Improved antigen-binding receptors

Abstract

The present invention generally relates to antigen-binding receptors capable of specifically binding to an Fc domain comprising the amino acid mutation P329G (EU numbering). The present invention also relates to T cells transduced with the antigen-binding receptor that are recruited by specifically binding to / interacting with the mutant Fc domain of a therapeutic antibody. Furthermore, the present invention relates to kits comprising the transduced T cells of the invention and / or nucleic acid molecules, vectors encoding the antigen-binding receptors of the invention, and tumor-targeting antibodies comprising the mutant Fc domain.

Description

[Technical Field]

[0001] Field of Invention The present invention generally relates to an antigen-binding receptor that can specifically bind to an Fc domain containing the amino acid mutation P329G by EU numbering. The present invention also relates to transduced T cells with an antigen-binding receptor that is recruited by specifically binding to / interacting with the mutant Fc domain of a therapeutic antibody. Furthermore, the present invention relates to a kit comprising a nucleic acid molecule, a vector, and a tumor-targeting antibody containing the transduced T cells and / or the antigen-binding receptor of the present invention. [Background technology]

[0002] background Adoptive T-cell therapy (ACT) is a powerful therapeutic approach that uses cancer-specific T cells (Rosenberg and Restifo, Science 348(6230)(2015), 62-68). ACT can use naturally occurring tumor-specific cells or T cells that have been genetically modified using T cells or chimeric antigen receptors (Rosenberg and Restifo, Science 348(6230)(2015), 62-68). ACT can successfully treat and induce remission even in patients with progressive and other treatment-resistant diseases, such as acute lymphoblastic leukemia, non-Hodgkin lymphoma, or melanoma (Dudley et al., J Clin Oncol 26(32)(2008), 5233-5239; Grupp et al., N Engl J Med 368(16)(2013), 1509-1518; Kochenderfer et al., J Clin Oncol.(2015)33(6):540-549, doi:10.1200 / JCO.2014.56.2025.Epub 2014 Aug 25).

[0003] However, despite impressive clinical efficacy, ACT is limited by treatment-related toxicity. The specificity of the manipulated T cells used in ACT, and the resulting on-target and off-target effects, are primarily driven by the tumor-targeting antigen-binding moieties implemented in the antigen-binding receptors. Non-exclusive expression of tumor antigens or temporal differences in expression levels can lead to serious side effects or even discontinuation of ACT due to unacceptable toxicity of the treatment.

[0004] Furthermore, the availability of tumor-specific T cells for efficient tumor cell lysis depends on the long-term survival and proliferation capacity of manipulated T cells in vivo. On the other hand, in vivo T cell survival and proliferation can also lead to undesirable long-term effects due to the persistence of an uncontrolled T cell response that can result in damage to healthy tissue (Grupp et al. 2013 N Engl J Med 368(16):1509-18, Maude et al. 2014 N Engl J Med 371(16):1507-17).

[0005] One approach to limit serious treatment-related toxicity and improve the safety of ACTs is to restrict T cell activation and proliferation by introducing adapter molecules into immune synapses. Such adapter molecules include small bimodal switches such as the recently described folate-FITC switch (Kim et al. J Am Chem Soc 2015;137:2832-2835). Further approaches have involved artificially modified antibodies containing tags that induce T cell specificity to target tumor cells (Ma et al. PNAS 2016;113(4):E450-458, Cao et al. Angew Chem 2016;128:1-6, Rogers et al. PNAS 2016;113(4):E459-468, Tamada et al. Clin Cancer Res 2012;18(23):6436-6445).

[0006] However, existing approaches have several limitations. Immunological synapses that rely on molecular switches require the introduction of additional elements that may trigger an immune response or result in nonspecific off-target effects. Furthermore, the complexity of such multi-component systems may limit therapeutic efficacy and tolerability. On the other hand, the introduction of tag structures into existing therapeutic monoclonal antibodies may affect the efficacy and safety profiles of these constructs. Moreover, tagging complicates the production of such antibodies and requires further modification and purification steps that necessitate further safety testing.

[0007] Furthermore, in vivo use of non-human or partially human antibodies can lead to the formation of anti-drug antibodies (ADAs), including anti-idiotypes or human anti-mouse antibodies (HAMAs) (Blanco et al Clin Immunol 17,96-106 (1997)). These ADAs may affect pharmacokinetic properties, safety, and functionality of administered antibodies, and humanization is applied to address this (Carter et al PNAS 89,4285-4289 (1992)). Similarly, ADAs have been observed for mouse-based CAR-T cells. Human anti-mouse IgG antibodies are known to occur with CAR-transduced T cells, but have been thought to have no adverse clinical consequences. Maus et al. first described anaphylaxis caused by CAR-modified T cells, most likely mediated by CAR-specific IgE antibodies. These results indicate that the potential immunogenicity of antigen-binding receptors derived from mouse antibodies can be a safety issue, especially when administered using intermittent dosing schedules (Maus et al Cancer Immunol Res 1,26-31(2013)). Therefore, to meet the needs of cancer patients, there is a need to improve targeted tumor therapies, particularly adoptive T-cell therapies. Thus, there remains a need to provide improved means that can improve the safety and efficacy of ACTs and overcome the aforementioned shortcomings. [Overview of the project]

[0008] Summary of the Invention The present invention provides an antigen-binding receptor having improved properties, in particular a humanized antigen-binding receptor that is stable and highly expressed in transduced cells.

[0009] An antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain, wherein the extracellular domain is (i) A heavy chain variable domain (VH) comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2 or SEQ ID NO: 40, and HCDR3 of SEQ ID NO: 3, (ii) Light chain variable domain (VL) including light chain complementarity determining region (LCDR)1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6 An antigen-binding receptor comprising an antigen-binding moiety is provided herein.

[0010] In one embodiment, the VH domain includes 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, SEQ ID NO: 41, SEQ ID NO: 44, and SEQ ID NO: 126.

[0011] In one embodiment, the VL domain includes 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: 9.

[0012] In one embodiment, the VL domain includes 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: 127.

[0013] In one embodiment, the anchoring transmembrane domain is a transmembrane domain selected from the group consisting of CD8, CD4, 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 CD8 transmembrane domain or a fragment thereof.

[0014] In one embodiment, the antigen-binding receptor comprises at least one stimulatory signaling domain and / or at least one co-stimulatory signaling domain.

[0015] 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, the intracellular domain of NKG2D, or a fragment thereof that retains stimulatory signaling activity. In particular, the at least one stimulatory signaling domain is the CD3z intracellular domain or a fragment thereof that retains CD3z stimulatory signaling activity.

[0016] In one embodiment, the at least one co-stimulatory 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 a fragment thereof that retains co-stimulatory signaling activity.

[0017] In one embodiment, the antigen-binding receptor comprises at least one CD28 co-stimulatory domain or a fragment thereof, and / or at least one CD137 co-stimulatory domain or a fragment thereof that retains CD28 co-stimulatory activity.

[0018] In one embodiment, the antigen-binding receptor comprises a stimulatory signaling domain that includes the intracellular domain of CD3z or a fragment thereof that retains CD3z stimulatory signaling activity, and a co-stimulatory signaling domain that includes the intracellular domain of CD28 or a fragment thereof that retains CD28 co-stimulatory signaling activity.

[0019] In one embodiment, the antigen-binding receptor comprises one stimulatory signaling domain containing the intracellular domain of CD3z or a fragment thereof retaining the CD3z stimulatory signaling activity, and one co-stimulatory signaling domain containing the intracellular domain of CD137 or a fragment thereof retaining the CD137 co-stimulatory signaling activity.

[0020] In one embodiment, the antigen-binding portion is linked, at its C-terminus, to the N-terminus of an anchoring transmembrane domain, optionally via a peptide linker.

[0021] In one embodiment, the light chain variable domain (VL) of the antigen-binding portion is linked, at its C-terminus, to the N-terminus of an anchoring transmembrane domain, optionally via a peptide linker, and / or the heavy chain variable domain (VH) is linked, at its C-terminus, to the N-terminus of the light chain variable domain (VL), optionally via a peptide linker.

[0022] In one embodiment, T cells capable of expressing the antigen-binding receptor described herein are provided.

[0023] In one embodiment, an isolated polynucleotide encoding the antigen-binding receptor described herein is provided.

[0024] In one embodiment, a vector, particularly an expression vector, containing the polynucleotide described herein is provided.

[0025] In one embodiment, (A) transduced T cells capable of expressing the antigen-binding receptor described herein, and (B) an antibody that binds to a target cell antigen and comprises an Fc domain containing the amino acid mutation P329G according to EU numbering are provided in a kit.

[0026] In one embodiment, (A) an isolated polynucleotide encoding the antigen-binding receptor described herein, and (B) An antibody that binds to a target cell antigen and contains an Fc domain including the amino acid mutation P329G by EU numbering. A kit is provided that includes the following.

[0027] In one embodiment, the target cell antigen is selected from the group consisting of fibroblast-activating protein (FAP), carcinoembryonic antigen (CEA), mesothelin (MSLN), CD20, folate receptor 1 (FOLR1), and tenascin (TNC).

[0028] In one embodiment, a kit described herein for use as a pharmaceutical is provided.

[0029] In one embodiment, a kit described herein is provided for use in the treatment of a disease, particularly for use in the treatment of cancer.

[0030] In one embodiment, transduced T cells as described herein are provided for use as a pharmaceutical, which are administered before, simultaneously with, or after administration of an antibody that binds to a target cell antigen and contains an Fc domain including an amino acid mutation P329G by EU numbering.

[0031] In one embodiment, the treatment includes the administration of transduced T cells before, concurrently with, or after the administration of an antibody that binds to a target cell antigen and contains an Fc domain including the amino acid mutation P329G by EU numbering.

[0032] In one embodiment, a method is provided for treating a disease in a subject, comprising administering transduced T cells capable of expressing the antigen-binding receptor described herein, and administering a therapeutically effective amount of an antibody that binds to a target cell antigen and contains an Fc domain including the EU-numbered amino acid mutation P329G before, simultaneously with, or after administration of the transduced T cells.

[0033] In one embodiment, a method is provided for inducing the lysis of target cells, comprising contacting the target cells with transduced T cells capable of expressing the antigen-binding receptor described herein in the presence of an antibody that binds to a target cell antigen and comprises an Fc domain containing an amino acid mutation P329G by EU numbering.

[0034] In one embodiment, the use of an antigen-binding receptor, a polynucleotide, or a transduced T cell as described herein is provided for the manufacture of a pharmaceutical product for the treatment of cancer. [Brief explanation of the drawing]

[0035] [Figure 1] Schematic diagram of a second-generation chimeric antigen-binding receptor having an anti-P329G binding moiety in scFv format. VH×VL scFv (Figure 1A) orientation and VL×VH (Figure 1B) orientation. Figures 1C and 1D show the DNA constructs encoding the antigen-binding receptors shown in Figures 1A and 1B, respectively. [Figure 2] Figure 2A shows CAR surface expression of different humanized scFv variants and correlated GFP expression (Figure 2B) that serves as a transduction control. [Figure 3] Evaluation of nonspecific signaling in anti-P329G CAR Jurkat reporter T cells using different humanized versions of the P329G binder as the binding site. Activation was assessed by quantifying the intensity of CD3 downstream signaling using the anti-P329G CAR Jurkat-NFAT reporter assay, either in the presence of antibodies with different Fc variants or under conditions containing the P329G Fc variant but without target cells. Triple-chain technical mean values ​​are shown, and error bars indicate SD. [Figure 4]Activation of anti-P329G CAR Jurkat reporter T cells in the presence of FolR1+ target cells with high (HeLa-FolR1), moderate (Skov3), and low (HT29) target expression levels, combined with antibodies having high (16D5), moderate (16D5 W96Y), or low (16D5 G49S / K53A) affinity for FolR1. Activation was assessed by quantifying the intensity of CD3 downstream signaling using the anti-P329G CAR Jurkat-NFAT reporter assay. Triple-chain technical mean values ​​are shown, and error bars indicate SD. [Figure 5] Activation of anti-P329G CAR Jurkat NFAT reporter T cells using different humanized versions of the P329G binder as the binding site. Reporter cell activity was evaluated in the presence of anti-FolR1(16D5)P329G IgG1 targeting IgG and HeLa(FolR1+) target cells (Figure 5A). Dose-dependent activation of the antibody was evaluated by quantifying the intensity of CD3 downstream signaling using the anti-P329G CAR Jurkat-NFAT reporter assay, and the area under the curve was calculated (Figure 5B). Triple technical mean values ​​are shown, and error bars indicate SD. [Figure 6] Activation of anti-P329G CAR Jurkat NFAT reporter T cells using different humanized versions of the P329G binder as the binding site. Reporter cell activity was evaluated in the presence of anti-HER2 (pertuzumab) P329G IgG1 targeting IgG and HeLa(HER2+) target cells (Figure 6A). Dose-dependent activation of the antibody was evaluated by quantifying the intensity of CD3 downstream signaling using the anti-P329G CAR Jurkat-NFAT reporter assay, and the area under the curve was calculated (Figure 6B). Triple technical mean values ​​are shown, and error bars indicate SD. [Figure 7] This shows the CAR surface expression of the disulfide-stabilized VHxVL1 scFv variant. [Modes for carrying out the invention]

[0036] Detailed explanation definition Terms are used herein in the manner they are commonly used in the art unless otherwise defined below.

[0037] For the purposes of this specification, “receptor human framework” means a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. A human immunoglobulin framework or human consensus framework “derived” acceptor human framework may contain the same amino acid sequence or may contain modifications of the amino acid sequence. In some embodiments, the number of amino acid modifications is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0038] An "activated Fc receptor" is an Fc receptor that, after being linked by the Fc domain of an antibody, triggers a signaling event that stimulates receptor-containing cells to exert its effector function. Examples of human activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0039] Antibody-dependent cell-mediated cytotoxicity ("ADCC") is an immune mechanism by which immune effector cells cause the lysis of antibody-coated target cells. Target cells are cells to which antibodies or derivatives containing an Fc domain specifically bind, typically via a protein portion that is N-terminus of the Fc domain. As used herein, the term "decreased ADCC" is defined as either a decrease in the number of target cells lysed at a given time by a given concentration of antibody in the culture medium surrounding the target cells, due to the ADCC mechanism as defined above, and / or an increase in the concentration of antibody in the culture medium surrounding the target cells required to achieve the lysis of a given number of target cells at a given time, due to the ADCC mechanism. ADCC reduction is compared to unmanipulated ADCC mediated by the same antibody produced by the same type of host cells using the same standard production, purification, formulation and storage methods (known to those skilled in the art). For example, an amino acid substitution that reduces ADCC, such as a reduction in ADCC mediated by an antibody containing its Fc domain, is compared to ADCC mediated by the same antibody that does not contain this amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, PCT International Publication No. 2006 / 082515 or No. 2012 / 130831).

[0040] The “effective amount” of a drug, such as a pharmaceutical composition, refers to the amount that is effective in the dosage and duration required to achieve the desired therapeutic or preventive outcome.

[0041] "Affinity" refers to the sum of the strength of non-covalent interactions between a single binding site between a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). D) can be expressed by. Affinity can be measured by methods common in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described below.

[0042] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function in a manner similar to natural amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same base chemical structure as natural amino acids, i.e., hydrogen, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same base chemical structure as naturally occurring amino acids. Amino acid mimes refer to compounds that have a structure different from the general or chemical structure of an amino acid, but function in a manner similar to a natural amino acid. Amino acids may be referred to herein by the commonly known three-letter or one-letter abbreviations of amino acids as recommended by the IUPAC-IUB Biochemical Nomenclature Committee.

[0043] As used herein, the term “amino acid mutation” encompasses amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be made to arrive at the final construct, insofar as the final construct has the desired characteristics, e.g., reduced binding to the Fc receptor or increased association with another peptide. Deletions and insertions of amino acid sequences include deletions and insertions of amino acids at the amino-terminus and / or carboxy-terminus. A specific amino acid mutation is an amino acid substitution. For example, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid with different structural and / or chemical properties, are particularly preferred for the purpose of altering the binding properties of the Fc region. Amino acid substitutions include substitutions with non-natural amino acids or substitutions with natural amino acid derivatives of 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be induced using genetic or chemical methods well known in the art. Genetic methods may include mutagenesis of specific sites, PCR, gene synthesis, etc. Methods other than genetic engineering to alter the side chain groups of amino acids, such as chemical modification, may also be useful. Various names can be used herein to describe the same amino acid mutation. For example, a substitution of proline to glycine at position 329 of the Fc domain is referred to as 329G, G329 329 It can be indicated as P329G or Pro329Gly.

[0044] The term "antibody" is used herein in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0045] An "antibody fragment" refers to a molecule other than an intact antibody, including a part 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 scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0046] The term "antigen-binding domain" refers to a portion of an antibody that specifically binds to some or all of a particular antigen and is complementary to some or all of that antigen. The antigen-binding domain may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). In particular, the antigen-binding domain includes an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0047] As used herein, the term “antigen-binding molecule” refers in its broadest sense to a molecule that specifically binds an antigenic determinant. Examples of antigen-binding molecules include immunoglobulins and their derivatives, such as their fragments, as well as antigen-binding receptors and their derivatives.

[0048] As used herein, the term “antigen-binding moiety” refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen-binding moiety can direct the entity to which it binds (e.g., a cell expressing an antigen-binding receptor containing the antigen-binding moiety) to a target site, such as a specific type of tumor cell or tumor stroma containing an antigenic determinant. The antigen-binding moiety includes an antibody and its fragments as further defined herein. A particular antigen-binding moiety includes the antigen-binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region (e.g., an scFv fragment). In a particular embodiment, the antigen-binding moiety may include an antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include any of two isotypes: κ and λ.

[0049] 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 containing at least one antigen-binding moiety. Antigen-binding receptors can be made from polypeptide moieties of different sources. Thus, antigen-binding receptors can also be understood as “fusion proteins” and / or “chimeric proteins.” Typically, a fusion protein is a protein created by the fusion of two or more genes (or preferably cDNAs) that originally encoded separate proteins. Translation of this fusion gene (or fusion cDNA) results in a single-stranded polypeptide having functional properties preferably derived from each of the original proteins. Recombinant fusion proteins are artificially created by recombinant DNA technology for use in biological research or therapeutics. Further details of the antigen-binding receptors of the present invention are described below herein. In the context of the present invention, a CAR (chimeric antigen receptor) is understood to be an antigen-binding receptor comprising an extracellular portion containing an antigen-binding moiety fused to an anchoring transmembrane domain, for example, fused to an intracellular signaling domain, by a spacer sequence.

[0050] An "antigen-binding site" refers to the part of an antigen-binding molecule that provides interaction with an antigen, i.e., one or more amino acid residues. For example, the antigen-binding site of an antibody contains amino acid residues from the complementarity-determining region (CDR). Natural immunoglobulin molecules typically contain two antigen-binding sites, while Fab molecules typically have one antigen-binding site.

[0051] The term "antigen-binding domain" refers to a portion of an antibody or antigen-binding receptor that includes a region that specifically binds to and is complementary to some or all of an antigen. The antigen-binding domain may be provided, for example, by one or more immunoglobulin variable domains (also called variable regions). In particular, the antigen-binding domain includes an immunoglobulin light chain variable domain (VL) and an immunoglobulin heavy chain variable domain (VH).

[0052] As used herein, the term “antigenic determinant” is synonymous with “antigen” and “epitope” and refers to a site on a polypeptide macromolecule to which an antigen-binding moiety binds, forming an antigen-antigen complex (e.g., a contiguous extension of amino acids or a conformational structure composed of discontinuous amino acids from different regions). Useful antigenic determinants may be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other affected cells, on the surface of immune cells, free in serum, and / or within the extracellular matrix (ECM). Unless otherwise specified, the protein referred to as an antigen herein may be any innate form of protein from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is a human protein. When referring to a particular protein of the present invention, the term encompasses “full-length” untreated proteins and any form of protein obtained from cell treatment. The term also encompasses naturally occurring protein variants, e.g., splice variants or allele variants.

[0053] The "antibody containing a mutant Fc domain" according to the present invention, i.e., a therapeutic antibody, may have one, two, three or more binding domains and may be monospecific, bispecific, or multispecific. The antibody may be full-length and derived from a single species, or it may be chimeric or humanized. For antibodies having more than two antigen-binding domains, some binding domains may be identical and / or have the same specificity.

[0054] As used herein, the term “ATD” refers to an “anchoring transmembrane domain” that defines a polypeptide chain that can be incorporated into the cell membrane (or cell membrane). ATMs can be fused to extracellular polypeptide domains and / or intracellular polypeptide domains, in which case these extracellular and / or intracellular polypeptide domains are confined to the cell membrane. In the context of the antigen-binding receptor of the present invention, the ATM provides the membrane attachment and confinement 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 containing an antigen-binding moiety. Furthermore, the ATM can be fused to an intracellular signaling domain.

[0055] "Specific binding" means that the binding is antigen-selective and can be distinguished from undesirable or nonspecific interactions. The ability of an antigen-binding moiety to bind to a specific antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques known to those skilled in the art, such as surface plasmon resonance (SPR) (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 degree of binding of an antigen-binding moiety to an unrelated protein is less than about 10% of the binding of the antigen-binding moiety to an antigen, for example, as measured by SPR. In certain embodiments, an antigen-binding moiety that binds to an antigen, or an antigen-binding molecule containing this antigen-binding moiety, has a dissociation constant (K D) is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M) is the answer.

[0056] The term "CDR" as used herein refers to the "complementarity-determining region," which is well known in the art. A CDR is a portion of an immunoglobulin or antigen-binding receptor that determines the specificity of a molecule and brings it into contact with a specific ligand. CDRs are the most variable parts of a molecule and contribute to the antigen-binding diversity of these molecules. Each V domain contains three CDR regions, namely CDR1, CDR2, and CDR3. CDR-H refers to the CDR region of the variable heavy chain, and CDR-L refers to the CDR region of the variable light chain. VH means the variable heavy chain, and VL means the variable light chain. The CDR region of the Ig-derived area can be determined as described in "Kabat" (Sequences of Proteins of Immunological Interest, 5th edit. 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).

[0057] The term "CD3z" refers to the T cell surface glycoprotein CD3 zeta chain, also known as "T cell receptor T3 zeta chain" and "CD247".

[0058] The terms "chimeric antigen receptor," "chimeric receptor," or "CAR" refer to an antigen-binding receptor composed of an extracellular portion of an antigen-binding moiety (e.g., a single-chain antibody domain) fused to an intracellular signaling / co-signaling domain (e.g., CD3z and CD28) by a spacer sequence.

[0059] The "class" of an antibody refers to the type of constant domain or constant region held by its heavy chain. Antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is the IgG1 isotype. In certain embodiments, the antibody is the IgG1 isotype with P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other embodiments, the antibody is the IgG2 isotype. In certain embodiments, the antibody is the IgG4 isotype with the S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0060] As used in this application, the terms “human-derived constant region” or “human constant region” refer to the constant heavy chain region and / or constant light chain kappa or lambda region of a human antibody of subclass IgG1, IgG2, IgG3, or IgG4. Such constant regions may be used in human or humanized antibodies, and are publicly known in the art, for example, as described in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, for example, Johnson, G., and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218; Kabat, EA, et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, the numbering of amino acid residues in the constant region follows the EU numbering system (also known as the Kabat EU index), as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0061] A "crossover" Fab molecule (also called "crossFab") refers to a Fab molecule in which the variable domains of the Fab heavy chain and light chain are exchanged (i.e., replaced by each other). Specifically, a crossover Fab molecule includes a peptide chain composed of a light chain variable domain VL and a heavy chain constant domain 1CH1 (VL-CH1, from the N-terminus to the C-terminus), and a peptide chain composed of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL, from the N-terminus to the C-terminus). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and Fab heavy chain are exchanged, the peptide chain containing the heavy chain constant domain 1CH1 is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0062] As used herein, the term "CSD" refers to the concurrent stimulus signaling domain.

[0063] "Effector function" refers to the biological activity resulting from the Fc region of an antibody, and it varies 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), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0064] As used herein, the terms “to manipulate,” “to be manipulated,” and “to manipulate” are understood to include any manipulation or post-translational modification of the peptide backbone of naturally occurring or recombinant polypeptides or fragments thereof. Manipulation includes modification of amino acid sequences, modification of glycosylation patterns, or modification of the side chain groups of individual amino acids, and combinations thereof.

[0065] The term "expression cassette" refers to a recombinant or synthetically produced polynucleotide having a set of specific nucleic acid elements capable of transcribing a particular nucleic acid in target cells. 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 includes, in particular, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the present invention includes a polynucleotide sequence or fragment thereof encoding the bispecific antigen-binding molecule of the present invention.

[0066] A "Fab molecule" refers to a protein consisting of the VH domain and CH1 domain of the immunoglobulin heavy chain ("Fab heavy chain") and the VL domain and CL domain of the immunoglobulin light chain ("Fab light chain").

[0067] The terms “Fc domain” or “Fc region” are used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes both the native sequence Fc region and the variant Fc region. While the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is typically defined to extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more amino acids, particularly one or two, from the C-terminus of the heavy chain. Thus, by expression of a particular nucleic acid molecule encoding a full-length heavy chain, antibodies produced by host cells may contain the full-length heavy chain or a cleaved variant of the full-length heavy chain (also referred to herein as a “cleaved variant heavy chain”). This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (K447) of the Fc domain may or may not be present. Unless otherwise specified, the amino acid sequence of the heavy chain containing the Fc domain (or a subunit of the Fc domain as defined herein) is shown herein without the C-terminal glycine-lysine dipeptide. In one embodiment of the present invention, the heavy chain containing the Fc domain subunit identified herein includes a further C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In another embodiment of the present invention, the heavy chain containing the Fc domain subunit identified herein includes a further C-terminal glycine residue (G446, numbered according to the Kabat EU index). The compositions of the present invention, such as pharmaceutical compositions described herein, comprise a collection of antigen-binding molecules of the present invention. The collection of antigen-binding molecules may include molecules containing full-length heavy chains and molecules containing cleaved variant heavy chains.The antigen-binding molecule assembly may consist of a mixture of molecules having full-length heavy chains and molecules having cleaved variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antigen-binding molecules have cleaved variant heavy chains. In one embodiment of the present invention, a composition comprising the antigen-binding molecule assembly of the present invention comprises an antigen-binding molecule comprising a heavy chain containing a subunit of the Fc domain specified herein, comprising a further C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of the present invention, a composition comprising the antigen-binding molecule assembly of the present invention comprises an immunoactivating Fc domain-binding molecule comprising a heavy chain containing a subunit of the Fc domain specified herein, comprising a further C-terminal glycine residue (G446, numbered according to the Kabat EU index). In one embodiment of the present invention, such a composition comprises an antigen-binding molecule comprising a molecule comprising a heavy chain comprising a subunit of the Fc domain specified herein, a molecule comprising a heavy chain comprising a subunit of the Fc domain specified herein, comprising a further C-terminal glycine residue (G446, numbered according to Kabat's EU index), and a molecule comprising a heavy chain comprising a subunit of the Fc domain specified herein, comprising a further C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to Kabat's EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also called the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). As used herein, the term "subunit" of the Fc domain means one of the two polypeptides that form the dimeric Fc domain, i.e., a polypeptide that contains the C-terminal constant region of the immunoglobulin heavy chain and is capable of stable self-assembly.For example, the subunits of the IgG Fc domain include the IgG CH2 and IgG CH3 constant domains.

[0068] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining region (CDR). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences generally appear in VH (or VL) in the following sequence: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0069] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein synonymously to refer to antibodies having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein.

[0070] "Fused" means that the constituent elements (e.g., the Fab and the transmembrane domain) are linked directly by peptide bonds or via one or more peptide linkers.

[0071] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and cells including the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring may contain mutations, although they may not have the exact same nucleic acid content as the parent cells. In this specification, offspring of mutants having the same function or biological activity as those screened or selected in the initially transformed cells are included.

[0072] A "human antibody" is defined as an antibody produced by a human or human cell, or an antibody that has an amino acid sequence corresponding to a non-human antibody that utilizes a sequence encoding a human antibody, such as a human antibody repertoire. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0073] The "Human Consensus Framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup Kappa I, as described in Kabat et al. above. In another embodiment, for VH, the subgroup is subgroup Kappa III, as described in Kabat et al. above.

[0074] A “humanized” antibody (e.g., a humanized scFv fragment) refers to a chimeric antibody containing amino acid residues derived from a non-human CDR and amino acid residues derived from a human FR. In certain embodiments, the humanized antibody substantially contains all of at least one, typically two, variable domains, in which all or substantially all of the CDRs correspond to the CDRs of a non-human antibody and all or substantially all of the FRs correspond to the FRs of a human antibody. The humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0075] As used herein, the terms “hypervariable region” or “HVR” mean each of the regions of an antibody variable domain that are hypervariable within a sequence and determine antigen-binding specificity, such as “complementarity-determining regions” (CDRs).

[0076] Generally, an antibody contains six CDRs, three of which are located in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Illustrative CDRs as used herein include: (a) Hypervariable loops arising at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)), (b) CDRs present in amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) is an example (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0077] Unless otherwise specified, the CDR is determined in accordance with Kabat et al. above. Those skilled in the art will understand that the notation of the CDR may be determined in accordance with Chothia above, McCallum above, or any other scientifically recognized nomenclature system.

[0078] An "immune conjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0079] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the individual or subject is a human.

[0080] An "isolated" antibody is one that has been separated from its natural environment. In some embodiments, antibodies are purified to a purity higher than 95% or 99%, as determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J.Chromatogr.B 848:79-87 (2007).

[0081] The term "immunoglobulin molecule" refers to a protein that has the structure of a naturally occurring antibody. For example, IgG class immunoglobulins are heterotetrameric glycoproteins with a weight of approximately 150,000 daltons, composed of two disulfide-linked light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy chain domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light chain domain or light chain variable region, followed by a constant light chain (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types called α(IgA), δ(IgD), ε(IgE), γ(IgG), or μ(IgM), some of which may be further divided into subtypes such as γ1(IgG1), γ2(IgG2), γ3(IgG3), γ4(IgG4), α1(IgA1), and α2(IgA2). The light chain of an immunoglobulin may be assigned to one of two types called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via an immunoglobulin hinge region.

[0082] The term "isolated nucleic acid" molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, isolated from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of this invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells, or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that originally contained polynucleotide molecules, but the polynucleotide molecules are extrachromosomal or located at chromosomal locations different from their natural chromosomal locations. Isolated RNA molecules include the in vivo or in vitro RNA transcripts of this invention, in positive and negative strand forms, and in double-stranded forms. Isolated polynucleotides or nucleic acids according to this invention further include such molecules produced by synthesis. In addition, polynucleotides or nucleic acids may be or contain regulatory elements such as promoters, ribosome binding sites, or transcription terminators.

[0083] The nucleic acids or polynucleotides having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention are intended to have a nucleotide sequence 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 the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with other nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. Such modifications to the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or at any position between these terminal positions, and may be individually scattered among residues in the reference sequence, or scattered as one or more consecutive 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 the nucleotide sequence of the present invention can be conventionally determined using known computer programs such as those discussed later for polypeptides (e.g., ALIGN-2).

[0084] The intended "isolated polypeptide" or its variant or derivative is a polypeptide not found in its natural environment. No specific level of purification is required. For example, isolated polypeptides can be taken from their natural or native environment. Recombinant-produced polypeptides and proteins expressed in host cells are considered to be isolated for the purposes of this invention, similar to native polypeptides or recombinant polypeptides, by any suitable technique, after being separated, fractionated, or partially or substantially purified.

[0085] "Modifications that promote the association of the first and second subunits of the Fc domain" are manipulations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent the association of a peptide containing an Fc domain subunit with an identical polypeptide for homodimer formation. When used herein, association-promoting modifications include distinct modifications for each of the two Fc domain subunits that are desired to associate (i.e., the first and second subunits of the Fc domain), and the modifications are complementary to each other so as to promote the association of the two Fc domain subunits. For example, the association-promoting modifications may alter the structure or charge of one or both of the Fc domain subunits so as to perform a sterically or electrostatically desired association. Thus, (hetero)dimerization occurs between a polypeptide containing a first Fc domain subunit and a polypeptide containing a second Fc domain subunit, and these may not be identical in the sense that the further components (e.g., antigen-binding moieties) that fuse to each subunit are not the same. In some embodiments, the association-promoting modifications include amino acid mutations within the Fc domain, specifically amino acid substitutions. In certain embodiments, the modifications that facilitate association include distinct amino acid mutations, specifically amino acid substitutions, in each of the two subunits of the Fc domain.

[0086] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bound to the same epitope, except for variant antibodies that include, for example, naturally occurring mutations or variant antibodies that may arise during the production of a monoclonal antibody preparation, such variants generally present in trace amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed toward different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed toward a single determinant on one antigen. Therefore, the modified term “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous collection of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies according to the present invention can be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, including but not limited to such methods and other exemplary methods for producing monoclonal antibodies described herein.

[0087] A "naked antibody" refers to an antibody that is not conjugated to a heterogeneous site (e.g., a cytotoxic site) or a radioactive label. Naked antibodies may be present in a pharmaceutical composition.

[0088] A "native antibody" refers to a native immunoglobulin molecule with various structures. For example, a native IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, containing two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant heavy domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light domain or light chain variable region, followed by a constant light (CL) domain.

[0089] "Percent (%) amino acid sequence identity" relative 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 for alignment purposes and introducing gaps if necessary to achieve the maximum sequence identity rate, without considering any conservative substitutions as part of the sequence identity. Alignment for determining the amino acid sequence identity rate can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or FASTA program packages. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. Alternatively, the identity rate value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code is filed in the user documentation of the US Copyright Office (Washington DC, 20559), registered under US Copyright Registration No. TXU510087, and published in International Publication No. 2001 / 007611.

[0090] Unless otherwise noted, for the purposes of this specification, amino acid sequence identity values ​​are generated using the ggsearch program in FASTA package version 36.3.8c, or subsequently using the BLOSUM50 comparison matrix. The FASTA program package is described by WRPearson and DJLipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; WRPearson (1996), "Effective protein sequence comparison," Meth.Enzymol.266:227-258; and Pearson et al. (1997), Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or http: / / www.ebi.ac.uk / Tools / sss / fasta. Alternatively, the ggsearch (Global Protein: Protein) program and its default options (BLOSUM50;open:-10;ext:-2;Ktup=2) can be used to compare sequences using a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi, ensuring that a global rather than local alignment is performed. The amino acid identity rate is given in the output alignment header. The term "nucleic acid molecule" refers to a sequence of bases, including purine and pyrimidine bases, contained by a polynucleotide, thereby representing the primary structure of the nucleic acid molecule. In this specification, the term nucleic acid molecule includes DNA, cDNA, genomic DNA, RNA, synthetic forms of DNA, and mixed polymers containing two or more of these molecules. In addition, the term nucleic acid molecule includes both sense and antisense strands. Furthermore, the nucleic acid molecules described herein may contain unnatural or derivatized nucleotide bases, as will be readily apparent to those skilled in the art.

[0091] The term “package insert” is used to refer to the instructions typically included on the market packaging of a therapeutic product, including information relating to indications, use, dosage, administration, combination therapy, contraindications, and / or warnings for such therapeutic products.

[0092] The term "pharmaceutical composition" refers to a preparation that is in a form that enables the biological activity of the active ingredient contained therein and does not contain any additional components that are unacceptably toxic to the subject to which the preparation is administered. A pharmaceutical composition typically contains one or more pharmaceutically acceptable carriers.

[0093] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical composition that is not the active ingredient and is not toxic to the target substance. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0094] 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).

[0095] The term “polypeptide” refers to any chain of two or more amino acids, and does not refer to a product of a specific length. Therefore, peptides, dipeptides, tripeptides, oligopeptides, “proteins,” “amino acid chains,” or any other terms used to refer to chains of two or more amino acids are included in the definition of “polypeptide,” and the term “polypeptide” may be used in place of or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, cleavage by proteolysis, or modification with amino acids not naturally occurring. Polypeptides may be derived from natural biosources or produced by recombinant techniques, but not necessarily translated from a specified nucleic acid sequence. Polypeptides may arise in any manner, including by chemical synthesis. The polypeptides of the present invention may have a size of approximately 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. Polypeptides may have a specified three-dimensional structure, but they do not necessarily have such a structure. Polypeptides having a specified three-dimensional structure are called folded, and polypeptides that do not have a specified three-dimensional structure but can rather adopt many different conformations are called unfolded.

[0096] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), viral RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or other types of bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term "nucleic acid molecule" refers to one or more arbitrary nucleic acid segments, such as DNA or RNA fragments present in a polynucleotide.

[0097] "Reduced binding," for example, reduced binding to the Fc receptor, refers to a decrease in affinity for each interaction, as measured, for example, by SPR. For clarity, this term also includes a reduction in affinity to zero (or below the detection limit of the analytical method), i.e., complete termination of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for individual interactions.

[0098] The term "regulatory sequence" refers to a DNA sequence necessary for the expression of a ligated coding sequence. The nature of such regulatory sequences varies depending on the host organism. In prokaryotes, regulatory sequences generally include promoters, ribosome binding sites, and terminators. In eukaryotes, regulatory sequences generally include promoters, terminators, and in some cases enhancers, transcription activators, or transcription factors. The term "regulatory sequence" is intended to include the presence of all components necessary for expression, at a minimum, and may also include additional advantageous components.

[0099] As used herein, the term “single-chain” refers to a molecule containing amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen-binding moieties is a single-chain Fab molecule, i.e., a Fab molecule in which a Fab light chain and a 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 connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule. In a preferred embodiment, the antigen-binding moiety is an scFv fragment.

[0100] As used herein, the term "SSD" refers to the "stimulus signaling domain."

[0101] As used herein, “treatment” (and its grammatical variations, e.g., “to treat” or “treating”) refers to a clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and may be carried out for preventive purposes or during the course of a clinicopathological disease. Desired effects of treatment include preventing the onset or recurrence of the disease, reducing symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, achieving remission or mitigation of the condition, and achieving a recovered or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of the disease or to slow the progression of the disease.

[0102] 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, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. The immunoactivating Fc domain-binding molecules 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.

[0103] The “therapeutic dose” of a drug, such as a pharmaceutical composition, refers to the effective amount in the dose and duration required to obtain the desired therapeutic or prophylactic outcome. For example, the therapeutic dose of a drug is effective in eliminating, reducing, delaying, minimizing, or preventing the side effects of a disease.

[0104] As used herein, the term "valence" refers to the presence of a specific number of antigen-binding sites within an antigen-binding molecule. Therefore, the term "monovalent binding to an antigen" refers to the presence of one (and not more than one) antigen-specific antigen-binding sites within an antigen-binding molecule.

[0105] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or antibody light chain that is involved in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) generally have a similar structure, and each domain includes four conserved framework regions (FRs) and three complementarity-determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6 th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, an antibody that binds a particular antigen may use the VH or VL domain of the antigen-binding antibody to screen, and isolate, a library of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0106] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes not only vectors as self-replicating nucleic acid structures but also vectors integrated into the genome of a host cell into which the vector has been introduced. A particular vector can direct the expression of a nucleic acid operably linked thereto. Such a vector is referred to herein as an "expression vector."

[0107] An antigen-binding receptor that can specifically bind to a mutant Fc domain The present invention relates to an antigen-binding receptor that can specifically bind to a mutant Fc domain of an antibody, such as a therapeutic antibody targeting cancer cells. In a preferred embodiment, the present invention relates to an antigen-binding receptor that can specifically bind to a mutant Fc domain containing the EU-numbered amino acid mutation P329G. The antigen-binding receptor of the present invention includes an extracellular domain containing at least one antigen-binding moiety that can specifically bind to the mutant Fc domain but cannot specifically bind to the parent non-mutant Fc domain. In a preferred embodiment, the antigen-binding moiety of the antigen-binding receptor is a humanized or human antigen-binding moiety, such as a humanized or human scFv. In a preferred embodiment, the amino acid mutation is P329G, and specific binding to the mutant Fc domain containing the EU-numbered amino acid mutation P329G is measured by SPR at 25°C.

[0108] 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, using the antigen-binding receptor described herein, and to the targeted recruitment of these cells to tumors, for example, by antibody molecules containing a mutant Fc domain (e.g., an Fc domain containing the amino acid mutation P329G by EU numbering), such as therapeutic antibodies. In one embodiment, the antibody can specifically bind to tumor-specific antigens naturally present on the surface of tumor cells.

[0109] As shown in the attached examples, as a proof of concept, an antigen-binding receptor (SEQ ID NO: 7, encoded by the DNA sequence shown in SEQ ID NO: 20) containing the anchoring transmembrane domain and humanized extracellular domain of the present invention was constructed, which can specifically bind to a therapeutic antibody containing the P329G mutation (represented by an anti-CD20 antibody containing the heavy chain of SEQ ID NO: 102 and the light chain of SEQ ID NO: 103). Transduced T cells (Jurkat NFAT T cells) expressing the VH3VL1-CD8ATD-CD137CSD-CD3zSSD fusion protein (SEQ ID NO: 7, encoded by the DNA sequence shown in SEQ ID NO: 20) could be strongly activated by co-incubation of CD20-positive tumor cells with an anti-CD20 antibody containing the P329G mutation in the Fc domain.

[0110] Therapy of tumor cells with antibodies against tumor antigens containing the P329G mutation, combined with transduced T cells expressing the VH3VL1-CD8ATD-CD137CSD-CD3zSSD fusion protein (sequence number 7, encoded by the DNA sequence shown in sequence number 20), surprisingly results in stronger activation of transduced T cells compared to transduced T cells expressing VL1VH3-CD8ATD-CD137CSD-CD3zSSD (sequence number 31, encoded by the DNA sequence shown in sequence number 33).

[0111] In the VH3VL1-CD8ATD-CD137CSD-CD3zSSD fusion protein, the VH domain (VH3) fuses at its C-terminus to the N-terminus of the VL domain (VL1) via a peptide linker, forming an scFv. The scFv then fuses at its C-terminus (the C-terminus of the VL domain) to the anchoring transmembrane domain (ATD) via a peptide linker. On the other hand, in the VL1VH3-CD8ATD-CD137CSD-CD3zSSD fusion protein, the VL domain (VL1) fuses at its C-terminus to the N-terminus of the VH domain (VH3) via a peptide linker, forming an scFv. The scFv then fuses at its C-terminus (the C-terminus of the VH domain) to the anchoring transmembrane domain (ATD) via a peptide linker. While not constrained by theory, the observation that the VH3VL1-CD8ATD-CD137CSD-CD3zSSD fusion protein results in stronger activation of transduced T cells compared to VL1VH3-CD28ATD-CD137CSD-CD3zSSD suggests that the fusion (via the peptide linker) of the VL domain and the anchoring domain results in a more potent antigen-binding receptor. This is unexpected and surprising.

[0112] The combination of the VH domain VH3 and the VL domain VL1 (both identified by the inventors) is particularly preferred because these variable domains are humanized antibody domains. While not bound by theory, humanized antibody domains are preferred because, when antigen-binding moieties containing such humanized antibody domains are applied to human patients, fewer side effects (e.g., less formation of anti-drug antibodies (ADAs)) can be expected. However, humanization can result in the loss of binding of antigen-binding moieties (e.g., those derived from non-human sources). As shown in the attached examples, the humanized VH3 and VL1 domains retain binding to the Fc domain containing the EU-numbered amino acid mutation P329G. This result is unexpected, as indicated by the fact that other humanized VH and VL domains cannot retain equivalent binding to the Fc domain containing the EU-numbered amino acid mutation P329G.

[0113] Accordingly, in a preferred embodiment of the present invention, an antigen-binding receptor is provided which includes a humanized antigen-binding moiety capable of specifically binding to an Fc domain containing the amino acid mutation P329G according to EU numbering. The concept of the present invention and its components (humanized antigen-binding receptor and therapeutic antibody) are described in further detail below herein.

[0114] According to the present invention, specific activation of T cells and subsequent lysis of tumor cells are obtained by pairing a tumor-specific antibody, i.e., a therapeutic antibody, containing a mutant Fc domain (e.g., an amino acid mutation P329G by EU numbering), with an antigen-binding receptor consisting of / containing an extracellular domain with an antigen-binding site that can specifically bind to the mutant Fc domain. This approach offers a significant safety advantage over conventional T cell-based approaches, as T cells may be inactive in the absence of the antibody containing the mutant Fc domain. Therefore, the present invention provides a versatile therapeutic platform in which an IgG-type antibody is used to mark or label tumor cells as guidance for T cells, and transduced T cells are specifically targeted to tumor cells by providing specificity for the mutant Fc domain of the IgG-type antibody. After binding to the mutant Fc domain of the antibody on the surface of tumor cells, the transduced T cells described herein are activated, and the tumor cells are subsequently lysed. The platform is flexible and specific by allowing the use of diverse (existing or newly developed) target antibodies, or the simultaneous application of multiple antibodies with different antigen specificities but containing the same mutation in the Fc domain (e.g., the P329G mutation). The degree of T cell activation can be further adjusted by adjusting the dosage of the co-applied therapeutic antibody, or by switching to a different antibody specificity or format. Transduced T cells according to the present invention are inactive without the simultaneous application of a targeted antibody containing the mutant Fc domain, since the mutation to the Fc domain described herein does not occur in natural or non-mutant immunoglobulins. Therefore, in one embodiment, the mutant Fc domain is not naturally present in (human) immunoglobulins.

[0115] Accordingly, the present invention relates to an antigen-binding receptor comprising an extracellular domain including at least one antigen-binding moiety capable of specifically binding to a mutant Fc domain, wherein at least one antigen-binding moiety cannot specifically bind to a parent non-mutant Fc domain. Since effector function can lead to serious side effects of antibody-based oncology, as further described herein, it may be particularly desirable to use therapeutic antibodies with reduced effector function in cancer therapy.

[0116] In the context of the present invention, the antigen-binding receptor includes an extracellular domain that is not naturally present in or on T cells. Therefore, the antigen-binding receptor can provide a binding specificity tailored to cells expressing the antigen-binding receptor of the present invention. Cells transduced to the antigen-binding receptor(s) of the present invention, such as T cells, become able to specifically bind to the mutant Fc domain but not to the non-mutant parental Fc domain. The specificity is provided by the antigen-binding moiety of the extracellular domain of the antigen-binding receptor. In the context of the present invention, as described herein, the antigen-binding moiety that can specifically bind to the mutant Fc domain binds to / interacts with the mutant Fc domain but does not bind to / the non-mutant parental Fc domain.

[0117] antigen binding part In an exemplary embodiment of the present invention, as a proof of concept, a humanized antigen-binding receptor capable of specifically binding to a mutant Fc domain containing the amino acid mutation P329G, and effector cells expressing the antigen-binding receptor are provided. The P329G mutation reduces binding to the Fcγ receptor and associated effector function. Therefore, the mutant Fc domain containing the P329G mutation binds to the Fcγ receptor with reduced or absent affinity compared to the non-mutant Fc domain.

[0118] In one embodiment, the antigen-binding moiety can specifically bind to a mutant Fc domain composed of first and second subunits capable of stable association. In one embodiment, the Fc domain is an IgG, specifically an IgG1 or IgG4 Fc domain. In one embodiment, the Fc domain is a human Fc domain. In one embodiment, the mutant Fc domain exhibits reduced binding affinity to the Fc receptor and / or reduced effector function compared to the natural IgG1 Fc domain. In one embodiment, the Fc domain contains one or more amino acid mutations that reduce binding to the Fc receptor and / or effector function.

[0119] In one preferred embodiment, the mutant Fc domain contains a P329G mutation. Therefore, the mutant Fc domain containing the P329G mutation binds to the Fcγ receptor with reduced or absent affinity compared to the non-mutant Fc domain.

[0120] In one embodiment, the antigen-binding receptor includes an extracellular domain containing an antigen-binding moiety. In one embodiment, the antigen-binding moiety can specifically bind to an Fc domain containing the amino acid mutation P329G according to EU numbering.

[0121] In one embodiment, the antigen-binding moiety comprises at least one of the following heavy chain variable domains (VH): (a) The amino acid sequence of the heavy chain complementarity determining region (CDR H) of RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequence of EITPDSSTINYAPSLKG (SEQ ID NO: 2) or EITPDSSTINYTPSLKG (SEQ ID NO: 40); and (c) Contains the CDR H3 amino acid sequence of PYDYGAWFAS (SEQ ID NO: 3).

[0122] In one embodiment, the antigen-binding moiety comprises at least one of the following light chain variable domains (VL): (d) Light chain (CDR L) 1 amino acid sequence of RSSTGAVTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence of GTNKRAP (SEQ ID NO: 5); and (f) Contains the CDR L3 amino acid sequence of ALWYSNHWV (SEQ ID NO: 6).

[0123] In one preferred embodiment, the antigen-binding moiety comprises a heavy chain variable domain (VH) including the following: (a) The amino acid sequence of the heavy chain complementarity determining region (CDR H) of RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequence of EITPDSSTINYAPSLKG (SEQ ID NO: 2) or EITPDSSTINYTPSLKG (SEQ ID NO: 40); (c) CDR H3 amino acid sequence of PYDYGAWFAS (SEQ ID NO: 3); And light chain variable domains (VLs) including the following: (d) Light chain (CDR L) 1 amino acid sequence of RSSTGAVTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence of GTNKRAP (SEQ ID NO: 5); and (f) Contains the CDR L3 amino acid sequence of ALWYSNHWV (SEQ ID NO: 6).

[0124] In one particular embodiment, the antigen-binding moiety includes a heavy chain variable domain (VH) comprising: (a) The amino acid sequence of the heavy chain complementarity determining region (CDR H) of RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequence of EITPDSSTINYAPSLKG (Sequence ID 2): (c) CDR H3 amino acid sequence of PYDYGAWFAS (SEQ ID NO: 3); And light chain variable domains (VLs) including the following: (d) Light chain (CDR L) 1 amino acid sequence of RSSTGAVTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence of GTNKRAP (SEQ ID NO: 5); and (f) Contains the CDR L3 amino acid sequence of ALWYSNHWV (SEQ ID NO: 6).

[0125] In other specific embodiments, the antigen-binding moiety includes a heavy chain variable domain (VH) comprising: (a) The amino acid sequence of the heavy chain complementarity determining region (CDR H) of RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequence of EITPDSSTINYTPSLKG (SEQ ID NO: 40); (c) CDR H3 amino acid sequence of PYDYGAWFAS (SEQ ID NO: 3); And light chain variable domains (VLs) including the following: (d) Light chain (CDR L) 1 amino acid sequence of RSSTGAVTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence of GTNKRAP (SEQ ID NO: 5); and (f) Contains the CDR L3 amino acid sequence of ALWYSNHWV (SEQ ID NO: 6).

[0126] In one embodiment, the antigen-binding portion includes a heavy chain variable domain (VH) containing 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 NOs: 8, SEQ ID NOs: 41, and SEQ ID NOs: 44.

[0127] In one embodiment, the antigen-binding portion includes a heavy chain variable domain (VH) having 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: 8.

[0128] In one embodiment, the antigen-binding portion includes a heavy chain variable domain (VH) containing 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: 41.

[0129] In one embodiment, the antigen-binding portion includes a heavy chain variable domain (VH) having 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: 44.

[0130] In one embodiment, the antigen-binding portion includes a heavy chain variable domain (VH) having 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: 126.

[0131] In one embodiment, the antigen-binding portion includes a light chain variable domain (VL) having 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: 9.

[0132] In one embodiment, the antigen-binding portion includes a light chain variable domain (VL) having 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: 127.

[0133] In one embodiment, the antigen-binding portion includes a heavy chain variable domain (VH) containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8, and a light chain variable domain (VL) containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9.

[0134] In one embodiment, the antigen-binding portion includes a heavy chain variable domain (VH) containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41, and a light chain variable domain (VL) containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9.

[0135] In one embodiment, the antigen-binding portion includes a heavy chain variable domain (VH) containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44, and a light chain variable domain (VL) containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9.

[0136] In other embodiments, the antigen-binding portion includes a heavy chain variable domain (VH) containing 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: 126, and a light chain variable domain (VL) containing 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: 127.

[0137] In a preferred embodiment, the antigen-binding portion includes a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 8 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 9.

[0138] In another preferred embodiment, the antigen-binding moiety includes a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 126 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 127.

[0139] In one embodiment, the antigen-binding portion is scFv or scFab. In a preferred embodiment, the antigen-binding portion is scFv.

[0140] In one embodiment, the antigen-binding moiety comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), where the VH domain is linked to the VL domain, particularly via a peptide linker. In one embodiment, the C-terminus of the VL domain is linked to the N-terminus of the VH domain, particularly via a peptide linker. In a preferred embodiment, the C-terminus of the VH domain is linked to the N-terminus of the VL domain, particularly via a peptide linker. In one embodiment, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 16).

[0141] In one embodiment, the antigen-binding portion is a polypeptide scFv comprising a heavy chain variable domain (VH), a light chain variable domain (VL), and a linker, wherein the variable domain and the linker have one of the following configurations in the direction from the N-terminus to the C-terminus: a) VH-linker-VL, or b) VL-linker-VH. In a preferred embodiment, the scFv has the configuration VH-linker-VL.

[0142] In one embodiment, the antigen-binding portion includes 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: 10, SEQ ID NO: 122, and SEQ ID NO: 124.

[0143] In one embodiment, the antigen-binding portion includes 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: 10.

[0144] In one embodiment, the antigen-binding portion includes 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: 122.

[0145] In one embodiment, the antigen-binding portion includes 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: 124.

[0146] Antigen-binding moieties comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), such as scFv and scFab fragments as described herein, can be further stabilized by introducing an interchain disulfide crosslink between the VH and VL domains. Thus, in one embodiment, the scFv fragment(s) and / or scFab fragment(s) included in the antigen-binding receptor according to the present invention are further stabilized by the formation of an interchain disulfide bond via the insertion of a cysteine ​​residue (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering). In one embodiment, one of the VH and / or VL sequences provided above is provided, comprising at least one substitution of an amino acid by cysteine ​​(in particular, position 44 in the variable heavy chain and / or position 100 in the variable light chain according to Kabat numbering).

[0147] In one embodiment, the antigen-binding portion includes 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: 128.

[0148] Anchoring transmembrane domain (ATD) In the context of the present invention, the anchoring transmembrane domain of the antigen-binding receptor of the present invention may be characterized by the absence of a cleavage site for mammalian proteases. In the context of the present invention, protease refers to a proteolytic enzyme capable of hydrolyzing the amino acid sequence of a transmembrane domain containing a protease cleavage site. The term protease includes both endopeptidases and exopeptidases. In the context of the present invention, any anchoring transmembrane domain of a transmembrane protein, as defined in particular by CD nomenclature, may be used to construct the antigen-binding receptor of the present invention.

[0149] Therefore, in the context of the present invention, the anchoring transmembrane domain may include a portion of a mouse or preferably human transmembrane domain. An example of such an anchoring transmembrane domain is the transmembrane domain of CD8 having an amino acid sequence such as that shown in SEQ ID NO: 11 herein (as encoded by the DNA sequence shown in SEQ ID NO: 24). In the context of the present invention, the anchoring transmembrane domain of the antigen-binding receptor of the present invention may include / compose an amino acid sequence such as that shown in SEQ ID NO: 11 (as encoded by the DNA sequence shown in SEQ ID NO: 24).

[0150] In another embodiment, the antigen-binding receptor provided herein is an amino acid 153-179, 154-179, 155-179, 156-179, 157-179, 158-179, 159-179, 160-179, 161- of the human full-length CD28 protein as shown in SEQ ID NO: 61 (encoded by the cDNA sequence shown in SEQ ID NO: 70). It may also include transmembrane domains of CD28 located at 179, 162-179, 163-179, 164-179, 165-179, 166-179, 167-179, 168-179, 169-179, 170-179, 171-179, 172-179, 173-179, 174-179, 175-179, 176-179, 177-179, or 178-179.

[0151] Alternatively, any protein having a transmembrane domain, as given by CD nomenclature, may be used as the anchoring transmembrane domain of the antigen-binding receptor protein of the present invention.

[0152] In some embodiments, the anchoring transmembrane domain includes one transmembrane domain from the group consisting of CD27 (sequence number 59, encoded by sequence number 58), CD137 (sequence number 67, encoded by sequence number 66), OX40 (sequence number 71, encoded by sequence number 70), ICOS (sequence number 75, encoded by sequence number 74), DAP10 (sequence number 80, encoded by sequence number 79), DAP12 (sequence number 83, encoded by sequence number 82), CD3z (sequence number 88, encoded by sequence number 87), FCGR3A (sequence number 90, encoded by sequence number 91), NKG2D (sequence number 94, encoded by sequence number 95), CD8 (sequence number 119, encoded by sequence number 120), or a transmembrane fragment thereof that has the ability to anchor an antigen-binding receptor to the membrane.

[0153] Human sequences may be beneficial in the common context of the invention, for example, because a portion of the anchoring transmembrane domain may be accessible from the extracellular space and thus the patient's immune system. In preferred embodiments, the anchoring transmembrane domain comprises a human sequence. In such embodiments, the anchoring transmembrane domain includes one transmembrane domain from the group consisting of human CD27 (sequence number 57 encoded by sequence number 56), human CD137 (sequence number 65 encoded by sequence number 64), human OX40 (sequence number 69 encoded by sequence number 68), human ICOS (sequence number 73 encoded by sequence number 72), human DAP10 (sequence number 78 encoded by sequence number 77), human DAP12 (sequence number 81 encoded by sequence number 80), human CD3z (sequence number 86 encoded by sequence number 85), human FCGR3A (sequence number 88 encoded by sequence number 89), human NKG2D (sequence number 92 encoded by sequence number 93), and human CD8 (sequence number 117 encoded by sequence number 118), or a transmembrane fragment thereof that has the ability to anchor an antigen-binding receptor to the membrane.

[0154] Stimulus signaling domain (SSD) and co-stimulus signaling domain (CSD) Preferably, the antigen-binding receptor of the present invention comprises at least one stimulus signaling domain and / or at least one co-stimulus signaling domain. Therefore, the antigen-binding receptor provided herein preferably comprises a stimulus signaling domain which gives rise to T cell activation. The antigen-binding receptors provided herein may include a stimulus signaling domain that is mouse / rat or human CD3z (UniProt registration for human CD3z is the fragment / polypeptide portion of P20963 (version number 177 of SEQ ID NO: 2), UniProt registration for mouse / rat CD3z is P24161 (primary citation acceptance number) or version number 143 and Q9D3G3 (secondary citation acceptance number) of SEQ ID NO: 1)), FCGR3A (UniProt registration for human FCGR3A is P08637 (version number 178 of SEQ ID NO: 2)), or NKG2D (UniProt registration for human NKG2D is P26718 (version number 151 of SEQ ID NO: 1), UniProt registration for mouse / rat NKG2D is O54709 (version number 132 of SEQ ID NO: 2)).

[0155] Therefore, the stimulus signaling domain included in the antigen-binding receptors provided herein may be the full-length fragment / polypeptide portion of CD3z, FCGR3A, or NKG2D. The amino acid sequences of mouse full-length CD3z or NKG2D are shown herein as SEQ ID NOs. 86(CD3z), 90(FCGR3A), or 94(NKG2D) (mouse encoded by the DNA sequence shown in SEQ ID NOs. 87(CD3z), 91(FCGR3A), or 95(NKG2D)). The amino acid sequences of human full-length CD3z, FCGR3A, or NKG2D are shown herein as SEQ ID NOs. 84(CD3z), 88(FCGR3A), or 92(NKG2D) (human encoded by the DNA sequence shown in SEQ ID NOs. 85(CD3z), 89(FCGR3A), or 93(NKG2D)). The antigen-binding receptor of the present invention may include a fragment of CD3z, FCGR3A, or NKG2D as a stimulating domain, provided that it includes at least one signaling domain. In particular, any part / fragment of CD3z, FCGR3A, or NKG2D is suitable as a stimulating domain insofar as it includes at least one signaling source. However, more preferably, the antigen-binding receptor of the present invention includes a polypeptide of human origin. Accordingly, more preferably, the antigen-binding receptor provided herein includes an amino acid sequence shown herein as SEQ ID NO: 84 (CD3z), 88 (FCGR3A), or 92 (NKG2D) (human, encoded by the DNA sequence shown in SEQ ID NO: 85 (CD3z), 89 (FCGR3A), or 93 (NKG2D)). In one embodiment, the antigen-binding receptor of the present invention may include, or consist of, the amino acid sequence shown in SEQ ID NO: 13 (encoded by the DNA sequence shown in SEQ ID NO: 26).In further embodiments, the antigen-binding receptor includes a sequence such as that shown 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 compared to SEQ ID NO: 13, and having stimulus signaling activity. Specific configurations of antigen-binding receptors including a stimulus signaling domain (SSD) are given herein and in examples and drawings. Stimulus signaling activity can be determined, for example, by enhancing cytokine (IL-2, IFNγ, TNFα) release as measured by ELISA, enhancing proliferation activity (as measured by cell number enhancement), or enhancing lytic activity as measured by an LDH release assay.

[0156] Furthermore, the antigen-binding receptors provided herein preferably include at least one co-stimulated signaling domain that provides further activity to T cells. The antigen-binding receptors provided herein include mouse / rat or human CD28 (UniProt registration for human CD28 is P10747 (version number 173 of SEQ ID NO: 1), and UniProt registration for mouse / rat CD28 is P31041 (version number 134 of SEQ ID NO: 2)), CD137 (UniProt registration for human CD137 is Q07011 (version number 145 of SEQ ID NO: 1), and UniProt registration for mouse / rat CD137 is P20334 (version number 1 of SEQ ID NO: 1) John number 139), OX40 (UniProt registration for human OX40 is P23510 (version number 138 of SEQ ID NO: 1), UniProt registration for mouse OX40 is P43488 (version number 119 of SEQ ID NO: 1), ICOS (UniProt registration for human ICOS is Q9Y6W8 (version number 126 of SEQ ID NO: 1), UniProt registration for mouse ICOS is Q9WV40 (primary citation acceptance number) or Q9JL17 (secondary citation acceptance number) , version number 102 and sequence version 2)), CD27 (UniProt registration for human CD27 is P26842 (version number 160 of sequence number 2), UniProt registration for mouse CD27 is P41272 (version number 137 of sequence version 1)), 4-1-BB (UniProt registration for mouse 4-1-BB is P20334 (version number 140 of sequence version 1), UniProt registration for human 4-1-BB is Q07011 (sequence version Version number 146)), DAP10 (UniProt registration for human DAP10 is Q9UBJ5 (version number 25 of sequence number 1), UniProt registration for mouse DAP10 is Q9QUJ0 (primary citation acceptance number) or Q9R1E7 (secondary citation acceptance number), version number 101 and sequence number 1)) or DAP12 (UniProt registration for human DAP12 is O43914 (version number 146 and sequence number 1), UniProt registration for mouse DAP12 is,The co-stimulated signaling domain may be O054885 (Primary Citation Acceptance Number) or Q9R1E7 (Secondary Citation Acceptance Number), and may include a fragment / polypeptide portion of version number 123 and Sequence ID No. 1). In certain embodiments of the present invention, the antigen-binding receptor of the present invention may include one or more co-stimulated signaling domains as defined herein, i.e., 1, 2, 3, 4, 5, 6, or 7. Thus, in the context of the present invention, the antigen-binding receptor of the present invention may include a fragment / polypeptide portion of mouse or preferably human CD137 as the first co-stimulated signaling domain, and the second co-stimulated signaling domain is selected from the group consisting of mouse or preferably human CD27, CD28, CD137, OX40, ICOS, DAP10, and DAP12, or fragments thereof. Preferably, the antigen-binding receptor of the present invention includes a co-stimulated signaling domain of human origin. Therefore, more preferably, the co-stimulation signaling domain(s) included in the antigen-binding receptor of the present invention may include or consist of an amino acid sequence such as that shown in SEQ ID NO: 12 (as encoded by the DNA sequence shown in SEQ ID NO: 25).

[0157] Therefore, any co-stimulation signaling domains that may be optionally included in the antigen-binding receptors provided herein are fragment / polypeptide portions of the full-length CD27, CD28, CD137, OX40, ICOS, DAP10, or DAP12. The amino acid sequences of the mouse full-length CD27, CD28, CD137, OX40, ICOS, CD27, DAP10, and DAP12 are shown herein as SEQ ID NOs. 59 (CD27), 63 (CD28), 67 (CD137), 71 (OX40), 75 (ICOS), 79 (DAP10), or 83 (DAP12) (as encoded by the DNA sequences shown in SEQ ID NOs. 58 (CD27), 62 (CD28), 66 (CD137), 70 (OX40), 74 (ICOS), 78 (DAP10), or 82 (DAP12)). However, since human sequences are most preferred in the context of the present invention, the co-stimulation signaling domains that may be optionally included in the antigen-binding receptor proteins provided herein are fragment / polypeptide portions 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 shown herein as SEQ ID NOs. 57, (CD27), 61 (CD28), 65 (CD137), 69 (OX40), 73 (ICOS), 77 (DAP10), or 81 (DAP12) (human sequences encoded by the DNA sequences shown in SEQ ID NOs. 56 (CD27), 60 (CD28), 64 (CD137), 68 (OX40), 72 (ICOS), 76 (DAP10), or 80 (DAP12)).

[0158] In one preferred embodiment, the antigen-binding receptor includes CD28 or a fragment thereof as a co-stimulated signaling domain. The antigen-binding receptors provided herein may include a fragment of CD28 as a co-stimulated signaling domain, provided that at least one signaling domain of CD28 is included. In particular, any portion / fragment of CD28 is suitable for the antigen-binding receptor of the present invention, insofar as it includes at least one of the signaling causes of CD28. The co-stimulated signaling domains PYAP (AA208-211 of CD28) and YMNM (AA191-194 of CD28) are beneficial for the functions and functional effects of the CD28 polypeptide listed above. The amino acid sequence of the YMNM domain is shown in SEQ ID NO: 96, and the amino acid sequence of the PYAP domain is shown in SEQ ID NO: 97. Therefore, in the antigen-binding receptor of the present invention, the CD28 polypeptide preferably includes a sequence derived from the intracellular domain of the CD28 polypeptide having the sequence YMNM (SEQ ID NO: 96) and / or the sequence PYAP (SEQ ID NO: 97). In other embodiments, in the antigen-binding receptor of the present invention, one or both of these domains are mutated to FMNM (SEQ ID NO: 98) and / or AYAA (SEQ ID NO: 99), respectively. Any of these mutations can be advantageously used to extend the viability of transduced cells, and therefore the therapeutic potential, by reducing their ability to release cytokines without affecting the ability of transduced cells containing the antigen-binding receptor to proliferate. Alternatively, in other words, such non-functional mutations preferably enhance the persistence of cells transduced by the antigen-binding receptor provided herein, in vivo. However, the causes of these signaling pathways may reside at any site within the intracellular domain of the antigen-binding receptor provided herein.

[0159] In another preferred embodiment, the antigen-binding receptor includes CD137 or a fragment thereof as a co-stimulated signaling domain. The antigen-binding receptors provided herein may include a fragment of CD137 as a co-stimulated signaling domain, provided that at least one signaling domain of CD137 is included. In particular, any portion / fragment of CD137 is suitable for the antigen-binding receptor of the present invention, insofar as it includes at least one of the signaling causes of CD137. In one preferred embodiment, the CD137 polypeptide contained in the antigen-binding receptor protein of the present invention contains or comprises the amino acid sequence shown in SEQ ID NO: 12 (encoded by the DNA sequence shown in SEQ ID NO: 25).

[0160] The specific stereochemistry of antigen-binding receptors including a co-stimulated signaling domain (CSD) is given herein and in the examples and drawings. Co-stimulated signaling activity can be determined, for example, by enhancing cytokine (IL-2, IFNγ, TNFα) release as measured by ELISA, enhancing proliferation activity (as measured by cell number enhancement), or enhancing lytic activity as measured by LDH release assay. As described above, in one embodiment of the present invention, the co-stimulated signaling domain of the antigen-binding receptor may be derived from human CD28 and / or CD137 gene T cell activity, defined as cytokine production, proliferation, and lytic activity of transdextrinsic cells described herein, such as transdextrinsic T cells. The activity of CD28 and / or CD137 is assessed by cytokine release by ELISA, or by T cell proliferation measured by flow cytometry of cytokines such as interferon-gamma (IFN-γ) or interleukin-2 (IL-2), e.g., by ki67 measurement, cell quantification by flow cytometry, or by lytic activity measured by real-time impedance measurement 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 US A. 99(5)(2002), 2983-2988; Duwell et al., Cell Death It can be measured using ICELLligence instruments such as those described in Differ.21(12)(2014),1825-1837, Erratum in:Cell Death, Differ.21(12)(2014),161.

[0161] Linkers and signal peptides Furthermore, the antigen-binding receptors provided herein may include at least one linker (or "spacer"). The linker is typically a peptide having a length of up to 20 amino acids. Therefore, in the context of the present invention, the linker 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 include a linker between extracellular domains, each containing at least one antigen-binding moiety capable of specifically binding to a variant Fc domain, an anchoring transmembrane domain, a co-stimulus signaling domain, and / or a stimulus signaling domain. Furthermore, the antigen-binding receptors provided herein may include a linker between the antigen-binding moiety, particularly between the immunoglobulin domains of the antigen-binding moiety (e.g., between the VH and VL domains of scFv). Such linkers have the advantage of increasing the probability that different polypeptides of the antigen-binding receptor (i.e., an extracellular domain containing at least one antigen-binding moiety, an anchoring transmembrane domain, a co-stimulus signaling domain, and / or a stimulus signaling domain) fold independently and behave as expected. Therefore, in the context of the present invention, an extracellular domain containing at least one antigen-binding moiety, an anchoring transmembrane domain, a co-stimulus signaling domain, and a stimulus signaling domain may be included in a single-stranded multifunctional polypeptide. A single-stranded fusion construct may consist of, for example, a polypeptide(s) containing at least one extracellular domain (may include multiple), an anchoring transmembrane domain (may include multiple), a co-stimulus signaling domain (may include multiple), and / or a stimulus signaling domain (may include multiple). Therefore, the antigen-binding moiety, the anchoring transmembrane domain, the co-stimulus signaling domain, and the stimulus signaling domain may be linked by one or more identical or different peptide linkers described herein. For example, in the antigen-binding receptor provided herein, the linker between the extracellular domain containing at least one antigen-binding moiety and the anchoring transmembrane domain may include or consist of the amino acid sequence shown in SEQ ID NO: 17.In another embodiment, the linker between the antigen-binding moiety and the anchoring transmembrane domain comprises or consists of the amino and amino acid sequences shown in SEQ ID NO: 19. Thus, the anchoring transmembrane domain, the co-stimulation signaling domain and / or the stimulating domain may be linked to each other by a peptide linker or, alternatively, by direct fusion of the domains.

[0162] In a preferred embodiment of the present invention, the antigen-binding portion included in the extracellular domain is a single-stranded variable fragment (scFv), which is a fusion protein of variable domains of the heavy chain (VH) and light chain (VL) of an antibody, linked by a short-chain linker peptide of 10 to about 25 amino acids. The linker is usually 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. In a preferred embodiment, the linker ligates the N-terminus of the VL domain to the C-terminus of the VH domain. For example, in the antigen-binding receptor provided herein, the linker may have the amino acid sequence shown in SEQ ID NO: 16. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96).

[0163] In some embodiments of the present invention, the antigen-binding portion included in the extracellular domain is a single-chain Fab fragment or scFab which is a polypeptide comprising 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 domain and the linker have one of the following sequences from the N-terminus to the 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; and the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragment is stabilized by a native disulfide bond between the CL domain and the CH1 domain.

[0164] The antigen-binding receptors or parts thereof provided herein may include a signal peptide. Such a signal peptide will result in a protein on the surface of the T cell membrane. For example, in the antigen-binding receptors provided herein, the signal peptide may have an amino acid sequence such as that shown in SEQ ID NO: 100 (encoded by the DNA sequence shown in SEQ ID NO: 101).

[0165] T cell-activating antigen-binding receptor that can specifically bind to the mutant Fc domain. The antigen-binding receptor components described herein can fuse with each other in various configurations to produce T cell-activating antigen-binding receptors.

[0166] In some embodiments, the antigen-binding receptor comprises an extracellular domain consisting of a heavy chain variable domain (VH) and a light chain variable domain (VL) bound to an anchoring transmembrane domain. In preferred 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 stimulus signaling domain and / or a co-stimulus signaling domain. In specific such embodiments, the antigen-binding receptor essentially comprises a VH domain and a VL domain bound by one or more peptide linkers, an anchoring transmembrane domain, and optionally a stimulus signaling domain, wherein the VH domain is fused at its C-terminus to the N-terminus of the VL domain, and 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 stimulus signaling domain. Optionally, the antigen-binding receptor further comprises a co-stimulus signaling domain. In one such specific embodiment, the antigen-binding receptor essentially comprises a VH domain and a VL domain, an anchoring transmembrane domain, a stimulus signaling domain, and a co-stimulus signaling domain bound 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 stimulus signaling domain, and the stimulus signaling domain is fused at its C-terminus to the N-terminus of the co-stimulus signaling domain. In an alternative embodiment, the co-stimulus signaling domain binds to the anchoring transmembrane domain instead of the stimulus signaling domain.In a preferred embodiment, the antigen-binding receptor essentially comprises a VH domain and a VL domain, an anchoring transmembrane domain, a co-stimulus signaling domain, and a stimulus signaling domain bound 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 co-stimulus signaling domain, and the co-stimulus signaling domain is fused at its C-terminus to the N-terminus of the stimulus signaling domain.

[0167] The antigen-binding domain, anchoring transmembrane domain, and co-stimulation signaling domain and / or co-stimulation signaling domain may be fused directly with each other or via one or more peptide linkers comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n Peptide linker, (SG4) n Peptide linker, (G4S) n Peptide linker or G4 (SG4) n Peptide linkers are listed, where "n" is generally a number from 1 to 10, typically from 2 to 4. A preferred peptide linker for linking the antigen-binding moiety to the anchoring transmembrane domain is GGGGS(G4S) according to SEQ ID NO: 17. Another preferred peptide linker for linking the antigen-binding moiety to the anchoring transmembrane domain is KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(CD8stalk) according to SEQ ID NO: 19. An exemplary peptide linker suitable for linking the variable heavy chain domain (VH) to the variable light chain domain (VL) is GGGSGGGSGGGSGGGS(G4S)4 according to SEQ ID NO: 16.

[0168] In addition, the linker may include (part of) the immunoglobulin hinge region. In particular, when the antigen-binding moiety fuses to the N-terminus of the anchoring transmembrane domain, it can fuse with or without an additional peptide linker via the immunoglobulin hinge region or a part thereof.

[0169] As described herein, the antigen-binding receptor of the present invention comprises an extracellular domain containing at least one antigen-binding moiety. Antigen-binding receptors having a single antigen-binding moiety capable of specifically binding to a target cell antigen are particularly useful and 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, having an antigen-binding receptor containing two or more antigen-binding moieties specific to the target cell antigen is advantageous for optimizing targeting to the target site or for enabling crosslinking of the target cell antigen.

[0170] In one particular embodiment, the antigen-binding receptor includes a single antigen-binding moiety that can specifically bind to a mutant Fc domain containing the P329G mutation (according to EU numbering), particularly to the IgG1 Fc domain. In one embodiment, the antigen-binding moiety that can specifically bind to the mutant Fc domain but not to the non-mutant parental Fc domain is scFv.

[0171] In one embodiment, the antigen-binding portion is optionally fused at the C-terminus of the scFv fragment to the N-terminus of the anchoring transmembrane domain via a peptide linker. In one embodiment, the peptide linker contains the amino acid sequence KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 19). In one embodiment, the anchoring transmembrane domain is a transmembrane domain selected from the group consisting of CD8, CD4, 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 the CD8 transmembrane domain or a fragment thereof. In a specific embodiment, the anchoring transmembrane domain contains or consists of the amino acid sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 11). In one embodiment, the antigen-binding receptor further includes a co-stimulation signaling domain (CSD). In one embodiment, the anchoring transmembrane domain of the antigen-binding receptor is fused at its C-terminus to the N-terminus of the co-stimulation signaling domain. In one embodiment, the co-stimulation 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 herein. In a preferred embodiment, the co-stimulation signaling domain is the intracellular domain of CD28 or a fragment thereof. In a preferred embodiment, the co-stimulation signaling domain includes the intracellular domain of CD28 or a fragment thereof that carries CD28 signaling. In another preferred embodiment, the co-stimulation signaling domain includes the intracellular domain of CD137 or a fragment thereof that carries CD137 signaling. In a particular embodiment, the co-stimulation signaling domain includes or comprises SEQ ID NO: 12. In one embodiment, the antigen-binding receptor further includes a stimulation signaling domain.In one embodiment, the co-stimulation signaling domain of the antigen-binding receptor is fused at its C-terminus to the N-terminus of the stimulation signaling domain. In one embodiment, at least one stimulation signaling domain is individually selected from the group consisting of intracellular domains of CD3z, FCGR3A, and NKG2D, or fragments thereof. In a preferred embodiment, the co-stimulation signaling domain is the intracellular domain of CD3z or a fragment thereof that carries CD3z signaling. In a particular embodiment, the co-stimulation signaling domain includes or comprises SEQ ID NO: 13.

[0172] 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 its C-terminus to the N-terminus of eGFP (enhanced green fluorescent protein) via an optionally specified peptide linker. In a preferred embodiment, the peptide linker is GEGRGSLLTCGDVEENPGP(T2A) according to SEQ ID NO: 18.

[0173] In a particular embodiment, the antigen-binding receptor comprises an anchoring transmembrane domain and an extracellular domain containing at least one antigen-binding moiety, the at least one antigen-binding moiety being an scFv that can specifically bind to a mutant Fc domain but not to a non-mutant parental Fc domain, and the mutant Fc domain contains the P329G mutation (according to EU numbering). 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 co-stimulus signaling domain (CSD), and a stimulus signaling domain (SSD). In such an embodiment, the antigen-binding receptor has the configuration scFv-ATD-CSD-SSD. In a preferred embodiment, the antigen-binding receptor has the configuration VH-VL-ATD-CSD-SSD. In a more specific embodiment, the antigen-binding receptor has the configuration VH-linker-VL-linker-ATD-CSD-SSD.

[0174] In one particular embodiment, the antigen-binding moiety is an scFv that can specifically bind to a mutant Fc domain containing the P329G mutation, and the antigen-binding moiety comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs: 1, 2, and 3, and at least one light chain CDR selected from the group consisting of SEQ ID NOs: 4, 5, and 6.

[0175] In another specific embodiment, the antigen-binding moiety is an scFv that can specifically bind to a mutant Fc domain containing the P329G mutation, and the antigen-binding moiety comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 40, and SEQ ID NO: 3, and at least one light chain CDR selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0176] In a preferred embodiment, the antigen-binding moiety is an scFv that can specifically bind to a mutant Fc domain containing the P329G mutation, and the antigen-binding moiety includes the complementarity-determining region (CDRH) 1 amino acid sequence RYWMN (SEQ ID NO: 1), the CDRH2 amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO: 2), the CDRH3 amino acid sequence PYDYGAWFAS (SEQ ID NO: 3), the light chain complementarity-determining region (CDRL) 1 amino acid sequence RSSTGAVTSNYAN (SEQ ID NO: 4), the CDRL2 amino acid sequence GTNKRAP (SEQ ID NO: 5), and the CDRL3 amino acid sequence ALWYSNHWV (SEQ ID NO: 6).

[0177] In one embodiment, the present invention provides an antigen-binding receptor comprising the following, in order from the N-terminus to the C-terminus: (i) Heavy chain variable domain (VH) including heavy chain complementarity determining region (CDR)1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 2, and heavy chain CDR3 of SEQ ID NO: 3, (ii) Peptide linkers, especially the peptide linker of SEQ ID NO: 16, (iii) Light chain variable domain (VL) including light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, and light chain CDR3 of SEQ ID NO: 6, (iv) Peptide linkers, especially the peptide linker of SEQ ID NO: 19, (v) Anchoring transmembrane domains, particularly the anchoring transmembrane domain of Sequence ID No. 11, (vi) Co-stimulus signaling domains, particularly the co-stimulus signaling domain of Sequence ID No. 12, and (vii) Stimulus signaling domains, particularly the stimulus signaling domain of SEQ ID NO: 13.

[0178] In one embodiment, the present invention provides an antigen-binding receptor comprising the following, in order from the N-terminus to the C-terminus: (i) Heavy chain variable domain (VH) including heavy chain complementarity determining region (CDR)1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 40, and heavy chain CDR3 of SEQ ID NO: 3, (ii) Peptide linkers, especially the peptide linker of SEQ ID NO: 16, (iii) Light chain variable domain (VL) including light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, and light chain CDR3 of SEQ ID NO: 6, (iv) Peptide linkers, especially the peptide linker of SEQ ID NO: 19, (v) Anchoring transmembrane domains, particularly the anchoring transmembrane domain of Sequence ID No. 11, (vi) Co-stimulus signaling domains, particularly the co-stimulus signaling domain of SEQ ID NO: 12, and (vii) Stimulus signaling domains, particularly the stimulus signaling domain of SEQ ID NO: 13.

[0179] In one embodiment, the present invention provides an antigen-binding receptor comprising the following, in order from the N-terminus to the C-terminus: (i) Heavy chain variable domain (VH), (ii) Peptide linkers, especially the peptide linker of SEQ ID NO: 16, (iii) A light chain variable domain (VL) which is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9, The VH and VL domains can form antigen-binding moieties that bind to the Fc domain containing the amino acid mutation P329G according to EU numbering. (iv) Peptide linkers, especially the peptide linker of SEQ ID NO: 19, (v) Anchoring transmembrane domains, in particular, anchoring transmembrane domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11, (vi) Co-stimulation signaling domains, in particular co-stimulation signaling domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12, and (vii) Stimulus signaling domains, in particular, stimulus signaling domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13.

[0180] In one embodiment, the present invention provides an antigen-binding receptor comprising the following, in order from the N-terminus to the C-terminus: (i) A heavy chain variable domain (VH) which is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8, (ii) Peptide linkers, especially the peptide linker of SEQ ID NO: 16, (iii) A light chain variable domain (VL) which is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9, (iv) Peptide linkers, especially the peptide linker of SEQ ID NO: 19, (v) Anchoring transmembrane domains, in particular, anchoring transmembrane domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11, (vi) Co-stimulation signaling domains, in particular co-stimulation signaling domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12, and (vii) Stimulus signaling domains, in particular, stimulus signaling domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13.

[0181] In one embodiment, the present invention provides an antigen-binding receptor comprising the following, in order from the N-terminus to the C-terminus: (i) A heavy chain variable domain (VH) which is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41, (ii) Peptide linkers, especially the peptide linker of SEQ ID NO: 16, (iii) A light chain variable domain (VL) which is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9, (iv) Peptide linkers, especially the peptide linker of SEQ ID NO: 19, (v) Anchoring transmembrane domains, in particular, anchoring transmembrane domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11, (vi) Co-stimulation signaling domains, in particular co-stimulation signaling domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12, and (vii) Stimulus signaling domains, in particular, stimulus signaling domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13.

[0182] In one embodiment, the present invention provides an antigen-binding receptor comprising the following, in order from the N-terminus to the C-terminus: (i) A heavy chain variable domain (VH) which is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44, (ii) Peptide linkers, especially the peptide linker of SEQ ID NO: 16, (iii) A light chain variable domain (VL) which is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9, (iv) Peptide linkers, especially the peptide linker of SEQ ID NO: 19, (v) Anchoring transmembrane domains, in particular, anchoring transmembrane domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11, (vi) Co-stimulation signaling domains, in particular co-stimulation signaling domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12, and (vii) Stimulus signaling domains, in particular, stimulus signaling domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13.

[0183] In one embodiment, the present invention provides an antigen-binding receptor comprising the following, in order from the N-terminus to the C-terminus: (i) A heavy chain variable domain (VH) which is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 126, (ii) Peptide linkers, especially the peptide linker of SEQ ID NO: 16, (iii) A light chain variable domain (VL) which is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 127, (iv) Peptide linkers, especially the peptide linker of SEQ ID NO: 19, (v) Anchoring transmembrane domains, in particular, anchoring transmembrane domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11, (vi) Co-stimulation signaling domains, in particular co-stimulation signaling domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12, and (vii) Stimulus signaling domains, in particular, stimulus signaling domains that are at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13.

[0184] In one embodiment, an antigen-binding receptor is provided that includes an amino acid sequence which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7.

[0185] In one embodiment, an antigen-binding receptor is provided that includes an amino acid sequence which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 121.

[0186] In one embodiment, an antigen-binding receptor is provided that includes an amino acid sequence which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 123.

[0187] In one embodiment, an antigen-binding receptor is provided that includes an amino acid sequence which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 125.

[0188] 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 its C-terminus to the N-terminus of eGFP (enhanced green fluorescent protein) via an optionally described peptide linker. In a preferred embodiment, the peptide linker is GEGRGSLLTCGDVEENPGP(T2A) of SEQ ID NO: 18.

[0189] Transduced cells capable of expressing the antigen-binding receptor of the present invention A further aspect of the present invention is a transduced T cell capable of expressing the antigen-binding receptor of the present invention. The antigen-binding receptor described herein relates to a molecule that is not naturally present in and / or on the surface of T cells and is not (endogenously) expressed in or on normal (untransformed) T cells. Therefore, the antigen-binding receptor of the present invention in and / or on T cells is artificially introduced into T cells. In the context of the present invention, the T cells, preferably CD8+ T cells, may be isolated / obtained from a subject to be treated as defined herein. Therefore, the antigen-binding receptor described herein, artificially introduced into and / or on the surface of the T cells and subsequently presented herein, comprises a domain containing one or more antigen-binding moieties accessible (in vitro or in vivo) to an (Ig-derived) immunoglobulin, preferably an antibody, particularly the Fc domain of the antibody. In the context of the present invention, these artificially introduced molecules are presented in and / or on the surface of the T cells after transduction (retroviral, lentiviral, or nonviral) as described below herein. Therefore, after transduction, the T cells according to the present invention can be activated in the presence of target cells by immunoglobulin, preferably an antibody (therapeutic) containing a mutation specific to the Fc domain as described herein.

[0190] The present invention also relates to transdextrinsed T cells expressing an antigen-binding receptor encoded by a nucleic acid molecule(s) encoding the antigen-binding receptor of the present invention. Therefore, in the context of the present invention, transdextrinsed cells may include a nucleic acid molecule encoding the antigen-binding receptor of the present invention or a vector of the present invention expressing the antigen-binding receptor of the present invention.

[0191] In the context of the present invention, the term “transformed T cell” refers to a genetically modified T cell (i.e., a T cell into which a nucleic acid molecule has been intentionally introduced). Transduced T cells provided herein may include the vector of the present invention. Preferably, transduced T cells provided herein include a nucleic acid molecule encoding the antigen-binding receptor of the present invention and / or the vector of the present invention. 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, the nucleic acid molecule encoding the antigen-binding receptor of the present invention can be stably incorporated into the genome of a T cell by retroviral or lentiviral transformation. Transient expression of the nucleic acid molecule encoding the antigen-binding receptor of the present invention can be achieved by mRNA transfection. Preferably, transduced T cells provided herein are genetically modified by introducing a nucleic acid molecule into a T cell via a viral vector (e.g., a retroviral vector or a lentiviral vector). Thus, the expression of the antigen-binding receptor may be constitutive, and the extracellular domain of the antigen-binding receptor may be detectable on the cell surface. This extracellular domain of the antigen-binding receptor may include, but may not include, the entire extracellular domain of the antigen-binding receptor as defined herein. The minimum size required is the antigen-binding site of the antigen-binding portion of the antigen-binding receptor.

[0192] When antigen-binding receptors are introduced into T cells under the control of an inductive or repressive promoter, expression may be conditional or inductive. Examples of such inductive or repressive promoters may include transcription systems containing the alcohol dehydrogenase I (alcA) gene promoter and the transactivating protein AlcR. Different agricultural alcohol-based formulations are used to control the expression of the target gene linked to the alcA promoter. Furthermore, tetracycline-responsive promoter systems can function to activate or repress gene expression systems in the presence of tetracycline. Some elements of the system include tetracycline repressor protein (TetR), tetracycline operator sequence (tetO), and tetracycline transcription activator fusion protein (tTA), which is a fusion of TetR and herpes simplex virus protein 16 (VP16) activating sequence. In addition, steroid-responsive promoters, metal-controlled, or pathogenicity-associated (PR) protein-associated promoters can be used.

[0193] Expression may be constitutive or constitutional, depending on the system used. The antigen-binding receptor of the present invention can be expressed on the surface of 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 co-stimulus signaling domain(s) and stimulus signaling domain(s)) cannot be detected on the cell surface. Detection of the extracellular domain of the antigen-binding receptor can be performed using an antibody that specifically binds to this extracellular domain, or by a mutant Fc domain to which the extracellular domain can bind. The extracellular domain can be detected by flow cytometry or microscopy using these antibodies or Fc domains.

[0194] Other cells can also be transduced with the antigen-binding receptor of the present invention and thereby directed toward target cells. These further cells include, but are not limited to, B cells, natural killer (NK) cells, innate lymphoid cells, macrophages, monocytes, dendritic cells, or neutrophils. Preferably, the immune cells are lymphocytes. Triggering the antigen-binding receptor of the present invention on the surface of leukocytes, in combination with a therapeutic antibody containing a mutant Fc domain, regardless of the cell lineage from which the cells originate, makes the cells cytotoxic to target cells. Cytotoxicity is intended to occur independently of selected or co-stimulated signaling domains for the antigen-binding receptor and does not rely on the exogenous supply of additional cytokines. Therefore, transduced cells of the present invention may be, for example, CD4+ T cells, CD8+ T cells, γδ T cells, natural killer (NK) T cells, natural killer (NK) cells, tumor-infiltrating lymphocytes (TILs), myeloid cells, or mesenchymal stem cells. Preferably, the transduced cells provided herein are T cells (e.g., autologous T cells), and 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 may be allogeneic cells, for example, allogeneic CD8+ T cells. The term allogeneic refers to cells derived from an unrelated donor individual / subject whose human leukocyte antigen (HLA) is compatible with the individual / subject treated with the antigen-binding receptor expressing transduced cells described herein, for example. Autologous cells refer to cells isolated / obtained as described above from a subject treated with the transduced cells described herein.

[0195] Transduced cells of the present invention can be simultaneously transduced with further nucleic acid molecules, such as nucleic acid molecules encoding cytokines.

[0196] The present invention also relates to a method for producing transduced T cells expressing the antigen-binding receptor of the present invention, comprising the steps of transducing the vector of the present invention into T cells, culturing the transduced T cells under conditions that express the antigen-binding receptor in or on the transduced cells, and recovering the transduced T cells.

[0197] In the context of the present invention, the transduced cells of the present invention are preferably produced by isolating cells (e.g., T cells, preferably CD8+ T cells) from a subject (preferably a human patient). Methods for isolating / obtaining cells (e.g., T cells, preferably CD8+ T cells) from a patient or donor are well known in the art, and for example, in the context of the cells (e.g., T cells, preferably CD8+ T cells) of the present invention from a patient or donor, the cells may be isolated by blood collection or bone marrow removal. After isolating / obtaining cells as a patient sample, the cells (e.g., T cells) are separated from other components of the sample. Several methods for separating cells (e.g., T cells) from a sample are known and not limited to, but include, for example, leukocyte apheresis for obtaining cells from a peripheral blood sample from a patient or donor, and isolating / obtaining cells using a FACS cell sorting device. The isolated / obtained T cells are then cultured and expanded, for example, by using anti-CD3 antibodies, by using anti-CD3 and anti-CD28 monoclonal antibodies, and / or by using anti-CD3 antibodies, anti-CD28 antibodies, and interleukin-2 (IL-2) (see, e.g., Dudley, Immunother. 26 (2003), 332-342 or Dudley, Clin. Oncol. 26 (2008), 5233-5239).

[0198] In subsequent steps, 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, when transducing nucleic acids or recombinant nucleic acids, include, but are not limited to, electroporation, calcium phosphate, cationic lipid, or liposome methods. The nucleic acids to be transduced can be transduced efficiently by conventional methods using commercially available transfection reagents, such as lipofectamine (Invitrogen, catalog number: 11668027). When using vectors, the vectors can be transduced in the same way as the nucleic acids described above, as long as they are plasmid vectors (i.e., vectors that are not viral vectors). In the context of the present invention, methods for transfecting cells (e.g., T cells) include retroviral or lentiviral T cell transfection, non-viral vectors (e.g., sleeping beauty minicircle vectors), and mRNA transfection. "MRNA transfection" in this example refers to a method well known to those skilled in the art for transiently expressing a target protein, such as the antigen-binding receptor of the present invention, in transfected 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.), and then cultured according to the standard cell (e.g., T cell) culture protocol described above (see Zhao et al., Mol Ther. 13(1)(2006), 151-159). Transfected cells of the present invention can be produced by lentiviral or, most preferably, retroviral transfection.

[0199] In this context, retroviral vectors suitable for transduction into cells are known in the art, for example, 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., Proc.Natl.Acad.Sci.USA 87 (1990), 473-477), LNL6 (Tiberghien et al., Blood 84 (1994), 1333-1341), pZipNEO (Chen et al., J. Immunol. 153 (1994), 3630-3638), LASN (Mullen et al., Hum. Gene Ther.7(1996),1123-1129),pG1XsNa(Taylor et 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),and LXSN(Sun et 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. al.,Cancer Immunol.Immunother.46(1998),61-66),pSTITCH(Weitjens et al.,Gene Ther 5(1998),1195-1203),pLZR(Yang et 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. al., J. Immunol. 174(2005), 4415-4423), or pMX(de Witte et al., J.Immunol. 181 (2008), 5128-5136. In the context of the present invention, suitable lentiviral vectors for transduction into cells (e.g., T cells) include, for example, 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 Catalogue No.: 22036), FUGW (Lois et al., Science 295(5556)(2002), 868-872), pLVX-EF1 (Addgene Catalogue No. 64368), and 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 Catalogue No.: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 This is pLEX (Addgene catalog number 27976).

[0200] The transformed cells of the present invention are preferably grown outside their natural environment under uncontrolled conditions. In particular, the term “culture” means growing cells (e.g., transdextrins of the present invention) derived from a multicellular eukaryote (preferably from a human patient) in vitro. Cell culture is an experimental technique that keeps cells alive that have been isolated from a natural tissue source. Hereinafter, the transformed cells of the present invention are cultured in or on the transformed cells under conditions that enable the expression of the antigen-binding receptor of the present invention. Conditions that enable the expression or transgene (i.e., of 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 the expression of the antigen-binding receptor of the present invention in cultured transduced cells (e.g., CD8+T), the transduced cells are harvested (i.e., re-extracted) from the culture (i.e., from the culture medium).

[0201] Therefore, transduced cells expressing antigen-binding receptors encoded by the nucleic acid molecules of the present invention, which can be obtained by the method of the present invention, preferably T cells, particularly CD8+ T cells, are also included in the present invention.

[0202] nucleic acid molecule Further embodiments of the present invention are nucleic acids and vectors encoding one or more antigen-binding receptors of the present invention. An exemplary nucleic acid molecule encoding an antigen-binding receptor of the present invention is shown in SEQ ID NO: 20. The nucleic acid molecule of the present invention may be under the control of a regulatory sequence. For example, a promoter, transcriptional enhancer, and / or sequence that enables induction of expression of the antigen-binding receptor of the present invention may be employed. In the context of the present invention, the nucleic acid molecule is expressed under the control of a constituent promoter or an inducible promoter. Appropriate 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), and the ACT5C promoter (Qin et al.). These include the TRE promoter (Qin et al., PLoS One 5(5)(2010), e10611), the Oct3 / 4 promoter, S367-S367 (Chang et al., Molecular Therapy 9(2004), S367-S367 (doi:10.1016 / j.ymthe.2004.06.904)), or the Nanog promoter (Wu et al., Cell Res. 15(5)(2005), 317-24). Accordingly, the present invention also relates to vectors(may) comprising the nucleic acid molecule(s) described in the present invention. In this specification, the term vector refers to a cyclic or linear nucleic acid molecule that can self-replicate in the cells into which it is introduced.Many suitable vectors, the selection of which will depend on the desired function, are known to those skilled in the art in molecular biology and include plasmids, cosmids, viruses, bacteriophages, and other vectors conventionally used in genetic engineering. Methods known to those skilled in the art can be used to construct various plasmid vectors; see, for example, the techniques described in Sambrook et al. (loc cit.) 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. Individual sequences of DNA were isolated using cloning vectors, as will be discussed in more detail below. The relevant sequences can be transferred into expression vectors that require the expression of a specific polypeptide. Typical cloning vectors include pBluescript SK, pGEM, pUC9, pBR322, pGA18, and pGBT9. Typical expression vectors include pTRE, pCAL-n-EK, pESP-1, and pOP13CAT.

[0203] The present invention also relates to vectors comprising nucleic acid molecules(or more) which are regulatory sequences operably ligated to nucleic acid molecules(or more) encoding antigen-binding receptors as defined herein. In the context of the present invention, the vector may be polycistronic. Such regulatory sequences (control elements) are known to those skilled in the art and may include promoters, splice cassettes, translation start codons, and translation and insertion sites for introducing inserts into the vector(or more). In the context of the present invention, nucleic acid molecules(or more) are ligated to act on expression control sequences that enable expression in eukaryotic or prokaryotic cells. The vector(or more) is assumed to be an expression vector(or more) comprising nucleic acid molecules(or more) encoding antigen-binding receptors as defined herein. "Operatably ligated" means that the components described in this way are in a relationship that allows them to function in the intended manner. The regulatory sequence operably ligated to the coding sequence is ligated in such a way that the expression of the coding sequence is achieved under conditions compatible with the regulatory sequence. When the regulatory sequence is a promoter, it is obvious to those skilled in the art that double-stranded nucleic acids are preferably used.

[0204] In the context of the present invention, the listed vectors are expression vectors. An expression vector is a construct that can be used to transform selected cells and prepares for the expression of a coding sequence in the selected cells. Expression vectors may be, for example, the cloning of vectors, binary vectors, or embedded vectors. Expression involves transcription from a nucleic acid molecule into preferably translatable mRNA. Regulatory elements that ensure expression in prokaryotic and / or eukaryotic cells are well known to those skilled in the art. In eukaryotic cells, the cell typically includes a promoter that ensures the initiation of transcription and, optionally, a poly(A) signal that ensures the termination of transcription and the stabilization of the transcript. Possible regulatory elements that enable expression in prokaryotic host cells include, for example, the PL promoter, lac promoter, trp promoter, or tac promoter in E. coli, while examples of regulatory elements that enable expression in eukaryotic host cells include the AOX1 promoter or GAL1 promoter in yeast, or the CMV promoter, SV40 promoter, RSV promoter (Roussarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian cells and other animal cells.

[0205] In addition to elements that initiate transcription, such regulatory elements may also include transcription termination signals downstream of the polynucleotide, such as the SV40-polyA site or the tk-polyA site. Furthermore, depending on the expression system used, signal peptides encoding the leader sequence, which can direct polypeptides into cell compartments or secrete polypeptides into the culture medium, may be added to the coding sequences of the enumerated nucleic acid sequences, as is well known in the art, and see, for example, the attached examples.

[0206] The leader sequence(s) polymerize in a suitable phase with the translation sequence, start sequence and stop sequence, and preferably a leader sequence that can direct the secretion of the translated protein or portion thereof into the pericellular lumen or extracellular culture medium. Optionally, heterogeneous sequences can encode an antigen-binding receptor containing an N-terminal specific peptide that confers a desired feature, e.g., stabilization or simplified purification of the expressed recombinant product. See above. In this context, suitable expression vectors known in the art include Okayama-Berg cDNA expression vector 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).

[0207] In the context of the present invention, the expression regulatory sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic cells, although regulatory sequences for prokaryotic cells may also be used. Once the vector is incorporated into suitable cells, the cells are maintained, as desired, under conditions suitable for high-level expression of the nucleotide sequence. Further regulatory elements may include transcriptional enhancers and translational enhancers. Advantageously, the vectors described above in the present invention include selectable markers and / or scoring markers. Selective marker genes useful for selecting transformed cells, such as plant tissues and plants, are well known to those skilled in the art, and include, for example, dhfr (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149) which confers resistance to methotrexate, npt (Herrera-Estrella, EMBO J. 2 (1983), 987-995) which confers resistance to the aminoglycosides neomycin, kanamycin, and paramycin, and hygro (Marsh, Gene 32 (1984), 481-485) which confers resistance to hygromycin, as a basis for selection of antimetabolite resistance. Further selectable genes have been described, namely trpB, which enables cells to utilize indole instead of tryptophan; hisD, which enables cells to utilize histinol instead of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate, which enables cells to utilize mannose (International Publication No. 94 / 20627); and ornithine decarboxylase inhibitors, such as ODC (ornithine decarboxylase) (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.) which confers resistance to 2-(difluoromethyl)-DL-ornithine and DFMO, or Aspergillus tereus-derived deaminase (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338).

[0208] Useful scoring markers are also known to those skilled in the art and are commercially available. Advantageously, these markers are genes encoding 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 listed vectors.

[0209] As described above, the enumerated nucleic acid molecules(s) can be used alone or as part of a vector(s) to express the antigen-binding receptors of the present invention in cells, for example, not only for adoptive T-cell therapy but also for gene therapy purposes. A nucleic acid molecule or vector(s) containing a DNA sequence(s) encoding any one of the antigen-binding receptors described herein is introduced into a cell, and the target polypeptide is produced. Gene therapy based on introducing therapeutic genes into cells by ex vivo or in vivo technology is one of the most important applications of gene transfer. Appropriate vectors, methods, or gene delivery systems for in vitro or in vivo gene therapy are 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 See Ther. 5 (1998), 692-699; Nabel, Ann. NYAcad. Sci. 811 (1997), 289-292; Verzeletti, Hum. Gene 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 listed nucleic acid molecules and vectors may be designed for direct introduction or for introduction into cells via liposomes or viral vectors (e.g., adenovirus, retrovirus).In the context of the present invention, the cell is a T cell, such as a CD8+ T cell, CD4+ T cell, CD3+ T cell, γδ T cell, or natural killer (NK) T cell, preferably a CD8+ T cell.

[0210] Accordingly, the present invention relates to a method for inducing vectors, particularly plasmids, cosmids, and bacteriophages, that have been conventionally used in genetic engineering, comprising nucleic acid molecules encoding polypeptide sequences of antigen-binding receptors as defined herein. In the context of the present invention, such vectors are expression vectors and / or gene transfer or targeting vectors. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpesviruses, or bovine papillomaviruses may be used to deliver the listed polynucleotides or vectors to a target cell population.

[0211] Recombinant vectors(s) can be constructed using methods well known to those skilled in the art. For example, see Sambrook et al. (loc cit.), Ausubel (1989, loc cit.), or other standard textbooks. Alternatively, the enumerated nucleic acid molecules and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing the nucleic acid molecules of the present invention can be transferred into host cells by different well-known methods depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation may be used for other cell hosts. See Sambrook above. The enumerated vectors may, among others, be 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.

[0212] The present invention also provides T cells transduced with the vectors described herein. These T cells can be produced by introducing at least one of the vectors or at least one of the nucleic acid molecules into T cells or their progenitor cells. The presence of the at least one vector or at least one nucleic acid molecule in the T cells mediates the expression of a gene encoding the antigen-binding receptor, which includes an extracellular domain containing an antigen-binding moiety capable of specifically binding to a mutant Fc domain. The vectors of the present invention may be polycistronic.

[0213] The described nucleic acid molecules or vectors introduced into T cells or their precursor cells can be incorporated into the cell's genome or maintained outside the chromosome.

[0214] target cell antigen As described above, the (Ig-derived) domain(s) of the antibodies described herein, which include a mutant Fc domain, particularly an Fc domain containing the amino acid mutation P329G (according to EU numbering), include an antigen interaction site having specificity for target cell surface molecules, such as tumor-specific antigens naturally present on the surface of tumor cells. In the context of the present invention, such an antibody brings transduced T cells described herein, which include the antigen-binding receptor of the present invention, into physical contact with target cells (e.g., tumor cells), and the transduced T cells are activated. The activation of the transduced T cells of the present invention preferentially results in the lysis of the target cells described herein.

[0215] Examples of target cell antigens (e.g., tumor markers) naturally occurring on the surface of target (e.g., tumor) cells are listed below herein, but are not limited to: FAP (fibroblast-activating 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, C Peptides bound to molecules of D33, CD38, CD52Flt3, folate receptor 1 (FOLR1), human trophoblast cell surface antigen 2 (Trop-2), cancer antigen 12-5 (CA-12-5), human leukocyte 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), carbon anhydrase IX (CA-IX), and / or human major histocompatibility complex (MHC).

[0216] Therefore, in the context of the present invention, the antigen-binding receptor described herein is an Fc domain containing the amino acid mutation P329G (according to EU numbering), namely FAP (fibroblast-activating 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, CD52Flt3, folate receptor 1 (FOLR1), The therapeutic antibody binds to antigens / markers naturally occurring on the surface of tumor cells, selected from the group consisting of human trophoblast cell surface antigen 2 (Trop-2), cancer antigen 12-5 (CA-12-5), human leukocyte 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), carbon anhydrase IX (CA-IX), and / or peptides bound to molecules of the human major histocompatibility complex (MHC).

[0217] A33 antigen, BCMA (B cell maturation antigen), cancer antigen 12-5 (CA-12-5), carbon anhydrase IX (CA-IX), CD19, CD20, CD22, CD33, CD38, CEA (carcinoembryonic antigen), EpCAM (epidermal cell adhesion molecule), FAP (fibroblast activating 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 the (human) members of (melanochondroitin sulfate proteoglycan), MUC-1 (mucin-1), PSMA (prostate-specific membrane), 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) are available in the UniProtKB / Swiss-Prot database and can be searched at http: / / www.uniprot.org / uniprot / ?query=reviewed%3Ayes. These (protein) sequences are also related to annotated modified sequences. The present invention also provides techniques and methods using homologous sequences and allele variants of the concise sequences provided herein. Preferably, such variants of the concise sequences provided herein are used. Preferably, such variants are genetic variants. Those skilled in the art can easily estimate 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(s) of (human) FAP (fibroblast-activating protein) can be obtained from Swiss-Prot database entry Q12884 (entry version 168, sequence version 5); the sequence(s) of (human) CEA (carcinoembryonic antigen) can be obtained from Swiss-Prot database entry P06731 (entry version 171, sequence version 3);The sequence(s)(multiple) of (human) EpCAM (epithelial cell adhesion molecule) can be obtained from Swiss-Prot database entry P16422 (entry version 117, sequence version 2); the sequence(s)(multiple) of (human) MSLN (mesothelin) can be obtained from UniProt entry number Q13421 (version number 132; sequence version 2); the sequence(s) of (human) FMS-like tyrosine kinase 3 (FLT-3) The sequence(s) can be obtained from Swiss-Prot database entry P36888 (primary citation accession number) or Q13414 (secondary accession number) in version 165 and sequence version 2; the sequence of (human) MCSP (melanoma chondroitin sulfate proteoglycan) can be obtained from UniProt entry number Q6UVK1 (version 118; sequence version 2); the sequence(s) can be obtained from (human) folate receptor 1 (FOLR1) in version 153 and sequence version 3 from UniProt entry number P15328 (primary citation accession number) or Q53EW2 (secondary accession number); the sequence(s) can be obtained from UniProt entry number P09758 (primary citation accession number) The sequence(s) of (human) PSCA (prostate stem cell antigen) can be obtained from UniProt entry number O43653 (primary citation accession number) or Q6UW92 (secondary accession number) in version 172 and sequence version 3; the sequence(s) of (human) HER-1 (epidermal growth factor receptor) can be obtained from Swiss-Prot database entry P00533 (entry version 177, sequence version 2); the sequence(s) 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(s)(multiple) for (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(s)(multiple) for (human) CD20 (B-lymphocyte antigen CD20) can be obtained from Swiss-Prot database entry P11836 (entry version 117, sequence version 1); the sequence(s)(multiple) for (human) CD22 (B-lymphocyte antigen CD22) can be obtained from Swiss-Prot database entry P20273 (entry version 135, sequence version 2); the sequence(s)(multiple) for (human) CD33 (B-lymphocyte antigen CD33) The sequence(s) can be obtained from Swiss-Prot database entry P20138 (entry version 129, sequence version 2); the sequence(s) of (human) CA-12-5 (mucin 16) can be obtained from Swiss-Prot database entry Q8WXI7 (entry version 66, sequence version 2); the sequence(s) of (human) HLA-DR The sequence(s) can be obtained from Swiss-Prot database entry Q29900 (entry version 59, sequence version 1); the sequence(s) of (human) MUC-1 (mucin-1) can be obtained from Swiss-Prot database entry P15941 (entry version 135, sequence version 3); the sequence(s) of (human) A33 (cell surface A33 antigen) can be obtained from Swiss-Prot database entry Q99795 (entry version 104, sequence version 1); (human) PSMA (glutamate carboxypeptidase);

[0218] 2) The sequence(s)(or more) can be obtained from Swiss-Prot database entry Q04609 (entry version 133, sequence version 1); the sequence(s)(or more) 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(s) of (human) TNC (tenascin) can be obtained from Swiss-Prot database entry P24821 (entry version 141, sequence version 3); or the sequence(s)(or more) of (human) CA-IX (carbonic anhydrase IX) can be obtained from Swiss-Prot database entry Q16790 (entry version 115, sequence version 2).

[0219] In preferred embodiments, the target cell antigen is selected from the group consisting of fibroblast-activating protein (FAP), carcinoembryonic antigen (CEA), mesothelin (MSLN), CD20, folate receptor 1 (FOLR1), and tenascin (TNC).

[0220] Antibodies capable of specifically binding to any of the above-mentioned target cell antigens can be generated using methods well known in the art, such as immunizing the mammalian immune system and / or phage display using a recombinant library.

[0221] The antibody used in accordance with the present invention comprises an Fc domain containing the P329G mutation (according to EU numbering). The P329G mutation reduces Fc receptor binding and / or effector function and can be used in combination with further Fc mutations that affect binding and / or effector function. Thus, in further embodiments, the mutant Fc domain of the antibody exhibits reduced binding affinity to the Fc receptor and / or reduced effector function compared to the native IgG1Fc domain. In such an embodiment, the mutant Fc domain (or the antibody containing the mutant Fc domain) exhibits a binding affinity to the Fc receptor of less than 50%, particularly less than 20%, more specifically less than 10%, and especially less than 5%, compared to the native IgG1Fc domain (or the antibody containing the native IgG1Fc domain), and / or exhibits effector function of less than 50%, particularly less than 20%, more specifically less than 10%, and especially less than 5%, compared to the native IgG1Fc domain (or the antibody containing the native IgG1Fc domain). In one embodiment, the mutant Fc domain (or an antibody containing the mutant Fc domain) does not substantially bind to the Fc receptor and / or induce effector function. In a particular embodiment, the Fc receptor is the Fcγ receptor. In one embodiment, the Fc receptor is the human Fc receptor. In one embodiment, the Fc receptor is the activated Fc receptor. In a specific embodiment, the Fc receptor is the activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, and most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the mutant Fc domain exhibits substantially modified binding affinity to the neonatal Fc receptor (FcRn) compared to the native IgG1Fc domain. In one embodiment, an antibody containing a mutant Fc domain exhibits a binding affinity of less than 20%, particularly less than 10%, and more specifically less than 5% to the Fc receptor compared to an antibody containing an unmodified Fc domain. In a particular embodiment, the Fc receptor is the Fcγ receptor.In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activated Fc receptor. In specific embodiments, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, and most specifically human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, the binding affinity to complementary components (specifically, the binding affinity to C1q) is also reduced.

[0222] In certain embodiments, the Fc domain of an antibody is mutated to reduce effector function compared to a non-mutant Fc domain. Reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cell-mediated cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent phagocytosis (ADCP), reduced cytokine secretion, reduced immunoconjugate-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-induced apoptosis, reduced cross-linking with target-binding 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 certain embodiments, the reduced effector function is reduced ADCC. In one embodiment, the reduction in ADCC is less than 20% of the ADCC induced by the unmodified Fc domain (or an antibody containing the unmodified Fc domain).

[0223] In one embodiment, the amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to the Fc receptor are amino acid substitutions. In one embodiment, the Fc domain includes amino acid substitutions at positions selected from the group E233, L234, L235, N297, and P331. In a more specific embodiment, the Fc domain includes amino acid substitutions at positions L234 and / or L235. In some embodiments, the Fc domain includes amino acid substitutions L234A and L235A. In one such embodiment, the Fc domain is an IgG1Fc domain, particularly a human IgG1Fc domain. In a more specific embodiment, further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a preferred embodiment, the Fc domain includes amino acid mutations L234A, L235A, and P329G ("P329G LALA") by EU numbering. In one such embodiment, the Fc domain is an IgG1Fc domain, particularly a human IgG1Fc domain. The amino acid substitution combination "P329G LALA" almost completely eliminates the Fcγ receptor (and complement) binding of the human IgIgG1Fc domain, as described in International Publication 2012 / 130831, which is incorporated herein by reference in its entirety. International Publication 2012 / 130831 also describes a method for preparing such mutant Fc domains and determining their properties, such as Fc receptor binding or effector function.

[0224] In certain embodiments, the N-glycosylation of the Fc domain is removed. In such embodiments, the Fc domain includes amino acid substitutions at position N297, particularly amino acid mutations that replace asparagine with alanine (N297A) or aspartic acid (N297D).

[0225] In addition to the Fc domains described herein and in PCT International Publication No. 2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function may also have mutations in one or more of the Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include those with mutations in two or more of the amino acid positions 265, 269, 270, and 297, and include the so-called "DANA" Fc variant in which residues 265 and 297 are mutated to alanine (U.S. Patent No. 7,332,581).

[0226] Mutant Fc domains can be prepared by deletion, substitution, insertion, or modification of amino acids using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of coding DNA sequences, PCR, gene synthesis, etc. Correct nucleotide changes can be confirmed, for example, by screening.

[0227] Binding to the Fc receptor can be easily determined, for example, by ELISA, or by surface plasmon resonance (SPR) using Fc receptors obtained by recombinant expression with standard equipment such as a BIAcore instrument (GE Healthcare). Alternatively, the binding affinity of the Fc domain or a cell-activating bispecific antigen-binding molecule containing an Fc domain to the Fc receptor may be evaluated using cell lines known to express a specific Fc receptor (e.g., human NK cells expressing the FcγIIIa receptor).

[0228] The effector function of an Fc domain or an antibody containing an Fc domain can be measured by methods known in the art. Other examples of in vitro assays for evaluating the ADCC activity of a molecule 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 (e.g., 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 in addition, the ADCC activity of the molecule of interest may be assessed in vivo in animal models such as those disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998), etc.

[0229] In some embodiments, the binding of the Fc domain to complementary components (specifically to C1q) is reduced. Therefore, in some embodiments where the Fc domain is modified to have reduced effector function, this reduced effector function includes reduced CDC. A C1q binding assay can be performed to determine whether the antibody can bind to C1q and thus possesses CDC activity. See, for example, C1q and C3c binding ELISAs in International Publications 2006 / 029879 and 2005 / 100402. A CDC assay may also 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)).

[0230] kit A further aspect of the present invention is a kit comprising or comprising (a) an antibody(s) containing a mutant Fc domain, wherein the antigen-binding receptor of the present invention can specifically bind to the mutant Fc domain.

[0231] therefore, (A) Transduced T cells capable of expressing the antigen-binding receptor of the present invention, (B) A kit is provided comprising an antibody that binds to a target cell antigen and contains an Fc domain containing the amino acid mutation P329G according to EU numbering.

[0232] moreover, (A) an isolated polynucleotide and / or vector encoding the antigen-binding receptor of the present invention, (B) A kit is provided comprising an antibody that binds to a target cell antigen and contains an Fc domain containing the amino acid mutation P329G according to EU numbering.

[0233] The kit of the present invention may comprise transduced T cells, isolated polynucleotides and / or vectors, and one or more antibodies comprising an Fc domain containing the amino acid mutation P329G by EU numbering. In certain embodiments, the antibody is a therapeutic antibody, such as a tumor-specific antibody as described above. Tumor-specific antigens are known in the art and are described above. In connection with the present invention, the antibody is administered before, concurrently with, or after administration of transduced T cells expressing the antigen-binding receptor of the present invention. The kit according to the present invention comprises transduced T cells or polynucleotides / vectors for generating transduced T cells. In this context, the transduced T cells are universal T cells because they are not specific to a given tumor and can target any tumor by the use of a therapeutic antibody containing the mutant Fc domain. While examples of antibodies containing an Fc domain with an amino acid mutation P329G according to EU numbering are provided herein (e.g., SEQ ID NOs: 102-115), any antibody containing an Fc domain with an amino acid mutation P329G according to EU numbering may be used in accordance with the present invention and may be included in the kits provided herein.

[0234] In one specific embodiment, an antibody containing a mutant Fc region can specifically bind to CD20 and includes the heavy chain sequence of SEQ ID NO: 102 and the light chain sequence of SEQ ID NO: 103. In another embodiment, an antibody containing a mutant Fc region can specifically bind to FAP and includes the heavy chain sequence of SEQ ID NO: 104 and the light chain sequence of SEQ ID NO: 105. In yet another embodiment, an antibody containing a mutant Fc region can specifically bind to CEA and includes the heavy chain sequence of SEQ ID NO: 106 and the light chain sequence of SEQ ID NO: 107, the heavy chain sequence of SEQ ID NO: 108 and the light chain sequence of SEQ ID NO: 109, the heavy chain sequence of SEQ ID NO: 110 and the light chain sequence of SEQ ID NO: 111, or the heavy chain sequence of SEQ ID NO: 112 and the light chain sequence of SEQ ID NO: 113. In yet another embodiment, an antibody containing a mutant Fc region can specifically bind to tenascin (TNC) and includes the heavy chain sequence of SEQ ID NO: 114 and the light chain sequence of SEQ ID NO: 115.

[0235] One embodiment of the present invention provides a kit comprising transduced T cells capable of expressing the amino acid sequence of SEQ ID NO: 7 ("VH3VL1-CD8ATD-CD137CSD-CD3zSSD"), or alternatively, the kit comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 7 in combination with an antibody comprising the heavy chain of SEQ ID NO: 102 and the light chain of SEQ ID NO: 103 (for example, the kit comprises a polynucleotide comprising the sequence of SEQ ID NO: 20). This kit can be used to treat CD20-positive cancer.

[0236] In another embodiment of the present invention, a kit is provided comprising transduced T cells capable of expressing the amino acid sequence of SEQ ID NO: 7 ("VH3VL1-CD8ATD-CD137CSD-CD3zSSD"), or alternatively, the kit comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 7 in combination with an antibody comprising the heavy chain of SEQ ID NO: 104 and the light chain of SEQ ID NO: 105 (for example, the kit comprises a polynucleotide comprising the sequence of SEQ ID NO: 20). This kit can be used for the treatment of FAP-positive cancer.

[0237] In another embodiment of the present invention, a kit is provided comprising transduced T cells capable of expressing the amino acid sequence of SEQ ID NO: 7 ("VH3VL1-CD8ATD-CD137CSD-CD3zSSD"), or alternatively, the kit comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 7 in combination with an antibody comprising the heavy chain of SEQ ID NO: 106 and the light chain of SEQ ID NO: 107 (for example, the kit comprises a polynucleotide comprising the sequence of SEQ ID NO: 20). Alternatively, a kit is provided comprising transduced T cells capable of expressing the amino acid sequence of SEQ ID NO: 7 ("VH3VL1-CD28ATD-CD137CSD-CD3zSSD"), or alternatively, the kit comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 7 in combination with an antibody comprising the heavy chain of SEQ ID NO: 108 and the light chain of SEQ ID NO: 109 (for example, the kit comprises a polynucleotide comprising the sequence of SEQ ID NO: 20). This kit can be used for the treatment of FAP-positive cancer. Alternatively, a kit is provided comprising transduced T cells capable of expressing the amino acid sequence of SEQ ID NO: 7 ("VH3VL1-CD28ATD-CD137CSD-CD3zSSD"), or alternatively, the kit comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 7 in combination with an antibody comprising the heavy chain of SEQ ID NO: 110 and the light chain of SEQ ID NO: 111 (for example, the kit comprises a polynucleotide comprising the sequence of SEQ ID NO: 20). In another embodiment, a kit is provided comprising transduced T cells capable of expressing the amino acid sequence of SEQ ID NO: 7 ("VH3VL1-CD8ATD-CD137CSD-CD3zSSD"), or alternatively, the kit comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 7 in combination with an antibody comprising the heavy chain of SEQ ID NO: 112 and the light chain of SEQ ID NO: 113 (for example, the kit comprises a polynucleotide comprising the sequence of SEQ ID NO: 20). These kits can be used to treat CEA-positive cancers.

[0238] In another embodiment of the present invention, a kit is provided comprising transduced T cells capable of expressing the amino acid sequence of SEQ ID NO: 7 ("VH3VL1-CD8ATD-CD137CSD-CD3zSSD"), or alternatively, the kit comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 7 in combination with an antibody comprising the heavy chain of SEQ ID NO: 114 and the light chain of SEQ ID NO: 115 (for example, the kit comprises a polynucleotide comprising the sequence of SEQ ID NO: 20). This kit can be used for the treatment of TNC-positive cancer.

[0239] Furthermore, the components of the kit of the present invention can be packaged individually in vials or bottles, or combined in containers or multi-container units. Furthermore, the kit of the present invention may include a (closed) bag cell incubation system capable of transducing patient cells, preferably T cells as described herein, with the antigen-binding receptor(s) of the present invention and incubating them under GMP (Good Manufacturing Practice) conditions (as described in the Guidelines for Good Manufacturing Practice published by the European Commission under http: / / ec.europa.eu / health / documents / eudralex / index_en.htm). Furthermore, the kit of the present invention includes a (closed) bag cell incubation system capable of transducing isolated / obtained patient T cells with the antigen-binding receptor(s) of the present invention and incubating them under GMP. Furthermore, in the context of the present invention, the kit may also include a vector encoding the antigen-binding receptor(s) described herein. The kit of the present invention can be advantageously used, in particular, to carry out the methods of the present invention and can be used in a variety of applications mentioned herein, e.g., research tools or medical tools. The preparation of the kit preferably follows standard procedures known to those skilled in the art.

[0240] In this context, patient-derived cells, preferably T cells, can be transduced using the above-described kit to the antigen-binding receptor of the present invention, which can specifically bind to the mutant Fc domain described herein. The extracellular domain containing the antigen-binding moiety that can specifically bind to the mutant Fc domain does not naturally exist inside or on T cells. Therefore, patient-derived cells transduced with the kit of the present invention acquire the ability to specifically bind to the mutant Fc domain of an antibody, such as a therapeutic antibody, and can induce the elimination / lysis of target cells through interaction with the therapeutic antibody containing the mutant Fc domain, and the therapeutic antibody can bind to tumor-specific antigens that naturally exist (endogenously expressed) on the surface of tumor cells. Binding of the extracellular domain of the antigen-binding receptor described herein activates the T cell, causing it to physically come into contact with the tumor cell via the therapeutic antibody containing the mutant Fc domain. Non-transduced T cells or endogenous T cells (e.g., CD8+ T cells) cannot bind to the mutant Fc domain of a therapeutic antibody containing the mutant Fc domain. Transduced T cells expressing an antigen-binding receptor containing an extracellular domain capable of specifically binding to a mutant Fc domain remain unaffected by therapeutic antibodies that do not contain mutations in the Fc domain as described herein. Therefore, T cells expressing the antigen-binding receptor molecule 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. The corresponding target cells include cells expressing surface molecules recognized by at least one, preferably two, binding domains of the therapeutic antibodies described herein, i.e., tumor-specific antigens naturally present on the surface of tumor cells. Such surface molecules are characterized herein as follows:

[0241] The lysis of target cells can be detected by methods known in the art. Such methods include, among other things, physiological in vitro assays. Such physiological assays may monitor cell death by, for example, 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, fluorescence-based europium release and calcein AM release assay). Further assays include, for example, monitoring of cell viability by photometric MTT, XTT, WST-1 and alamarBlue assays, radiometric 3H-Thd uptake assay, clonality assays measuring cell division activity, and fluorescence-based rhodamine 123 assays measuring mitochondrial transmembrane gradients. Furthermore, apoptosis may be monitored by, for example, FACS-based phosphatidylserine exposure assays, ELISA-based TUNEL tests, caspase activity assays (photometric, fluorescence-based, or ELISA-based) or analysis of altered cell morphology (contraction, membrane blebbing).

[0242] Therapeutic use and treatment methods The molecules or constructs provided herein (e.g., antigen-binding receptors, transduced T cells, and kits) are particularly useful in clinical practice, especially in the treatment of cancer. For example, tumors can be treated with transduced T cells expressing the antigen-binding receptor of the present invention, in conjunction with a therapeutic antibody containing a mutant Fc domain (i.e., an Fc domain containing the P329G mutation according to EU numbering) that binds to a target antigen on tumor cells. Thus, in certain embodiments, the antigen-binding receptor, transduced T cells, or kits are used in the treatment of cancer, particularly cancers of epithelial, endothelial, or mesothelial origin and hematological cancers.

[0243] The tumor specificity of the treatment is provided by a therapeutic antibody that binds to the target cell antigen, and the antibody is administered before, concurrently with, or after the administration of transduced T cells expressing the antigen-binding receptor of the present invention. In this context, the transduced T cells are universal T cells because, although not specific to a given tumor, they can target any tumor depending on the specificity of the therapeutic antibody used according to the present invention.

[0244] Cancer may be a cancer / carcinoma of epithelial, endothelial, or mesothelial origin, or a hematological cancer. In one embodiment, 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 lymphoma and T-cell lymphoma, myeloid leukemia, ovarian cancer, leukemia, lymphocytic leukemia, nasopharyngeal carcinoma, colon cancer, prostate cancer, renal cell carcinoma, head and neck cancer, skin cancer (melanoma), urogenital tract cancer, such as testicular cancer, ovarian cancer, endothelial cancer, cervical cancer and kidney cancer, bile duct cancer, esophageal cancer, salivary gland cancer and thyroid cancer, or other neoplastic diseases such as hematological malignancies, gliomas, sarcomas or osteosarcomas.

[0245] For example, neoplastic diseases and / or lymphomas can be treated with specific constructs for these medical indications. For instance, gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer and / or oral cancer can be treated with antibodies against (human) EpCAM (as a tumor-specific antigen naturally present on the surface of tumor cells).

[0246] Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, and / or oral cancer may be treated with transdextrinsed T cells of the present invention administered before, simultaneously with, or after the administration of a therapeutic antibody against HER1, preferably 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 transdextrinsed T cells of the present invention administered before, simultaneously with, or after the administration of a therapeutic antibody against MCSP, preferably human MCSP. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma, and / or oral cancer may be treated with transdextrinsed T cells of the present invention administered before, simultaneously with, or after the administration of a therapeutic antibody against FOLR1, preferably human FOLR1. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma, and / or oral cancer may be treated with transdextrinsed T cells of the present invention administered before, simultaneously with, or after the administration of a therapeutic antibody against Trop-2, preferably 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 transdextrinsed T cells of the present invention administered before, simultaneously with, or after the administration of a therapeutic antibody against PSCA, preferably human PSCA. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma, and / or oral cancer may be treated with transdextrinsed T cells of the present invention administered before, simultaneously with, or after the administration of a therapeutic antibody against EGFRvIII, preferably human EGFRvIII. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma, and / or oral cancer may be treated with transdextrinsed T cells of the present invention administered before, simultaneously with, or after the administration of a therapeutic antibody against MSLN, preferably human MSLN. Gastric cancer, breast cancer, and / or cervical cancer may be treated with transdextrinsed T cells of the present invention administered before, simultaneously with, or after the administration of a therapeutic antibody against HER2, preferably human HER2. Gastric cancer and / or lung cancer may be treated with transdextrinsed T cells of the present invention administered before, simultaneously with, or after the administration of a therapeutic antibody against HER3, preferably human HER3.B-cell lymphoma and / or T-cell lymphoma may be treated with transduced T cells of the present invention administered before, concurrently with, or after administration of a therapeutic antibody against CD20, preferably human CD20. B-cell lymphoma and / or T-cell lymphoma may be treated with transduced T cells of the present invention administered before, concurrently with, or after administration of a therapeutic antibody against CD22, preferably human CD22. Myeloid leukemia may be treated with transduced T cells of the present invention administered before, concurrently with, or after administration of a therapeutic antibody against CD33, preferably human CD33. Ovarian cancer, lung cancer, breast cancer and / or gastrointestinal cancer may be treated with transduced T cells of the present invention administered before, concurrently with, or after administration of a therapeutic antibody against CA12-5, preferably human CA12-5. Gastrointestinal cancer, leukemia, and / or nasopharyngeal carcinoma may be treated with transdextrinsed T cells of the present invention administered before, simultaneously with, or after the administration of a therapeutic antibody against HLA-DR, preferably human HLA-DR. Colon cancer, breast cancer, ovarian cancer, lung cancer, and / or pancreatic cancer may be accompanied by transdextrinsed T cells of the present invention administered before, simultaneously with, or after the administration of a therapeutic antibody against MUC-1, preferably human MUC-1. Colon cancer may be treated with transdextrinsed T cells of the present invention administered before, simultaneously with, or after the administration of a therapeutic antibody against A33, preferably human A33. Prostate cancer may be treated with transdextrinsed T cells of the present invention administered before, simultaneously with, or after the administration of a therapeutic antibody against PSMA, preferably human PSMA. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, and / or oral cancer may be treated with transdextrin T cells of the present invention administered before, concurrently with, or after the administration of a therapeutic agent against the transferrin receptor, preferably human transdextrin receptor. Pancreatic cancer, lung cancer, and / or breast cancer may be treated with transdextrin T cells of the present invention administered before, concurrently with, or after the administration of a therapeutic antibody against the transferrin receptor, preferably transferrin receptor. Renal cancer may be treated with transdextrin T cells of the present invention administered before, concurrently with, or after the administration of a therapeutic antibody against CA-IX, preferably human CA-IX.

[0247] The present invention also relates to a method of treating a disease, a malignant disease, such as cancer of epithelial, endothelial or mesothelial origin and / or blood cancer. In the context of the present invention, the subject is a human. In the context of the present invention, a specific method for treating a disease comprises the following steps: (a) isolating T cells, preferably CD8+ T cells, from the subject; (b) transducing the isolated T cells, preferably CD8+ T cells, with an antigen-binding receptor described herein; and (c) administering the transduced T cells, preferably CD8+ T cells, to the subject.

[0248] In the context of the present invention, the transduced T cells, preferably CD8+ T cells, and / or the therapeutic antibody(ies) are co-administered to the subject by intravenous infusion.

[0249] Furthermore, in the context of the present invention, the present invention provides a method for treating a disease comprising the following steps: (a) isolating T cells, preferably CD8+ T cells, from the subject; (b) transducing the isolated T cells, preferably CD8+ T cells, with an antigen-binding receptor 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 antibody and anti-CD28 antibody; and (e) administering the transduced T cells, preferably CD8+ T cells, to the subject.

[0250] Step (d) above (referring to the step of proliferating T cells such as TILs with an anti-CD3 antibody and / or an anti-CD28 antibody) can also be carried out in the presence (stimulation) of cytokines such as interleukin-2 and / or interleukin-15 (IL-15). In the context of the present invention, step (d) above (referring to the step of proliferating T cells such as TILs with an anti-CD3 antibody and / or an anti-CD28 antibody) can also be carried out in the presence of interleukin-12 (IL-12), interleukin-7 (IL-7), and / or interleukin-21 (IL-21).

[0251] The treatment method further comprises the administration of an antibody used in accordance with the present invention. The antibody may be administered before, simultaneously with, or after the administration of transduced T cells. In connection with the present invention, the administration of transduced T cells is carried out by intravenous infusion. In the context of the present invention, transduced T cells are isolated / obtained from the subject to be treated.

[0252] The present invention further envisions a co-administration protocol with other compounds, such as molecules capable of providing activation signals for immune effector cells, cell proliferation, or cell stimulation. Such molecules may be, for example, further primary activation signals for T cells (e.g., further co-stimulating molecules: molecules of the B7 family, Ox40L, 4.1 BBL, CD40L, anti-CTLA-4, anti-PD-1), or further cytokine interleukins (e.g., IL-2).

[0253] The compositions of the present invention described above may also optionally further include means and methods for detection as diagnostic compositions.

[0254] composition Furthermore, the present invention provides a composition (pharmaceutical) comprising antibody molecules(or more) having mutant Fc domains, and / or transduced T cells(or more) containing the antigen-binding receptor of the present invention, and / or nucleic acid molecules(or more), and vectors(or more) encoding the antigen-binding receptor of the present invention. Furthermore, the present invention provides a kit comprising one or more of the compositions. In the context of the present invention, a composition is a pharmaceutical composition optionally further comprising a suitable formulation of a carrier, stabilizer, and / or excipient. Accordingly, in the context of the present invention, a pharmaceutical composition (pharmaceutical) is provided comprising antibody molecules containing mutant Fc domains as defined herein, which are administered in combination with transduced T cells containing the antigen-binding receptor described herein and / or a composition comprising the transduced T cells, wherein the antibody molecules are administered before, concurrently with, or after administration of transduced T cells containing the antigen-binding receptor of the present invention.

[0255] The use of the term “in combination” does not limit the order in which the components of a therapeutic regimen are administered to a subject. Accordingly, the pharmaceutical compositions / pharmaceuticals described herein include the administration of antibodies as defined herein, before, concurrently with, or after the administration of transduced T cells containing the antigen-binding receptor of the present invention. The term “in combination” as used herein also does not limit the timing between the administration of antibodies as defined herein and transduced T cells containing the antigen-binding receptor as defined herein. Accordingly, if the two components are not administered simultaneously with / concurrently, the administrations may 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 by any appropriate time difference readily determined by those skilled in the art and / or described herein.

[0256] In the context of the present invention, the term “combined” also encompasses situations in which transduced T cells comprising the antibody as defined herein and the antigen-binding receptor according to the present invention are pre-incubated together before administration to a subject. Thus, the two components may be pre-incubated before administration for, for example, 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, or 1 hour, or any appropriate time readily determined by those skilled in the art. In another preferred embodiment, the present invention relates to a therapeutic regime in which transduced T cells comprising the antibody as defined herein and the antigen-binding receptor as defined herein are administered simultaneously / together. In the context of the present invention, the antibody as defined herein may be administered after the transduced T cells comprising the antigen-binding receptor have been administered.

[0257] Furthermore, as used herein, “combined” does not limit the disclosed therapeutic regimens to the immediate sequence of administration of the antibody as defined herein and transduced T cells, preferably CD8+ T cells, comprising the antigen-binding receptor of the present invention (i.e., administering one of the two components, then (with a time interval) administering the other, in between, without administering and / or performing any other therapeutic protocol). Thus, the therapeutic regimens also encompass separate administrations of the antibody molecule as defined herein and transduced T cells, preferably CD8+ T cells, comprising the antigen-binding receptor according to the present invention, and the administrations are separated by one or more therapeutic protocols that are necessary and / or preferred for the treatment or prevention of the disease or its symptoms. Examples of such intervening therapeutic protocols include, but are not limited to, the administration of analgesics; the administration of chemotherapeutic agents; and surgical treatment of the disease or its symptoms. Accordingly, the therapeutic regimens disclosed herein include administering, one or more of, an antibody as defined herein and a transduced T cell, preferably CD8+ T cell, comprising an antigen-binding receptor as defined herein, without a therapeutic protocol suitable for the treatment or prevention of a disease or its symptoms described herein or known in the art.

[0258] The pharmaceutical composition(s) / pharmaceutical(s) are particularly intended to be administered to a patient by infusion or injection. In the context of the present invention, the transduced T cells containing the antigen-binding receptor described herein should be administered to a patient by infusion or injection. The appropriate composition / pharmaceutical may be administered by different methods, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.

[0259] The pharmaceutical composition / pharmaceutical of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to a subject in an appropriate dose. The administration regimen is determined by the attending physician and clinical factors. As is well known in the medical field, the dose for any one patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, overall health, and other drugs administered together. Generally, a regimen as a regular administration of a pharmaceutical composition should be in the range of 1 μg to 5 g units per day. However, a more preferred dose for continuous infusion may be in the range of 0.01 μg to 2 mg, preferably 0.01 μg to 1 mg, more preferably 0.01 μg to 100 μg, even more preferably 0.01 μg to 50 μg, and most preferably 0.01 μg to 10 μg units / kilogram body weight / hour. Particularly preferred doses are listed below. Progress can be monitored by periodic evaluation. Although the doses vary, a preferred dose for intravenous administration of DNA is about 10 times the amount of DNA molecules. 6 ~10 12 It's a copy.

[0260] The compositions of the present invention can be administered topically or systematically. Administration is generally parenterally, for example, intravenously; transduced T cells can also be administered to a target site, for example, intra-arterially by a catheter. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, and include saline and buffering media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution, or fixative oils. Intravenous vehicles include liquids and nutritional supplements, electrolyte supplements (e.g., those based on ringer's dextrose), etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. Furthermore, the pharmaceutical compositions of the present invention may preferably include a proteinaceous carrier of human origin, such as serum albumin or immunoglobulin. In addition to a proteinaceous antibody construct or a nucleic acid molecule or vector encoding it (as described in the present invention) and / or cells, the pharmaceutical compositions of the present invention may include further biologically active agents, depending on the intended use of the pharmaceutical composition. Such agents may include drugs that act on the gastrointestinal system, drugs that act as cell proliferation inhibitors, drugs that prevent hyperuricemia, drugs that inhibit immune responses (e.g., corticosteroids), drugs that act on the circulatory system and / or agents such as T cell co-stimulating molecules or cytokines known in the art.

[0261] Exemplary Embodiments 1. An antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain, wherein the extracellular domain is (i) A heavy chain variable domain (VH) comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2 or SEQ ID NO: 40, and HCDR3 of SEQ ID NO: 3, (ii) An antigen-binding receptor comprising an antigen-binding moiety including a light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 4, LCDR 2 of SEQ ID NO: 5, and a light chain variable domain (VL) including LCDR 3 of SEQ ID NO: 6.

[0262] 2. The antigen-binding receptor according to Embodiment 1, wherein the VH domain 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: 8, SEQ ID NO: 41, SEQ ID NO: 44, and SEQ ID NO: 126.

[0263] 3. The antigen-binding receptor according to Embodiment 1 or 2, wherein the VL domain 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: 9 or SEQ ID NO: 127.

[0264] 4. An antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain, wherein the extracellular domain comprises an antigen-binding moiety comprising the heavy chain variable domain (VH) of SEQ ID NO: 8 and the light chain variable domain (VL) of SEQ ID NO: 9; or an antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain, wherein the extracellular domain comprises an antigen-binding moiety comprising the heavy chain variable domain (VH) of SEQ ID NO: 126 and the light chain variable domain (VL) of SEQ ID NO: 127.

[0265] 5. An antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain, wherein the extracellular domain comprises an antigen-binding moiety comprising the heavy chain variable domain (VH) of SEQ ID NO: 41 and the light chain variable domain (VL) of SEQ ID NO: 9.

[0266] 6. An antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain, wherein the extracellular domain comprises an antigen-binding moiety comprising the heavy chain variable domain (VH) of SEQ ID NO: 44 and the light chain variable domain (VL) of SEQ ID NO: 9.

[0267] 7. The antigen-binding receptor according to any one of embodiments 1 to 6, wherein the antigen-binding portion is a scFv.

[0268] 8. The antigen-binding receptor according to any one of embodiments 1 to 7, wherein the antigen-binding portion 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: 10, SEQ ID NO: 122, SEQ ID NO: 124 or SEQ ID NO: 128.

[0269] 9. An antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain, wherein the extracellular domain comprises an antigen-binding portion, and the antigen-binding portion comprises the amino acid sequence of SEQ ID NO: 10, or an antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain, wherein the extracellular domain comprises an antigen-binding portion, and the antigen-binding portion comprises the amino acid sequence of SEQ ID NO: 128.

[0270] 10. An antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain, wherein the extracellular domain comprises an antigen-binding portion, and the antigen-binding portion comprises the amino acid sequence of SEQ ID NO: 122.

[0271] 11. An antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain, wherein the extracellular domain comprises an antigen-binding portion, and the antigen-binding portion comprises the amino acid sequence of SEQ ID NO: 124.

[0272] 12. The antigen-binding receptor according to any one of embodiments 1 to 11, wherein the anchoring transmembrane domain is a transmembrane domain selected from the group consisting of the CD8, CD4, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10 or DAP12 transmembrane domain or a fragment thereof.

[0273] 13. An antigen-binding receptor according to any one of Embodiments 1 to 12, wherein the anchoring transmembrane domain is a CD8 transmembrane domain, and in particular the anchoring transmembrane domain contains the amino acid sequence of SEQ ID NO: 11.

[0274] 14. An antigen-binding receptor according to any one of Embodiments 1 to 13, further comprising at least one stimulus signaling domain and / or at least one co-stimulus signaling domain.

[0275] 15. An antigen-binding receptor according to any one of Embodiments 1 to 14, wherein at least one stimulus 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 that retain stimulus signaling activity.

[0276] 16. An antigen-binding receptor according to any one of Embodiments 1 to 15, wherein at least one stimulus signaling domain is the intracellular domain of CD3z or a fragment thereof that retains stimulus signaling activity, and in particular, at least one stimulus signaling domain comprises the amino acid sequence of SEQ ID NO: 13.

[0277] 17. An antigen-binding receptor according to any one of Embodiments 1 to 16, wherein at least one co-stimulation 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 that retain co-stimulation signaling activity.

[0278] 18. An antigen-binding receptor according to any one of Embodiments 1 to 17, comprising a CD137 co-stimulation domain or a fragment thereof that retains CD137 co-stimulation activity, and in particular comprising a co-stimulation signaling domain comprising the amino acid sequence of SEQ ID NO: 12.

[0279] 19. An antigen-binding receptor according to any one of Embodiments 1 to 18, wherein at least one co-stimulation signaling domain is a CD28 intracellular domain or a fragment thereof that retains CD28 co-stimulation activity.

[0280] 20. An antigen-binding receptor according to any one of Embodiments 1 to 19, comprising a stimulus signaling domain containing the intracellular domain of CD3z or a fragment thereof that retains CD3z stimulus signaling activity, and comprising a co-stimulation signaling domain containing the intracellular domain of CD28 or a fragment thereof that retains CD28 co-stimulation signaling activity.

[0281] 21. The antigen-binding receptor according to Embodiment 20, wherein the stimulus signaling domain comprises the amino acid sequence of SEQ ID NO: 13.

[0282] 22. An antigen-binding receptor according to any one of Embodiments 1 to 18, comprising one stimulus signaling domain containing the intracellular domain of CD3z or a fragment thereof that retains CD3z stimulus signaling activity, and one co-stimulation signaling domain containing the intracellular domain of CD137 or a fragment thereof that retains CD137 co-stimulation signaling activity.

[0283] 23. The antigen-binding receptor according to Embodiment 22, wherein the stimulus signaling domain comprises the amino acid sequence of SEQ ID NO: 13 and the co-stimulus signaling domain comprises the amino acid sequence of SEQ ID NO: 12.

[0284] 24. An antigen-binding receptor according to any one of Embodiments 1 to 23, wherein the extracellular domain is optionally linked to the anchoring transmembrane domain via a peptide linker.

[0285] 25. The antigen-binding receptor according to Embodiment 24, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 19.

[0286] 26. An antigen-binding receptor according to any one of Embodiments 14 to 25, wherein the anchoring transmembrane domain is optionally linked to a co-signaling domain or a stimulus-signaling domain via a peptide linker.

[0287] 27. An antigen-binding receptor according to any one of Embodiments 1 to 26, wherein the signaling domain and / or co-signaling domain are optionally linked via at least one peptide linker.

[0288] 28. An antigen-binding receptor according to any one of Embodiments 1 to 27, wherein the antigen-binding portion is optionally linked at the C-terminus to the N-terminus of the anchoring transmembrane domain via a peptide linker.

[0289] 29. An antigen-binding receptor according to any one of Embodiments 1 to 28, wherein the light chain variable domain (VL) is optionally linked at the C-terminus to the N-terminus of an anchoring transmembrane toxin via a peptide linker.

[0290] 30. An antigen-binding receptor according to any one of Embodiments 1 to 29, wherein the heavy chain variable domain (VH) is optionally linked at its C-terminus to the N-terminus of the light chain variable domain (VL) via a peptide linker.

[0291] 31. The antigen-binding receptor according to any one of embodiments 14 to 30, wherein the antigen-binding receptor comprises one simultaneous signaling domain, the simultaneous signaling domain being ligated at its N-terminus to the C-terminus of an anchoring transmembrane domain.

[0292] 32. The antigen-binding receptor according to Embodiment 31, wherein the antigen-binding receptor further comprises one stimulus signaling domain, the stimulus signaling domain being ligated at its N-terminus to the C-terminus of a co-stimulus signaling domain.

[0293] 33. The antigen-binding receptor according to any one of Embodiments 1 to 32, wherein 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: 7 or SEQ ID NO: 125.

[0294] 34. An antigen-binding receptor containing the amino acid sequence of SEQ ID NO: 7; or an antigen-binding receptor containing the amino acid sequence of SEQ ID NO: 125.

[0295] 35. An isolated polynucleotide encoding an antigen-binding receptor according to any one of Embodiments 1 to 34.

[0296] 36. A polypeptide encoded by the isolated polynucleotide described in Embodiment 35.

[0297] 37. A vector, particularly an expression vector, comprising the polynucleotide described in Embodiment 35.

[0298] 38. Transduced T cells comprising the polynucleotide described in Embodiment 35 or the vector described in Embodiment 37.

[0299] 39. Transduced T cells capable of expressing the antigen-binding receptor described in any one of Embodiments 1 to 34.

[0300] 40. It is a kit, (A) Transduced T cells capable of expressing the antigen-binding receptor described in any one of Embodiments 1 to 34, (B) A kit comprising an antibody that binds to a target cell antigen and contains an Fc domain including the amino acid mutation P329G according to EU numbering.

[0301] 41. It is a kit, (A) An isolated polynucleotide encoding an antigen-binding receptor as described in any one of Embodiments 1 to 34, (B) A kit comprising an antibody that binds to a target cell antigen and contains an Fc domain including the amino acid mutation P329G according to EU numbering.

[0302] 42. It is a kit, (A) an isolated polynucleotide as described in Embodiment 35 or a vector as described in Embodiment 37, (B) A kit comprising an antibody that binds to a target cell antigen and contains an Fc domain including the amino acid mutation P329G according to EU numbering.

[0303] 43. A kit according to any one of Embodiments 40 to 42, wherein the Fc domain is an IgG1 or IgG4 Fc domain, particularly a human IgG1 Fc domain.

[0304] 44. The kit according to any one of Embodiments 40 to 43, wherein the target cell antigen is selected from the group consisting of fibroblast-activating protein (FAP), carcinoembryonic antigen (CEA), mesothelin (MSLN), CD20, folate receptor 1 (FOLR1), and tenascin (TNC).

[0305] 45. A kit according to any one of embodiments 40 to 44 for use as a pharmaceutical.

[0306] 46. ​​An antigen-binding receptor according to any one of Embodiments 1 to 34 or a transduced T cell according to any one of Embodiments 38 to 39, for use as a pharmaceutical, wherein transduced T cells expressing an antigen-binding receptor bind to a target cell antigen (particularly a cancer cell antigen) and are administered before, simultaneously with, or after administration of an antibody containing an amino acid mutation P329G by EU numbering.

[0307] 47. A kit according to any one of embodiments 40 to 45, for use in the treatment of a disease, particularly for use in the treatment of cancer.

[0308] 48. An antigen-binding receptor according to any one of Embodiments 1 to 34 or a transduced T cell according to Embodiment 38 or 39 for use in the treatment of cancer, wherein the treatment comprises the administration of transduced T cells expressing the antigen-binding receptor before, simultaneously with, or after the administration of an antibody that binds to a cancer cell antigen and contains an Fc domain including an amino acid mutation P329G according to EU numbering.

[0309] 49. An antigen-binding receptor, transduced T cell, or kit for use according to Embodiment 47 or 48, wherein the cancer is selected from epithelial, endothelial, or mesothelial cancers and hematological cancers.

[0310] 50. An antigen-binding receptor, transduced T cell, or kit for use according to Embodiment 49, wherein the cancer antigen is selected from the group consisting of fibroblast-activating protein (FAP), carcinoembryonic antigen (CEA), mesothelin (MSLN), CD20, folate receptor 1 (FOLR1), and tenascin (TNC).

[0311] 51. An antigen-binding receptor, transduced T cell, or kit for use according to any one of embodiments 48-50, wherein the transduced T cells are derived from cells isolated from a subject to be treated.

[0312] 52. An antigen-binding receptor, transduced T cell, or kit for use according to any one of embodiments 48 to 51, wherein the transduced T cells are not derived from cells isolated from the subject to be treated.

[0313] 53. A method for treating a disease in a subject, comprising administering transduced T cells capable of expressing an antigen-binding receptor as described in any one of Embodiments 1 to 34, and administering a therapeutically effective amount of an antibody that binds to a target cell antigen and contains an Fc domain including the amino acid mutation P329G according to EU numbering, before, simultaneously with, or after administration of the transduced T cells.

[0314] 54. The method according to Embodiment 48, further comprising isolating T cells from a subject and generating transduced T cells by transducing the isolated T cells with the polynucleotide of Embodiment 35 or the vector of Embodiment 37.

[0315] 55. The method according to Embodiment 54, wherein T cells are transduced with a retroviral or lentiviral vector construct or a non-viral vector construct.

[0316] 56. The method according to any one of embodiments 53 to 55, wherein transduced T cells are administered to a subject by intravenous injection.

[0317] 57. The method according to any one of embodiments 53 to 56, wherein transduced T cells are contacted with an anti-CD3 antibody and / or an anti-CD28 antibody before administration to a target.

[0318] 58. The method according to any one of Embodiments 53 to 57, wherein transduced T cells are brought into contact 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, before administration to a subject.

[0319] 59. The method according to any one of embodiments 53 to 58, wherein the disease is cancer.

[0320] 60. The method according to Embodiment 59, wherein the cancer is selected from cancers of epithelial, endothelial, or mesothelial origin and hematological cancers.

[0321] 61. A method for inducing the lysis of target cells, comprising contacting the target cells with transduced T cells capable of expressing an antigen-binding receptor according to any one of Embodiments 1 to 34, in the presence of an antibody that binds to a target cell antigen and comprises an Fc domain containing an amino acid mutation P329G by EU numbering.

[0322] 62. The method according to embodiment 61, wherein the target cells are cancer cells.

[0323] 63. The method according to Embodiment 61 or 62, wherein the target cells express an antigen selected from the group consisting of fibroblast-activating protein (FAP), carcinoembryonic antigen (CEA), mesothelin (MSLN), CD20, folate receptor 1 (FOLR1), and tenascin (TNC).

[0324] 64. Use of an antigen-binding receptor according to any one of Embodiments 1 to 34, a polynucleotide according to Embodiment 35, or a transduced T cell according to Embodiment 38 or 39 for the manufacture of a pharmaceutical product.

[0325] 65. The use according to Embodiment 64, wherein the pharmaceutical is for the treatment of cancer.

[0326] 66. The use according to Embodiment 65, characterized in that the cancer is selected from epithelial, endothelial, or mesothelial cancers and hematological cancers.

[0327] 67. An invention as described above.

[0328] These and other embodiments are disclosed and included in the specification and examples of the present invention. Further literature relating to any one of the antibodies, methods, uses, and compounds employed by the present invention can be searched from public libraries and databases, for example, using electronic devices. For example, the public database "Medline," available on the Internet, can be accessed, for example, at http: / / www.ncbi.nlm.nih.gov / PubMed / medline.html. Further databases and addresses such as http: / / www.ncbi.nlm.nih.gov / , http: / / www.infobiogen.fr / , http: / / www.fmi.ch / biology / research_tools.html, and http: / / www.tigr.org / are known to those skilled in the art and can also be obtained, for example, using http: / / www.lycos.com.

[0329] Example sequence [Table 1] TIFF0007848182000002.tif63170 [Table 2] TIFF0007848182000004.tif248170TIFF0007848182000005.tif253170TIFF0007848182000006.tif253170TIFF0007848182000007.tif13170 [Table 3] [Table 4] TIFF0007848182000010.tif249170TIFF0007848182000011.tif253170TIFF0007848182000012.tif23170 [Table 5] TIFF0007848182000014.tif250170TIFF0007848182000015.tif251170TIFF0007848182000016.tif251170TIFF0007848182000017.tif131170 [Table 6] [Table 7] TIFF0007848182000020.tif253170TIFF0007848182000021.tif253170TIFF0007848182000022.tif253170TIFF000 7848182000023.tif253170TIFF0007848182000024.tif253170TIFF0007848182000025.tif249170TIFF00078481820 00026.tif253170TIFF0007848182000027.tif253170TIFF0007848182000028.tif245170TIFF0007848182000029.t if252170TIFF0007848182000030.tif253170TIFF0007848182000031.tif249170TIFF0007848182000032.tif140170 [Table 8] [Table 9] [Table 10] [Examples]

[0330] The following are examples of the methods and compositions of the present invention. Given the general description given above, it will be understood that various other embodiments may be implemented.

[0331] Recombinant DNA technology DNA was manipulated using standard methods, as described in Sambrook et al., Molecular Cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions. General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains was found in Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, 5 th It is described in ed., NIH Publication No. 91-3242.

[0332] DNA sequencing The DNA sequence was determined by double-strand sequencing.

[0333] gene synthesis Where necessary, the desired gene segments were generated by PCR using appropriate templates, or synthesized by automated gene synthesis from synthetic oligonucleotides and PCR products by Geneart AG (Regensburg, Germany). Where accurate gene sequences were unavailable, oligonucleotide primers were designed based on the sequence of the nearest homolog, and the gene was isolated by RT-PCR from RNA derived from appropriate tissues. Gene segments adjacent to a single restriction endonuclease cleavage site were cloned into standard cloning / sequencing vectors. Plasmid DNA was purified from transformed bacteria, and its concentration was measured by UV spectroscopy. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene segments with appropriate restriction sites were designed to enable subcloning into their respective expression vectors. All constructs were designed to include a 5' terminal DNA sequence encoding a leader peptide targeting a protein for secretion in eukaryotic cells.

[0334] Production of IgG-like proteins in HEK293 EBNA or CHO EBNA cells Antibodies and bispecific antibodies were generated by transient transfection of HEK293 EBNA cells or CHO EBNA cells. The cells were centrifuged, and the culture medium was replaced with pre-warmed CD CHO medium (Thermo Fisher, catalog no. 10743029). The expression vector was mixed in CD CHO medium, PEI (polyethyleneimine, Polysciences, Inc., catalog no. 23966-1) was added, and the solution was vortexed and incubated at room temperature for 10 minutes. Subsequently, cells (2 MiO / mL) were mixed with the vector / PEI solution, transferred to a flask, and incubated in a shaking incubator under a 5% CO2 atmosphere at 37°C for 3 hours. After incubation, Excell medium containing supplements (80% of the total volume) was added (W. Zhou and A. Kantardjieff, Mammalian Cell Cultures for Biologics Manufacturing, DOI:10.1007 / 978-3-642-54050-9;2014). One day after transfection, supplements (Feed, 12% of the total volume) were added. After 7 days, the cell supernatant was collected by centrifugation and subsequent filtration (0.2 μm filter), and the collected supernatant was purified by the standard method described below.

[0335] Production of IgG-like protein in CHO K1 cells Alternatively, using a proprietary vector system with conventional (non-PCR-based) cloning techniques, and suspension-adapted CHO K1 cells (originally commissioned by ATCC and adapted by Evitria for serum-free growth in suspension culture), Evitria prepared the antibodies described herein and bispecific antibodies. For production, Evitria used proprietary animal component-free serum-free media (eviGrow and eviMake2) and proprietary transfection reagents (eviFect). The supernatant was collected by centrifugation and subsequent filtration (0.2 μm filter), and the proteins were purified from the collected supernatant by standard methods.

[0336] Purification of IgG-like proteins Following a standard protocol, proteins were purified from the filtered cell culture supernatant. Briefly, Fc-containing proteins were purified from the cell culture supernatant by protein A affinity chromatography (equilibrium buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was achieved at pH 3.0, and the sample was immediately neutralized. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15 (item number: UFC903096)), and aggregated proteins were separated from monomeric proteins by size exclusion chromatography in 20 mM histidine, 140 mM sodium chloride, pH 6.0.

[0337] Analysis of IgG-like proteins The concentration of purified protein was measured by measuring the absorption at 280 nm using a mass decay coefficient calculated based on the amino acid sequence according to Pace, et al., Protein Science, 1995, 4, 2411-1423. Protein purity and molecular weight were analyzed by CE-SDS in the presence and absence of a reducing agent using LabChip GXII or LabChip GX Touch (Perkin Elmer). Aggregate content was determined by HPLC chromatography at 25°C using an analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated in running buffer (200 mM KH2PO4, 250 mM KCl pH 6.2, 0.02% NaN3).

[0338] Preparation of lentiviral supernatant and transduction of Jurkat-NFAT cells Using approximately 80% confluent Hek293T cells (ATCC CRL3216), a CAR-encoding transfer vector, and packaging vectors pCAG-VSVG and psPAX2 in a 2:2:1 molar ratio, lipofectamine LTX (商標)Transfection was performed based on the following (Giry-Laterriere M, et al Methods Mol Biol. 2011;737:183-209, Myburgh R, et al Mol Ther Nucleic Acids. 2014). After 66 hours, the supernatant was collected, centrifuged at 350 × g for 5 minutes, and filtered through a 0.45 μm polyethersulfone filter to collect and purify the virus particles. The virus particles were used directly or concentrated (Lenti-x-Concentrator, Takara) and used for spinfection of Jurkat NFAT T cells (loResponse Jurkat NFAT-RE-luc2P, Promega #CS176501) at 900 × g for 2 hours and 31°C.

[0339] Jurkat NFAT Activation Assay The Jurkat NFAT activation assay measures T cell activation in a human acute lymphoblastic leukemia reporter cell line (GloResponse Jurkat NFAT-RE-luc2P, Promega#CS176501). This immortalized T cell line is genetically engineered to stably express a luciferase reporter driven by an NFAT response element (NFAT-RE). Furthermore, the cell line expresses a chimeric antigen receptor (CAR) construct containing a CD3z signaling domain. Binding of the CAR to an immobilized adapter molecule (e.g., a tumor antigen-binding adapter molecule) results in CAR crosslinking, leading to T cell activation and luciferase expression. After substrate addition, cellular changes in NFAT activity can be measured as relative light units (Darowski et al. Protein Engineering, Design and Selection, Volume 32, Issue 5, May 2019, Pages 207-218, https: / / doi.org / 10.1093 / protein / gzz027). Generally, assays were performed in 384-well plates (Falcon #353963 white, clear bottom). Target cells (CAR-Jurkat-NFAT cells) and effector cells were triple-seed in RPMI-1640 + 10% FCS + 1% Glutamax (growth medium) in a 1:5 ratio of 10 μl each (2000 target cells and 10000 effector cells). Furthermore, serial dilutions of the antibody of interest were prepared in growth medium to obtain final concentrations ranging from 67 nM to 0.000067 nM in the assay plate, resulting in a total final volume of 30 μl per well. The 384-well plates were centrifuged at 300 g and RT for 1 minute and incubated in a humid atmosphere at 37°C and 5% CO2. After 7 hours of incubation, 20% of the final volume of ONE-Glo® luciferase assay (E6120, Promega) was added, and the plate was centrifuged at 350 × g for 1 minute. Relative luminescence units (RLU) per well were then immediately measured using a Tecan microplate reader. Concentration-response curves were fitted and EC was performed using GraphPadPrism version 7. 50The values ​​were calculated. For the p-values, the New England Journal of Medicine style was used, as listed in GraphPadPrism 7: *=P≦0.033;**=P≦0.002;***=P≦0.001.

[0340] Example 1 Production and Characterization of Humanized Anti-P329G Antibodies Parental anti-P329G antibodies and humanized anti-P329G antibodies were produced in HEK cells and purified by Protein A affinity chromatography and size exclusion chromatography. All antibodies were purified to good quality (Table). 11 ). [Table 11]

[0341] Binding of the parent and six humanized variants of the anti-P329G binder M-1.7.24 to human Fc(P329G). Measurement method: Biacore T200 Tip: CM5 (#772) Fc1~4: Anti-human Fab specific (GE Healthcare 28-9583-25) Capture: 50nM IgG over 60 seconds Analyte: Human Fc (P329G) (P1AD9000-004) Running buffer: HBS-EP T°:25°C Dilution: 2-fold dilution in HBS-EP at concentrations of 0.59 nM to 37.5 nM. Flow rate: 30μl / min Meeting: 240 seconds Dissociation: 800 seconds Regeneration: 10 mM glycine pH 2.1, 2 x 60 seconds

[0342] SPR experiments were performed on a Biacore T200 using HBS-EP+ (0.01M HEPES pH 7.4, 0.15M NaCl, 0.005% surfactant P20 (BR-1006-69, GE Healthcare)) as the running buffer. An anti-human Fab-specific antibody (GE Healthcare 28-9583-25) was directly immobilized on a CM5 chip (GE Healthcare) by amine coupling. IgG was captured at 50 nM for 60 seconds. A two-fold dilution series of human Fc (P329G) was passed over the ligand at 30 μl / min for 240 seconds, and the association phase was recorded. The dissociation phase was monitored for 800 seconds and triggered by switching from the sample solution to HBS-EP+. 10 mM glycine pH 2.1 was injected twice for 60 seconds, and the chip surface was regenerated after each cycle. The bulk refractive index difference was corrected by subtracting the response obtained in reference flow cell 1. The affinity constant was derived from the kinetic rate constant by fitting it to a 1:1 Langmuir bond using Biaeval software (GE Healthcare). Measurements were performed in triplicate using independent dilution series.

[0343] The following samples were analyzed for binding to human Fc(P329G) (Table). 12 ). [Table 12]

[0344] Human Fc(P329G) was prepared by plasmin digestion of human IgG1 followed by affinity purification with Protein A and size exclusion chromatography.

[0345] Binding of the parent and six humanized variants of the anti-P329G binder M-1.7.24 to human Fc(P329G). The dissociation phase was fitted to a single curve to help characterize the dissociation rate. The ratio of coupling to the capture response level was calculated. (Table) 13 ). [Table 13]

[0346] Affinity of the parent and three humanized variants of the anti-P329G binder M-1.7.24 to human Fc(P329G) Three humanized variants with similar bonding patterns to the parent were evaluated in more detail. The kinetic constants of the 1:1 Langmuir bond are expressed. 14 To summarize: [Table 14]

[0347] conclusion Six human variants were generated. Three of them (VH4VL1, VH1VL2, VH1VL3) showed reduced binding to human Fc(P329G) compared to the parent M-1.7.24. The other three humanized variants (VH1VL1, VH2VL1, VH3VL1) had binding kinetics very similar to the parent binder and did not lose affinity through humanization.

[0348] Example 2 Preparation of humanized anti-P329G antigen-binding receptors To evaluate the functionality of humanized P329G variants, different variable domains of heavy chain (VH) and light chain (VL) DNA sequences encoding binders specific to the P329G Fc mutation were cloned as single-stranded variable fragment (scFv) binding sites and used as antigen-binding domains for second-generation chimeric antigen receptors (CARs).

[0349] Different humanized variants of the P329G binder contain an Ig heavy chain variable main domain (VL) and an Ig light chain variable domain (VL). VH and VL are linked via a (G4S)4 linker. The scFv antigen-binding domain was fused to the anchoring transmembrane domain (ATD) CD8a (Uniprot P01732[183-203]), which was then fused to the intracellular simultaneous stimulus signaling domain (CSD) CD137 (Uniprot Q07011AA214-255), and subsequently to the stimulus signaling domain (SSD) CD3ζ (Uniprot P20963 AA52-164). The scFv of anti-P329G CARs was constructed with two different orientations: VHxVL (Figure 1A) or VLxVH (Figure 1B). Figure 1C shows a graph of an example expression construct (including a GFP reporter) for the VHVL configuration, and Figure 1D shows the VLVH configuration.

[0350] Example 3 Expression of anti-P329G antigen-binding receptor in Jurkat-NFAT cells Different humanized anti-P329G antigen-binding receptors were introduced into Jurkat (GloResponse Jurkat NFAT-RE-luc2P, Promega#CS176501) cells via viral transduction.

[0351] Anti-P329G antigen-binding receptor expression was evaluated by flow cytometry. Jurkat cells using different humanized anti-P329G antigen-binding receptors were harvested, washed with PBS, and seeded at 50,000 cells / well in 96-well flat-bottom plates. After staining with antibodies containing the P329G mutation in the Fc domain at different concentrations (1:5 serial dilutions from 500 nM to 0 nM) in the dark and refrigerated (4-8°C) for 45 minutes, the samples were washed three times with FACS buffer (2% FBS, 10% 0.5 M EDTA, pH 8, and PBS containing 0.5 g / L NaN3). Subsequently, the samples were stained with 2.5 μg / mL polyclonal anti-human IgG Fcγ fragment-specific and PE-conjugated AffiniPure F(ab')2 goat fragment antibody in the dark and refrigerated for 30 minutes and analyzed by flow cytometry (Fortessa BD). Furthermore, the anti-P329G antigen-binding receptor contained an intracellular GFP reporter (see Figure 1C).

[0352] While GFP expression is comparable, the original unhumanized binder exhibits weak CAR labeling on the cell surface compared to the humanized versions of the P329G binder (VH1VL1, VH2VL1, and VH3VL1) (Figure 2A). Interestingly, the VL1VH1 construct (see Figure 1D) shows high GFP expression but also exhibits weak CAR labeling on the cell surface, indicating that this is an undesirable confirmation of the binder.

[0353] Overall, unexpectedly, the VH3VL1 version exhibits the highest GFP expression and CAR surface expression. Furthermore, all tested constructs in the VHVL confirm (VH1VL1, VH2VL1, and VH3VL1) show enhanced GFP signaling upon transduction into Jurkat T cells compared to the original non-humanized P329G antigen-binding receptor and, interestingly, the construct in the VLVH confirm (VL1VH3).

[0354] In conclusion, VHVL confirmation appears to be advantageous for determining the expression level of antigen-binding receptors and for correct targeting to the cell surface.

[0355] Furthermore, different tests were conducted to characterize the selectivity, specificity, and safety of the humanized anti-P329G antigen-binding receptor.

[0356] Example 4 Specific T cell activation in the presence of a targeted antibody containing the P329G mutation in the Fc domain. To eliminate nonspecific binding of different humanized anti-P329G-scFv variants, Jurkat NFAT cells expressing antigen-binding receptors containing these variants were evaluated for their activation in the presence of CD20-positive WSUDLCL2 target cells and anti-CD20(GA101) antibodies with different Fc variants (Fc wild-type, Fc P329G mutation, LALA mutation, D246A mutation, or combinations thereof). The CAR-Jurkat NFAT activation assay was performed as described above, and the possibility of nonspecific binding was evaluated using anti-CD20(GA101) wild-type IgG1 (Figure 3A), anti-CD20(GA101) P329G LALA IgG1 (Figure 3B), anti-CD20(GA101) LALA IgG1 (Figure 3D), anti-CD20(GA101) D246A P329G IgG1 (Figure 3F), or nonspecific DP-47 P329G LALA IgG1 (Figure 3E). Nonspecific anti-P329G CAR activation could not be detected for anti-CD20(GA101) wild-type IgG1 (Figure 3A), anti-CD20(GA101) LALA IgG1 (Figure 3D), or nonspecific DP-47 P329G LALA IgG1 (Figure 3E).

[0357] Specific anti-P329G CAR activation was detected in the presence of anti-CD20(GA101)P329G LALA IgG1 (Figure 3B) and anti-CD20(GA101)D246A P329G IgG1 (Figure 3F). The evaluated EC 50 This is equivalent among all humanized anti-P329G variants, and the EC of the original binder is the same. 50 It was no different.

[0358] Interestingly, antigen-binding receptors containing the scFv binder in the VHVL conformation result in stronger activation of Jurkat NFAT T cells compared to the original non-humanized binder and the humanized binder in the VLVH conformation. Higher plateaus (see, e.g., Figure 3F) may be attributed to improved expression levels and / or improved transport to the cell surface of the antigen-binding receptor, leading to stronger activation. Furthermore, conformation may influence binding to the P329G mutation.

[0359] To investigate the risk of clustering of potential antigen-binding domains leading to T cell tonic signaling or nonspecific activation, the Jurkat NFAT activation assay was performed as described above, but with increased initial antibody concentrations, serial dilutions initiated with 100 nM GA101 P329G LALA IgG1, and no target cell seeding.

[0360] As shown in Figure 3C, no activation was detected for any of the tested humanized P329G variants, and they showed detectable receptor clustering or nonspecific activation in the absence of target cells.

[0361] Example 5 Sensitivity of various humanized P329G antigen-binding receptor variants as assessed by T cell activation against target cells expressing various levels of antigen. Furthermore, to characterize the sensitivity and selectivity of the humanized anti-P329G antigen-binding receptor, the Jurkat NFAT activation assay was performed as described above.

[0362] Jurkat NFAT reporter cells expressing different humanized anti-P329G-scFv variant antigen-binding receptors were evaluated for their ability to identify high (HeLa-FolR1), moderate (Skov3), and low (HT29) FolR1-positive target cells. Different variants of anti-P329G binders were used as scFv antigen recognition scaffolds in Jurkat-Reporter cell lines, combined with antibodies that yielded high (16D5) (Figure 4A, D, G), moderate (16D5 W96Y) (Figure 4B, E, H), or low (16D5 G49S / K53A) (Figure 4C, F, I) affinity for FolR1. Highly expressed target cells HeLa-FolR1, combined with high anti-FolR1 16D5 (Figure 4A), moderate anti-FolR1 16D5 W96Y (Figure 4B), and low affinity adapter IgG anti-FolR1 G49S K53A (Figure 4C), showed dose-dependent activation. Medium-expression target cells Skov3, combined with high anti-FolR1 16D5 (Figure 4D), moderate anti-FolR1 16D5 W96Y (Figure 4E), and low affinity adapter IgG anti-FolR1 G49S K53A (Figure 4F), also showed dose-dependent activation. Low-expression target cells HT29, combined with different affinity binders anti-FolR1 16D5 (Figure 4G), anti-FolR1 16D5 W96Y (Figure 4H), or low affinity adapter IgG anti-FolR1 G49S K53A (Figure 4I), did not show any signal. Furthermore, interestingly, the VHVL-format antigen-binding receptor resulted in higher activation of Jurkat NFAT T cells compared to the original unhumanized binder and the VLVH-format humanized binder. The humanized variant VH3VL1 scFv binder yielded the highest signal intensity among all constructs (Figures 4A-F).

[0363] Furthermore, the Jurkat NFAT activation assay was used in combination with either anti-FolR1 16D5 P329G LALA IgG1 (Figure 5) or anti-HER2 P329G LALA IgG1 (Figure 6) to test for HeLa(FolR1 + and HER2 +The tests were performed on cells. Both studies support the finding that VHVL orientation is superior to VLVH orientation. The humanized variant VH3VL1 resulted in the strongest activation of Jurkat NFAT T cells.

[0364] Example 6 Further expression of anti-P329G antigen-binding receptors in Jurkat-NFAT cells Disulfide-humanized anti-P329G antigen-binding receptors were virally transduced into Jurkat (GloResponse Jurkat NFAT-RE-luc2P, Promega#CS176501) cells. Anti-P329G antigen-binding receptor expression was evaluated by flow cytometry. Jurkat cells using disulfide-stabilized humanized anti-P329G antigen-binding receptors were harvested, washed with PBS, and seeded at 50,000 cells / well in 96-well flat-bottom plates. After staining with antibodies containing the P329G mutation in the Fc domain at different concentrations (1:10 serial dilutions from 600 nM to 0 nM) in the dark and refrigerated (4-8°C) for 45 minutes, the samples were washed three times with FACS buffer (PBS containing 2% FBS, 10% 0.5 M EDTA, pH 8, and 0.5 g / L NaN3). Next, the samples were stained with 2.5 μg / mL of polyclonal anti-human IgG Fcγ fragment-specific and PE-conjugated AffiniPure F(ab')2 goat fragment antibody in the dark in a refrigerator for 30 minutes and analyzed by flow cytometry (Fortessa BD). Furthermore, the anti-P329G antigen-binding receptor contained an intracellular GFP reporter (see Figure 1C). CAR expression was normalized to the GFP signal.

[0365] Compared to the humanized versions VH3VL1 and VL1VH3, the disulfide-stabilized P329G binder exhibits CAR labeling comparable to that on the cell surface (Figure 7).

Claims

1. An antigen-binding receptor comprising an anchoring transmembrane domain and an extracellular domain, wherein the extracellular domain includes an antigen-binding portion that binds to an antibody comprising an Fc domain containing the amino acid mutation P329G according to EU numbering, and the antigen-binding portion (i) A heavy chain variable domain (VH) comprising the heavy chain complementarity determination region (HCDR) 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2 or SEQ ID NO: 40, and HCDR 3 of SEQ ID NO: 3, (ii) Light chain variable domain (VL) including light chain complementarity determination region (LCDR) 1 of SEQ ID NO: 4, LCDR 2 of SEQ ID NO: 5, and LCDR 3 of SEQ ID NO: 6 Antigen-binding receptors, including those mentioned above.

2. The antigen-binding receptor according to claim 1, wherein the VH domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 41, SEQ ID NO: 44, and SEQ ID NO:

126.

3. The antigen-binding receptor according to claim 1 or 2, wherein the VL domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO:

127.

4. The antigen-binding receptor according to any one of claims 1 to 3, wherein the anchoring transmembrane domain is a transmembrane domain selected from the group consisting of CD8, CD4, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10, or DAP12 transmembrane domains.

5. The antigen-binding receptor according to any one of claims 1 to 4, further comprising at least one stimulus signaling domain and / or at least one co-stimulus signaling domain.

6. The antigen-binding receptor according to claim 5, wherein the at least one stimulus 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 that retain stimulus signaling activity.

7. The antigen-binding receptor according to claim 5, wherein the at least one co-stimulation 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 that retain co-stimulation signaling activity.

8. An antigen-binding receptor according to any one of claims 1 to 7, comprising at least one CD28 co-stimulating domain or a fragment thereof that retains CD28 co-stimulating activity, and / or at least one CD137 co-stimulating domain or a fragment thereof that retains CD137 co-stimulating activity.

9. An antigen-binding receptor according to any one of claims 1 to 8, comprising a stimulus signaling domain containing the intracellular domain of CD3z or a fragment thereof that retains CD3z stimulus signaling activity, and comprising a co-stimulation signaling domain containing the intracellular domain of CD28 or a fragment thereof that retains CD28 co-stimulation signaling activity.

10. An antigen-binding receptor according to any one of claims 1 to 8, comprising one stimulus signaling domain containing the intracellular domain of CD3z or a fragment thereof that retains CD3z stimulus signaling activity, and comprising one co-stimulation signaling domain containing the intracellular domain of CD137 or a fragment thereof that retains CD137 co-stimulation signaling activity.

11. The antigen-binding receptor according to any one of claims 1 to 10, wherein the antigen-binding portion is linked at the C-terminus to the N-terminus of the anchoring transmembrane domain.

12. The antigen-binding receptor according to any one of claims 1 to 11, wherein the light chain variable domain (VL) of the antigen-binding portion is ligated at its C-terminus to the N-terminus of the anchoring transmembrane domain, and / or the heavy chain variable domain (VH) is ligated at its C-terminus to the N-terminus of the light chain variable domain (VL).

13. Transduced T cells capable of expressing the antigen-binding receptor described in any one of claims 1 to 12.

14. An isolated polynucleotide encoding an antigen-binding receptor according to any one of claims 1 to 12.

15. A vector, particularly an expression vector, comprising the polynucleotide described in claim 14.

16. (A) Transduced T cells capable of expressing the antigen-binding receptor described in any one of claims 1 to 12, (B) An antibody that binds to a target cell antigen and contains an Fc domain containing the amino acid mutation P329G according to EU numbering. Pharmaceuticals, including

17. (A) an isolated polynucleotide encoding an antigen-binding receptor according to any one of claims 1 to 12, (B) An antibody that binds to a target cell antigen and contains an Fc domain containing the amino acid mutation P329G according to EU numbering. Pharmaceuticals, including

18. The pharmaceutical product according to claim 16 or 17, wherein the target cell antigen is selected from the group consisting of fibroblast-activating protein (FAP), carcinoembryonic antigen (CEA), mesothelin (MSLN), CD20, folate receptor 1 (FOLR1), and tenascin (TNC).

19. A pharmaceutical product according to any one of claims 16 to 18, for use in the treatment of a disease.

20. Transduced T cells according to claim 13, for use as a pharmaceutical, to be administered before, simultaneously with, or after administration of an antibody that binds to a target cell antigen and contains an Fc domain including an amino acid mutation P329G by EU numbering.

21. Transduced T cells according to claim 13 for use in the treatment of cancer, wherein the treatment comprises administering the transduced T cells before, simultaneously with, or after administration of an antibody that binds to a target cell antigen and contains an Fc domain including an amino acid mutation P329G by EU numbering.

22. A pharmaceutical product comprising transduced T cells capable of expressing an antigen-binding receptor according to any one of claims 1 to 12 for the treatment of a disease in a target, wherein a therapeutically effective amount of an antibody comprising an Fc domain that binds to a target cell antigen and includes an amino acid mutation P329G according to EU numbering is administered before, simultaneously with, or after administration of the transduced T cells.

23. A composition comprising transduced T cells capable of expressing an antigen-binding receptor according to any one of claims 1 to 12 for inducing the lysis of target cells, wherein the target cells are in contact with the transduced T cells in the presence of an antibody that binds to a target cell antigen and comprises an Fc domain containing an amino acid mutation P329G by EU numbering.

24. Use of an antigen-binding receptor according to any one of claims 1 to 12, a polynucleotide according to claim 14, or a transduced T cell according to claim 13 for the manufacture of a pharmaceutical product for the treatment of cancer.

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