Improved Chimeric Receptors

Abstract

The present invention generally relates to a chimeric receptor comprising an extracellular domain that comprises a variant Fc domain. The present invention also relates to transduced immune cells expressing the chimeric receptor of the invention and / or a nucleic acid molecule encoding the chimeric receptor of the invention. Furthermore, kits comprising such cells and / or a nucleic acid molecule encoding the chimeric receptor, as well as an antibody capable of binding to the chimeric receptor, are also provided.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention generally relates to a chimeric receptor comprising an extracellular domain that comprises a variant Fc domain. The present invention also relates to transduced immune cells expressing the chimeric receptor of the invention and / or a nucleic acid molecule encoding the chimeric receptor of the invention. Furthermore, kits comprising such cells and / or a nucleic acid molecule encoding the chimeric receptor, as well as an antibody capable of binding to the chimeric receptor, are also provided. [Background technology]

[0002] background Adoptive T cell therapy (ACT) is a powerful treatment approach using cancer-specific T cells (Rosenberg and Restifo, Science 348(6230)2015:62-68). ACT may use naturally occurring tumor-specific cells or T cells made tumor-specific by genetic engineering with 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 aggressive or otherwise treatment-resistant diseases, such as acute lymphoblastic leukemia, non-Hodgkin's 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.33(6)2015:540-549).

[0003] However, ACT is limited by treatment-related toxicity, despite its remarkable clinical efficacy. The specificity of engineered T cells used in ACT and the resulting on-target and off-target effects are mainly driven by the tumor-targeting antigen-binding moiety introduced into the antigen-binding receptor (Hinrichs et al., Nat Biotechnol. 31(11)2013, 999-1008). Non-exclusive expression of tumor antigens or time lag in expression levels may cause severe side effects or even lead to the discontinuation of ACT due to unacceptable toxicity of treatment. In fact, the progress of conventional CAR-T cells, which target epithelial tumor antigens, except for some lineage-specific antigens such as CD19, CD20 or BCMA, which are also frequently expressed in normal tissues and result in on-target / off-tumor toxicity, has been slow, especially in solid tumors, which has been shown, for example, in the case of CAR-T cells targeting HER2 (Morgan et al., Mol Ther 18(4)1020:843-851). Therefore, methods to make CAR-T cells more tumor-specific have great therapeutic potential.

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

[0005] One approach to suppress severe treatment-related toxicity and improve the safety of ACT is to limit T cell activation and proliferation by introducing adaptor molecules that control the formation of immune synapses. Such adaptor molecules include, for example, small molecule bimodal switches, such as the recently described folate-FITC switch (Kim et al. J Am Chem Soc 2015;137:2832-2835). Further approaches included artificially modified antibodies containing tags that induce the specificity of T cells 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. The immune synapse, which relies on molecular switches, requires the introduction of additional elements that may induce immune responses or have nonspecific off-target effects. Meanwhile, the introduction of tag structures into existing therapeutic monoclonal antibodies may affect the efficacy and safety profile of these constructs. Moreover, the addition of tags requires additional modification and purification steps that make the production of such antibodies more complicated, as well as additional safety testing.

[0007] Another limitation of existing approaches is the possibility of on-target / off-tumor effects. In most indications, the antigen of interest is highly expressed on tumor cells but also on healthy tissues, so the perfect target has not been found. To reduce side effects and increase the options of target antigens that can lead to the development of new drugs, it is desirable to activate only cancer-specific T cells locally. To increase the selectivity of CAR T cells to activate only in the tumor, various approaches have been developed, for example, oxygen-sensitive CARs (Juillerat A et al.Sci Rep.2017;7:39833.Published 2017 Jan 20.doi:10.1038 / srep39833). In addition, direct CARs with protease-sensitive linkers have also been developed to improve the safety of conventional ACTs (Han X et al. Mol Ther. 2017;25(1):274-284. doi:10.1016 / j.ymthe.2016.10.011). However, tumor-activated CARs are complex and often depend on tumor-specific activation triggers, such as tumor-associated proteases. Due to the heterogeneity of cancer, the presence of these activation triggers can vary greatly among different tumor lesions, cancer types, and patients.

[0008] Adapter molecules, otherwise biologically inactive, can specifically interact with engineered chimeric receptors, allowing dose-dependent control of T cell activation. Thus, such adapter approaches provide an alternative, comprehensive strategy to control therapy-related side effects. However, engineered orthogonal ligand / receptor systems are often based on mutated ligand-receptor interfaces that carry the risk of immunogenicity when administered to patients. This can lead to the formation of anti-drug antibodies that can potentially result in loss of exposure of the therapeutically active components of cell therapy.

[0009] Therefore, improved therapies to address the challenges of ACT are needed. Summary of the Invention

[0010] Summary of the Invention The present invention provides chimeric receptors with improved properties as described herein below.

[0011] In this specification, (i) an extracellular domain comprising a variant Fc domain or a fragment thereof; (ii) a transmembrane domain; A chimeric receptor is provided, comprising:

[0012] In one embodiment, the chimeric receptor further comprises (iii) at least one intracellular stimulatory signaling domain and / or at least one costimulatory signaling domain.

[0013] In one embodiment, the extracellular domain comprises a mutated CH2 domain and / or a mutated CH3 domain.

[0014] In one embodiment, the extracellular domain comprises a mutated CH2 domain and / or a mutated CH3 domain, wherein the mutated CH2 domain and / or the mutated CH3 domain has the following amino acid substitutions (numbering according to Kabat EU index): (a) I253A, H310A and H435A, (b) I253A, (c) I253A and H310A, (d) I253A and H435A, (e) H310A and H435A, (f) H310A, (g) H435A, (h) L234A, L235A, and P329G, or (i) L234A, L235A, I253A, H310A, P329G and H435A Includes.

[0015] In one embodiment, the transmembrane domain is selected from the group consisting of a CD8, CD4, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10 or DAP12 transmembrane domain or a fragment thereof.

[0016] In one embodiment, the stimulatory signaling domain is selected from the group consisting of the intracellular domains of CD3z, FCGR3A and NKG2D, or fragments thereof that retain stimulatory signaling activity.

[0017] In one embodiment, the costimulatory signaling domain is selected from the group consisting of the intracellular domains of CD27, CD28, CD137, OX40, ICOS, DAP10, and DAP12, or fragments thereof that retain costimulatory signaling activity.

[0018] In one embodiment, the costimulatory signaling domain is the CD28 intracellular domain or a fragment thereof that retains CD28 costimulatory activity.

[0019] In one embodiment, the chimeric receptor comprises one stimulatory signaling domain comprising the intracellular domain of CD3z or a fragment thereof that retains CD3z stimulatory signaling activity, and the chimeric receptor comprises one costimulatory signaling domain comprising the intracellular domain of CD137 or a fragment thereof that retains CD137 costimulatory signaling activity.

[0020] In one embodiment, the stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:13 and the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:12.

[0021] In one embodiment, the chimeric receptor comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:87, SEQ ID NO:106, SEQ ID NO:125 and SEQ ID NO:127.

[0022] In one embodiment, a chimeric receptor is provided comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:87, SEQ ID NO:106, SEQ ID NO:125 and SEQ ID NO:127.

[0023] In one embodiment, an isolated polynucleotide encoding the chimeric receptor described hereinabove is provided.

[0024] In one embodiment, there is provided a polypeptide encoded by the isolated polynucleotide described herein above.

[0025] In one embodiment, there is provided a vector, particularly an expression vector, comprising a polynucleotide as hereinbefore described.

[0026] In one embodiment there is provided a transduced T cell comprising a polynucleotide as described herein above or a vector as described herein above.

[0027] In one embodiment, a transduced T cell capable of expressing a chimeric receptor as described herein above is provided.

[0028] In one embodiment, (A) a transduced T cell capable of expressing a chimeric receptor as described herein above, wherein the chimeric receptor comprises an extracellular domain that comprises a variant Fc domain or a fragment thereof; (B) an antibody comprising at least one antigen-binding moiety capable of binding to the variant Fc domain but incapable of binding to the non-variant parent Fc domain, the antibody comprising at least one effector moiety.

[0029] In one embodiment, (A) an isolated polynucleotide encoding a chimeric receptor as described herein above, wherein the chimeric receptor comprises an extracellular domain that comprises a variant Fc domain or a fragment thereof; (B) an antibody comprising at least one antigen-binding moiety capable of binding to the variant Fc domain but incapable of binding to the non-variant parent Fc domain, the antibody comprising at least one effector moiety. In one embodiment, the effector moiety is (i) an antigen-binding moiety capable of binding to a target cell antigen, or (ii) and an immune activating moiety, in particular a cytokine.

[0030] In one embodiment, there is provided a kit as described herein above for use as a medicament.

[0031] In one embodiment there is provided a chimeric receptor as hereinbefore described, or a transduced T cell as hereinbefore described, for use as a medicament, wherein the chimeric receptor comprises an extracellular domain comprising a mutated Fc domain or a fragment thereof, and wherein the transduced T cell expressing the chimeric receptor is administered prior to, simultaneously with, or following administration of an antibody comprising at least one antigen binding moiety capable of binding to a target cell antigen and at least one antigen binding moiety capable of binding to the mutated Fc domain but not to the non-mutated parent Fc domain.

[0032] In one embodiment, there is provided a kit as described hereinabove for use in the treatment of disease, in particular for use in the treatment of cancer.

[0033] In one embodiment, there is provided a method of treating a disease in a subject, comprising administering to the subject transduced T cells capable of expressing a chimeric receptor as hereinbefore described, wherein the chimeric receptor comprises an extracellular domain comprising a mutated Fc domain or a fragment thereof, and administering, prior to, simultaneously with, or after administration of the transduced T cells, a therapeutically effective amount of an antibody comprising at least one antigen binding moiety capable of binding to the mutated Fc domain but not to the non-mutated parent Fc domain, wherein the antibody comprises at least one effector moiety. In one embodiment, the effector moiety is (i) an antigen binding moiety capable of binding to a target cell antigen, or (ii) and an immune activating moiety, in particular a cytokine.

[0034] In one embodiment there is provided the use of a chimeric receptor as described herein, a polynucleotide as described herein or a transduced T cell as described herein for the manufacture of a medicament.

[0035] In one embodiment, the medicament is for the treatment of cancer. In one embodiment, the cancer is selected from cancers of epithelial, endothelial or mesothelial origin and hematological cancers. [Brief description of the drawings]

[0036] [Figure 1] Schematic diagram of a chimeric receptor of the invention comprising a CH2 domain containing a P329G substitution (A) or a CH2-CH3 domain containing the substitutions I253A, H310A, P329R (CH2 domain) and H435A (CH3 domain) (B) and a schematic diagram of the corresponding DNA construct (C). [Diagram 2]Expression of CH2 P329G constructs in Jurkat TCRab KO CD4+ cells (Promega) after lentiviral transduction and pooled selection of eGFP positive cells. Constructs were detected with anti-P329G VH3xVL1 (clone M-1.7.24) humanized IgG labeled with AF647. Schematic of the assay (A). Surface expression of CH2 P329G constructs (B) and corresponding eGFP expression (C). Jurkat TCRab KO CD4+ wild type cells served as negative control. [Diagram 3] Activation of sorted pools of Jurkat TCRab KO CD4+ cells transduced with CH2 P329G construct (SEQ ID NO: 87) in the presence of FolR1+ target cells with high (HeLa), medium (Ovcar3) and low (HT-29) target expression levels upon stimulation with anti-FolR1 x anti-CH2_P329G bispecific antibodies (SEQ ID NO: 108, 109, 110, 111). Activation was assessed by quantification of the intensity of CD3 downstream signaling reported by NFAT promoter controlled luciferase expression. Schematic of the assay (A). Dose-dependent activation of Jurkat cells in the presence of HT29 (B), Ovcar-3 (C) or HeLa (D) as target cells. Mock-transduced cells were used as negative control. Shown are the mean of triplicates of technical values, error bars indicate SD. [Figure 4] Binding of anti-FolR1 x anti-CH2-CH3 AAA P329R bispecific antibodies (SEQ ID NO: 108, 112, 113, 114) to unselected Jurkat NFAT cells (Promega) transduced with the CH2-CH3 AAA P329R construct (SEQ ID NO: 105) via lentivirus. Binding was assessed via secondary R-phycoerythrin (PE) AffiniPure F(ab')2 fragment goat anti-human IgG (F(ab')2 fragment specific). Schematic of the assay (A). Dose-dependent binding to CH2-CH3 AAA P329R construct-transduced Jurkat cells (B). Jurkat NFAT wild type cells were used as a negative control. [Diagram 5]Expression of CH2-CH3 AAA P329R construct (SEQ ID NO: 105) in Jurkat NFAT cells after lentiviral transduction. The construct was detected with anti-AAAxFolR1 2+1 bispecific antibody (SEQ ID NO: 108, 112, 113, 114) and secondary PE AffiniPure F(ab')2 fragment goat anti-human IgG (F(ab')2 fragment specific). Schematic of the assay (A). Surface expression of CH2-CH3 AAA P329R construct (B) and corresponding eGFP expression (C). Jurkat NFAT wild type cells served as negative control. [Figure 6] Activation of unsorted Jurkat NFAT cells transduced with CH2-CH3 AAA P329R construct (SEQ ID NO: 105) in the presence of FolR1+ target cells with high target expression levels (HeLa) in combination with anti-FolR1xAAA 2+1 bispecific antibody (SEQ ID NO: 108, 112, 113, 114). Activation was assessed by quantification of the intensity of CD3 downstream signaling reported by NFAT promoter controlled luciferase expression. Schematic of the assay (A). Dose-dependent activation of Jurkat cells in the presence of HeLa as target cells (B). Jurkat NFAT wild type cells served as negative control. Shown are the mean of triplicates of technical values, error bars indicate SD. [Figure 7] FIG. 7 shows a schematic diagram of the combined use of a chimeric receptor of the invention and an exemplary immunostimulatory Fc binding molecule having an IL2v (cytokine) effector moiety. [Figure 8A-8D] Figures 8A-8B show schematic diagrams of chimeric receptors of the invention comprising a CH2 domain containing the P329G mutation (Figure 8A) or a CH2-CH3 domain containing the mutations I253A, H310A, P329G (CH2 domain) and H435A (CH3 domain) (Figure 8B). Figures 8C-8D show schematic diagrams of the corresponding DNA constructs. Furthermore, the DNA constructs used in the appended examples contained an eGFP-encoding gene separated from the chimeric receptor via a 2A self-cleaving peptide. [Figure 9A-9B]Primary T cells expressing the chimeric receptors of the invention. Human CD4 and CD8 T cells were transduced with either the CH2-CH3 AAA P329G CAR (SEQ ID NO: 127), the CH2-CH3 AAA P329R CAR (SEQ ID NO: 125) or the CH2 P329G CAR (SEQ ID NO: 87), respectively. Expression of the constructs was assessed by eGFP expression and surface staining of the CAR. For the latter, staining was performed with either anti-PG VH3xVL1 huIgG-AF647 (CH2-CH3 AAA P329G CAR, CH2 P329G CAR) (Figure 9A) or anti-AAAxFolR1 2+1 TCB and secondary APC-F(ab)2 fragment anti-huIgG (Jackson ImmunoResearch, #109-136-097) (CH2-CH3 AAA P329G CAR, CH2-CH3 AAA P329R CAR) (Figure 9B). 86-89% of primary T cells expressed the chimeric receptor. [Figure 10A-10F] T cell-mediated killing. CH2-CH3 AAA P329G CAR (SEQ ID NO: 127, Figures 10A-B) / CH2-CH3 AAA P329R CAR (SEQ ID NO: 125, Figures 10C-D) or CH2 P329G CAR T cells (SEQ ID NO: 87, Figures 10E-F) were incubated with HeLa NLR (NucLight Red) cells. Anti-AAAxFolRI 2+1 / Anti-AAAxFolRI 1+1 or Anti-PGxFolRI 2+1 / Anti-PGxFolRI 1+1 antibodies served as adapter molecules. Red blood cell counts were monitored in an Incucyte® system over a period of 6 days. Wells without adapter molecules served as negative controls and 10 nM anti-FolR1 TCB served as a positive control. A dose-dependent reduction in red blood cell counts (cancer cell killing) or cancer cell growth was observed. Technical average values ​​from replicates are shown, error bars indicate SD. [Figures 11A-11C]T cell-mediated killing. CH2-CH3 AAA P329G CAR (SEQ ID NO: 127, FIG. 11A) / CH2-CH3 AAA P329R CAR (SEQ ID NO: 125, FIG. 11B) or CH2 P329G CAR T cells (SEQ ID NO: 87, FIG. 11C) were incubated with MKN45 NLR (NucLight Red) cells. Anti-AAA×CEA 2+1 or anti-PG×CEA 2+1 antibodies served as adapter molecules. Wells without adapter molecules served as negative controls and 10 nM anti-CEA TCB served as positive controls. A dose-dependent reduction in red blood cell counts (cancer cell killing) or cancer cell growth was observed. Technical mean values ​​from replicates are shown and error bars indicate SD. [Figure 12A-12B] CH2-CH3 AAA P329G CAR (SEQ ID NO: 127) / CH2-CH3 AAA P329R CAR (SEQ ID NO: 125) or CH2 P329G CAR (SEQ ID NO: 87) T cells were stained with anti-PG and anti-AAA antibodies to evaluate whether the AAA and P329G mutations can be bound simultaneously (Figure 12A). Figure 12B shows double positive (BV421+, AF647+) CAR-T cells, indicating that the AAA and P329G mutations can be bound simultaneously. CH2-CH3 AAA P329R CAR or CH2 P329G CAR T cells showed only single staining of each, as expected. [Figure 13A-13D]STAT5 phosphorylation (pSTAT5) of primary T cells transduced with CH2-CH3 AAA P329G CAR (SEQ ID NO: 127) / CH2-CH3 AAA P329R CAR (SEQ ID NO: 125) or CH2 P329G CAR (SEQ ID NO: 87) after stimulation with anti-P329G-IL2v. Median pSTAT5 fluorescence intensity after gating on eGFP+ (CAR+) or eGFP- (CAR-) cells is shown. Targeting of anti-P329G-IL2v to CAR T cells via the P329G mutation results in an approximately 50-fold (CH2 PG CAR, FIG. 13A) or 225-fold (CH2 CH3 AAA PG CAR, FIG. 13B) difference in EC50 compared to eGFP- cells in the same well. This effect was not observed in CH2 CH3 AAA PR CAR T cells as expected (FIG. 13C). pSTAT5 MFI of cells incubated with Proleukin was comparable between cell products and between eGFP+ and eGFP- (Figure 13D). Technical average values ​​from replicates are shown, error bars indicate SD. EC50 values ​​were calculated using GraphPad Prism 8.4.2 (log(agonist) vs. response--variable slope (4 parameters)). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description definition Unless otherwise defined below, terms are used herein as commonly used in the art.

[0038] For purposes herein, an "acceptor human framework" is a framework that comprises 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. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes 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 identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0039] An "activating Fc receptor" is an Fc receptor that, following binding of the Fc domain of an antibody, triggers signaling events that stimulate a receptor-bearing cell to carry out an effector function. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32) and FcαRI (CD89).

[0040] "Antibody-dependent cell-mediated cytotoxicity" ("ADCC") is an immune mechanism that leads to the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which an antibody or derivative thereof that contains an Fc region specifically binds, usually via a protein portion that is N-terminal to the Fc region. As used herein, the term "reduced ADCC" is defined as either a reduction in the number of target cells that are lysed in a given time at a given antibody concentration in the medium surrounding the target cells by the mechanism of ADCC as defined above, and / or an increase in the antibody concentration in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time by the mechanism of ADCC. The reduced ADCC is relative to the ADCC mediated by the same antibody, but not engineered, produced by the same type of host cell, using the same standard production, purification, formulation and storage methods, which are known to those skilled in the art. For example, the reduced ADCC mediated by an antibody that contains an ADCC-reducing amino acid substitution in the Fc domain is relative to the 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, WO 2006 / 082515 or WO 2012 / 130831).

[0041] An "effective amount" of an agent, eg, a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0042] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of 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 a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K DAffinity can be measured by common methods known in the art, including those described herein. Specific illustrative exemplary methods for measuring binding affinity are described below.

[0043] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that differs from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid. Amino acids may be represented herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0044] The term "amino acid mutation" as used herein is meant to encompass amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct has the desired characteristics (e.g., reduced binding to Fc receptors or increased association with another peptide). Deletions and insertions in the amino acid sequence include amino- and / or carboxy-terminal deletions and insertions of amino acids. Particular amino acid mutations are amino acid substitutions. For example, to alter the binding properties of the Fc region, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include replacement with non-naturally occurring amino acids or with naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that methods of modifying the side chain groups of amino acids other than genetic engineering, such as chemical modification, may also be useful. Various names may be used herein to indicate the same amino acid mutation. For example, a proline to glycine substitution at position 329 of the Fc domain is represented by 329G, G329, G 329 , P329G or Pro329Gly.

[0045] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0046] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an 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 (dAbs), and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0047] The term "antigen-binding domain" refers to a part of an antibody that comprises an area that specifically binds to and is complementary to part or all of an antigen. An antigen-binding domain may, for example, be provided by one or more antibody variable domains (also called antibody variable regions). In particular, an antigen-binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0048] As used herein, the term "antigen-binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are immunoglobulins and derivatives thereof, such as fragments thereof, and antigen-binding receptors and derivatives thereof.

[0049] As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding moiety can direct the entity to which it binds to a target site, for example, to a particular type of tumor cell or tumor stroma that bears the antigenic determinant. Antigen-binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen-binding moieties include the antigen-binding domain of an antibody (e.g., an scFv fragment), which includes an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, an 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 the five isotypes: alpha, delta, epsilon, gamma, or mu. Useful light chain constant regions include any of the two isotypes: kappa and lambda.

[0050] In the context of the present invention, the term "chimeric receptor" relates to an engineered receptor comprising an extracellular domain, including a variant Fc domain, in particular a variant CH2 and / or CH3 domain, a transmembrane domain, and at least one intracellular stimulatory signaling domain and / or at least one costimulatory signaling domain. Chimeric receptors can be made from polypeptide moieties from different sources. Thus, antigen-binding receptors can also be understood as "fusion proteins" and / or "chimeric proteins". Typically, a fusion protein is a protein made by joining two or more genes (preferably cDNAs) that originally encoded separate proteins. Translation of this fusion gene (or fusion cDNA) preferably results in a single polypeptide with functional properties derived from each of the original proteins. Recombinant fusion proteins are artificially made by recombinant DNA technology for use in biological research or therapy. Further details of the chimeric receptors of the present invention are given later in this specification.

[0051] "Antigen-binding site" refers to a site 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). A naturally occurring immunoglobulin molecule typically has two antigen-binding sites, and a Fab molecule typically has a single antigen-binding site.

[0052] The term "antigen-binding domain" refers to a portion of an antibody or antigen-binding receptor that comprises an area that specifically binds to and is complementary to a part or all of an antigen. An antigen-binding domain may be provided, for example, by one or more immunoglobulin variable domains (also called variable regions). In particular, antigen-binding domains include immunoglobulin light chain variable domains (VL) and immunoglobulin heavy chain variable domains (VH).

[0053] 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 to form an antigen-binding moiety-antigen complex (e.g., a conformational configuration composed of a contiguous stretch of amino acids or distinct regions of non-contiguous amino acids). Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virally infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise specified, a protein referred to herein as an antigen refers to the native form of the protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is a human protein. When a particular protein is referred to herein, the term encompasses not only the "full-length," unprocessed protein, but also any form of the protein that results from processing within the cell. The term also encompasses naturally occurring protein variants, such as splice variants or allelic variants.

[0054] An "antibody" according to the invention, i.e. a therapeutic antibody, may have one, two, three or more binding domains and may be monospecific, bispecific or multispecific. Antibodies may be full-length from a single species or may be chimerized or humanized. In the case of antibodies with more than two antigen-binding domains, some of the binding domains may be identical and / or have the same specificity.

[0055] The term "transmembrane domain" or "TD" for short as used herein defines a polypeptide stretch that can be integrated into the cell membrane (or plasma membrane) of a cell. The transmembrane domain can be fused to an extracellular and / or intracellular polypeptide domain, which is localized to the cell membrane. In the context of the chimeric receptor of the present invention, the transmembrane domain provides membrane attachment and localization of the chimeric receptor of the present invention. The chimeric receptor of the present invention comprises at least one transmembrane domain and an extracellular domain comprising a CH2 domain. Furthermore, the transmembrane domain can be fused to an intracellular signaling domain.

[0056] The ability of an antigen-binding moiety to bind to a particular antigenic determinant can be measured by either enzyme-linked immunosorbent assay (ELISA) or other techniques known to those of skill in the art, such as surface plasmon resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of the antigen-binding moiety to an unrelated protein is less than about 10% of the binding of the antigen-binding moiety to the antigen as measured, for example, by SPR. In certain embodiments, an antigen-binding moiety that binds to an antigen, or an antigen-binding molecule comprising an antigen-binding moiety, has an affinity for an antigen of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9M~10 -13 Dissociation constant (K D ).

[0057] The term "CDR" as used herein relates to "complementarity determining region" as known in the art. CDRs are the parts of an immunoglobulin or antigen-binding receptor that determine the specificity of the molecule and contact a specific ligand. CDRs are the most variable parts of the molecule and contribute to the antigen-binding diversity of such molecules. In each V domain, there are three CDR regions CDR1, CDR2 and CDR3. CDR-H represents the CDR region of the variable heavy chain and CDR-L represents the CDR region of the variable light chain. VH means variable heavy chain and VL means variable light chain. The CDR regions of the Ig-derived regions can be determined as described in "Kabat" (Sequences of Proteins of Immunological Interest", 5th ed., NIH Publication no. 91-3242 USDepartment of Health and Human Services (1991); Chothia J. Mol. Biol. 196 (1987), 901-917) or "Chothia" (Nature 342 (1989), 877-883).

[0058] The term "CD3z" refers to the T cell surface glycoprotein CD3 zeta chain, also known as the "T cell receptor T3 zeta chain" and "CD247."

[0059] The term "chimeric antigen receptor" or "CAR" refers to an antigen-binding receptor composed of an extracellular portion of an antigen-binding moiety (e.g., a single-chain antibody domain) fused to an intracellular / co-signaling domain (e.g., those of CD3z and CD28) by a spacer sequence. The chimeric receptor according to the invention comprises a different extracellular domain compared to a CAR, i.e., the extracellular domain of the chimeric receptor according to the invention does not comprise a target antigen-binding moiety (e.g., the chimeric receptor according to the invention does not comprise an scFv). However, the transmembrane domain and intracellular signaling domain comprised in the chimeric receptor according to the invention are similar or identical to the transmembrane domain and intracellular signaling domain that may be used in a CAR.

[0060] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is of the IgG1 isotype. In certain embodiments, the antibody is of the IgG1 isotype with P329G, L234A, and L235A substitutions to reduce Fc region effector function. In other embodiments, the antibody is of the IgG2 isotype. In certain embodiments, the antibody is of the IgG4 isotype with S228P substitutions in the hinge region to improve the stability of IgG4 antibodies. The heavy chain constant domains corresponding to the 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) and lambda (λ), based on the amino acid sequence of its constant domain.

[0061] The term "constant region of human origin" or "human constant region" as used in this application refers to the constant heavy chain region of a human antibody of subclass IgG1, IgG2, IgG3 or IgG4, and / or the constant light chain kappa or lambda region. Such constant regions can be used in human or humanized antibodies and are well known in the art and described, for example, 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, numbering of amino acid residues in the constant region is according to the EU numbering system (also called the Kabat EU index), as described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0062] By "crossover" Fab molecule (also called "Crossfab") is meant a Fab molecule in which the variable domains of the Fab heavy chain and the variable domains of the Fab light chain are exchanged (i.e. replaced by each other), i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1CH1 (VL-CH1, in the N-terminal to C-terminal direction) and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in the N-terminal to C-terminal direction). For the sake of clarity, in the crossover Fab molecule in which the variable domains of the Fab light chain and the variable domains of the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1CH1 is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0063] As used herein, the term "CSD" refers to a costimulatory signaling domain. An example of a costimulatory signaling domain is the intracellular domain of CD137 or a fragment thereof.

[0064] "Effector function" refers to the biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0065] As used herein, the terms "engineer, engineered, engineering" are intended to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modification of the amino acid sequence, modification of the glycosylation pattern or modification of the side groups of individual amino acids, as well as combinations of these techniques.

[0066] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide having a set of specific nucleic acid elements that allow transcription of a specific nucleic acid in a target cell. A recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, an expression cassette of the invention comprises a polynucleotide sequence encoding a bispecific antigen-binding molecule of the invention or a fragment thereof.

[0067] A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain (a "Fab heavy chain") and the VL and CL domains of a light chain (a "Fab light chain").

[0068] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226 or from Pro230 to the carboxy-terminus of the heavy chain. However, antibodies produced by a host cell may undergo post-translational truncation of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, upon expression of a particular nucleic acid molecule encoding a full-length heavy chain, an antibody produced by a host cell may contain a full-length heavy chain or a truncated variant of the full-length heavy chain (also referred to herein as a "truncated variant heavy chain"). This is the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present. The amino acid sequence of a heavy chain comprising an Fc domain (or a subunit of an Fc domain as defined herein) is shown herein without the C-terminal glycine-lysine dipeptide, unless otherwise indicated. In one embodiment of the invention, a heavy chain comprising a subunit of an Fc domain as defined herein comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment of the invention, a heavy chain comprising a subunit of an Fc domain as defined herein comprises an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). The compositions of the invention, such as the pharmaceutical compositions described herein, comprise a population of antigen-binding molecules of the invention. The population of antigen-binding molecules may comprise molecules having full-length heavy chains and molecules having truncated variant heavy chains.The population of antigen-binding molecules may be composed of a mixture of molecules with full-length heavy chains and molecules with truncated variant heavy chains, where at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the antigen-binding molecules have truncated variant heavy chains. In one embodiment of the invention, a composition comprising a population of antigen-binding molecules of the invention comprises antigen-binding molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment of the invention, a composition comprising a population of antigen-binding molecules of the invention comprises immunostimulatory Fc domain-binding molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). In one embodiment of the invention, such compositions comprise a population of antigen-binding molecules comprised of molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein; molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to the EU index of Kabat); and molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the EU index of Kabat). Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system, also referred to as 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 supra). As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association.For example, the subunits of the IgG Fc domain include the IgG CH2 and IgG CH3 constant domains.

[0069] "Framework" or "FR" refers to variable domain residues other than the complementarity determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, the CDR and FR sequences generally appear in the VH (or VL) in the following sequence: FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0070] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or an antibody having a heavy chain that includes an Fc region as defined herein.

[0071] By "fused" it is meant that the components (eg, a Fab and a transmembrane domain) are linked together by a peptide bond, either directly or via one or more peptide linkers.

[0072] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0073] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or an antibody derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0074] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is derived from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda MD (1991), 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0075] A "humanized" antibody (e.g., a humanized scFv fragment) refers to a chimeric antibody comprising amino acid residues of non-human CDRs and amino acid residues of human FRs. In certain aspects, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDRs corresponding to the CDRs of a non-human antibody and all or substantially all of the FRs corresponding to the FRs of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a humanized antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been subjected to humanization.

[0076] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is hypervariable in sequence and which determines antigen-binding specificity, e.g., the "complementarity determining regions" (CDRs).

[0077] Generally, an antibody comprises six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include: (a) hypervariable loops located 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 located at 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 sites located at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0078] Unless otherwise specified, CDRs are determined according to Kabat et al., supra. Those skilled in the art will appreciate that the nomenclature of CDRs may also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature.

[0079] As used herein, an "immunostimulatory moiety" refers to one or more polypeptides that induce activation of an immune cell (e.g., a T cell) upon interaction with an antigen, receptor or ligand on the immune cell (or other element of the cell that induces activation). Exemplary immune activating moieties are antigen binding moieties capable of binding to a cytokine (e.g., IL2), a costimulatory T cell antigen (e.g., CD28, 4-1BB) or a costimulatory ligand (e.g., 4-1BBL) as described herein and in WO 2021 / 255138.

[0080] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent.

[0081] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, an individual or subject is human.

[0082] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0083] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons composed of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called the variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called the variable light domain or light chain variable region, followed by a constant light domain (CL), also called the light chain constant region. The heavy chains of immunoglobulins can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which can be further classified into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1) (IgA1) and α2) (IgA2). The light chains of immunoglobulins may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain. Immunoglobulins essentially consist of two Fab molecules linked via an immunoglobulin hinge region and an Fc domain.

[0084] An "isolated nucleic acid" molecule or polynucleotide is a DNA or RNA that is a nucleic acid molecule that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or purified (partially or substantially) in solution. An isolated polynucleotide includes a polynucleotide molecule contained in a cell that normally contains the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that differs from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative stranded forms, and double-stranded forms. Additionally, isolated polynucleotides or nucleic acids of the present invention include such molecules produced synthetically. In addition, the polynucleotide or nucleic acid may be or may include a regulatory element, such as a promoter, a ribosome binding site, or a transcription terminator.

[0085] A nucleic acid or polynucleotide having a nucleotide sequence that is, for example, at least 95% "identical" to a reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the nucleotide sequence of the polynucleotide 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 replaced with another nucleotide, or up to 5% of the number of nucleotides in the reference sequence may be inserted into the reference sequence. Such modifications of 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, either individually interspersed among residues in the reference sequence or interspersed in one or more contiguous groups in the reference sequence. As a practical matter, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be conventionally determined using known computer programs such as those discussed below for polypeptides (e.g., ALIGN-2).

[0086] An "isolated" polypeptide or variant or derivative thereof refers to a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposes of the present invention, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0087] A "modification that promotes the association of a first and a second subunit of an Fc domain" is a peptide backbone manipulation or a post-translational modification of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form homodimers. As used herein, a modification that promotes association particularly includes separate modifications made to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to be associated, which are complementary to each other to promote the association of the two Fc domain subunits. For example, a modification that promotes association can change the structure or charge of one or both of the Fc domain subunits to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may not be identical in the sense that the further components (e.g., antigen-binding moieties) fused to each of the subunits are not the same. In some embodiments, a modification that promotes association includes amino acid mutations, particularly amino acid substitutions, within the Fc domain. In certain embodiments, the association-promoting modifications comprise separate amino acid mutations, specifically amino acid substitutions, in each of the two subunits of the Fc domain.

[0088] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up the population are identical and / or bind the same epitope, except for possible variant antibodies that are generally present in small amounts, including, for example, naturally occurring mutations or mutations that arise during the manufacture of the monoclonal antibody preparation. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not constructed to require production of the antibody by any particular method. For example, monoclonal antibodies according to the 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 the human immunoglobulin loci, such methods, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0089] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0090] "Native antibody" refers to naturally occurring immunoglobulin molecules with various structures. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called the variable heavy domain or heavy chain variable region, followed by three constant heavy chain domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called the variable light domain or light chain variable region, followed by a constant light chain (CL) domain.

[0091] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity for alignment. Alignment to determine percent amino acid sequence identity can be achieved using a variety of methods within the skill of the art, such as 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 aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. Alternatively, percent identity values ​​can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc. and the source code is on file in the user documentation of the U.S. Copyright Office, Washington DC, 20559, registered under U.S. Copyright Registration No. TXU510087, and described in WO 2001 / 007611.

[0092] Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c, or subsequent BLOSUM50 comparison matrices. The FASTA program package is written by WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448, WR Pearson (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, sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch (global protein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2) to ensure a global, rather than local, alignment. The percent amino acid identity is given in the output alignment header. The term "nucleic acid molecule" refers to the sequence of bases, including purine and pyrimidine bases, that constitute a polynucleotide, which bases represent the primary structure of the nucleic acid molecule. Here, 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. The term nucleic acid molecule also includes both sense and antisense strands. Additionally, the nucleic acid molecules described herein may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art.

[0093] The term "package insert" is used to refer to instructions typically included in commercial packaging of a therapeutic product that contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings for the use of such therapeutic product.

[0094] The term "pharmaceutical composition" refers to a formulation in a form that effectively utilizes the biological activity of the active ingredient contained therein and does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. A pharmaceutical composition further comprises one or more pharma- ceutically acceptable carriers.

[0095] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, additives, stabilizers, or preservatives.

[0096] As used herein, the term "polypeptide" refers to a molecule consisting of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds).

[0097] The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other terms referring to a chain of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides may be obtained from natural biological sources or produced by recombinant technology, but are not necessarily translated from a designated nucleic acid sequence. Polypeptides may be generated by any method, including chemical synthesis. Polypeptides of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, or 2000 or more amino acids. Polypeptides may, but do not necessarily, have a clearly defined three-dimensional structure: Polypeptides that have a clearly defined three-dimensional structure are said to be "folded" and polypeptides that do not have a clearly defined three-dimensional structure and can adopt a number of different conformations are said to be "unfolded."

[0098] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), viral-derived RNA, or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds as found in peptide nucleic acids (PNA)). The term nucleic acid molecule refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0099] "Reduced binding", e.g., reduced binding to Fc receptors, refers to a reduced affinity for the respective interaction, e.g., as measured by SPR. For clarity, the term also includes a reduction in affinity to zero (or below the detection limit of the analytical method), i.e., a complete loss of interaction. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0100] The term "regulatory sequence" refers to DNA sequences necessary to effect expression of a ligated coding sequence. The nature of such regulatory sequences varies depending on the host organism. In prokaryotes, regulatory sequences usually include a promoter, a ribosome binding site, and a terminator. In eukaryotes, regulatory sequences usually include a promoter, a terminator, and optionally an enhancer, transactivator, or transcription factor. The term "regulatory sequence" is intended to include, at a minimum, all components necessary for expression, and may also include additional advantageous components.

[0101] As used herein, the term "single chain" refers to a molecule that comprises 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 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 in the single chain Fab molecule is linked to the N-terminus of the Fab heavy chain.

[0102] As used herein, the term “SSD” refers to a “co-stimulatory signaling domain.” An example of a stimulatory signaling domain is the intracellular domain of CD3z or a fragment thereof.

[0103] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and may be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and remission or improving prognosis. In some aspects, the antibodies of the invention are used to delay the onset of disease or to slow the progression of the disease.

[0104] As used herein, "T cell activation" refers to one or more cellular responses selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers of T lymphocytes, particularly cytotoxic T lymphocytes. The immunoactivating Fc domain antigen binding molecules of the present invention can induce T cell activation. Suitable assays for measuring T cell activation are known in the art and described herein.

[0105] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the side effects of a disease.

[0106] The term "valency" as used herein indicates that a particular number of antigen-binding sites are present within an antigen-binding molecule. Thus, the term "monovalent binding to an antigen" indicates that one (and not more than one) antigen-binding site specific for that antigen is present within the antigen-binding molecule.

[0107] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have a similar structure, and each domain contains four conserved framework regions (FR) and three complementarity determining regions (CDR). (See, for example, Kindt et al. Kuby Immunology, 6 th ed., W. H. Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind a particular antigen can be isolated using the VH or VL domain from an antibody that binds that antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0108] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0109] Chimeric Receptors of the Invention The present invention relates to chimeric receptors that comprise an extracellular domain that comprises a variant Fc domain or a fragment thereof (such as a CH2 and / or CH3 domain).

[0110] Provided herein is a chimeric receptor comprising a variant Fc domain or fragment thereof and a transmembrane domain.

[0111] The variant Fc domain can specifically interact with an antigen binding molecule, e.g., an antibody according to the invention, to target cells expressing the chimeric receptor of the invention to target cells (e.g., tumor cells) or deliver effector molecules (e.g., cytokines) to cells expressing the chimeric receptor of the invention.

[0112] In one embodiment, the extracellular domain comprises a mutated CH2 domain and / or a mutated CH3 domain. In one embodiment, the extracellular domain comprises a mutated CH2 domain.

[0113] The intracellular portion of a chimeric receptor can confer stimulatory and costimulatory activity to a cell (e.g., a T cell) that contains the chimeric receptor. Accordingly, further provided herein are chimeric receptors that contain at least one intracellular stimulatory signaling domain and / or at least one costimulatory signaling domain.

[0114] The chimeric receptors of the present invention are useful in a number of therapeutic and non-therapeutic applications.

[0115] For example, the chimeric receptors of the invention can be used to direct (or target) immune cells (such as T cells) to target cells via a therapeutic antibody that comprises at least one antigen-binding moiety that can bind to the mutated Fc domain of the chimeric receptor but not to the non-mutated parent Fc domain. In a preferred embodiment, the antibody is a multispecific antibody, i.e., the antibody comprises at least one antigen-binding moiety that can bind to a target cell antigen and at least one antigen-binding moiety that can bind to the mutated Fc domain of the chimeric receptor. The therapeutic antibody can simultaneously bind to the target cell and to the immune cell that contains the chimeric receptor on the immune cell, after which the immune cell is activated (see, e.g., Figures 3A and 6A).

[0116] According to this concept, chimeric receptors comprising variant Fc domains are used to direct effector cells to target cells based on the target specificity of therapeutic antibodies. Chimeric receptors are inactive in the absence of therapeutic antibodies, and importantly, show improved safety profiles. Chimeric receptors of the present invention comprising variant Fc domains cannot bind, for example, in patient tissues. This is in contrast to CARs comprising antigen-binding moieties (e.g., scFvs) that may show off-target or on-target / off-tumor binding, which may result in undesirable toxicity.

[0117] A further exemplary use of the chimeric receptors of the present invention is to specifically target effector molecules (e.g., cytokines) to cells, particularly T cells, that contain the chimeric receptors of the present invention (see, e.g., FIG. 7).

[0118] Notably, CH2 domains containing the P329G substitution (EU numbering) have been tested in clinical trials without inducing anti-drug antibodies. Moreover, the P329G substitution prevents interaction with activating Fc gamma receptors, which are abundantly expressed by various innate immune cells.

[0119] Thus, the present invention provides improved chimeric receptors for the improved treatment of, for example, cancer.

[0120] 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, natural killer (NK) cells, NKT cells or macrophages, preferably CD8+ T cells, with the chimeric receptors described herein and their targeted recruitment, e.g. to tumors, by antibody molecules, e.g. therapeutic antibodies capable of binding to a variant Fc domain.

[0121] As shown in the appended examples, as a proof of concept, a chimeric receptor (encoded by the DNA sequence shown in SEQ ID NO: 96 and shown in FIG. 1A) was constructed that comprises a CH2 domain containing a P329G substitution according to the present invention, capable of binding to a therapeutic antibody as described herein. Transduced T cells (Jurkat NFAT T cells) expressing a construct according to SEQ ID NO: 87 could be strongly activated by co-incubation with an antibody that comprises two antigen-binding moieties capable of binding to FolR1 and one antigen-binding moiety capable of binding to a CH2 domain containing a P329G substitution but not capable of binding to a non-mutated parent Fc domain (see, for example, FIG. 3). The antibody may further comprise a substitution in the Fc domain at position P329 other than glycine (G) to prevent multimerization of the therapeutic antibody (for example, the antibody may further comprise a P329R substitution).

[0122] As a further proof of concept, a chimeric receptor was constructed (encoded by the DNA sequence shown in SEQ ID NO: 107 and shown in FIG. 1B) comprising a CH2-CH3 domain containing AAA substitutions according to the invention capable of binding to a therapeutic antibody described herein. Transduced T cells expressing a construct according to SEQ ID NO: 105 (Jurkat NFAT T cells) could be strongly activated by co-incubation with an antibody comprising two antigen-binding moieties capable of binding to FolR1 and one antigen-binding moiety capable of binding to the CH2-CH3 domain containing I253A, H310A, H435A substitutions but unable to bind to the non-mutated parent Fc domain (see, for example, FIG. 6).

[0123] The inventive concept and its components are described in more detail below.

[0124] In one aspect of the invention, pairing of a tumor-specific antibody described herein with a T cell transduced with a chimeric receptor described herein results in specific activation of the T cell and subsequent lysis of the tumor cell. As previously described herein, this approach has significant safety advantages over conventional T cell-based approaches. Importantly, T cells are inactive in the absence of an antibody capable of binding to the mutant Fc domain.

[0125] For many antigen-binding moieties, it is difficult to completely avoid non-specific binding in patients, which may result in undesirable toxicity. The present invention provides a two-component system in which the antigen-binding moiety-bearing molecule (e.g., therapeutic antibody) has a defined half-life time in vivo. This is in contrast to, for example, CAR-T cells, which are permanently present in patients after application. Thus, the treatment of undesirable toxicity in CAR-T approaches is limited. In contrast, the chimeric receptors provided herein do not contain an antigen-binding moiety, but contain a mutated Fc domain that shows a very well-known activity profile in vivo. This approach offers the possibility of significantly reducing side effects and increasing the safety of treatments based on this.

[0126] The degree of T cell activation can be further modulated by adjusting the dosage of the co-administered therapeutic antibody or by switching to a different antibody specificity or format.

[0127] Mutant Fc domain or fragment thereof In one aspect, a chimeric receptor comprising a variant Fc domain or a fragment thereof is provided. Fc domains and subdomains thereof are well known in the art and are described herein. An Fc domain consists of a pair of polypeptide chains comprising a heavy chain constant domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises a CH2 and a CH3 IgG heavy chain constant domain. The two subunits of an Fc domain are capable of stable association with each other. An extracellular domain according to the present invention comprises one or both subunits of an IgG Fc domain or a fragment thereof (or a dimer of a fragment thereof, e.g., a CH2 dimer). In one aspect, the extracellular domain comprises an IgG Fc domain or a fragment thereof. In a particular embodiment, the extracellular domain comprises an IgG1 Fc domain or a fragment thereof, or an IgG4 Fc domain or a fragment thereof.

[0128] In one embodiment, the variant Fc domain is a variant CH2 domain and / or a variant CH3 domain. In one embodiment, the variant Fc domain is a variant CH2 domain. In one embodiment, the extracellular domain comprises a variant CH2 domain and / or a variant CH3 domain. In one embodiment, the extracellular domain comprises a variant CH2 domain.

[0129] In one embodiment, the variant Fc domain comprises at least one amino acid substitution at a position selected from the group consisting of L234, L235, I253, H310, P331, P329 and H435 (numbering according to Kabat EU index), in particular the amino acid substitutions are L234A, L235A, I253A, N297A, H310A, P329G and / or H435A.

[0130] In a preferred embodiment, the extracellular domain comprises a mutated CH2 domain, in particular the mutated CH2 domain comprises the amino acid substitution P329G (numbering according to Kabat EU index).

[0131] In another preferred embodiment, the extracellular domain comprises a mutated CH2 domain and / or a mutated CH3 domain, in particular a mutated CH2 domain and / or a mutated CH3 domain comprising the following amino acid substitutions (numbering according to Kabat EU index): (a) I253A, H310A and H435A, (b)I253A (c) I253A and H310A (d) I253A and H435A (e) H310A and H435A (f) H310A, or (g)H435A Includes.

[0132] In another preferred embodiment, the extracellular domain comprises a mutated CH2 domain and / or a mutated CH3 domain, in particular a mutated CH2 domain and / or a mutated CH3 domain comprising the following amino acid substitutions (numbering according to Kabat EU index): (a) I253A, H310A and H435A, (b)I253A (c) I253A and H310A (d) I253A and H435A (e) H310A and H435A (f) H310A, (g) H435A, (h) L234A, L235A, and P329G, or (i) L234A, L235A, I253A, H310A, P329G and H435A Includes.

[0133] In one embodiment, the extracellular domain comprising a mutant CH2 domain and / or a mutant CH3 domain further comprises an amino acid substitution selected from the group consisting of P329G, P329L, P329I, P329R and P329A (numbering according to Kabat EU index).

[0134] In a preferred embodiment, the extracellular domain comprises a mutated CH2 domain and a mutated CH3 domain, the mutated CH2 domain and the mutated CH3 domain comprising the amino acid substitutions I253A, H310A, P329G and H435A (numbering according to Kabat EU index). In one such embodiment, the chimeric receptor is combined with a therapeutic antibody as described herein that comprises a P329R substitution (numbering according to Kabat), and an antigen binding moiety capable of binding to a CH2 domain that comprises a P329R substitution.

[0135] In another preferred embodiment, the extracellular domain comprises a mutated CH2 domain and a mutated CH3 domain, wherein the mutated CH2 domain and the mutated CH3 domain comprise the amino acid substitutions I253A, H310A, P329R and H435A (numbering according to Kabat EU index). In one such embodiment, the chimeric receptor is combined with a therapeutic antibody as described herein that comprises a P329G substitution (numbering according to Kabat) and an antigen binding moiety capable of binding to a CH2 domain that comprises a P329R substitution.

[0136] In one embodiment, the extracellular domain does not include a VH domain and / or a VL domain.

[0137] In the context of the present invention, an antigen-binding receptor comprises an extracellular domain that is not naturally present in or on a T cell. Thus, the antigen-binding receptor can confer tailored binding specificity to a cell expressing the antigen-binding receptor according to the present invention. In one embodiment, the extracellular domain comprises a mutant CH2 domain. In one embodiment, the extracellular domain comprises a mutant Fc domain or a fragment thereof, which comprises or consists of one of the mutant CH2 domains (1) to (18) below.

[0138] (1) In one embodiment, the variant CH2 domain comprises or consists of 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: 20, and the variant CH2 domain comprises the amino acid G329 (numbering according to Kabat).

[0139] (2) In one embodiment, the variant CH2 domain comprises or consists of 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:20, wherein the variant CH2 domain comprises a glycine (G) at position 99, where the numbering is relative to the sequence of SEQ ID NO:20.

[0140] (3) In one embodiment, the variant CH2 domain comprises or consists of the amino acid sequence of SEQ ID NO:20.

[0141] (4) In one embodiment, the variant CH2 domain comprises or consists of 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: 21, and the variant CH2 domain comprises amino acid L329 (numbering according to Kabat).

[0142] (5) In one embodiment, the variant CH2 domain comprises or consists of 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:21, wherein the variant CH2 domain comprises a leucine (L) at position 99, where the numbering is relative to the sequence of SEQ ID NO:21.

[0143] (6) In one embodiment, the variant CH2 domain comprises or consists of the amino acid sequence of SEQ ID NO:21.

[0144] (7) In one embodiment, the variant CH2 domain comprises or consists of 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: 22, and the variant CH2 domain comprises amino acid I329 (numbering according to Kabat).

[0145] (8) In one embodiment, the variant CH2 domain comprises or consists of an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:22, wherein the variant CH2 domain comprises an isoleucine (I) at position 99, where the numbering is relative to the sequence of SEQ ID NO:22.

[0146] (9) In one embodiment, the variant CH2 domain comprises or consists of the amino acid sequence of SEQ ID NO: 22.

[0147] (10) In one embodiment, the variant CH2 domain comprises or consists of 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: 23, and the variant CH2 domain comprises amino acid R329 (numbering according to Kabat).

[0148] (11) In one embodiment, the variant CH2 domain comprises or consists of an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23, wherein the variant CH2 domain comprises an arginine (R) at position 99, where the numbering is relative to the sequence of SEQ ID NO: 23.

[0149] (12) In one embodiment, the variant CH2 domain comprises or consists of the amino acid sequence of SEQ ID NO: 23.

[0150] (13) In one embodiment, the variant CH2 domain comprises or consists of 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: 24, and the variant CH2 domain comprises amino acid A329 (numbering according to Kabat).

[0151] (14) In one embodiment, the variant CH2 domain comprises or consists of 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:24, wherein the variant CH2 domain comprises an alanine (A) at position 99, where the numbering is relative to the sequence of SEQ ID NO:24.

[0152] (15) In one embodiment, the mutant CH2 domain comprises or consists of the amino acid sequence of SEQ ID NO: 24.

[0153] (16) In one embodiment, the variant CH2 domain comprises or consists of 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: 86, and the variant CH2 domain comprises amino acids A234, A235 and G329 (numbering according to Kabat).

[0154] (17) In one embodiment, the variant CH2 domain comprises or consists of 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: 86, wherein the variant CH2 domain comprises an alanine (A) at positions 4 and 5 and a glycine (G) at position 99, the numbering being relative to the sequence of SEQ ID NO: 86.

[0155] (18) In a preferred embodiment, the mutant CH2 domain comprises or consists of the amino acid sequence of SEQ ID NO: 86.

[0156] In a particularly preferred embodiment, a chimeric receptor is provided that comprises an extracellular domain that includes a mutated CH2 domain that comprises or consists of the amino acid sequence of SEQ ID NO:86, and a transmembrane domain.

[0157] Optionally, the mutant CH2 domain described herein above further comprises a CH3 domain.As can be seen in Example 7, the inclusion of a CH3 domain can result in a stronger effect (even when the antibody capable of binding to the chimeric receptor of the present invention is directed against the mutant CH2 domain).Without being bound by theory, the inventors believe that the inclusion of a CH2-CH3 domain in the extracellular domain results in a stronger expression of the chimeric receptor.The expression of the chimeric receptor can be measured using methods known in the art, as described in the appended examples.

[0158] In one embodiment, the CH3 domain is a mutated CH3 domain. In one embodiment, the extracellular domain comprises a mutated CH2-CH3 domain (i.e., a CH2 domain fused to a CH3 domain, optionally via a peptide linker).

[0159] In one embodiment, the extracellular domain comprises a mutant Fc domain or a fragment thereof, and the mutant Fc domain or a fragment thereof comprises or consists of one of the mutant CH2-CH3 domains (1) to (4) below.

[0160] (1) In one embodiment, the mutant CH2-CH3 domain comprises or consists of the amino acid sequence of SEQ ID NO:27.

[0161] (2) In one embodiment, the mutant CH2-CH3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 105.

[0162] (3) In one embodiment, the mutant CH2-CH3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 124.

[0163] (4) In one embodiment, the mutant CH2-CH3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 126.

[0164] Transmembrane domain (TD) In the context of the present invention, any transmembrane domain of a transmembrane protein, especially as defined by the CD nomenclature, can be used to generate the chimeric receptors of the present invention.

[0165] Thus, in the context of the present invention, the transmembrane domain may comprise a part of the murine / mouse transmembrane domain or, preferably, a part of the human transmembrane domain. An example of such a transmembrane domain is, for example, the transmembrane domain of CD8, which has the amino acid sequence shown herein in SEQ ID NO: 89 (encoded by the DNA sequence shown in SEQ ID NO: 98). In the context of the present invention, the transmembrane domain of the chimeric receptor of the present invention may comprise / consist of the amino acid sequence shown in SEQ ID NO: 89 (encoded by the DNA sequence shown in SEQ ID NO: 98).

[0166] In another embodiment, the chimeric receptor provided herein comprises a chimeric receptor selected from amino acids 153-179, 154-179, 155-179, 156-179, 157-179, 158-179, 159-179, 160-179, 161-179, 162-179, 163-179, 164-179, 165-179, 166-179, 167-179, 168-179, 169-200, 170-170, 171-170, 172-170, 173-170, 174-170, 175-170, 176-170, 177-170, 178-170, 179-200, 179-200, 180-170, 181-170, 182-170, 183-170, 184-170, 185-170, 186-170, 187-170, 188-170, 189-200, 190-191, 191-192, 192-193, 193-194, 194-195, 195-196, 196-197, 197-198, 198-199, 199-200, 200-201, 201-202, 202-203, 202-204, 202-205, 202-206, 202-2 9, 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 (encoded by the cDNA shown in SEQ ID NO:40).

[0167] Alternatively, any protein having a transmembrane domain, among others, as provided by the CD nomenclature, can be used as the transmembrane domain of the chimeric receptor proteins of the present invention.

[0168] In some embodiments, the transmembrane domain comprises a transmembrane domain of any one of the group consisting of CD27 (SEQ ID NO:37 encoded by SEQ ID NO:36), CD137 (SEQ ID NO:45 encoded by SEQ ID NO:44), OX40 (SEQ ID NO:49 encoded by SEQ ID NO:48), ICOS (SEQ ID NO:53 encoded by SEQ ID NO:52), DAP10 (SEQ ID NO:57 encoded by SEQ ID NO:56), DAP12 (SEQ ID NO:61 encoded by SEQ ID NO:60), CD3z (SEQ ID NO:64 encoded by SEQ ID NO:65), FCGR3A (SEQ ID NO:68 encoded by SEQ ID NO:69), NKG2D (SEQ ID NO:72 encoded by SEQ ID NO:73), CD8 (SEQ ID NO:82 encoded by SEQ ID NO:83), or a transmembrane fragment thereof that retains the ability to anchor the chimeric receptor to a membrane.

[0169] Human sequences may be beneficial in the general invention context, since, for example, (part of) the transmembrane domain may be accessible from the extracellular space and thus accessible to the patient's immune system.

[0170] Stimulatory signaling domains (SSD) and costimulatory signaling domains (CSD) Preferably, the chimeric receptors of the invention comprise at least one stimulatory signaling domain and / or at least one costimulatory signaling domain. Thus, the chimeric receptors provided herein preferably comprise a stimulatory signaling domain that results in T cell activation. Chimeric receptors provided herein can comprise a stimulatory signaling domain that is a fragment / polypeptide portion of murine / mouse or human CD3z (the UniProt entry for human CD3z is P20963 (version number 177, SEQ ID NO:2); the UniProt entry for mouse CD3z is P24161 (primary citable accession number) or Q9D3G3 (secondary citable accession number), version number 143, SEQ ID NO:1)), FCGR3A (the UniProt entry for human FCGR3A is P08637 (version number 178, SEQ ID NO:2)), or NKG2D (the UniProt entry for human NKG2D is P26718 (version number 151, SEQ ID NO:1); the UniProt entry for mouse NKG2D is O54709 (version number 132, SEQ ID NO:2)).

[0171] Thus, the stimulatory signaling domain included in the chimeric receptors provided herein may be a full-length fragment / polypeptide portion of CD3z, FCGR3A, or NKG2D. The amino acid sequence of murine / mouse full-length CD3z or NKG2D is set forth herein as SEQ ID NO: 66 (CD3z), 70 (FCGR3A), or 74 (NKG2D) (murine / mouse is encoded by the DNA sequence set forth in SEQ ID NO: 67 (CD3z), 71 (FCGR3A), or 75 (NKG2D)). The amino acid sequence of human full-length CD3z, FCGR3A, or NKG2D is set forth herein as SEQ ID NO: 64 (CD3z), 68 (FCGR3A), or 72 (NKG2D) (human is encoded by the DNA sequence set forth in SEQ ID NO: 65 (CD3z), 69 (FCGR3A), or 73 (NKG2D)). The chimeric receptors of the present invention may comprise a fragment of CD3z, FCGR3A or NKG2D as a stimulatory domain, provided that it comprises at least one signaling domain. In particular, any part / fragment of CD3z, FCGR3A or NKG2D is suitable as a stimulatory domain, as long as it comprises at least one signaling motif. However, more preferably, the chimeric receptors of the present invention comprise a polypeptide obtained from human origin.

[0172] In preferred embodiments, the one or more stimulatory signaling domains comprised in the chimeric receptor comprise or consist of the amino acid sequence set forth in SEQ ID NO: 91 (encoded by the DNA sequence set forth in SEQ ID NO: 100). In further embodiments, the chimeric receptor comprises a sequence set forth in SEQ ID NO: 91 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: 91, and characterized in that it has (or retains) stimulatory signaling activity. Exemplary structures of chimeric receptors that include stimulatory signaling domains (SSDs) are shown herein below and in the Examples and Figures. Stimulatory signaling activity can be determined, for example, by enhanced cytokine release (measured by ELISA) (IL-2, IFNγ, TNFα), enhanced proliferative activity (measured by increased cell number) or enhanced lytic activity (measured by LDH release assay).

[0173] Additionally, the chimeric receptors provided herein preferably contain at least one costimulatory signaling domain that provides additional activity to T cells. The chimeric receptors provided herein include murine / mouse or human CD28 (the UniProt entry for human CD28 is P10747 (version number 173, SEQ ID NO: 1) and the UniProt entry for mouse CD28 is P31041 (version number 134, SEQ ID NO: 2)), CD137 (the UniProt entry for human CD137 is Q07011 (version number 145, SEQ ID NO: 1) and the UniProt entry for mouse CD137 is P20334 (version number 146, SEQ ID NO: 1)), and / or human CD28 (the UniProt entry for human CD28 is P10747 (version number 173, SEQ ID NO: 1) and the UniProt entry for mouse CD28 is P31041 (version number 134, SEQ ID NO: 2)), CD137 (the UniProt entry for human CD137 is Q07011 (version number 145, SEQ ID NO: 1) and the UniProt entry for mouse CD137 is P20334 (version number 145, SEQ ID NO: 1)). OX40 (the UniProt entry for human OX40 is P23510 (version number 138, SEQ ID NO: 1) and the UniProt entry for mouse OX40 is P43488 (version number 119, SEQ ID NO: 1)); ICOS (the UniProt entry for human ICOS is Q9Y6W8 (SEQ ID NO: 1, version number 126)); and the UniProt entry for mouse ICOS is Q9WV40 (primary citable accession number) or Q9JL17 (secondary citation number). available accession number, version number 102, sequence version 2), CD27 (the UniProt entry for human CD27 is P26842 (version number 160, SEQ ID NO: 2), and the UniProt entry for mouse CD27 is P41272 (version number 137, sequence version 1)), 4-1-BB (the UniProt entry for mouse 4-1-BB is P20334 (version number 140, sequence version 1), and the UniProt entry for human 4-1-BB is Q07 011 (version number 146, sequence version)), DAP10 (the UniProt entry for human DAP10 is Q9UBJ5 (version number 25, SEQ ID NO: 1), and the UniProt entry for mouse DAP10 is Q9QUJ0 (primary citable accession number) or Q9R1E7 (secondary citable accession number), version number 101, SEQ ID NO: 1)) or DAP12 (the UniProt entry for human DAP12 is O43914 (version number 146, SEQ ID NO: 1),The UniProt entry for mouse DAP12 is O054885 (primary citable accession number) or Q9R1E7 (secondary citable accession number), version number 123, SEQ ID NO: 1). In a particular embodiment of the invention, the chimeric receptor of the invention may comprise one or more, i.e. 1, 2, 3, 4, 5, 6 or 7, costimulatory signaling domains as described herein. Thus, in the context of the present invention, the chimeric receptor of the invention may comprise as a first costimulatory signaling domain a fragment / polypeptide portion of murine / mouse CD137 or, preferably, a fragment / polypeptide portion of human CD137, and the second costimulatory 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 chimeric receptor of the invention comprises a costimulatory signaling domain obtained from human origin.

[0174] In a preferred embodiment, the stimulatory signaling domain contained in a chimeric receptor of the invention may comprise or consist of the amino acid sequence set forth in SEQ ID NO:90 (encoded by the DNA sequence set forth in SEQ ID NO:99).

[0175] Thus, the costimulatory signaling domains optionally included in the chimeric receptors provided herein are fragments / polypeptide portions of full length CD27, CD28, CD137, OX40, ICOS, DAP10, or DAP12. The amino acid sequences of murine / mouse full length CD27, CD28, CD137, OX40, ICOS, CD27, DAP10, and DAP12 are set forth herein as SEQ ID NO: 39 (CD27), 43 (CD28), 47 (CD137), 51 (OX40), 55 (ICOS), 59 (DAP10), or 63 (DAP12) (mouse are encoded by the DNA sequences set forth in SEQ ID NO: 38 (CD27), 42 (CD28), 46 (CD137), 50 (OX40), 54 (ICOS), 58 (DAP10), or 62 (DAP12)). However, since human sequences are most preferred in the context of the present invention, the costimulatory signaling domains that may optionally be included in the chimeric receptor proteins provided herein are fragments / 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 set forth herein as SEQ ID NO: 37 (CD27), 41 (CD28), 45 (CD137), 49 (OX40), 53 (ICOS), 57 (DAP10), or 61 (DAP12) (human is encoded by the DNA sequence set forth in SEQ ID NO: 36 (CD27), 40 (CD28), 44 (CD137), 48 (OX40), 52 (ICOS), 56 (DAP10), or 60 (DAP12)).

[0176] In one embodiment, the chimeric receptor comprises CD28 or a fragment thereof as a costimulatory signaling domain. The chimeric receptors provided herein may comprise a fragment of CD28 as a costimulatory signaling domain, provided that at least one signaling domain of CD28 is included. In particular, any part / fragment of CD28 is suitable for the chimeric receptor of the present invention, as long as at least one signaling motif of CD28 is included. The costimulatory signaling domains PYAP (AA208 to 211 of CD28) and YMNM (AA191 to 194 of CD28) are beneficial for the function of the CD28 polypeptide and the functional effects listed above. The amino acid sequence of the YMNM domain is shown in SEQ ID NO: 76, and the amino acid sequence of the PYAP domain is shown in SEQ ID NO: 77. Thus, in the chimeric receptor of the present invention, the CD28 polypeptide preferably comprises a sequence from the intracellular domain of the CD28 polypeptide having the sequence YMNM (SEQ ID NO: 76) and / or PYAP (SEQ ID NO: 77). In other embodiments, one or both of these domains are mutated to FMNM (SEQ ID NO: 78) and / or AYAA (SEQ ID NO: 79), respectively, in the chimeric receptors of the present invention. Any of these mutations can be used to reduce the ability of transduced cells containing the chimeric receptor to release cytokines without affecting their own proliferation capacity, and advantageously to prolong the survival of the transduced cells, and thus enhance their therapeutic potential. In other words, such non-functional mutations preferably enhance the persistence of cells transduced in vivo with the chimeric receptors provided herein. However, these signaling motifs can be present anywhere within the intracellular domain of the chimeric receptors provided herein.

[0177] In a preferred embodiment, the chimeric receptor comprises CD137 or a fragment thereof as a costimulatory signaling domain. The chimeric receptors provided herein may comprise a fragment of CD137 as a costimulatory signaling domain, provided that at least one signaling domain of CD137 is included. In particular, any part / fragment of CD137 is suitable for the chimeric receptor of the present invention, as long as at least one signaling motif of CD137 is included. In a preferred embodiment, the CD137 polypeptide comprised in the chimeric receptor protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 90 (encoded by the DNA sequence shown in SEQ ID NO: 99).

[0178] Exemplary structures of chimeric receptors containing a costimulatory signaling domain (CSD) are provided herein below and in the Examples and Figures. Costimulatory signaling activity can be determined, for example, by enhanced cytokine release (measured by ELISA) (IL-2, IFNγ, TNFα), enhanced proliferative activity (measured by increased cell number) or enhanced lytic activity (measured by LDH release assay). CD28 and / or CD137 activity can be measured by cytokine release by ELISA or flow cytometry for cytokines such as interferon-gamma (IFN-γ) or interleukin 2 (IL-2), T cell proliferation measured by, for example, ki67 measurement, cell quantification by flow cytometry, or lytic activity assessed by real-time impedance measurement of target cells (see, for example, 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 Differ. 21(12)(2014), 1825-1837, Erratum in: Cell Death Differ. 21(12)(2014), 161).

[0179] Further linkers and signal peptides Furthermore, the chimeric receptors provided herein may include at least one linker (or "spacer"). A linker is usually a peptide of up to 20 amino acids in length. Thus, in the context of the present invention, a linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. For example, the chimeric receptors provided herein may include a linker between the extracellular domain, the transmembrane domain, the costimulatory signaling domain, and / or the stimulatory signaling domain. Such a linker may have the advantage that the different polypeptides (i.e., the extracellular domain, the transmembrane domain, the costimulatory signaling domain, and / or the stimulatory signaling domain) of the chimeric receptor may be folded independently and may function as expected. In the context of the present invention, the extracellular domain, the transmembrane domain, the costimulatory signaling domain, and the stimulatory signaling domain may be included in a single chain multifunctional polypeptide. The single chain fusion construct may be composed of, for example, an extracellular domain, a transmembrane domain, a costimulatory signaling domain, and / or a stimulatory signaling domain. Thus, the extracellular domain, the transmembrane domain, the costimulatory signaling domain, and the stimulatory signaling domain may be connected by one or more identical or different peptide linkers as described herein. For example, in the chimeric receptors provided herein, the linker between the extracellular domain and the transmembrane domain may comprise or consist of the amino acid and amino acid sequence shown in SEQ ID NO: 97. Thus, the transmembrane domain, the costimulatory signaling domain, and / or the stimulatory domain may be linked to each other by a peptide linker or by direct fusion of the domains.

[0180] The chimeric receptors provided herein or portions thereof can include a signal peptide. Such a signal peptide will deliver the protein to the surface of the T cell membrane. For example, in the chimeric receptors provided herein, the signal peptide can have the amino acid sequence set forth in SEQ ID NO:80 (encoded by the DNA sequence set forth in SEQ ID NO:81).

[0181] Specific chimeric receptors capable of binding mutant Fc domains The components of the chimeric receptors described herein above can be fused to each other in a variety of configurations.

[0182] In some embodiments, the chimeric receptor comprises an extracellular domain comprising a mutated Fc domain. In one embodiment, the chimeric receptor comprises a mutated CH2 domain and / or a mutated CH3 domain as described herein above. In a preferred embodiment, the Fc domain is fused C-terminally to the N-terminus of the transmembrane domain, optionally via a peptide linker. In other embodiments, the chimeric receptor further comprises a stimulatory signaling domain and / or a costimulatory signaling domain.

[0183] In a specific such embodiment, the chimeric receptor comprises (or essentially consists of) an extracellular domain (e.g., a CH2 and / or CH3 domain) comprising a mutant Fc domain fused at its C-terminus to the N-terminus of a transmembrane domain, which is fused at its C-terminus to the N-terminus of a stimulatory signaling domain.

[0184] Optionally, the chimeric receptor further comprises a costimulatory signaling domain. In one specific such embodiment, the chimeric receptor comprises (or consists essentially of) an extracellular domain (e.g., a CH2 and / or CH3 domain) comprising a variant Fc domain fused at its C-terminus to the N-terminus of a transmembrane domain, which is fused at its C-terminus to the N-terminus of the stimulatory signaling domain, which is fused at its C-terminus to the N-terminus of the costimulatory signaling domain.

[0185] In another embodiment, the costimulatory signaling domain is linked to the transmembrane domain instead of the stimulatory signaling domain. In a specific such embodiment, the chimeric receptor comprises (or consists essentially of) an extracellular domain (e.g., a CH2 and / or CH3 domain) comprising a variant Fc domain fused at its C-terminus to the N-terminus of the transmembrane domain, the transmembrane domain is fused at its C-terminus to the N-terminus of the costimulatory signaling domain, and the costimulatory signaling domain is fused at its C-terminus to the N-terminus of the stimulatory signaling domain.

[0186] In a preferred embodiment, the chimeric receptor comprises (or consists essentially of), in N-terminus to C-terminus, an extracellular domain comprising a variant Fc domain (e.g., a CH2 and / or CH3 domain), a transmembrane domain, a costimulatory signaling domain, and a stimulatory signaling domain, linked in that order by one or more peptide linkers. In a preferred embodiment, the variant Fc domain is a CH2 domain.

[0187] The Fc domain, transmembrane domain, and stimulatory signaling and / or costimulatory signaling domain may be fused directly to one another or via one or more peptide linkers comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n , (SG4) n , (G4S) n or G4 (SG4) n Peptide linkers are included, where "n" is usually a number between 1 and 10, typically between 2 and 4. A preferred peptide linker for linking the antigen-binding moiety and the anchoring transmembrane moiety is (G4S). Another preferred peptide linker for linking the antigen-binding moiety and the anchoring transmembrane moiety is the amino acid sequence of SEQ ID NO: 88 (CD8 stalk). Additionally, the linker may comprise (part of) an immunoglobulin hinge region.

[0188] In one embodiment, the Fc domain (e.g., a variant CH2 domain) is an IgG Fc domain or a fragment thereof, in particular an IgG1 or IgG4 Fc domain or a fragment thereof. In one embodiment, the extracellular domain comprises a CH2 domain, a CH3 domain and / or a CH4 domain, preferably a CH2 domain. In one embodiment, the Fc domain comprises at least one amino acid substitution compared to a native Fc domain. In one embodiment, the at least one amino acid substitution reduces binding to an Fc receptor and / or reduces effector function. In one embodiment, the at least one amino acid substitution is at a position selected from the list consisting of 233, 234, 235, 238, 253, 265, 269, 270, 297, 310, 331, 327, 329 and 435 (numbering according to Kabat EU index). In one embodiment, the at least one amino acid substitution comprises a substitution at position P329 (numbering according to Kabat EU index). In one embodiment, the at least one amino acid substitution comprises a substitution at position P329 (numbering according to Kabat EU index) with an amino acid selected from the list consisting of alanine (A), arginine (R), leucine (L), isoleucine (I) and proline (P). In one embodiment, the at least one amino acid substitution comprises the amino acid substitution P329G (numbering according to Kabat EU index).

[0189] In one particular embodiment, the chimeric receptor comprises a mutated Fc domain, in particular an IgG1 Fc domain, comprising a P329G substitution (according to EU numbering).

[0190] In one embodiment, the Fc domain comprises a P329G substitution (according to EU numbering). In one embodiment, the Fc domain is fused at the C-terminus to the N-terminus of the transmembrane domain, optionally via a peptide linker. In one embodiment, the peptide linker comprises the amino acid sequence of SEQ ID NO: 88. In one embodiment, the transmembrane domain is a transmembrane domain or a fragment thereof selected from the group consisting of CD8, CD4, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10 or DAP12 transmembrane domains. In a preferred embodiment, the transmembrane domain is a CD8 transmembrane domain or a fragment thereof. In a particular embodiment, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 89. In one embodiment, the chimeric receptor further comprises a costimulatory signaling domain (CSD). In one embodiment, the transmembrane domain of the chimeric receptor is fused at the C-terminus to the N-terminus of the costimulatory signaling domain. In one embodiment, the costimulatory signaling domain is individually selected from the group consisting of the intracellular domains of CD27, CD28, CD137, OX40, ICOS, DAP10, and DAP12, or fragments thereof, as previously described herein. In one embodiment, the costimulatory signaling domain is the intracellular domain of CD28, or a fragment thereof. In one embodiment, the costimulatory signaling domain comprises the intracellular domain of CD28, or a fragment thereof that retains CD28 signaling. In a preferred embodiment, the costimulatory signaling domain comprises the intracellular domain of CD137, or a fragment thereof that retains CD137 signaling. In certain such embodiments, the costimulatory signaling domain comprises or consists of SEQ ID NO: 90. In one embodiment, the chimeric receptor further comprises a stimulatory signaling domain. In one embodiment, the costimulatory signaling domain of the chimeric receptor is fused C-terminally to the N-terminus of the stimulatory signaling domain. In one embodiment, at least one stimulatory signaling domain is individually selected from the group consisting of the intracellular domains of CD3z, FCGR3A, and NKG2D, or a fragment thereof. In a preferred embodiment, the stimulatory signaling domain is the intracellular domain of CD3z, or a fragment thereof that retains CD3z signaling.In certain embodiments, the stimulatory signaling domain comprises or consists of SEQ ID NO:91.

[0191] In one embodiment, the chimeric receptor is fused to a reporter protein, in particular GFP or an enhanced analogue thereof. In one embodiment, the chimeric receptor is fused at the C-terminus to the N-terminus of eGFP (enhanced green fluorescent protein), optionally via a peptide linker as described herein. In a preferred embodiment, the peptide linker is GEGRGSLTCGDVEENPGP (T2A) according to sequence number 95.

[0192] In a particular embodiment, the chimeric receptor comprises a transmembrane domain and an extracellular domain comprising a mutated CH2 domain, the mutated CH2 domain comprising a P329G substitution (according to EU numbering). The P329G substitution reduces Fcγ receptor binding. In one embodiment, the chimeric receptor of the present invention comprises a transmembrane domain (TD), a costimulatory signaling domain (CSD) and a stimulatory signaling domain (SSD). In one such embodiment, the chimeric receptor has the structure CH2-TD-CSD-SSD. In another embodiment, the chimeric receptor has the structure CH2-TD-SSD-CSD. In a preferred embodiment, the chimeric receptor has the structure CH2(P329G)-TD-CSD-SSD. In a more particular such embodiment, the chimeric receptor has the structure CH2(P329G)-linker-TD-CSD-SSD, where "linker" is a linker as described herein above.

[0193] In one embodiment, the invention provides a chimeric receptor comprising, or consisting of, in N-terminal to C-terminal order, an extracellular domain comprising a CH2 domain, the CH2 domain comprising the amino acid substitution P329G (numbering according to Kabat EU index), and a transmembrane domain. In a particular embodiment, the CH2 domain comprises or consists of the sequence of SEQ ID NO:86.

[0194] In one embodiment, the present invention provides a method for the preparation of a polypeptide comprising, in order from the N-terminus to the C-terminus: (i) an extracellular domain comprising a CH2 domain, the CH2 domain comprising the amino acid substitution P329G (numbering according to Kabat EU index); (ii) a transmembrane domain; and (iii) providing a chimeric receptor comprising at least one intracellular stimulatory signaling domain and / or at least one costimulatory signaling domain.

[0195] In one embodiment, the present invention provides a method for the preparation of a polypeptide comprising, in order from the N-terminus to the C-terminus: (i) an extracellular domain comprising a CH2 domain of SEQ ID NO: 86 and, optionally, a linker, in particular a linker of SEQ ID NO: 88; (ii) a transmembrane domain, in particular a transmembrane domain of SEQ ID NO: 89, (iii) a costimulatory signaling domain, particularly a costimulatory signaling domain of SEQ ID NO: 90; (iv) a chimeric receptor comprising a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:91.

[0196] In one embodiment, a chimeric receptor is provided that 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: 87. In one embodiment, a chimeric receptor is provided that comprises or consists of the amino acid sequence of SEQ ID NO: 87.

[0197] In one embodiment, the invention provides a chimeric receptor comprising, in N-terminal to C-terminal order, an extracellular domain comprising a CH2-CH3 domain, the CH2-CH3 domain comprising the amino acid substitutions I253A, H310A and H435A (numbering according to Kabat EU index), and a transmembrane domain. In a particular embodiment, the CH2-CH3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 105.

[0198] In one embodiment, the present invention provides a method for the preparation of a polypeptide comprising, in order from the N-terminus to the C-terminus: (i) an extracellular domain comprising a CH2-CH3 domain, the CH2-CH3 domain comprising the amino acid substitutions I253A, H310A and H435A (numbering according to Kabat EU index); (ii) a transmembrane domain; and (iii) providing a chimeric receptor comprising at least one intracellular stimulatory signaling domain and / or at least one costimulatory signaling domain.

[0199] In one embodiment, the present invention provides a method for the preparation of a polypeptide comprising, in order from the N-terminus to the C-terminus: (i) an extracellular domain comprising the CH2-CH3 domain of SEQ ID NO: 105 and, optionally, a linker, in particular a linker of SEQ ID NO: 88; (ii) a transmembrane domain, in particular a transmembrane domain of SEQ ID NO: 89, (iii) a costimulatory signaling domain, particularly a costimulatory signaling domain of SEQ ID NO: 90; (iv) a chimeric receptor comprising a stimulatory signaling domain, in particular the stimulatory signaling domain of SEQ ID NO:91.

[0200] In one embodiment, a chimeric receptor is provided that 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:106: In one embodiment, a chimeric receptor is provided that comprises or consists of the amino acid sequence of SEQ ID NO:106.

[0201] In one embodiment, a chimeric receptor is provided that 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: 125: In one embodiment, a chimeric receptor is provided that comprises or consists of the amino acid sequence of SEQ ID NO: 125.

[0202] In one embodiment, a chimeric receptor is provided that 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: 126: In one embodiment, a chimeric receptor is provided that comprises or consists of the amino acid sequence of SEQ ID NO: 126.

[0203] Transduced Cells Capable of Expressing the Chimeric Receptors of the Invention A further aspect of the present invention is a transduced T cell capable of expressing the chimeric receptor of the present invention. A chimeric receptor as described herein refers to a molecule that is not naturally contained within and / or on the surface of a T cell and is not expressed (endogenously) in or on a normal (non-transduced) T cell. Thus, the chimeric receptor of the present invention within and / or on the surface of a T cell is artificially introduced into the T cell. In the context of the present invention, said T cell, preferably a CD8+ T cell, may be isolated / obtained from a subject to be treated as defined herein. In the context of the present invention, the chimeric receptor is presented within and / or on the surface of said T cell after (retroviral, lentiviral or non-viral) transduction as described herein below. Thus, after transduction, the T cell according to the present invention may be activated by an immunoglobulin, preferably a (therapeutic) antibody comprising at least one antigen-binding moiety capable of binding to a target cell antigen and at least one antigen-binding moiety capable of binding to a mutated Fc domain but not capable of binding to the respective non-mutated parent Fc domain.

[0204] The present invention also relates to a transduced T cell expressing a chimeric receptor encoded by a nucleic acid molecule encoding a chimeric receptor of the invention. Thus, in the context of the present invention, a transduced cell may comprise a nucleic acid molecule encoding a chimeric receptor of the invention or a vector of the invention expressing a chimeric receptor of the invention.

[0205] In the context of the present invention, the term "transduced T cells" relates to genetically modified T cells (i.e., T cells into which a nucleic acid molecule has been intentionally introduced). The transduced T cells provided herein may comprise a vector of the present invention. Preferably, the transduced T cells provided herein comprise a nucleic acid molecule encoding a chimeric receptor of the present invention and / or a vector of the present invention. The transduced T cells of the present invention may be T cells that transiently or stably express foreign DNA (i.e., a nucleic acid molecule introduced into the T cell). In particular, the nucleic acid molecule encoding the chimeric receptor of the present invention can be stably integrated into the genome of the T cell by using retroviral or lentiviral transduction. The nucleic acid molecule encoding the chimeric receptor of the present invention can be transiently expressed by using mRNA transfection. Preferably, the transduced T cells provided herein are genetically modified by introducing a nucleic acid molecule into the T cell via a viral vector (e.g., a retroviral or lentiviral vector). Thus, the expression of the chimeric receptor may be constitutive and the extracellular domain (and the variant Fc domain) of the chimeric receptor may be detectable on the cell surface.

[0206] When the chimeric receptor is introduced into T cells under the control of an inducible or repressible promoter, expression may be conditional or inducible. An example of such an inducible or repressible promoter may be a transcription system comprising the alcohol dehydrogenase I (alcA) gene promoter and the transactivator protein AlcR. Different agricultural alcohol-based formulations are used to control the expression of the gene of interest linked to the alcA promoter. Furthermore, tetracycline-responsive promoter systems may function to either activate or repress the gene expression system in the presence of tetracycline. Elements of these systems include the tetracycline repressor protein (TetR), the tetracycline operator sequence (tetO), and the tetracycline transactivator fusion protein (tTA), which is a fusion of TetR with the herpes simplex virus protein 16 (VP16) activation sequence. Furthermore, steroid-responsive promoters, metal-regulated promoters, or virulence-related (PR) protein-related promoters may be used.

[0207] This expression can be constitutive or constitutive, depending on the system used. The chimeric receptor of the present invention can be expressed on the surface of the transduced T cells provided herein. The extracellular portion of the chimeric receptor (i.e., the extracellular domain of the chimeric receptor) can be detectable on the cell surface, while the intracellular portion (i.e., the costimulatory signaling domain and the stimulatory signaling domain) is not detectable on the cell surface. Detection of the extracellular domain of the chimeric receptor can be performed, for example, by flow cytometry or microscopy, by using an antibody that specifically binds to the extracellular domain and / or the variant Fc domain.

[0208] Other cells can also be transduced with the chimeric receptor of the invention and thereby directed to the target cell. These additional cells include, but are not limited to, B cells, natural killer (NK) cells, innate lymphoid cells, macrophages, monocytes, dendritic cells or neutrophils. Preferably, said immune cells will be lymphocytes. The chimeric receptor of the invention on the surface of the leukocyte triggers the cell to be cytotoxic to the target cell, regardless of the lineage from which the cell originates, in conjunction with the therapeutic antibodies described herein. Cytotoxicity occurs regardless of the stimulatory or costimulatory signaling domains selected for the chimeric receptor and is not dependent on exogenous supply of additional cytokines. Thus, the transduced cells of the invention can be, for example, CD4+ T cells, CD8+ T cells, γδ T cells, natural killer (NK) T cells, natural killer (NK) cells, tumor infiltrating lymphocytes (TIL), myeloid cells or mesenchymal stem cells. Preferably, the transduced cells provided herein are T cells (e.g., autologous T cells), more preferably, the transduced cells are CD8+ T cells. Thus, 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. Thus, 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. Thus, in the context of the present invention, the transduced cells may be allogeneic cells, such as allogeneic CD8+ T cells. The term allogeneic refers to cells derived from an unrelated donor individual / subject that is human leukocyte antigen (HLA)-matched with the individual / subject to be treated with the chimeric receptor-expressing transduced cells described herein. Autologous cells refer to cells isolated / obtained as described above from the subject to be treated with the transduced cells described herein.

[0209] The transduced cells of the invention can be co-transduced with additional nucleic acid molecules, for example, nucleic acid molecules encoding cytokines.

[0210] The invention also relates to a method for producing transduced T cells expressing a chimeric receptor of the invention, comprising the steps of transducing T cells with a vector of the invention, culturing said transduced T cells under conditions that allow expression of the chimeric receptor in or on said transduced cells, and recovering said transduced T cells.

[0211] 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 from a donor are well known in the art, and in the context of the present cells (e.g., T cells, preferably CD8+ T cells) from a patient or from a donor, for example, the cells can be isolated by blood collection or removal of bone marrow. After isolating / obtaining the cells as a patient sample, the cells (e.g., T cells) are separated from other components of the sample. Several methods of isolating cells (e.g., T cells) from a sample are known, including, but not limited to, leukapheresis to obtain cells from a peripheral blood sample of a patient or donor, isolating / obtaining cells by using a FACS cell sorter, etc. The isolated / obtained T cells are then cultured and expanded, for example, by using an anti-CD3 antibody, by using anti-CD3 and anti-CD28 monoclonal antibodies, and / or by using an anti-CD3 antibody, an anti-CD28 antibody and interleukin 2 (IL-2) (see, for example, Dudley, Immunother. 26 (2003), 332-342 or Dudley, Clin. Oncol. 26 (2008), 5233-5239).

[0212] In a subsequent step, cells (e.g., T cells) are artificially / genetically modified / transduced by methods known in the art (see, e.g., Lemoine, J Gene Med 6 (2004), 374-386). Methods for transducing cells (e.g., T cells) are known in the art and include, but are not limited to, electroporation, calcium phosphate, cationic lipid or liposome methods when nucleic acids or recombinant nucleic acids are transduced. The nucleic acid to be introduced can be transduced with high efficiency as conventionally by using commercially available transfection reagents, such as Lipofectamine (Invitrogen, Catalog No.: 11668027). When a vector is used, the vector can be transduced in the same manner as the above-mentioned nucleic acids, as long as the vector is a plasmid vector (i.e., a vector that is not a viral vector). In the context of the present invention, methods for transducing cells (e.g., T cells) include retroviral or lentiviral T cell transduction, non-viral vectors (e.g., Sleeping Beauty minicircle vector), as well as mRNA transfection. "mRNA transfection" refers to a method well known to those skilled in the art for transiently expressing a protein of interest, e.g., the chimeric receptor of the present invention, in the cells to be transduced. Briefly, cells can be electroporated with mRNA encoding the chimeric receptor of the present invention by using an electroporation system (e.g., Gene Pulser, Bio-Rad), and then cultured by standard cell (e.g., T cell) culture protocols as described above (see Zhao et al., Mol Ther. 13(1) (2006), 151-159). The transduced cells of the present invention can be generated by lentiviral transduction, or most preferably, retroviral transduction.

[0213] In this regard, suitable retroviral vectors for transducing cells are known in the art and include, 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 transducing cells (e.g., T cells) are, for example, the PL-SIN lentiviral vector (Hotta et al., Nat Methods. 6(5)(2009), 370-376), p156RRL-sinPPT-CMV-GFP-PRE / NheI (Campeau et al., PLoS One 4(8)(2009), e6529), pCMVR8.74 (Addgene Catalogue No.: 22036), FUGW (Lois et al., Science 295(5556)(2002), 868-872), pLVX-EF1 (Addgene Catalogue No.: 64368), pLVE (Brunger et al., Proc Natl Acad Sci USA 111(9)(2014),E798-806), pCDH1-MCS1-EF1(Hu et al., Mol Cancer Res.7(11)(2009),1756-1770), pSLIK(Wang et al., Nat Cell Biol.16(4)(2014),345-356), pLJM1(Solomon et al. al., Nat Genet.45(12)(2013),1428-30), pLX302(Kang et al., Sci Signal.6(287)(2013),rs13), pHR-IG(Xie et al.,J Cereb Blood Flow Metab.33(12)(2013),1875-85), pRRLSIN(Addgene Catalogue No.:62053), pLS(Miyoshi et al.,J Virol.72(10)(1998),8150-8157), pLL3.7(Lazebnik et al.,Biol Chem.283(7)(2008),11078-82), FRIG(Raissi et al.,Mol Cell Neurosci.57(2013),23-32), pWPT(Ritz-Laser et al. al., Diabetologia.46(6)(2003),810-821), pBOB(Marr et al., J Mol Neurosci.22(1-2)(2004),5-11), or pLEX (Addgene catalog number 27976).

[0214] The transduced cells of the invention are preferably grown under controlled conditions outside their natural environment. In particular, the term "culture" refers to the in vitro growth of cells (e.g., the transduced cells of the invention) derived from a multicellular eukaryote (preferably from a human patient). Cell culture is an experimental technique for keeping cells alive that have been separated from their original tissue source. Here, the transduced cells of the invention are cultured under conditions that allow the expression of the chimeric receptor of the invention in or on said transduced cells. Conditions that allow the expression of the transgene (i.e., the chimeric receptor of the 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). After the chimeric receptor of the invention is expressed in the cultured transduced cells (eg, CD8+ T), the transduced cells are harvested (ie, re-extracted) from the culture (ie, the culture medium).

[0215] Thus, the present invention also includes transduced cells, preferably T cells, in particular CD8+ T cells, expressing a chimeric receptor encoded by a nucleic acid molecule of the invention obtainable by the method of the invention.

[0216] nucleic acid molecule Further aspects of the present invention are nucleic acids and vectors encoding one or more chimeric receptors of the present invention. Exemplary nucleic acid molecules encoding the chimeric receptors of the present invention are shown in SEQ ID NO: 96 or SEQ ID NO: 107. The nucleic acid molecules of the present invention can be under the control of regulatory sequences. For example, promoters, transcriptional enhancers and / or sequences that allow inducible expression of the chimeric receptors of the present invention can be employed. In the context of the present invention, the nucleic acid molecules are expressed under the control of constitutive or inducible promoters. Suitable promoters include, for example, the CMV promoter (Qin et al., PLoS One 5(5)(2010), e10611), the UBC promoter (Qin et al., PLoS One 5(5)(2010), e10611), the PGK (Qin et al., PLoS One 5(5)(2010), e10611), the EF1A promoter (Qin et al., PLoS One 5(5)(2010), e10611), the CAGG promoter (Qin et al., PLoS One 5(5)(2010), e10611), the SV40 promoter (Qin et al., PLoS One 5(5)(2010), e10611), the COPIA promoter (Qin et al., PLoS One 5(5)(2010), e10611), the ACT5C ... al., PLoS One 5(5)(2010), e10611), TRE promoter (Qin et al., PLoS One.5(5)(2010), e10611), Oct3 / 4 promoter (Chang et al., Molecular Therapy 9(2004), S367-S367(doi:10.1016 / j.ymthe.2004.06.904)), or Nanog promoter (Wu et al., Cell Res.15(5)(2005), 317-24). The present invention therefore also relates to a vector comprising a nucleic acid molecule according to the present invention. The term vector herein refers to a circular or linear nucleic acid molecule capable of autonomously replicating in a cell into which it is introduced.Many suitable vectors are known to those skilled in the art of molecular biology, the choice of which depends on the desired function and includes plasmids, cosmids, viruses, bacteriophages, and other vectors conventionally used in genetic engineering. Various plasmids and vectors can be constructed using methods well known to those skilled in the art; see, for example, the techniques described in Sambrook et al. (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 invention can be reconstituted into liposomes for delivery to target cells. As will be described in more detail below, cloning vectors have been used to isolate individual sequences of DNA. When expression of a particular polypeptide is required, the relevant sequence can be introduced into an expression vector. Exemplary cloning vectors include pBluescript SK, pGEM, pUC9, pBR322, pGA18, and pGBT9. Exemplary expression vectors include pTRE, pCAL-n-EK, pESP-1, and pOP13CAT.

[0217] The present invention also relates to a vector comprising a nucleic acid molecule which is a regulatory sequence operably linked to said nucleic acid molecule encoding a chimeric receptor 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 initiation codons, translation and insertion sites for introducing an insert into the vector. In the context of the present invention, said nucleic acid molecule is operably linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells. It is envisaged that said vector is an expression vector comprising a nucleic acid molecule encoding a chimeric receptor as defined herein. Operably linked refers to a juxtaposition in which the components so described are in a relationship permitting them to function in their intended manner. A control sequence operably linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequences. It will be apparent to those skilled in the art that when the control sequence is a promoter, a double-stranded nucleic acid is preferably used.

[0218] In the context of the present invention, the vectors described are expression vectors. An expression vector is a construct that can be used to transform a selected cell, providing expression of a coding sequence in the selected cell. An expression vector can be, for example, a cloning vector, a binary vector, or an integrative vector. Expression preferably involves transcription of a nucleic acid molecule into a translatable mRNA. Regulatory elements ensuring expression in prokaryotes and / or eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells, they usually include a promoter ensuring initiation of transcription and, optionally, a polyA signal ensuring termination of transcription and stabilization of the transcript. Possible regulatory elements allowing expression in prokaryotic host cells include, for example, the PL, lac, trp or tac promoters of E. coli, while examples of regulatory elements allowing expression in eukaryotic host cells include the AOX1 or GAL1 promoters of yeast, or the CMV, SV40, RSV promoters (Rous sarcoma virus), CMV enhancer, SV40 enhancer, or globin introns in mammalian and other animal cells.

[0219] Besides elements responsible for the initiation of transcription, such regulatory elements may also include transcription termination signals, such as the SV40-polyA site or the tk-polyA site downstream of the polynucleotide. Furthermore, depending on the expression system used, leader sequences encoding signal peptides capable of directing the polypeptide to a cellular compartment or secreting it into the medium can be added to the coding sequence of the described nucleic acid sequences, which are well known in the art; see also the accompanying examples, for example.

[0220] The leader sequence is assembled in appropriate step with the translation, initiation and termination sequences and is preferably a leader sequence capable of directing secretion of the translated protein or a part thereof into the periplasmic space or into the extracellular medium. Optionally, the heterologous sequence can code for a chimeric receptor comprising an N-terminal identification peptide that confers desired properties, such as stabilization or convenient purification of the expressed recombinant product; see supra. In this context, suitable expression vectors are known in the art, such as the Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (In-vitrogene), pEF-DHFR, pEF-ADA or pEF-neo (Raum et al. Cancer Immunol Immunother 50 (2001), 141-150) or pSPORT1 (GIBCO BRL).

[0221] In the context of the present invention, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic cells, although control sequences for prokaryotic cells may also be used. Once the vector is incorporated into a suitable cell, the cell is maintained as desired under conditions suitable for high level expression of the nucleotide sequence. Additional regulatory elements may include transcriptional enhancers as well as translational enhancers. Advantageously, the above vectors of the present invention include selectable and / or scorable markers. Selectable marker genes useful for the selection of transformed cells and, for example, plant tissues and plants, are well known to those of skill in the art and include, for example, antimetabolite resistance as the basis of selection, such as dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149), npt, which confers resistance to the aminoglycosides neomycin, kanamycin, and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995), and hygro, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481-485). Additional selection genes have been described, namely trpB, which allows cells to utilize indole instead of tryptophan; hisD, which allows cells to utilize histinol instead of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate isomerase, which allows cells to utilize mannose (WO 94 / 20627) and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.) or deaminase from Aspergillus terreus, which confers resistance to blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338).

[0222] Useful scorable markers are also known to those skilled in the art and are commercially available. Advantageously, said markers are genes encoding luciferase (Diacomin, 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 described vectors.

[0223] As mentioned above, the described nucleic acid molecules can be used alone or as part of a vector to express the chimeric receptor of the invention in cells, for example for adoptive T cell therapy, but also for gene therapy purposes. A nucleic acid molecule or vector containing a DNA sequence encoding any one of the chimeric receptors described herein is introduced into a cell, which then produces the polypeptide of interest. Gene therapy is based on the introduction of therapeutic genes into cells by ex vivo or in vivo techniques and is one of the most important applications of gene transfer. Suitable vectors, methods or gene delivery systems for in vitro or in vivo gene therapy are described in the literature and are known to those skilled in the art; see, for example, Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma, Nature 389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodera, Blood 91 (1998), 30-36; Verma, Gene Ther. 5 (1998), 692-699; Nabel, Ann. NY Acad. 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; U.S. Patent No. 5,580,859; U.S. Patent No. 5,589,466; or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640. The nucleic acid molecules and vectors described can be designed for direct introduction into cells or for introduction via liposomes or viral vectors (e.g., adenovirus, retrovirus).In the context of the present invention, said cell is a T cell, such as a CD8+ T cell, a CD4+ T cell, a CD3+ T cell, a γδ T cell or a natural killer (NK) T cell, preferably a CD8+ T cell.

[0224] In accordance with the above, the present invention relates to a method for deriving vectors, in particular plasmids, cosmids and bacteriophages, conventionally used in genetic engineering, which contain a nucleic acid molecule encoding a polypeptide sequence of a chimeric receptor as defined herein. In the context of the present invention, said vectors are expression vectors and / or gene transfer or targeting vectors. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses or bovine papilloma viruses can be used for the delivery of said polynucleotides or vectors to a target cell population.

[0225] Methods well known to those skilled in the art can be used to construct recombinant vectors, see for example the techniques described in Sambrook et al. (loc cit.), Ausubel (1989, loc cit.) or other standard textbooks. Alternatively, the above nucleic acid molecules and vectors can be reconstituted into liposomes for delivery to target cells. The vectors containing the nucleic acid molecules of the invention can be introduced into host cells by well-known methods that vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation is used for other cellular hosts (see Sambrook, supra). The vectors described can be, inter alia, pEF-DHFR, pEF-ADA or pEF-neo. The vectors pEF-DHFR, pEF-ADA and pEF-neo have been described in the art, for example in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995), 7021-7025 and Raum et al. Cancer Immunol Immunother 50 (2001), 141-150.

[0226] The present invention also provides a T cell transduced with a vector as described herein. The T cell may be produced by introducing at least one of the vectors or at least one of the nucleic acid molecules into a T cell or a precursor thereof. The presence of the at least one vector or at least one nucleic acid molecule in the T cell mediates the expression of a gene encoding the chimeric receptor as described herein above. The vector of the present invention may be polycistronic.

[0227] The described nucleic acid molecules or vectors introduced into a T cell or its precursor cell can be integrated into the genome of the cell or maintained extrachromosomally.

[0228] Antibodies capable of binding to the chimeric receptors of the present invention The chimeric receptors of the present invention are useful in methods for treating or slowing the progression of various diseases (e.g., cancer), for example, particularly in combination with antibodies capable of binding to the chimeric receptors of the present invention.

[0229] In one embodiment, a method of treating a disease in a subject is provided comprising administering to the subject transduced T cells capable of expressing a chimeric receptor as described herein, where the chimeric receptor comprises an extracellular domain that comprises a mutated Fc domain or a fragment thereof, and administering prior to, concomitantly with, or following administration of the transduced T cells, a therapeutically effective amount of a molecule (e.g., an antibody) that comprises at least one antigen binding moiety that is capable of binding to the mutated Fc domain but is incapable of binding to the non-mutated parent Fc domain. In one embodiment, the antibody comprises at least one effector moiety.

[0230] In one embodiment, the effector moiety is an antigen-binding moiety capable of binding to a target cell antigen.

[0231] In one embodiment, the effector moiety is an immune activating moiety. In one embodiment, the immune activating moiety is a cytokine (e.g., IL2). In a particular embodiment, the cytokine is IL2 or a derivative thereof. In one embodiment, the immune activating moiety is a costimulatory T cell antigen (e.g., CD28, 4-1BB) or a costimulatory ligand (e.g., 4-1BBL).

[0232] Antigen-binding moieties, target antigens, and immunoactivating moieties that can be bound to variant Fc domains are further described herein below.

[0233] Antigen-binding moieties capable of binding to variant Fc domains The present invention further provides antibodies comprising at least one antigen-binding moiety capable of binding to a variant Fc domain (e.g., comprising a P329G substitution according to EU numbering). Antigen-binding moieties capable of binding to a (modified / engineered) Fc domain or fragment thereof can be generated, for example, by immunization of the immune system of a mammal with an Fc domain or fragment thereof comprising the appropriate mutation. Such methods are known in the art and are described, for example, in Burns in Methods in Molecular Biology 295:1-12 (2005). Alternatively, the antigen-binding moieties of the present invention can be isolated by screening combinatorial libraries for antibodies with the desired activity. Methods for screening combinatorial libraries are reviewed, for example, in Lerner et al. in Nature Reviews 16:498-508 (2016). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antigen-binding moieties with desired binding properties.Such methods are reviewed, for example, in Frenzel et al. in mAbs 8:1177-1194 (2016), Bazan et al. in Human Vaccines and Immunotherapeutics 8:1817-1828 (2012), and Zhao et al. in Critical Reviews in Biotechnology 36:276-289 (2016), as well as Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991), Marks et al. al., J.Mol.Biol.222:581-597(1992), and Marks and Bradbury in Methods in Molecular Biology 248:161-175(Lo,ed., Human Press, Totowa, NJ, 2003), Sidhu et al., J.Mol.Biol.338(2):299-310(2004), Lee et al. al., J.Mol.Biol.340(5):1073-1093(2004), Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004), and Lee et al.,J.Immunol.Methods 284(1-2):119-132 (2004). In one type of phage display method, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR), randomly recombined in phage libraries, and then screened for antigen-binding phage as described in Winter et al., Annual Review of Immunology 12:433-455 (1994). The phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high affinity antigen-binding moieties without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antigen-binding moieties for a wide range of antigens without immunization, as described by Griffiths et al., EMBO Journal 12:725-734 (1993). Additionally, naive libraries can be synthesized by cloning unsequenced V gene segments from stem cells and using PCR primers containing random sequences that code for highly variable CDR3 regions to achieve sequence alteration in vitro, as described by Hoogenboom and Winter, Journal of Molecular Biology 227:381-388 (1992). Patent publications describing human antibody phage libraries can include, for example, U.S. Patent Nos. 5,750,373, 7,985,840, 7,785,903, and 8,679,490, as well as U.S. Patent Application Publication Nos. 2005 / 0079574, 2007 / 0117126, 2007 / 0237764, and 2007 / 0292936 and 2009 / 0002360. Further examples of methods known in the art for screening combinatorial libraries for antigen-binding moieties having one or more desired activities include ribosome and mRNA display, as well as methods for display and selection of antibodies in bacteria, mammalian cells, insect cells, or yeast cells. Yeast surface display methods are reviewed, for example, in Scholler et al., Methods in Molecular Biology 503:135-56 (2012), Cherf et al., Methods in Molecular Biology 1319:155-175 (2015), and Zhao et al., Methods in Molecular Biology 889:73-84 (2012).Methods for ribosome display are described, for example, in He et al. in Nucleic Acids Research 25:5132-5134 (1997) and Hanes et al. in PNAS 94:4937-4942 (1997).

[0234] In an exemplary embodiment of the invention, as proof of concept, an antigen-binding moiety capable of binding to a mutant Fc domain comprising the amino acid substitution P329G is provided.

[0235] More generally, there is provided an antibody capable of binding to a variant Fc domain comprising a substitution at position P329 (numbering according to Kabat EU index). In one embodiment, the at least one amino acid substitution comprises a substitution at position P329 (numbering according to Kabat EU index) with an amino acid selected from the list consisting of alanine (A), arginine (R), leucine (L), isoleucine (I) and proline (P). In one embodiment, the at least one amino acid substitution comprises the amino acid substitution P329G (numbering according to Kabat EU index).

[0236] In one embodiment, the antigen binding portion is capable of binding to a variant Fc domain. 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 variant Fc domain exhibits reduced binding affinity to an Fc receptor and / or reduced effector function compared to a native IgG1 Fc domain. In one embodiment, the Fc domain comprises one or more amino acid mutations that reduce binding to an Fc receptor and / or effector function.

[0237] In a preferred embodiment, the variant Fc domain comprises a P329G substitution.

[0238] In one embodiment, an antibody is provided that has at least one antigen-binding moiety that is capable of binding to a mutated Fc domain comprising a P329G substitution (EU numbering) but is incapable of binding to the non-mutated parent Fc domain.

[0239] In one embodiment, the antigen binding portion comprises: (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SEQ ID NO: 1; (b) a CDR H2 amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:12, or SEQ ID NO:15, and (c) a heavy chain variable domain (VH) comprising at least one of the CDR H3 amino acid sequences of SEQ ID NO:3.

[0240] In one embodiment, the antigen binding portion comprises: (d) the light chain (CDR L) 1 amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 18; (e) the CDR L2 amino acid sequence of SEQ ID NO:5, and (f) a light chain variable domain (VL) comprising at least one of the CDR L3 amino acid sequences of SEQ ID NO:6.

[0241] In a preferred embodiment, the antigen-binding portion comprises: (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SEQ ID NO: 1; (b) a CDR H2 amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:12, or SEQ ID NO:15, and (c) a heavy chain variable domain (VH) comprising the CDR H3 amino acid sequence of SEQ ID NO: 3; (d) the light chain (CDR L) 1 amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 18; (e) the CDR L2 amino acid sequence of SEQ ID NO:5, and (f) a light chain variable domain (VL) comprising the CDR L3 amino acid sequence of SEQ ID NO:6.

[0242] In one particular embodiment, the antigen binding portion comprises: (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SEQ ID NO: 1; (b) the CDR H2 amino acid sequence of SEQ ID NO:2, and (c) a heavy chain variable domain (VH) comprising the CDR H3 amino acid sequence of SEQ ID NO: 3; (d) the light chain (CDR L) 1 amino acid sequence of SEQ ID NO: 4; (e) the CDR L2 amino acid sequence of SEQ ID NO:5, and (f) a light chain variable domain (VL) comprising the CDR L3 amino acid sequence of SEQ ID NO:6.

[0243] In another particular embodiment, the antigen binding portion is (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SEQ ID NO: 1; (b) the CDR H2 amino acid sequence of SEQ ID NO: 9, and (c) a heavy chain variable domain (VH) comprising the CDR H3 amino acid sequence of SEQ ID NO: 3; (d) the light chain (CDR L) 1 amino acid sequence of SEQ ID NO: 4; (e) the CDR L2 amino acid sequence of SEQ ID NO:5, and (f) a light chain variable domain (VL) comprising the CDR L3 amino acid sequence of SEQ ID NO:6.

[0244] In another particular embodiment, the antigen binding portion is (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SEQ ID NO: 1; (b) the CDR H2 amino acid sequence of SEQ ID NO: 12, and (c) a heavy chain variable domain (VH) comprising the CDR H3 amino acid sequence of SEQ ID NO: 3; (d) the light chain (CDR L) 1 amino acid sequence of SEQ ID NO: 4; (e) the CDR L2 amino acid sequence of SEQ ID NO:5, and (f) a light chain variable domain (VL) comprising the CDR L3 amino acid sequence of SEQ ID NO:6.

[0245] In another particular embodiment, the antigen binding portion is (g) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SEQ ID NO: 1; (h) the CDR H2 amino acid sequence of SEQ ID NO: 15, and (i) a heavy chain variable domain (VH) comprising the CDR H3 amino acid sequence of SEQ ID NO: 3; (j) the light chain (CDR L) 1 amino acid sequence of SEQ ID NO: 4; (k) the CDR L2 amino acid sequence of SEQ ID NO:5, and (l) a light chain variable domain (VL) comprising the CDR L3 amino acid sequence of SEQ ID NO:6.

[0246] In another particular embodiment, the antigen binding portion is (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SEQ ID NO: 1; (b) the CDR H2 amino acid sequence of SEQ ID NO: 9, and (c) a heavy chain variable domain (VH) comprising the CDR H3 amino acid sequence of SEQ ID NO: 3; (d) the light chain (CDR L) 1 amino acid sequence of SEQ ID NO: 18; (e) the CDR L2 amino acid sequence of SEQ ID NO:5, and (f) a light chain variable domain (VL) comprising the CDR L3 amino acid sequence of SEQ ID NO:6.

[0247] In one embodiment, the antigen-binding portion comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:16.

[0248] In one embodiment, the antigen-binding portion comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7.

[0249] In one embodiment, the antigen-binding portion comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:10.

[0250] In one embodiment, the antigen-binding portion comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:13.

[0251] In one embodiment, the antigen-binding portion comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:14.

[0252] In one embodiment, the antigen-binding portion comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:16.

[0253] In one embodiment, the antigen-binding portion comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:17 and SEQ ID NO:19.

[0254] In one embodiment, the antigen-binding portion comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8.

[0255] In one embodiment, the antigen-binding portion comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:11.

[0256] In one embodiment, the antigen-binding portion comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:17.

[0257] In one embodiment, the antigen-binding portion comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:19.

[0258] In one embodiment, the antigen-binding portion comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8.

[0259] In one embodiment, the antigen-binding portion comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0260] In one embodiment, the antigen-binding portion comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13, and a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0261] In one embodiment, the antigen-binding portion comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14, and a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0262] In one embodiment, the antigen-binding portion comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16, and a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0263] In one embodiment, the antigen-binding portion comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17.

[0264] In one embodiment, the antigen-binding portion comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19.

[0265] In a preferred embodiment, the antigen-binding portion comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO:14 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO:11.

[0266] In one embodiment, the variant Fc domain comprises I253A, H310A and H435A ("AAA") substitutions. In one embodiment, an antibody is provided that comprises at least one antigen-binding moiety that is capable of binding to a variant Fc domain that comprises I253A, H310A and H435A substitutions (EU numbering) but is incapable of binding to the non-variant parent Fc domain.

[0267] In one embodiment, the antigen binding portion comprises: (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SEQ ID NO: 28; (b) the CDR H2 amino acid sequence of SEQ ID NO: 29, and (c) the CDR H3 amino acid sequence of SEQ ID NO: 30).

[0268] In one embodiment, the antigen binding portion comprises: (d) the light chain (CDR L) 1 amino acid sequence of SEQ ID NO: 31; (e) the CDR L2 amino acid sequence of SEQ ID NO: 32, and (f) comprises at least one of the CDR L3 amino acid sequences of SEQ ID NO: 33.

[0269] In one embodiment, the antigen-binding portion comprises at least one heavy chain complementarity determining region (CDR) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, and at least one light chain CDR selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33.

[0270] In one embodiment, the antigen binding portion comprises the heavy chain complementarity determining regions (CDRs) of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, and the light chain CDRs of SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33.

[0271] In one embodiment, the antigen binding portion comprises: (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SEQ ID NO: 28; (b) the CDR H2 amino acid sequence of SEQ ID NO: 29, and (c) a heavy chain variable region comprising the CDR H3 amino acid sequence of SEQ ID NO: 30; (d) the light chain (CDR L) 1 amino acid sequence of SEQ ID NO: 31; (e) the CDR L2 amino acid sequence of SEQ ID NO: 32, and (f) a light chain variable region comprising the CDR L3 amino acid sequence of SEQ ID NO: 33.

[0272] In one embodiment, the antigen-binding portion comprises a heavy chain variable region (VH) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO: 34, and a light chain variable region (VL) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NO: 35.

[0273] In one embodiment, the antigen-binding portion comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:34 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:35.

[0274] In one embodiment, the antigen binding portion comprises: (d) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SEQ ID NO: 130; (e) the CDR H2 amino acid sequence of SEQ ID NO: 131, and (f) a heavy chain variable region comprising at least one of the CDR H3 amino acid sequences of SEQ ID NO: 132.

[0275] In one embodiment, the antigen binding portion comprises: (g) the light chain (CDR L) 1 amino acid sequence of SEQ ID NO: 133; (h) the CDR L2 amino acid sequence of SEQ ID NO: 134, and (i) comprises at least one of the CDR L3 amino acid sequences of SEQ ID NO: 135.

[0276] In one embodiment, the antigen-binding portion comprises at least one heavy chain complementarity determining region (CDR) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:130, SEQ ID NO:131 and SEQ ID NO:132, and at least one light chain CDR selected from the group consisting of SEQ ID NO:133, SEQ ID NO:134 and SEQ ID NO:135.

[0277] In one embodiment, the antigen binding portion comprises the heavy chain complementarity determining regions (CDRs) of SEQ ID NO:130, SEQ ID NO:131, and SEQ ID NO:132, and the light chain CDRs of SEQ ID NO:133, SEQ ID NO:134, and SEQ ID NO:135.

[0278] In one embodiment, the antigen binding portion comprises: (g) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SEQ ID NO: 130; (h) the CDR H2 amino acid sequence of SEQ ID NO: 131, and (i) a heavy chain variable region comprising the CDR H3 amino acid sequence of SEQ ID NO: 132; (j) the light chain (CDR L) 1 amino acid sequence of SEQ ID NO: 133; (k) the CDR L2 amino acid sequence of SEQ ID NO: 134, and (l) a light chain variable region comprising the CDR L3 amino acid sequence of SEQ ID NO: 135.

[0279] In one embodiment, the antigen-binding portion comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 136, and a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NO:35.

[0280] In one embodiment, the antigen-binding portion comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 136, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:35.

[0281] target cell antigen As mentioned above, the (therapeutic) antibodies described herein may comprise at least one antigen-binding portion capable of binding to a target cell antigen (e.g., a tumor-specific antigen naturally present on the surface of a tumor cell) in addition to an antigen-binding portion capable of binding to a mutated Fc domain but not to a non-mutated parent Fc domain (i.e., capable of binding to a chimeric receptor of the invention). In the context of the present invention, such an antibody physically contacts a transduced T cell described herein comprising a chimeric receptor of the invention with a target cell (e.g., a tumor cell), and the transduced T cell is activated. Activation of the transduced T cell of the present invention preferentially leads to lysis of the target cell, as described herein.

[0282] Examples of target cell antigens (e.g., tumor markers) that naturally occur on the surface of target cells (e.g., tumors) are provided herein below and include, but are not limited to, FAP (fibroblast activation protein), CEA (carcinoembryonic antigen), p95 (p95HER2), BCMA (B cell maturation antigen), EpCAM (epithelial cell adhesion molecule), MSLN (mesothelin), MCSP (melanoma chondroitin sulfate proteoglycan), HER-1 (human epidermal growth factor 1), HER-2 (human epidermal growth factor 2), HER-3 (human epidermal growth factor 3), CD19, CD20, CD22, CD33, CD38, These include CD52Flt3, folate receptor 1 (FOLR1), human trophoblast cell surface antigen 2 (Trop-2), cancer antigen 12-5 (CA-12-5), human leukocyte antigen-antigen D-related (HLA-DR), MUC-1 (mucin-1), A33 antigen, PSMA (prostate specific membrane antigen), FMS-like tyrosine kinase 3 (FLT-3), PSMA (prostate specific membrane antigen), PSCA (prostate stem cell antigen), transferrin receptor, TNC (tenascin), carbonic anhydrase IX (CA-IX), and / or peptides bound to molecules of the human major histocompatibility complex (MHC).

[0283] A33 antigen, BCMA (B cell maturation antigen), cancer antigen 12-5 (CA-12-5), carbonic anhydrase IX (CA-IX), CD19, CD20, CD22, CD33, CD38, CEA (carcinoembryonic antigen), EpCAM (epithelial cell adhesion molecule), FAP (fibroblast activation protein), FMS-like tyrosine kinase 3 (FLT-3), folate receptor 1 (FOLR1), HER-1 (human epidermal growth factor 1), HER-2 (human epidermal growth factor 2), HER-3 (human epidermal growth factor 3), human leukocyte antigen-antigen D-related (HLA-DR), MSLN (mesothelin), MCS Sequences of (human) members of P (melanoma chondroitin sulfate proteoglycan), MUC-1 (mucin-1), PSMA (prostate specific membrane antigen), PSMA (prostate specific membrane antigen), PSCA (prostate stem cell antigen), p95 (p95HER2), transferrin receptor, TNC (tenascin), human trophoblast cell surface antigen 2 (Trop-2) are available in the UniProtKB / Swiss-Prot database and can be obtained from http: / / www.uniprot.org / uniprot / ?query=reviewed%3Ayes. These (protein) sequences are also associated with annotated modified sequences. The present invention also provides techniques and methods in which homologous sequences of the concise sequences provided herein, and genetic allelic variants, etc. are used. Preferably, such variants of the concise sequences herein are used. Preferably, such variants are genetic variants. The skilled artisan can easily deduce the relevant coding regions of these (protein) sequences from the entries in these databanks, which contain entries for genomic DNA as well as mRNA / cDNA: the sequence of (human) FAP (fibroblast activation protein) can be taken from Swiss-Prot database entry Q12884 (entry version 168, sequence version 5); the sequence of (human) CEA (carcinoembryonic antigen) can be taken from Swiss-Prot database entry P06731 (entry version 171, sequence version 3); the sequence of (human) EpCAM (epithelial cell adhesion molecule) can be taken from Swiss-Prot database entry P16422 (entry version 117, sequence version 2);The sequence of (human) MSLN (mesothelin) can be obtained from UniProt entry number Q13421 (version number 132, sequence version 2); the sequence of (human) FMS-like tyrosine kinase 3 (FLT-3) can be obtained from Swiss-Prot database entry P36888 (primary citable accession number) or Q13414 (secondary accession number), version number 165, sequence version 2; the sequence of (human) MCSP (melanoma chondroitin sulfate proteoglycan) can be obtained from UniProt entry number Q6 The sequence of (human) folate receptor 1 (FOLR1) can be obtained from UniProt entry no. P15328 (primary citable accession number) or Q53EW2 (secondary accession number), version no. 153 and sequence version 3; the sequence of (human) trophoblast surface antigen 2 (Trop-2) can be obtained from UniProt entry no. P09758 (primary citable accession number) or Q15658 (secondary accession number), version no. 172 and sequence version 3. The sequence of (human) PSCA (prostate stem cell antigen) can be obtained from UniProt entry number O43653 (primary citable accession number) or Q6UW92 (secondary accession number), version number 134 and sequence version 1; the sequence of (human) HER-1 (epidermal growth factor receptor) can be obtained from Swiss-Prot database entry P00533 (entry version 177, sequence version 2); the sequence of (human) HER-2 (receptor tyrosine protein kinase erbB-2) can be obtained from Swiss-Prot database entry P00533 (entry version 177, sequence version 2). can be obtained from Swiss-Prot database entry P04626 (entry version 161, sequence version 1); the sequence of (human) HER-3 (receptor tyrosine protein kinase erbB-3) can be obtained from Swiss-Prot database entry P21860 (entry version 140, sequence version 1); the sequence of (human) CD20 (B lymphocyte antigen CD20) can be obtained from Swiss-Prot database entry P11836 (entry version 117, sequence version 1);The sequence of (human) CD22 (B-lymphocyte antigen CD22) can be obtained from Swiss-Prot database entry P20273 (entry version 135, sequence version 2); the sequence of (human) CD33 (B-lymphocyte antigen CD33) can be obtained from Swiss-Prot database entry P20138 (entry version 129, sequence version 2); the sequence of (human) CA-12-5 (mucin 16) can be obtained from Swiss-Prot database entry The sequence of (human) HLA-DR can be obtained from Swiss-Prot database entry Q29900 (entry version 59, sequence version 1); the sequence of (human) MUC-1 (mucin-1) can be obtained from Swiss-Prot database entry P15941 (entry version 135, sequence version 3); the sequence of (human) A33 (cell surface A33 antigen) can be obtained from Swiss-Prot database entry P15941 (entry version 135, sequence version 3); , can be obtained from Swiss-Prot database entry Q99795 (entry version 104, sequence version 1); the sequence of (human) PSMA (glutamic acid carboxypeptidase 2) can be obtained from Swiss-Prot database entry Q04609 (entry version 133, sequence version 1); the sequence of (human) transferrin receptor can be obtained from Swiss-Prot database entries Q9UP52 (entry version 99, sequence version 1) and P02786 (entry version 152, sequence version 2); the sequence of (human) TNC (tenascin) can be obtained from Swiss-Prot database entry P24821 (entry version 141, sequence version 3); or the sequence of (human) CA-IX (carbonic anhydrase IX) can be obtained from Swiss-Prot database entry Q16790 (entry version 115, sequence version 2).

[0284] In a specific embodiment, the target cell antigen is selected from the group consisting of fibroblast activation protein (FAP), carcinoembryonic antigen (CEA), mesothelin (MSLN), CD20, folate receptor 1 (FOLR1) and tenascin (TNC).

[0285] Antibodies capable of binding to any of the above target cell antigens can be generated using methods well known in the art, such as immunizing a mammalian immune system and / or phage display using recombinant libraries as described hereinabove.

[0286] Immune activation Fc binding molecule The chimeric receptors of the invention can be combined with immunostimulatory Fc binding molecules capable of binding to the variant Fc domain. Suitable molecules are described herein below and in WO 2021 / 255138.

[0287] In one embodiment, there is provided a chimeric receptor as described herein above for use in a method for treating or delaying the progression of cancer, wherein the chimeric receptor is used in combination with any of the immunostimulatory Fc binding molecules described in WO 2021 / 255138.

[0288] In one embodiment, there is provided a chimeric receptor as described herein for use in a method for treating or delaying the progression of cancer, wherein the chimeric receptor is used in combination with a molecule described in Figure 29A and Example 5 of WO 2021 / 255138.

[0289] kit A further aspect of the present invention is a kit comprising or consisting of a nucleic acid encoding a chimeric receptor of the invention and / or a cell, preferably a T cell, for transducing / transduced with a chimeric receptor of the invention, and optionally one or several (therapeutic) antibodies as described herein above.

[0290] Thus, there is provided a kit comprising a transduced T cell capable of expressing a chimeric receptor of the invention, wherein the chimeric receptor comprises an extracellular domain comprising a mutated Fc domain or a fragment thereof, and an antibody, in particular a therapeutic antibody, comprising at least one antigen-binding moiety capable of binding to the mutated Fc domain but not to the non-mutated parent Fc domain, said antibody comprising at least one effector moiety as hereinbefore described.

[0291] Further provided is a kit comprising an isolated polynucleotide encoding a chimeric receptor of the present invention.

[0292] In one embodiment, the effector moiety is an antigen-binding moiety capable of binding to a target cell antigen as previously described herein.

[0293] In one embodiment, the effector moiety is an immunostimulatory moiety as described herein above, in particular a cytokine (eg, IL2 or a derivative thereof).

[0294] In one embodiment, the chimeric receptor comprises a variant Fc domain comprising a P329G substitution (numbering according to Kabat EU index). In a preferred such embodiment, the therapeutic antibody does not comprise a P329G substitution (numbering according to Kabat EU index). This prevents dimerization of the therapeutic antibody comprising an antigen-binding portion capable of specific binding to the P329G substitution. In one embodiment, the therapeutic antibody comprises an amino acid substitution selected from the group consisting of P329G, P329L, P329I, P329R and P329A (numbering according to Kabat EU index). In a preferred embodiment, the therapeutic antibody comprises a P329R substitution (numbering according to Kabat EU index).

[0295] In another preferred embodiment, the chimeric receptor comprises a variant Fc domain comprising the amino acid substitutions I253A, H310A and H435A (numbering according to Kabat EU index).In a preferred such embodiment, the therapeutic antibody comprises a P329G substitution (numbering according to Kabat EU index).

[0296] In a particular embodiment, the antibody is a therapeutic antibody, such as a tumor-specific antibody or an immune-activating antibody as described herein above. Tumor-specific antigens and immune-activating moieties are known in the art and have been described herein above. In the context of the present invention, the antibody is administered before, simultaneously with, or after administration of transduced T cells expressing the chimeric receptor of the present invention.

[0297] In one embodiment of the invention, a kit is provided comprising transduced T cells capable of expressing the amino acid sequence of SEQ ID NO: 87, or alternatively, a kit is provided comprising a polynucleotide encoding the amino acid sequence of SEQ ID NO: 87 (e.g., the kit comprises a polynucleotide comprising the sequence of SEQ ID NO: 96) in combination with an antibody comprising a heavy chain of SEQ ID NO: 109, a heavy chain of SEQ ID NO: 110, a light chain of SEQ ID NO: 11 and two light chains of SEQ ID NO: 108. This kit can be used for the treatment of FolR positive cancer.

[0298] In one embodiment of the invention, a kit is provided comprising transduced T cells capable of expressing the amino acid sequence of SEQ ID NO: 106, or alternatively, a kit is provided comprising a polynucleotide encoding the amino acid sequence of SEQ ID NO: 106 (e.g., the kit comprises a polynucleotide comprising the sequence of SEQ ID NO: 107) in combination with an antibody comprising a heavy chain of SEQ ID NO: 112, a heavy chain of SEQ ID NO: 113, a light chain of SEQ ID NO: 114 and two light chains of SEQ ID NO: 108. This kit can be used for the treatment of FolR1 positive cancer.

[0299] In one embodiment of the invention, a kit is provided comprising transduced T cells capable of expressing the amino acid sequence of SEQ ID NO: 87, or alternatively, a kit is provided comprising a polynucleotide encoding the amino acid sequence of SEQ ID NO: 87 (e.g., the kit comprises a polynucleotide comprising the sequence of SEQ ID NO: 96) in combination with an antibody comprising a heavy chain of SEQ ID NO: 109, a heavy chain of SEQ ID NO: 115, a light chain of SEQ ID NO: 108, and a light chain of SEQ ID NO: 111. This kit can be used for the treatment of FolR1 positive cancer.

[0300] In one embodiment of the invention, a kit is provided that comprises transduced T cells capable of expressing the amino acid sequence of SEQ ID NO: 106, or alternatively, a kit is provided that comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 106 (e.g., the kit comprises a polynucleotide comprising the sequence of SEQ ID NO: 107) in combination with an antibody comprising a heavy chain of SEQ ID NO: 112, a heavy chain of SEQ ID NO: 116, a light chain of SEQ ID NO: 114, and a light chain of SEQ ID NO: 108. This kit can be used for the treatment of FolR1 positive cancer.

[0301] In one embodiment of the invention, a kit is provided that comprises transduced T cells capable of expressing the amino acid sequence of SEQ ID NO: 87, or alternatively, a kit is provided that comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 87 (e.g., the kit comprises a polynucleotide comprising the sequence of SEQ ID NO: 96) in combination with an antibody that comprises a heavy chain of SEQ ID NO: 118, a heavy chain of SEQ ID NO: 119, a light chain of SEQ ID NO: 117, and two light chains of SEQ ID NO: 120. This kit can be used for the treatment of CEA positive cancer.

[0302] In one embodiment of the invention, a kit is provided that comprises transduced T cells capable of expressing the amino acid sequence of SEQ ID NO: 106, or alternatively, a kit is provided that comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 106 (e.g., the kit comprises a polynucleotide comprising the sequence of SEQ ID NO: 107) in combination with an antibody comprising a heavy chain of SEQ ID NO: 122, a heavy chain of SEQ ID NO: 123, a light chain of SEQ ID NO: 121 and two light chains of SEQ ID NO: 114. This kit can be used for the treatment of CEA positive cancer.

[0303] Furthermore, the parts of the kit of the invention can be packaged individually in vials or bottles or packaged together in a container or multicontainer unit. Furthermore, the kit of the invention can comprise a (closed) bag cell incubation system in which the patient cells, preferably the T cells as described herein, can be transduced with the chimeric receptor of the invention and incubated under GMP (Good Manufacturing Practice 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) conditions. Furthermore, the kit of the invention comprises a (closed) bag cell incubation system in which the isolated / obtained T cells of the patient can be transduced with the chimeric receptor of the invention and incubated under GMP. Furthermore, in the context of the present invention, the kit can also comprise a vector encoding the chimeric receptor as described herein. The kit of the invention can in particular be advantageously used for carrying out the method of the invention and can be employed for various applications as mentioned herein, for example as a research tool or medical tool. The manufacture of the kit preferably follows standard procedures known to the person skilled in the art.

[0304] In this context, patient-derived cells, preferably T cells, can be transduced with the chimeric receptors described herein using the kits described above. The extracellular domain, including the variant Fc domain, is not naturally present in or on T cells. Thus, patient-derived cells transduced with the kits of the invention acquire the ability to interact with a therapeutic antibody and can induce target cell elimination / lysis through interaction with the therapeutic antibody, which can bind to a tumor-specific antigen that is naturally present (endogenously expressed) on the surface of tumor cells. Binding of the extracellular domain of the chimeric receptor described herein activates the T cell, bringing it into physical contact with the tumor cell via the therapeutic antibody. Non-transduced T cells or endogenous T cells (e.g., CD8+ T cells) are unable to bind to the therapeutic antibody. Thus, T cells expressing the chimeric receptor molecules of the invention have the ability to lyse target cells in vivo and / or in vitro in the presence of the antibodies described herein. The corresponding target cells include cells expressing a surface molecule recognized by at least one, and preferably two, binding domains of the therapeutic antibodies described herein, i.e., a tumor-specific antigen that is naturally present on the surface of tumor cells.

[0305] Lysis of target cells can be detected by methods known in the art. Such methods therefore include, in particular, physiological in vitro assays. Such physiological assays can monitor cell death, for example, due to loss of cell membrane integrity (e.g., FACS-based propidium iodide assay, trypan blue influx assay, photometric enzyme release assay (LDH), radiometric 51Cr release assay, fluorometric Europium release assay and calcein AM release assay). Further assays include, for example, photometric MTT, XTT, WST-1 and Alamar Blue assays, radioactive 3H-Thd incorporation assays, clonogenic assays measuring cell division activity, and fluorescent rhodamine 123 assays measuring mitochondrial transmembrane gradients to monitor cell viability. Furthermore, apoptosis can be monitored, for example, by FACS-based phosphatidylserine exposure assays, ELISA-based TUNEL tests, caspase activity assays (photometric, fluorimetric or ELISA-based) or by analyzing altered cell morphology (shrinkage, membrane blebbing).

[0306] Exemplary Therapeutic Uses and Methods of Treatment The molecules or constructs provided herein (e.g., chimeric receptors, transduced T cells and kits) are particularly useful in medical settings, especially for the treatment of cancer. For example, tumors can be treated with transduced T cells expressing the chimeric receptors of the present invention in conjunction with therapeutic antibodies that contain at least one antigen-binding moiety capable of binding to a target cell antigen and at least one antigen-binding moiety capable of binding to a mutated Fc domain (e.g., containing a P329G substitution according to EU numbering) but not capable of binding to a non-mutated parent Fc domain. Thus, in certain embodiments, the chimeric receptors, transduced T cells or kits are used in the treatment of cancer, especially cancers of epithelial, endothelial or mesothelial origin and hematological cancers.

[0307] The tumor specificity of the treatment is provided by a therapeutic antibody that binds to a target cell antigen, which is administered before, simultaneously with or after the administration of the transduced T cells expressing the chimeric receptor of the invention. In this context, the transduced T cells are universal T cells, since they are not specific for a given tumor, but can target any tumor depending on the specificity of the therapeutic antibody used according to the invention.

[0308] The cancer may be of epithelial, endothelial or mesothelial origin or of blood. In one embodiment, the cancer is selected from the group consisting of gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, oral cancer, gastric cancer, cervical cancer, B and T cell lymphoma, myeloid leukemia, ovarian cancer, leukemia, lymphocytic leukemia, nasopharyngeal cancer, colon cancer, prostate cancer, renal cell cancer, head and neck cancer, skin cancer (melanoma), cancer of the genitourinary tract, such as testicular cancer, ovarian cancer, endothelial cancer, cervical cancer, and kidney cancer, cancer of the bile duct, cancer of the esophagus, cancer of the salivary gland, and cancer of the thyroid gland, or other neoplastic diseases such as blood tumors, gliomas, sarcomas, or osteosarcomas.

[0309] For example, neoplastic diseases and / or lymphomas can be treated with specific constructs directed to these medical indications. For example, gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, and / or oral cancer can be treated with antibodies directed against (human) EpCAM (as a tumor-specific antigen naturally occurring on the surface of tumor cells).

[0310] Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, and / or oral cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against HER1, preferably human HER1. Furthermore, gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma, and / or oral cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against MCSP, preferably human MCSP. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma, and / or oral cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against FOLR1, preferably human FOLR1. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma, and / or oral cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed 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 can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against PSCA, preferably human PSCA. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma, and / or oral cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against EGFRvIII, preferably human EGFRvIII. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, glioblastoma, and / or oral cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against MSLN, preferably human MSLN. Gastric cancer, breast cancer, and / or cervical cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against HER2, preferably human HER2.Gastric and / or lung cancers can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against HER3, preferably human HER3. B-cell and / or T-cell lymphomas can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against CD20, preferably human CD20. B-cell and / or T-cell lymphomas can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against CD22, preferably human CD22. Myeloid leukemias can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against CD33, preferably human CD33. Ovarian, lung, breast and / or gastrointestinal cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against CA12-5, preferably human CA12-5. Gastrointestinal, leukemia and / or nasopharyngeal cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against HLA-DR, preferably human HLA-DR. Colon, breast, ovarian, lung and / or pancreatic cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against MUC-1, preferably human MUC-1. Colon cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against A33, preferably human A33. Prostate cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against PSMA, preferably human PSMA. Gastrointestinal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, breast cancer, ovarian cancer, skin cancer, and / or oral cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against the transferrin receptor, preferably the human transferrin receptor.Pancreatic cancer, lung cancer, and / or breast cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against the transferrin receptor, preferably the human receptor. Renal cancer can be treated with the transduced T cells of the invention administered before, simultaneously or after administration of a therapeutic antibody directed against CA-IX, preferably human CA-IX.

[0311] The invention also relates to a method for the treatment of diseases, for example malignant diseases such as cancers of epithelial, endothelial or mesothelial origin and / or hematological cancers.In the context of the present invention, the subject is a human being.

[0312] In the context of the present invention, a particular method for the treatment of a disease is (a) isolating T cells, preferably CD8+ T cells, from a subject; (b) transducing isolated T cells, preferably CD8+ T cells, with a chimeric receptor as described herein; (c) administering to the subject the transduced T cells, preferably CD8+ T cells.

[0313] In the context of the present invention, the transduced T cells, preferably CD8+ T cells, and / or one or more therapeutic antibodies are co-administered to the subject by intravenous infusion.

[0314] Furthermore, in the context of the present invention, the present invention relates to a method for treating a disease, comprising the steps of: (a) isolating T cells, preferably CD8+ T cells, from a subject; (b) transducing isolated T cells, preferably CD8+ T cells, with a chimeric receptor as described herein; (c) expanding T cells, preferably CD8+ T cells, with anti-CD3 and anti-CD28 antibodies; (d) administering the transduced T cells, preferably CD8+ T cells, to the subject.

[0315] The above mentioned step (c) (referring to the step of expanding T cells such as TILs by anti-CD3 and / or anti-CD28 antibodies) can also be carried out in the presence of (stimulatory) cytokines such as interleukin 2 and / or interleukin 15 (IL-15). In the context of the present invention, the above mentioned step (d) (referring to the step of expanding T cells such as TILs by anti-CD3 and / or anti-CD28 antibodies) can also be carried out in the presence of interleukin 12 (IL-12), interleukin 7 (IL-7) and / or interleukin 21 (IL-21).

[0316] The method of treatment further comprises the administration of an antibody used according to the invention. Said antibody may be administered before, simultaneously or after the administration of the transduced T cells. In the context of the present invention, the administration of the transduced T cells will be performed by intravenous infusion. In the context of the present invention, the transduced T cells are isolated / obtained from the subject to be treated.

[0317] The present invention further contemplates co-administration protocols with other compounds, such as molecules capable of providing activation signals for immune effector cells, cell proliferation or cell stimulation, which may be, for example, additional primary activation signals for T cells (e.g., additional costimulatory molecules: molecules of the B7 family, Ox40L, 4.1BBL, CD40L, anti-CTLA-4, anti-PD-1), or additional cytokines interleukins (e.g., IL-2).

[0318] The above-mentioned compositions of the present invention may also be diagnostic compositions, optionally further comprising means and methods for detection.

[0319] composition Furthermore, the present invention provides compositions (medicaments) comprising the antibody molecules and / or transduced T cells comprising the chimeric receptor of the present invention and / or nucleic acid molecules and vectors encoding the chimeric receptor according to the present invention. Furthermore, the present invention provides kits comprising one or more of the above compositions. In the context of the present invention, the composition is a pharmaceutical composition optionally further comprising suitable formulations that are carriers, stabilizers and / or additives. Thus, in the context of the present invention, pharmaceutical compositions (medicaments) are provided comprising the antibody molecules described herein administered in combination with transduced T cells comprising the chimeric receptor as described herein and / or compositions comprising said transduced T cells, where said antibody molecules are administered before, simultaneously or after administration of the transduced T cells comprising the chimeric receptor of the present invention.

[0320] The use of the term "in combination" does not limit the order in which the components of a treatment regimen are administered to a subject. Thus, the pharmaceutical compositions / medicaments described herein encompass administration of an antibody as defined herein before, simultaneously with, or after administration of transduced T cells comprising a chimeric receptor of the invention. "In combination" as used herein also does not limit the timing of administration of an antibody as defined above and a transduced T cell comprising a chimeric receptor as defined herein. Thus, when the two components are not administered simultaneously / together, their administration may be spaced apart by 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours, or any suitable time period that is readily determined by the skilled artisan and / or as described herein.

[0321] In the context of the present invention, the term "in combination" also encompasses the situation where an antibody as defined herein and a transduced T cell comprising a chimeric receptor according to the present invention are pre-incubated together before administration to a subject. Thus, these two components may be pre-incubated for, for example, 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes or 1 hour before administration, or any suitable time that can be easily determined by a person skilled in the art. In another embodiment, the present invention relates to a treatment regimen in which an antibody as defined herein and a transduced T cell comprising a chimeric receptor as defined herein are administered simultaneously / together. In the context of the present invention, an antibody as defined herein may be administered prior to administration of a transduced T cell comprising a chimeric receptor.

[0322] Moreover, "in combination" as used herein does not limit the disclosed treatment regimen to the administration of the antibody as defined herein and the transduced T cells (preferably CD8+ T cells) comprising the chimeric receptor of the invention in sequential order (i.e., administration of one of these two components followed (at a certain time interval) by the other without administration and / or implementation of any other treatment protocol in between). Thus, the treatment regimen also encompasses the separate administration of the antibody molecule as defined herein and the transduced T cells (preferably CD8+ T cells) comprising the chimeric receptor according to the invention, separated by one or more treatment protocols necessary and / or suitable for the treatment or prevention of the disease or its symptoms. Examples of such intervening treatment protocols include, but are not limited to, administration of analgesics, administration of chemotherapeutic agents, surgical treatment of the disease or its symptoms, etc. Thus, the treatment regimens disclosed herein comprise the administration of an antibody as defined herein and a transduced T cell (preferably a CD8+ T cell) comprising a chimeric receptor as defined herein, together with zero, one or more treatment protocols suitable for treating or preventing a disease or a symptom thereof, as described herein or as known in the art.

[0323] In particular, it is envisaged that said pharmaceutical composition / medicine is administered to the patient via infusion or injection. In the context of the present invention, the transduced T cells comprising the chimeric receptors described herein will be administered to the patient via infusion or injection. The administration of suitable compositions / medicines can be performed by different methods: intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0324] The pharmaceutical composition / medicine of the present invention may further comprise a pharma- ceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline, water, emulsions such as oil-in-water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions containing such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to subjects in appropriate doses. The administration regimen is determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for one patient depends on many factors, including the patient's size, body surface area, age, the specific compound administered, sex, time and route of administration, general condition, and other drugs administered at the same time. Usually, the regimen for regular administration of the pharmaceutical composition is preferably in the range of 1 μg to 5 g units per day. However, more preferred dosages for continuous infusion would be in the range of 0.01 μg to 2 mg per kg of body weight per hour, 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 increments. Particularly preferred dosages are described hereinafter. Progress can be monitored by periodic evaluation. Dosages vary, but a preferred dosage for intravenous administration of DNA is approximately 10 of the DNA molecule. 6 From 10 12 It's a copy.

[0325] The compositions of the invention may be administered locally or systemically. Typically, administration is parenteral (e.g., intravenously), and the transduced T cells may be administered, for example, by catheter, directly to the target site, or to a site in an artery. 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, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the present invention may comprise a proteinaceous carrier, such as, for example, serum albumin or immunoglobulin (preferably of human origin). It is envisaged that the pharmaceutical composition of the present invention, in addition to the proteinaceous antibody construct (as described in the present invention) or the nucleic acid molecule or vector encoding it, and / or the cells, may comprise further biologically active agents depending on the intended use of the pharmaceutical composition. Such agents may be drugs acting on the gastrointestinal system, drugs acting as cytostatica, drugs preventing hyperurikemia, drugs inhibiting immune responses (e.g. corticosteroids), drugs acting on the cardiovascular system, and / or drugs such as T cell costimulatory molecules or cytokines known in the art.

[0326] Further exemplary embodiments: 1. (i) an extracellular domain comprising a variant Fc domain or a fragment thereof; (ii) a transmembrane domain; A chimeric receptor comprising:

[0327] 2.(iii) The chimeric receptor of embodiment 1, further comprising at least one intracellular stimulatory signaling domain and / or at least one costimulatory signaling domain.

[0328] 3. The chimeric receptor of embodiment 1 or 2, wherein the extracellular domain comprises a mutated CH2 domain and / or a mutated CH3 domain.

[0329] 4. The chimeric receptor of any one of embodiments 1 to 3, wherein the extracellular domain comprises a mutated CH2 domain.

[0330] 5. The chimeric receptor according to any one of embodiments 1 to 4, wherein the variant Fc domain comprises at least one amino acid substitution at a position selected from the group consisting of L234, L235, I253, H310, P331, P329 and H435 (numbering according to Kabat EU index), in particular the amino acid substitution is selected from the group consisting of L234A, L235A, I253A, N297A, H310A, P329G and / or H435A.

[0331] 6. The chimeric receptor of any one of embodiments 1 to 5, wherein the extracellular domain comprises a mutated CH2 domain, and wherein the mutated CH2 domain comprises the amino acid substitution P329G (numbering according to Kabat EU index).

[0332] 7. The extracellular domain comprises a variant CH2 domain and / or a variant CH3 domain, and the variant CH2 domain and / or the variant CH3 domain have an amino acid substitution (numbering according to Kabat EU index): (a) I253A, H310A and H435A, (b)I253A (c) I253A and H310A (d) I253A and H435A (e) H310A and H435A (f) H310A, (g) H435A, (h) L234A, L235A, and P329G, or (i) L234A, L235A, I253A, H310A, P329G and H435A 7. The chimeric receptor according to any one of embodiments 1 to 6, comprising:

[0333] 8. The chimeric receptor of any one of embodiments 1 to 7, wherein the extracellular domain does not comprise a heavy chain variable domain (VH) and / or a light chain variable domain (VL).

[0334] 9. The chimeric receptor of any one of embodiments 1 to 8, wherein the transmembrane domain is selected from the group consisting of a CD8, CD4, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10 or DAP12 transmembrane domain or a fragment thereof.

[0335] 10. The chimeric receptor of any one of embodiments 2 to 9, wherein the transmembrane domain is a CD8 transmembrane domain, in particular, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 11.

[0336] 11. The chimeric receptor of any one of embodiments 2-10, wherein the stimulatory signaling domain is 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 stimulatory signaling activity.

[0337] 12. The chimeric receptor of any one of embodiments 2 to 11, wherein the stimulatory signaling domain is the intracellular domain of CD3z or a fragment thereof that retains stimulatory signaling activity, in particular, wherein the stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 13.

[0338] 13. The chimeric receptor of any one of embodiments 2 to 12, wherein the costimulatory signaling domain is selected from the group consisting of the intracellular domains of CD27, CD28, CD137, OX40, ICOS, DAP10 and DAP12 or fragments thereof that retain costimulatory signaling activity.

[0339] 14. The chimeric receptor of any one of embodiments 2 to 13, wherein the costimulatory signaling domain is the CD137 intracellular domain or a fragment thereof that retains CD137 costimulatory activity, in particular, wherein the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 12.

[0340] 15. The chimeric receptor of any one of embodiments 2-14, wherein the costimulatory signaling domain is the CD28 intracellular domain or a fragment thereof that retains CD28 costimulatory activity.

[0341] 16. A chimeric receptor according to any one of embodiments 2 to 15, comprising one stimulatory signalling domain comprising the intracellular domain of CD3z or a fragment thereof that retains CD3z stimulatory signalling activity, and one costimulatory signalling domain comprising the intracellular domain of CD137 or a fragment thereof that retains CD137 costimulatory signalling activity.

[0342] 17. The chimeric receptor of any one of embodiments 2 to 16, wherein the stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 13 and the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 12.

[0343] 18. A chimeric receptor according to any one of embodiments 1 to 17, wherein the extracellular domain is linked to the transmembrane domain, optionally via a peptide linker.

[0344] 19. The chimeric receptor of any one of embodiments 1 to 18, wherein the transmembrane domain is linked at its N-terminus to the C-terminus of the extracellular domain.

[0345] 20. The chimeric receptor of any one of embodiments 2 to 19, wherein the transmembrane domain is linked, optionally via a peptide linker, to the costimulatory signaling domain or the stimulatory signaling domain.

[0346] 21. The chimeric receptor of any one of embodiments 2 to 20, wherein the costimulatory signaling domain is linked at its N-terminus to the C-terminus of the transmembrane domain.

[0347] 22. The chimeric receptor of embodiment 21, comprising a stimulatory signaling domain, wherein the stimulatory signaling domain is linked at its N-terminus to the C-terminus of the costimulatory signaling domain.

[0348] 23. The chimeric receptor of any one of embodiments 1 to 22, wherein the chimeric 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.

[0349] 24. A chimeric receptor comprising the amino acid sequence of SEQ ID NO:7, or a chimeric receptor comprising the amino acid sequence of SEQ ID NO:125.

[0350] 25. A chimeric receptor described in any one of embodiments 1 to 22, comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:87, SEQ ID NO:106, SEQ ID NO:125 and SEQ ID NO:126.

[0351] 26. A chimeric receptor comprising or consisting of the amino acid sequence of SEQ ID NO:87.

[0352] 27. A chimeric receptor comprising or consisting of the amino acid sequence of SEQ ID NO: 106.

[0353] 28. A chimeric receptor comprising or consisting of the amino acid sequence of SEQ ID NO: 125.

[0354] 29. A chimeric receptor comprising or consisting of the amino acid sequence of SEQ ID NO: 127.

[0355] 30. An isolated polynucleotide encoding a chimeric receptor according to any one of embodiments 1 to 29.

[0356] 31. A polypeptide encoded by the isolated polynucleotide of embodiment 30.

[0357] 32. A vector, particularly an expression vector, comprising a polynucleotide according to embodiment 30.

[0358] 33. A transduced T cell comprising a polynucleotide according to embodiment 30 or a vector according to embodiment 32.

[0359] 34. A transduced T cell capable of expressing a chimeric receptor according to any one of embodiments 1 to 29.

[0360] 35. (A) a transduced T cell capable of expressing a chimeric receptor according to any one of embodiments 1 to 30, wherein the chimeric receptor comprises an extracellular domain comprising a mutant Fc domain or a fragment thereof; (B) an antibody comprising at least one antigen-binding portion capable of binding to the variant Fc domain but incapable of binding to the non-variant parent Fc domain, the antibody comprising at least one effector portion.

[0361] 36. (A) an isolated polynucleotide encoding the chimeric receptor of any one of embodiments 1 to 30, wherein the chimeric receptor comprises an extracellular domain comprising a variant Fc domain or a fragment thereof; (B) an antibody comprising at least one antigen-binding portion capable of binding to the variant Fc domain but incapable of binding to the non-variant parent Fc domain, the antibody comprising at least one effector portion.

[0362] 37. The kit according to embodiment 36 or 37, wherein the effector moiety is (i) an antigen-binding moiety capable of binding to a target cell antigen, or (ii) and an immune activating moiety, in particular a cytokine.

[0363] 38. A kit according to any one of embodiments 35 to 37 for use as a medicament.

[0364] 39. A chimeric receptor according to any one of embodiments 1 to 29, or a transduced T cell according to any one of embodiments 33 or 34, for use as a medicament, wherein the chimeric receptor comprises an extracellular domain comprising a mutated Fc domain or a fragment thereof, and the transduced T cell expressing the chimeric receptor is administered prior to, simultaneously with, or following administration of an antibody comprising at least one antigen-binding moiety capable of binding to a target cell antigen and at least one antigen-binding moiety capable of binding to the mutated Fc domain but not to the non-mutated parent Fc domain.

[0365] 40. A kit according to any one of embodiments 35 to 36 for use in the treatment of a disease, in particular for use in the treatment of cancer.

[0366] 41. A method of treating a disease in a subject, the method comprising administering to the subject transduced T cells capable of expressing a chimeric receptor according to any one of embodiments 1-30, wherein the chimeric receptor comprises an extracellular domain comprising a mutated Fc domain or a fragment thereof, and administering prior to, simultaneously with, or after administration of the transduced T cells, a therapeutically effective amount of an antibody comprising at least one antigen-binding moiety capable of binding to the mutated Fc domain but not to the non-mutated parent Fc domain, wherein the antibody comprises at least one effector moiety.

[0367] 42. The method according to embodiment 41, wherein the effector moiety is (i) an antigen-binding moiety capable of binding to a target cell antigen, or (ii) and an immune activating moiety, in particular a cytokine.

[0368] 43. Use of a chimeric receptor according to any one of embodiments 1 to 29, a polynucleotide according to embodiment 30 or a transduced T cell according to embodiment 33 or 34 for the manufacture of a medicament.

[0369] 44. The use according to embodiment 43, wherein the medicament is for the treatment of cancer.

[0370] 45. The use according to embodiment 44, wherein said cancer is selected from cancers of epithelial, endothelial or mesothelial origin and hematological cancers.

[0371] 46. ​​The invention as hereinbefore described.

[0372] These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature on any one of the antibodies, methods, uses and compounds employed according to the present invention can be retrieved from public libraries and databases, for example, using electronic devices. For example, the public database "Medline" available on the Internet can be accessed 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, http: / / www.tigr.org / , etc., are known to those skilled in the art and can be accessed at, for example, http: / / www.lycos.com.

[0373] Exemplary Sequences The definitions of CDRs in the sequence listing are according to Kabat. [Table 1] TIFF2025512797000002.tif255170TIFF2025512797000003.tif243170 [Table 2]

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

Table 10

Table 11

Table 12

Table 13

Table 14

[0374] The following are examples of methods and compositions of the present invention. Given the general description provided above, it will be understood that other various embodiments may be practiced.

[0375] Recombinant DNA Technology Standard methods were used to manipulate DNA as described by Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions. General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242.

[0376] DNA sequencing DNA sequences were determined by double-stranded Sanger sequencing.

[0377] Gene synthesis Desired gene segments were generated by PCR using appropriate templates, as appropriate, or synthesized by Genscript Biotech (NJ, USA) from synthetic oligonucleotides and PCR products by automated gene synthesis. Gene segments flanked by single restriction endonuclease cleavage sites were cloned into standard cloning / sequencing vectors. Plasmid DNA was purified from transformed bacteria and concentrations were measured by UV spectroscopy. DNA sequences of subcloned gene fragments were confirmed by DNA sequencing. Gene segments were designed with appropriate restriction sites to allow subcloning into the respective expression vectors. All constructs were designed with a 5'-terminal DNA sequence encoding a leader peptide that targets the protein for secretion in eukaryotic cells.

[0378] Production of IgG-like protein in Expi293F cells Antibodies and bispecific antibodies were produced by transient transfection of Expi293F cells. Cells were seeded at a density of 2.5x106 / ml in Expi293 medium (Gibco, Cat. No. 1435101). Expression vector and ExpiFectamine (Gibco, ExpiFectamine Transfection Kit, Cat. No. 13385544) were mixed separately in OptiMEM (Gibco, Cat. No. 11520386). After 5 min, both solutions were combined, mixed by pipette and incubated for 25 min at room temperature. Cells were added to the vector / ExpiFectamine solution and incubated for 24 h at 37°C in a shaking incubator with a 5% CO2 atmosphere. One day after transfection, supplements (Enhancer 1+2, ExpiFectamine Transfection Kit) were added. After 4-5 days, the cell supernatant was harvested by centrifugation followed by filtration (0.2 μm filter), and the protein was purified from the harvested supernatant by standard methods as follows.

[0379] Production of IgG-like protein in CHO K1 cells Alternatively, the antibodies and bispecific antibodies described herein were prepared by Evitria using a proprietary vector system and conventional (non-PCR-based) cloning techniques in suspension-adapted CHO K1 cells (originally acquired from ATCC and adapted by Evitria to serum-free growth in suspension culture). For production, Evitria used proprietary animal component-free serum-free media (eviGrow and eviMake2) and a proprietary transfection reagent (eviFect). Supernatants were harvested by centrifugation followed by filtration (0.2 μm filter) and proteins were purified from the harvested supernatants by standard methods.

[0380] Purification of IgG-like proteins Proteins were purified from filtered cell culture supernatants with reference to standard protocols. Briefly, Fc-containing proteins were purified from cell culture supernatants by Protein A-affinity chromatography (equilibration 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, followed immediately by neutralization of the pH of the sample. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15 (Art.Nr.:UFC903096)) and aggregated proteins were separated from monomeric proteins by size-exclusion chromatography in 20 mM histidine, 140 mM sodium chloride, pH 6.0.

[0381] Analysis of IgG-like proteins The concentration of purified proteins was determined by measuring the absorbance at 280 nm using the mass extinction 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 reducing agents using LabChip GXII or LabChip GX Touch (Perkin Elmer). Determination of aggregate content was performed 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).

[0382] Preparation of lentiviral supernatant and transduction into Jurkat cells Lipofectamine LTX™ based transfection was performed using approximately 70% confluent Lenti-X™ 293T cells (Takara, #632180) with chimeric receptor encoding transfer vector and packaging vectors pCAG-VSVG and psPAX2 in a 2:1:2 molar ratio (Giry-Laterriere M,et al Methods Mol Biol.2011;737:183-209, Myburgh R et al Mol Ther Nucleic Acids.2014). As a control for all experiments, mock virus-like particles (VLPs) were also produced using only packaging vector and no transfer vector. After 48 hours, the supernatant was collected and centrifuged at 350×g for 5 min to remove remaining cells and the viral particles were purified. VLPs were used directly or concentrated 10-fold (Lenti-x-Concentrator, Takara, #631231) and added to 24-well plates with 8ug / ml polybrene (Sigma Aldrich) and Lentiboost P (1:100) (Sirion Biotech, #SB-P-LV-101-12) for spinfection of Jurkat NFAT (GloResponse Jurkat NFAT-RE-luc2P, Promega #CS176501) or Jurkat TCRab-KO CD4+ (T-cell activation bioassay (TCRαβ-KO), Promega, #GA1172) cells at 1100×g for 99 minutes at 31° C. Cells were incubated for at least 72 hours before transduction was confirmed by flow cytometry.

[0383] Selection of transduced cells If the percentage of transduced cells was less than 90%, the cells were sorted for eGFP-positive cells. 3,000,000–10,000,000 cells were collected and spun down (400 × g, 4 min). The medium was removed and the cells were resuspended in MACS buffer (Miltenyi Biotec, #130-091-222) supplemented with 5% BSA (Miltenyi Biotec, #130-091-376). The cells were pool-sorted for eGFP-positive cells on a BD FACSAriaIII. For sorting of Jurkat cells, a 100 micron nozzle was used and the 4-way purity precision mode was applied.

[0384] Jurkat NFAT activation assay The Jurkat activation assay measures T cell activation in a human acute lymphoblastic leukemia reporter cell line (GloResponse Jurkat NFAT-RE-luc2P, Promega, #CS176501 or T cell activation bioassay TCRαβ-KO CD4+, Promega, #GA1172). This immortalized T cell line is engineered to stably express a luciferase reporter that is driven by T cell activation via CD3. After transduction, the cell line expresses a chimeric antigen receptor construct with a CD3z signaling domain. Binding of the construct to an immobilized adaptor molecule (such as a tumor antigen binding adaptor molecule) results in cross-linking, leading to T cell activation and expression of luciferase. After addition of substrate, cellular changes in NFAT activity can be measured as relative luminescence units. The assay was performed in a 384 plate (Falcon, #353988, flat white, white bottom). Target cells (transduced Jurkat cells) and effector cells were seeded in triplicate in a 2.5:1 ratio (20000 effector cells and 8000 target cells) in RPMI-1640+10% FCS+1% Glutamax (growth medium) at 20μl or 10ul, respectively. For the Jurkat NFAT assay, the readout was performed using GloSensor cAMP Reagent (Promega, #E1291) and 2% of the final volume (here 40ul, resulting in 0.8ul / well) was added to the wells. Additionally, serial dilutions of the antibody of interest were prepared in growth medium and 10ul were added to the wells to obtain final concentrations ranging from 20nM to 0.00122nM in the assay plate, with a total final volume of 40μl per well. The 384-well plate was centrifuged at 300g for 1 minute at room temperature and incubated at 37°C and 5% CO2 in a humidified atmosphere. After 4-6 hours of incubation, plates were allowed to equilibrate to room temperature for 10 minutes before measurement. For Jurkat TCRab-KO CD4+ cells, 100% of the final volume of Bio-Glo-NL reagent (Promega, #J3082) was added, plates were centrifuged at 350×g for 1 minute, and incubated for 5-10 minutes.For assays using Jurkat NFAT cells, cAMP reagent was added already at the time of cell plating (see above). Relative luminescence units (RLU) per second / well were then immediately measured using a Tecan microplate reader. Concentration-response curves were fitted using GraphPad Prism version 8 to determine EC. 50 The value was calculated.

[0385] Isolation of primary T cells from buffy coats Buffy coats were ordered from Blutspende Zurich (Rutistrasse 19, 8952 Schlieren). Leucosep tubes containing 15 mL of room temperature Histopaque density gradient medium (Sigma-Aldrich, #10771) were prepared and centrifuged at 400 × g for 5 min until the Histopaque passed through the filter. Blood was transferred to a T75 flask and an equal volume of DPBS was added. 30 ml of blood / buffer mixture was added to the Leucosep tube and centrifuged at high speed for 20 min at 1200 × g for separation. The band containing peripheral blood mononuclear cells (PBMCs) was carefully pipetted into a new 50 ml Falcon tube and topped up to 50 ml with DPBS. The tubes were centrifuged at 350 × g for 10 min (at low speed trituration) and then the supernatant was discarded. The cells were washed three more times with DPBS (1st. 250×g, 10 min; 2nd. 250×g, 10 min; 3rd. 120×g, 10 min) before being resuspended and counted. CD4+ and CD8+ T cell isolation was performed by negative selection using CD4+ or CD8+ T cell isolation kits (Miltenyi, #130-096-533, #130-096-495) according to the manufacturer's instructions. Cells were either frozen or used immediately after isolation. Cells were cultured in modified RPMI (Gibco, #11530446), 10% FBS (Sigma, #F4135-500ML), 1% Glutamax (Gibco, #35050-038), 50 IU / ml Proleukin (Novartis), 25 ng / ml IL-7 (Miltenyi, #130-095-364) and 50 ng / ml IL-15 (Miltenyi, #130-095-766) (T cell medium).

[0386] Preparation of virus-like particles (VLPs) Lipofectamine LTX™-based transfection (Thermo Fisher, #15338-100) was performed using approximately 70% confluent Lenti-X™ 293T cells (Takara #632180) with the transfer vector-encoding construct and the packaging vectors pCAG-VSVG and psPAX2 in a molar ratio of 2:1:2 (Giry-Laterriere M, et al Methods Mol Biol. 2011;737:183-209, Myburgh R, et al Mol Ther Nucleic Acids. 2014). As a control for all experiments, mock virus-like particles (VLPs) were generated using only the packaging vector and no transfer vector. After 48 hours, the supernatant was collected and centrifuged at 500×g for 10 minutes to remove remaining cells. VLPs were concentrated 10-fold using a Lenti-X Concentrator (Takara #631231) according to the manufacturer's instructions. For storage, VLPs were aliquoted into Eppendorf tubes, flash frozen in liquid nitrogen, and then stored at -80°C.

[0387] Transduction of primary T cells CD4+ and CD8+ T cells were thawed, mixed 1:1, and directly activated for 24 hours using ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator (StemCell, #10990) according to the manufacturer's manual. Activated cells were then resuspended, counted, and seeded at 1,000,000 cells / well / ml into 24-well plates. VLPs were thawed at 37°C, and 150 μl was added to the activated cells in 24-well plates with 8 μg / ml polybrene (Sigma Aldrich) and Lentiboost P (1:100) (Sirion Biotech, #SB-P-LV-101-12). After incubating the cells for 24 hours at 37°C, 5% CO2, they were resuspended in 4 ml of fresh T cell medium and added to gREX plates (Wilson Wolf, #MSPP-80192M) for expansion. 72 hours after transduction, expression of the constructs was confirmed by flow cytometry.

[0388] STAT5 phosphorylation assay Untransduced and transduced T cells were spun down, washed with DPBS, and plated in cytokine-free medium (modified RPMI, 10% FBS, 1x Glutamax (assay medium)) to starve (4 hours to overnight). Fixation buffer I (BD, #557870) was placed at 37°C and Perm buffer III (BD, #558050) was placed at -20°C prior to the assay. Starved T cells were spun down and resuspended in assay medium. Depending on the expression rate of the transduced T cell pool, T cells were mixed with untransduced cells to ensure a similar distribution of untransduced (eGFP-) and construct expressing cells (eGFP+) in each well. 150,000-300,000 cells were plated in 25 μl in a 96-well V-bottom plate. Cytokines or molecules were diluted in assay medium and 50 μl of the dilution was added to the T cells in duplicate. Plates were incubated at 37°C for 15 min, after which 75 μl of Fixation Buffer I was added to the wells. Plates were incubated at 37°C for an additional 30 min, after which the plates were spun down (400×g, 3 min) and the supernatant was discarded. Cells were resuspended in 100 μl of ice-cold Perm Buffer III and incubated on ice for 30 min. At this stage, cells were either stained directly or stored at -20°C for up to 2 weeks.

[0389] For staining, the plates were washed twice with DPBS (400×g, 3 min) and then the cells were resuspended in 50 μl of diluted AF647 mouse anti-Stat5 (pY694) staining antibody (1:20 in FACS buffer, BD, #562076) and incubated for 1 h at 4° C. The cells were then washed twice with FACS buffer before the samples were acquired on a BD FACS Fortessa. To compare the effect of the cytokine fusion molecules on non-transduced (eGFP−) cells with its effect on transduced (eGFP+) cells, the median fluorescence intensity of the two populations was analyzed.

[0390] Incucyte® Immune Cell Killing Assay Cancer cell lines (HeLa and MKN45) were transduced in-house with Incucyte® NucLight Red lentivirus (EF1α, Puro, #4476) and stable cell lines were generated under puromycin selection (HeLa NLR, MKN45 NLR). Human CD4+ / CD8+ T cells were isolated and transduced with the desired constructs as described above herein. For the assay, HeLa NLR or MKN45 NLR cells were resuspended in RPMI-1640 (Gibco, #42401-018) + 2% FCS (Sigma, #F4135-500ML) + 1% Glutamax (Gibco, #35050-038) (killing medium) and 10,000 cells were seeded in 100 μl per well of a flat-bottom 96-well plate. Plates were incubated at 37°C, 5% CO2 for 4-24 hours until cells attached. Transduced T cells were counted and adjusted to 10,000 eGFP+ cells / 50μl of killing medium and added to the attached cancer cells. Adaptor and control antibodies were diluted to the desired concentrations in killing medium and 50μl was added to the wells. All conditions were pipetted in duplicate. Air bubbles were removed from the well surface and the plate was placed in an Incucyte® S3 machine. Five images of each well were taken every 4 hours over a period of 5-6 days. Reduction in cancer cell numbers was quantified using an analysis mask that counts the amount of red blood cells.

[0391] Example 1 Generation and characterization of humanized anti-P329G antibodies Parental and humanized anti-P329G antibodies were produced in HEK cells and purified by Protein A affinity and size exclusion chromatography. All antibodies were purified to high quality (Table 13). [Table 13]

[0392] Binding of the parent and six humanized variants of anti-P329G binder M-1.7.24 to human Fc (P329G) Equipment: Biacore T200 Chip: CM5(#772) Fc1~4: Anti-human Fab specific (GE Healthcare 28-9583-25) Capture: 50nM IgG in 60 seconds Analyte: Human Fc (P329G) (P1AD9000-004) Running buffer: HBS-EP Temperature: 25℃ Dilution: 2-fold dilution from 0.59 to 37.5 nM in HBS-EP Flow rate: 30μl / min Meeting: 240 seconds Dissociation: 800 seconds Regeneration: 10mM glycine pH 2.1 2x60 seconds

[0393] SPR experiments were performed on a Biacore T200 using HBS-EP+ (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20 (BR-1006-69, GE Healthcare)) as running buffer. Anti-human Fab specific antibodies (GE Healthcare 28-9583-25) were directly immobilized on a CM5 chip (GE Healthcare) by amine coupling. IgG was captured at 50 nM for 60 s. A two-fold dilution series of human Fc (P329G) was passed over the ligand at 30 μl / min for 240 s and the association phase was recorded. The dissociation phase was monitored for 800 s and triggered by switching from the sample solution to HBS-EP+. The chip surface was regenerated after each cycle with two injections of 10 mM glycine (pH 2.1) for 60 s. Bulk refractive index differences were corrected by subtracting the response obtained with the reference flow cell 1. Affinity constants were derived from the rate constants by fitting to 1:1 Langmuir binding using Biaeval software (GE Healthcare). Measurements were performed in triplicate with independent dilution series.

[0394] The following samples were analyzed for binding to human Fc(P329G) (Table 14). [Table 14]

[0395] Human Fc(P329G) was prepared by plasmin digestion of human IgG1, followed by affinity purification with Protein A and size exclusion chromatography.

[0396] Binding of the parent and six humanized variants of anti-P329G binder M-1.7.24 to human Fc (P329G) The dissociation phase was fitted with a curve to help characterize the off-rate, and the ratio of bound to captured response levels was calculated (Table 15). [Table 15]

[0397] 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 binding patterns to the parent were evaluated in more detail. The 1:1 Langmuir binding rate constants are summarized in Table 16. [Table 16]

[0398] conclusion Six humanized variants were generated, three of which (VH4VL1, VH1VL2, VH1VL3) showed reduced binding to human Fc (P329G) compared to the parent M-1.7.24, while the other three humanized variants (VH1VL1, VH2VL1, VH3VL1) showed binding kinetics very similar to the parent binder and no affinity was lost upon humanization.

[0399] Example 2 Preparation of CH2 or CH2-CH3 chimeric antigen receptors DNA sequences encoding either the constant heavy chain 2 (CH2) with a P329G substitution or a CH2-constant heavy chain 3 (CH3) fusion containing P329R and AAA substitutions (I253A, H310A, H435A) were cloned and used as the targeting domain of the adapter molecule in the chimeric receptor according to the invention.

[0400] The C-terminus of CH2 or CH2-CH3 is linked via a G4S linker to the stalk and transmembrane domain of CD8a (Uniprot P01732[183-203]), which is fused to the intracellular costimulatory signaling domain (CSD) CD137 (Uniprot Q07011AA 214-255) which is in turn fused to the stimulatory signaling domain (SSD) CD3ζ (Uniprot P20963 AA 52-164) (Figure 1A and Figure 1B). Graphical representations of exemplary expression constructs, such as the eGFP reporter and RQR8 purification tag, are shown in Figure 1C for the CH2 P329G construct and in Figure 1D for the CH2-CH3 AAA construct.

[0401] Example 3 Expression of CH2 or CH2-CH3 chimeric antigen receptors in Jurkat-NFAT or Jurkat TCRab KO CD4+ cells CH2 or CH2-CH3 receptors were virally transduced into Jurkat NFAT (GloResponse Jurkat NFAT-RE-luc2P, Promega, #CS176501) or Jurkat TCRab-KO CD4+ (T cell activation bioassay (TCRαβ-KO), Promega, #GA1172) cells.

[0402] Expression of chimeric receptors was assessed by flow cytometry. Jurkat cells featuring either the CH2 P329G construct or the CH2-CH3 AAA construct were harvested, washed with PBS and plated at 100,000 cells / well in 96-well U-bottom plates. Cells were stained with LIVE / DEAD™ Fixable Near-IR Dead (Invitrogen, #L34976) dye (1:1000 in PBS) for 20 min at 4°C and washed twice with FACS buffer (1×PBS, 2% FBS, 5 mM EDTA pH 8.0, 0.05% NaN3). Cells transduced with the CH2-CH3 AAA construct were incubated with anti-AAA×FolR1 2+1 antibody at different concentrations (500 nM to 0 nM serial dilution 1:4) and incubated at 4°C for 30 min. The samples were washed twice with FACS buffer and then stained with R-Phycoerythrin AffiniPure F(ab')2 fragment goat anti-human IgG, F(ab')2 fragment specific antibody (1:50 dilution) for 30 minutes at 4°C in the dark (Figures 4A and 4B). For the analysis of receptor expression, anti-AAAxFolR1 2+1 antibody was chosen at a concentration of 2 nM and is shown in Figure 5B. The expression of the CH2 P329G construct was also analyzed by FACS staining. Therefore, cells were harvested, washed and stained for live cells as described above. The CH2 P329G construct was detected by staining with 100 nM anti-P329G VH3xVL1 AF647-labeled IgG (Figures 2A and 2B). In addition, the intracellular eGFP reporter was analyzed (see Figures 2C and 5C).

[0403] Both the CH2 P329G construct and the CH2-CH3 AAA construct were shown to be expressed on the surface of Jurkat cells, and their expression was shown to correlate with intracellular eGFP expression.

[0404] To test the functionality of the CARs, they were assessed in a Jurkat activation assay.

[0405] Example 4 Specific T cell activation in the presence of targeting antibodies including anti-P329G or anti-AAA binders To assess specific T cell activation, CH2 P329G Jurkat cells and CH2-CH3AAA Jurkat cells were assessed for their activation in the presence of FolR1 positive target cells and anti-PGxFolRI 2+1 antibody (Figure 3A) or anti-AAAxFolRI 2+1 antibody (Figure 6A). CH2 P329G Jurkat cells were tested with high (HeLa), medium (Ovcar-3) and low (HT29) FolR1 positive target cells. Mock-transduced Jurkat cells (transduced with VLPs without transgene vector) served as negative control. Dose- and antigen level-dependent activation of CH2 P329G Jurkat cells was observed. Mock-transduced control cells did not show activation by anti-PGxFolRI 2+1 antibody in any of the tested target cells (Figures 3B-D). CH2-CH3 AAA Jurkat cells were tested in an activation assay using high (HeLa) FolR1 positive target cells. Jurkat NFAT wild type cells served as a negative control. Again, dose-dependent activation of CH2-CH3 AAA Jurkat cells, but not Jurkat NFAT wild type cells, was observed (Figure 6B).

[0406] These results lead to the conclusion that the CH2 P329G construct as well as the CH2-CH3 AAA construct can be expressed in T cells and both are functional and can be selectively activated by antibodies, including cognate antibody binders.

[0407] Example 5 Expression of CH2-CH3 AAA P329G CAR, CH2-CH3 AAA P329R CAR or CH2 P329G CAR in primary T cells The CH2-CH3 AAA P329G CAR (Figure 8B, sequence number 127), CH2-CH3 AAA P329R CAR (Figure 1B, sequence number 125) or CH2 P329G CAR (Figure 1A, sequence number 87) receptors were transduced into human T cells (1:1 CD4+:CD8+) using virus-like particles (VLPs) as described above.

[0408] Chimeric receptor expression was assessed and compared by flow cytometry 7 days after transduction as follows: Transduced T cells were harvested, washed with DPBS and seeded at 100.000 cells / well in 96-well U-bottom plates. T cells transduced with CAR containing the P329G mutation were stained with LIVE / DEAD™ Fixable Near-IR Dead (Invitrogen, #L34976) dye (1:1000 in DPBS) and 50 nM anti-PG VH3xVL1 huIgG-AF647 for 20 min at 4° C. Cells were then washed twice with FACS buffer (1×DPBS, 2% FBS, 5 mM EDTA pH 8.0, 0.05% NaN3), fixed (BD CytoFix, #554655) and analyzed by FACS.

[0409] Cells transduced with CAR containing AAA mutations were stained with LIVE / DEAD™ Fixable Near-IR Dead (Invitrogen, #L34976) dye (1:1000 in DPBS) and 200 nM anti-AAA×FolR1 2+1 TCB (20 min at 4° C.). Cells were then washed twice with FACS buffer. Cell pellets were resuspended in 50 ul of secondary antibody diluted in FACS buffer (1:100, APC-F(ab)2 fragment anti-huIgG (Jackson ImmunoResearch, #109-136-097)) and incubated for 30 min at 4° C. After two further washing steps, cells were fixed (BD CytoFix, #554655) and analyzed by FACS.

[0410] Intracellular eGFP expression and anti-PG / anti-AAA staining are shown in Figure 9A and Figure 9B. 87-89% of the cells were eGFP positive and expressed the CAR construct on the cell surface.

[0411] To assess whether the P329G and AAA mutations could be bound simultaneously by each molecule, a second staining was performed as follows (Figure 12A). Cells were incubated with LIVE / DEAD™ Fixable Near-IR Dead (Invitrogen, #L34976) dye (1:1000 in DPBS) and 50 nM of anti-PG VH3xVL1 huIgG-AF647 for 20 min at 4°C. Cells were then washed twice with FACS buffer. In a second step, cells were incubated with 100 nM of anti-AAA x FolR1-Avi-Biotin 1+1 TCB and 500 nM of Fc_P329LALA (20 min at 4°C). Fc_P329GLALA served as a blocking molecule to prevent binding of anti-PG VH3xVL1 huIgG-AF647 to the PG mutations in the anti-AAAx FolR1-Avi-Biotin 1+1 TCB. After incubation, cells were washed twice with FACS buffer. In a third step, cells were resuspended in BV421 streptavidin (Biolegend, #405225) and 500 nM Fc_P329LALA (20 min, 4°C). After two further washing steps, cells were fixed (BD CytoFix, #554655) and analyzed by FACS.

[0412] CH2-CH3 AAA P329G CAR T cells showed double staining (BV421+, AF647+), indicating that AAA and P329G mutations can be targeted simultaneously in one CAR construct with anti-AAA and anti-PG adaptors (Figure 12B). CH2-CH3 AAA P329R CAR or CH2 P329G CAR T cells showed only single staining of each, as expected.

[0413] To test the functionality of the constructs, they were evaluated in an Incucyte® immune cell killing assay. To further evaluate cytokine targeting in cis to CAR T cells via the P329G mutation, a STAT5 phosphorylation assay was performed.

[0414] Example 6 Incucyte® immune cell killing assay using CEA+ or FolR1+ target cells A killing assay was performed to evaluate the cytotoxicity of the CH2-CH3 AAA P329G CAR (Figure 8B, SEQ ID NO: 127), CH2-CH3 AAA P329R CAR (Figure 1B, SEQ ID NO: 125) or CH2 P329G CAR (Figure 1A, SEQ ID NO: 87) receptors. To compare different constructs, the amount of T cells per well was normalized to the same percentage of eGFP+ cells, resulting in 10,000 target cells / well and 10,000 eGFP+ T cells / well. The killing assay was performed as described above.

[0415] Anti-AAAxFolR1 2+1, anti-AAAxFolR1 1+1, anti-PGxFolRI 2+1 or anti-PGxFolRI 1+1 antibodies were titrated from 0 pM to 10 nM (1:10) as adaptor molecules for HeLa NLR. 10 nM anti-FolR1 2+1 TCB was used as a positive killing control.

[0416] Dose-dependent killing was observed for all CAR constructs incubated with their corresponding adaptor molecules (Figure 10A, Figure 10B, Figure 10C, Figure 10F). Incubation of T cells expressing CH2-CH3 AAA P329R CAR with anti-PGxFolR1 antibody did not result in any killing as expected (Figure 10D). The same was observed for CH2 P329G CAR T cells incubated with anti-AAAxFolR1 antibody (Figure 10E).

[0417] In both approaches (anti-PG or anti-AAA), the 2+1 format showed more sensitive killing compared to the 1+1 format, independent of the CAR construct (Figures 10A, 10B, 10C, 10F). In a direct comparison, the anti-AAA x FolRI molecule showed higher sensitivity compared to the anti-PG x FolRI adaptor.

[0418] MKN45 NLR cells were targeted with anti-AAAxCEA 2+1 or anti-PGxCEA 2+1 antibodies (0 pM-10 nM, (1:10)). Dose-dependent growth inhibition was observed for all CAR constructs incubated with their corresponding adapter molecules (Figure 11A, Figure 11B (top), Figure 11C (bottom)). As previously observed, addition of adapter molecules with binders that do not match the mutations present in the CAR constructs did not result in any killing (Figure 11B (bottom) and 11C (top)). Consistent with the HeLa NLR killing data, the anti-AAAxCEA molecule showed higher sensitivity compared to the anti-PGxCEA adapter.

[0419] Example 7 STAT5 phosphorylation assay using anti-P329G targeting IL2v To evaluate the selective targeting of cytokines to the CH2-CH3 AAA P329G CAR (Figure 8B, SEQ ID NO: 127) or CH2 P329G CAR (Figure 1A, SEQ ID NO: 87), a STAT5 phosphorylation assay was performed. Non-transduced and transduced cells were mixed 1:1 for this assay to ensure a similar distribution of non-transduced (eGFP-) and construct-expressing cells (eGFP+) in each well (>90% of transduced cells were expressing the construct). Anti-P329G-IL2v or Proleukin (Novartis) was titrated onto the cells starting at 100 nM (1:10). The assay was performed as described above. Samples were acquired on a BD FACS Fortessa and the median fluorescence intensity (MFI) of the pSTAT5 signal of the non-transduced (eGFP-) versus transduced (eGFP+) populations was plotted in GraphPad Prism 8.

[0420] Figure 13 shows the results of the pSTAT5 assay. In samples containing CH2 P329G CAR T cells, a 50-fold increase in pSTAT5 MFI was observed in the eGFP+ population compared to the eGFP- population (Figure 13A). This effect was even greater when T cells expressed the CH2-CH3 AAA P329G CAR (250-fold, Figure 13B), indicating very strong cis-targeting of IL2v to the eGFP+ population. No cis-effect was observed in CH2-CH3 AAA P329R CAR T cells, confirming that this effect is indeed due to P329G targeting. Looking at the data obtained with Proleukin, all samples and populations show comparable STAT5 phosphorylation.

Claims

1. (i) an extracellular domain comprising a variant Fc domain or a fragment thereof; (ii) a transmembrane domain; A chimeric receptor comprising:

2. The chimeric receptor of claim 1, further comprising (iii) at least one intracellular stimulatory signaling domain and / or at least one costimulatory signaling domain.

3. The chimeric receptor of claim 1 or 2, wherein the extracellular domain comprises a mutated CH2 domain and / or a mutated CH3 domain.

4. wherein the extracellular domain comprises a variant CH2 domain and / or a variant CH3 domain, and the variant CH2 domain and / or the variant CH3 domain have an amino acid substitution (numbering according to Kabat EU index): (a) I253A, H310A and H435A, (b) I253A, (c) I253A and H310A, (d) I253A and H435A, (e) H310A and H435A, (f) H310A, (g) H435A, (h) L234A, L235A, and P329G, or (i) L234A, L235A, I253A, H310A, P329G, and H435A The chimeric receptor of any one of claims 1 to 3, comprising:

5. 5. The chimeric receptor of any one of claims 1 to 4, wherein the transmembrane domain is selected from the group consisting of a CD8, CD4, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10 or DAP12 transmembrane domain or a fragment thereof.

6. 6. The chimeric receptor of any one of claims 2 to 5, wherein the stimulatory signaling domain is 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 stimulatory signaling activity.

7. 7. The chimeric receptor of any one of claims 2 to 6, wherein the costimulatory signaling domain is selected from the group consisting of the intracellular domains of CD27, CD28, CD137, OX40, ICOS, DAP10 and DAP12 or fragments thereof that retain costimulatory signaling activity.

8. The chimeric receptor of any one of claims 2 to 7, wherein the costimulatory signaling domain is the CD28 intracellular domain or a fragment thereof that retains CD28 costimulatory activity.

9. 9. The chimeric receptor of any one of claims 2 to 8, comprising one stimulatory signalling domain comprising the intracellular domain of CD3z or a fragment thereof that retains CD3z stimulatory signalling activity, and one costimulatory signalling domain comprising the intracellular domain of CD137 or a fragment thereof that retains CD137 costimulatory signalling activity.

10. 10. The chimeric receptor of any one of claims 2 to 9, wherein the stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 13 and the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:

12.

11. The chimeric receptor of any one of claims 1 to 10, comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:87, SEQ ID NO:106, SEQ ID NO:125 and SEQ ID NO:

127.

12. A chimeric receptor comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:87, SEQ ID NO:106, SEQ ID NO:125 and SEQ ID NO:

127.

13. An isolated polynucleotide encoding the chimeric receptor of any one of claims 1 to 12.

14. A polypeptide encoded by the isolated polynucleotide of claim 13.

15. A vector, particularly an expression vector, comprising the polynucleotide of claim 13.

16. A transduced T cell comprising the polynucleotide of claim 13 or the vector of claim 15.

17. A transduced T cell capable of expressing a chimeric receptor according to any one of claims 1 to 12.

18. (A) a transduced T cell capable of expressing a chimeric receptor according to any one of claims 1 to 12, wherein the chimeric receptor comprises an extracellular domain comprising a variant Fc domain or a fragment thereof; (B) an antibody comprising at least one antigen-binding portion capable of binding to the variant Fc domain but incapable of binding to a non-variant parent Fc domain, the antibody comprising at least one effector portion.

19. (A) an isolated polynucleotide encoding the chimeric receptor of any one of claims 1 to 12, wherein the chimeric receptor comprises an extracellular domain comprising a variant Fc domain or a fragment thereof; and (B) an antibody comprising at least one antigen-binding portion capable of binding to the variant Fc domain but incapable of binding to a non-variant parent Fc domain, the antibody comprising at least one effector portion.

20. 20. The kit according to claim 18 or 19, wherein the effector moiety is (i) an antigen-binding moiety capable of binding to a target cell antigen, or (ii) and an immune activating moiety, in particular a cytokine.

21. A kit according to any one of claims 18 to 20 for use as a medicine.

22. 18. The chimeric receptor of any one of claims 1 to 12 or the transduced T cell of any one of claims 16 or 17 for use as a medicament, wherein the chimeric receptor comprises an extracellular domain comprising a mutated Fc domain or a fragment thereof, and wherein the transduced T cell expressing the chimeric receptor is administered prior to, simultaneously with or after administration of an antibody comprising at least one antigen binding moiety capable of binding to a target cell antigen and at least one antigen binding moiety capable of binding to the mutated Fc domain but not to the non-mutated parent Fc domain.

23. A kit according to any one of claims 18 to 20 for use in the treatment of a disease, in particular for use in the treatment of cancer.

24. 13. A method of treating a disease in a subject, comprising administering to the subject transduced T cells capable of expressing a chimeric receptor according to any one of claims 1 to 12, wherein the chimeric receptor comprises an extracellular domain that comprises a mutated Fc domain or a fragment thereof, and administering prior to, simultaneously with, or after administration of the transduced T cells, a therapeutically effective amount of an antibody comprising at least one antigen binding moiety that is capable of binding to the mutated Fc domain but not to a non-mutated parent Fc domain, wherein the antibody comprises at least one effector moiety.

25. 25. The method of claim 24, wherein the effector moiety is (i) an antigen-binding moiety capable of binding to a target cell antigen, or (ii) and an immune activating moiety, in particular a cytokine.

26. Use of a chimeric receptor according to any one of claims 1 to 12, a polynucleotide according to claim 13 or a transduced T cell according to claim 16 or 17 for the manufacture of a medicament.

27. 27. The use according to claim 26, wherein the medicament is for the treatment of cancer.

28. 28. Use according to claim 27, characterized in that the cancer is selected from cancers of epithelial, endothelial or mesothelial origin and hematological cancers.

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