Multichain antigen-specific receptors for cell-based immunotherapy

Abstract

The present invention is in the field of cell-based immunotherapy. In particular, the present invention provides modified cells comprising a first and a second polypeptide forming an antigen-binding site on the outside of the cell and a polypeptide comprising a signaling domain, where when the antigen-binding site binds to a corresponding antigen, the signaling domain triggers a process within the cell that allows the cell to promote the death of a target cell that comprises said antigen on the cell surface. The present invention also provides medical applications of the modified cells, in particular the use in the treatment of disease. Furthermore, the present invention provides kits comprising at least one nucleic acid molecule encoding said polypeptides, and methods for producing the modified cells of the present invention. Furthermore, the present invention provides chimeric polypeptides and nucleic acid molecules encoding the chimeric polypeptides.

Description

[Technical field]

[0001] The present invention is in the field of cell-based immunotherapy. In particular, the present invention provides modified cells comprising a first and a second polypeptide forming an antigen-binding site on the outside of the cell and a polypeptide comprising a signaling domain, where when the antigen-binding site binds to a corresponding antigen, the signaling domain triggers a process within the cell that allows the cell to promote the killing of a target cell that comprises said antigen on the cell surface. The present invention also provides medical applications of the modified cells, in particular the use in the treatment of disease. Furthermore, the present invention provides kits comprising at least one nucleic acid molecule encoding said polypeptides, and methods for producing the modified cells of the present invention. Furthermore, the present invention provides chimeric polypeptides and nucleic acid molecules encoding the chimeric polypeptides. [Background technology]

[0002] Cellular therapies utilize genetically modified or non-modified immune cells to target or retarget pathogenic cells, e.g. cancer cells. Most of these therapies rely on receptors, e.g. chimeric antigen receptors (CARs), that direct immune cells to target cells. These CARs are usually introduced into T cells, resulting in so-called CAR T cells.

[0003] The general concept of recombinant chimeric receptors for immune cells that confer antigen specificity via an extracellular binding domain (i.e., single-chain variable fragments (scFv)) and trigger immune signaling via cytoplasmic signaling motifs has been the basis of CAR development since the early days of the field. The design of such receptors initially began with efforts to reprogram the specificity of the T cell receptor (TCR) (Kuwana, et al. 1987). It then moved to single-chain variants, culminating in clinically approved CAR-T cell therapies (Jayaraman, et al. 2020). Notably, single-chain receptors offered reduced complexity in cellular applications. In the first implementation of CARs, Eshhar's "T-bodies," scFvs were fused directly to the CD3ζ (CD3 zeta or CD247) chain of the T cell receptor complex (Eshhar, et al. 1993; Gross, et al. 1989; Yanez-Munoz and Grupp 2018). Since then, many coactivation domains, hinge domains, and transmembrane domains have been added or exchanged to further enhance effector cell expansion, persistence, and antitumor activity (Weinkove, et al. 2019). However, the basic single-chain receptor architecture, characterized by the antigen-targeting scFv, one hinge domain, one transmembrane domain, one or two cofactor domains, and CD3ζ, has hardly evolved (Jayaraman, et al. 2020).

[0004] Thus, CAR construction is still based on single gene constructs encoding different protein domains derived from multiple sources, resulting in single-chain chimeric receptors. The most common common feature of CARs is the N-terminal and C-terminal domains. Typically, CARs contain an N-terminal single-chain variable fragment (scFv) as the target antigen binder and a C-terminal CD3 zeta chain as the cell activation signaling domain. There are no defined standards yet for the linking spacer, hinge, transmembrane domain, and costimulatory domain of CARs. Although the CAR approach has been shown to provide an effective treatment for patients with end-stage lymphoma and myeloma, it suffers from at least three fundamental problems.

[0005] First, in terms of modularity, each domain swap may lead to loss of receptor surface localization and function, requiring tedious construct optimization. In particular, current CAR designs require adjustments to the scFv and hinge domains to switch target epitopes. This has the disadvantage of having to make completely new proteins, which often leads to expression problems (Jayaraman, et al. 2020). In addition, because the hinge domain is also associated with CAR activity, extensive research has been conducted on the identification of superior hinge domains and their lengths. As a result, it was found that shorter hinge domains tend to have higher activation abilities. However, short hinge domains need to be adjusted for each target, as they may prevent the scFv from reaching membrane-proximal epitopes.

[0006] Second, mispairing of scFvs may cross-link multiple receptors on the effector cell surface, resulting in undesirable tonic signaling in CAR-T cells (Zajc, et al. 2021). In particular, the predisposition of scFv-based receptors to cross-assemble limits the potential cell surface density of functional protein. This can result in low efficacy and exhaustion of effector cells despite good receptor expression levels.

[0007] Third, compared to the signaling domains of natural receptors, the costimulatory and activation domains are placed in-line in current chimeric antigen receptors, thus altering the plasma membrane distance of these domains. This is known to negatively affect the activity of the signaling domains in the vicinity of the membrane and is associated with poor functionality. Furthermore, in single-chain multi-activation domain receptors, the plasma membrane distance of the signaling domains is not optimal, which requires them to be placed in-line consecutively, limiting the maximum multiplicity of ITAMs. For example, only up to six ITAMs have been used so far, and only in the case of CAR dimers (Feucht, et al. 2019; Jayaraman, et al. 2020; Zajc, et al. 2021). Nearly all current CAR approaches use only one transmembrane domain with one slot for the signaling / activation domain. In some cases, up to two costimulatory domains (CD28, 41BB, OX40) and one activation domain (CD3z or FceRIg) have been combined in the CAR (Pfefferle and Huntington 2020). However, such domain stacking in third-generation CARs has more or less failed as no significant increase in cytotoxic activity was observed (Huang, et al. 2020). In one case, two activation domains (DAP10 and CD3z) are combined in one ligand CAR, which cannot be easily adapted to another target epitope. Moreover, DAP10 cannot be replaced (Chang, et al. 2013).

[0008] Furthermore, prior art CAR constructs are typically delivered to T cells and are not optimized for other cell types.

[0009] Natural killer (NK) cells are another class of cytotoxic cells that are safer and more suitable for allogeneic transplantation than T cells. Natural killer cells play a pivotal role in immune surveillance through the recognition and elimination of cancer cells, virus-infected cells, and other disease cells (Abel, et al. 2018; Correia, et al. 2021; Iannello and Raulet 2013; Pech, et al. 2019; Vivier, et al. 2008). NK cells belong to the innate immune system and therefore do not possess antigen-specific receptors. Therefore, NK cells are nonspecific in nature and cannot specifically recognize target antigens. Unlike antigen-specific cytotoxic cells such as CD8+ T cells, NK cells tightly integrate multiple signals from antigen-nonspecific activating and inactivating receptors. Most of these nonspecific receptors must rely on synergistic coactivation to induce sufficient signals to trigger a cytotoxic response in NK cells (Bryceson, et al. 2006).

[0010] However, CD16 is a low-affinity receptor for the crystallizable fragment (Fc) region of immunoglobulin gamma (IgG) expressed on a subset of NK cells, and as such, functions to form a link between adaptive and innate immune responses, conferring antibody-supported antigen specificity to NK cells (Cooper, et al. 2001). This link is formed by the binding of soluble IgG antibodies bound to antigens at the cell surface to CD16, inducing antibody-dependent cellular cytotoxicity (ADCC) through the activation, polarization, and degranulation of NK cells (Capuano, et al. 2021; Nimmerjahn and Ravetch 2006; Wang, et al. 2015b). Induction of ADCC of NK cells by monoclonal antibodies contributes to the efficacy of the antibodies (Muntasell, et al. 2017). For example, the antitumor effect of monoclonal IgG antibodies such as trastuzumab / Herceptin is in part due to ADCC (Lee, et al. 2020; Liang, et al. 2018). One of the first steps in this process involves the signaling domains of CD16-associated CD247 (CD3ζ (CD3zeta)) and FcεRIγ (FceRIg) that contain immunoreceptor tyrosine-based activation motifs (ITAMs). Phosphorylation of ITAMs leads to potent activation of NK cells, tipping the balance of activating and inactivating signals in favor of NK cell activation and resulting cytotoxicity (Blazquez-Moreno, et al. 2017).

[0011] The antigen-specific cytotoxic activation ability of NK cells has been exploited in clinical research and applications. For example, the combination of NK-92 cells expressing a high-affinity mutant of CD16 and avelumab targeting PDL1 was used to treat Merkel cell carcinoma in clinical trials (Park, et al. 2021).

[0012] However, three elements are required for injecting NK cells to trigger ADCC against tumors: NK cells, soluble antibodies, and tumors. As a result, its effectiveness is limited by the availability of NK cells and soluble antibodies at the tumor site. In particular, the contact between soluble antibodies and target antigens relies on a passive process of diffusion, which can significantly inhibit the efficiency of ADCC. In fact, ADCC by a combination of NK cells and soluble antibodies against solid tumors has been shown to be inferior to ADCC by T cells with antigen-specific receptors (Szoor, et al. 2020) due to the insufficient availability of soluble antibodies in the tumor (Thurber, et al. 2008).

[0013] In some studies, CARs used in the context of T cells have also been applied to NK cells. However, these CARs have not been optimized for NK cells and may not be the best way to boost NK cells with antigen specificity. Indeed, the low efficacy of engineered NK cells is often cited as an obstacle to their widespread acceptance as a treatment.

[0014] Further approaches have been developed for CAR cell-based immunotherapy. For example, CAR-like single-chain receptors were recruited by B cells with the goal of inducing B cell proliferation upon encountering cognate antigen (Pesch, et al. 2019). Furthermore, CAR-based multi-chain receptors were engineered to tether signaling-deficient CARs to endogenous signaling chains via their transmembrane domains and harness their signaling capabilities (Wang et al. 2015a). However, many or all of the limitations and shortcomings of CARs mentioned above still apply to these studies.

[0015] Furthermore, reprogramming T cell specificity via TCR engineering remains an area of ​​ongoing pursuit in the development of T cell therapeutics. The latter approach ranges from engineering TCRs with binding domain candidates identified in mice with a fully diverse human TCRαβ repertoire (Li, et al. 2010) to scFv-decorated TCRs (TRuCs) (Baeuerle, et al. 2019). However, only the antigen-binding portion is engineered, while the signaling domain remains an endogenous TCR component. Furthermore, retaining endogenous αβ or γδ TCR antigen-binding domains in T cells augmented with engineered binding domains may cause crosstalk and lead to graft-versus-host disease (GvHd) (Bendle, et al. 2010).

[0016] Thus, there is a need for improved tools and methods for cell-based immunotherapy. Summary of the Invention

[0017] In particular, the invention relates to the embodiments as characterized in the claims and described hereinafter. [Brief description of the drawings]

[0018] [Figure 1] Comparison of canonical ADCC and cis-ADCC. a, Schematic diagram of canonical ADCC elicited by CD16POS NK cells loaded with soluble IgG1 antibody against target antigen-positive cancer cells. b, Schematic diagram of cis-ADCC against target antigen-positive cancer cells according to the present invention. Legend indicates the displayed elements. [Diagram 2]Transduction of cis-ADCC against Her2 positive target cells. a, Schematic of constructs used to prepare lentiviral vectors for transduction of NK-92 cells. Constructs for EF1A-driven constitutive expression of CD16, membrane-bound anti-Her2 immunoglobulin gamma 1 (mIgG1), and CD79 with fluorescent markers SBFP2, mScarlet, and mCerulean, respectively, are shown. The color code of the various building blocks (more visible in priority application EP21217757.0) is used throughout the remainder of the examples for consistency. b, Schematic and description of proteins and protein domains used to perform cis-ADCC. The term "CD79A / B" as used throughout the specification and figures means "CD79A and CD79B". As used herein and throughout the drawings, the term "CD3z" is synonymous with "CD3ζ:CD247", the term "FCERIG" is synonymous with "FcεRIγ", the term "CD16" is synonymous with "CD16:FCGR3A", the term "SK-BR-3" is synonymous with "SK-BR-3-Luc-Cit", and the term "MDA-MB-468" is synonymous with "MDA-MB-468-Luc-Cit". cl, specific lysis quantified using LCA co-incubation assay after 4 hours of NK-92 cell line variants. Cell line names (see Table 1) and schematic diagrams of cell surface modifications are displayed above the respective dose-response charts. The charts show the extent of specific cell lysis (y-axis) as a function of increasing effector to target cell ratios (E:T cell ratios) (x-axis). Red indicates effect on Her2 positive SK-BR-3 cells and blue indicates effect on Her2 negative MDA-MB-468 cells. Shown are the mean +- SD of biological triplicates (extrapolated as shaded areas between discrete E:T ratios). *** indicates p-value <0.001 for statistical significance between cytotoxic effect on Her2 positive and Her2 negative target cells at a given E:T ratio. [Diagram 3]Characterization of an NK-92 derivative encoding Her2-tIgG1. a, Schematic diagram of the assembly of anti-Her2 immunoglobulin, CD79 heterodimer, CD16 CD3, and CD26 tethered to the cell membrane with a (GGGGS)11x linker. All domains within each protein are labeled with the exact protein fragment used. b, Schematic diagram of the lentiviral vector. Constructs for EF1A-driven constitutive expression of CD16, membrane-bound anti-Her2 immunoglobulin g1 (tIgG1), and CD79 with the fluorescent markers SBFP2 (note: mTagBFP2 can be used equivalently), mScarlet, and mCerulean, respectively, are shown. The color code in (a) and (b) (better visible in the priority application EP21217757.0) corresponds to the illustrations in panels (c), (e) and (g). c, e, g, Fluorescence microscopy images taken after 4 h of co-incubation (i.e., 4-h killing assay) of NK-92 variants (schematically depicted in the pictorial diagram above) with Her2-positive SK-BR-3 cells at an E:T cell ratio of 10:1. mScarlet (red pseudocolor), SBFP2 (blue pseudocolor), and mCerulean (turquoise pseudocolor) indicate antibody, CD16, and CD79 protein expression, respectively. mCitrine is an indicator of SK-BR-3 target cells (the LUT for mScarlet was adjusted between (c), (e) 300-8000 and (g) 300-3000 due to low clustering of NK-92 in (g)). d, f, Specific lysis quantified using LCA co-incubation assay after 4 h for NK-92 variants depicted in panels (c) and (e), respectively, against Her2-positive SK-BR-3 (red) and Her2-negative MDA-MB-468 cells (blue) at different E:T cell ratios (x-axis). Shown are the mean values ​​+- SD of biological triplicates (extrapolated as shaded areas between discrete E:T ratios). h, i, Summary of specific data obtained for all NK-92 variants (different cell lines shown in different colors according to legend) eliciting ADCC in the presence of overexpressed CD16 at different E:T cell ratios (x-axis) against Her2-positive SK-BR-3 cells (i) and Her2-negative MDA-MB-468 cells (h).*** indicates p-value <0.001 for statistical significance of cytotoxic effect on Her2-positive and Her2-negative cell lines. Note that "Trastuzumab" is the same as "Herceptin." [Figure 4] CD79-CD3 zeta chimeric polypeptide fusions in multichain receptors. a, d, g, Schematic representation of surface expression of membrane-bound immunoglobulins complexed with CD79-CD3 zeta chimeric polypeptides. All domains within each protein are labeled to indicate the exact protein fragment. b, e, h, Evaluation of expression and surface localization of stably integrated constructs shown in (a), (d), and (g), respectively. Surface expression of membrane-bound immunoglobulins was determined by confocal microscopy and flow cytometry after immunostaining against immunoglobulin constant domains. One cell is shown per photograph. Histograms show expression levels in cell populations (right-shifted distribution) compared to unstained controls (left-shifted distribution). c, f, i, Specific lysis calculated from LCA co-incubation assays after 4 h of NK-92 variants indicated in panels (a), (d) and (g), respectively, at different E:T cell ratios (x-axis) against Her2-positive SK-BR-3 cells (red) and Her2-negative MDA-MB-468 cells (blue). Shown are the mean values ​​+- SD of biological triplicates (extrapolated as shaded areas between discrete E:T ratios). *** indicates p-value <0.001 for statistical significance between cytotoxic effects against Her2-positive and Her2-negative cell lines. [Diagram 5]Incorporate two ADCC signal transduction domains into the receptor. a, d, g, Schematic diagrams of FcγIγ complexed with CD79-CD3 zeta chimeric polypeptide and of native, surface-expressed, C-terminally modified membrane-bound immunoglobulin γ1 (mIgG1). In the schematic diagrams, all domains within each protein are labeled with the exact protein fragments used. b, e, h, Evaluation of the expression and surface localization of the stably incorporated constructs shown in (a), (d), (g), respectively. Surface expression of membrane-bound immunoglobulin was determined by confocal microscopy and flow cytometry after immunostaining against the immunoglobulin constant domain. One cell is shown per photograph. Histograms show the expression levels in cell populations (shifted to the right distribution) compared to unstained controls (shifted to the left distribution). c, f, i, Specific lysis calculated from the LCA co-incubation assay after 4 hours of the NK-92 variants shown in (a), (d), (g) at different E:T cell ratios (x-axis) against Her2-positive SK-BR-3 cells (red) and Her2-negative MDA-MB-468 cells (blue). Shown are the mean values ± SD of biological triplicates (extrapolated as the hatched area between discrete E:T ratios). *** indicates a p-value of < 0.001 for the statistical significance between the cytotoxic effects against Her2-positive and Her2-negative cell lines. [Figure 6]Surface expression of trastuzumab-derived mIgG1 in HeLa cells. In panels a-c, the schematic on the left shows the structure and localization of the receptor. The central diagram shows the transfected construct. The photomicrographs on the right show fixed HeLa cells, with Brightfield 10x labels indicating the brightfield channel, red pseudocolor indicating expression of the transfection control mCherry (panels a and b) or the antibody chains (panel c), green pseudocolor indicating the intensity of anti-IgG staining and antibody surface expression, and turquoise pseudocolor indicating mCerulean, a surrogate for CD79 expression (panel c only). Scale bars are 100 μm. a, Plasmid transfection of constitutively driven antibody heavy and light chains and transfection control. b, Plasmid transfection of constructs encoding antibody heavy and light chains on a continuous scaffold and CD79A and CD79B proteins encoded on two separate plasmids. c, Transfection of lentiviral-packaged adapted polycistronic constructs encoding antibody chains with an mScarlet fluorescent reporter and lentiviral-adapted polycistronic constructs encoding CD79A and CD79B with an mCerulean fluorescent reporter. The green pseudocolor intensity cannot be directly compared between panels (a) and (b) and panel (c) because the primary antibody used to stain the samples in panel c is from a different lot with stronger staining compared to the lot used in panels (a) and (b). [Figure 7]Generation of stably transduced NK-92 cells. a, SBFP2-mCerulean flow cytometry scatter plot of NK-92 cells stably transduced with lv-EF1A-CD79 (mCerulean) and / or lv-EF1A-CD16 (SBFP2), showing the cell sorting gates and the population frequencies of sorted cells. NK-92-WT cells (left plot) were used as a control and were not sorted. The names of the cell lines according to the sorting results are indicated above the scatter plot. b, mScarlet-FCS flow cytometry scatter plot of the cell lines whose sorting was described in panel a, stably transduced with lv-EF1A-mIgG1 / Her2 or lv-EF1A-mIgG1 / Pollen, showing the sorting window and population frequency. Each plot in this panel shows the transduction results of cells pre-sorted according to the expression of a BFP, i.e., SBFP2, and Cerulean, as shown in the plot immediately above panel a. The names of the cell lines resulting from sorting are shown above the scatter plots. ck, Antibody surface staining of membrane-bound immunoglobulins and CD16 in transduced NK-92 cell lines. Histograms of membrane-bound immunoglobulin (light) and CD16 (dark) surface expression are shown. The names of the cell lines (Table 1) and indications of cell surface modifications are shown in the histograms above. l, Bar graphs showing the median expression intensity (i.e., median fluorescence) of membrane-bound immunoglobulin (light) and CD16 (dark) surface expression corresponding to histograms in (d)–(k). m, Percentage of lysis of SK-BR-3 cells when co-cultured (i.e., 4-h incubation) with NK-92 cells transduced with the viral vectors shown below the bar graphs at a 5:1 E:T cell ratio or, alternatively, when indicated, when fed with 10 μg / mL Herceptin (canonical ADCC) or 2% TritonX-100 (positive control for cell lysis). Shown are means +- SD of biological triplicates; ***, **, * represent p-values ​​<0.001, <0.01, <0.05 for statistical significance of the difference in effect size between compared conditions, respectively. See Figure 12 for details of plasmids and viral vectors and Table 1 for details of cell lines. [Figure 8] Generation and characterization of target cells. In panels (a) and (b), green pseudocolor indicates mCitrine and red pseudocolor indicates APC, an indicator of Her2 / ErbB2 surface expression (see Methods). a, Characterization of MDA-MB-468-LUC-CIT cell line. b, Characterization of SK-BR-3-LUC-CIT cell line. Scale bars are 50 μm. c, Correlation between SK-BR-3-LUC-CIT cell number and total luminescence signal. Shown are the mean values ​​+- SD of biological triplicates. d, Total luminescence of SK-BR-3-LUC-CIT cells with TritonX-100 added for 30 min. Shown are six independent biological replicates. [Figure 9]Sorting of NK-92 cells stably transduced with tIgG1: a, mScarlet -FSC flow cytometry scatter plot showing mScarlet expression in the cell lines described in Fig. 7a after lv-EF1A-tIgG1 / Her2 transduction, with cell sorting gates and population frequencies of sorted cells indicated. The names of cell lines according to sorting results are indicated above the scatter plot. b, Antibody surface staining for immunoglobulins and CD16 in transduced and sorted NK-92 cell lines. Histograms of membrane-bound immunoglobulins (light) and CD16 (dark) surface expression are shown. The names of cell lines (Table 1) and indication of cell surface modifications are shown in the histograms above. f, Bar graphs showing the median membrane-bound immunoglobulins (light) and CD16 (dark) surface expression corresponding to the histograms in (b)-(e). g, Percentage of lysis of SK-BR-3 cells when co-cultured (i.e., 4 h culture) at a 5:1 E:T cell ratio with NK-92 cells transformed with the viral vectors indicated below the bar graph, or alternatively fed with 10 μg / mL Herceptin (canonical ADCC) or 2% TritonX-100 (positive control for cell lysis) where indicated. Shown are the mean values ​​+- SD of biological triplicates, and ***, **, * represent p values ​​of statistical significance in the difference in effect size between the compared conditions, <0.001, <0.01, <0.05, respectively. See Figure 12 for details of plasmids and viral vectors and Table 1 for details of cell lines. [Figure 10]Sorting of NK-92 cells stably transduced with CD79-CD3 fusions. a, mScarlet over mCerulean flow cytometry scatter plots showing cell sorting gates and population frequencies of sorted cells. a, NK-92-WT and NK-92-mIgG1 / Her2 cells stably transduced with lv-EF1A-CD79-CD3. NK-92-WT cells (left plot) were used as control and were not sorted. b, NK-92-CD79-CD3 cells transduced with lv-EF1A-mIgG1 / Pollen. The names of cell lines according to sorting results are indicated above the scatter plots. c, Percentage of lysis of SK-BR-3 cells when co-cultured with NK-92 cells transduced with the viral vectors indicated below the bars at a 5:1 E:T cell ratio, or alternatively fed with 10 μg / mL Herceptin (canonical ADCC) or 2% TritonX-100 (positive control for cell lysis) where indicated. Shown are the mean values ​​+- SD of biological triplicates, and ***, **, * represent statistical significance between effect sizes between compared conditions with p values ​​of <0.001, <0.01, <0.05, respectively. See Figure 12 for details of plasmids and viral vectors and Table 1 for details of cell lines. d, mScarlet over mCerulean flow cytometry scatter plot of NK-92-CD79-CD3 additionally transduced with lv-EF1A-mIgM / Her2 showing cell sorting gates and population frequencies of sorted cells. e, Antibody surface staining for membrane-bound immunoglobulin (light green) in NK-92 cells transduced with lv-EF1A-mIgM / Her2 or lv-EF1A-CD79-CD3. f, Median membrane-bound immunoglobulin (light green) surface expression from the histograms in (e) is summarized in a bar graph. See Figure 12 for details of plasmids and viral vectors, and Table 1 for details of cell lines. [Figure 11]Sorting of NK-92 cells stably transduced with mIgG1-FcεRIγ. a, mScarlet over mCerulean flow cytometry scatter plots of NK-92-WT or NK-92-CD79-CD3 transduced with lv-EF1A-mIgG1 / Her2-FceRIg or lv-EF1A-mIgG1 / Pollen-FceRIg show the cell sorting gates and population frequencies of sorted cells. b, Percentage of lysis of SK-BR-3 cells when co-cultured with NK-92 cells transduced with the viral vectors indicated below the bars at a 5:1 E:T cell ratio or, where indicated, when alternatively fed with 10 μg / mL Herceptin (canonical ADCC) or 2% TritonX-100 (positive control for cell lysis). Shown are means +- SD of biological triplicates; ***, **, * denote statistical significance between effect sizes between compared conditions with p-values ​​of <0.001, <0.01, <0.05, respectively. See Figure 12 for details of plasmids and viral vectors and Table 1 for details of cell lines. [Figure 12] Designation of constructs and lentiviral vectors [Figure 13] Exchange of receptor target specificity. a, b, c, and d, Specific lysis (y-axis) of CD19-positive Raji and CD20-positive Raji target cells (black line) after 4 h of co-culture with NK-92 cells modified with ASIMut receptors targeting HER2 (a), CD19 (b, c), and CD20 (d) at different E:T cell ratios (x-axis). Description and structure of the corresponding receptors are shown above each panel. Shown are the mean +- SD of biological triplicates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Accordingly, the present invention relates to a modified mammalian cell comprising: (I) a first and a second polypeptide, each of which comprises a variable region, wherein the variable region of the first polypeptide and the variable region of the second polypeptide form an antigen-binding site on the outside of a cell; wherein the first polypeptide further comprises a membrane domain that is located within the membrane of the cell; and, wherein the first and second polypeptides are (i) the alpha and beta chains of the T cell receptor (TCR), or (ii) the gamma and delta chains of the TCR; is not, or preferably does not include, And (II) at least one polypeptide, e.g., the first polypeptide and / or at least one further polypeptide, comprising an intracellular domain that comprises at least one signaling domain; and Here, when the antigen-binding site binds to a corresponding antigen, at least one of the signaling domains triggers a process within the cell that enables the cell to promote the killing of a target cell that contains the antigen on its cell surface.

[0020] In particular, said first polypeptide, said second polypeptide and (if present) said at least one further polypeptide comprising an intracellular domain comprising at least one signalling domain (e.g. a third CD79A-like and / or a fourth CD79B-like polypeptide) form a multi-chain antigen receptor (i.e. a multi-chain antigen-specific receptor) according to the invention, as described herein.

[0021] Furthermore, said multi-chain antigen receptor may comprise one or more further polypeptides that bind to or interact with said polypeptide, such as a CD16-like polypeptide, as described herein in the context of the present invention. In particular, the term "multiple chains" refers to the presence of multiple polypeptides (ie, multiple amino acid chains).

[0022] The multi-chain antigen receptor of the present invention may be referred to herein simply as the "antigen receptor" or "antigen-specific receptor." Sometimes, particularly when at least one further polypeptide as described herein binds to or interacts with said first and said second polypeptides, the multi-chain antigen receptor is also considered herein and in the context of the present invention as a multi-chain antigen receptor complex, e.g. a BCR-like complex.

[0023] Here, in the context of the present invention, one or more of the polypeptides comprising an intracellular domain that comprises at least one signaling domain (e.g., the first polypeptide, the third CD79A-like polypeptide and / or the fourth CD79B-like polypeptide) may be a chimeric polypeptide as described herein, e.g., the extracellular domain and membrane domain are from one protein (e.g., CD79A or CD79B) and the intracellular domain is from another protein (e.g., CD3 zeta).

[0024] In some embodiments, the multi-chain antigen receptor of the present invention is referred to herein and in the context of the present invention as an "antigen-specific synthetic immunoglobulin-based multi-chain receptor ( A Ntigen-specific S Synthetic I Munoglobulin-based Mu As described herein and exemplified in the Examples, the multi-chain antigen receptors of the present invention can be highly modular and versatile, and therefore can also be considered herein and in the context of the present invention as a platform, e.g., the ASIMut platform.

[0025] Exemplary modified mammalian cells according to the present invention are modified NK cells as described and exemplified in the accompanying Examples. Exemplary first and second polypeptides according to the present invention are membrane-bound anti-Her2 antibodies expressed on the surface of NK cells as described and exemplified in the accompanying Examples. Accordingly, corresponding exemplary target cells are Her2-expressing target cells employed in the accompanying Examples.

[0026] Furthermore, an exemplary polypeptide comprising an intracellular domain comprising at least one signaling domain is the CD79A or CD79B polypeptide employed in the attached examples. An exemplary polypeptide comprising an intracellular domain comprising at least one signaling domain can further comprise the CD3 zeta and / or FceRIg polypeptide employed in the attached examples, particularly when the modified cell further comprises a CD16-like polypeptide exemplified by the CD16 polypeptide employed in the attached examples. An exemplary intracellular domain or signaling domain is the CD3 zeta, FceRIG, CD79A, CD79B or IgG1 intracellular domain or signaling domain employed in the attached examples. An exemplary process that allows the modified cell to promote the killing of target cells is cis-ADCC, as exemplified in the attached examples. The modularity and versatility of the multi-chain antigen receptor of the present invention are shown, for example, in Examples 5 and 6. For example, exemplary chimeric polypeptides that can be used in the multi-chain antigen receptor of the present invention are shown in these examples.

[0027] However, the invention is in no way limited to the accompanying examples or to these exemplary cells, polypeptides, domains or processes.

[0028] The present invention is based, at least in part, on the surprising discovery that antibodies, e.g. IgG1 or IgM, can also be expressed on the surface of mammalian cells other than B cells, e.g. natural killer (NK) cells or cancer cells such as HeLa cells, even in the absence of CD79, as shown in the accompanying examples. Furthermore, the inventors unexpectedly found that NK cells expressing antibodies, e.g. antibodies against Her2, on their cell surface can eliminate Her2-positive target cells (Fig. 2h). This suggests that the expression of membrane-bound antibodies in NK cells induces antibody-dependent cellular cytotoxicity (ADCC) against target cells in cis, i.e., in the absence of soluble antibodies (see, e.g., Fig. 1b). Remarkably and quite unexpectedly, NK cells expressing anti-Her2 antibodies on their cell surface but lacking CD16 still eliminated up to 27% of Her2-positive target cells (Fig. 2h, i), indicating that membrane-bound antibodies may function alone.

[0029] Even more surprisingly, we found that cis-ADCC induced by expressing membrane-bound anti-Her2 antibody in CD16-expressing NK cells was as effective in killing target cells as canonical trans-ADCC induced by adding the corresponding soluble anti-Her2 antibody to control NK cells that did not express the antibody (compare Fig. 2g and j).

[0030] This further indicates that in the context of the present invention, mammalian cells such as NK cells can be endowed with selective cytotoxicity against target cells expressing an antigen. Moreover, this indicates that immobilizing antibodies on the surface of mammalian cells such as NK cells (FIG. 1b) can circumvent the need for soluble antibodies to contact the target antigen to induce trans-ADCC (FIG. 1a). Notably, inducing ADCC in cis according to the present invention (FIG. 1b) does not depend on the diffusion of soluble antibodies, which is often a limiting factor for ADCC, and thus can significantly improve ADCC efficiency in many therapeutic applications, at least in the treatment of tumors.

[0031] However, the present invention is not limited to the use of antibodies, but also includes the use of proteins that share some similarity with antibodies, as described herein, particularly in the context of the first and second polypeptides of the present invention.Preferably, the protein used in the context of the present invention for antigen binding comprises at least two polypeptides, each of which comprises a part of an antigen binding site, at least one of which has a membrane domain.In other words, it is preferred in the context of the present invention to use an antigen binding site that is divided into at least two, for example two polypeptides, as in the case of antibodies.

[0032] The use of multi-chain antigen binding sites and multi-chain antigen receptors, e.g. multi-chain antigen receptors formed by or comprising a first and a second polypeptide of the invention, such as antibodies (immunoglobulins), makes it possible to overcome many of the disadvantages associated with single-chain chimeric antigen receptors (CARs).

[0033] For example, the use of full-length membrane-bound antibodies or antibody-like multi-chain proteins as or for antigen receptors allows for more robust expression of antigen receptors in mammalian cells, avoiding undesirable clustering of antigen receptors on the cell surface and tonic signaling, as is often observed with CARs. In particular, full-length immunoglobulins cannot be mispaired. Moreover, immunoglobulins appear to have an optimal length for activating cells, since they were designed for this purpose in nature. Thus, multi-chain antigen receptors similar to or including immunoglobulins (antibodies), particularly those formed by or including the first and second polypeptides according to the invention, are considered to be advantageous for activating mammalian cells in cis for this additional reason.

[0034] Moreover, due to its particularly high modularity, the antigen receptor according to the invention can be constructed in a simple manner on the basis of existing monoclonal antibodies. In particular, as shown in Example 6 and FIG. 13, the antigen-binding site, which may for example be formed by the CDRs of the heavy and light chains of an antibody, can be easily switched and / or adapted to the target antigen, without changing the rest of the antigen receptor protein. Furthermore, as shown in the appended examples, the non-antigen-binding part of a polypeptide involved in antigen binding (for example the first polypeptide of the invention), for example a polypeptide similar to the heavy chain of a membrane-bound antibody, can also be easily switched and / or adapted for a specific purpose. For example, as shown in the appended examples, when a chimeric CD79-CD3 zeta polypeptide was co-expressed in NK cells, the non-variable region of the heavy chain of membrane-bound IgG1 could be replaced by the non-variable region of the heavy chain of membrane-bound IgM without adversely affecting the killing of the target cells (see for example FIG. 4). Furthermore, by using antigen-binding polypeptides from different immunoglobulin classes, it is possible to couple (for example IgG1) or decouple (for example IgM) the antigen receptor response from other endogenous receptors, such as CD16. Furthermore, in the context of the present invention, it is also possible to exchange the portions similar to the constant regions of IgG antibodies with less immunogenic or non-immunogenic constant regions from IgM.

[0035] Thus, the multi-chain antigen receptor according to the invention, i.e., multi-chain antigen receptor formed by or comprising the first and second polypeptides of the invention, can offer greater modularity and adaptability than conventional CARs. In addition, the use of multi-chain antigen receptors according to the invention, which may form dimers, multimers and / or complexes and may have several membrane and intracellular domains, allows the addition of intracellular signaling or activation domains not only in series ("in-line") in one polypeptide chain as in conventional CARs, but also in parallel, i.e., in different polypeptide chains of the antigen receptor. As described herein, the use of multiple signaling or activation domains in parallel can avoid undesirable problems due to non-optimal distance of the domains from the cell membrane, which may be related to insufficient or undesirable activation of signaling pathways, as observed with CARs (Feucht, et al. 2019). Thus, the multi-chain antigen receptor or antigen receptor complex according to the invention can have a higher efficacy than conventional CARs, at least because multiple signaling and / or co-stimulatory domains can be arranged in an optimal way, e.g., in parallel, at an optimal distance and orientation relative to the cell membrane. Furthermore, activation motifs such as ITAMs can be enriched in the multi-chain antigen receptor complexes of the present invention compared to prior art CARs, and increasing the number of ITAMs per antigen receptor can lower the activation threshold of cytotoxic cells for a given concentration of target epitope.

[0036] Furthermore, the multi-chain antigen receptor according to the invention, i.e. the receptor formed by or comprising the first and second polypeptides of the invention, can interact with further polypeptides in mammalian cells, such as CD16, as shown in the accompanying examples.

[0037] Furthermore, the present inventors have found that co-expression of CD79 increases the surface expression of antibodies in mammalian cells, e.g., NK cells or cancer cells such as HeLa cells, as shown in the accompanying examples. Surprisingly, as shown in the accompanying examples, when the antibodies are expressed in combination with CD79 in NK cells, the NK cells are effective in killing target cells. Without being bound by theory, the antibodies may have formed multimeric B cell receptor (BCR)-like complexes in these mammalian cells.

[0038] Thus, the antigen receptor according to the present invention, i.e. the first and second polypeptides, can also form a BCR-like complex with CD79 in mammalian cells that are not B cells, such as cytotoxic cells. The combination of CD79A and / or CD79B, or polypeptides similar to CD79 in at least some aspects, in particular the CD79A-like and / or CD79B-like polypeptides described herein, has the additional advantage that more signaling domains can be present in the multi-chain antigen receptor complex. Again, the signaling domains can be added in parallel within the intracellular domains of different polypeptides (e.g., a polypeptide involved in antigen binding, such as the first polypeptide of the present invention, and a CD79-like polypeptide, such as the third and / or fourth polypeptide of the present invention), rather than being present only in series within one polypeptide chain as in conventional CARs.

[0039] The extracellular and membrane domains of the polypeptides involved in antigen binding, e.g. the first polypeptide of the invention, may resemble the extracellular and membrane domains of the heavy chain of a membrane-bound antibody, and the extracellular and membrane domains of the CD79-like polypeptide may resemble the extracellular and membrane domains of CD79A and / or CD79B, while the intracellular domains of these antibody heavy chain-like or CD79-like polypeptides may be entirely different. Thus, in certain embodiments, the polypeptides of the invention, e.g. the first, third and / or fourth polypeptides, are considered chimeric polypeptides. For example, the intracellular domain of such chimeric polypeptides according to the invention may resemble the intracellular domain and / or ITAM of CD3 zeta and / or FceRIg. Thus, the multi-chain antigen receptor according to the invention may comprise chimeric polypeptides, e.g. the extracellular and / or membrane domains are from different proteins (e.g. antibodies or CD79) and the intracellular domain is from another protein (e.g. CD3 zeta or FceRIg).

[0040] Very surprisingly, as shown in the appended examples, the inventors further found that NK cells expressing a membrane-bound anti-Her2 antibody, e.g. IgG1 or IgM, and a chimeric CD79-CD3ζ (CD3 zeta) polypeptide comprising the extracellular and membrane domains of CD79A or CD79B, and the intracellular domain of CD3 zeta (i.e. CD3 zeta ITAM), kill target cells expressing Her2 much more efficiently, i.e. with a much higher efficiency, than the canonical trans-ADCC induced by the addition of a corresponding soluble anti-Her2 antibody to NK cells expressing CD16 but not the antibody (see e.g. Figures 4c, 4i, 2g and Example 5). Remarkably, the presence of CD16 in the NK cells bearing the antibody and the CD79-CD3 zeta chimeric polypeptide was not necessary to achieve this significantly improved target cell lysis efficiency. Furthermore, this indicates that chimeric polypeptides according to the invention, for example polypeptides comprising an extracellular and / or membrane domain similar to that of CD79A or CD79B and the intracellular domain of CD3 zeta, can further improve the multi-chain antigen receptor according to the invention and may be particularly useful for killing target cells.

[0041] Moreover, and very surprisingly, the inventors further found that NK cells expressing a membrane-bound chimeric anti-Her2 antibody-like protein containing the extracellular and membrane domains of the membrane-bound antibody and the intracellular domain of FceRIg killed Her2-expressing target cells much more efficiently than canonical trans-ADCC induced by adding the corresponding soluble anti-Her2 antibody to NK cells expressing CD16 but not antibody (see, for example, Figs. 5c, 2g, 3i, and Example 5). Surprisingly, this chimeric antibody was well expressed on the surface of NK cells and efficiently killed target cells even in the absence of CD16 and CD79.

[0042] More surprisingly, the inventors found that the combined expression of chimeric antibody-FceRIg polypeptide and chimeric CD79-CD3zeta polypeptide further increased the surface expression of the antibody and the efficiency of the killing process, with 96% of the target cells being killed at a NK cell:target cell ratio of 10:1 (see, for example, Fig. 5d-f and Example 5). This response strongly exceeded the response achieved by canonical trans-ADCC. This further indicates that the BCR-like complex domain according to the present invention, including CD3ζ (CD3zeta) and / or FcεRIγ (FceRIg) signaling, can confer a very high level of antigen-specific cytotoxicity to mammalian cells against antigen-expressing cells.

[0043] Furthermore, as shown in Example 6 and Figure 13, it has been confirmed that the antigen specificity of the multi-chain antigen receptors of the present invention, such as those comprising chimeric antibody-FceRIg polypeptides and chimeric CD79-CD3zeta polypeptides, can be easily switched to kill different target cells. For example, a multi-chain antigen receptor comprising binding sites for CD19 and CD20 has been found to very efficiently kill Raji tumor cells expressing CD19 and CD20.

[0044] However, the intracellular domain may also be derived from the same polypeptide as the extracellular and / or membrane domain, e.g. the entire polypeptide may resemble the heavy chain of a membrane-bound antibody, or CD79A or CD79B.As already mentioned above and shown in the appended examples, the non-chimeric antibodies alone or in combination with CD16 and / or non-chimeric CD79 proteins also achieved considerable killing efficiency against target cells.

[0045] Moreover, as shown in the appended Examples, the inventors further surprisingly found that introduction of a flexible peptide linker into the membrane proximal part of the extracellular domain significantly increased surface expression of the antibody on NK cells and improved lysis of target cells compared to antibodies without such a linker, particularly in the presence of CD16 or CD16 and CD79 (Example 4). Remarkably and unexpectedly, tethering of antibodies to the cell membrane by a flexible linker in combination with CD16 or CD16 and CD79 eliminated target cells much more efficiently than canonical ADCC and as efficiently as membrane-bound antibodies (without linker) in combination with a chimeric CD79-CD3 zeta polypeptide bearing an ITAM from CD3 zeta.

[0046] This again demonstrates that the multi-chain receptor structures according to the invention exhibit superior cytotoxicity and further highlights the advantage of synergy between multiple extracellular and intracellular domains arranged at an optimal distance from each other and / or in an optimal orientation relative to the cell membrane.

[0047] Overall, our findings show that the antigen receptor or antigen receptor complex according to the present invention, which may also be called antigen-specific synthetic immunoglobulin-based multichain receptor (ASIMut receptor), shows high surface expression and induces potent ADCC against target cells in an antigen-specific manner. Thus, the present invention further provides a flexible and programmable framework and platform for the development of additional therapeutic antigen-specific mammalian cells, such as NK cells. Furthermore, the present invention provides a new class of mammalian cell (e.g., NK cell)-based cell therapy that may complement or even surpass existing antigen-specific cell therapies, such as CAR-T cell therapies, in the treatment of cancer and other diseases. Furthermore, the therapeutic use of modified mammalian cells, such as NK cells, according to the present invention may have additional advantages, such as the possibility of using allogeneic cell material due to the absence of GvHd (Xie, et al. 2020).

[0048] Thus, as described herein, the present invention relates to a modified mammalian cell comprising: (I) a first and a second polypeptide, each of which comprises a variable region, wherein the variable region of the first polypeptide and the variable region of the second polypeptide form an antigen-binding site outside the cell; wherein the first polypeptide further comprises a membrane domain that is located within the membrane of the cell; and (II) at least one polypeptide, e.g., said first polypeptide and / or at least one further polypeptide, comprising an intracellular domain that comprises at least one signaling domain.

[0049] In particular, said first and second polypeptides are not (i) the alpha and beta chains of a T cell receptor (TCR), or (ii) the gamma and delta chains of a TCR. In particular, when the modified cell is a T cell, it comprises a polypeptide as defined in (I) and (II) above in addition to, or instead of, a conventional, e.g. endogenous, T cell receptor.

[0050] In particular, herein, the modified cell of the present invention can promote the killing of a target cell that contains an antigen on the cell surface to which the antigen-binding site can bind.In particular, herein and in the context of the present invention, at least one of the signaling domains, when the antigen-binding site binds to, for example, a corresponding antigen on the surface of the target cell, triggers a process in the cell that allows the cell to promote the killing of a target cell that contains the antigen on the cell surface.Preferably, herein and in the context of the present invention, the modified cell is not a B cell that needs to interact with another immune cell type to promote the killing of a target cell.In some embodiments, the modified cell of the present invention is not a B cell.

[0051] The modified cell used herein and in the context of the present invention is a mammalian cell. The modified mammalian cell according to the present invention is not limited to a particular cell type or mammalian species. Preferably, the mammalian cell is a human cell, but may be a cell of other mammalian species, such as mouse, rat, hamster, monkey, horse, pig, cow or sheep. In some embodiments of the present invention, the modified cell is not a HEK393 cell.

[0052] Preferably, herein and in the context of the present invention, the modified cells are cytotoxic lymphocytes, in particular natural killer (NK) cells or T cells, most preferably NK cells. T cells can be CD8+ T cells, such as cytotoxic T cells, or CD4+ T cells, such as helper T cells or regulatory T cells. Preferably, T cells are CD8+ T cells, preferably cytotoxic CD8+ T cells.

[0053] Furthermore, the modified cells of the present invention may be primary cells or cell lines, as is generally understood in the art. Furthermore, the modified cells of the present invention may be derived from umbilical cord blood or peripheral blood, and may be obtained by differentiation of pluripotent cells, such as induced pluripotent stem cells, or by reprogramming of other cell types.

[0054] Polypeptide as used herein includes or refers to an amino acid chain, which is preferably at least 5, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or 10000, preferably at least 50, 100, 200, 300, 400 or 500, more preferably at least 200, 300, 400 or 500 amino acids long. Furthermore, the polypeptide is preferably less than 50000, 20000, 10000, 5000, 4000, 3000, 2000 or 1000 amino acids long. Polypeptide may also be referred to as protein herein, and since polypeptide as used herein is considered to be a single chain protein, protein may be referred to as polypeptide. However, in contrast to a polypeptide, a protein can also refer to a complex of multiple polypeptides.

[0055] Generally, different polypeptides refer to separate amino acid chains in the present specification and in the context of the present invention, unless otherwise expressly indicated.However, when two or more polypeptides are expressly indicated to be covalently linked, for example, via peptide bonds, these two or more polypeptides form one amino acid chain and are therefore exceptionally considered to be one polypeptide.

[0056] Preferably, and in most embodiments of the present invention, the modified mammalian cell comprises a first and a second polypeptide, each comprising a variable region, the variable region of the first polypeptide and the variable region of the second polypeptide forming an antigen binding site outside the cell, and the first polypeptide further comprises a membrane domain located within the membrane of the cell. Furthermore, the first polypeptide may comprise an intracellular domain. The second polypeptide may also comprise a membrane domain and optionally an intracellular domain, or may be located entirely outside the cell. Preferably, the first and second polypeptides are expressed from one or more nucleic acids in the modified mammalian cell. Thus, the modified cell may comprise one or more nucleic acid molecules from which the first and second polypeptides are expressed.

[0057] Thus, the modified mammalian cells of the invention preferably comprise a multi-chain antigen-binding site, where one part of the antigen-binding site is contained in the variable region of a first polypeptide and another part of the antigen-binding site is contained in the variable region of a second polypeptide.

[0058] The amino acid sequence of variable region used herein and in the context of the present invention depends on antigen specificity.Variable region used herein may correspond to the variable region of antibody, but is not limited thereto.For example, it may include only one or more CDRs of the variable region of antibody, and / or other sequences that allow specific binding to antigen.

[0059] As used herein, and in the context of the present invention, an antigen-binding site is capable of specifically binding to an antigen or epitope, as is generally understood in the art. Preferably, the first and second polypeptides according to the present invention, considered as antigen receptors, bind to an antigen through their common antigen-binding site with similar strength and / or similar specificity as an antibody, preferably a monoclonal antibody used for therapeutic and / or detection purposes. For example, the first and second polypeptides may bind to an antigen with a common antigen-binding site of 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10-8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or 10 -13 M or less, preferably 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 It can bind to an antigen with a dissociation constant (KD) of less than or equal to M.

[0060] In particular, in the context of the present specification and the present invention, the first and second polypeptides form or are part of an antigen receptor or antigen receptor complex, i.e., a multi-chain antigen receptor or a multi-chain antigen receptor complex, as described herein. The terms "antigen receptor" and "antigen-specific receptor" may be used interchangeably herein.

[0061] As used herein, a membrane domain may be, but is not limited to, a transmembrane domain, as is commonly understood in the art. As used herein, a membrane of a cell refers to the plasma membrane of a cell, as is commonly understood in the art.

[0062] The modified mammalian cells according to the invention are not unmodified B cells or unmodified T cells.

[0063] The modified cells of the invention may be modified T cells, but do not solely comprise a conventional T cell receptor (TCR) as an antigen receptor. In particular, the modified T cells of the invention comprise, instead of or in addition to the TCR, a first and second polypeptide as described herein.

[0064] Therefore, preferably, herein and in most aspects of the present invention, the first and second polypeptides are not (i) the alpha and beta chains of the TCR, or (ii) the gamma and delta chains of the TCR. More preferably, the first and second polypeptides do not include (i) the alpha and beta chains of the TCR, or (ii) the gamma and delta chains of the TCR. In particular, the first polypeptide does not include the alpha chain of the TCR and the second polypeptide does not include the beta chain of the TCR, or vice versa, and / or the first polypeptide does not include the gamma chain of the TCR and the second polypeptide does not include the delta chain of the TCR, or vice versa. In particular, the first and second polypeptides described herein do not form a conventional T cell receptor as generally understood in the art. Moreover, a person skilled in the art can easily recognize whether a polypeptide is a conventional TCR alpha chain, beta chain, gamma chain or delta chain, based on conventional general knowledge, using common sense, even if the alpha chain, beta chain, gamma chain or delta chain of the TCR is slightly modified. However, the first and second peptides according to the invention may be or comprise a significantly modified TCR alpha, beta, gamma or delta chain, e.g. a chimeric polypeptide and / or a polypeptide having significantly different functionality than the unmodified alpha, beta, gamma or delta chain.

[0065] Furthermore, the modified cell according to the present invention comprises at least one polypeptide comprising an intracellular domain comprising at least one signaling domain as described herein. The signaling domain can be comprised in the first polypeptide according to the present invention and / or in another polypeptide, such as the third and / or fourth polypeptide, comprised in the modified cell. Thus, said at least one polypeptide comprising an intracellular domain comprising at least one signaling domain may comprise the first polypeptide described herein and / or at least one other polypeptide, such as the third and / or fourth polypeptide described herein.

[0066] A signaling domain, sometimes referred to herein as an "activation domain", has the ability to induce or enhance a process in a mammalian cell, such as a modified cell of the invention, that enables the cell to promote the death of a target cell. A target cell, as used herein and in the context of the present invention, is a cell, preferably a mammalian cell, such as a cell from the same species as the modified cell of the invention, that contains an antigen to which the antigen-binding site of a modified cell according to the invention can bind, i.e. specifically bind, as described herein. In particular, the target cell expresses or presents said antigen on the cell surface.

[0067] In particular, in the context of the present invention, as described herein, when the antigen-binding site binds to a corresponding antigen, the signaling domain induces or enhances a process in the modified cell of the present invention that allows the cell to promote the death of a target cell that contains said antigen on the cell surface. In particular, the modified cell of the present invention kills a target cell upon binding to an antigen, e.g., when the antigen-binding site binds or binds to an antigen on the cell surface of the target cell. Preferably, the signaling domain does not induce or enhance said death-promoting process in the cell when the antigen-binding site is not or does not bind to its cognate antigen. At least, when the antigen-binding site binds or binds to the antigen, said death-promoting process is induced or enhanced to a greater extent. For example, the modified cell of the present invention can kill a target cell when it binds to an antigen on the surface of the target cell via the antigen-binding site formed by the first and second polypeptides according to the present invention.

[0068] The modified cells are capable of promoting the killing of target cells directly or indirectly, preferably in a direct manner, when the antigen-binding site binds to an antigen according to the present invention.

[0069] Thus, the promotion of target cell killing when the first and second polypeptides, i.e., the antigen receptors of the invention, bind to an antigen, in particular the surface of a target cell, may in at least some respects resemble antibody-dependent cellular cytotoxicity (ADCC), in particular cis-ADCC, as described herein and exemplified in the accompanying examples, although the first and second polypeptides may differ from conventional antibodies. In particular, the promotion of target cell killing, when CD16 is involved, may in at least some respects resemble ADCC, i.e., cis-ADCC, as described herein and exemplified in the accompanying examples.

[0070] Without being bound by theory, the modified cells of the present invention bind to antigens on the surface of target cells through the antigen-binding site, forming an immunological synapse. Considering the kinetic separation model as discussed in the art, upon antigen binding, the phosphatase in the modified cells that protrudes into the extracellular space and constantly dephosphorylates the signaling domain, e.g., ITAMs contained in the signaling domain, may be dephosphorylated, i.e., separated from the phosphatase, resulting in phosphorylation of the signaling domain. When the signaling domain is phosphorylated, a process may be induced or enhanced in the modified cells that may kill the target cell, e.g., via a cis-ADCC-like mechanism. Thus, it is important that the modified cells contain an antigen-binding site on the outside of the cell and at least one signaling domain on the inside of the cell. However, it is not so important whether the signaling domain is contained in the same polypeptide as the polypeptide that forms the antigen-binding site or in another polypeptide in the modified cell. For example, the first and second polypeptides that form the antigen-binding site and the other polypeptide that has the signaling domain may be in direct contact or may not be together in a membrane raft in the modified cell.

[0071] Preferably, the modified cells of the present invention do not need to interact with another immune cell type to promote the killing of the target cell. In other words, the modified cells of the present invention preferably kill the target cell in a direct manner, e.g., in at least some aspects, by a process similar to cis-ADCC as described herein and illustrated in the accompanying examples. Furthermore, the modified cells of the present invention are also considered as effector cells, as described herein and illustrated in the accompanying examples.

[0072] As is well known in the art, conventional B cells cannot promote the killing of target cells without the need to interact with at least other immune cell types, and in particular, conventional B cells cannot directly kill target cells.

[0073] Thus, in certain embodiments of the invention, the modified cells are not B cells, which need to interact with other immune cell types to promote killing of the target cell.

[0074] However, the modified cell of the invention may also be a B cell modified according to the invention, e.g., a B cell having the ability to promote killing of a target cell without the need to interact with other immune cell types, such as in a cis-ADCC manner as described herein. Furthermore, the modified cell of the invention may be a B cell comprising, instead of or in addition to an unmodified or conventional membrane-bound antibody, a first and a second polypeptide and / or an antigen receptor according to the invention, wherein said first and second polypeptides and said antigen receptor are distinct from a conventional membrane-bound antibody, e.g., the membrane-bound antibody of an unmodified B cell.

[0075] Furthermore, the modified cell of the invention may be a B cell comprising an antigen receptor complex according to the invention, e.g. a BCR-like complex, e.g. a first and second polypeptide as described herein, and a third CD79A-like and / or a fourth CD79B-like polypeptide, wherein said antigen receptor complex is distinct from a conventional BCR, e.g. a BCR of an unmodified B cell. For example, the modified B cell of the invention may lack at least one or all components of an endogenous BCR, e.g. a membrane-bound antibody, and / or a CD79A or CD79B gene may be knocked out, and at least one nucleic acid encoding the first and second polypeptide of the invention as described herein, and / or a CD79A-like third and / or a CD79B-like fourth polypeptide may be introduced.

[0076] Additionally, the modified B cells of the invention may be B cells that further comprise CD16 and / or a fifth polypeptide of the invention, as described herein.

[0077] Additionally, the modified cells of the invention, such as modified B cells of the invention, may comprise a chimeric antigen-binding polypeptide, or a chimeric CD79 peptide, as described herein.

[0078] However, in some embodiments of the invention, the mammalian cell is not a B cell.

[0079] In the present context, the variable regions of the first and second polypeptides may comprise at least one, preferably at least three, and preferably all, complementarity determining regions (CDRs) of an antibody. Preferably, the first and second polypeptides each comprise at least one, preferably three CDR(s) of an antibody.

[0080] In particular, the first polypeptide may comprise the complementarity determining regions (CDRs) of the heavy chain of an antibody, i.e., CDR-H1, CDR-H2 and CDR-H3, and / or the variable region of the second polypeptide may comprise the CDRs of the light chain of said antibody, i.e., CDR-L1, CDR-L2 and CDR-L3.

[0081] However, it is also possible that the first polypeptide comprises the complementarity determining regions (CDRs) of the light chain of the antibody, i.e. CDR-L1, CDR-L2 and CDR-L3, and / or the variable region of the second polypeptide comprises the CDRs of the heavy chain of said antibody, i.e. CDR-H1, CDR-H2 and CDR-H3.

[0082] Furthermore, the first polypeptide according to the invention may comprise the variable region of a heavy chain of an antibody, and / or the second polypeptide may comprise the variable region of a light chain of an antibody.

[0083] It is well known to those skilled in the art that complementarity determining regions (CDRs) determine the binding specificity of an antibody. The CDR regions of antibodies or Ig-derived regions are described in Kabat (1991), Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 US Department of Health and Human Services, and / or Chothia (1987), J. Mol.Biol. 196, 901-917; and Chothia (1989) Nature, 342, 877-883. The CDRs provided herein above were determined by the Kabat system. Due to its widespread use and reliability, the Kabat numbering system may be preferred.

[0084] Suitable methods for determining the sequence of an antibody, eg, a monoclonal antibody, are also readily available in the art.

[0085] The variable regions of an antibody can be divided into CDRs and framework regions (FRs), inter alia, using the Kabat or Chothia numbering system.

[0086] Thus, the CDRs or variable regions of an antibody can be readily determined by methods known in the art, for example, by employing the Kabat system.

[0087] The terms "antibody" and "immunoglobulin" are used interchangeably herein and are as generally understood in the art. Preferably, the antibody used herein is a monoclonal antibody. The antibody may also be a CDR-grafted antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0088] In the context of the antibody according to the invention, i.e. the CDRs and / or variable regions of the antibody, there is no limitation to a particular antibody. Indeed, any existing or future antibody can be used in the context of the present invention. In particular, the first and / or second polypeptide of the present invention can comprise the CDRs and / or variable regions from any existing or future antibody. The choice of the CDRs or variable regions of the antibody depends mainly on the antigen to be recognized and / or the target cell to be killed. For example, as shown in the appended examples, an anti-Her2 antibody, i.e. an anti-Her2 antibody derived from Trastuzumab / Herceptin, has been expressed on the surface of cells modified according to the present invention in order to kill target cells expressing Her2. However, the present invention is in no way limited to anti-Her2 antibodies or Her2-expressing target cells. It is further confirmed in Example 6 and Figure 13 that the CDR or variable region of trastuzumab in the multi-chain antigen receptor of the present invention can be easily replaced with the CDR or variable region of other antibodies, such as anti-CD19.1 antibody (i.e., FMC63), anti-CD19.2 antibody (i.e., inebilizumab) or anti-CD20 antibody (i.e., rituximab). In fact, it has been found that effector cells expressing such modified multi-chain antigen receptors efficiently killed target cells expressing CD19 or CD20 (e.g., Raji) cells.

[0089] Further suitable antibodies that can be used in the context of the present invention are in particular: 3F8, abagovomab, abciximab, avituzumab, abrezekimab, abrilumab, actotumab, adalimumab, adecatumumab, aducanumab, afacevicumab, afelimomab, alacizumab pegol, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, amivantamab, anatumomab mafenatox, andecaliximab, anetumab ravtansine, anifrolumab, ansuvimab, anrukinzumab (= IMA-638), apolizumab, apurutumab Ixadotin, Arcitumomab, Asclinvacumab, Acelizumab, Atezolizumab, Atidortoxumab, Atinumab, Atortivimab, Atortivimab / Maftivimab / Odesivimab, Atorlimumab, Avelumab, Azintuxizumab Vedotin, Bamlanivimab, Bapineuzumab, Basiliximab, Bavituximab, BCD-100, Bectumomab, Begelomab, Belantamab Mahodotin, belimumab, bemarituzumab, benralizumab, berlimatoxumab, bermekimab, berusanlimab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, virutamimab, bivatuzumab, bleselumab blinatumomab, brontuvemab, brosozumab, bococizumab, brazikumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, broticutuzumab, burosumab, cabilalizumab, camidanlumab tesirin, camrelizumab, canakinumab, cantuzumab mertansine, cantuzumab Lovetansine, Caplacizumab, Casirivimab, Capromab, Carlumab, Carotuximab, Catumaxomab, cBR96-doxorubicin immunoconjugate, Cedelizumab, Cemiplimab, Sergituzumab Amnaleukin, Certolizumab pegol, Cetrelimab, Cetuximab, Civisatamab, Sirmutuzumab, SitamuzumabBogatox, Cixutumumab, Clazakizumab, Clenoliximab, Crivatuzumab tetraxetan, Codrituzumab, Cofetuzumab peridotin, Cortuximab ravtansine, Conatumumab, Concizumab, Cosfrobiximab, Crenezumab, Crizanlizumab, Cloteduzumab, CR6261, Cusatuzumab, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirorizumab pegol, Daratumumab, Dectrecumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Depatuxizumab mafodotin, Dellotuximab biotin, Detumomab, Dezamizumab, Dinutuximab, Dinutuximab Beta, dilidabumab, domagrozumab, dorlimomab alitox, dostarlimab, drozitumab, DS-8201, durigotuzumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezanumab, elgemtuzumab, elotuzumab, ersilimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enapotamab vedotin, enavatuzumab, enfortumab vedotin, enlimomab Pegol, enoblitzumab, enokizumab, enoticumab, encituximab, epcolitamab, epitumomab situxetan, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etesevimab, etigilimab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, faricimab, faretuzumab, sinumab, FBTA05, felvizumab, fezakinumab, fibatuzumab, ficratuzumab figitumumab, filibumab, framvotumab, fretikumab, flotetuzumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, frobocimab, fulnevetomab, fulranumab, futuximab, galcanezumab, galiximab, gancotamab, ganitumab, gantenerumab, gatipotuzumab, gavilimomab, gedibumab, gemtuzumab ozogamicin, gevokizumab, gilvetomab, gimsilumab, direntuximab, glenbatumumabVedotin, Golimumab, Gomiliximab, Goslanemab, Guselkumab, Ianalumab, Ibalizumab, Sintilimab, Ibritumomab Tiuxetan, Icrucumab, Idarucizumab, Ifavotuzumab, Igovomab, Iradatuzumab Vedotin, Imalumab, Imaprelimab, Imusiromab, Imdevimab, Imgatuzumab, Incracumab, Indatuximab Ravtansine, Indusatumab Vedotin, Inebilizumab, Infliximab, Intetumumab, Inolimomab, Inotuzumab Ozogamicin, ipilimumab, iomab-B, iratumumab, isatuximab, iscalimumab, istiratumab, itolizumab, ixekizumab, keliximab, labetuzumab, lacinotuzumab, ladiratuzumab vedotin, lampalizumab, lanadelumab, landgrozumab, laprituximab emtansine, ralcaviximab, lebrikizumab, remaresomab, lendalizumab, lenbervimab, lenzilumab, lerdelimumab, leronlimab, lesofabumab, letolizumab, lexatumumab, ribivirumab, rifatuzumab Vedotin, ligelizumab, roncastaximab tesirin, rosatuxizumab vedotin, rilotomab satetratetan, lintozumab, lirilumab, roderucizumab, lokivetomab, lorvotuzumab mertansine, lucatumumab, lurizumab pegol, rumiliximab, lumuletuzumab, rupartumab, rupartumab amadotin, rutikizumab, maftivimab, mapatumumab, margetuximab, marstacimab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimuab, milatuzumab, minretumomab, mirikizumab, mirvetuximab Soravtansine, mitumomab, modotuximab, mogamulizumab, monalizumab, morolimumab, mosunetuzumab, motavizumab, moxetumomab-pasudotox, muromonab-CD3, nacolomab-tafenatox, namilumab, naptumomab-estafenatox, naratuximab-emtansine, narunatumab, natalizumab, navicizumab, naxitamab, nebacumab, necitumumab, nemolizumab, NEOD001, nerelimomab, nesvacumab, netakimab, nimotuzumab, nirsevimab, nivolumab, nofetumomabMerpentan, obiltoxin, obinutuzumab, ocaratuzumab, ocrelizumab, odesivimab, odurimomab, ofatumumab, olaratumab, oleculab, orendalizumab, olokizumab, omalizumab, omburtamab, OMS721, onartuzumab, ontuxizumab, ombatilimab, opicinumab, oportuzumab Monatox, oregovomab, olticumab, otelixizumab, otilimab, otlertuzumab, oselumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, pancomab, panobacumab, palsatuzumab, pascolizumab, pasotuximab, pateclizumab, patritumab, PDR001, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placumab, prezalumab, prozalizumab, pogalizumab, polatuzumab Vedotin, ponezumab, polgabiximab, prasinezumab, prezarizumab, priliximab, plitoxiaximab, pritumumab, PRO 140, kirisumab, racotumomab, radletumab, rafivirumab, ralpanzizumab, ramucirumab, ranevetomab, ranibizumab, raxibacumab, ravagalimab, ravavurizumab, refanezumab, regavirumab, regdanvimab, relatorimab, lemtolumab, reslizumab, rilotumumab, rinukumab, risankizumab, rituximab, rivavazumab Pegol, lobatumumab, Rmab, loretumumab, romilkimab, romosozumab, rontalizumab, rosmantizumab, rovalpituzumab tesirin, rovelizumab, rozanolixizumab, ruplizumab, SA237, sacituzumab govitecan, samaryzumab, samrotamab vedotin, sarilumab, satralizumab, satumomab pendetide, secukinumab, seliclerumab, serivanchumab, setoxaximab, setoruzumab, sevirumab, sibrotuzumab, SGN-CD19A, SHP647, sifalimumab, siltoximab, simtuzumab, siplizumab, siltratumab vedotin, sirukumab, sofituzumabVedotin, solanezumab, solitomab, soneptizumab, sontuzumab, sotrovimab, spartalizumab, stamulumab, sulesomab, sputabumab, stimulimab, subizumab, sublatoxumab, tabalumab, tacatuzumab Tetraxetan, tadocizumab, tafasitamab, talacotuzumab, talizumab, talquetamab, tamtubetomab, tanezumab, taplitumomab Paptox, talectumab, taborimab, teclistamab, tefibazumab, terimomab Alitox, terisotuzumab, terisotuzumab Vedotin, Tenatumomab, Teneliximab, Teplizumab, Tepositamab, Teprotumumab, Tesidolumab, Tetulomab, Tezepelumab, TGN1412, Tiburizumab, Tildrakizumab, Tigatuzumab, Timigtuzumab, Timolumab, Tiragolumab, Tilaggotumab, Tislelizumab, Tisotumab Vedotin, TNX-650, Tocilizumab, Tomzotuximab, Toralizumab, Tosatoxumab, Tositumomab, Tobetumab, Tralokinumab, Trastuzumab, Trastuzumab Duocarmazine, Trastuzumab Emtansine, TRBS07, Tregalizumab, Tremelimumab, Trevoglumab, Tucotuzumab Celmoleukin, tubilumab, ublituximab, urocupulumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadastoximab-talilline, banalimab, bundetuzumab-vedotin, vantitumab, vanucizumab, bapaliximab, balisakumab, varlilumab, batelizumab, vedolizumab, veltuzumab, bepalimomab, besencumab, visilizumab, bovalilizumab, volociximab, bonlerolizumab, bopratelimab, borsetuzumab mafodotin, votumumab, bunakizumab, zentuzumab, XMAB-5574, zalutumumab, zanolimumab, zatuximab, zenoctuzumab, diralimumab, zolbetuximab, or zolimomab alitox.

[0090] Furthermore, the present invention relates to a kit comprising one or more nucleic acid molecules encoding the first and second polypeptides according to the invention, and optionally at least one further polypeptide.

[0091] Thus, the present invention further relates to a kit comprising one or more nucleic acid molecules, wherein said nucleic acid molecules comprise: (I) a first coding sequence encoding a first polypeptide, and a second coding sequence encoding a second polypeptide, wherein the first and second polypeptides each comprise a variable region, and the first polypeptide further comprises a membrane domain; wherein the variable region of the first polypeptide and the variable region of the second polypeptide are capable of forming an antigen-binding site when expressed together in the modified mammalian cell; and the first and second polypeptides are (i) the alpha and beta chains of the T cell receptor (TCR), or (ii) the gamma and delta chains of the TCR; is not, or preferably does not include, And (II) at least one coding sequence encoding at least one polypeptide, e.g., the first polypeptide and / or at least one further polypeptide, comprising an intracellular domain that includes at least one signaling domain; and wherein in a modified cell comprising first and second polypeptides, and at least one polypeptide comprising an intracellular domain comprising at least one signaling domain, when the antigen binding site binds to a corresponding antigen, at least one of the signaling domains triggers an intracellular process that enables the cell to promote the killing of a target cell that comprises the antigen on its cell surface.

[0092] For example, as described herein in the context of the modified cells of the invention with respect to the first and second polypeptides, antigen binding sites, modified cells, intracellular domains, signaling domains, antigens, target cells and death promoting processes, apply equally to the kits of the invention.

[0093] Here, in the context of the present invention, the first polypeptide may comprise an intracellular domain comprising at least one signaling domain, as described herein.

[0094] Furthermore, the modified cell of the present invention may comprise a third and / or fourth polypeptide, at least one of the third and fourth polypeptides comprising an intracellular domain comprising at least one signaling domain as described herein. Preferably, each of the third and fourth polypeptides comprises an intracellular domain comprising at least one signaling domain. Furthermore, the third and / or fourth polypeptide comprising an intracellular domain comprising at least one signaling domain can interact and / or bind with the first polypeptide in the modified cell of the present invention, i.e.

[0095] Further, the modified cell may comprise a fifth polypeptide, wherein the fifth polypeptide is capable of interacting and / or binding to the first polypeptide, and the third and / or fourth polypeptide comprises an intracellular domain comprising at least one signaling domain as described herein, i.e., in the modified cell of the present invention.

[0096] Thus, the nucleic acid molecule(s) may comprise a third coding sequence encoding a third polypeptide and / or a fourth coding sequence encoding a fourth polypeptide, e.g., in the context of a kit of the invention, e.g., in the context of a modified cell of the invention, as described herein. Additionally, the nucleic acid molecule(s) may further comprise a fifth coding sequence encoding a fifth polypeptide, e.g., in the context of a kit of the invention, e.g., in the context of a modified cell of the invention, as described herein.

[0097] In the context of the present invention, the first polypeptide is considered to be an antibody heavy chain-like polypeptide and the second polypeptide is considered to be an antibody light chain-like polypeptide. Furthermore, the first and second polypeptides together can be considered to be an antibody-like protein.

[0098] Further, the third polypeptide is believed to be a CD79A-like polypeptide, and the fourth polypeptide is believed to be a CD79B-like polypeptide.

[0099] Additionally, the fifth polypeptide is believed to be a CD16-like polypeptide.

[0100] However, in some embodiments of the invention, i.e., where a fifth CD16-like polypeptide is involved, the third polypeptide is considered to be a CD3 zeta-like polypeptide and / or the fourth polypeptide is considered to be an FceRIg-like polypeptide.

[0101] In particular, the first and second polypeptides as described herein, optionally in combination with a third, fourth and / or fifth polypeptide as described herein in the context of the present invention, form a multi-chain antigen receptor as described herein in accordance with the present invention.

[0102] Thus, the first and second polypeptides, in combination with at least one further polypeptide according to the invention, such as a third and / or fourth polypeptide, or a fifth polypeptide, are also considered as an antigen receptor complex, or simply an antigen receptor. However, the first and second polypeptides can also form the antigen receptor of the invention by themselves. The combination of the first and second polypeptides with a third CD79A-like and / or a fourth CD79B-like polypeptide is further considered as a BCR-like protein or BCR-like complex.

[0103] Generally, herein and in the context of the present invention, a polypeptide may comprise an amino acid sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, preferably at least 80%, 85%, 90%, 95% or 100%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in a particular SEQ ID NO. Generally, the higher the % of sequence identity, the more preferred the amino acid sequence is. For example, a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:4 may be more preferred than a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO:4. Moreover, a sequence having 100% sequence identity to SEQ ID NO:4, i.e., the sequence set forth in SEQ ID NO:4 itself, may be most preferred in this example. The same logic applies herein and in the context of the present invention to essentially all amino acid sequences defined by sequence identity to an amino acid sequence defined in a particular SEQ ID NO. However, it should be noted that the present invention is in no way limited to high sequence identity, and any sequence identity, such as those described above, may be considered.

[0104] The term "sequence identity", as used herein and in the context of the present invention, has essentially the same meaning as commonly used and understood by those of skill in the art.

[0105] In particular, the term "sequence identity" is used herein to describe the sequence relationships between two or more amino acid sequences, proteins (or fragments thereof), or polypeptides (or fragments thereof). This term may be understood in the context of and in combination with terms including (a) reference sequence, (b) comparison window, (c) sequence identity, (d) percentage of sequence identity, and (e) substantial identity or "homology," as described below.

[0106] In particular, a "reference sequence," eg, a sequence as set forth in a particular SEQ ID NO:, is a defined sequence used as a basis for sequence comparison.

[0107] In particular, a "comparison window" includes reference to a contiguous designated segment of an amino acid / polypeptide / protein sequence, where the amino acid / polypeptide / protein sequence can be compared to a reference sequence. The portion of the amino acid / polypeptide / protein sequence within the comparison window may contain additions, substitutions, or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions, substitutions, or deletions) for optimal alignment of the two sequences. For example, the comparison window may be about 20, 50, 100, or 200 amino acid residues or longer in length. Those skilled in the art will understand that gap penalties can be introduced and subtracted from the number of matches to avoid misleadingly high similarity to the reference sequence due to the inclusion of gaps in the polynucleotide or polypeptide sequence.

[0108] Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison may be achieved using the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2: 482, 1981; the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 8: 2444, 1988; computerized implementations of these algorithms, including, but not limited to, CLUSTAL in the PC / Gene program of Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 7 Science Dr., Madison, Wisc., USA; the CLUSTAL program is a program developed by Higgins and Sharp (1988) Gene The CLUSTAL program is well described in: Corpet et al. (1988) Nucleic Acids Research 16: 881-90; Huang, et al. (1992) Computer Applications in the Biosciences, 8:1-6; and Pearson, et al. (1994) Methods in Molecular Biology, 24:7-331. The BLAST family of programs that can be used for similarity searching of databases include: BLASTN for nucleotide query sequences against nucleotide database sequences, BLASTX for nucleotide query sequences against protein database sequences, BLASTP for protein query sequences against protein database sequences, TBLASTN for protein query sequences against nucleotide database sequences, and TBLASTX for nucleotide query sequences against nucleotide database sequences.See Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York, 1995. Newer versions of the above programs, or entirely new programs, will no doubt become available in the future and can be used in conjunction with the present invention.

[0109] Unless otherwise stated, the sequence identity / similarity values ​​provided herein may refer to values ​​obtained using the BLAST 2.0 family of programs or its successors, using default parameters. Altschul et al. (1997) Nucleic Acids Res, 2:3389-3402. It should be understood that the default settings of these parameters can be easily changed in the future as necessary. Obviously, for comparison of amino acid / protein / polypeptide sequences, an algorithm / program directed to alignment of amino acid / protein / polypeptide sequences, such as BLASTP, should be used. As will be appreciated by those skilled in the art, BLAST searches assume that proteins or nucleic acids can be modeled as random sequences. However, many real proteins and nucleic acids contain regions of non-random sequence, such as homopolymer tracts, short period repeats, or regions enriched with one or more amino acids or nucleic acids. Such low-complexity regions may be aligned between unrelated proteins, even if other regions of the proteins or nucleic acids are not similar at all. To reduce such low-complexity alignments, many low-complexity filter programs can be used. For example, the low complexity filters SEG (Wooten et al. (1993) Comput. Chem. 17:149-163) and XNU (Claverie et al. (1993) Comput. Chem. 17:191-1) can be used alone or in combination.

[0110] "Sequence identity" in the context of two polypeptide / protein sequences specifically includes reference to residues in the two sequences that are identical when aligned for maximum correspondence over a specified comparison window, and may take into account additions, deletions, and substitutions. When percentages of sequence identity are used in the context of proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, where an amino acid residue is replaced with another amino acid residue that has similar chemical properties (e.g., charge or hydrophobicity) and thus does not adversely affect the functional properties of the molecule. When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions can be said to have sequence similarity. Methods for making this adjustment are well known to those of skill in the art. Typically, this involves scoring conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, conservative substitutions are given a score between 0 and 1. Conservative substitution scores are calculated, for example, according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17, 1988, for example as implemented in the program PC / GENE (Intelligenetics, Mountain View, Calif., USA).

[0111] "Percentage of sequence identity" refers in particular to a value determined by comparing two optimally aligned sequences over a comparison window, where the portion of the amino acid / peptide / protein sequence in the comparison window may contain additions, substitutions, or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the two sequences (which does not contain additions, substitutions, or deletions). The percentage may be calculated by determining the number of positions where identical amino acid residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0112] Furthermore, the third polypeptide and / or the fourth polypeptide of the present invention may comprise a membrane domain as described herein. In particular, the membrane domain is located in the membrane of the modified cell of the present invention. Preferably, each of the third and fourth polypeptides comprises a membrane domain.

[0113] The third, i.e., membrane domain of the CD79A-like polypeptide according to the invention, comprises the sequence motif "EX (10) -P" (i.e., EXXXXXXXXXXP), or a sequence having at least 80% sequence identity to SEQ ID NO: 1. Additionally, the fourth, i.e., membrane domain of the CD79B-like polypeptide may comprise the sequence motif "QXXXXXXXP" (i.e., EXXXXXXXXXXP), or a sequence having at least 80% sequence identity to SEQ ID NO: 1. (10) -P" (i.e., QXXXXXXXXXP), or a sequence having at least 80% sequence identity to SEQ ID NO:2.

[0114] Furthermore, the third, i.e., membrane domain of the CD79A-like polypeptide may comprise a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 4. Furthermore, the fourth, i.e., membrane domain of the CD79B-like polypeptide may comprise a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 6. Furthermore, the third, i.e., membrane domain of the CD79A-like polypeptide may comprise a sequence having at least 50% sequence identity to the membrane domain of the CD79A protein and / or the sequence set forth in SEQ ID NO: 8. Furthermore, the fourth, i.e., membrane domain of the CD79B-like polypeptide may comprise a sequence having at least 50% sequence identity to the membrane domain of the CD79B protein and / or the sequence set forth in SEQ ID NO: 10.

[0115] Obviously, herein and in the context of the present invention, a particular domain or combination of domains (e.g., membrane domain, extracellular domain or intracellular domain, or combination thereof) defined by multiple larger and / or smaller sequences and / or sequence motifs derived from the same protein or its corresponding domain(s) may contain these sequences and / or motifs in an overlapping (i.e. nested) manner. In particular, in such cases, the larger sequence usually contains the smaller sequence. Here, a particular domain or combination of domains (e.g., membrane domain, extracellular domain or intracellular domain, or combination thereof) may be described in a preferred or more preferred manner by sequences and / or motifs derived from the same protein or its corresponding domain(s) or corresponding sequences (defined by a certain % sequence identity). In particular, both higher sequence identity and greater overlap with a reference protein or domain(s), as well as the presence of important motifs, may indicate a higher level of preference. With regard to the level of preference, in particular the similarity (structural and functional) to a reference protein (e.g. CD79A) or its corresponding reference domain (e.g. the membrane domain of CD79A, or a combination of the extracellular and membrane domains of CD79A) may be taken into account.

[0116] In an exemplary embodiment, the membrane domain of the CD79A-like third polypeptide comprises (i) a sequence having at least 50% sequence identity with the sequence set forth in SEQ ID NO:8, which may be considered as a membrane domain derived from the CD79A protein, (ii) a sequence having at least 50% sequence identity with the sequence set forth in SEQ ID NO:4, which may be considered as a part of the membrane domain derived from CD79A, and (iii) a sequence motif "EX (10) In this example, the sequence defined in (ii) is specifically contained within the larger sequence defined in (i). Furthermore, the sequence motif "EX (10) "-P" is specifically present in the sequence defined by (i) and (ii), and "E" and "P" are present in the sequence defined by (i) and (ii), and the positions of "E" and "P" are defined by ten "X"s between them.

[0117] Similar logic can be applied to other domains, such as membrane domains, constant domains, intracellular domains, and combinations thereof, as described herein and in the context of the present invention.

[0118] Furthermore, the third polypeptide and / or the fourth polypeptide according to the present invention may comprise an extracellular domain. In particular, the extracellular domain is located outside the modified cell of the present invention. Preferably, each of the third and fourth polypeptides comprises an extracellular domain.

[0119] The third, i.e., extracellular domain of the CD79A-like polypeptide may comprise a sequence having at least 50% sequence identity to the extracellular domain of the CD79A protein and / or the sequence set forth in SEQ ID NO: 12 or 174, such as a sequence having at least about 70% sequence identity to SEQ ID NO: 12. Furthermore, the fourth, i.e., extracellular domain of the CD79B-like polypeptide may comprise a sequence having at least 50% sequence identity to the extracellular domain of the CD79B protein and / or the sequence set forth in SEQ ID NO: 14 or 177, such as a sequence having at least about 70%, preferably at least 80%, sequence identity to SEQ ID NO: 14.

[0120] Furthermore, the third, i.e., CD79A-like, polypeptide may comprise a sequence having at least 50% sequence identity to a CD79A protein, and / or a sequence set forth in SEQ ID NO: 16 or 175, such as a sequence having at least 80% sequence identity to SEQ ID NO: 16. Furthermore, the fourth, i.e., CD79B-like, polypeptide may comprise a sequence having at least 50% sequence identity to a CD79B protein, and / or a sequence set forth in SEQ ID NO: 18 or 178, such as a sequence having at least 80% sequence identity to SEQ ID NO: 18.

[0121] Preferably, the third, i.e., CD79A-like polypeptide and the fourth, i.e., CD79B-like polypeptide, are capable of interacting and / or binding to each other, i.e., in the modified cells of the invention.

[0122] In particular, in the context of the present specification and the present invention, it is not necessary for CD79A or the extracellular domain of CD79A, such as set forth in SEQ ID NO: 16 or 12, or as contained in SEQ ID NO: 110, or a corresponding sequence (e.g. having about 50% sequence identity thereto), to contain a leader sequence, such as the sequence set forth in SEQ ID NO: 173 (i.e. MPGGPGVLQALPATIFLLFLLSAVYLGPGCQA). Thus, the leader sequence, i.e. SEQ ID NO: 173 or a sequence corresponding thereto, may be omitted in SEQ ID NOs: 12, 16 and 110, or may be omitted in the corresponding sequences, such as set forth in SEQ ID NOs: 174, 175 and 184, respectively.

[0123] For example, it is further possible for a sequence having about 70%, about 75%, or about 80% sequence identity to SEQ ID NO:12 to be free of, or essentially free of, a leader sequence (e.g., a leader sequence as set forth in SEQ ID NO:173).

[0124] Thus, the third, i.e., the extracellular and membrane domain of the CD79A-like polypeptide may comprise a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO:202.

[0125] Similarly, in the context of the present specification and the present invention, it is not necessary for CD79B or the extracellular domain of CD79B, in particular as set out in SEQ ID NO: 18 or 14, or as contained in SEQ ID NO: 112, or a corresponding sequence (e.g. having about 50% sequence identity thereto), to comprise a leader sequence, such as the sequence set out in SEQ ID NO: 176 (i.e. MARLALSPVPSHWMVALLLLLSAEPVPA). Thus, the leader sequence, i.e. SEQ ID NO: 176 or a sequence corresponding thereto, may be omitted in SEQ ID NOs: 14, 18 and 112, or may be omitted in the corresponding sequences, such as set out in SEQ ID NOs: 177, 178 and 185, respectively.

[0126] It is further possible that a sequence having, for example, about 80% sequence identity to SEQ ID NO:14 does not include, or is essentially free of, a leader sequence (e.g., a leader sequence as set forth in SEQ ID NO:176).

[0127] Thus, the fourth, i.e., the extracellular and membrane domain of the CD79B-like polypeptide, may comprise a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO:203.

[0128] It has previously been determined that the amino acid sequence motif “YS” in the membrane domain of membrane-bound antibodies is sufficient for the interaction of membrane-bound antibodies with CD79 in the context of the B cell receptor; Gottwick (2019), PNAS,116 (27).

[0129] Thus, in the present specification and in the context of the present invention, the membrane domain of the first polypeptide may comprise the sequence motif "YS". Furthermore, the membrane domain of the first polypeptide may comprise the larger sequence motif "WXXXXXFXXLFXLXXXYSXXXT" (SEQ ID NO: 19), or a sequence having at least 80% sequence identity to SEQ ID NO: 19. Furthermore, the membrane domain of the first polypeptide may comprise a sequence having at least 50% sequence identity to the membrane domain of a membrane-bound immunoglobulin, and / or a sequence according to SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, 29, 31, 37 or 38, preferably SEQ ID NO: 20, 21, 22, 29 or 31.

[0130] Furthermore, the first polypeptide according to the present invention may further comprise a constant region as described herein.In particular, the constant region is located on the outside of the modified cell of the present invention.As described herein, the constant region of the first polypeptide may be derived from the constant region of an antibody, the Fc fragment of an antibody, or the constant region or Fc fragment of an antibody, as generally understood in the art, but the constant region of the first polypeptide is not limited thereto.

[0131] Furthermore, the membrane domain and / or the constant region of the first polypeptide according to the invention can interact and / or bind with the membrane domain and / or the extracellular domain of at least one polypeptide selected from the group consisting of CD79A protein, CD79B protein, the third i.e. CD79A-like polypeptide according to the invention and the fourth i.e. CD79B-like polypeptide according to the invention. Furthermore, the membrane domain and / or the extracellular domain of the third i.e. CD79A-like polypeptide of the invention and / or the membrane domain and / or the extracellular domain of the fourth i.e. CD79B-like polypeptide of the invention can interact and / or bind with the membrane domain and / or the constant region of at least one polypeptide selected from the group consisting of membrane-bound immunoglobulins and the first polypeptide of the invention. Preferably, the membrane domain of the first polypeptide can interact and / or bind with the membrane domain of at least one polypeptide selected from the group consisting of CD79A protein, CD79B protein, the third i.e. CD79A-like polypeptide and the fourth i.e. CD79B-like polypeptide. Also preferably, the membrane domain of the third, i.e., CD79A-like polypeptide and / or the membrane domain of the fourth, i.e., CD79B-like polypeptide is capable of interacting and / or binding to a membrane domain of at least one polypeptide selected from the group consisting of a membrane-bound immunoglobulin and the first polypeptide of the invention.

[0132] Furthermore, the fifth, i.e. CD16-like polypeptide may comprise a membrane domain. In particular, the membrane domain is located in the membrane of the modified cell of the invention. The membrane domain of the fifth polypeptide may comprise the sequence motif "FXXDT" or "FXXNT", i.e. "FXX(D / N)T". Furthermore, the membrane domain of the fifth polypeptide may comprise a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 34. Furthermore, the membrane domain of the fifth polypeptide may comprise a sequence having at least 50% sequence identity to the membrane domain of the CD16 protein and / or the sequence set forth in SEQ ID NO: 36.

[0133] Furthermore, the fifth or CD16-like polypeptide according to the present invention may comprise an extracellular domain. In particular, the extracellular domain is located on the outside of the modified cell of the present invention. Sequence motifs of CD16 that can interact with antibodies have been identified; see, for example, Sondermann (2000), Nature 406. In particular, it is known that the extracellular domain of an antibody, i.e., the constant region or Fc region, can interact with the extracellular domain of CD16.

[0134] Thus, the extracellular domain of the fifth, i.e., CD16-like polypeptide may comprise a sequence motif set forth in SEQ ID NO: 39, or a sequence having at least 80% sequence identity to SEQ ID NO: 39. Furthermore, the extracellular domain of the fifth polypeptide may comprise a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 41. Furthermore, the extracellular domain of the fifth polypeptide may comprise the extracellular domain of the CD16 protein, and / or a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 43 or 45, such as a sequence having at least 80%, preferably at least 90% sequence identity to SEQ ID NO: 43.

[0135] Furthermore, in the context of the present specification and the present invention, for example when CD16 and / or the fifth polypeptide of the invention are concerned, the constant region of the first polypeptide may comprise a sequence motif as set forth in SEQ ID NO: 50 or a sequence having at least 80% sequence identity to SEQ ID NO: 50. Furthermore, the constant region of the first polypeptide may comprise a sequence having at least 50% sequence identity to the sequence as set forth in SEQ ID NO: 52. Furthermore, the constant region of the first polypeptide of the present invention may comprise a constant domain of an immunoglobulin, such as C H 1. C H 2. C H 3 or C H4, and / or a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 62, 64, 66 or 68. Further, the constant region of the first polypeptide may comprise a sequence having at least 50% sequence identity to a constant region of an immunoglobulin and / or a sequence set forth in SEQ ID NO: 54.

[0136] Furthermore, in the context of the present invention, the constant region and / or membrane domain of the first, i.e. antibody heavy chain-like polypeptide can interact and / or bind to the extracellular domain and / or membrane domain of at least one polypeptide selected from the group consisting of: Fc receptors, CD16 protein and the fifth, i.e. CD16-like polypeptide of the present invention. Furthermore, the extracellular domain and / or membrane domain of the fifth polypeptide of the present invention can interact and / or bind to the extracellular domain and / or membrane domain of a membrane-bound immunoglobulin or at least one constant domain thereof, e.g. H 1. C H 2. C H 3 or C H 4, and the first polypeptide of the present invention.

[0137] Preferably, the constant region of the first polypeptide of the invention is capable of interacting with and / or binding to the extracellular domain of at least one polypeptide selected from the group consisting of an Fc receptor protein, a CD16 protein, and the fifth polypeptide of the invention. Also preferably, the extracellular domain of the fifth polypeptide of the invention is capable of interacting with and / or binding to the extracellular domain of a membrane-bound immunoglobulin or at least one constant domain thereof, such as a C H 1. C H 2. C H 3 or C H 4, and the first polypeptide according to the present invention.

[0138] Furthermore, in the context of the present specification and the present invention, for example when a fifth polypeptide and / or CD16 is involved, the third, i.e., CD3 zeta-like polypeptide may comprise a sequence having at least 50% sequence identity to the CD3 zeta protein and / or the sequence set forth in SEQ ID NO: 56. Furthermore, in the context of the present specification and the present invention, for example when a fifth polypeptide and / or CD16 is involved, the fourth, i.e., FceRIg-like polypeptide may comprise a sequence having at least 50% sequence identity to the FceRIg protein and / or the sequence set forth in SEQ ID NO: 58.

[0139] In the context of the present invention, the membrane domain and / or the extracellular domain of the fifth, i.e. CD16-like polypeptide can interact and / or bind with the membrane domain and / or the extracellular domain of at least one polypeptide selected from the group consisting of CD3 zeta protein, FceRIg protein, the third, i.e. CD3 zeta-like polypeptide, and the fourth, i.e. FceRIg-like polypeptide. Furthermore, the membrane domain and / or the extracellular domain of the third, i.e. CD3 zeta-like, polypeptide and / or the membrane domain and / or the extracellular domain of the fourth, i.e. FceRIg-like, polypeptide can interact and / or bind with the membrane domain and / or the extracellular domain of at least one polypeptide selected from the group consisting of CD16 protein and the fifth polypeptide according to the present invention.

[0140] Preferably, the membrane domain of the fifth polypeptide is capable of interacting with and / or binding to a membrane domain of at least one polypeptide selected from the group consisting of CD3 zeta protein, FceRIg protein, a third, i.e. CD3 zeta-like polypeptide, and a fourth, i.e. FceRIg-like polypeptide. Also preferably, the membrane domain of the third, i.e. CD3 zeta-like, polypeptide and / or the membrane domain of the fourth, i.e. FceRIg-like, polypeptide is capable of interacting with and / or binding to a membrane domain of at least one polypeptide selected from the group consisting of CD16 protein and the fifth polypeptide according to the invention.

[0141] In certain embodiments of the invention, the modified cell comprises a third, i.e., CD79A-like polypeptide and a fourth, i.e., CD79B-like polypeptide as described herein, and the first and second polypeptides of the invention. In particular, the first, second, third and fourth polypeptides are capable of forming a protein complex, i.e., a BCR-like complex and / or an antigen receptor complex as described herein, i.e., in the modified cell of the invention.

[0142] It is further possible, i.e. in the context of these embodiments, that the modified cells do not comprise a CD16 protein.It is further possible, i.e. in the context of these embodiments, that the modified cells do not comprise a polypeptide having at least 90% sequence identity to the sequence set forth in SEQ ID NO:47.

[0143] In certain embodiments of the invention, the modified cells comprise, as well as the first and second polypeptides of the invention, a third, i.e., CD3 zeta-like polypeptide and / or a fourth, i.e., FceRIg-like polypeptide as described herein.

[0144] Furthermore, in the context of the present invention, it is also possible that the modified cells do not comprise a CD79A protein, a CD79B protein, or a polypeptide having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 16 or 18. It is also possible that the modified cells do not comprise a polypeptide having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 175 or 178.

[0145] Here, in the context of the present invention, the second polypeptide may further comprise a constant region of the light chain of the antibody and / or a constant region comprising a sequence having at least 50% sequence identity with the sequence set forth in SEQ ID NO: 60. In particular, the constant region of the second polypeptide is located outside the modified cell of the present invention. Furthermore, the second polypeptide may be located entirely outside the cell.

[0146] Furthermore, herein and in the context of the present invention, a first polypeptide and a second polypeptide can form a Y-shaped protein comprising two first polypeptide chains linked to each other, e.g., by disulfide bonds, and two second polypeptide chains, each of the first polypeptide chains linked to the second polypeptide chain, e.g., by disulfide bonds.

[0147] Furthermore, the first polypeptide according to the present invention may comprise a dimerization domain. In particular, the first polypeptide according to the present invention may form a homodimer, i.e., via the dimerization domain. Preferably, the dimerization domain is comprised in the extracellular domain and / or membrane domain of the first polypeptide, preferably in the extracellular domain. In particular, the dimerization domain may be comprised in the constant region of the first polypeptide of the present invention. Furthermore, the dimerization domain may be or may be derived from the constant region or Fc portion of an antibody. Dimerization domains are well known in the art, and any of them may be employed in the context of the present invention.

[0148] Furthermore, in the context of the present specification and the present invention, the first polypeptide may further comprise a linker region between the variable region and the membrane domain. In particular, the linker region forms a flexible linker. In particular, the first polypeptide of the present invention may comprise a linker region between the constant region and the membrane domain. Furthermore, the linker region according to the present invention may comprise about 10 to about 100 amino acids, preferably about 50 amino acids. Preferably, the linker is a glycine-serine linker. For example, the linker region may comprise 2 to 20 repeats of the amino acid sequence GGGGS (SEQ ID NO: 69). Furthermore, the linker region may have, at its N-terminus, for example, the sequence SGGGGS (SEQ ID NO: 70) as set forth in SEQ ID NO: 72. Furthermore, the linker may have a length of about 5 to 50 nm, preferably about 20 nm.

[0149] Particularly herein and in the context of the present invention, the first, third and / or fourth polypeptide may comprise an intracellular domain comprising at least one signaling domain, and the signaling domain(s) may be the same or different between the first, third and / or fourth polypeptides. In particular, the at least one signaling domain is located on the internal side of the modified cell of the present invention.

[0150] In the context of the present invention, and as described herein, a signaling domain can include, e.g., in the first, third and / or fourth polypeptide, at least one signaling or activation motif or region, e.g., an ITAM, ITAM region or ITSM, of a protein selected from the group consisting of: CD3 zeta, FcεRIγ (FceRIg), CD16A, CD16B, NKp30, NKp46, KIR2DS1-2, KIR2DS3-6, KIR3DS1, NKG2C, NKG2D, 2B4 (CD24 4), CD2, CRACC, NTB-A (SLAMF6), DNAM-1 (CD226), CD7, CD59, BY55, KIR2DL4 (CD158d), CD44, TNFRSF9 (4-1BB), SLAMF1 (CD150), CD28, TMIGD2 (CD28H), SLAMF7 (CD319), TNFRSF18 (CD357), CD84, HCST (DAP10), TYROB (DAP12), FCRL3, TNFRSF13C (BAFF), and polypeptides having at least 50% sequence identity to any of the foregoing proteins.

[0151] For example, a signaling domain according to the invention can comprise, e.g., in the first, third and / or fourth polypeptide, at least one signaling or activation motif or region, e.g., an ITAM or ITAM region, of a protein selected from the group consisting of: CD3 zeta, FcεRIγ (FceRIg), CD16A, CD16B, NKp30, NKp46, KIR2DS1-2, KIR2DS3-6, KIR3DS1, NKG2C, NKG2D, 2B4, CD2, CRACC, NTB-A, DNAM-1, CD7, CD59, BY55, KIR2DL4, CD44, and a polypeptide having at least 50% sequence identity to any of the foregoing proteins.

[0152] Furthermore, the signaling domain according to the invention may comprise, for example, in the first, third and / or fourth polypeptide, an ITAM consensus motif, i.e., the sequence motif "Y-XX-I or LX (6~12)X may include any amino acid, such as "X-Y-XX-I or L", e.g., as set forth in SEQ ID NO: 73, 74, 48 or 49, or a sequence having at least 80% sequence identity to SEQ ID NO: 73, 74, 48 or 49, where X means "any amino acid", and X (6~12) means 6 to 12 amino acids.

[0153] Furthermore, the signaling domain according to the invention, e.g. in the first, third and / or fourth polypeptide, may comprise at least one immunoreceptor tyrosine-based activation motif (ITAM) of CD3 zeta protein, a sequence having at least 50% sequence identity with the sequence set forth in SEQ ID NO: 76, a sequence having at least 50% sequence identity with the sequence set forth in SEQ ID NO: 78, and / or a sequence having at least 50% sequence identity with the sequence set forth in SEQ ID NO: 80.

[0154] Furthermore, a signaling domain according to the present invention may comprise, e.g., in the first, third and / or fourth polypeptide, at least one ITAM region of a CD3 zeta protein and / or a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO:82.

[0155] Furthermore, in the context of the present specification and the present invention, the intracellular domain of the first, third and / or fourth polypeptide may comprise a sequence having at least 50% sequence identity to the intracellular domain of the CD3 zeta protein, and / or the sequence set forth in SEQ ID NO:84.

[0156] Furthermore, a signaling domain according to the present invention may comprise, for example, in the first, third and / or fourth polypeptide, at least one immunoreceptor tyrosine-based activation motif (ITAM) of an FceRIg protein, and / or a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO:86.

[0157] Furthermore, in the present specification and in the context of the present invention, the intracellular domain of the first, third and / or fourth polypeptide may comprise an intracellular domain of an FceRIg protein and / or a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 88, or 183, preferably SEQ ID NO: 88.

[0158] Furthermore, in the context of the present specification and the present invention, the intracellular domain of the first, third and / or fourth polypeptide, in particular the first polypeptide, may comprise a sequence having at least 50% sequence identity to the intracellular domain of a membrane-bound immunoglobulin, such as IgG1, and / or the sequence set forth in SEQ ID NO: 90.

[0159] Furthermore, a signaling domain according to the present invention may comprise, e.g., in the first, third and / or fourth polypeptide, at least one ITAM of a CD79A protein, and / or a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO:92.

[0160] Furthermore, in the context of the present specification and the present invention, the intracellular domain of the first, third and / or fourth polypeptide, in particular the third polypeptide, may comprise a sequence having at least 50% sequence identity to the intracellular domain of the CD79A protein, and / or the sequence set forth in SEQ ID NO:96.

[0161] Furthermore, a signaling domain according to the present invention may comprise, for example, in the first, third and / or fourth polypeptide, at least one ITAM of a CD79B protein, and / or a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO:94.

[0162] Furthermore, in the context of the present specification and the present invention, the first, third and / or fourth polypeptide, in particular the intracellular domain of the fourth polypeptide, may comprise a sequence having at least 50% sequence identity to the intracellular domain of CD79B protein, and / or the sequence set forth in SEQ ID NO:98.

[0163] Below, further signalling and intracellular domains according to the invention are described which may in particular be comprised in the first, third and / or fourth polypeptide of the invention:

[0164] Furthermore, herein and in the context of the present invention, the signaling domain may comprise an immunoreceptor tyrosine-based switch motif (ITSM) consensus motif, i.e., TXYXX(V / I), e.g., "TXYXXV" or "TXYXXI".

[0165] Furthermore, a signaling domain according to the invention may comprise an ITSM from SLAMF1, in particular the motif "TIYAQV" (SEQ ID NO: 187) or a sequence having at least 50% sequence identity thereto. Furthermore, a signaling domain according to the invention may comprise an extended motif from SLAMF1, in particular the motif set forth in SEQ ID NO: 188, or a sequence having at least 50% sequence identity thereto. Furthermore, an intracellular domain according to the invention may comprise an intracellular domain from SLAMF1, in particular a sequence having at least 50% sequence identity thereto.

[0166] Furthermore, a signaling domain according to the invention may comprise at least one ITSM from SLAMF6, in particular the motif "TVYASV" (SEQ ID NO:200) or a sequence having at least 50% sequence identity thereto, and / or the motif "ITIYSTI" (SEQ ID NO:201) or a sequence having at least 50% sequence identity thereto. Furthermore, an intracellular domain according to the invention may comprise an intracellular domain from SLAMF6, in particular a sequence having at least 50% sequence identity thereto, SEQ ID NO:235.

[0167] Furthermore, the signaling domain according to the invention may comprise at least one ITSM from CD244, in particular the motif "TLYSLI" (SEQ ID NO: 190) or a sequence having at least 50% sequence identity thereto, and / or the motif "TIYEVI" (SEQ ID NO: 191) or a sequence having at least 50% sequence identity thereto. Furthermore, the intracellular domain according to the invention may comprise an intracellular domain from CD244, in particular a sequence having at least 50% sequence identity thereto with SEQ ID NO: 213.

[0168] Furthermore, the signaling domain according to the invention may comprise at least one ITSM from CD244, in particular the motif "TLYSLI" (SEQ ID NO: 190) or a sequence having at least 50% sequence identity thereto, and / or the motif "TIYEVI" (SEQ ID NO: 191) or a sequence having at least 50% sequence identity thereto. Furthermore, the intracellular domain according to the invention may comprise an intracellular domain from CD244, in particular a sequence having at least 50% sequence identity thereto with SEQ ID NO: 213.

[0169] Furthermore, the intracellular domain according to the invention may comprise a sequence having at least 50% sequence identity with the intracellular domain from TNFRSF9, in particular SEQ ID NO:205.

[0170] Furthermore, the intracellular domain according to the invention may comprise a sequence having at least 50% sequence identity to the intracellular domain from KIR2DL4, in particular SEQ ID NO:209.

[0171] Furthermore, the signaling domain according to the invention may comprise a motif from CD226, in particular an immunoreceptor tyrosine tail (ITT)-like motif, in particular the motif "EDIYVN" (SEQ ID NO: 189) or a sequence having at least 50% sequence identity thereto. Furthermore, the intracellular domain according to the invention may comprise an intracellular domain from CD226, in particular a sequence having at least 50% sequence identity thereto.

[0172] Furthermore, the signaling domain according to the invention may comprise at least one motif from CD28, in particular the motif "YMNM" (SEQ ID NO: 192) or a sequence having at least 50% sequence identity thereto, and / or the motif "PYAP" (SEQ ID NO: 193) or a sequence having at least 50% sequence identity thereto. Furthermore, the intracellular domain according to the invention may comprise an intracellular domain from CD28, in particular a sequence having at least 50% sequence identity thereto with SEQ ID NO: 215.

[0173] Furthermore, the signaling domain according to the invention may comprise at least one motif from TMIGD2, in particular the motif "YXN", e.g. "YSN", and / or the proline-rich motif "PSPRPCPSPRPGHP" (SEQ ID NO: 194), or a sequence having at least 50% sequence identity thereto. Furthermore, the intracellular domain according to the invention may comprise an intracellular domain from TMIGD2, in particular a sequence having at least 50% sequence identity to SEQ ID NO: 217.

[0174] Furthermore, the intracellular domain according to the invention may comprise a sequence having at least 50% sequence identity with the intracellular domain from TNFRSF18, in particular SEQ ID NO:221.

[0175] Furthermore, the intracellular domain according to the invention may comprise an intracellular domain from CD44, in particular a sequence having at least 50% sequence identity with SEQ ID NO:223.

[0176] Furthermore, the signaling domain according to the invention may comprise a motif from CD7, in particular the motif "YEDM" (SEQ ID NO: 197) or a sequence having at least 50% sequence identity thereto. Furthermore, the intracellular domain according to the invention may comprise an intracellular domain from CD7, in particular a sequence having at least 50% sequence identity thereto.

[0177] Furthermore, the intracellular domain according to the invention may comprise a sequence having at least 50% sequence identity to the intracellular domain from CD84, in particular SEQ ID NO:227.

[0178] Furthermore, the signaling domain according to the invention may comprise at least one motif from HCST (DAP10), in particular the motif "YXXM", such as "YINM" (SEQ ID NO: 198). Furthermore, the intracellular domain according to the invention may comprise a sequence having at least 50% sequence identity with the intracellular domain from HCST, in particular SEQ ID NO: 229.

[0179] Furthermore, a signalling domain according to the invention may comprise a motif from TYROB, in particular an ITAM, in particular the motif "YQELQGQRSDVYSDL" (SEQ ID NO: 197) or a sequence having at least 50% sequence identity thereto. Furthermore, an intracellular domain according to the invention may comprise an intracellular domain from TYROB, in particular a sequence having at least 50% sequence identity thereto with SEQ ID NO: 231.

[0180] Furthermore, an intracellular domain according to the invention may comprise an intracellular domain from FCRL3, in particular a sequence having at least 50% sequence identity with SEQ ID NO:233.

[0181] Furthermore, the intracellular domain according to the present invention may comprise an intracellular domain from TNFRSF13C, in particular a sequence having at least 50% sequence identity to SEQ ID NO:237.

[0182] As described herein, the present invention also relates to chimeric polypeptides. In particular, the first, third and / or fourth polypeptides of the present invention may also be chimeric polypeptides. For example, a chimeric polypeptide (e.g., a third CD79A-like polypeptide) according to the present invention may have (i) an extracellular domain and / or a membrane domain from one protein (e.g., CD79A or CD79B) and (ii) an intracellular domain from another protein (e.g., CD3 zeta) and / or an intracellular domain that includes a signaling domain that includes one or more motifs from another protein (e.g., one or more ITAMs from CD3 zeta). As described herein and illustrated in the accompanying examples, chimeric polypeptides may be advantageous in the context of the present invention. For example, the chimeric polypeptides of the present invention may enhance the efficacy, particularly the killing activity, of modified cells of the present invention.

[0183] In some embodiments of the present invention, for example in the context of the chimeric polypeptide according to the present invention, the first polypeptide comprises a constant region as described herein and a membrane domain as defined herein, for example the first polypeptide may comprise a sequence as described in SEQ ID NO: 106, or at positions 1 to 371 of SEQ ID NO: 100. Furthermore, particularly in the context of these embodiments, the first polypeptide may comprise a linker region as described herein between the constant region and the membrane domain. Furthermore, particularly in the context of these embodiments, the first polypeptide may further comprise an intracellular domain as described herein, for example the first polypeptide may comprise a sequence as described in SEQ ID NO: 100 or 102. Furthermore, for example in the context of these embodiments, the first polypeptide may comprise an intracellular domain comprising an ITAM as described herein in the context of FceRIg and / or an intracellular domain as described herein in the context of FceRIg, for example as described in SEQ ID NO: 114. Further, for example, in the context of these embodiments, the first polypeptide can include at least one ITAM, or an ITAM region as described herein in the context of CD3 zeta, and / or an intracellular domain including an intracellular domain as described herein in the context of CD3 zeta.

[0184] In certain embodiments of the invention, for example in the context of a chimeric polypeptide according to the invention, the third polypeptide comprises an extracellular domain as described herein in the context of a third CD79A-like polypeptide, and a membrane domain as described herein in the context of a CD79A-like third polypeptide. Further, for example in the context of these embodiments, the third CD79-like polypeptide may comprise an intracellular domain comprising an ITAM as described herein in the context of CD79A and / or an intracellular domain as described herein in the context of CD79A, for example as set forth in SEQ ID NO: 16 or 175. Further, for example in the context of these embodiments, the third CD79-like polypeptide may comprise an intracellular domain comprising at least one ITAM, or an ITAM region as described herein in the context of CD3 zeta, and / or an intracellular domain as described herein in the context of CD3 zeta, for example as set forth in SEQ ID NO: 110 or 184. Further, for example, in the context of these embodiments, the third CD79-like polypeptide may comprise an intracellular domain that includes an ITAM as described herein in the context of FceRIg, and / or an intracellular domain as described herein in the context of FceRIg.

[0185] In certain embodiments of the invention, for example in the context of a chimeric polypeptide according to the invention, the fourth CD79B-like polypeptide comprises an extracellular domain as described in the context of a fourth CD79B-like polypeptide, and a membrane domain as described herein in the context of a fourth CD79B-like polypeptide. Furthermore, for example in the context of these embodiments, the fourth CD79B-like polypeptide may further comprise an intracellular domain comprising an ITAM and / or an intracellular domain as described herein in the context of CD79B, for example as set forth in SEQ ID NO: 18 or 178. Furthermore, for example in the context of these embodiments, the fourth CD79B-like polypeptide may comprise an intracellular domain comprising at least one ITAM or ITAM region as described herein in the context of CD3 zeta, and / or an intracellular domain as described herein in the context of CD3 zeta, for example as set forth in SEQ ID NO: 112 or 185. Further, for example, in the context of these embodiments, the fourth polypeptide can include an intracellular domain that includes an ITAM as described herein in the context of FceRIg, and / or an intracellular domain as described herein in the context of FceRIg.

[0186] Furthermore, a chimeric polypeptide of the invention (e.g., a third CD79A-like polypeptide of the invention and / or a fourth CD79B-like polypeptide of the invention) may comprise (i) an extracellular domain and a membrane domain from CD79A or CD79B, in particular a sequence having at least 50%, preferably at least 80%, sequence identity to SEQ ID NO: 202 or SEQ ID NO: 203, and (ii) a signaling domain (e.g., comprising one or more motifs such as ITAM) and / or an intracellular domain from another protein as described herein, e.g., an intracellular domain from CD3 zeta, FceRIg, TNFRSF9, SLAMF1, KIR2DL4, CD226, CD244, CD28, TMIGD2, SLAMF7, TNFRSF18, CD44, CD7, CD84, HCST, TYROB, FCRL3, SLAMF6, TNFRSF13C.

[0187] In particular, the intracellular domain from CD3 zeta may have at least 50% sequence identity to SEQ ID NO:84, the intracellular domain from FceRIg may have at least 50% sequence identity to SEQ ID NO:88, the intracellular domain from TNFRSF9 may have at least 50% sequence identity to SEQ ID NO:205, the intracellular domain from SLAMF1 may have at least 50% sequence identity to SEQ ID NO:207, the intracellular domain from KIR2DL4 may have at least 50% sequence identity to SEQ ID NO:209, the intracellular domain from CD226 may have at least 50% sequence identity to SEQ ID NO:211, the intracellular domain from CD244 may have at least 50% sequence identity to SEQ ID NO:213, the intracellular domain from CD28 may have at least 50% sequence identity to SEQ ID NO:215, and the intracellular domain from TMIGD2 may have at least 50% sequence identity to SEQ ID NO:217. The intracellular domain from SLAMF7 may have at least 50% sequence identity to SEQ ID NO: 219, and the intracellular domain from TNFRSF18 may have at least 50% sequence identity to SEQ ID NO: 221. The intracellular domain from CD44 may have at least 50% sequence identity to SEQ ID NO: 223, the intracellular domain from CD7 may have at least 50% sequence identity to SEQ ID NO: 225, and the intracellular domain from CD84 may have at least 50% sequence identity to SEQ ID NO: 227. The intracellular domain from HCST may have at least 50% sequence identity to SEQ ID NO: 229, the intracellular domain from TYROB may have at least 50% sequence identity to SEQ ID NO: 231, and the intracellular domain from FCRL3 may have at least 50% sequence identity to SEQ ID NO: 233. The intracellular domain from SLAMF6 may have at least 50% sequence identity to SEQ ID NO:235, and the intracellular domain from TNFRSF13C may have at least 50% sequence identity to SEQ ID NO:237.

[0188] Corresponding signaling domains, in particular signaling domains comprising one or more motifs, e.g. ITAM or ITSM, within said intracellular domain, are also described herein and can be used to define the chimeric polypeptides of the invention, including the corresponding embodiments of the first, third and / or fourth polypeptides of the invention.

[0189] The extracellular domain from CD79A or CD79B may or may not comprise an N-terminal leader sequence, in particular a sequence as set forth in SEQ ID NO: 173 and 176, respectively, or a sequence having at least 50% sequence identity thereto. Thus, the extracellular and membrane domain from CD79A may also refer to the constituent sequence defined by SEQ ID NO: 12, or the sequence immediately following at the C-terminus by SEQ ID NO: 8, or a sequence having at least 50% sequence identity thereto. Furthermore, the extracellular and membrane domain from CD79B may also refer to the constituent sequence defined by SEQ ID NO: 14, or the sequence immediately following at the C-terminus by SEQ ID NO: 10, or a sequence having at least 50% sequence identity thereto.

[0190] Moreover, two or more chimeric polypeptides of the invention can be combined, e.g., in a modified cell of the invention, such as a first, third and / or fourth polypeptide described herein in the context of a chimeric polypeptide.

[0191] Thus, two or more signaling domains and / or intracellular domains may be combined, for example, by using multiple chimeric polypeptides. A preferred combination herein and in the context of the present invention is (i) a signaling domain and / or intracellular domain from FceRIg as described herein, for example in the context of a first polypeptide of the invention, and (ii) a signaling domain and / or intracellular domain from CD3 zeta as described herein, for example in the context of a third or fourth polypeptide of the invention.

[0192] Furthermore, the chimeric CD79A-like polypeptides described herein and the chimeric CD79B-like polypeptides described herein can be used to combine two or more signaling domains and / or intracellular domains. Particular combinations are shown in SEQ ID NOs: 238 to 431. These sequences are designated by names having the following exemplary structure: "CD79A(EC-TM)-CD357(CYT)-CD79B(EC-TM)-CD44(CYT)". The term "CD79A(EC-TM)" refers to the extracellular (EC) domain and the membrane domain (TM; i.e., transmembrane domain) from CD79A, and the term "CD79B(EC-TM)" refers to the extracellular (EC) domain and the membrane domain (TM) from CD79B. The term "CYT" refers to the intracellular domain (i.e., the cytoplasmic domain). Thus, in this example, the term "CD357(CYT)" refers to the intracellular domain of CD357 (i.e., TNFRSF18), and the term "CD44(CYT)" refers to the intracellular domain of CD44. Thus, this exemplary sequence combines the intracellular domains from TNFRSF18 and CD44.

[0193] In the sequences shown in SEQ ID NOs: 238 to 431, the sequence of the first chimeric polypeptide (CD79A(EC-TM)-CD357(CYT) in the above example) and the sequence of the second chimeric polypeptide (CD79B(EC-TM)-CD44(CYT) in the above example) are separated by a 2A sequence (i.e., as shown in SEQ ID NO: 439 for the DNA and SEQ ID NO: 440 for the polypeptide, respectively). Since translation is skipped at the 2A sequence, two separate polypeptides (e.g., the third polypeptide of the invention and the fourth polypeptide of the invention) are produced. Thus, in the sequences shown in SEQ ID NOs: 238 to 431, the N-terminal polypeptide (or the corresponding DNA sequence) extends, in particular, from position 1 as shown in SEQ ID NOs: 439 and 440 to the last position before the 2A sequence. Furthermore, in these sequences, the C-terminal polypeptide (or the corresponding DNA sequence) extends, in particular, from the first position after the 2A sequence as shown in SEQ ID NOs: 439 and 440 to the last position of the entire sequence.

[0194] Further combinations of chimeric CD79-like polypeptides as set forth in SEQ ID NOs: 238-431 are provided below:

[0195] JPEG2025503499000002.jpg255141

[0196] JPEG2025503499000003.jpg255141

[0197] JPEG2025503499000004.jpg113145

[0198] Additionally, for example, in the context of a chimeric polypeptide, CD79A(EC-TM) and CD79B(EC-TM) may be exchanged for one another. Furthermore, the amino acid sequences of the individual domains, for example the extracellular and / or membrane domains, and various signaling and / or intracellular domains of CD79A or CD79B, may have at least 50% sequence identity to the corresponding amino acid sequences described herein in the context of the domains listed above and shown in SEQ ID NOs: 238-431.

[0199] Now, in the context of the present invention, in a cell, in particular in a modified cell of the present invention, the process that allows the cell to promote the death of a target cell may include the activation of at least one signaling pathway. In particular, when the antigen-binding site binds to the corresponding antigen, at least one of the signaling domains activates at least one signaling pathway in the cell, allowing the cell to promote the death of a target cell that contains said antigen on the cell surface. For example, the signaling pathway(s) in the context of the present invention may include or involve Ca2+ signaling, and / or at least one protein selected from the group consisting of at least one Src family kinase, at least one Syk family kinase, PLCG1, PI3K, Vav1, at least one Rho family GTPase, ERK1 / 2, and NFAT. In particular, the modified cell of the present invention is capable of killing a target cell that contains the corresponding antigen on the cell surface, when a death-promoting process according to the present invention is triggered and / or when said at least one signaling pathway according to the present invention is activated.

[0200] In addition, the signaling or intracellular domains of the following proteins have been reported to exert the following effects on cells, particularly NK cells (or T cells, where indicated):

[0201] TNFRSF9: costimulation / proliferation; SLAMF1: costimulation / co-stimulation; KIR2DL4: activation / inhibition; CD226: activation / cell adhesion; CD244: strong costimulation; CD28 (commonly used in the art): costimulation; TMIGD2: costimulation; SLAMF7: costimulation; TNFRSF18: activation, proliferation, cytokine production (in T cells); CD44: activation, recirculation, homing; CD7: costimulation; CD84: costimulation / cell adhesion; HCST: one of the most common NK cell activation signals / strong activation; TYROB: one of the most common NK activation signals / strong activation; FCRL3: costimulation / co-inhibition; SLAMF6: costimulation; TNFRSF13C: very potent activator in T cells.

[0202] Of note, certain inhibitory effects on cells (eg, reduced overstimulation) may also lead to a general activation of the cells that enhances their efficiency in promoting target cell killing.

[0203] Thus, the combination of multiple signaling domains in multiple polypeptides of the multi-chain antigen receptor of the invention (e.g., the first, third and / or fourth polypeptides of the invention) may further increase the efficiency of promoting target cell killing, as also shown in the accompanying examples, e.g., Example 5. In particular, the combination of multiple signaling domains may promote or enhance the overall activation of the modified cells with an increased efficiency of promoting target cell killing. Furthermore, the increased activation of the cells may be characterized, in particular, by increased proliferation, increased secretion of cytokines (e.g., immunostimulatory cytokines), and / or increased antigen-specific killing activity.

[0204] Furthermore, the presence of certain signaling domains (or activity motifs thereof) may confer additional effects on the cell upon antigen binding, such as altered or increased costimulation, altered cell adhesion, or altered recirculation and / or homing, as indicated above.

[0205] The modified cells of the present invention can secrete cytotoxic compounds and / or promote or kill the target cells by contacting the target cells with cytotoxic compounds upon binding of the antigen-binding site to the corresponding antigen, particularly to the antigen on the surface of the target cells. Furthermore, the modified cells can secrete granzymes and / or perforin upon binding of the antigen-binding site to the corresponding antigen, particularly to the antigen on the surface of the target cells. Furthermore, the modified cells of the present invention can secrete at least one cytokine upon binding of the antigen-binding site to the corresponding antigen.

[0206] Furthermore, herein and in the context of the present invention, the modified cells are capable of expressing and / or secreting IL-2 and / or IL-15.

[0207] Additionally, herein and in the context of the present invention, the modified cells may express a kill switch protein that causes the modified cells to die upon binding of a small molecule. Kill switch proteins and their corresponding small molecules are well known in the art and include, for example, the caspase 9 kill switch.

[0208] Additionally, the modified cells of the present invention can be used in disease treatments and / or methods of treatment, as described herein.

[0209] 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 the individual being treated. Desirable effects of treatment include, but are not limited to, prevention, prevention of the onset or recurrence of a disease or symptoms associated with a disease, alleviation of symptoms, reduction of the direct or indirect pathological consequences of a disease, slowing the rate of disease progression, amelioration or alleviation of the disease state, improved prognosis, and cure.

[0210] Diseases that may be treated in the context of the present invention include, inter alia, cancer, metabolic diseases, cardiovascular diseases, infectious diseases, respiratory diseases, hematological diseases, immune diseases, autoimmune diseases, neurological diseases, muscular diseases, or skeletal diseases.

[0211] Therefore, the present invention further relates to the modified cells of the present invention for use in treating disease in a mammalian subject.Preferably, the subject in the context of this specification and the present invention is a human.However, the subject may also be any other mammal, such as a horse, a dog, a cat, a cow, a pig, a sheep, a goat, a monkey, or a polar bear.

[0212] Furthermore, the present invention relates to a modified cell of the present invention for use in the treatment of a disease caused by and / or associated with a pathogenic target cell, said modified cell promoting the killing of said pathogenic target cell, in particular by binding of the antigen-binding site with a corresponding antigen, for example binding of the antigen on the surface of the target cell. In particular, the pathogenic cell expresses, i.e. on the cell surface, an antigen that is recognized by the antigen-binding site of the modified cell of the present invention. Here, in the context of the present invention, the pathogenic target cell may be a tumor cell or a pathogenic lymphocyte associated with and / or causing an autoimmune disease.

[0213] Therefore, the present invention also relates to the modified cells of the present invention for use in the treatment of cancer in mammalian subjects, preferably humans.Cancer is not particularly limited and can be any cancer, including liquid tumors and solid tumors.For example, cancer can be, in particular, breast cancer, liver cancer, such as hepatocellular carcinoma, skin cancer, such as melanoma, prostate cancer, blood cancer or leukemia, brain tumor, such as glioblastoma, lung cancer, etc.

[0214] Furthermore, the present invention relates to the modified cells of the present invention for use in the treatment of an autoimmune disease in a mammalian subject, preferably a human, such as, inter alia, rheumatoid arthritis, lupus, inflammatory bowel disease, multiple sclerosis, diabetes, Guillain-Barre syndrome, psoriasis, chronic inflammatory demyelinating polyneuropathy, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, or vasculitis.

[0215] Furthermore, the present invention relates to the modified cells of the invention for use in the immunotherapy of a mammal, preferably a human.

[0216] Here, in the context of the present invention, for example in the context of medical applications, the modified cells may be allogeneic or autologous cells. Preferably, the modified cells of the present invention are allogeneic cells.

[0217] Additionally, the kit of the present invention may include: (i) at least one nucleic acid molecule, each comprising a coding sequence for either the first, second, third or fourth polypeptide, or optionally a fifth polypeptide; (ii) at least one nucleic acid molecule, each of which comprises a coding sequence for two of a first, a second, a third and a fourth polypeptide, and optionally a fifth polypeptide, e.g., one nucleic acid molecule comprises a coding sequence for the first and second polypeptides, and another nucleic acid molecule comprises a coding sequence for the third and fourth polypeptides; and / or (iii) at least one nucleic acid molecule comprising a coding sequence for at least three, four or all of the first, second, third and fourth polypeptides, and optionally a fifth polypeptide.

[0218] In options (ii) and / or (iii) above, the at least two coding sequences may be separated by at least one 2A or IRES sequence and are not separated by a stop codon.

[0219] Furthermore, the kit of the invention may comprise at least one plasmid or viral vector comprising the nucleic acid molecule of option (i), (ii) or (iii) above, respectively.

[0220] Furthermore, a plasmid or viral vector comprising a nucleic acid molecule according to option (ii) or (iii) above may comprise a promoter capable of producing, in particular in a mammalian cell, an mRNA comprising at least two coding sequences, said mRNA being capable of being translated into at least two polypeptides, in particular in a mammalian cell such as the modified cell of the invention.

[0221] Furthermore, a plasmid or viral vector comprising a nucleic acid molecule according to option (ii) or (iii) above may comprise multiple promoters, each promoter being capable of producing, in particular in a mammalian cell such as the modified cell of the invention, an mRNA comprising one of the at least two coding sequences.

[0222] Here, and in the present invention, for example in the context of the kit of the present invention, the nucleic acid molecule may be a DNA molecule or an RNA molecule. Furthermore, the viral vector of the present invention may be a lentiviral vector, preferably a baboon pseudotyped lentivirus.

[0223] Furthermore, the present invention relates to a method of producing a modified cell of the present invention, the method comprising the step of introducing a nucleic acid molecule(s) described herein, e.g. in the context of a kit of the present invention, or a plasmid or viral vector described herein, into a mammalian cell.

[0224] With regard to mammalian cells, the same applies as described herein, for example in the context of the modified cells of the invention. Thus, the mammalian cells may in particular be NK cells or T cells.

[0225] Furthermore, the production method of the present invention may further comprise the step of activating the mammalian cells, for example by contacting the mammalian cells with a cytokine such as IL-2.

[0226] In certain embodiments of the invention, such as in the context of the modified cells of the invention, the first and second polypeptides of the invention are covalently linked, preferably by a peptide bond. Preferably, in the context of these embodiments, the modified cells of the invention further comprise a third CD79A-like polypeptide of the invention, a fourth CD79B-like polypeptide of the invention and / or a fifth CD16-like polypeptide of the invention.

[0227] Thus, the present invention further relates to a modified mammal comprising: (I) a first and a second polypeptide of the invention, and (II)(i) a third CD79A-like and / or a fourth CD79B-like polypeptide of the invention, and / or (ii) a fifth CD16-like polypeptide of the invention; Optionally, wherein the first and second polypeptides of the invention are covalently linked, for example by a peptide bond.

[0228] Moreover, the modified mammalian cells can have the properties of the modified cells of the invention generally described herein, for example with respect to promoting the killing of target cells.

[0229] Thus, in certain embodiments of the invention, the first and second coding sequences form a contiguous nucleic acid sequence that encodes a polypeptide comprising the amino acid sequence of the first and second polypeptides of the invention.

[0230] Thus, the present invention further relates to a kit comprising at least one nucleic acid molecule comprising: (I) a first coding sequence encoding a first polypeptide according to the invention, and a second coding sequence encoding a second polypeptide according to the invention, and (II)(i) a third and / or a fourth coding sequence encoding a third CD79A-like and / or a fourth CD79B-like polypeptide of the invention, and / or (ii) a fifth coding sequence encoding a fifth CD16-like polypeptide of the invention; Optionally, wherein the first and second coding sequences form a contiguous nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of the first and second polypeptides of the invention.

[0231] Furthermore, the present invention relates to chimeric polypeptides, such as those described herein above and below. Furthermore, the present invention relates to nucleic acid molecules, such as DNA or RNA, described herein that encode the chimeric polypeptides of the present invention, as well as to plasmids or viral vectors comprising said nucleic acid molecules described herein.

[0232] Thus, the present invention relates to a polypeptide comprising: (a) (I) an extracellular domain of a CD79A protein, and / or an extracellular domain comprising a sequence having at least 50% sequence identity to a sequence set forth in SEQ ID NO: 12 or 174, such as a sequence having at least about 70% sequence identity to SEQ ID NO: 12; and / or Membrane domains containing (i) a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO:4, and / or (ii) a sequence having at least 50% sequence identity to the membrane domain of CD79A protein and / or the sequence set forth in SEQ ID NO:8; and (II) an intracellular domain comprising: (i) at least one immunoreceptor tyrosine-based activation motif (ITAM) of CD3 zeta protein, a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 76, a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 78, and / or a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 80; (ii) at least one ITAM region of the CD3 zeta protein, and / or a sequence having at least 50% sequence identity with the sequence set forth in SEQ ID NO: 82, and / or (iii) a sequence having at least 50% sequence identity to the intracellular domain of the CD3 zeta protein and / or the sequence set forth in SEQ ID NO:84; and / or (b) a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 110 or 184.

[0233] Moreover, said polypeptide, in particular its membrane domain, is capable of interacting and / or binding to the membrane domain of a membrane-bound immunoglobulin in a mammalian cell. Furthermore, the present invention relates to a nucleic acid molecule comprising a coding sequence encoding said polypeptide. Said nucleic acid molecule may be DNA or RNA. Furthermore, the present invention relates to a viral vector comprising said nucleic acid molecule.

[0234] Furthermore, the present invention relates to a polypeptide comprising: (a) (I) an extracellular domain of a CD79B protein, and / or an extracellular domain comprising a sequence having at least 50% sequence identity to a sequence set forth in SEQ ID NO: 14 or 177, such as a sequence having at least about 70% sequence identity to SEQ ID NO: 14; and / or Membrane domains containing (i) a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO:6, and / or (ii) a sequence having at least 50% sequence identity to the membrane domain of CD79B protein and / or the sequence set forth in SEQ ID NO: 10; and (II) an intracellular domain comprising: (i) at least one immunoreceptor tyrosine-based activation motif (ITAM) of CD3 zeta protein, a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 76, a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 78, and / or a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 80; (ii) at least one ITAM region of the CD3 zeta protein, and / or a sequence having at least 50% sequence identity with the sequence set forth in SEQ ID NO: 82, and / or (iii) a sequence having at least 50% sequence identity to the intracellular domain of the CD3 zeta protein and / or the sequence set forth in SEQ ID NO:84; and / or (b) a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 112 or 185. Furthermore, said polypeptide, i.e. its membrane domain, is capable of interacting and / or binding to the membrane domain of a membrane-bound immunoglobulin in a mammalian cell. Furthermore, the present invention relates to a nucleic acid molecule comprising a coding sequence encoding said polypeptide. Said nucleic acid molecule may be DNA or RNA. Furthermore, the present invention relates to a viral vector comprising said nucleic acid molecule.

[0235] Furthermore, the present invention relates to a polypeptide comprising: (a) (I) a constant region comprising: (i) a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 52; (ii) an immunoglobulin constant domain, e.g., C H 1. C H 2. C H 3 or C H 4, and / or a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 62, 64, 66 or 68, and / or (iii) an immunoglobulin constant region and / or a sequence as set forth in SEQ ID NO: 54; and / or a membrane domain comprising a sequence having at least 50% sequence identity with a membrane domain of a membrane-bound immunoglobulin and / or a sequence as set forth in SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, 29 or 31, preferably SEQ ID NO: 20, 21, 22, 29 or 31; and (II) an intracellular domain comprising: (i) at least one immunoreceptor tyrosine-based activation motif (ITAM) of an FceRIg protein, and / or a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 86, and / or (ii) an intracellular domain of the FceRIg protein, and / or a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO:88; and / or (b) a sequence having at least 50% sequence identity to the sequence set forth in SEQ ID NO: 114.

[0236] Furthermore, said polypeptide, i.e. its membrane domain, can interact and / or bind to the membrane domain of CD79A and / or CD79B in mammalian cells. Furthermore, said polypeptide, i.e. its constant region, can interact and / or bind to the extracellular domain of an Fc receptor and / or to the CD16 protein in mammalian cells. Furthermore, the present invention relates to a nucleic acid molecule comprising a coding sequence encoding said polypeptide. Said nucleic acid molecule may be DNA or RNA. Furthermore, the present invention relates to a viral vector comprising said nucleic acid molecule.

[0237] Furthermore, the present invention relates to at least one of the polypeptides of the invention provided herein, such as at least one of the chimeric polypeptides of the invention, and / or to a mammalian cell comprising at least one nucleic acid molecule encoding at least one of said polypeptides, such as at least one of the chimeric polypeptides of the invention.

[0238] References The following detailed references relate to references outlined hereinabove and below as well as in the accompanying Examples.

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Dane Wittrup 2008 Antibody tumor penetration: Transport opposed by systemic and antigen-mediated clearance. Advanced Drug Delivery Reviews 60(12):1421-1434. Tiscornia, Gustavo, Oded Singer, and Inder M. Verma 2006 Production and purification of lentiviral vectors. Nature Protocols 1(1):241-245. Trempe, G. L. 1976 Human Breast Cancer in Culture. Pp. 33-41: Springer Berlin Heidelberg. Tsourkas, P. K., et al. 2007 Mechanisms of B-cell synapse formation predicted by Monte Carlo simulation. Biophys J 92(12):4196-208. Venkitaraman, Ashok R., et al. 1991 The B-cell antigen receptor of the five immunoglobulin classes. Nature 352(6338):777-781. Vivier, Eric, et al. 2008 Functions of natural killer cells. Nature Immunology 9(5):503-510. Wang, Enxiu, et al. 2015a Generation of Potent T-cell Immunotherapy for Cancer Using DAP12-Based, Multichain, Chimeric Immunoreceptors. Cancer Immunology Research 3(7):815-826. Wang, W., et al. 2015b NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Cancer Immunotherapy. Front Immunol 6:368. Weinkove, Robert, et al. 2019 Selecting costimulatory domains for chimeric antigen receptors: functional and clinical considerations. Clinical & Translational Immunology 8(5). Xie, Guozhu, et al. 2020 CAR-NK cells: A promising cellular immunotherapy for cancer. EBioMedicine 59:102975. Yanez-Muñoz, Rafael J., and Stephan A. Grupp 2018 CAR-T in the clinic: drive with care. Gene Therapy 25(3):157-161. Zajc, Charlotte U., et al. 2021 Driving CARs with alternative navigation tools - the potential of engineered binding scaffolds. The FEBS Journal 288(7):2103-2118.

[0240] The present invention is also characterized by the following figures, illustrations, and the following non-limiting examples. EXAMPLES

[0241] Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0242] Example 1: Materials and Methods

[0243] material

[0244] JPEG2025503499000005.jpg55170

[0245] JPEG2025503499000006.jpg175170

[0246] JPEG2025503499000007.jpg77170

[0247] JPEG2025503499000008.jpg110170

[0248] [Table 1-1]

[0249] [Table 1-2]

[0250] For further details on lentiviral vectors, see FIG.

[0251] [Table 2-1]

[0252] [Table 2-2]

[0253] [Table 2-3]

[0254]

Table 3

[0255] method Cell culture SK-BR-3 cells (American Type Culture Collection, Cat# HTB-30, LOT# 70022931), HEK293T cells (ATCC, Cat# CRL-11268), and HeLa cells (ATCC, Cat# CCL-2, Lot# 58930571) were cultured at 37°C and 5% CO2 in DMEM medium (Gibco, Cat# 41966-029) supplemented with 10% fetal bovine serum (Gibco, Cat# 10270-106), penicillin (100 U / mL), and streptomycin (100 μg / mL) (Gibco, Cat# 15140-148). MDA-MB-468 cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; Cat# ACC738, Lot#5) were cultured in Leibovitz medium (Gibco, Cat# 11415-064) supplemented with 10% FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL) at 37°C in atmospheric CO2. NK-92 cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; Cat# ACC488, Lot# 11) were cultured in 100 mL of 100% CO2-free medium supplemented with 2.2 g / L sodium bicarbonate (Sigma, Cat# S5761), 0.2 mM Myo-Inositol (Sigma, Cat# I-7508), 0.1 mM 2-mercaptoethanol (Gibco, Cat# 21985-023), 0.02 mM folic acid (Sigma, Cat# F-8758), 12.5% ​​horse serum (Gibco, Cat# 16050122), 12.5% ​​fetal bovine serum (100 U / mL), streptomycin (100 μg / mL), and 10 ng / mL recombinant human IL-2 (Gibco, Cat# The cells were cultured at 37°C, 5% CO2 in αMEM without ribonucleosides (Thermofisher, Cat# 12000-063), solubilized in HO (Gibco, Cat# 10977-035) supplemented with ribonucleoside (PHC0026), and cultured at 37°C, 5% CO2.All media were sterile filtered at 0.22 μm (TPP, Cat#99950, Lot#20210129). Adherent cell lines were cultured in T-75 flasks with filter caps (Greiner bio one; Cat#658175) and suspension cell lines were cultured in T-75 flasks with filter caps (Greiner bio one; Cat#658195). All cell lines were negative for mycoplasma contamination.

[0256] Lentiviral vector production HEK293T cells were cultured at 5.5 × 10 per T75 plate (Greiner Bio One, Cat# 658175). 6The cells were seeded and incubated at 37°C, 5% CO2 for 20 hours. DMEM supplemented with 10% FBS was used for culturing the lentivirus production cells without antibiotics. DNA-Opti-MEM mix was prepared by mixing the following components: 34.2 μg of transfer plasmids (pFS312, pFS322, pFS331, pFS335, pFS349, pFS350, pFS353, pFS354, pFS355, pFS357, pBA1037; see FIG. 12 and Table 4), 22.9 μg of pJD14 (see Table 4), 4.5 μg of pJD15 for VSV-G pseudotype or pFS295 for baboon pseudotype (see Table 4), and 2.3 μg of pJD16 (see Table 4) to a final volume of 500 μL in Opti-MEM. Meanwhile, 128 μl of Lipofectamine2000 (Invitrogen, Cat#11668019) was added to Opti-MEM so that the ratio of DNA (μg):Lipofectamine2000 (μl) remained 1:2 to prepare 500 μL of Lipofectamine2000-Opti-MEM mix. Then, Lipofectamine2000-Opti-MEM mix was gently added dropwise to DNA-Opti-MEM mix and incubated at room temperature for 15 min. The transfection mix was added dropwise to HEK293T packaging cells and incubated for 10 h. After that, the medium was gently aspirated and fed with 15 mL of pre-warmed fresh medium. The lentivirus present in the supernatant (medium) was collected at 48 h and stored at 4 °C, after which the cells were fed with 15 mL of pre-warmed fresh medium. The same was repeated at 72 h post-transfection. Lentivirus harvested at 48 and 72 hours were pooled together. The pooled lentivirus was centrifuged at 500xg for 5 minutes and filtered using a 0.45mm filter (Sartorius, Cat#16555-K). The viral supernatant was loaded onto an Amicon Ultra-15 centrifugal filter unit (Merck Millipore, Cat#UFC910096) and concentrated and buffer exchanged according to the manufacturer's instructions. The buffer was exchanged into sterile PBS-MK buffer.The titration of lentivirus was performed according to Tiscornia et al. (Tiscornia, et al. 2006): 5.0 × 10. 5 HEK293T cells were seeded in 24-well plates (Thermo Scientific, Cat#142475) in a final volume of 500 μl of DMEM medium per well. Plates were incubated at 37°C and 5% CO2. After 24 hours, virus stocks were diluted from stock to 10 in PBS-MK. -3 10-fold serial dilutions were made until dilution. 20 μl of each virus dilution was added to the cells, mixed thoroughly but gently, and the cells were incubated at 37° C. The cells were grown for 48 hours. The medium was removed and discarded. The cells were suspended in 150 μL Accutase (ThermoFisher, Cat#A11105-0) and incubated for 5 minutes at RT. Flow cytometry analysis was performed to determine the percentage of fluorescent reporter positive cells. Calculate the biological titer (BT = TU / ml, transfection units) according to the following formula: TU / μl = (P × N / 100 × V) × 1 / DF, where P = % Fluorophore + cells, N = number of cells at transfection = 1 × 10 5 , V = volume of dilution added to each well = 20 μl, DF = dilution factor = 1 (undiluted), 10 -1 (1 / 10 dilution), 10 -2 (1 / 100 dilution), use cells from a dilution that results in less than 40% transduction efficiency (if available).

[0257] [Table 4-1]

[0258] [Table 4-2]

[0259] [Table 4-3]

[0260] [Table 4-4]

[0261] [Table 4-5]

[0262] [Table 4-6]

[0263] [Table 4-7]

[0264] It should be noted that mTagBFP2 may be used as an equivalent in the present specification and context of the present invention, for example in the Examples, instead of SBFP2.

[0265] Lentiviral transduction of target cells Three million MDA-MB-468 or SK-BR-3 cells were seeded in T-75 flasks (Greiner bio one; Cat# 658175) and supplied with VSV-G pseudotyped lentivirus carrying EF1A-Luciferase-P2A-mCitrine genes (pBA1037) at MOI 3. Cells were incubated for 3 days in the corresponding culture conditions (see cell culture methods). For sorting, cells were detached with 2 mL of Trypsin-EDTA (Gibco; Cat#25200072) for 5 min. The reaction was stopped by adding 8 mL of the appropriate culture medium. Cells were centrifuged at 350xg for 5 min. The supernatant was discarded and cells were resuspended in 2 mL of sterile-filtered PBS+5%FBS. Cells were sorted using a FACSMelody (BD Biosciences) (Ex: 488 nm, Em: 527 / 32 nm).

[0266] Lentiviral transduction of NK cells NK-92 cells were activated by adding 10ng / mL fresh IL-2 (Gibco, Cat# PHC0026) 2 hours prior to transduction. After the incubation period, cells were counted using a Neubauer counter. 100000 cells were transferred to a sterile 1.5mL Eppendorf tube. Cells were centrifuged at 350xg for 5 minutes. The supernatant was discarded and cells were resuspended in baboon pseudotype lentivirus at MOI 100. The volume of the cell mix was adjusted to 1mL with the appropriate medium. This cell mix was spinfected at 1000xg for 30 minutes at RT. After spinfection, the transduced NK cells were transferred to a 12-well plate and supplemented with 1mL of NK cell medium (see Cell Culture). Cells were then cultured at 37°C and 5% CO2. After 2 days, cells were expanded for sorting by transferring them to T75 flasks (Greiner bio one; Cat# 658195) and feeding with 15 mL of NK cell medium. Cells were expanded for an additional 7 days before sorting. Cells were sorted using a BD Aria sorter (BD biosciences) for SBFP2 (Ex: 405 nm, Em: 450 / 50) and mCerulean (Ex: 405 nm, Em: 510 / 50) transduced cells, and a BD FACSMelody (BD biosciences) for mScarlet transduced cells (Ex: 561 nm, Em: 613 / 18 nm).

[0267] Staining and imaging of antibody surface expression on HeLa cells HeLa cells were transfected into 8-well microslides (ibidi, Cat# 80827) using Lipofectamine2000 transfection reagent (Invitrogen, Cat# 11668-027). 24 hours prior to transfection, 2.5 × 10 cells were transfected per well to achieve a culture density of 80-90% at the time of transfection. 4Cells were seeded at a density of 100 μL / well. Up to 175 ng of plasmid was mixed with Opti-MEM (ThermoFisher, Cat# 31985-062) in a final volume of 12.5 μL. An appropriate amount of lipofectamine2000 was mixed with Opti-MEM to achieve a DNA:Lipofectamine2000 ratio of 1:2 in a final volume of 12.5 μL and added dropwise to the samples after 20 min incubation. The medium was removed 48 h after transfection. Cells were washed three times with 300 μL PBS (Gibco, Cat# 10010-023) and fixed for 15 min using 200 μL Image-iT (Invitrogen, Cat# FB002). After removing the fixative, cells were washed three times with 300 μL PBS. Primary antibody targeting human IgG1 (SouthernBiotech; Cat#2040-08; Lot#C1316-PM87D) was diluted 1:500 in PBS+5%FBS (Gibco, Cat#10270-106). 250 μL of this dilution was added per well and incubated for 30 min at RT. After removing the antibody mixture, cells were washed 3 times with 300 μL PBS + 5%FBS. Streptavidin-FITC (SouthernBiotech; Cat#7100-02S; Lot#D1017-TL27D) was diluted 1:500 in PBS + 5%FBS. 250 μL of this dilution was added per well and incubated for 30 min in the dark. After removing the staining mixture, cells were washed 3 times with 300 μL PBS + 5%FBS. 200 μl of PBS+5% FBS was added per well and stored until imaging. Cells were imaged with a Nikon Eclipse Ti microscope (see Methods: Fluorescence Microscopy).

[0268] Recombinant DNA methods The various kits used were according to the manufacturer's instructions unless otherwise specified. Plasmids were constructed using standard cloning techniques. DNA amplification was performed using Phusion High Fidelity DNA Polymerase (NEB, Cat#M0530). Desalted primers / oligonucleotides (Table 2) were ordered from IDT / Sigma Aldrich. Gene fragments and gBlocks were ordered from IDT or Twist Biosciences (Table 3). Digested fragments were purified using the MinElute PCR purification kit (QIAGEN, Cat#28006) or Qiaquick PCR purification kit (QIAGEN, Cat#28106). Gel extraction and purification were performed using the MinElute Gel purification kit (QIAGEN, Cat#28606) or Qiaquick Gel Extraction kit (QIAGEN, Cat#28706). Restriction enzyme digestion was performed at 65°C for BstBI, 50°C for SfiI, 70°C for BtgZI, and 37°C for the other enzymes. Ligation reactions were performed using T4 DNA ligase (NEB, Cat#M0202). Chemically competent cells Top10 (ThermoFisher, Cat#C404010) were prepared using Mix and Go E.coli transformation kit (Zymo, Cat#T3001). In-house prepared Mach1 electrocompetent cells (ThermoFisher, Cat#C862003) and chemically competent Stbl3 cells (ThermoFisher, Cat#C737303) were also used for cloning. Screening of positive clones was performed using restriction digestion or colony PCR using Quick-Load Taq 2x Master Mix (NEB, Cat#M0271). Plasmid isolation from positive clones was performed using the GenElute Plasmid Mini-prep kit (Sigma Aldrich, Cat#PLN350-1KT).All plasmids were verified using Sanger sequencing services provided by Microsynth AG (Switzerland). Transformed bacteria were grown in Difco LB broth, Miller (BD, Cat#244610) supplemented with 100mg / mL ampicillin (Sigma Aldrich, Cat#A9518). Plasmids were isolated and purified using the PureYield Plasmid Midi-prep System (Promega, Cat# A2495). Endotoxin Removal kit (Norgen, Cat#52200) was used to remove endotoxins from purified plasmids. Assembly as per Gibson et al. (2009) was performed for 1 h at 50°C by mixing vector (50 ng) and insert (5 molar equivalents) in 1x Gibson assembly buffer (0.1 M Tris-HCl, pH 7.5, 0.01 M MgCl2, 0.2 mM dGTP, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dCTP, 0.01 M DTT, 5% (w / v) PEG-8000, 1 mM NAD), 0.04 units of T5 exonuclease (NEB, Cat# M0363), 0.25 units of Phusion DNA polymerase (NEB, Cat# M0530) and 40 units of Taq DNA ligase (NEB, Cat# M0208) in a final volume of 20 mL. The negative control for Gibson assembly was vector alone.

[0269] Staining of mIgs and CD16 in NK-92 cells NK cells were counted using a Neubauer counter. Three million cells per condition were transferred to a 15 mL Falcon tube (Greiner bio one; Cat# 188261) and centrifuged at 350xg for 5 min at 4°C. After centrifugation, the supernatant was discarded and the cells were washed three times with 3 mL of ice-cold PBS. The cells were then resuspended in 250 μL of a 1:500 dilution (cold PBS + 5% FBS) of biotinylated goat anti-human IgG antibody (Invitrogen, Cat# 13-4998-83, Lot# 2311211) to stain for IgG, and resuspended in a 1:500 dilution (cold PBS + 5% FBS) of biotinylated donkey anti-human Ig antibody (Invitrogen; Cat# 31782, Lot# WE3278964) to stain for IgM. The mixture was incubated on ice in the dark for 45 minutes. The mixture was then centrifuged at 350xg for 5 minutes. The supernatant was removed and the cells were washed with 3mL of ice-cold PBS + 5% FBS. The cell pellet was resuspended in 250μl of a 1:500 dilution of anti-human CD16 antibody (Invitrogen; Cat# 56-0168-41, Lot# 2072513) in cold PBS + 5% FBS and a 1:500 dilution of Streptavidin-BB515 (BD Bioscience; Cat# 564453; Lot# 1025848) in cold PBS + 5% FBS. The cells were incubated on ice in the dark for 45 minutes. The cells were then centrifuged at 350xg for 5 minutes at 4℃. After centrifugation, the supernatant was discarded and the cells were washed three times with 3mL of ice-cold PBS. After the final wash, the supernatant was discarded and the cells were resuspended in 200 μL of Image-iT fixative (Invitrogen, Cat# FB002) for 15 min. After removing the fixative, the cells were washed three times with 1 mL of RT PBS. Stained NK cells were used for flow cytometry or confocal microscopy.

[0270] Flow cytometry To analyze stained NK cells (see mIgs and CD16 staining of NK-92 cells) by flow cytometer, samples were taken, transferred to 1.5 mL Eppendorf LoBind Tubes (Eppendorf, Cat#022431021) and centrifuged at 350xg for 3 min at RT. The supernatant was discarded and cells were resuspended in PBS and stored on ice until measurement. For adherent cells, the medium was removed and cells were washed with 500 μL PBS and detached with Accutase (ThermoFisher, Cat#A11105-01) in a total volume of 150 μL. Cells were then resuspended and transferred to microdilution tubes (Cat#02-1412-0000, Life Systems Design). After this, cells were analyzed using a BD LSR Fortessa II Cell Analyzer (BD Biosciences). The instrument was calibrated with Sphero Rainbow Calibration Particles 8-peak beads (Spherotech, Cat#PCP-30-5A) before use. The excitation laser (Ex) and emission filter (Em) used for each fluorescent protein measurement were as follows: SBFP2 (Ex: 405 nm, Em: 450 / 50 nm), mCerulean / CFP (Ex: 445 nm, Em: 473 / 10 nm), FITC (Ex: 488 nm, Em: 530 / 30 nm, long pass filter 505 nm), mScarlet (Ex: 561 nm, Em: 610 / 20 nm, long pass filter 600 nm), and AlexaFlour 700 (Ex: 640, Em: 730 / 45). The mV values ​​of the photomultiplier tubes used were FSC-A: 450, SSC-A: 270, SBFP2: 550, mCerulean: 1000, mScarlet: 600, FITC: 650, and Alexafluor700: 500.

[0271] Fluorescence microscopy Unless specified as "confocal imaging," images were acquired using a Nikon Eclipse Ti microscope equipped with a mechanized stage and a temperature-controlled chamber maintained at 37 °C. Excitation light was generated by a Nikon IntensiLight C-HGFI mercury lamp or LED light source and filtered through a set of optimized Semrock filter cubes. Images were collected using a 10x objective with a Hammamatsu, ORCA R2, Flash4, or Prime BSI Express camera. To minimize crosstalk between the different fluorescent channels, the following optimal excitation (Ex), emission (Em), and dichroic (Dc) filter sets were used: mScarlet (Ex 562 / 40 nm or 575 nm LED, 10% intensity, Em 624 / 40 nm, Dc 593 nm), mCitrine (Ex 500 / 24 ​​nm or 475 nm LED, 10% intensity, Em 542 / 27 nm, Dc 520 nm), GFP (Ex 500 / 24 ​​nm or 475 nm LED, 10% intensity, Em 542 / 27 nm, Dc 520 nm), CFP / mCerulean (Ex 438 / 24 or 438 nm LED, 10% intensity, Em 483 / 32 nm, Dc 458 nm), SBFP2 (Ex 370 / 36 nm or 390 nm). LED, 10% intensity, Em 483 / 32 nm, Dc 458 nm). Image processing for the figures was performed using Fiji software (https: / / imagej.net / ).

[0272] Confocal microscopy 2 μL of pre-stained and fixed NK-92 cell line (see Methods: Staining of mIgs and CD16 on NK-92 cells) was transferred to a microscope slide and covered with a coverslip. Images were taken using a Leica SP8-Falcon point-scanning confocal microscope (Leica) equipped with a Leica DMI 8 base, a Leica TCS Tandem scanner, 2 PMT + 2 HyD detectors, and a HC PL APO CS2 63x / 1.40 oil immersion objective. The light sources used were a 442 nm diode laser, an argon laser (30% power), and a white light laser (85% power, 80 MHz). Images were acquired in sequential mode: Array 1: mCerulean (Ex: 442 nm, AOBS at 10%; Em: HyD SMD2 448-483 nm) and mScarlet (Ex: white light laser 561 nm, AOBS at 10%; Em: HyD SMD4 571-674 nm). Array 2: BD BB515 (Ex: argon laser 488 nm line, AOBS at 5%; Em: HyD SMD2 495-553 nm). Images were captured with a field of view of 2048 × 2048 pixels, scanned unidirectionally at a rate of 400 Hz, pixel size of 90 nm, and pixel dwell time of 0.79 μs. The confocal pinhole was set to 95.5 μm and the z-step was 0.3 μm. No averaging or summation of frames was performed.

[0273] Stained target cells on ibidi coverslips were used directly (see Methods: Staining of target cells). Images were taken with a Nikon A1 microscope equipped with a Nikon Eclipse Ti2-E base, a Nikon A1 H25 scan head with galvanometer scanner, and a 2x GaAsP + 3x PMT detector, using an S Plan Fluor ELWD 20x ph ADM objective (NA 0.45, Nikon). Images were acquired in sequential channel mode: sequence 1: mCitrine (Ex: 488 nm laser at 8%; Em: 525 / 50 nm, Gain: 32 mV), sequence 2: APC (Ex: 640 nm laser at 10%; Em: 700 / 75 nm, Gain: 70 mV). Images were taken with a field of view of 1024 × 1024 pixels, unidirectional scanning with zoom 1.821, pixel size 340 nm, and pixel dwell time 10.2 μs. The confocal pinhole was set to 46 μm for both channels and a line integration of 8 was applied. Image processing for figure production was performed using Fiji software (https: / / imagej.net / ).

[0274] Killing assay The killing capacity of the NK-92 cell array was assessed by a luminescence-based cytolytic assay (LCA) (Brown, et al. 2005) using luciferase-modified target cells and luciferase activity as an indicator of cell viability. The luminescence signal resulting from the treatment of luciferin with ATP by firefly luciferase-expressing target cells is used as the readout of the assay. Dead cells or cells with compromised membrane integrity are unable to maintain intracellular ATP levels. This reduction leads to a decrease in the observed luminescence signal. For this purpose, SK-BR-3 and MDA-MB-468 target cells were cultured at 20 × 10 per well in 96-well plates (Greiner bio one, Cat#655094). 5and seeded in 100 μl of the appropriate medium for the cell line 24 h prior to the experiment. NK-92 derived cell lines were activated by feeding them with fresh 10 ng / mL IL-2 24 h prior to the experiment. On the day of the experiment, viable cells of the target and NK-92 cell lines were counted using a Neubauer counter. NK-92 cell numbers for effector-to-target cell ratios of 10:1, 5:1, 2:5:1, and 1.25:1 were transferred to 15 mL Falcon tubes and centrifuged at 350xg for 5 min. The supernatant was removed and the NK cells were resuspended in the target cell culture medium. The target cell medium was then replaced with 100 μL of the appropriate effector cell mix. For 2% Triton X100 controls, target cells were fed with 100 μL of cell line appropriate medium containing 2% Triton X 100 (Carl Roth GmbH + Co. KG, Cat#9002-93-1). For no-kill controls, 100 μL of cell line appropriate medium was added. Killing assays were incubated for 4 h at 37°C and 5% CO2 in a darkened cell culture incubator or incubated for 4 h at 37°C and 5% CO2 in the incubation chamber of a darkened Nikon Ti2 microscope (see Methods: Fluorescence Microscopy). mScarlet: 500 ms exposure time, mCitrine: 500 ms exposure time, mCerulean: 500 ms exposure time, SBFP2: 500 ms exposure time. 15 min before the end of the incubation period, 5 μl of 15 mg / mL luciferin (Promega, E1605) in PBS was added to each well. After the incubation period, luminescence was measured using a Tecan Infinite pro M1000 plate reader.

[0275] The specific lysis value was calculated as follows:

[0276] JPEG2025503499000022.jpg28113

[0277] Correlation between target cell death and luminescence 35,000 SK-BR-3-LUC-CIT cells were seeded into each well of a 96-well plate (Greiner bio one, Cat#655094) in 100 μL DMEM medium (Gibco, Cat#41966-029) supplemented with 10% fetal bovine serum (Gibco, Cat#10270-106), penicillin (100 U / mL), and streptomycin (100 μg / mL) (Gibco, Cat#15140-148) and incubated at 37°C, 5% CO2 for 24 h. The next day, dilutions of TritonX-100 (SigmaAldrich; Cat#X100-100ML) were prepared in DMEM. The cell medium of the cells in the 96-well plate was replaced with 100 μL of TritonX-100 dilution (six replicates each) and incubated for 15 min at 37°C and 5% CO2. Then, 5 μL of 15 mg / mL luciferin (Promega, E1605) in PBS was added to each well and the plate was incubated for another 15 min at 37°C and 5% CO2. The luminescence of the samples was then measured on a Tecan Infinite pro M1000 plate reader.

[0278] Correlation between target cell number and luminescence SK-BR-3-LUC-CIT cells were seeded at the indicated cell numbers in wells of a 96-well plate (Greiner bio one, Cat#655094) in DMEM medium (Gibco, Cat#41966-029) supplemented with 10% fetal bovine serum (Gibco, Cat#10270-106), penicillin (100 U / mL), streptomycin (100 μg / mL) (Gibco, Cat#15140-148). After 4 h of adhesion time at 37 °C and 5% CO2, cells were fed with 5 μL of 15 mg / mL luciferin (Promega, E1605) in PBS. The luminescence of the samples was then measured on a Tecan Infinite pro M1000 plate reader. Afterwards, the cell medium was removed, and the cells were washed with 200 μL PBS (Gibco, Cat# 10010-023) and detached with 250 μL Accutase (ThermoFisher, Cat#A11105-01) for 10 min at RT. The total volume of each well containing all the cells was transferred to a BD Trucount tube (BD, Cat#340334) and run on a BD LSR Fortessa II Cell Analyzer (BD Biosciences) and counted according to the manufacturer's protocol. SK-BR-3 cells were identified using mCitrine (Ex: 488 nm, Em: 530 / 30 nm, long pass filter 505 nm).

[0279] Staining of Her2 on target cells SK-BR-3-LUC-CIT and MDA-MB-468-LUC-CIT cells were seeded in 8-well microslides (ibidi, Cat# 80827) 24 hours prior to staining and incubated at 37°C, 5% CO2. Cells were washed three times with 300 μL PBS (Gibco, Cat# 10010-023) and fixed for 15 minutes with 200 μL Image-iT (Invitrogen, Cat# FB002). After removing the fixative, cells were washed three times with 300 μL PBS. Antibody targeting human Her2 (Invitrogen; Cat#2040-08; Lot#C1316-PM87D) was diluted 1:500 in PBS + 5% FBS (Gibco, Cat#10270-106). 250 μl of this dilution was added per well and incubated for 30 min at RT. After removing the antibody mixture, cells were washed 3 times with 300 μL PBS. 200 μL PBS + 5% FBS was added per well and stored until imaging. Cells were imaged with a Nikon A1 point-scan microscope (see Confocal Microscopy).

[0280] statistical analysis Statistical analysis was performed in Microsoft Excel. Student's t test was used to compare quantitative differences (mean ± SD) between samples; P values ​​were two-sided, and P < 0.05 was considered significant.

[0281] Example 2: Surface expression of membrane-bound IgG1 immunoglobulin from polycistronic constructs. Conventional antibody-dependent cellular cytotoxicity (ADCC) involves the signaling receptor CD16 expressed on the effector cell surface, which recognizes the Fc domain of soluble antibodies bound to antigens on target cells. Thus, ADCC traditionally relies on the presence of soluble antibodies (Gauthier, et al. 2021; Gomez Roman, et al. 2014), (Figure 1a). However, according to the present invention, ADCC can be triggered by cis interactions, particularly between membrane-bound / tethered antibodies and the CD16 receptor on the cell surface of effector cells, such as NK cells (Figure 1b). We used (i) the cell line NK-92, which does not naturally express CD16 but can be supplemented with exogenous CD16 by lentiviral transduction, (ii) HER2 / neu (Her2; ErbB2) as a model antigen on the surface of target cells, (iii) Her2-expressing and non-expressing breast cancer cell lines to represent potential target cells, and (iv) the Her2 monoclonal antibody Trastuzumab as the basis for antigen-specific recognition.

[0282] Surprisingly, we were able to bind the antibody to the cell surface of NK-92 cells. Initially, we evaluated many tethering approaches using HeLa cells, which are easy to genetically manipulate. Trastuzumab is a soluble antibody, and like other antibodies, it is derived from membrane-bound immunoglobulins (mIg) via an alternative splicing event that removes the transmembrane domain. We therefore cloned the constant domain, transmembrane domain, and cytoplasmic domain of the human genomic IGHG1 locus into the antigen-binding heavy chain variable fragment (VIL) of trastuzumab. H) to generate an mIg version of the trastuzumab heavy chain (Dodev et al. 2014). However, transfection of HeLa cells with constructs expressing this modified heavy chain and the trastuzumab kappa light chain (Dodev, et al. 2014), each under the control of the constitutive EF1A promoter, failed to result in surface expression of trastuzumab, as evidenced by the absence of surface staining for the IgG1 region (Figure 6a). It has previously been shown that several classes of Ig require the presence of the BCR cofactors CD79A and CD79B for efficient surface expression (Dylke, et al. 2007; Venkitaraman, et al. 1991). These studies show that surface localization of mIgG1 is independent of CD79, but also show that coexpression of CD79 and mIgG1 results in the formation of mIgG1-CD79 BCR complexes (Venkitaraman, et al. 1991). Therefore, based on the prior art knowledge, it was unclear whether this dimerization would enhance the surface expression of mIgG1.Surprisingly, the present inventors found that co-transfection of genes encoding mIgG1 heavy and light chains with genes encoding CD79A and CD79B into HeLa cells significantly increased the surface localization of mIgG1 (Figure 6b).

[0283] Although the above experiments were performed with plasmids and transient transfection, we considered that stable modification of NK cells might be necessary for clinical application. These modifications are usually performed with the help of retroviral vectors (Colamartino, et al.), and the number of vectors needs to be reduced to simplify the manufacturing process. We solved this problem by adapting the construct to lentiviral vector encoding, while minimizing the number of vectors. To this end, we restricted the transcription of anti-Her2 mIgG1 heavy chain and κ light chain on the one hand, and CD79A and CD79B on the other hand, each in a single open reading frame, with the protein coding sequences separated by a 2A ribosomal skipping site (Liu, et al. 2017; Ryan, et al.). To simplify the selection of transduced NK cells, we added the coding sequences for the fluorescent reporter proteins mScarlet and mCerulean to the 5' ends of the anti-Her2 kappa light chain / mIgG1 heavy chain construct and the CD79A / CD79B construct, respectively. To overexpress CD16 in NK-92 cells, a third construct was constructed encoding CD16 in combination with the fluorescent reporter SBFP2 (Fig. 2a). Unexpectedly, transient transfection of HeLa cells with constructs encoding mIgG1 and CD79A / CD79B resulted in surface expression of mIgG1 (Fig. 6c), suggesting that the polycistronic cassette incorporated into the lentiviral backbone was functional.

[0284] Overall, these surprising results demonstrate that a soluble monoclonal antibody can be engineered into a membrane-bound immunoglobulin and suggest that, at least in HeLa cells, the extent of its surface expression can be increased by co-expression of CD79A and CD79B (see Figure 12 for details of constructs and lentiviral vectors).

[0285] Example 3: Stable NK-92 cell line transduction After the surprising discovery that antibodies could be immobilized on the cell membrane of HeLa cells, we considered an NK cell model, namely the NK-92 cell line, as a benchmark cell line for ADCC studies (Clemenceau, et al. 2013). All constructs were packaged in baboon pseudotyped lentiviral vectors that transduce NK-92 cells with high efficiency (Colamartino, et al.). Since NK-92 cells do not endogenously express the Fcγ receptor CD16 required for ADCC (Gong, et al. 1994), it was initially unclear whether exogenous CD16 overexpression was required in addition to the antibody and CD79A / CD79B elements (Figure 2b) to recapitulate cis ADCC (Colamartino, et al. 2019). To investigate this question, we transduced all possible subsets of the three constructs, generating seven variants of NK-92 cells. NK-92 cells transduced with a vector expressing constitutive mScarlet were used as a negative control. An additional negative control was generated by exchanging the variable fragment of the antibody of NK-92-mIgG1 / Her2-CD16-CD79 with an anti-Pollen antigen variable fragment (Fv) (Dodev, et al. 2014). To generate stable cell lines, we first transduced NK-92 cells with lentiviruses encoding CD79 and CD16 (as appropriate) and selected for high expression of mCerulean and / or SBFP2, respectively (Figure 7a). In a second step, wild-type NK-92 cells and three sorted cell lines were transduced with the antibody-encoding constructs and either selected for high mScarlet expression (Figure 7b) or used as is (see Table 1 for details of cell lines).

[0286] To evaluate the functionality of the lentiviral vectors, we measured the surface expression of mIgG1 and CD16 on the transduced NK-92 cell line (Fig. 7c-l). Based on the mIgG1 expression test in HeLa cells (Fig. B), surface expression of mIgG1 was expected only upon co-expression with the CD79 cofactor. However, very surprisingly, we found a population of cells positive for mIgG1 surface expression among NK-92 cells transduced with only the mIgG1-encoding vector (Fig. 7g), even though NK-92 cells are not known to endogenously express either CD79A or CD79B (Chu and Arber 2001). Nevertheless, we observed that the surface expression of mIgG1 was increased by the co-expression of CD79 (Fig. 7h). As an aside, when CD16 and mIgG1 were co-expressed, the surface localization of mIgG1 was reduced, and interference between the immunoglobulin and CD16 was observed (Fig. 7i). However, all NK-92 cells transduced with vectors encoding mIgG1 and / or CD16 showed detectable surface expression of these proteins and cis-ADCC was consistently observed, indicating that such potential interference was not a problem.

[0287] Example 3: Efficient cis-ADCC by mIgG1 and CD16-transduced NK-92 cells We assessed the cytotoxicity of modified NK-92 cell lines by a luminescence-based cytolytic assay (LCA) using luciferase-modified target cells and luciferase activity as an indicator of cell viability (Brown, et al. 2005). To this end, NK-92-resistant breast cancer cell lines SK-BR-3 (Her2 positive) (Trempe 1976) and MDA-MB-468 (Her2 negative) (Chavez, et al. 2011) were stably transduced with a VSV-G lentiviral vector constitutively driving firefly luciferase and mCitrine coding sequences linked by a 2A linker, sorted for Citrine expression, and stained to confirm Her2 surface expression (Fig. 8a, b). A linear correlation between cell number and luciferase signal was confirmed as well (Fig. 8c, d). The cytotoxic activity of modified NK-92 cells was evaluated after 4 h of co-culture of target cells with NK-92 cells at effector / target cell ratios (E:T ratios) of 10:1, 5:1, 2.5:1, and 1.25:1 (Fig. 2c-l). At an E:T ratio of 10:1, NK-92-mIgG1 / Her2-CD16 and NK-92-mIgG1 / Her2-CD16-CD79 cells reached lysis levels of 76% and 67%, respectively (Fig. 2j, k). Strikingly, these results were comparable to the 71% lysis measured in the control conditions of canonical ADCC, where NK-92-CD16 cells were supplied with soluble trastuzumab at 10 μg / mL at an E:T ratio of 10:1 (Fig. 2g). For all cytotoxic cell lines, lysis increased in an effector cell number-dependent manner. Neither the mIgG1 negative control cell lines (NK-92-mScarlet, NK-92-CD79, NK-92-CD16, NK-92-CD16-CD79) (Fig. 2c-f), nor the control cell line NK-92-mIgG1 / Pollen-CD16-CD79 (Fig. 2l) caused substantial lysis of SK-BR-3 cells at any effector / target cell ratio tested. Cell lines that were cytotoxic against Her2 positive cells were inactive against the Her2 negative cell line MDA-MB-468, with less than 10% lysis even at the highest E:T ratio (Fig. 2g,j,k), confirming the antigen specificity of the effector cells.Surprisingly and unexpectedly, NK-92-mIgG1 / Her2 and NK-92-mIgG1 / Her2-CD79 cells, which express mIgG1 / Her2 but lack CD16, eliminated up to 27% of Her2-positive target cells (Fig. 2h, i), indicating that mIgG1 can function by itself.

[0288] These surprising results indicate that mIgG1 / Her2 is expressed on the surface of NK-92 cells and can cooperate with CD16 to induce strong antigen-specific cis ADCC against target cells. It is thought that the strongly reactive cell lines rely for their cytotoxicity on the CD3ζ and FcεRIγ signaling domains associated with overexpressed CD16. However, our surprising findings also suggest that in CD16-negative cells, these domains may not be engaged and therefore these cells may rely on signaling domains that are typically active in B cells, such as the mIgG1 cytoplasmic tail and CD79 ITAMs, present in the transduced construct and able to signal with the help of the associated Syk kinase (Dal Porto, et al. 2004), resulting in a less pronounced but still significant and measurable cytotoxicity.

[0289] Example 4: Increasing immunoglobulin membrane distance using tethering GS linkers Traditionally, under physiological conditions, ADCC is triggered at the onset of contact of NK cells with antibody-coated target cells through the interaction of membrane-bound antibodies with CD16 (Figure 1a). In the context of the present invention, we observed that the surface localization of mIgG1 in NK-92 cells is reduced when co-transfected with CD16. Therefore, we wondered whether this might be caused by non-specific interactions between mIgG1 and CD16 that could lead to mIgG1 internalization (Al Qaraghuli, et al. 2020; Caballero, et al. 2006; Sun, et al. 2021). However, it was entirely unclear whether we could increase mIg surface localization and promote efficient effector-target cell interaction and, consequently, target cell lysis by increasing the distance between the mIgG1 constant domain and CD16. To address this question, we developed a flexible (GGGGS) IgG1 (IgG1 ... 11The linker was chosen because it is comparable to twice the size of the extracellular vertical protrusions of the Fcγ-IgG complex (Chen, et al. 2013; Patel, et al.). In the context of the present invention, this construct is also called "tethered IgG1" (tIgG1, Fig. 3a, b). In light of the surprising finding that all four mIgG1 / Her2-expressing NK-92 cell lines showed cytotoxic activity (Fig. 2h-k), we constructed similar cell lines in which mIgG1 / Her2 was replaced by tIgG1 / Her2 using a similar cell transduction and sorting strategy (Fig. 9a). Surprisingly, all four tIgG1-transduced NK-92 cell lines (NK-92-tIgG1 / Her2, NK-92-tIgG1 / Her2-CD16, NK-92-tIgG1 / Her2-CD79, and NK-92-tIgG1 / Her2-CD16-CD79) showed strong surface expression of tIgG1 (Fig. 9b-f). Moreover, surprisingly, CD16- and tIgG1-positive NK-92 cell lines showed high cytotoxicity (Fig. 3c-f, and Fig. 9g). NK-92-tIgG1 / Her2-CD16 cells showed 84% lysis at an E:T ratio of 10:1 (Fig. 3c, d), while NK-92-tIgG1 / Her2-CD16-CD79 cells eliminated 91% of SK-BR-3 cells at the same E:T ratio (Fig. 3e, f). Both of these cell lines showed background activity only against MDA-MB-468 (Fig. 3d, f). We analyzed surface expression in NK-92-tIgG1 / Her2-CD16 and NK-92-mIgG1 / Her2-CD16 in similar mScarlet expression modes of 6747 relative fluorescence units (rfu) and 6516 r.fu (data not shown), respectively, with tIgG1 surface expression mode at 8045 r.fu and mIgG1 surface expression mode at 2514 r.fu (compare Fig. 7i and Fig. 9d). This difference in surface expression translated into a 10% increase in cytotoxicity (p-value=0.00015) (Fig. 3h, i). In the case of NK-92-mIgG1 / Her2-CD16-CD79 and NK-92-tIgG1 / Her2-CD16-CD79, similar comparisons may not be entirely without issue, since mScarlet levels are low in NK-92-mIgG1 / Her2.Nevertheless, 89% cell lysis was observed in tIgG1-encoded cells compared to 67% in mIgG1-encoded cells. Remarkably and unexpectedly, in NK-92-tIgG1 / Her2-CD16-CD79, despite the tIgG1 surface expression mode being only 4045r.fu, we measured 45% target cell lysis at an E:T of 1.25:1, which exceeded the canonical ADCC with NK92-CD16 and 10μg / mL trastuzumab by a factor of 2 (Figure 3i). In NK-92 cells lacking CD16 (NK-92-tIgG1 / Her2 and NK-92-tIgG1 / Her2-CD79), the addition of the linker did not result in an increased cytotoxic response compared to the non-linker IgG1 version (compare Figures 2h-I at E:T 5:1 with bars 2 and 4 in Figure 9g).

[0290] These surprising results show that a flexible GS linker can be added between the transmembrane and constant domains of IgG1 antibodies. Unexpectedly, this linker-conjugated IgG1 resulted in stronger immunoglobulin surface expression and elicited a significantly stronger cytotoxic response, at least for NK-92-tIgG1-CD16, than its mIgG1 / Her2 counterpart at comparable antibody expression levels. Moreover, and unexpectedly, the Her2-specific cytotoxicity obtained with cis-ADCC on NK-92-tIgG1-CD16-CD79 dramatically exceeded that of canonical ADCC (Fig. 3h, i).

[0291] Example 5: Recapitulation of ADCC from a modular receptor architecture We observed a moderate cytotoxic effect by NK-92-mIgG1 / Her2-CD79 cells (Fig. 2i). The combination of mIgG1 and CD79A / CD79B reconstitutes the B-cell receptor (BCR) complex. The surprising findings presented in Examples 1-4 led us to wonder whether BCR-like complexes, when expressed in NK cells, could induce cytotoxicity without the need for CD16 (Gong et al. 1994). We then addressed the question of whether it would be possible to convert these complexes into multichain antigen receptor structures that would elicit strong cytotoxicity that is CD16-independent and comparable to CD16-expressing NK cells. We envision that this antigen-specific synthetic immunoglobulin-based multichain (ASIMut) receptor platform would fulfill two requirements: i) modular control of signaling domains to fine-tune downstream responses, and ii) high modularity of immunoglobulin constant modules to allow control of the potential for interaction with Fc receptors such as CD16. As a proof of concept, we decided to endow this receptor scaffold with the singling domains normally associated with CD16 in order to induce ADCC-like cytotoxicity in NK-92 cells without overexpressing CD16 itself.

[0292] First, we investigated whether it was possible to replace the cytoplasmic domain of CD79 with ITAMs from CD3ζ to induce a potent cytotoxic response without the need for CD16. 179-226 and CD79B 185-229 The cytoplasmic domain of CD3ζ, which contains three ITAM motifs, 61-164 Domain replacement and fusion construct CD79A 1-179 ::CD3ζ 61-164 and CD79B 1-185 ::CD3ζ 61-164 (CD79-CD3), i.e., the CD79-CD3 zeta chimeric polypeptide was obtained (Figure 4a).

[0293] Modification of the cytoplasmic tails of CD79A and CD79B did not prevent surface localization of mIgG1 / Her2. That is, NK-92 cells transduced with mIgG1 / Her2 and CD79-CD3ζ (CD3 zeta) chimeric polypeptides (Fig. 10a) showed similar mIgG1 surface expression (Fig. 4b) as NK-92-mIgG1-CD79 cells (Fig. 7h). Of note, in these experiments we relied on mIgG1 and not tIgG1 to ensure the correct formation of the immunological synapse (Tsourkas, et al. 2007). Strikingly, cells expressing the hybrid receptor caused 87% lysis of SK-BR3 cells at E:T 10:1 (Figure 4c), comparable to NK-92-tIgG1 / Her2-CD16-CD79 (compare Figure 4c with Figure 3i), with cytotoxicity levels against MDA-MB-468 below 5% (Figure 4c). This cytotoxic response indicates that it is possible to exchange the CD79A and CD79B cytoplasmic chains with other signaling domains to increase cytotoxicity while eliminating the need for CD16 overexpression, thus fulfilling our first requirement. To validate the Her2 antigen specificity of this receptor, an identical receptor containing an anti-Pollen variable fragment was constructed and transduced into NK-92 cells (Figure 4d-e, Figure 10b). Although this cell line had comparable surface expression of mIgG1 (compare Figure 4b with Figure 4e), it lysed less than 5% of SK-BR-3 cells (Figure 4f). A cell line expressing the CD79-CD3 zeta fusion alone lysed less than 10% of the target cells (FIG. 10c).

[0294] Next, we evaluated the modularity of the constant immunoglobulin domain. All previous NK-92 mutants used in the above experiments were dependent on CD16 and therefore likely restricted to the IgG class of immunoglobulins due to the presumed ADCC dependency on the IgG constant domain for CD16 binding. We suspected that the removal of the CD16 component would eliminate the need for the IgG constant domain. Thus, we used the CD16 POSWe investigated whether the use of non-IgG constant domains in NK cells would allow for greater design flexibility and avoid receptor-CD16 interactions without compromising receptor functionality. As a proof of concept, we exchanged the heavy chain constant domain of mIgG1 in the mIgG1 / Her2 construct with an IgM class domain (mIgM / Her2) (Figure 4g). Another reason for testing IgM was to assess whether efficacy would be maintained even with longer proteins and increased distances between effector and target cells (Schroeder and Cavacini 2010), especially since reduced efficacy was observed under similar circumstances with other synthetic immune receptors (Hombach, et al. 2007). Finally, it is known that IgM class immunoglobulins are more dependent on the CD79 cofactor for efficient surface expression compared to mIgG1 (Venkitaraman, et al. 1991). However, it was unclear whether this would translate into increased control of immunoglobulin surface expression via the CD79 component.

[0295] First, we transduced NK-92 cells with a vector encoding constitutive mIgM / Her2 (Fig. 10d). Immunostaining assays of these cells for IgM showed that only 15% were positive for immunoglobulin surface expression (Fig. 10e-f). However, remarkably, when these cells were co-transduced with lv-EF1A-CD79-CD3 zeta chimeric polypeptide, the surface localization of IgM increased to 93% (Fig. 4h). Even more remarkably, in a killing assay with these NK-92-mIgM / Her2-CD79-CD3 zeta cells, 92% lysis of SK-BR-3 was measured at an E:T cell ratio of 10:1 (Fig. 4i). Moreover, the 43% lysis at an E:T ratio of 1.25:1 was one of the strongest responses measured for all receptors.

[0296] Similar to using the cytoplasmic domain of CD79 as an attachment point for signaling domains, we further investigated whether the cytoplasmic domain of the antibody construct could be used as a "slot" to introduce additional domains to potentially enhance the overall effect. Keeping in mind the fact that ADCC and CD16 also rely on FcεRIγ (sometimes in synergy with the CD3 zeta domain), we decided to address this question by appending the FcεRIγ signaling domain to the antibody. This domain was used as part of the CAR of NK-92 cells by Clemenceau et al. (Clemenceau, et al. 2015), but the combination of FcεRIγ and CD3ζ in one antigen-specific receptor in NK-92 cells has not been evaluated so far. To this end, we introduced the FcεRIγ ITAM domain immediately after the -KVK motif at the transition from the mIgG1-Her2 transmembrane domain to the cytoplasmic domain (Figure 5a). We left the -KVK motif intact, especially since it is the conserved minimal cytoplasmic tail of various mIg classes and thus may circumvent the problem of mIgG1 surface expression (Cambier and Campbell 1989). We first transduced NK-92 cells with lv-EF1A-mIgG1-FceRIg / Her2 chimeric polypeptide (Fig. 11a, b) and quantified the surface expression of fusion mIgG1. Surprisingly, 24% of NK-92 cells transduced with FcεRIγ-fusion mIgG1 chimeric polypeptide were positive for mIgG1 surface localization without co-expression of CD79 (Fig. 5b). Moreover, surprisingly, expression of this FcεRIγ fusion antibody in NK-92 cells was sufficient to induce a substantial cytotoxic response against target cells (Fig. 5c). At an E:T ratio of 10:1, 84% lysis was observed, and at 1.25:1, 32% lysis was observed, with cell death remaining lower than with NK-92-tIgG1 / Her2-CD16-CD79 (compare Fig. 5c with Fig. 3i), but unexpectedly still higher than with canonical ADCC (compare Fig. 3i).Surprisingly and unexpectedly, additional introduction of CD79-CD3 zeta chimeric polypeptide (Fig. 11a) into this cell line further increased immunoglobulin surface expression to 95% (Fig. 5d, e) and lysis to 96% at an E:T ratio of 10:1 (Fig. 5f). Considering the cytotoxic potential especially at an E:T ratio of 1.25:1, this was the strongest response seen across all receptor designs, lysing 54% of target cells (Fig. 5f). Her2 specificity was maintained, as co-culture with MDA-MB-468 did not result in target cell lysis above background levels (Fig. 5f). Fab fragment exchange from anti-Her2 to anti-Pollen Fab fragments (Fig. 11a) resulted in similar surface expression (Fig. 5g, h), but co-culture with SK-BR-3 abolished target cell lysis (Fig. 5i), suggesting antigen specificity.

[0297] Example 6. Altering the target specificity of modular multi-chain receptors To further confirm that the multi-chain receptors of the invention, for example those based on the ASIMut receptor platform described in Example 5, can be switched to kill different target cells if desired, we replaced the variable fragments of the receptors shown in Figure 5d with variable fragments of antibodies against CD19.1 (i.e. FMC63), CD19.2 (i.e. Inebilizumab) or CD20 (i.e. Rituximab); see SEQ ID NOs: 433, 435 and 437 and Figure 12. To this end, we targeted CD19 and CD20 positive Raji cells derived from B-cell malignancies using the original anti-HER2 ASIMut receptors shown in Figure 5d (see Figure 13a) or these modified receptors (with specificity for CD19.1, CD19.2 or CD20; see Figures 13b-d). The experiments were performed as described herein, particularly in the context of Figures 5, c, f and i.

[0298] We found that multi-chain antigen receptors bearing the variable domains of anti-CD19.1, anti-CD19.2, or anti-CD20 antibodies killed target cells, i.e., Raji cells, much more efficiently and more efficiently than anti-HER2 receptors bearing the variable domains of trastuzumab. Of note, the measured killing activity of anti-HER2 multi-chain antigen receptors against Raji cells (Figure 13a) is not mediated through antigen-specific binding (i.e., HER2 binding) but is rather likely related to the intrinsic killing activity of NK cells against these cells, i.e., background target cell lysis in this experiment.

[0299] Thus, these experiments confirm that the specificity of multi-chain antigen receptors, such as the ASIMut receptor, can be altered and that engineered cells (e.g. NK cells) expressing altered receptors containing the corresponding antigen-binding sites can be used to kill tumor or cancer cells expressing other antigens.

[0300] Sequence list (partial) For a complete sequence listing, please see the attached sequence listing in accordance with WIPO St. 26.

[0301] JPEG2025503499000023.jpg231170

[0302] JPEG2025503499000024.jpg252170

[0303] JPEG2025503499000025.jpg248170

[0304] JPEG2025503499000026.jpg227153

[0305] JPEG2025503499000027.jpg255170

[0306] JPEG2025503499000028.jpg255168

[0307] JPEG2025503499000029.jpg230151

[0308] Any amino acid sequence having a designated name herein may be encoded by a DNA sequence having the same designated name herein. Furthermore, any amino acid sequence encoded by a DNA sequence disclosed herein, i.e., a polypeptide, is also disclosed herein, particularly in the context of the present invention.

[0309] Further references to the sequences set forth in SEQ ID NOs: 115-172 are set forth in Tables 2 and 3 herein.

Claims

1. A modified mammalian cell, comprising: (I) first and second polypeptides, each comprising a variable region; wherein the variable region of the first polypeptide and the variable region of the second polypeptide form an antigen-binding site on the outside of the cell; wherein the first polypeptide further comprises a membrane domain located within the membrane of the cell, and wherein the first and second polypeptides are (i) the alpha and beta chains of the T cell receptor (TCR), or (ii) gamma and delta chains of the TCR; Instead, and, (II) a third polypeptide comprising a membrane domain or an extracellular domain, wherein: (i) the membrane domain of the third polypeptide comprises a sequence having at least 80% sequence identity to the membrane domain of a CD79A protein; (ii) the extracellular domain of the third polypeptide comprises a sequence having at least 80% sequence identity to the extracellular domain of a CD79A protein; (iii) the membrane domain of the third polypeptide comprises a sequence having at least 80% sequence identity to the membrane domain of a CD79B protein; or (iv) the extracellular domain of the third polypeptide comprises a sequence having at least 80% sequence identity to the extracellular domain of a CD79B protein; wherein the third polypeptide further comprises an intracellular domain comprising at least one signaling domain, and wherein the modified cell is not a B cell, which needs to interact with other immune cell types to promote killing of the target cell.

2. The modified cell of claim 1, comprising a fourth polypeptide, wherein the fourth polypeptide comprises a membrane domain or an extracellular domain, wherein: (i) the membrane domain of the fourth polypeptide comprises a sequence having at least 80% sequence identity to the membrane domain of a CD79B protein; (ii) the extracellular domain of the fourth polypeptide comprises a sequence having at least 80% sequence identity to the extracellular domain of a CD79B protein; and wherein: (i) the membrane domain of the third polypeptide comprises a sequence having at least 80% sequence identity to the membrane domain of a CD79A protein, or (ii) the extracellular domain of the third polypeptide comprises a sequence having at least 80% sequence identity to the extracellular domain of a CD79A protein. The modified cell of claim 1.

3. The modified cell of claim 1, wherein: (i) the variable region of the first polypeptide comprises antibody heavy chain complementarity determining regions (CDRs) CDR-H1, CDR-H2, and CDR-H3; and the variable region of the second polypeptide comprises CDR-L1, CDR-L2, and CDR-L3 of the light chain of the antibody; (ii) the variable region of the first polypeptide comprises CDR-L1, CDR-L2, and CDR-L3 of the light chain of the antibody, and the variable region of the second polypeptide comprises CDR-H1, CDR-H2, and CDR-H3 of the heavy chain of the antibody; The modified cell of claim 1.

4. The modified cell described in claim 3, wherein the first polypeptide further comprises a constant region.

5. The modified cell of claim 1, wherein the modified cell is a natural killer (NK) cell or a T cell.

6. The modified cell described in claim 5, wherein the T cell is a CD8+ T cell, a CD4+ cell, a helper T cell or a regulatory T cell. (a) the membrane domain of the third polypeptide comprises the sequence motif "EXXXXXXXXXXP" and the membrane domain of the fourth polypeptide comprises the sequence motif "QXXXXXXXXXXP", (b) the membrane domain of the third polypeptide comprises a sequence having at least 80% sequence identity to SEQ ID NO:4, and the membrane domain of the fourth polypeptide comprises a sequence having at least 80% sequence identity to SEQ ID NO:6; or (c) the membrane domain of the third polypeptide comprises a sequence having at least 80% sequence identity to SEQ ID NO:8, and the membrane domain of the fourth polypeptide comprises a sequence having at least 80% sequence identity to SEQ ID NO:10; The modified cell of claim 2. (a) the extracellular domain of the third polypeptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 12 or 174; and the extracellular domain of the fourth polypeptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 14 or 177; or (b) the third polypeptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 16 or 175; and the fourth polypeptide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 18 or 178. The modified cell of claim 2.

9. The modified cell described in claim 2, wherein the third polypeptide comprises both an extracellular domain and a membrane domain, and wherein the fourth polypeptide comprises both an extracellular domain and a membrane domain.

10. The modified cell of claim 1, wherein the membrane domain of the first polypeptide comprises the sequence motif "YS" and / or wherein the membrane domain of the first polypeptide comprises the sequence motif "WXXXXXFXXLFXLXXXYSXXXT" (sequence number 19).

11. The modified cell of claim 1, wherein the membrane domain of the first polypeptide comprises a sequence having at least 80% sequence identity to the membrane domain of the sequence set forth in SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, 29, 31, 37 or 38.

12. 2. The modified cell of claim 1, wherein the membrane domain of the first polypeptide is capable of binding to the membrane domain of a CD79A protein or a CD79B protein.

13. The modified cell of claim 1, wherein the modified cell does not contain CD16 protein.

14. The modified cell described in claim 1, wherein the first polypeptide further comprises a linker region between the variable region and the membrane domain, or between the constant region and the membrane domain of the first polypeptide, wherein the linker region forms a flexible linker.

15. The modified cell of claim 1, wherein at least one signaling domain comprises an immunoreceptor tyrosine-based activation motif of a protein selected from the group consisting of CD3 zeta, FceRIg, CD16A, CD16B, NKp30, NKp46, KIR2DS1-2, KIR2DS3-6, KIR3DS1, NKG2C, NKG2D, 2B4, CD2, CRACC, NTB-A, DNAM-1, CD7, CD59, BY55, KIR2DL4, CD44, and a polypeptide having at least 80% sequence identity to any of the foregoing proteins.

16. The signaling domain(s) contain the sequence motif "Y-XX-I or LX" (6～12) 16. The modified cell of claim 15, wherein the signaling domain(s) comprise the sequence motif set forth in SEQ ID NO: 73, 48, 49 or 74, and / or wherein the signaling domain(s) comprise the sequence motif set forth in SEQ ID NO: 73, 48, 49 or 74.

17. The modified cell described in claim 1, wherein at least one signaling domain comprises an ITAM region of CD3 zeta protein or a sequence having at least 80% sequence identity to SEQ ID NO:

82.

18. The modified cell described in claim 1, wherein the intracellular domain of the first polypeptide comprises a signaling domain comprising at least one immunoreceptor tyrosine-based activation motif (ITAM) of an FceRIg protein, or a sequence having at least 80% sequence identity to SEQ ID NO:

86. (I) The intracellular domain of the third polypeptide comprises: (a) at least one ITAM of the CD79A protein, or a sequence having at least 80% sequence identity to SEQ ID NO: 92; or (b) a sequence having at least 80% sequence identity to SEQ ID NO: 96 and (II) the intracellular domain of the fourth polypeptide is (a) at least one ITAM of a CD79B protein, or a sequence having at least 80% sequence identity to SEQ ID NO: 94; or (b) a sequence having at least 80% sequence identity to SEQ ID NO: 98 3. The modified cell of claim 2, comprising:

20. 2. The modified cell of claim 1, wherein, upon binding of the antigen-binding site to a corresponding antigen, at least one of the signaling domains activates at least one signaling pathway within the cell that enables the cell to promote the killing of a target cell that contains the antigen on its cell surface.

21. The modified cell of claim 20, wherein the signaling pathway comprises Ca2+ signaling or at least one protein selected from the group consisting of at least one Src family kinase, at least one Syk family kinase, PLCG1, PI3K, Vav1, at least one Rho family GTPase, ERK1 / 2, and NFAT.

22. 22. The modified cell of any one of claims 1 to 21 for use in treating a disease in a mammalian subject.

23. The modified cell for use according to claim 22, wherein the mammalian subject is a human.

24. The modified cells for use according to claim 22, wherein the disease is cancer or an autoimmune disease.

25. A set of one or more nucleic acid molecules, wherein the nucleic acid molecule(s) comprises: (I) a first coding sequence encoding a first polypeptide, and a second coding sequence encoding a second polypeptide, wherein the first and second polypeptides each comprise a variable region; wherein the variable region of the first polypeptide and the variable region of the second polypeptide are capable of forming an antigen-binding site when expressed together in the modified mammalian cell; and wherein the first and second polypeptides are (i) the alpha and beta chains of the T cell receptor (TCR), or (ii) gamma and delta chains of the TCR; Instead, and (II) a third coding sequence encoding a third polypeptide, wherein the third polypeptide comprises a membrane domain or an extracellular domain, wherein: (i) the membrane domain of the third polypeptide comprises a sequence having at least 80% sequence identity to the membrane domain of a CD79A protein; (ii) the extracellular domain of the third polypeptide comprises a sequence having at least 80% sequence identity to the extracellular domain of a CD79A protein; (iii) the membrane domain of the third polypeptide comprises a sequence having at least 80% sequence identity to the membrane domain of a CD79B protein; or (iv) the extracellular domain of the third polypeptide comprises a sequence having at least 80% sequence identity to the extracellular domain of a CD79B protein; wherein the third polypeptide further comprises an intracellular domain comprising at least one signaling domain.

26. A method for producing a modified cell, comprising the step of introducing a set of nucleic acid molecules (which may be multiple) described in claim 25 into a mammalian cell, thereby producing the modified cell.

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