion comprising: (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequence of TIFF0007642010000020.tif4128, (c) CDR H3 amino acid sequence of TIFF0007642010000021.tif4128 a heavy chain variable region comprising (d) Light chain (CDR L) 1 amino acid sequence of TIFF0007642010000022.tif4128; (e) the CDR L2 amino acid sequence of GTNKRAP (SEQ ID NO:5), and (f) CDR L3 amino acid sequence of TIFF0007642010000023.tif4128 a light chain variable region comprising Includes:

[0136] In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising a P329G mutation, wherein the antigen-binding portion comprises a heavy chain variable region (VH) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO:8 and SEQ ID NO:32, and a light chain variable region (VL) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO:9 and SEQ ID NO:33.

[0137] In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising a P329G mutation, wherein the antigen-binding portion comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from SEQ ID NO:8 and SEQ ID NO:32, and a light chain variable region (VL) comprising an amino acid sequence selected from SEQ ID NO:9 and SEQ ID NO:33.

[0138] In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising a P329G mutation, wherein the antigen-binding portion comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:33.

[0139] In a preferred embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising a P329G mutation, wherein the antigen-binding portion comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:8 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:9.

[0140] In one embodiment, at least one antigen-binding portion is an scFv, Fab, crossFab, or scFab fragment. In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising a P329G mutation, and said antigen-binding portion is a Fab fragment.

[0141] In a preferred embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising a P329G mutation, wherein the Fab fragment comprises a heavy chain of SEQ ID NO:40 and a light chain of SEQ ID NO:41.

[0142] In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising a P329G mutation, and at least one antigen-binding portion is an scFv fragment, which is a polypeptide consisting of a heavy chain variable domain (VH), a light chain variable domain (VL), and a linker, wherein the variable domains and linker have, from N-terminus to C-terminus, one of the following configurations: a) VH-linker-VL or b) VL-linker-VH. In a preferred embodiment, the scFv fragment has the configuration VH-linker-VL.

[0143] In a preferred embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising a P329G mutation, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO:10.

[0144] In alternative specific embodiments, the variant Fc domain comprises I253A, H310A, and H435A ("AAA") mutations. The AAA mutations reduce binding to the neonatal Fc receptor (FcRn). Thus, variant Fc domains comprising AAA mutations bind to FcRn with reduced or eliminated affinity compared to the non-variant Fc domain.

[0145] Thus, in one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising the I253A, H310A, and H435A mutations, said antigen-binding portion comprising: (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SYGMS (SEQ ID NO:53); (b) the CDR H2 amino acid sequence of SSGGSY (SEQ ID NO:54), and (c) CDR H3 amino acid sequence of TIFF0007642010000024.tif4128 The heavy chain variable region comprises at least one of:

[0146] In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising I253A, H310A, and H435A mutations, said antigen-binding portion comprising: (d) Light chain (CDR L) 1 amino acid sequence of TIFF0007642010000025.tif4128; (e) the CDR L2 amino acid sequence of KVSNRFS (SEQ ID NO:57), and (f) CDR L3 amino acid sequence of TIFF0007642010000026.tif4128 The light chain variable region comprises at least one of:

[0147] In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising I253A, H310A, and H435A mutations, wherein said antigen-binding portion comprises at least one heavy chain complementarity-determining region (CDR) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55, and at least one light chain CDR selected from the group of SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58.

[0148] In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising a P329G mutation, wherein the antigen-binding portion comprises the heavy chain complementarity-determining regions (CDRs) of SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55 and the light chain CDRs of SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58.

[0149] In a preferred embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising I253A, H310A, and H435A mutations, said antigen-binding portion comprising: (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SYGMS (SEQ ID NO:53); (b) the CDR H2 amino acid sequence of SSGGSY (SEQ ID NO:54); (c) CDR H3 amino acid sequence of TIFF0007642010000027.tif4128 a heavy chain variable region comprising (d) Light chain (CDR L) 1 amino acid sequence of TIFF0007642010000028.tif4128; (e) the CDR L2 amino acid sequence of KVSNRFS (SEQ ID NO:57), and (f) CDR L3 amino acid sequence of TIFF0007642010000029.tif4128 a light chain variable region comprising Includes:

[0150] In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising I253A, H310A, and H435A mutations, wherein the antigen-binding portion comprises a heavy chain variable region (VH) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:61, and a light chain variable region (VL) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to a selected amino acid sequence of SEQ ID NO:62.

[0151] In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising I253A, H310A, and H435A mutations, wherein the antigen-binding portion comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:61 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:62.

[0152] In one embodiment, at least one antigen-binding portion is an scFv, Fab, crossFab, or scFab fragment. In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising I253A, H310A, and H435A mutations, and at least the antigen-binding portion is a Fab fragment. In a particular embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising I253A, H310A, and H435A mutations, and wherein the Fab fragment comprises a heavy chain of SEQ ID NO:64 and a light chain of SEQ ID NO:65.

[0153] In one embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising the I253A, H310A, and H435A mutations, and at least one antigen-binding portion is an scFv fragment. In a particular embodiment, the extracellular domain of the antigen-binding receptor comprises an antigen-binding portion capable of specifically binding to an Fc domain comprising the I253A, H310A, and H435A mutations, and wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO:60.

[0154] In a further embodiment of the invention, the antigen-binding portion comprised in the extracellular domain is a single chain Fab fragment, or scFab.

[0155] Fab fragments and scFab fragments are stabilized by a natural disulfide bond between the CL domain and the CH1 domain. Antigen-binding moieties comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), such as the Fab fragments, crossFab fragments, scFv fragments, and scFab fragments described herein, can be further stabilized by introducing an interchain disulfide bridge between the VH and VL domains. Thus, in one embodiment, the Fab fragments, crossFab fragments, scFv fragments, and / or scFab fragments contained in the antigen-binding receptors of the present invention can be further stabilized by the creation of an interchain disulfide bond through the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain according to the Kabat numbering). Such stabilized antigen-binding moieties are referred to by the term "ds" in the accompanying examples and figures.

[0156] Anchoring transmembrane domain In the context of the present invention, the anchoring transmembrane domain of the antigen-binding receptor of the present invention may be characterized in that it does not have a cleavage site for a mammalian protease. In the context of the present invention, a protease refers to a proteolytic enzyme capable of hydrolyzing the amino acid sequence of a transmembrane domain that contains a cleavage site for the protease. The term protease encompasses both endopeptidases and exopeptidases. In the context of the present invention, any anchoring transmembrane domain of a transmembrane protein, particularly one defined by the CD nomenclature, may be used to generate an antigen-binding receptor of the present invention that activates T cells, preferably CD8+ T cells, when bound to a variant Fc domain as defined herein.

[0157] Thus, in the context of the present invention, the anchoring transmembrane domain may comprise parts of a murine / mouse transmembrane domain, or preferably parts of a human transmembrane domain. An example of such an anchoring transmembrane domain is the transmembrane domain of CD28, e.g., having the amino acid sequence shown in SEQ ID NO:11 herein (encoded by the DNA sequence shown in SEQ ID NO:24). In the context of the present invention, the transmembrane domain of an antigen-binding receptor of the invention may comprise / consist of the amino acid sequence shown in SEQ ID NO:11 (encoded by the DNA sequence shown in SEQ ID NO:24).

[0158] In an exemplary embodiment of the present invention, as a proof of concept, there is provided an antigen-binding receptor comprising an antigen-binding portion comprising the amino acid sequence of SEQ ID NO:10 (encoded by the DNA sequence shown in SEQ ID NO:22) and a fragment / polypeptide part of CD28 (the Uniprot entry number for human CD28 is P10747 (version number 173, version 1 of the sequence)), herein shown as SEQ ID NO:71 (encoded by the DNA sequence shown in SEQ ID NO:70). Alternatively, any protein having a transmembrane domain, especially one given by the CD nomenclature, may be used as the anchoring transmembrane domain of the antigen-binding receptor protein of the present invention. As mentioned above, the antigen-binding receptor provided herein is composed of the human full-length CD28 protein shown in SEQ ID NO:71 (encoded by the DNA sequence shown in SEQ ID NO:70). The CD28 anchoring transmembrane domain may comprise the CD28 anchoring transmembrane domain located at amino acids 153 to 179, 154 to 179, 155 to 179, 156 to 179, 157 to 179, 158 to 179, 159 to 179, 160 to 179, 161 to 179, 162 to 179, 163 to 179, 164 to 179, 165 to 179, 166 to 179, 167 to 179, 168 to 179, 169 to 179, 170 to 179, 171 to 179, 172 to 179, 173 to 179, 174 to 179, 175 to 179, 176 to 179, 177 to 179, or 178 to 179. Thus, in the context of the present invention, the anchoring transmembrane domain may comprise / consist of the amino acid sequence shown in SEQ ID NO:11 (encoded by the DNA sequence shown in SEQ ID NO:24).

[0159] In one embodiment, the antibody comprises an anchoring transmembrane domain and an extracellular domain comprising a Fab fragment capable of specifically binding to an Fc domain comprising the I253A, H310A, and H435A mutations; (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:64 fused at its C-terminus to the N-terminus of an anchoring transmembrane domain of SEQ ID NO:11, optionally via a peptide linker of SEQ ID NO:17; (b) a light chain comprising the amino acid sequence of SEQ ID NO:65; An antigen-binding receptor is provided, comprising:

[0160] In one embodiment, the antibody comprises an anchoring transmembrane domain and an extracellular domain comprising a Fab fragment capable of specifically binding to an Fc domain comprising a P329G mutation, (a) a heavy chain comprising an amino acid sequence selected from SEQ ID NO:40 and SEQ ID NO:49 fused at its C-terminus to the N-terminus of an anchoring transmembrane domain of SEQ ID NO:11, optionally via a peptide linker of SEQ ID NO:17; (b) a light chain comprising an amino acid sequence selected from SEQ ID NO:41 and SEQ ID NO:50; An antigen-binding receptor is provided, comprising:

[0161] In one embodiment, the antibody comprises an anchoring transmembrane domain and an extracellular domain comprising a Fab fragment capable of specifically binding to an Fc domain comprising a P329G mutation, (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:40 fused at its C-terminus to the N-terminus of an anchoring transmembrane domain of SEQ ID NO:11, optionally via a peptide linker of SEQ ID NO:17; (b) a light chain comprising the amino acid sequence of SEQ ID NO:41; An antigen-binding receptor is provided, comprising:

[0162] In one preferred embodiment, the antibody comprises an anchoring transmembrane domain and an extracellular domain comprising a Fab fragment capable of specifically binding to an Fc domain comprising a P329G mutation, (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:49 fused at its C-terminus to the N-terminus of an anchoring transmembrane domain of SEQ ID NO:11, optionally via a peptide linker of SEQ ID NO:17; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; An antigen-binding receptor is provided, comprising:

[0163] In one aspect, an antigen-binding receptor is provided that comprises an anchoring transmembrane domain and an extracellular domain comprising an scFv fragment that can specifically bind to an Fc domain comprising the I253A, H310A, and H435A mutations but cannot specifically bind to the non-mutated parent Fc domain, and that comprises the amino acids of SEQ ID NO:60 fused at the C-terminus to the N-terminus of the anchoring transmembrane domain of SEQ ID NO:11, optionally via a peptide linker of SEQ ID NO:17.

[0164] In one aspect, an antigen-binding receptor is provided, comprising an anchoring transmembrane domain and an extracellular domain comprising an scFv fragment capable of specifically binding to an Fc domain comprising a P329G mutation but incapable of specifically binding to a non-mutated parent Fc domain, the antigen-binding receptor comprising an amino acid sequence selected from SEQ ID NO:10 and SEQ ID NO:34 fused at its C-terminus to the N-terminus of the anchoring transmembrane domain of SEQ ID NO:11, optionally via a peptide linker of SEQ ID NO:17.

[0165] In one preferred embodiment, an antigen-binding receptor is provided, comprising an anchoring transmembrane domain and an extracellular domain comprising an scFv fragment capable of specifically binding to an Fc domain comprising a P329G mutation but incapable of specifically binding to a non-mutated parent Fc domain, the antigen-binding receptor comprising the amino acid sequence of SEQ ID NO:10 fused at its C-terminus to the N-terminus of the anchoring transmembrane domain of SEQ ID NO:11, optionally via a peptide linker of SEQ ID NO:17.

[0166] In one aspect, there is provided an antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain comprising an scFab fragment capable of specifically binding to an Fc domain comprising a P329G mutation but incapable of specifically binding to a non-mutated parent Fc domain, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO:34 fused at its C-terminus to the N-terminus of the anchoring transmembrane domain of SEQ ID NO:11, optionally via a peptide linker of SEQ ID NO:17.

[0167] Stimulatory signaling domain (SSD) and costimulatory signaling domain (CSD) Preferably, the antigen-binding receptors of the present invention comprise at least one stimulatory signaling domain and / or at least one costimulatory signaling domain. Thus, the antigen-binding receptors provided herein preferably comprise a stimulatory signaling domain that results in T cell activation. The antigen binding receptors provided herein can comprise a stimulatory signaling domain that is a fragment / polypeptide part of murine / mouse or human CD3z (the UniProt entry for human CD3z is P20963 (version number 177, SEQ ID NO: 2); the UniProt entry for murine / mouse CD3z is P24161 (primary citable accession number) or Q9D3G3 (secondary citable accession number), version number 143, SEQ ID NO: 1), FCGR3A (the UniProt entry for human FCGR3A is P08637 (version number 178, SEQ ID NO: 2)), or NKG2D (the UniProt entry for human NKG2D is P26718 (version number 151, SEQ ID NO: 1); the UniProt entry for murine / mouse NKG2D is O54709 (version number 132, SEQ ID NO: 2)).

[0168] Thus, the stimulatory signaling domains contained in the antigen-binding receptors provided herein can be fragments / polypeptide parts of full-length CD3z, FCGR3A, and NKG2D. The amino acid sequences of murine / mouse full-length CD3z or NKG2D are set forth herein as SEQ ID NO:96 (CD3z), 100 (FCGR3A), or 104 (NKG2D) (encoded by the DNA sequences set forth in murine / mouse, SEQ ID NO:97 (CD3z), 101 (FCGR3A), or 105 (NKG2D)). The amino acid sequences of human full-length CD3z, FCGR3A, or NKG2D are set forth herein as SEQ ID NO:94 (CD3z), 98 (FCGR3A), or 102 (NKG2D) (human, encoded by the DNA sequence set forth in SEQ ID NO:95 (CD3z), 99 (FCGR3A), or 103 (NKG2D)). The antigen-binding receptors of the present invention may comprise a fragment of CD3z, FCGR3A, or NKG2D as a stimulatory domain, provided that at least one signaling domain is included. In particular, any part / fragment of CD3z, FCGR3A, or NKG2D is suitable as a stimulatory domain, provided that at least one signaling motif is included. However, more preferably, the antigen-binding receptors of the present invention comprise a polypeptide derived from human origin. Thus, more preferably, the antigen-binding receptors provided herein comprise the amino acid sequence set forth herein as SEQ ID NO:94 (CD3z), 98 (FCGR3A), or 102 (NKG2D) (human, encoded by the DNA sequence set forth in SEQ ID NO:95 (CD3z), 99 (FCGR3A), or 103 (NKG2D)). For example, a fragment / polypeptide part of human CD3z that may be included in an antigen-binding receptor of the present invention may comprise or consist of the amino acid sequence set forth in SEQ ID NO:13 (encoded by the DNA sequence set forth in SEQ ID NO:26).Thus, in one embodiment, the antigen-binding receptor comprises the sequence set forth in SEQ ID NO:13, or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 substitutions, deletions, or insertions relative to SEQ ID NO:13, wherein the sequence is characterized as having stimulatory signaling activity. Specific configurations of antigen-binding receptors that include a stimulatory signaling domain (SSD) are described below and in the Examples and Figures. Stimulatory signaling activity can be determined, for example, by enhanced cytokine release (IL-2, IFNγ, TNFα) as measured by ELISA, enhanced proliferative activity (as measured by increased cell number), or enhanced lytic activity as measured by LDH release assay.

[0169] Furthermore, the antigen-binding receptors provided herein preferably contain at least one costimulatory signaling domain that confers additional activity to T cells. Antigen binding receptors provided herein include murine / mouse or human CD28 (the UniProt entry for human CD28 is P10747 (version number 173, SEQ ID NO: 1); the UniProt entry for murine / mouse CD28 is P31041 (version number 134, SEQ ID NO: 2)), CD137 (the UniProt entry for human CD137 is Q07011 (version number 145, SEQ ID NO: 1); the UniProt entry for murine / mouse CD137 is P20334 (version number 139, SEQ ID NO: 1)), OX40 (the UniProt entry for human OX40 is P23510 (version number 138, SEQ ID NO: 1); the UniProt entry for murine / mouse OX40 is P43488 (version number 119, SEQ ID NO: 1)), ICOS (the UniProt entry for human ICOS is Q9Y6W8 (version number 126, SEQ ID NO: 1)), and / or human CD28 (the UniProt entry for human ICOS is Q9Y6W9 (version number 138, SEQ ID NO: 1)). column number 1); the UniProt entry for murine / mouse ICOS is Q9WV40 (primary citable accession number) or Q9JL17 (secondary citable accession number), version number 102 and sequence version 2), CD27 (the UniProt entry for human CD27 is P26842 (version number 160, SEQ ID NO: 2); the Uniprot entry for murine / mouse CD27 is P41272 (version number 137, sequence version 1)), 4-1-BB (the UniProt entry for murine / mouse 4-1-BB is P20334 (version number 140, sequence version 1); the UniProt entry for human 4-1-BB is Q07011 (version number 146, sequence version)), DAP10 (the UniProt entry for human DAP10 is Q9UBJ5 (version number 25, SEQ ID NO: 1);The UniProt entry for murine / mouse DAP10 is Q9QUJ0 (primary citable accession number) or Q9R1E7 (secondary citable accession number), version number 101 and SEQ ID NO: 1) or DAP12 (the UniProt entry for human DAP12 is O43914 (version number 146 and SEQ ID NO: 1); the UniProt entry for murine / mouse DAP12 is O054885 (primary citable accession number) or Q9R1E7 (secondary citable accession number), version number 123 and SEQ ID NO: 1). In certain embodiments of the invention, the antigen-binding receptor of the invention may comprise one or more, i.e., 1, 2, 3, 4, 5, 6 or 7, of the costimulatory signaling domains defined herein. Thus, in the context of the present invention, the antigen-binding receptor of the present invention can comprise a fragment / polypeptide part of murine / mouse CD28 or preferably human CD28 as a first costimulatory signaling domain, and the second costimulatory signaling domain is selected from the group consisting of murine / mouse or preferably human CD27, CD28, CD137, OX40, ICOS, DAP10 and DAP12 or fragments thereof. Preferably, the antigen-binding receptor of the present invention comprises a costimulatory signaling domain derived from human origin. Thus, more preferably, the costimulatory signaling domain comprised in the antigen-binding receptor of the present invention can comprise or consist of the amino acid sequence shown in SEQ ID NO:12 (encoded by the DNA sequence shown in SEQ ID NO:25).

[0170] Thus, the costimulatory signaling domains that can optionally be included in the antigen-binding receptors provided herein are fragments / polypeptide parts of full-length CD27, CD28, CD137, OX40, ICOS, DAP10, and DAP12. The amino acid sequences of murine / mouse full-length CD27, CD28, CD137, OX40, ICOS, CD27, DAP10, or DAP12 are set forth herein as SEQ ID NO:69 (CD27), 73 (CD28), 77 (CD137), 81 (OX40), 85 (ICOS), 89 (DAP10), or 93 (DAP12) (encoded by the DNA sequence set forth in murine / mouse, SEQ ID NO:68 (CD27), 72 (CD28), 76 (CD137), 80 (OX40), 84 (ICOS), 88 (DAP10), or 92 (DAP12)). However, since human sequences are most preferred in the context of the present invention, the costimulatory signaling domains that may optionally be included in the antigen-binding receptor proteins provided herein are fragments / polypeptide parts of human full-length CD27, CD28, CD137, OX40, ICOS, DAP10, or DAP12. The amino acid sequences of human full-length CD27, CD28, CD137, OX40, ICOS, DAP10, or DAP12 are set forth herein as SEQ ID NO:67 (CD27), 71 (CD28), 75 (CD137), 79 (OX40), 83 (ICOS), 87 (DAP10), or 91 (DAP12) (human, encoded by the DNA sequence set forth in SEQ ID NO:66 (CD27), 70 (CD28), 74 (CD137), 78 (OX40), 82 (ICOS), 86 (DAP10), or 90 (DAP12)).

[0171] In a preferred embodiment, the antigen-binding receptor comprises CD28 or a fragment thereof as a costimulatory signaling domain. The antigen-binding receptors provided herein may comprise a fragment of CD28 as a costimulatory signaling domain, provided that at least one signaling domain of CD28 is included. In particular, any part / fragment of CD28 is suitable for the antigen-binding receptor of the present invention, as long as it contains at least one signaling motif of CD28. For example, the CD28 polypeptide comprised in the antigen-binding receptor protein of the present invention may comprise or consist of the amino acid sequence shown in SEQ ID NO:12 (encoded by the DNA sequence shown in SEQ ID NO:25). In the present invention, the intracellular domain of CD28 functioning as a costimulatory signaling domain may comprise a sequence derived from the intracellular domain of a CD28 polypeptide having the sequence YMNM (SEQ ID NO:106) and / or PYAP (SEQ ID NO:107). Preferably, the antigen-binding receptor of the present invention comprises a polypeptide derived from human origin. For example, a fragment / polypeptide part of human CD28 that can be included in an antigen-binding receptor of the present invention can comprise or consist of the amino acid sequence shown in SEQ ID NO:12 (encoded by the DNA sequence shown in SEQ ID NO:25). Therefore, in the context of the present invention, an antigen-binding receptor includes a sequence that comprises the sequence shown in SEQ ID NO:12 or has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, deletions, or insertions compared to SEQ ID NO:12, and is characterized by having costimulatory signaling activity. Specific configurations of antigen-binding receptors that include a costimulatory signaling domain (CSD) are described below and in the Examples and Figures. Costimulatory signaling activity can be determined, for example, by enhanced cytokine release (IL-2, IFNγ, TNFα) measured by ELISA, enhanced proliferative activity (measured by increased cell number), or enhanced lytic activity measured by LDH release assay.

[0172] As noted above, in embodiments of the invention, the costimulatory signaling domain of the antigen binding receptor can be derived from the human CD28 gene (UniProt entry number: P10747 (accession number, entry version: 173 and sequence version 1)) and confers CD28 activity, defined as cytokine production, proliferation, and lytic activity of transduced cells described herein, such as transduced T cells. CD28 activity can be measured by cytokine release by ELISA or flow cytometry for cytokines such as interferon-gamma (IFN-γ) or interleukin 2 (IL-2), T cell proliferation measured by, for example, KI67 measurement, cell quantification by flow cytometry, or lytic activity assessed by real-time impedance measurements of target cells (e.g., Thakur et al., Biosens Bioelectron. 35(1)(2012), 503-506; Krutzik et al., Methods Mol Biol. 699(2011), 179-202; Ekkens et al., Infect Immun. 75(5)(2007), 2291-2296; Ge et al., Proc Natl Acad Sci USA. 99(5)(2002), 2983-2988; Duewell et al., Cell Death Differ. 21(12)(2014), 1825-1837; Erratum in: Cell Death Differ. 21(12)(2014), 161, etc. The costimulatory signaling domains PYAP (AA208-211 of SEQ ID NO: 107) and YMNM (AA191-194 of SEQ ID NO: 106) are beneficial for the function of the CD28 polypeptide and the functional effects listed above. The amino acid sequence of the YMNM domain is shown in SEQ ID NO: 106, and the amino acid sequence of the PYAP domain is shown in SEQ ID NO: 107.Thus, in the antigen-binding receptors of the present invention, the CD28 polypeptide preferably comprises a sequence derived from the intracellular domain of a CD28 polypeptide having the sequence YMNM (SEQ ID NO:106) and / or PYAP (SEQ ID NO:107). In the context of the present invention, the intracellular domain of a CD28 polypeptide having the sequence YMNM (SEQ ID NO:106) and / or PYAP (SEQ ID NO:107) is characterized by CD28 activity, defined as cytokine production, proliferation, and lytic activity of transduced cells as described herein, such as transduced T cells. Thus, in the context of the present invention, the costimulatory signaling domain of the antigen-binding receptors of the present invention has the amino acid sequence (human) of SEQ ID NO:12 (encoded by the DNA sequence shown in SEQ ID NO:25). However, in the antigen-binding receptors of the present invention, one or both of these domains may be mutated to FMNM (SEQ ID NO:108) and / or AYAA (SEQ ID NO:109), respectively. Both of these mutations reduce the cytokine release capacity of transduced cells containing the antigen-binding receptor without affecting their proliferative capacity, and can be advantageously used to prolong the viability and, therefore, the therapeutic potential of the transduced cells. In other words, such non-functional mutations preferably increase the in vivo persistence of cells transduced with the antigen-binding receptors provided herein. However, these signaling motifs may be present anywhere within the intracellular domain of the antigen-binding receptors provided herein.

[0173] Linker and signal peptide Furthermore, the antigen-binding receptors provided herein may comprise at least one linker (or "spacer"). Linkers are typically peptides having a length of up to 20 amino acids. Thus, in the context of the present invention, linkers may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. For example, the antigen-binding receptors provided herein may comprise a linker between the extracellular domain comprising at least one antigen-binding moiety capable of specific binding to a variant Fc domain, the anchoring transmembrane domain, the costimulatory signaling domain, and / or the stimulatory signaling domain. Such linkers have the advantage that they increase the probability that the different polypeptides of the antigen-binding receptor (i.e., the extracellular domain comprising at least one antigen-binding moiety capable of specific binding to a variant Fc domain, the anchoring transmembrane domain, the costimulatory signaling domain, and / or the stimulatory signaling domain) will fold independently and behave predictably. Thus, in the context of the present invention, a single-chain multifunctional polypeptide can include an extracellular domain comprising at least one antigen-binding moiety capable of specifically binding to a variant Fc domain, an anchoring transmembrane domain lacking a cleavage site for a mammalian protease, a costimulatory signaling domain, and a stimulatory signaling domain. A single-chain fusion construct can, for example, consist of a polypeptide comprising an extracellular domain comprising at least one antigen-binding moiety capable of specifically binding to a variant Fc domain, an anchoring transmembrane domain, a costimulatory signaling domain, and / or a stimulatory signaling domain. In alternative embodiments, the antigen-binding receptor comprises an antigen-binding moiety that is not a single-chain fusion construct. That is, the antigen-binding moiety is a Fab or crossFab fragment. In such embodiments, the antigen-binding receptor is not a single-chain fusion construct comprising only one polypeptide chain. Preferably, such a construct would comprise a single-chain heavy chain fusion polypeptide combined with an immunoglobulin light chain, as described herein.For example, a heavy chain fusion polypeptide may comprise an immunoglobulin heavy chain, an anchoring transmembrane domain, a costimulatory signaling domain, and / or a stimulatory signaling domain, combined with an immunoglobulin light chain. Thus, the antigen-binding portion, the anchoring transmembrane domain, the costimulatory signaling domain, and the stimulatory signaling domain may be linked by one or more identical or different peptide linkers, as described herein. For example, in the antigen-binding receptors provided herein, the linker between the extracellular domain comprising at least one antigen-binding portion capable of specifically binding to a variant Fc domain and the anchoring transmembrane domain may comprise or consist of the amino acid sequence set forth in SEQ ID NO:17. Thus, the anchoring transmembrane domain, the costimulatory signaling domain, and / or the stimulatory domain may be linked to each other by a peptide linker or by direct fusion of the domains.

[0174] In some embodiments of the present invention, the antigen-binding portion contained in the extracellular domain is a single-chain variable fragment (scFv), which is a fusion protein of an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) linked by a short linker peptide of 10 to approximately 25 amino acids. The linker is typically glycine-rich for flexibility and serine- or threonine-rich for solubility, and can link the N-terminus of VH to the C-terminus of VL, or vice versa. For example, in the antigen-binding receptor provided herein, the linker can have the amino acid sequence shown in SEQ ID NO:16. The scFv antigen-binding portion described herein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96.

[0175] In some embodiments of the present invention, the antigen-binding portion contained in the extracellular domain is a single-chain Fab fragment, or scFab, which is a polypeptide consisting of a heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and a linker. The antibody domains and linker are arranged in one of the following orders from N- to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL, where the linker is a polypeptide of at least 30 amino acids, preferably 32-50 amino acids. The single-chain Fab fragment is stabilized by a native disulfide bond between the CL and CH1 domains.

[0176] In some embodiments of the present invention, the antigen-binding portion included in the extracellular domain is a crossover single-chain Fab fragment, which is a polypeptide consisting of a heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and linker have one of the following orders from N-terminus to C-terminus: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL, wherein the VH and VL together form an antigen-binding site that specifically binds to an antigen, and the linker is a polypeptide of at least 30 amino acids.

[0177] The antigen-binding receptors provided herein or parts thereof may contain a signal peptide. Such a signal peptide is thought to direct the protein to the surface of the T cell membrane. For example, in the antigen-binding receptors provided herein, the signal peptide may have the amino acid sequence shown in SEQ ID NO:110 (encoded by the DNA sequence shown in SEQ ID NO:111).

[0178] T-cell activating antigen-binding receptor capable of specifically binding to a variant Fc domain Components of the antigen-binding receptors described herein can be fused to each other in a variety of configurations to create T cell activating antigen-binding receptors.

[0179] In some embodiments, the antigen-binding receptor comprises an extracellular domain composed of a heavy chain variable domain (VH) and a light chain variable domain (VL) linked to an anchoring transmembrane domain. In some embodiments, the VH domain is fused at its C-terminus to the N-terminus of the VL domain, optionally via a peptide linker. In other embodiments, the antigen-binding receptor further comprises a stimulatory signaling domain and / or a costimulatory signaling domain. In specific such embodiments, the antigen-binding receptor consists essentially of a VH domain and a VL domain, an anchoring transmembrane domain, and optionally a stimulatory signaling domain, linked by one or more peptide linkers, wherein the VH domain is fused at its C-terminus to the N-terminus of the VL domain, the VL domain is fused at its C-terminus to the N-terminus of the anchoring transmembrane domain, and the anchoring transmembrane domain is fused at its C-terminus to the N-terminus of the stimulatory signaling domain. Optionally, the antigen-binding receptor further comprises a costimulatory signaling domain. In one such specific embodiment, the antigen-binding receptor consists essentially of VH and VL domains, an anchoring transmembrane domain, a stimulatory signaling domain, and a costimulatory signaling domain, linked by one or more peptide linkers, where the VH domain is fused at its C-terminus to the N-terminus of the VL domain, the VL domain is fused at its C-terminus to the N-terminus of the anchoring transmembrane domain, the anchoring transmembrane domain is fused at its C-terminus to the N-terminus of the stimulatory signaling domain, and the stimulatory signaling domain is fused at its C-terminus to the N-terminus of the costimulatory signaling domain. In an alternative embodiment, the costimulatory signaling domain is linked to the anchoring transmembrane domain but not to the stimulatory signaling domain.In a preferred embodiment, the antigen-binding receptor consists essentially of a VH domain and a VL domain, an anchoring transmembrane domain, a costimulatory signaling domain, and a stimulatory signaling domain, linked by one or more peptide linkers, wherein the VH domain is fused at its C-terminus to the N-terminus of the VL domain, the VL domain is fused at its C-terminus to the N-terminus of the anchoring transmembrane domain, the anchoring transmembrane domain is fused at its C-terminus to the N-terminus of the costimulatory signaling domain, and the costimulatory signaling domain is fused at its C-terminus to the N-terminus of the stimulatory signaling domain.

[0180] In a preferred embodiment, one of the binding moieties is a Fab fragment or a crossFab fragment. In a preferred embodiment, the antigen-binding moiety is fused at the C-terminus of the Fab or crossFab heavy chain to the N-terminus of the anchoring transmembrane domain, optionally via a peptide linker. In an alternative embodiment, the antigen-binding moiety is fused at the C-terminus of the Fab or crossFab light chain to the N-terminus of the anchoring transmembrane domain, optionally via a peptide linker. In another embodiment, the antigen-binding receptor further comprises a stimulatory signaling domain and / or a costimulatory signaling domain. In a specific such embodiment, the antigen-binding receptor consists essentially of a Fab or crossFab fragment, an anchoring transmembrane domain, and optionally a stimulatory signaling domain linked by one or more peptide linkers, wherein the Fab or crossFab fragment is fused at the C-terminus of the heavy chain or light chain to the N-terminus of the anchoring transmembrane domain, and the anchoring transmembrane domain is fused at the C-terminus to the N-terminus of the stimulatory signaling domain. Preferably, the antigen-binding receptor further comprises a costimulatory signaling domain. In one such embodiment, the antigen-binding receptor consists essentially of a Fab or crossFab fragment, an anchoring transmembrane domain, a stimulatory signaling domain, and a costimulatory signaling domain, linked by one or more peptide linkers, where the Fab or crossFab fragment is fused at the C-terminus of the heavy or light chain to the N-terminus of the anchoring transmembrane domain, and the stimulatory signaling domain is fused at the C-terminus of the heavy or light chain to the N-terminus of the costimulatory signaling domain. In a preferred embodiment, the costimulatory signaling domain is linked to the anchoring transmembrane domain but not to the stimulatory signaling domain.In a most preferred embodiment, the antigen-binding receptor consists essentially of a Fab or crossFab fragment, an anchoring transmembrane domain, a costimulatory signaling domain, and a stimulatory signaling domain, wherein the Fab or crossFab fragment is fused at the C-terminus of the heavy chain to the N-terminus of the anchoring transmembrane domain via a peptide linker, the anchoring transmembrane domain is fused at the C-terminus to the N-terminus of the costimulatory signaling domain, and the costimulatory signaling domain is fused at the C-terminus to the N-terminus of the stimulatory signaling domain.

[0181] The antigen-binding portion, the anchoring transmembrane domain, and the stimulatory and / or costimulatory signaling domains can be fused to each other directly or via one or more peptide linkers comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n , (SG4) n , (G4S) n or G4 (SG4) n Peptide linkers include those in which "n" is generally a number 1 to 10, typically 2 to 4. A preferred peptide linker for linking the antigen-binding moiety and the anchoring transmembrane moiety is GGGGS (G4S) of SEQ ID NO: 17. An exemplary peptide linker suitable for linking the variable heavy chain (VH) and the variable light chain (VL) is GGGGS (G4S) of SEQ ID NO: 16. The file is TIFF0007642010000030.tif4128.

[0182] In addition, the linker may comprise (a portion of) an immunoglobulin hinge region. In particular, when the antigen-binding moiety is fused to the N-terminus of the anchoring transmembrane domain, it can be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

[0183] As described herein, the antigen-binding receptor of the present invention comprises an extracellular domain comprising at least one antigen-binding moiety. Antigen-binding receptors with a single antigen-binding moiety capable of specifically binding to a target cell antigen are useful and are particularly preferred when high expression of the antigen-binding receptor is required. In such cases, the presence of two or more antigen-binding moieties specific to the target cell antigen may limit the expression efficiency of the antigen-binding receptor. However, in other cases, it may be advantageous to have an antigen-binding receptor comprising two or more antigen-binding moieties specific to a target cell antigen, for example, to optimize targeting to a target site or to enable cross-linking of the target cell antigen.

[0184] In one particular embodiment, the antigen-binding receptor comprises one antigen-binding moiety capable of specifically binding to a variant Fc domain comprising the P329G mutation, in particular to an IgG1 Fc domain. In one embodiment, the antigen-binding moiety capable of specifically binding to the variant Fc domain but not to the non-variant parent Fc domain is an scFv, Fab or crossFab.

[0185] In one embodiment, the antigen-binding portion is fused to the N-terminus of the anchoring transmembrane domain at the C-terminus of the scFv fragment or the C-terminus of the Fab heavy chain or crossFab heavy chain, optionally via a peptide linker. In one embodiment, the peptide linker comprises the amino acid sequence GGGGS (SEQ ID NO: 16). In one embodiment, the anchoring transmembrane domain is a transmembrane domain selected from the group consisting of CD8, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10, or DAP12 transmembrane domains, or fragments thereof. In a preferred embodiment, the anchoring transmembrane domain is a CD28 transmembrane domain or a fragment thereof. In a particular embodiment, the anchoring transmembrane domain is TIFF0007642010000031.tif4128. In one embodiment, the antigen-binding receptor further comprises a costimulatory signaling domain (CSD). In one embodiment, the anchoring transmembrane domain of the antigen-binding receptor is fused at the C-terminus to the N-terminus of the costimulatory signaling domain. In one embodiment, the costimulatory signaling domain is individually selected from the group consisting of the intracellular domains of CD27, CD28, CD137, OX40, ICOS, DAP10, and DAP12, or fragments thereof, as described above. In a preferred embodiment, the costimulatory signaling domain is the intracellular domain of CD28, or a fragment thereof. In a specific embodiment, the costimulatory signaling domain is a CSD of the sequence TIFF0007642010000032.tif4147 or consists of the sequence. In one embodiment, the antigen-binding receptor further comprises a stimulatory signaling domain. In one embodiment, the costimulatory signaling domain of the antigen-binding receptor is fused at the C-terminus to the N-terminus of the stimulatory signaling domain. In one embodiment, at least one stimulatory signaling domain is individually selected from the group consisting of the intracellular domains of CD3z, FCGR3A, and NKG2D, or fragments thereof. In a preferred embodiment, the costimulatory signaling domain is the intracellular domain of CD3z, or a fragment thereof. In a particular embodiment, the costimulatory signaling domain comprises the sequence Contains or consists of the sequence TIFF0007642010000033.tif19160.

[0186] In one embodiment, the antigen-binding receptor is fused to a reporter protein, particularly GFP or an enhanced analog thereof. In one embodiment, the antigen-binding receptor is fused at the C-terminus to the N-terminus of eGFP (enhanced green fluorescent protein), optionally via a peptide linker as described herein. In a preferred embodiment, the peptide linker is the sequence of SEQ ID NO: 18 The file is TIFF0007642010000034.tif4128.

[0187] In a specific embodiment, the antigen-binding receptor comprises an anchoring transmembrane domain and an extracellular domain comprising at least one antigen-binding moiety, wherein the at least one antigen-binding moiety is an scFv fragment capable of specifically binding to a mutated Fc domain but not to a non-mutated parent Fc domain, and the mutated Fc domain comprises a P329G mutation. The P329G mutation reduces Fcγ receptor binding. In one embodiment, the antigen-binding receptor of the present invention comprises an anchoring transmembrane domain (ATD), a costimulatory signaling domain (CSD), and a stimulatory signaling domain (SSD). In one such embodiment, the antigen-binding receptor has the following structure: scFv-ATD-CSD-SSD. In a preferred embodiment, the antigen-binding receptor has the following structure: scFv-G4S-ATD-CSD-SSD, where G4S is a linker comprising the sequence GGGGS of SEQ ID NO: 17. Optionally, a reporter protein can be added to the C-terminus of the antigen-binding receptor, optionally via a peptide linker.

[0188] In a particular embodiment, the antigen-binding portion is an scFv fragment capable of specifically binding to a mutant Fc domain comprising a P329G mutation, wherein the antigen-binding portion comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 and at least one light chain CDR selected from the group of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.

[0189] In a preferred embodiment, the antigen-binding portion is an scFv capable of specifically binding to a mutant Fc domain comprising a P329G mutation, wherein the antigen-binding portion comprises a complementarity-determining region (CDR H) 1 amino acid sequence RYWMN (SEQ ID NO:1), a CDR H2 amino acid sequence TIFF0007642010000035.tif4128CDR H3 amino acid sequence TIFF0007642010000036.tif4128 Light chain complementarity determining region (CDR L) 1 amino acid sequence TIFF0007642010000037.tif4128CDR L2 amino acid sequence GTNKRAP (SEQ ID NO:5) and CDR L3 amino acid sequence Includes TIFF0007642010000038.tif4128.

[0190] In one aspect, the present invention provides a method for producing a medicament comprising, in order from the N-terminus to the C-terminus: (i) an antigen-binding portion which is an scFv fragment capable of specifically binding to a mutant Fc domain comprising a P329G mutation, the scFv fragment comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3, and a light chain variable region (VH) comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6; (ii) a peptide linker, in particular the peptide linker of SEQ ID NO: 17; (iii) an anchoring transmembrane domain, in particular the anchoring transmembrane domain of SEQ ID NO: 11; (iii) a costimulatory signaling domain, particularly the costimulatory signaling domain of SEQ ID NO: 12, and (iv) a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:13 The present invention provides an antigen-binding receptor comprising:

[0191] In one aspect, the present invention provides a method for producing a medicament comprising, in order from the N-terminus to the C-terminus: (i) an antigen-binding portion that is an scFv molecule capable of specifically binding to a variant Fc domain comprising a P329G mutation, the variant Fc domain comprising a heavy chain variable domain (VH) selected from SEQ ID NO:8 and SEQ ID NO:32 and a light chain variable domain (VL) selected from SEQ ID NO:9 and SEQ ID NO:33; (ii) a peptide linker, in particular the peptide linker of SEQ ID NO: 17; (iii) an anchoring transmembrane domain, in particular the anchoring transmembrane domain of SEQ ID NO: 11; (iii) a costimulatory signaling domain, particularly the costimulatory signaling domain of SEQ ID NO: 12, and (iv) a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:13 The present invention provides an antigen-binding receptor comprising:

[0192] In a preferred embodiment, the present invention provides a method for producing a polypeptide comprising, in order from the N-terminus to the C-terminus: (i) an antigen-binding portion that is an scFv molecule that is capable of specifically binding to a variant Fc domain containing a P329G mutation, the variant Fc domain comprising a heavy chain variable domain (VH) SEQ ID NO:8 and a light chain variable domain (VL) SEQ ID NO:9; (ii) a peptide linker, in particular the peptide linker of SEQ ID NO: 17; (iii) an anchoring transmembrane domain, in particular the anchoring transmembrane domain of SEQ ID NO: 11; (iii) a costimulatory signaling domain, particularly the costimulatory signaling domain of SEQ ID NO: 12, and (iv) a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:13 The present invention provides an antigen-binding receptor comprising:

[0193] In a preferred embodiment, the present invention provides a method for producing a polypeptide comprising, in order from the N-terminus to the C-terminus: (i) an antigen-binding portion that is an scFv molecule capable of specifically binding to a mutant Fc domain containing a P329G mutation, comprising the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:34; (ii) a peptide linker, in particular the peptide linker of SEQ ID NO: 17; (iii) an anchoring transmembrane domain, in particular the anchoring transmembrane domain of SEQ ID NO: 11; (iii) a costimulatory signaling domain, particularly the costimulatory signaling domain of SEQ ID NO: 12, and (iv) a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:13 The present invention provides an antigen-binding receptor comprising:

[0194] In certain embodiments, the antigen-binding portion can specifically bind to a mutant Fc domain comprising a P329G mutation, and the antigen-binding receptor comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:31.

[0195] In a preferred embodiment, the antigen-binding portion is capable of specifically binding to a mutant Fc domain comprising a P329G mutation, and the antigen-binding receptor comprises the amino acid sequence of SEQ ID NO:31.

[0196] In a preferred embodiment, the antigen-binding portion is a Fab fragment. In one embodiment, the antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the anchoring transmembrane domain. In one embodiment, the anchoring transmembrane domain is a transmembrane domain selected from the group consisting of CD8, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10, or DAP12 transmembrane domains, or fragments thereof. In a preferred embodiment, the anchoring transmembrane domain is a CD28 transmembrane domain or a fragment thereof. In a specific embodiment, the anchoring transmembrane domain is TIFF0007642010000039.tif4128. In one embodiment, the antigen-binding receptor further comprises a costimulatory signaling domain (CSD). In one embodiment, the anchoring transmembrane domain of the antigen-binding receptor is fused at the C-terminus to the N-terminus of the costimulatory signaling domain. In one embodiment, the costimulatory signaling domain is individually selected from the group consisting of the intracellular domains of CD27, CD28, CD137, OX40, ICOS, DAP10 and DAP12 or fragments thereof, as described above. In a preferred embodiment, the costimulatory signaling domain is the intracellular domain of CD28 or a fragment thereof. In a particular embodiment, the costimulatory signaling domain has the sequence TIFF0007642010000040.tif4147 or consists of the sequence. In one embodiment, the antigen-binding receptor further comprises a stimulatory signaling domain. In one embodiment, the costimulatory signaling domain of the antigen-binding receptor is fused at the C-terminus to the N-terminus of the stimulatory signaling domain. In one embodiment, at least one stimulatory signaling domain is individually selected from the group consisting of the intracellular domains of CD3z, FCGR3A, and NKG2D, or fragments thereof. In a preferred embodiment, the costimulatory signaling domain is the intracellular domain of CD3z, or a fragment thereof. In a particular embodiment, the costimulatory signaling domain comprises the sequence Contains or consists of the sequence TIFF0007642010000041.tif19160.

[0197] In one embodiment, the antigen-binding receptor is fused to a reporter protein, particularly GFP or an enhanced analog thereof. In one embodiment, the antigen-binding receptor is fused at the C-terminus to the N-terminus of eGFP (enhanced green fluorescent protein), optionally via a peptide linker as described herein. In a preferred embodiment, the peptide linker is the sequence of SEQ ID NO: 18 The file is TIFF0007642010000042.tif4128.

[0198] In a specific embodiment, the antigen-binding receptor comprises an anchoring transmembrane domain and an extracellular domain comprising at least one antigen-binding moiety, wherein the at least one antigen-binding moiety is a Fab fragment capable of specifically binding to a mutated Fc domain but not to a non-mutated parent Fc domain, wherein the mutated Fc domain comprises a P329G mutation, which reduces Fcγ receptor binding. In one embodiment, the antigen-binding receptor of the present invention comprises an anchoring transmembrane domain (ATD), a costimulatory signaling domain (CSD), and a stimulatory signaling domain (SSD). In one such embodiment, the antigen-binding receptor has the following structure: Fab-ATD-CSD-SSD. In a preferred embodiment, the antigen-binding receptor has the following structure: Fab-G4S-ATD-CSD-SSD, where G4S is a linker comprising the sequence GGGGS of SEQ ID NO: 17. Optionally, a reporter protein can be attached to the C-terminus of the antigen-binding receptor, optionally via a peptide linker.

[0199] In certain embodiments, the antigen-binding portion is capable of specifically binding to a mutant Fc domain comprising a P329G mutation, and the antigen-binding portion is a Fab fragment comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and at least one light chain CDR selected from the group of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.

[0200] In a preferred embodiment, the antigen-binding portion is a Fab fragment capable of specifically binding to a mutant Fc domain comprising a P329G mutation, wherein the antigen-binding portion comprises the complementarity-determining region (CDR H) 1 amino acid sequence RYWMN (SEQ ID NO:1), the CDR H2 amino acid sequence TIFF0007642010000043.tif4128CDR H3 amino acid sequence TIFF0007642010000044.tif4128 Light chain complementarity determining region (CDR L) 1 amino acid sequence TIFF0007642010000045.tif4128CDR L2 amino acid sequence GTNKRAP (SEQ ID NO:5) and CDR L3 amino acid sequence Includes TIFF0007642010000046.tif4128.

[0201] In one aspect, the present invention provides a method for producing a medicament comprising, in order from the N-terminus to the C-terminus: (i) an antigen-binding portion that is a Fab molecule capable of specifically binding to a mutant Fc domain comprising a P329G mutation, the mutant Fc domain comprising a heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:1, a heavy chain CDR2 of SEQ ID NO:2, a heavy chain CDR3 of SEQ ID NO:3, a light chain CDR1 of SEQ ID NO:4, a light chain CDR2 of SEQ ID NO:5, and a light chain CDR3 of SEQ ID NO:6; (ii) a peptide linker, in particular the peptide linker of SEQ ID NO: 17; (iii) an anchoring transmembrane domain, in particular the anchoring transmembrane domain of SEQ ID NO: 11; (iii) a costimulatory signaling domain, particularly the costimulatory signaling domain of SEQ ID NO: 12, and (iv) a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:13 The present invention provides an antigen-binding receptor comprising:

[0202] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) From the N-terminus to the C-terminus, (i) a heavy chain comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3; (ii) a peptide linker, in particular the peptide linker of SEQ ID NO: 17; (iii) an anchoring transmembrane domain, in particular the anchoring transmembrane domain of SEQ ID NO: 11; (iii) a costimulatory signaling domain, particularly the costimulatory signaling domain of SEQ ID NO: 12, and (iv) a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:13 a heavy chain fusion polypeptide comprising: b) a light chain comprising a light chain CDR1 of SEQ ID NO:4, a light chain CDR2 of SEQ ID NO:5, and a light chain CDR3 of SEQ ID NO:6; The present invention provides an antigen-binding receptor comprising:

[0203] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) From the N-terminus to the C-terminus, (i) a heavy chain variable domain (VH) of SEQ ID NO:8; (ii) a peptide linker, in particular the peptide linker of SEQ ID NO: 17; (iii) an anchoring transmembrane domain, in particular the anchoring transmembrane domain of SEQ ID NO: 11; (iii) a costimulatory signaling domain, particularly the costimulatory signaling domain of SEQ ID NO: 12, and (iv) a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:13 a heavy chain fusion polypeptide comprising: b) the light chain variable domain (VL) of SEQ ID NO:9; The present invention provides an antigen-binding receptor comprising:

[0204] In one embodiment, the antigen-binding portion is a Fab fragment comprising a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:40 or SEQ ID NO:49 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:50. In a preferred embodiment, the antigen-binding portion is a Fab fragment comprising a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:40 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:41.

[0205] In certain embodiments, the antigen-binding portion is a Fab fragment capable of specifically binding to a mutant Fc domain comprising a P329G mutation, and the antigen-binding receptor comprises a heavy chain fusion polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group of SEQ ID NO:39 and SEQ ID NO:48, and a light chain polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group of SEQ ID NO:41 and SEQ ID NO:50.

[0206] In a preferred embodiment, the antigen-binding portion is a Fab fragment capable of specifically binding to a mutant Fc domain comprising a P329G mutation, and the antigen-binding receptor comprises a heavy chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO:39 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:41.

[0207] In an alternative embodiment, the antigen-binding receptor comprises one antigen-binding moiety capable of specifically binding to a variant Fc domain comprising the mutations I253A, H310A, and H435A ("AAA"), particularly to an IgG1 Fc domain. In one embodiment, the antigen-binding moiety capable of specifically binding to the variant Fc domain but not to the non-variant parent Fc domain is an scFv, Fab, or crossFab.

[0208] In one embodiment, the antigen-binding portion is fused to the N-terminus of the anchoring transmembrane domain at the C-terminus of the scFv fragment or the C-terminus of the Fab heavy chain or crossFab heavy chain, optionally via a peptide linker. In one embodiment, the peptide linker comprises the amino acid sequence GGGGS (SEQ ID NO: 16). In one embodiment, the anchoring transmembrane domain is a transmembrane domain selected from the group consisting of CD8, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10, or DAP12 transmembrane domains, or fragments thereof. In a preferred embodiment, the anchoring transmembrane domain is a CD28 transmembrane domain or a fragment thereof. In a particular embodiment, the anchoring transmembrane domain is TIFF0007642010000047.tif4128. In one embodiment, the antigen-binding receptor further comprises a costimulatory signaling domain (CSD). In one embodiment, the anchoring transmembrane domain of the antigen-binding receptor is fused at the C-terminus to the N-terminus of the costimulatory signaling domain. In one embodiment, the costimulatory signaling domain is individually selected from the group consisting of the intracellular domains of CD27, CD28, CD137, OX40, ICOS, DAP10, and DAP12, or fragments thereof, as described above. In a preferred embodiment, the costimulatory signaling domain is the intracellular domain of CD28, or a fragment thereof. In a specific embodiment, the costimulatory signaling domain is a CSD of the sequence TIFF0007642010000048.tif4147 or consists of the sequence. In one embodiment, the antigen-binding receptor further comprises a stimulatory signaling domain. In one embodiment, the costimulatory signaling domain of the antigen-binding receptor is fused at the C-terminus to the N-terminus of the stimulatory signaling domain. In one embodiment, at least one stimulatory signaling domain is individually selected from the group consisting of the intracellular domains of CD3z, FCGR3A, and NKG2D, or fragments thereof. In a preferred embodiment, the costimulatory signaling domain is the intracellular domain of CD3z, or a fragment thereof. In a particular embodiment, the costimulatory signaling domain comprises the sequence Contains or consists of the sequence TIFF0007642010000049.tif19160.

[0209] In one embodiment, the antigen-binding receptor is fused to a reporter protein, particularly GFP or an enhanced analog thereof. In one embodiment, the antigen-binding receptor is fused at the C-terminus to the N-terminus of eGFP (enhanced green fluorescent protein), optionally via a peptide linker as described herein. In a preferred embodiment, the peptide linker is the sequence of SEQ ID NO: 18 The file is TIFF0007642010000050.tif4128.

[0210] In a specific embodiment, the antigen-binding receptor comprises an anchoring transmembrane domain and an extracellular domain comprising at least one antigen-binding portion, wherein the at least one antigen-binding portion is an scFv fragment capable of specifically binding to a mutated Fc domain but not to a non-mutated parent Fc domain, wherein the mutated Fc domain comprises I253A, H310A, and H435A mutations. The I253A, H310A, and H435A mutations reduce FcRn receptor binding. In one embodiment, the antigen-binding receptor of the present invention comprises an anchoring transmembrane domain (ATD), a costimulatory signaling domain (CSD), and a stimulatory signaling domain (SSD). In one such embodiment, the antigen-binding receptor has the following structure: scFv-ATD-CSD-SSD. In a preferred embodiment, the antigen-binding receptor has the following structure: scFv-G4S-ATD-CSD-SSD, where G4S is a linker comprising the sequence GGGGS of SEQ ID NO: 17. Optionally, a reporter protein can be attached to the C-terminus of the antigen-binding receptor, optionally via a peptide linker.

[0211] In certain embodiments, the antigen-binding portion is an scFv fragment capable of specifically binding to a mutant Fc domain comprising I253A, H310A, and H435A mutations, wherein the antigen-binding portion comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55, and at least one light chain CDR selected from the group of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58.

[0212] In a preferred embodiment, the antigen-binding portion is an scFv capable of specifically binding to a mutant Fc domain comprising the mutations I253A, H310A, and H435A, wherein the antigen-binding portion comprises a complementarity-determining region (CDR H) 1 amino acid sequence SYGMS (SEQ ID NO:53), a CDR H2 amino acid sequence SSGGSY (SEQ ID NO:54), a CDR H3 amino acid sequence TIFF0007642010000051.tif4128 Light chain complementarity determining region (CDR L) 1 amino acid sequence TIFF0007642010000052.tif4128CDR L2 amino acid sequence KVSNRFS (SEQ ID NO: 57) and CDR L3 amino acid sequence Includes TIFF0007642010000053.tif4128.

[0213] In one aspect, the present invention provides a method for producing a medicament comprising, in order from the N-terminus to the C-terminus: (i) an antigen-binding portion which is an scFv fragment capable of specifically binding to a mutant Fc domain comprising I253A, H310A, and H435A mutations, the scFv fragment comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 53, a heavy chain CDR2 of SEQ ID NO: 54, a heavy chain CDR3 of SEQ ID NO: 55, and a light chain variable region (VH) comprising a light chain CDR1 of SEQ ID NO: 56, a light chain CDR2 of SEQ ID NO: 57, and a light chain CDR3 of SEQ ID NO: 58; (ii) a peptide linker, in particular the peptide linker of SEQ ID NO: 17; (iii) an anchoring transmembrane domain, in particular the anchoring transmembrane domain of SEQ ID NO: 11; (iii) a costimulatory signaling domain, particularly the costimulatory signaling domain of SEQ ID NO: 12, and (iv) a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:13 The present invention provides an antigen-binding receptor comprising:

[0214] In one aspect, the present invention provides a method for producing a medicament comprising, in order from the N-terminus to the C-terminus: (i) an antigen-binding portion that is an scFv molecule that is capable of specifically binding to a variant Fc domain comprising the I253A, H310A, and H435A mutations, comprising a heavy chain variable domain (VH) of SEQ ID NO:61 and a light chain variable domain (VL) of SEQ ID NO:62; (ii) a peptide linker, in particular the peptide linker of SEQ ID NO: 17; (iii) an anchoring transmembrane domain, in particular the anchoring transmembrane domain of SEQ ID NO: 11; (iii) a costimulatory signaling domain, particularly the costimulatory signaling domain of SEQ ID NO: 12, and (iv) a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:13 The present invention provides an antigen-binding receptor comprising:

[0215] In one aspect, the present invention provides a method for producing a medicament comprising, in order from the N-terminus to the C-terminus: (i) an antigen-binding portion that is an scFv molecule capable of specifically binding to a mutant Fc domain comprising the I253A, H310A, and H435A mutations, comprising the amino acid sequence of SEQ ID NO:60; (ii) a peptide linker, in particular the peptide linker of SEQ ID NO: 17; (iii) an anchoring transmembrane domain, in particular the anchoring transmembrane domain of SEQ ID NO: 11; (iii) a costimulatory signaling domain, particularly the costimulatory signaling domain of SEQ ID NO: 12, and (iv) a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:13 The present invention provides an antigen-binding receptor comprising:

[0216] In certain embodiments, the antigen-binding portion can specifically bind to a mutant Fc domain comprising I253A, H310A, and H435A mutations, and the antigen-binding receptor comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:59.

[0217] In a preferred embodiment, the antigen-binding portion is capable of specifically binding to a mutant Fc domain comprising I253A, H310A, and H435A mutations, and the antigen-binding receptor comprises the amino acid sequence of SEQ ID NO:595.

[0218] In a preferred embodiment, the antigen-binding portion is a Fab fragment. In one embodiment, the antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the anchoring transmembrane domain. In one embodiment, the anchoring transmembrane domain is a transmembrane domain selected from the group consisting of CD8, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10, or DAP12 transmembrane domains, or fragments thereof. In a preferred embodiment, the anchoring transmembrane domain is a CD28 transmembrane domain or a fragment thereof. In a particular embodiment, the anchoring transmembrane domain is TIFF0007642010000054.tif4128. In one embodiment, the antigen-binding receptor further comprises a costimulatory signaling domain (CSD). In one embodiment, the anchoring transmembrane domain of the antigen-binding receptor is fused at the C-terminus to the N-terminus of the costimulatory signaling domain. In one embodiment, the costimulatory signaling domain is individually selected from the group consisting of the intracellular domains of CD27, CD28, CD137, OX40, ICOS, DAP10 and DAP12 or fragments thereof, as described above. In a preferred embodiment, the costimulatory signaling domain is the intracellular domain of CD28 or a fragment thereof. In a particular embodiment, the costimulatory signaling domain has the sequence TIFF0007642010000055.tif4147 or consists of the sequence. In one embodiment, the antigen-binding receptor further comprises a stimulatory signaling domain. In one embodiment, the costimulatory signaling domain of the antigen-binding receptor is fused at the C-terminus to the N-terminus of the stimulatory signaling domain. In one embodiment, at least one stimulatory signaling domain is individually selected from the group consisting of the intracellular domains of CD3z, FCGR3A, and NKG2D, or fragments thereof. In a preferred embodiment, the costimulatory signaling domain is the intracellular domain of CD3z, or a fragment thereof. In a particular embodiment, the costimulatory signaling domain comprises the sequence Contains or consists of the sequence TIFF0007642010000056.tif19160.

[0219] In one embodiment, the antigen-binding receptor is fused to a reporter protein, particularly GFP or an enhanced analog thereof. In one embodiment, the antigen-binding receptor is fused at the C-terminus to the N-terminus of eGFP (enhanced green fluorescent protein), optionally via a peptide linker as described herein. In a preferred embodiment, the peptide linker is the sequence of SEQ ID NO: 18 The file is TIFF0007642010000057.tif4128.

[0220] In a specific embodiment, the antigen-binding receptor comprises an anchoring transmembrane domain and an extracellular domain comprising at least one antigen-binding moiety, wherein the at least one antigen-binding moiety is a Fab fragment capable of specifically binding to a mutant Fc domain but not to a non-mutated parent Fc domain, wherein the mutant Fc domain comprises I253A, H310A, and H435A mutations, and the I253A, H310A, and H435A mutations reduce FcRn receptor binding. In one embodiment, the antigen-binding receptor of the present invention comprises an anchoring transmembrane domain (ATD), a costimulatory signaling domain (CSD), and a stimulatory signaling domain (SSD). In one such embodiment, the antigen-binding receptor has the following configuration: Fab-ATD-CSD-SSD. In a preferred embodiment, the antigen-binding receptor has the following configuration: Fab-G4S-ATD-CSD-SSD, wherein G4S is a linker comprising the sequence GGGGS of SEQ ID NO: 17. Optionally, a reporter protein can be attached to the C-terminus of the antigen-binding receptor, optionally via a peptide linker.

[0221] In certain embodiments, the antigen-binding portion can specifically bind to a mutant Fc domain comprising the I253A, H310A, and H435A mutations, and the antigen-binding portion is a Fab fragment comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55, and at least one light chain CDR selected from the group of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58.

[0222] In a preferred embodiment, the antigen-binding portion is a Fab fragment capable of specifically binding to a mutant Fc domain comprising the mutations I253A, H310A, and H435A, wherein the antigen-binding portion comprises the complementarity-determining region (CDR H) 1 amino acid sequence SYGMS (SEQ ID NO:53), the CDR H2 amino acid sequence SSGGSY (SEQ ID NO:54), the CDR H3 amino acid sequence TIFF0007642010000058.tif4128Light chain complementarity determining region (CDR L) 1 amino acid sequence TIFF0007642010000059.tif4128CDR L2 amino acid sequence KVSNRFS (SEQ ID NO: 57) and CDR L3 amino acid sequence Includes TIFF0007642010000060.tif4128.

[0223] In one aspect, the present invention provides a method for producing a medicament comprising, in order from the N-terminus to the C-terminus: (i) an antigen-binding portion that is a Fab molecule capable of specifically binding to a mutant Fc domain comprising I253A, H310A, and H435A mutations, the mutant Fc domain comprising heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 53, heavy chain CDR2 of SEQ ID NO: 54, heavy chain CDR3 of SEQ ID NO: 55, light chain CDR1 of SEQ ID NO: 56, light chain CDR2 of SEQ ID NO: 57, and light chain CDR3 of SEQ ID NO: 58; (ii) a peptide linker, in particular the peptide linker of SEQ ID NO: 17; (iii) an anchoring transmembrane domain, in particular the anchoring transmembrane domain of SEQ ID NO: 11; (iii) a costimulatory signaling domain, particularly the costimulatory signaling domain of SEQ ID NO: 12, and (iv) a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:13 The present invention provides an antigen-binding receptor comprising:

[0224] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) From the N-terminus to the C-terminus, (i) a heavy chain comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 53, a heavy chain CDR2 of SEQ ID NO: 54, and a heavy chain CDR3 of SEQ ID NO: 55; (ii) a peptide linker, in particular the peptide linker of SEQ ID NO: 17; (iii) an anchoring transmembrane domain, in particular the anchoring transmembrane domain of SEQ ID NO: 11; (iii) a costimulatory signaling domain, particularly the costimulatory signaling domain of SEQ ID NO: 12, and (iv) a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:13 a heavy chain fusion polypeptide comprising: b) a light chain comprising a light chain CDR1 of SEQ ID NO:56, a light chain CDR2 of SEQ ID NO:57, and a light chain CDR3 of SEQ ID NO:58; The present invention provides an antigen-binding receptor comprising:

[0225] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) From the N-terminus to the C-terminus, (i) a heavy chain variable domain (VH) of SEQ ID NO:61; (ii) a peptide linker, in particular the peptide linker of SEQ ID NO: 17; (iii) an anchoring transmembrane domain, in particular the anchoring transmembrane domain of SEQ ID NO: 11; (iii) a costimulatory signaling domain, particularly the costimulatory signaling domain of SEQ ID NO: 12, and (iv) a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:13 a heavy chain fusion polypeptide comprising: b) the light chain variable domain (VL) of SEQ ID NO:62; The present invention provides an antigen-binding receptor comprising:

[0226] In one particular embodiment, the antigen-binding portion is a Fab fragment comprising a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:64 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:65.

[0227] In certain embodiments, the antigen-binding portion is a Fab fragment capable of specifically binding to a mutant Fc domain comprising I253A, H310A, and H435A mutations, and the antigen-binding receptor comprises a heavy chain fusion polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:63, and a light chain polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:65.

[0228] In a preferred embodiment, the antigen-binding portion is a Fab fragment capable of specifically binding to a mutant Fc domain comprising the I253A, H310A, and H435A mutations, and the antigen-binding receptor comprises a heavy chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO:63 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:65.

[0229] In certain alternative embodiments, the antigen-binding receptor, Fab light chain polypeptide, and Fab heavy chain fusion polypeptide of the invention are fused to each other, optionally via a linker peptide. Fusion of the Fab heavy and light chains can improve pairing of the Fab heavy and light chains and reduce the number of plasmids required to express some antigen-binding receptors of the invention. An alternative strategy for reducing the number of plasmids required to express an antigen-binding receptor is to use an internal ribosomal entry side, such that both heavy and light chain constructs can be expressed from the same plasmid, as illustrated, for example, in Figure 2.

[0230] In certain embodiments, the antigen-binding receptor comprises a polypeptide in which the Fab light chain variable region of the antigen-binding portion shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the antigen-binding portion (i.e., the antigen-binding portion comprises a crossFab heavy chain in which the heavy chain variable region is replaced with a light chain variable region), and the polypeptide shares a carboxy-terminal peptide bond with an anchoring transmembrane domain (VL (1) -CH1 (1) In some embodiments, the antigen-binding receptor comprises a polypeptide in which the Fab heavy chain variable region of the first antigen-binding moiety shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first antigen-binding moiety (VH (1) -CL (1) ). In certain embodiments, the polypeptides are covalently linked, e.g., by a disulfide bond. In alternative embodiments, the antigen-binding receptor comprises a polypeptide in which the Fab heavy chain variable region of the antigen-binding portion shares a carboxy-terminal peptide bond with the Fab light chain constant region of the antigen-binding portion (i.e., the antigen-binding portion comprises a crossFab heavy chain in which the heavy chain constant region is replaced with a light chain constant region), and the polypeptide shares a carboxy-terminal peptide bond with an anchoring transmembrane domain (VH (1) -CL (1) In some embodiments, the antigen-binding receptor is a polypeptide in which the Fab light chain variable region of the antigen-binding portion shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the antigen-binding portion (VL (1) -CH1 (1) In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

[0231] In any of the above embodiments, the components of the antigen-binding receptor (e.g., VH and VL, antigen-binding portion, anchoring transmembrane domain, costimulatory signaling domain, stimulatory signaling domain) can be fused directly or via a variety of linkers, particularly peptide linkers comprising one or more amino acids, typically about 2-20 amino acids, as described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, (G4S) n , (SG4) n , (G4S) n or G4 (SG4) n Peptide linkers are included, where n is generally a number from 1 to 10, preferably from 1 to 4.

[0232] Exemplary T Cell Activation Antigen Binding Receptors As illustrated in the accompanying Examples and Figure 1A, as a proof-of-concept of the present invention, an antigen-binding receptor "anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD pETR17096" (SEQ ID NO:7) was constructed, which comprises one stabilized scFv antigen-binding moiety that binds to / directs to / interacts with or at an antibody comprising a P329G mutation in its Fc domain. This construct further comprises a CD28 transmembrane domain, a fragment of CD28 as a costimulatory signaling domain, and a fragment of CD3z as a stimulatory signaling domain. The sequence (amino acid and cDNA) of the antibody-binding molecule "anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD pETR17096" is shown in Tables 2 and 3.

[0233] Furthermore, as illustrated in FIG. 1B , as a further proof-of-concept of the present invention, an antigen-binding receptor "anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD pETR17100" (SEQ ID NOs: 39, 41) was constructed, which contains one stabilized Fab antigen-binding portion that binds to / directs to / interacts with or at an antibody comprising a P329G mutation in the Fc domain. This construct further contains a CD28 transmembrane domain, a fragment of CD28 as a costimulatory signaling domain, and a fragment of CD3z as a stimulatory signaling domain. The sequence (amino acid and DNA) of the antigen-binding receptor "anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD pETR17100" is shown in Tables 4 and 5.

[0234] As a further proof-of-concept of the present invention, an antigen-binding receptor "anti-P329G-Fab-CD28ATD-CD28CSD-CD3zSSD pETR17594" (SEQ ID NOs: 48, 50) was constructed that contains one Fab antigen-binding portion that binds to / directs to / interacts with or in an antibody comprising a P329G mutation in the Fc domain. This construct further contains a CD28 transmembrane domain, a fragment of CD28 as a costimulatory signaling domain, and a fragment of CD3z as a stimulatory signaling domain. The sequence (amino acid and DNA) of the antigen-binding receptor "anti-P329G-Fab-CD28ATD-CD28CSD-CD3zSSD pETR17594" is shown in Tables 6 and 7.

[0235] As a further proof-of-concept of the present invention, an antigen-binding receptor "anti-AAA scFv" (SEQ ID NO:59) was constructed that contains one scFv antigen-binding portion that binds to / directs to / interacts with or at an antibody containing the I253A, H310A, and H435A mutations in the Fc domain. This construct further contains a CD28 transmembrane domain, a fragment of CD28 as a costimulatory signaling domain, and a fragment of CD3z as a stimulatory signaling domain. The sequence (amino acid and cDNA) of the antibody-binding molecule "anti-AAA scFv" is shown in Tables 8 and 9.

[0236] As a further proof-of-concept of the present invention, an antigen-binding receptor "anti-AAA Fab" (SEQ ID NO: 63, 65) was constructed that contains one Fab antigen-binding portion that binds to / directs to / interacts with or at an antibody containing the I253A, H310A, and H435A mutations in the Fc domain. This construct further contains a CD28 transmembrane domain, a fragment of CD28 as a costimulatory signaling domain, and a fragment of CD3z as a stimulatory signaling domain. The sequence (amino acid and cDNA) of the antibody-binding molecule "anti-AAA scFv" is shown in Tables 10 and 11.

[0237] The present invention also provides nucleic acid molecules encoding the antigen-binding receptors of the invention described herein. Also encompassed by the present invention are kits comprising nucleic acid molecules encoding the antigen-binding receptors of the invention, and the nucleic acid molecules of the invention as further described herein.

[0238] kit A further aspect of the present invention is a kit comprising or consisting of a nucleic acid encoding an antigen-binding receptor of the invention and / or a cell, preferably a T cell, transduced with an antigen-binding receptor of the invention, and optionally one or more antibodies comprising a variant Fc domain, wherein the antigen-binding receptor is capable of specific binding to the variant Fc domain.

[0239] therefore, (A) a transduced T cell capable of expressing an antigen-binding receptor of the present invention; (B) Antibodies containing variant Fc domains A kit comprising: the antigen-binding receptor is capable of specifically binding to the variant Fc domain but is incapable of specifically binding to the non-variant parent Fc domain; A kit is provided.

[0240] moreover, (A) an isolated polynucleotide and / or vector encoding an antigen-binding receptor of the present invention; (B) Antibodies containing variant Fc domains A kit comprising: the antigen-binding receptor is capable of specifically binding to the variant Fc domain but is incapable of specifically binding to the non-variant parent Fc domain; A kit is provided.

[0241] In the context of the present invention, kits of the present invention may include transduced T cells, isolated polynucleotides and / or vectors, and one or more antibodies comprising a variant Fc domain. In certain embodiments, the antibody is a therapeutic antibody, e.g., a tumor-specific antibody. Tumor-specific antigens are known in the art and are described herein. In the context of the present invention, the antibody is administered before, simultaneously with, or after administration of transduced T cells expressing an antigen-binding receptor of the present invention. Kits of the present invention include transduced T cells or polynucleotides / vectors for producing transduced T cells. In this case, the transduced T cells are not specific to a given tumor but are general T cells because they can be directed to any tumor depending on the therapeutic antibody comprising the variant Fc domain. Examples of antibodies comprising a variant Fc domain are listed herein, and any antibody comprising a variant Fc domain described herein can be included in the kits provided herein. In certain embodiments, a variant Fc domain of an antibody exhibits reduced binding affinity to an Fc receptor and / or reduced effector function compared to a native IgG1 Fc domain. In one such embodiment, the variant Fc domain (or an antibody comprising said Fc variant domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the binding affinity to an Fc receptor compared to a native IgG1 Fc domain (or an antibody comprising a native IgG1 Fc domain), and / or less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the effector function compared to a native IgG1 Fc domain (or an antibody comprising a native IgG1 Fc domain). In one embodiment, the variant Fc domain (or an antibody comprising said variant Fc domain) does not substantially bind to an Fc receptor and / or induce effector function. In a certain embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor.In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the variant Fc domain exhibits substantially altered binding affinity to the neonatal Fc receptor (FcRn) compared to a native IgG1 Fc domain. In one embodiment, an antibody comprising the variant Fc domain exhibits less than 20%, particularly less than 10%, particularly less than 5% of the binding affinity to the Fc receptor compared to an antibody comprising an unmodified Fc domain. In a particular embodiment, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to complement components, particularly C1q, is also reduced.

[0242] In certain embodiments, the Fc domain of the antibody is mutated to have reduced effector function compared to the non-mutated Fc domain. The reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling to induce apoptosis, reduced cross-linking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, the reduced effector function is one or more selected from the group consisting of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a specific embodiment, the reduced effector function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by an unmodified Fc domain (or an antibody comprising an unmodified Fc domain).

[0243] In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or the effector function is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331, and P329. In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235, and P329. In some embodiments, the Fc domain comprises amino acid substitutions L234A and L235A. In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297, and P331. In more specific embodiments, said further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235. In one embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA"). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. The amino acid substitution combination "P329G LALA" almost completely abolishes Fcγ receptor (and complement) binding of human IgG1 Fc domains, as described in PCT Publication No. WO 2012 / 130831, which is incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods for preparing such mutant Fc domains and determining their properties, such as Fc receptor binding or effector function.

[0244] In one particular embodiment, the Fc domain that exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain is a human IgG1 Fc domain comprising the amino acid mutations L234A, L235A, and optionally P329G, or a human IgG4 Fc domain comprising the amino acid mutations S228P, L235E, and optionally P329G.

[0245] In certain embodiments, N-glycosylation of the Fc domain is eliminated, hi one such embodiment, the Fc domain comprises an amino acid mutation at position N297, specifically replacing asparagine with alanine (N297A) or aspartic acid (N297D).

[0246] In addition to the Fc domains described above and in PCT Publication No. WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or reduced effector function also include those with mutations at one or more of residues 238, 265, 269, 270, 297, 327, and 329 of the Fc domain (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with mutations at two or more of amino acids 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with mutations at residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0247] Variant Fc domains can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods include site-directed mutagenesis of the coding DNA sequence, PCR, gene synthesis, etc. Correct nucleotide changes can be verified, for example, by sequencing.

[0248] Binding to Fc receptors can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors can be obtained by recombinant expression. Alternatively, the binding affinity of an Fc domain, or of a cell-activating bispecific antigen-binding molecule containing an Fc domain, for an Fc receptor can be assessed using cell lines known to express specific Fc receptors, such as human NK cells expressing the FcγIIIa receptor.

[0249] The effector function of Fc domain or antibody comprising Fc domain can be measured by methods known in the art.Other examples of in vitro assays for evaluating the ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362, Hellstrom et al. Proc Natl Acad Sci USA 83,7059-7063(1986) and Hellstrom et al., Proc Natl Acad Sci USA 82,1499-1502(1985), U.S. Patent No. 5,821,337, Bruggemann et al., J Exp Med 166,1351-1361(1987). Alternatively, non-radioactive assay methods may be used (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA) and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0250] In some embodiments, the binding of the Fc domain to complement components, particularly C1q, is reduced. Thus, in some embodiments in which the Fc domain is engineered to have reduced effector function, the reduced effector function includes reduced CDC. To determine whether an antibody can bind to C1q and therefore has CDC activity, a C1q binding assay can be performed. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. A CDC assay can be performed to assess complement activation (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0251] In one embodiment, the binding affinity to the neonatal Fc receptor (FcRn) is reduced. In a particular embodiment, the variant Fc domain of the present invention exhibits reduced binding affinity to the FcRn receptor compared to a native IgG1 Fc domain. In one such embodiment, the Fc domain (or an antibody comprising the Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the binding affinity to the neonatal Fc receptor compared to a native IgG1 Fc domain (or an antibody comprising a native IgG1 Fc domain), and / or less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the effector function compared to a native IgG1 Fc domain (or an antibody comprising a native IgG1 Fc domain). In one embodiment, the variant Fc domain (or an antibody comprising the variant Fc domain) does not substantially bind to the neonatal Fc receptor. In a particular embodiment, the Fc receptor is an FcRn receptor. In one embodiment, the Fc receptor is a human FcRn receptor. In certain embodiments, the Fc domain comprises amino acid substitutions at positions 1253, H310, and H435. In more specific embodiments, the Fc domain comprises amino acid mutations at 1253A, H310A, and H435A ("AAA"). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. The amino acid substitution combination "AAA" almost completely abolishes the FcRn receptor binding of the human IgG1 Fc domain.

[0252] In a specific embodiment, the antibody comprising a variant Fc region is capable of specifically binding to CD20 and comprises the heavy chain sequence of SEQ ID NO:112 and the light chain sequence of SEQ ID NO:113. In one embodiment, the antibody comprising a variant Fc region is capable of specifically binding to FAP and comprises the heavy chain sequence of SEQ ID NO:114 and the light chain sequence of SEQ ID NO:115. In one embodiment, the antibody comprising a variant Fc region is capable of specifically binding to CEA and comprises the heavy chain sequence of SEQ ID NO:116 and the light chain sequence of SEQ ID NO:117, the heavy chain sequence of SEQ ID NO:118 and the light chain sequence of SEQ ID NO:119, the heavy chain sequence of SEQ ID NO:120 and the light chain sequence of SEQ ID NO:121, or the heavy chain sequence of SEQ ID NO:122 and the light chain sequence of SEQ ID NO:123. In a further embodiment, the antibody comprising the variant Fc region is capable of specifically binding to tenascin (TNC) and comprises the heavy chain sequence of SEQ ID NO:124 and the light chain sequence of SEQ ID NO:125.

[0253] In further embodiments, the antibody comprising a variant Fc region is a bispecific antibody, e.g., a T cell activation bispecific antibody. In one such embodiment, the bispecific antibody comprises a first binding moiety capable of specifically binding to a T cell activation target, in particular CD3, and a second binding moiety capable of specifically binding to a tumor antigen described herein.

[0254] In one embodiment, the antibody comprising the variant Fc region is bispecific and capable of specifically binding to Her2, the bispecific antibody comprising a first heavy chain sequence of SEQ ID NO:126, a first light chain sequence of SEQ ID NO:127, a second heavy chain sequence of SEQ ID NO:128, and a second light chain sequence of SEQ ID NO:129.

[0255] In an exemplary embodiment of the invention, as proof of concept, a kit is provided comprising the amino acid sequence shown in SEQ ID NO:7 ("anti-P329G-ds-scFv-CD28ATD-CD28CSD-CD3zSSD" (encoded by the DNA sequence shown in SEQ ID NO:19)) in combination with an antibody comprising a heavy chain of SEQ ID NO:112 and a light chain of SEQ ID NO:113. Alternatively, the kit may comprise the amino acid sequence shown in SEQ ID NO:31 ("anti-P329G-scFv-CD28ATD-CD28CSD-CD3zSSD" (encoded by the DNA sequence shown in SEQ ID NO:35)) in combination with an antibody comprising a heavy chain of SEQ ID NO:112 and a light chain of SEQ ID NO:113. Furthermore, in the context of the present invention, a kit may comprise the amino acid sequence shown in SEQ ID NO:39 ("anti-P329G-ds-Fab-CD28ATD-CD28CSD-CD3zSSD" (encoded by the DNA sequence shown in SEQ ID NO:44)) in combination with an antibody comprising a heavy chain of SEQ ID NO:112 and a light chain of SEQ ID NO:113. Alternatively, a kit may comprise the amino acid sequence shown in SEQ ID NO:48 ("anti-P329G-Fab-CD28ATD-CD28CSD-CD3zSSD" (encoded by the DNA sequence shown in SEQ ID NO:51)) in combination with an antibody comprising a heavy chain of SEQ ID NO:112 and a light chain of SEQ ID NO:113. Alternatively, the kit may comprise the amino acid sequence shown in SEQ ID NO:59 in combination with an antibody comprising a heavy chain of SEQ ID NO:112 and a light chain of SEQ ID NO:113 ("anti-AAA-scFv-CD28ATD-CD28CSD-CD3zSSD"). Furthermore, in the context of the present invention, the kit may comprise the amino acid sequence shown in SEQ ID NO:63 in combination with an antibody comprising a heavy chain of SEQ ID NO:112 and a light chain of SEQ ID NO:113 ("anti-AAA-Fab-CD28ATD-CD28CSD-CD3zSSD").Furthermore, in the context of the present invention, a kit may comprise at least one antibody molecule comprising a heavy chain and a light chain selected from the group consisting of SEQ ID NO:112 and SEQ ID NO:113, SEQ ID NO:114 and SEQ ID NO:115, SEQ ID NO:116 and SEQ ID NO:117, SEQ ID NO:118 and SEQ ID NO:119, SEQ ID NO:120 and SEQ ID NO:121, SEQ ID NO:122 and SEQ ID NO:123, and SEQ ID NO:124 and SEQ ID NO:125. Furthermore, in the context of the present invention, a kit may comprise a bispecific antibody molecule, in particular a bispecific antibody comprising a first heavy chain of SEQ ID NO:128, a first light chain of SEQ ID NO:129, a second heavy chain of SEQ ID NO:130, and a second light chain of SEQ ID NO:131.

[0256] Furthermore, the parts of the kit of the present invention can be packaged individually in vials or bottles, or combined and packaged in a container or multi-container unit. In addition, the kit of the present invention may also comprise a (closed) bag cell incubation system capable of transducing and incubating patient cells, preferably T cells as described herein, with the antigen-binding receptor of the present invention under GMP (Good Manufacturing Practice as set forth in the guidelines on Good Manufacturing Practice published by the European Commission at http: / / ec.europa.eu / health / documents / eudralex / index_en.htm) conditions. Furthermore, the kit of the present invention comprises a (closed) bag cell incubation system capable of transducing and incubating isolated / obtained patient T cells with the antigen-binding receptor of the present invention under GMP conditions. Furthermore, in the context of the present invention, the kit may also comprise a vector encoding the antigen-binding receptor described herein. The kit of the present invention can be advantageously used, inter alia, to carry out the method of the present invention and can be used in various applications mentioned herein, for example, as a research tool or a medical tool. The production of the kit preferably follows standard procedures known to those skilled in the art.

[0257] In this regard, patient-derived cells, preferably T cells, can be transduced with an antigen-binding receptor of the present invention capable of specifically binding to the variant Fc domain described herein using the kit described above. The extracellular domain containing the antigen-binding portion capable of specifically binding to the variant Fc domain is not naturally found in or on T cells. Thus, patient-derived cells transduced using the kit of the present invention will acquire the ability to specifically bind to the variant Fc domain of an antibody, e.g., a therapeutic antibody, and will be able to induce target cell elimination / lysis through interaction with a therapeutic antibody comprising the variant Fc domain that can bind to a tumor-specific antigen naturally present (i.e., endogenously expressed) on the surface of tumor cells. Binding of the extracellular domain of the antigen-binding receptor described herein activates the T cell and brings it into physical contact with tumor cells via the therapeutic antibody comprising the variant Fc domain. Non-transduced T cells, i.e., endogenous T cells (e.g., CD8+ T cells), are unable to bind to the variant Fc domain of a therapeutic antibody comprising the variant Fc domain. Transduced T cells expressing an antigen-binding receptor comprising an extracellular domain capable of specifically binding to a variant Fc domain are unaffected by therapeutic antibodies that do not contain mutations in the Fc domain described herein. Thus, T cells expressing the antigen-binding receptor molecules of the present invention have the ability to lyse target cells in vivo and / or in vitro in the presence of antibodies containing mutations in the Fc domain as described herein. Corresponding target cells include cells that express naturally occurring surface molecules, i.e., tumor-specific antigens, on the surface of tumor cells that are recognized by at least one, and preferably two, binding domains of the therapeutic antibodies described herein. Such surface molecules are characterized later in this specification.

[0258] Lysis of target cells can be detected by methods known in the art. Such methods therefore include, inter alia, physiological in vitro assays. Such physiological assays may monitor cell death, for example, by loss of cell membrane integrity (e.g., FACS-based propidium iodide assay, trypan blue influx assay, photometric enzyme release assay (LDH), radiometric 51Cr release assay, fluorometric europium release and calcein AM release assay). Additional assays include monitoring cell viability, for example, by photometric MTT, XTT, WST-1 and alamarBlue assays, radiometric 3H-Thd incorporation assays, clonogenic assays measuring mitotic activity, and fluorometric rhodamine 123 assays measuring mitochondrial transmembrane gradients. Apoptosis can also be monitored, for example, by FACS-based phosphatidylserine exposure assays, ELISA-based TUNEL tests, caspase activity assays (photometric, fluorometric or ELISA-based assays), or by analyzing altered cell morphology (shrinkage, membrane blebbing).

[0259] Transduced T cells capable of expressing the antigen-binding receptor of the present invention A further aspect of the present invention is transduced T cells capable of expressing the antigen-binding receptor of the present invention. The antigen-binding receptors described herein relate to molecules that are not naturally contained in and / or on the surface of T cells and are not (endogenously) expressed in or on normal (non-transduced) T cells. Thus, the antigen-binding receptors of the present invention in and / or on T cells are artificially introduced into T cells. In the context of the present invention, T cells, preferably CD8+ T cells, can be isolated / obtained from a subject to be treated as defined herein. Thus, the antigen-binding receptors described herein that are artificially introduced into and / or on the surface of T cells and then presented comprise a domain comprising one or more antigen-binding moieties that can access (in vitro or in vivo) an (Ig-derived) immunoglobulin, preferably an antibody, in particular the Fc domain of an antibody. In the context of the present invention, these artificially introduced molecules are presented in and / or on the surface of T cells after (retroviral or lentiviral) transduction as described below. Thus, the transduced T cells of the invention can be activated by immunoglobulins, preferably (therapeutic) antibodies containing specific mutations in the Fc domain as described herein.

[0260] The present invention also relates to transduced T cells that express an antigen-binding receptor encoded by a nucleic acid molecule encoding the antigen-binding receptor of the present invention. Thus, in the context of the present invention, the transduced cell may comprise a nucleic acid molecule encoding the antigen-binding receptor of the present invention or a vector of the present invention that expresses the antigen-binding receptor of the present invention.

[0261] In the context of the present invention, the term "transduced T cells" relates to genetically modified T cells (i.e., T cells into which a nucleic acid molecule has been intentionally introduced). The transduced T cells provided herein may comprise a vector of the present invention. Preferably, the transduced T cells provided herein comprise a nucleic acid molecule encoding an antigen-binding receptor of the present invention and / or a vector of the present invention. The transduced T cells of the present invention may be T cells that transiently or stably express foreign DNA (i.e., a nucleic acid molecule introduced into the T cell). In particular, a nucleic acid molecule encoding an antigen-binding receptor of the present invention can be stably integrated into the genome of a T cell using retroviral or lentiviral transduction. A nucleic acid molecule encoding an antigen-binding receptor of the present invention may also be transiently expressed using mRNA transduction. Preferably, the transduced T cells provided herein are genetically modified by introducing a nucleic acid molecule into the T cell via a viral vector (e.g., a retroviral or lentiviral vector). Thus, expression of the antigen-binding receptor can be constitutive, and the extracellular domain of the antigen-binding receptor can be detectable on the cell surface. This extracellular domain of the antigen-binding receptor may include the entire extracellular domain of the antigen-binding receptor as defined herein, but may also include parts thereof. The minimum required size is the antigen-binding site of the antigen-binding portion in the antigen-binding receptor.

[0262] Expression can also be conditional or inducible when the antigen-binding receptor is introduced into T cells under the control of an inducible or repressible promoter. An example of such an inducible or repressible promoter is a transcription system containing the alcohol dehydrogenase I (alcA) gene promoter and the transcriptional activator protein AlcR. Various agricultural alcohol-based formulations are used to control the expression of a gene of interest linked to the alcA promoter. Furthermore, a tetracycline-responsive promoter system can function to activate or repress the gene expression system in the presence of tetracycline. Some elements of this system include the tetracycline repressor protein (TetR), the tetracycline operator sequence (tetO), and the tetracycline transactivator fusion protein (tTA), which is a fusion of TetR and the herpes simplex virus protein 16 (VP16) activation sequence. Additionally, steroid-responsive promoters, metal-regulated promoters, or virulence-related (PR) protein-associated promoters can be used.

[0263] Expression can be constitutive or constitutive, depending on the system used. The antigen-binding receptor of the present invention can be expressed on the surface of the transduced T cells provided herein. The extracellular portion of the antigen-binding receptor (i.e., the extracellular domain of the antigen-binding receptor) can be detected on the cell surface, while the intracellular portion (i.e., the costimulatory signaling domain and the stimulatory signaling domain) cannot be detected on the cell surface. Detection of the extracellular domain of the antigen-binding receptor can be carried out by using an antibody that specifically binds to this extracellular domain or by a variant Fc domain to which the extracellular domain can bind. The extracellular domain can be detected by flow cytometry or microscopy using these antibodies or the Fc domain.

[0264] The transduced cells of the present invention can be any immune cell, including, but not limited to, B cells, T cells, natural killer (NK) cells, natural killer (NK) T cells, gamma-delta T cells, innate lymphoid cells, macrophages, monocytes, dendritic cells, or neutrophils. Preferably, the immune cell is a lymphocyte, preferably an NK cell or a T cell. The T cells include CD4 T cells and CD8 T cells. Triggering of the antigen-binding receptor of the present invention on the surface of a leukocyte, in combination with a therapeutic antibody comprising a variant Fc domain, is believed to render the cell cytotoxic to target cells, regardless of the lineage from which the cell originates. Cytotoxicity occurs regardless of the stimulatory or costimulatory signaling domain selected for the antigen-binding receptor and is not dependent on the exogenous supply of additional cytokines. Therefore, the transduced cells of the present invention can be, for example, CD4+ T cells, CD8+ T cells, γδ T cells, natural killer (NK) T cells, natural killer (NK) cells, tumor-infiltrating (TIL) cells, myeloid cells, or mesenchymal stem cells. Preferably, the transduced cells provided herein are T cells (e.g., autologous T cells), more preferably, the transduced cells are CD8+ T cells. Therefore, in the context of the present invention, the transduced cells are CD8+ T cells. Furthermore, in the context of the present invention, the transduced cells are autologous T cells. Therefore, in the context of the present invention, the transduced cells are preferably autologous CD8+ T cells. In addition to the use of autologous cells (e.g., T cells) isolated from a subject, the present invention also encompasses the use of allogeneic cells. Therefore, in the context of the present invention, the transduced cells can also be allogeneic cells, for example, allogeneic CD8+ T cells. The use of allogeneic cells is based on the fact that if a foreign antigen-presenting cell (APC) expresses an MHC molecule class I or class II to which a specific responder cell population, i.e., a T cell population, is restricted, along with the antigen epitope recognized by the T cell, the cell, preferably a T cell, can recognize the specific antigen epitope presented by the APC.Thus, the term allogeneic refers to cells obtained from an unrelated donor individual / subject who is human leukocyte antigen (HLA) compatible with the individual / subject to be treated with, for example, the antigen-binding receptor-expressing transduced cells described herein. Autologous cells refer to cells isolated / obtained as described above from the subject to be treated with the transduced cells described herein.

[0265] The transduced cells of the invention can be co-transduced with additional nucleic acid molecules, for example, nucleic acid molecules encoding T cell receptors.

[0266] The present invention also relates to a method for producing transduced T cells expressing an antigen-binding receptor of the present invention, comprising the steps of transducing T cells with a vector of the present invention, culturing the transduced T cells under conditions that allow expression of the antigen-binding receptor in or on the transduced cells, and recovering the transduced T cells.

[0267] In the context of the present invention, the transduced cells of the present invention are preferably produced by the following process: cells (e.g., T cells, preferably CD8+ T cells) are isolated / obtained from a subject (preferably a human patient). Methods for isolating / obtaining cells (e.g., T cells, preferably CD8+ T cells) from patients or donors are well known in the art, and in the context of the present invention, cells (e.g., T cells, preferably CD8+ T cells) from patients or donors can be isolated by blood collection or bone marrow collection. After isolating / obtaining the cells as a patient sample, the cells (e.g., T cells) are separated from other components of the sample. Several methods are known for isolating cells (e.g., T cells) from a sample, including, but not limited to, leukapheresis to obtain cells from the peripheral blood of a patient or donor, isolation / harvesting of cells by using a FACSort instrument, and manual or micromanipulator removal of live cells from dead cells in a fresh biopsy sample containing live cells (see, for example, Dudley, Immunother. 26 (2003), 332-342; Robbins, Clin. Oncol. 29 (2011), 917-924 or Leisegang, J. Mol. Med. 86 (2008), 573-58). The isolated / obtained cells, e.g., T cells, preferably CD8+ T cells, are then cultured and expanded, e.g., by using an anti-CD3 antibody, by using an anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibody, and / or by using an anti-CD3 antibody, an anti-CD28 antibody and interleukin 2 (IL-2) (see, e.g., Dudley, Immunother. 26 (2003), 332-342 or Dudley, Clin. Oncol. 26 (2008), 5233-5239).

[0268] In the next step, cells (e.g., T cells) are artificially / genetically modified / transduced by methods known in the art (see, for example, Lemoine, J Gene Med 6 (2004), 374-386). Methods for transducing cells (e.g., T cells) are known in the art, and include, but are not limited to, electroporation, calcium phosphate, cationic lipid, or liposome methods when nucleic acids or recombinant nucleic acids are transduced. The nucleic acid to be transduced can be conventionally transduced with high efficiency using commercially available transfection reagents, such as Lipofectamine (Invitrogen, catalog number: 11668027). When a vector is used, as long as the vector is a plasmid vector (i.e., a vector that is not a viral vector), the vector can be transduced in the same manner as the nucleic acid described above. In the context of the present invention, methods for transducing cells (e.g., T cells) include retroviral or lentiviral T cell transduction, non-viral vectors (e.g., Sleeping Beauty minicircle vector), and mRNA transfection. "MRNA transfection" refers to methods well known to those skilled in the art for transiently expressing a protein of interest, in this case, an antigen-binding receptor of the present invention, in transduced cells. Briefly, mRNA encoding the antigen-binding receptor of the present invention can be electroporated into cells using an electroporation system (e.g., Gene-Pulser, Bio-Rad, etc.), followed by culture using standard cell (e.g., T cell) culture protocols as described above (see Zhao et al., Mol Ther. 13(1) (2006), 151-159). The transduced cells of the present invention are T cells, most preferably CD8+ T cells, and are generated by lentiviral or, most preferably, retroviral T cell transduction.

[0269] It is a great way to get a smile on your face when you are SAMEN CMV / SRa(Clay et al.,J.Immunol.163(1999),507-513)、LZRS-id3-IHRES(Heemskerk et al.,J.Exp.Med.186(1997),1597-1602)、FeLV(Neil et al.,Nature 308(1984),814-820), SAX(Kantoff et al.,Proc.Natl.Acad.Sci.USA 83(1986),6563-6567), pDOL(Desiderio,J.Exp.Med.167(1988),372-388), N2(Kasid et al al.,Proc.Natl.Acad.Sci.USA 87(1990),473-477);LNL6(Tiberghien et al.,Blood 84(1994),1333-1341)、pZipNEO(Chen et al al.,J.Immunol.153(1994),3630-3638), LASN(Mullen et al.,Hum.Gene Ther.7(1996),1123-1129), pG1XsNa(Taylor et al al.,J.Exp.Med.184(1996),2031-2036), LCNX(Sun et al.,Hum.Gene Ther.8(1997),1041-1048), SFG(Gallardo et al.,Blood 90(1997). al.,Hum.Gene Ther.8(1997),1041-1048), SFG(Gallardo et al.,Blood 90(1997),952-957), HMB-Hb-Hu(Vieillard et al.,Proc.Natl.Acad.Sci.USA). 94(1997),11595-11600), pMV7(Cochlovius et al.,Cancer Immunol.Immunother.46(1998),61-66), pSTITCH(Weitjens et al.,Gene Ther 5(1998),1195-1203), pLZR(Yang et al al.,Hum.Gene Ther.10(1999),123-132;pBAG(Wu et al.,Hum.Gene Ther.10(1999),977-982), rKat.43.267bn(Gilham et al., J.Immunother.25(2002),139-151), pLGSN(Engels et al.,Hum.Gene Ther.14(2003),1155-1168), pMP71(Engels et al.,Hum.Gene Ther.14(2003),1155-1168), pGCSAM(Morgan et al.,J.Immunol.171(2003),3287-3295), pMSGV(Zhao et al.,J.Immunol.174(2005),4415-4423), or pMX(de Witte et al. al., J. Immunol. 181 (2008), 5128-5136) and the like are known in the art. In the context of the present invention, suitable lentiviral vectors for transducing cells (e.g., T cells) include, for example, the PL-SIN lentiviral vector (Hotta et al., Nat Methods. 6(5)(2009), 370-376), p156RRL-sinPPT-CMV-GFP-PRE / NheI (Campeau et al., PLoS One 4(8)(2009), e6529), pCMVR8.74 (Addgene Catalog No.: 22036), FUGW (Lois et al., Science 295(5556)(2002), 868-872, pLVX-EF1 (Addgene Catalog No.: 64368), pLVE (Brunger et al., Proc Natl Acad Sci USA 111(9)(2014),E798-806), pCDH1-MCS1-EF1(Hu et al., Mol Cancer Res.7(11)(2009),1756-1770), pSLIK(Wang et al., Nat Cell Biol.16(4)(2014),345-356), pLJM1(Solomon et al. al., Nat Genet.45(12)(2013),1428-30), pLX302(Kang et al.,Sci Signal.6(287)(2013),rs13), pHR-IG(Xie et al.,J Cereb Blood Flow Metab.33(12)(2013),1875-85), pRRLSIN(Addgene catalog number: 62053), pLS(Miyoshi et al., J Virol.72(10)(1998),8150-8157), pLL3.7(Lazebnik et al., J Biol Chem.283(7)(2008),11078-82), FRIG(Raissi et al., Mol Cell Neurosci.57(2013),23-32), pWPT(Ritz-Laser et al., Diabetologia.46(6)(2003),810-821), pBOB(Marr et al., J Mol Neurosci.22(1-2)(2004), 5-11), or pLEX (Addgene catalog number: 27976). .

[0270] Transduced T cells / T cells of the present invention are preferably grown outside their natural environment under controlled conditions. In particular, the term "culturing" refers to the in vitro growth of cells (e.g., transduced cells of the present invention) derived from a multicellular eukaryote (preferably a human patient). Cell culture is an experimental technique that keeps cells alive after isolation from their original tissue source. Here, transduced cells of the present invention are cultured under conditions that allow expression of the antigen-binding receptor of the present invention in or on the transduced cells. Conditions that allow expression of the transgene (i.e., the antigen-binding receptor of the present invention) are generally known in the art and include, for example, the addition of agonistic anti-CD3 and anti-CD28 antibodies, and cytokines such as interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), and / or interleukin-15 (IL-15). Following expression of the antigen-binding receptor of the invention in the cultured transduced cells (eg, CD8+ T cells), the transduced cells are harvested (ie, re-extracted) from the culture (ie, from the culture medium).

[0271] Thus, the present invention also encompasses transduced cells, preferably T cells, in particular CD8+ T cells, that express an antigen-binding receptor encoded by a nucleic acid molecule of the present invention, obtainable by the method of the present invention.

[0272] nucleic acid molecule Further aspects of the present invention are nucleic acids and vectors encoding one or more antigen-binding receptors of the present invention. Exemplary nucleic acid molecules encoding antigen-binding receptors of the present invention are set forth in SEQ ID NOs: 19, 30, 35, 38, 44, 47, 51, and 52. The nucleic acid molecules of the present invention can be under the control of regulatory sequences. For example, promoters, transcriptional enhancers, and / or sequences that allow inducible expression of the antigen-binding receptors of the present invention can be used. In the context of the present invention, the nucleic acid molecules are expressed under the control of a constitutive promoter or an inducible promoter. Suitable promoters include, for example, the CMV promoter (Qin et al., PLoS One 5(5)(2010), e10611), the UBC promoter (Qin et al., PLoS One 5(5)(2010), e10611), the PGK promoter (Qin et al., PLoS One 5(5)(2010), e10611), the EF1A promoter (Qin et al., PLoS One 5(5)(2010), e10611), the CAGG promoter (Qin et al., PLoS One 5(5)(2010), e10611), the SV40 promoter (Qin et al., PLoS One 5(5)(2010), e10611), the COPIA promoter (Qin et al., PLoS One 5(5)(2010), e10611), the ACT5C ... al., PLoS One 5(5)(2010), e10611), TRE promoter (Qin et al., PLoS One.5(5)(2010), e10611), Oct3 / 4 promoter (Chang et al., Molecular Therapy 9(2004), S367-S367(doi:10.1016 / j.ymthe.2004.06.904)), or Nanog promoter (Wu et al., Cell Res.15(5)(2005), 317-24). Therefore, the present invention also relates to a vector comprising a nucleic acid molecule according to the present invention. As used herein, the term vector refers to a circular or linear nucleic acid molecule that can autonomously replicate in a host cell into which it has been introduced (i.e., in a transduced cell).Many suitable vectors are known to those skilled in the art of molecular biology, and their selection depends on the desired function, including plasmids, cosmids, viruses, bacteriophages, and other vectors traditionally used in genetic engineering. Various plasmids and vectors can be constructed using methods well known to those skilled in the art. See, for example, the techniques described in Sambrook et al. (ibid.) and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1989), (1994). Alternatively, the polynucleotides and vectors of the present invention can be reconstituted into liposomes for delivery to target cells. As described in more detail below, cloning vectors have been used to isolate individual DNA sequences. Where expression of a specific polypeptide is desired, the relevant sequence can be transferred into an expression vector. Exemplary cloning vectors include pBluescript SK, pGEM, pUC9, pBR322, pGA18, and pGBT9. Typical expression vectors include pTRE, pCAL-n-EK, pESP-1, and pOP13CAT.

[0273] The present invention also relates to a vector comprising a nucleic acid molecule that is a regulatory sequence operably linked to a nucleic acid molecule encoding an antigen-binding receptor as defined herein. In the context of the present invention, the vector can be polycistronic. Such regulatory sequences (control elements) are known to those skilled in the art and may include a promoter, a splice cassette, a translation initiation codon, a translation, and an insertion site for introducing an insert into the vector. In the context of the present invention, the nucleic acid molecule is operably linked to an expression control sequence that allows expression in eukaryotic or prokaryotic cells. The vector is considered to be an expression vector comprising a nucleic acid molecule encoding an antigen-binding receptor as defined herein. "Operably linked" refers to a juxtaposition of the components so described, in a relationship allowing them to function as intended. A control sequence operably linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequences. When the control sequence is a promoter, it will be apparent to those skilled in the art that double-stranded nucleic acids are preferably used.

[0274] In the context of the present invention, the vectors referred to are expression vectors. An expression vector is a construct that can be used to transform a selected cell, resulting in the expression of a coding sequence in the selected cell. Expression vectors can be, for example, cloning vectors, binary vectors, or integrating vectors. Expression involves transcription of a nucleic acid molecule, preferably into a translatable mRNA. Regulatory elements ensuring expression in prokaryotes and / or eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells, they usually include a promoter ensuring initiation of transcription and, optionally, a poly(A) signal ensuring termination of transcription and stabilization of the transcript. Possible regulatory elements enabling expression in prokaryotic host cells include, for example, the PL, lac, trp, or tac promoters in E. coli. Examples of regulatory elements enabling expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast, or the CMV promoter, SV40 promoter, RSV promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian and other animal cells.

[0275] In addition to elements responsible for transcription initiation, such regulatory elements may also include transcription termination signals, such as the SV40 polyA site or tk polyA site, downstream of the polynucleotide. Furthermore, depending on the expression system used, leader sequences encoding signal peptides that can direct the polypeptide into an intracellular compartment or secrete the polypeptide into the medium can be added to the coding sequence of the nucleic acid sequences provided, and are well known in the art. See, e.g., the accompanying examples.

[0276] The leader sequence is assembled in an appropriate stage with translation, initiation and termination sequences, and preferably with a leader sequence capable of directing secretion of the translated protein or a portion thereof into the periplasmic space or the extracellular medium. Optionally, the heterologous sequence can encode an antigen-binding receptor containing an N-terminal identification peptide that confers desirable characteristics, such as stabilization or simplified purification of the expressed recombinant product. See above. In this regard, suitable expression vectors are known in the art, such as the Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogene), pEF-DHFR, pEF-ADA, or pEF-neo (Raum et al., Cancer Immunol Immunother 50 (2001), 141-150), or pSPORT1 (GIBCO BRL).

[0277] In the context of the present invention, expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic cells, although control sequences for prokaryotic cells may also be used. Once the vector has been incorporated into an appropriate cell, the cell is maintained under conditions suitable for high-level expression of the nucleotide sequence, as desired. Additional regulatory elements may include transcriptional and translational enhancers. Advantageously, the vectors of the present invention described above include selectable and / or scorable markers. Selectable marker genes useful for the selection of transformed cells and, for example, plant tissues and plants, are well known to those skilled in the art and include, for example, antimetabolite resistance as the basis for selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149), npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995), and hygro, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481-485).Additional selectable genes, namely trpB, which allows cells to utilize indole instead of tryptophan; hisD, which allows cells to utilize histinol instead of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate isomerase, which allows cells to utilize mannose (WO 94 / 20627); and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.), or Aspergillus terreus, which confers resistance to blasticidin S. A deaminase from Bacillus terreus (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338) has also been described.

[0278] Useful scorable markers are also known to those skilled in the art and are commercially available. Advantageously, the marker encodes luciferase (Giacomin, Pl. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44-47), or β-glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907). This embodiment is particularly useful for simple and rapid screening of cells, tissues, and organisms containing the vectors described herein.

[0279] As mentioned above, the nucleic acid molecules provided herein, alone or as part of a vector, can be used to express the antigen-binding receptors of the present invention in cells, for example, for adoptive T cell therapy, and also for gene therapy purposes. When a nucleic acid molecule or vector containing a DNA sequence encoding any one of the antigen-binding receptors described herein is introduced into a cell, the cell produces the polypeptide of interest. Gene therapy, which relies on the introduction of a therapeutic gene into cells by ex vivo or in vivo techniques, is one of the most important applications of gene transfer. Suitable vectors, methods, or gene delivery systems for in vitro or in vivo gene therapy have been described in the literature and are known to those skilled in the art. For example, Giordano, Nature Medicine 2(1996), 534-539, Schaper, Circ.Res.79(1996), 911-919, Anderson, Science 256(1992), 808-813, Verma, Nature 389(1994), 239, Isner, Lancet. 348(1996), 370-374, Muhlhauser, Circ. Res. 77(1995), 1077-1086, Onodera, Blood 91(1998), 30-36, Verma, Gene Ther.5(1998),692-699, Nabel,Ann.NYAcad.Sci.811(1997),289-292,Verzeletti,Hum.Gene See Ther. 9 (1998), 2243-51; Wang, Nature Medicine 2 (1996), 714-716; WO 94 / 29469; WO 97 / 00957; US 5,580,859; US 5,589,466; or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640. The nucleic acid molecules and vectors provided herein can be designed for direct introduction into cells or for introduction into cells via liposomes or viral vectors (e.g., adenovirus, retrovirus). In the context of the present invention, the cells are T cells, such as CD8+ T cells, CD4+ T cells, CD3+ T cells, γδ T cells, or natural killer (NK) T cells, preferably CD8+ T cells.

[0280] In accordance with the above, the present invention relates to methods for obtaining vectors, particularly plasmids, cosmids and bacteriophages, conventionally used in genetic engineering, which contain nucleic acid molecules encoding the polypeptide sequences of antigen-binding receptors as defined herein. In the context of the present invention, the vectors are expression vectors and / or gene transfer or gene targeting vectors. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses may be used to deliver the expressing polynucleotide or vector to a targeted cell population.

[0281] Methods well known to those skilled in the art can be used to construct recombinant vectors. See, for example, the techniques described in Sambrook et al. (ibid.), Ausubel (1989, ibid.), or other standard textbooks. Alternatively, the provided nucleic acid molecules and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing the nucleic acid molecules of the invention can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is often utilized for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cellular hosts. See Sambrook, supra. The provided vectors can be, inter alia, pEF-DHFR, pEF-ADA, or pEF-neo. The vectors pEF-DHFR, pEF-ADA and pEF-neo have been described in the art, for example in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995), 7021-7025 and Raum et al. Cancer Immunol Immunother 50 (2001), 141-150.

[0282] The present invention also provides T cells transformed or transfected with the vectors described herein. The T cells can be produced by introducing at least one of the vectors or at least one of the nucleic acid molecules described above into T cells or their precursor cells. The presence of the at least one vector or nucleic acid molecule in the T cells can mediate the expression of a gene encoding the antigen-binding receptor described above, which comprises an extracellular domain comprising an antigen-binding portion capable of specifically binding to a variant Fc domain. The vectors of the present invention can be polycistronic.

[0283] The described nucleic acid molecules or vectors introduced into T cells or their precursor cells can be integrated into the genome of the cells or maintained extrachromosomally.

[0284] tumor-specific antigens As noted above, the (Ig-derived) domain of the antibodies described herein, including variant Fc domains, can contain an antigen-interaction site with specificity for a cell surface molecule naturally present on the surface of tumor cells, i.e., a tumor-specific antigen. In the context of the present invention, such antibodies bring transduced T cells described herein, comprising an antigen-binding receptor of the present invention, into physical contact with tumor cells, whereupon the transduced T cells become activated. Activation of the transduced T cells of the present invention can result in tumor cell lysis, as described herein.

[0285] Examples of tumor markers that are naturally present on the surface of tumor cells include FAP (fibroblast activation protein), CEA (carcinoembryonic antigen), p95 (p95HER2), BCMA (B-cell maturation antigen), EpCAM (epithelial cell adhesion molecule), MSLN (mesothelin), MCSP (melanoma chondroitin sulfate proteoglycan), HER-1 (human epidermal growth factor 1), HER-2 (human epidermal growth factor 2), HER-3 (human epidermal growth factor 3), CD19, CD20, CD22, CD33, CD38, and CD52F1. These include, but are not limited to, peptides bound to t3, folate receptor 1 (FOLR1), human trophoblast cell surface antigen 2 (Trop-2), cancer antigen 12-5 (CA-12-5), human leukocyte antigen-antigen D-related (HLA-DR), MUC-1 (mucin-1), A33 antigen, PSMA (prostate-specific membrane antigen), FMS-like tyrosine kinase 3 (FLT-3), PSMA (prostate-specific membrane antigen), PSCA (prostate stem cell antigen), transferrin receptor, TNC (tenascin), carbonic anhydrase IX (CA-IX), and / or molecules of the human major histocompatibility complex (MHC).

[0286] Thus, in the context of the present invention, the antigen binding receptors described herein bind to a variant Fc domain of an antibody, i.e., to a therapeutic antibody capable of specifically binding to: FAP (fibroblast activation protein), CEA (carcinoembryonic antigen), p95 (p95HER2), BCMA (B cell maturation antigen), EpCAM (epithelial cell adhesion molecule), MSLN (mesothelin), MCSP (melanoma chondroitin sulfate proteoglycan), HER-1 (human epidermal growth factor 1), HER-2 (human epidermal growth factor 2), HER-3 (human epidermal growth factor 3), CD19, CD20, CD22, CD33, CD38, CD and / or a peptide bound to a molecule of the human major histocompatibility complex (MHC).

[0287] A33 antigen, BCMA (B cell maturation antigen), cancer antigen 12-5 (CA-12-5), carbonic anhydrase IX (CA-IX), CD19, CD20, CD22, CD33, CD38, CEA (carcinoembryonic antigen), EpCAM (epithelial cell adhesion molecule), FAP (fibroblast activation protein), FMS-like tyrosine kinase 3 (FLT-3), folate receptor 1 (FOLR1), HER-1 (human epidermal growth factor 1), HER-2 (human epidermal growth factor 2), HER-3 (human epidermal growth factor 3), human leukocyte antigen-antigen D-related (HLA-DR), MSLN (mesothelin), MCSP ( Sequences of (human) members of the following proteins can be found in the UniProtKB / Swiss-Prot database and can be searched at http: / / www.uniprot.org / uniprot / ?query=reviewed%3Ayes: melanoma chondroitin sulfate proteoglycan, MUC-1 (mucin-1), PSMA (prostate-specific membrane antigen), PSCA (prostate stem cell antigen), p95 (p95HER2), transferrin receptor, TNC (tenascin), and human trophoblast cell surface antigen 2 (Trop-2). These (protein) sequences also relate to annotated modified sequences. The present invention also provides techniques and methods for using homologous sequences and genetic allelic variants, etc., of the concise sequences provided herein. Preferably, such variants, etc., of the concise sequences herein are used. Preferably, such variants are genetic variants. Those skilled in the art can easily deduce the relevant coding regions of these (protein) sequences in these databank entries, which may also include entries for genomic DNA and mRNA / cDNA. The sequence of (human) FAP (fibroblast activation protein) can be obtained from Swiss-Prot database entry Q12884 (entry version 168, sequence version 5), the sequence of (human) CEA (carcinoembryonic antigen) can be obtained from Swiss-Prot database entry P06731 (entry version 171, sequence version 3),The sequence of (human) EpCAM (epithelial cell adhesion molecule) can be obtained from Swiss-Prot database entry P16422 (entry version 117, sequence version 2), the sequence of (human) MSLN (mesothelin) can be obtained from UniProt entry number Q13421 (version number 132, sequence version 2), and the sequence of (human) FMS-like tyrosine kinase 3 (FLT-3) can be obtained from Swiss-Prot database entry P36888 (primary citable accession number or Q13414 (secondary accession number), version number 165 and sequence version 2; the sequence of (human) MCSP (melanoma chondroitin sulfate proteoglycan) can be obtained from UniProt entry number Q6UVK1 (version number 118, sequence version 2); and the sequence of (human) folate receptor 1 (FOLR1) can be obtained from UniProt entry number P15328 (primary citable accession number) or Q53EW2 (secondary accession number). , version number 153 and sequence version 3; the sequence of (human) trophoblast cell surface antigen 2 (Trop-2) can be obtained from UniProt entry number P09758 (primary citable accession number) or Q15658 (secondary accession number), version number 172 and sequence version 3; the sequence of (human) PSCA (prostate stem cell antigen) can be obtained from UniProt entry number O43653 (primary citable accession number) or Q6UW92 (secondary accession number), version number 134 and sequence version 1; the sequence of (human) HER-1 (epidermal growth factor receptor) can be obtained from Swiss-Prot database entry P00533 (entry version 177, sequence version 2); and the sequence of (human) HER-2 (receptor tyrosine protein kinase erbB-2) can be obtained from Swiss-Prot database entry P04626 (entry version 161, sequence version 1);The sequence of (human) HER-3 (receptor tyrosine protein kinase erbB-3) can be obtained from Swiss-Prot database entry P21860 (entry version 140, sequence version 1), the sequence of (human) CD20 (B lymphocyte antigen CD20) can be obtained from Swiss-Prot database entry P11836 (entry version 117, sequence version 1), and the sequence of (human) CD22 (B lymphocyte antigen CD22) can be obtained from Swiss-Prot database entry P20273 (entry The sequence of (human) CD33 (B lymphocyte antigen CD33) can be obtained from Swiss-Prot database entry P20138 (entry version 129, sequence version 2), the sequence of (human) CA-12-5 (mucin 16) can be obtained from Swiss-Prot database entry Q8WXI7 (entry version 66, sequence version 2), and the sequence of (human) HLA-DR can be obtained from Swiss-Prot database entry Q29900 ( The sequence of (human) MUC-1 (mucin-1) can be obtained from Swiss-Prot database entry P15941 (entry version 135, sequence version 3), the sequence of (human) A33 (cell surface A33 antigen) can be obtained from Swiss-Prot database entry Q99795 (entry version 104, sequence version 1), and the sequence of (human) PSMA (glutamate carboxypeptidase 2) can be obtained from Swiss-Prot database entry Q99795 (entry version 104, sequence version 1). The sequence of the (human) transferrin receptor can be obtained from Swiss-Prot database entry Q04609 (entry version 133, sequence version 1), the sequence of the (human) transferrin receptor can be obtained from Swiss-Prot database entries Q9UP52 (entry version 99, sequence version 1) and P02786 (entry version 152, sequence version 2), the sequence of the (human) TNC (tenascin) can be obtained from Swiss-Prot database entry P24821 (entry version 141, sequence version 3),The sequence of (human) CA-IX (carbonic anhydrase IX) can be obtained from Swiss-Prot database entry Q16790 (entry version 115, sequence version 2).

[0288] Therapeutic Uses and Methods of Treatment The molecules or constructs (i.e., antigen-binding receptors, transduced T cells, and kits) provided herein are particularly useful in medical settings, especially for the treatment of malignant diseases. For example, tumors can be treated using transduced T cells expressing the antigen-binding receptor of the present invention in combination with a therapeutic antibody specific for tumor cells and comprising a variant Fc domain. Thus, in certain embodiments, the antigen-binding receptor, transduced T cells, or kits are used in the treatment of malignant diseases, particularly those selected from cancers of epithelial, endothelial, or mesothelial origin and hematological cancers.

[0289] Tumor specificity of the treatment is provided by a therapeutic antibody comprising a variant Fc domain, which is administered before, simultaneously with, or after administration of transduced T cells expressing an antigen-binding receptor of the invention. In this case, the transduced T cells are not specific to a given tumor but are general T cells, since they can be directed to any tumor depending on the therapeutic antibody comprising a variant Fc domain used in accordance with the invention.

[0290] In this context, the malignant disease may be a cancer / carcinoma of epithelial, endothelial or mesothelial origin or a blood cancer. In the context of the present invention, the cancer / carcinoma is selected from the group consisting of gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, oral cancer, stomach cancer, cervical cancer, B-cell and T-cell lymphoma, myeloid leukemia, ovarian cancer, leukemia, lymphocytic leukemia, nasopharyngeal cancer, colon cancer, prostate cancer, renal cell carcinoma, head and neck cancer, skin cancer (melanoma), cancer of the genitourinary tract, e.g. testicular cancer, ovarian cancer, endothelial cancer, cervical cancer and kidney cancer, cancer of the bile duct, cancer of the esophagus, cancer of the salivary glands and cancer of the thyroid gland, or other neoplastic diseases such as blood tumors, gliomas, sarcomas or osteosarcomas.

[0291] For example, neoplastic diseases and / or lymphomas may be treated with specific constructs directed to these medical indications. The indication of the transduced T cells of the present invention combined with therapeutic antibodies comprising a variant Fc domain is given by the specificity of the therapeutic antibody for the tumor antigen. For example, gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, and / or oral cancer may be treated with antibodies comprising a variant Fc domain directed against (human) EpCAM (as a tumor-specific antigen naturally present on the surface of tumor cells).

[0292] Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, and / or oral cancer may be treated with the transduced T cells of the invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain, the therapeutic antibody being directed against HER1, preferably against human HER1. Furthermore, gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma, and / or oral cancer may be treated with the transduced T cells of the invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain, the therapeutic antibody being directed against MCSP, preferably against human MCSP. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma and / or oral cancer may be treated with the transduced T cells of the invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain, the therapeutic antibody being directed against FOLR1, preferably against human FOLR1. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma and / or oral cancer may be treated with the transduced T cells of the invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain, the therapeutic antibody being directed against Trop-2, preferably against human Trop-2. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma and / or oral cancer may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain, the therapeutic antibody being directed against PSCA, preferably against human PSCA. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma and / or oral cancer may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain, the therapeutic antibody being directed against EGFRvIII, preferably against human EGFRvIII.Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma, and / or oral cancer may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain against MSLN, preferably against human MSLN. Gastric cancer, breast cancer, and / or cervical cancer may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain against HER2, preferably against human HER2. Gastric cancer and / or lung cancer may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain against HER3, preferably against human HER3. B-cell lymphomas and / or T-cell lymphomas may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain, the therapeutic antibody being directed against CD20, preferably against human CD20. B-cell lymphomas and / or T-cell lymphomas may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain, the therapeutic antibody being directed against CD22, preferably against human CD22. Myeloid leukemias may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain, the therapeutic antibody being directed against CD33, preferably against human CD33. Ovarian cancer, lung cancer, breast cancer and / or gastrointestinal cancer may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain, the therapeutic antibody being directed against CA12-5, preferably against human CA12-5. Gastrointestinal cancer, leukemia and / or nasopharyngeal cancer may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain, the therapeutic antibody being directed against HLA-DR, preferably against human HLA-DR.Colon cancer, breast cancer, ovarian cancer, lung cancer, and / or pancreatic cancer may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain, the therapeutic antibody being directed against MUC-1, preferably against human MUC-1. Colon cancer may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain, the therapeutic antibody being directed against A33, preferably against human A33. Prostate cancer may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain, the therapeutic antibody being directed against PSMA, preferably against human PSMA. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, and / or oral cancer may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain against the transferrin receptor, preferably against the human transferrin receptor. Pancreatic cancer, lung cancer, and / or breast cancer may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain against the transferrin receptor, preferably against the human transferrin receptor. Renal cancer may be treated with the transduced T cells of the present invention administered before, simultaneously with, or after administration of a therapeutic antibody comprising a variant Fc domain against CA-IX, preferably against human CA-IX.

[0293] The present invention therefore also relates to a method for the treatment of diseases, malignancies, such as cancers of epithelial, endothelial or mesothelial origin and / or hematological cancers.In the context of the present invention, the subject is a human being.

[0294] In the context of the present invention, one particular method for treating a disease comprises the following steps: (a) isolating T cells, preferably CD8+ T cells, from a subject; (b) transducing isolated T cells, preferably CD8+ T cells, with an antigen-binding receptor as described herein; and (c) administering the transduced T cells, preferably CD8+ T cells, to said subject.

[0295] In the context of the present invention, the transduced T cells, preferably CD8+ T cells, and / or one or more therapeutic antibodies are co-administered to the subject by intravenous infusion.

[0296] Furthermore, in the context of the present invention, the present invention provides a method for treating a disease, comprising the steps of: (a) isolating T cells, preferably CD8+ T cells, from a subject; (b) transducing isolated T cells, preferably CD8+ T cells, with an antigen-binding receptor as described herein; (c) optionally co-transducing the isolated T cells, preferably CD8+ T cells, with a T cell receptor; (d) expanding the T cells, preferably CD8+ T cells, with anti-CD3 and anti-CD28 antibodies; and (e) administering the transduced T cells, preferably CD8+ T cells, to the subject.

[0297] The above-mentioned step (d) (referred to as the step of expanding T cells such as TILs with anti-CD3 antibodies and / or anti-CD28 antibodies) can also be carried out in the presence of (stimulatory) cytokines such as interleukin 2 and / or interleukin 15 (IL-15). In the context of the present invention, the above-mentioned step (d) (referred to as the step of expanding T cells such as TILs with anti-CD3 antibodies and / or anti-CD28 antibodies) can also be carried out in the presence of interleukin 12 (IL-12), interleukin 7 (IL-7) and / or interleukin 21 (IL-21).

[0298] The treatment method further comprises administering an antibody used according to the present invention. The antibody can be administered before, simultaneously with, or after the transduced T cells are administered. In the context of the present invention, the transduced T cells are administered by intravenous infusion. In the context of the present invention, the transduced T cells are isolated / obtained from the subject to be treated.

[0299] composition Furthermore, the present invention provides compositions (medicaments) comprising antibody molecules with variant Fc domains, transduced T cells comprising the antigen-binding receptor of the present invention, nucleic acid molecules and vectors encoding the antigen-binding receptor of the present invention, and / or kits comprising one or more of the compositions. In the context of the present invention, compositions are pharmaceutical compositions, optionally further comprising an appropriate formulation of carriers, stabilizers, and / or excipients. Thus, in the context of the present invention, there is provided a pharmaceutical composition (medicament) comprising an antibody molecule comprising a variant Fc domain as defined herein, administered in combination with transduced T cells comprising the antigen-binding receptor described herein and / or a composition comprising the transduced T cells, wherein the antibody molecule is administered before, simultaneously with, or after the administration of the transduced T cells comprising the antigen-binding receptor of the present invention.

[0300] According to the present invention, the term "pharmaceutical composition" relates to a composition for administration to a patient, preferably a human patient. Furthermore, in the context of the present invention, the patient suffers from a disease, which is a malignant disease, in particular a cancer / carcinoma of epithelial, endothelial, or mesothelial origin or a blood cancer. In the context of the present invention, the cancer / carcinoma is selected from the group consisting of gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, oral cancer, stomach cancer, cervical cancer, B-cell and T-cell lymphoma, myeloid leukemia, ovarian cancer, leukemia, lymphocytic leukemia, nasopharyngeal cancer, colon cancer, prostate cancer, renal cell carcinoma, head and neck cancer, skin cancer (melanoma), cancer of the genitourinary tract, e.g., testicular cancer, endothelial cancer, cervical cancer and kidney cancer, cancer of the bile duct, cancer of the esophagus, cancer of the salivary glands and cancer of the thyroid gland, or other neoplastic diseases such as blood tumors, gliomas, sarcomas or osteosarcomas.

[0301] In a preferred embodiment, the pharmaceutical composition / medicament comprises an antibody and / or transduced T cells as defined herein for parenteral, transdermal, intraluminal, intraarterial, intravenous, intrathecal administration, or direct injection into a tissue or into a tumor. In the context of the present invention, the composition / medicament comprises an antibody comprising a variant Fc domain as defined herein, administered before, simultaneously with, or after administration of transduced T cells comprising an antigen-binding receptor as defined herein. In the context of the present invention, the pharmaceutical composition / medicament comprising an antibody as defined herein is administered in combination with a composition / medicament comprising transduced T cells comprising an antigen-binding receptor as defined herein, the T cells being obtained from the subject to be treated.

[0302] The use of the term "in combination" does not limit the order in which components of a treatment regimen are administered to a subject. Thus, the pharmaceutical compositions / medicines described herein encompass the administration of an antibody as defined herein before, simultaneously with, or after the administration of transduced T cells comprising the antigen-binding receptor of the present invention. As used herein, "in combination" also does not limit the timing between the administration of an antibody as defined herein above and the administration of transduced T cells comprising the antigen-binding receptor as defined herein. Thus, when the two components are not administered simultaneously, the administrations can be separated by 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours, or any suitable time interval that can be easily determined by a person skilled in the art and / or described herein.

[0303] In the context of the present invention, the term "in combination" also encompasses the situation where an antibody defined herein and transduced T cells comprising an antigen-binding receptor of the present invention are preincubated together before administration to a subject. Thus, the two components can be preincubated for, for example, 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, or 1 hour before administration, or any suitable time period easily determined by one skilled in the art. In another preferred embodiment, the present invention relates to a treatment regimen in which an antibody defined herein and transduced T cells comprising an antigen-binding receptor defined herein are administered simultaneously. In the context of the present invention, an antibody defined herein can be administered after transduced T cells comprising an antigen-binding receptor have been administered.

[0304] Furthermore, as used herein, "in combination" is not limited to adjacent sequential administration (i.e., administration of one of the two components followed (after a time interval) by administration of the other without the administration and / or implementation of any other treatment protocol) of an antibody molecule as defined herein and transduced T cells, preferably CD8+ T cells, comprising an antigen-binding receptor of the present invention. Thus, the present treatment regimen also encompasses separate administration of an antibody molecule as defined herein and transduced T cells, preferably CD8+ T cells, comprising an antigen-binding receptor of the present invention, separated by one or more treatment protocols necessary and / or suitable for treating or preventing a disease or its symptoms. Examples of such intervening treatment protocols include, but are not limited to, administration of analgesics, administration of chemotherapeutic agents, and surgical management of a disease or its symptoms. Thus, the treatment regimens disclosed herein involve administering an antibody as defined herein and transduced T cells, preferably CD8+ T cells, comprising an antigen-binding receptor as defined herein, together with one or more treatment protocols suitable for treating or preventing a disease or symptom thereof described herein or known in the art.

[0305] It is particularly contemplated that the pharmaceutical composition / medicament is one that is administered to a patient by infusion or injection. In the context of the present invention, the transduced T cells comprising the antigen-binding receptors described herein are one that is administered to a patient by infusion or injection. Administration of suitable compositions / medicaments can be achieved by various methods: intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0306] The pharmaceutical compositions / medicaments of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art, including phosphate-buffered saline solution, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, etc. Compositions containing ...

Claims

1. 1. An antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain comprising an antigen-binding portion, the antigen-binding portion is capable of specifically binding to the mutated fragment crystallizable (Fc) domain but is incapable of specifically binding to the non-mutated parent Fc domain; the antigen-binding portion is an scFv, the non-mutated parent Fc domain is a human IgG1 Fc domain; the variant Fc domain is a human IgG1 Fc domain containing only the amino acid mutations L234A, L235A, and P329G according to EU numbering; the antigen-binding receptor further comprises (i) at least one stimulatory signaling domain, or (ii) at least one stimulatory signaling domain and at least one costimulatory signaling domain; The antigen-binding receptor.

2. 2. The antigen-binding receptor of claim 1, wherein the anchoring transmembrane domain is a transmembrane domain selected from the group consisting of CD8, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10, or DAP12 transmembrane domains.

3. The antigen-binding receptor of claim 2, wherein the anchoring transmembrane domain is the transmembrane domain of CD28.

4. The antigen-binding receptor of any one of claims 1 to 3, wherein at least one stimulatory signaling domain is the intracellular domain of CD3z.

5. 5. The antigen-binding receptor of any one of claims 1 to 4, wherein at least one costimulatory signaling domain is individually selected from the group consisting of the intracellular domain of CD27, the intracellular domain of CD28, the intracellular domain of CD137, the intracellular domain of OX40, the intracellular domain of ICOS, the intracellular domain of DAP10, and the intracellular domain of DAP12.

6. The antigen-binding receptor described in claim 5, wherein at least one costimulatory signaling domain is the intracellular domain of CD28.

7. 7. The antigen-binding receptor of any one of claims 1 to 6, comprising one costimulatory signaling domain comprising the intracellular domain of CD28 and one stimulatory signaling domain comprising the intracellular domain of CD3z.

8. The antigen-binding receptor of any one of claims 1 to 7, wherein the scFv fragment is linked at its C-terminus to the N-terminus of the anchoring transmembrane domain via a peptide linker.

9. 9. The antigen-binding receptor of any one of claims 1 to 8, wherein the scFv is stabilized by the formation of interchain disulfide bonds by the insertion of cysteine ​​residues.

10. The antigen-binding portion comprises: (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence RYWMN (SEQ ID NO:1); (b) CDR H2 amino acid sequence , and (c) CDR H3 amino acid sequence A heavy chain variable region (VH) comprising (ii) (d) light chain complementarity determining region (CDR L) 1 amino acid sequence (e) the CDR L2 amino acid sequence GTNKRAP (SEQ ID NO:5), and (f) CDR L3 amino acid sequence and a light chain variable region (VL) comprising The antigen-binding receptor of any one of claims 1 to 9, comprising:

11. A transduced T cell expressing an antigen-binding receptor according to any one of claims 1 to 10.

12. An isolated polynucleotide encoding the antigen-binding receptor of any one of claims 1 to 10.

13. A vector comprising the polynucleotide of claim 12.

14. The vector of claim 13, which is an expression vector.

15. (A) a transduced T cell expressing an antigen-binding receptor according to any one of claims 1 to 10; (B) An antibody comprising a mutant Fc domain, which is a human IgG1 Fc domain containing only the amino acid mutations L234A, L235A, and P329G according to EU numbering. Including the kit.

16. (A) an isolated polynucleotide encoding an antigen-binding receptor according to any one of claims 1 to 10; (B) An antibody comprising a mutant Fc domain, which is a human IgG1 Fc domain containing only the amino acid mutations L234A, L235A, and P329G according to EU numbering. Including the kit.

17. A kit described in any one of claims 15 or 16, wherein the antibody comprising the mutant Fc domain is capable of specifically binding to an antigen expressed on the surface of a tumor cell.

18. The kit of any one of claims 15 to 17, wherein the antibody comprising a variant Fc domain is capable of specifically binding to an antigen selected from the group consisting of fibroblast activation protein (FAP), carcinoembryonic antigen (CEA), mesothelin (MSLN), CD20, folate receptor 1 (FOLR1), and tenascin (TNC).

19. A kit according to any one of claims 15 to 18 for use as a medicine.

20. A kit according to any one of claims 17 or 18 for use in the treatment of a malignant disease.

21. A pharmaceutical comprising the transduced T cells of claim 11. A pharmaceutical agent administered in combination with an antibody comprising a variant Fc domain, the variant Fc domain is a human IgG1 Fc domain containing only the amino acid mutations L234A, L235A, and P329G according to EU numbering; a medicament is administered prior to, simultaneously with, or following administration of an antibody comprising a variant Fc domain; The said medicine.

22. A medicament for the treatment of a malignant disease comprising the transduced T cells of claim 11, the treatment comprises administering a medicament in combination with an antibody comprising a variant Fc domain, the antibody being capable of specifically binding to an antigen expressed on the surface of a tumor cell; the variant Fc domain is a human IgG1 Fc domain containing only the amino acid mutations L234A, L235A, and P329G according to EU numbering; a medicament is administered prior to, simultaneously with, or following administration of an antibody comprising a variant Fc domain; The said medicine.

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