Novel CD20 protein

Abstract

The present invention relates to human CD20 proteins having modifications to one or more intracellular domains sufficient to reduce or abrogate intracellular signaling when bound to or associated with the membrane of cells such as (for example) T cells, natural killer cells, B cells, myeloid cells, pluripotent stem cells, and hematopoietic stem cells. The present invention further contemplates nucleic acids encoding the modified human CD20 proteins described herein, and the utility of the modified human CD20 proteins as safety switches in chimeric antigen receptor T cell therapy or as selection markers for gene therapy.

Description

[Technical Field]

[0001] The present invention relates to human CD20 proteins containing at least one mutation designed to reduce or abrogate intracellular signaling when bound to or associated with the membrane of cells, including, for example, T cells, natural killer cells, B cells, myeloid cells, hematopoietic stem cells, non-hematopoietic stem cells, pluripotent stem cells, and human cell lines. In particular, the present invention provides engineered human CD20 proteins containing at least one mutation in at least one intracellular domain comprising amino acid residues 1-56 or 210-297 as defined by SEQ ID NO:1. [Background technology]

[0002] The following contains information that may be helpful in understanding the present invention. None of the information, publications, or documents referenced herein, expressly or implicitly, is admitted to be prior art or essential to the presently described or claimed invention. All publications and patents mentioned herein are incorporated herein by reference in their entirety.

[0003] It is estimated that by 2025, the US Food and Drug Administration (FDA) will approve 10–20 gene therapies each year.[1] The rapid expansion of this field, targeting a wide range of diseases including blindness, immune and neurological disorders, and cancer,[2] highlights the urgent need for safe mechanisms to address potential short- and long-term adverse effects (e.g., cytotoxicity in CAR T-cell therapy).

[0004] Adverse effects resulting from gene therapy can be controlled by incorporating safety switches, such as suicide genes, into the genetic construct or by including cell elimination markers not normally present on the modified cells, thereby enabling antibody-mediated cytotoxicity. [3] The use of clinically approved antibodies, such as cetuximab (anti-EGFR) [4] or rituximab (anti-CD20) [5], offers advantages over other types of control for cell surface elimination markers. The Federal Drug Agency (FDA) and the European Medicines Agency (EMA) recommend assessing the cellular kinetics, biodistribution, and persistence of genetically engineered cells, as well as monitoring for late events, including secondary malignancies, in long-term follow-up. The use of a single protein that can serve as both a safety switch and a detection marker offers a solution. [6, 7] Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention seeks to address this clinical need by providing engineered CD20 proteins in which the intracellular domain has been modified to abrogate CD20-mediated signaling while retaining detection and killing by anti-CD20 antibodies, including, for example, rituximab, obinutuzumab, and ocrelizumab. [Means for solving the problem]

[0006] The invention described and claimed herein has many attributes and embodiments, including but not limited to those shown or described or referenced in the Summary of the Invention. It is not intended to be all-inclusive, and the invention described and claimed herein is not limited to or by the features or embodiments identified in the Summary of the Invention, which are included for purposes of illustration only and not limitation.

[0007] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) at least one mutation that results in truncation to any one or more of amino acid residues 1 to 56 set forth in SEQ ID NO: 1; (ii) at least one mutation that results in truncation to any one or more of amino acid residues 210 to 297 set forth in SEQ ID NO: 1; (iii) at least one mutation to threonine or serine located within amino acid residues 1 to 56 set forth in SEQ ID NO: 1; (iv) at least one mutation to threonine or serine located within amino acid residues 210 to 297 of SEQ ID NO: 1; or (v) A combination including any one of (i) to (iv). 1. A modified human CD20 protein comprising: The present invention provides a modified human CD20 protein, wherein at least one mutation defined by any one of (i) to (v) causes a reduction or abrogation of intracellular signaling when the modified CD20 protein is bound to or associated with the membrane of a cell.

[0008] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) at least one mutation that results in truncation to any one or more of amino acid residues 1 to 56 set forth in SEQ ID NO: 1; and (ii) at least one mutation to threonine or serine located within amino acid residues 210 to 297 of SEQ ID NO: 1; 1. A modified human CD20 protein comprising: The present invention provides a modified human CD20 protein, wherein at least one mutation defined by (i) and (ii) causes reduced or abrogated intracellular signaling when the modified CD20 protein is bound to or associated with the membrane of a cell.

[0009] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) at least one mutation to threonine or serine located within amino acid residues 1 to 56 of SEQ ID NO: 1; and (ii) at least one mutation that causes truncation to any one or more of the amino acid residues 210 to 297 set forth in SEQ ID NO: 1; 1. A modified human CD20 protein comprising: The present invention provides a modified human CD20 protein, wherein at least one mutation defined by (i) and (ii) causes reduced or abrogated intracellular signaling when the modified CD20 protein is bound to or associated with the membrane of a cell.

[0010] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) at least one mutation to threonine or serine located within amino acid residues 1 to 56 of SEQ ID NO: 1; and (ii) at least one mutation to threonine or serine located within amino acid residues 210 to 297 of SEQ ID NO: 1; 1. A modified human CD20 protein comprising: The present invention provides a modified human CD20 protein, wherein at least one mutation defined by (i) and (ii) causes reduced or abrogated intracellular signaling when the modified CD20 protein is bound to or associated with the membrane of a cell.

[0011] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) truncation of amino acid residues 1 to 50 shown in SEQ ID NO: 1; and (ii) at least one mutation to threonine or serine located within amino acid residues 210 to 297 of SEQ ID NO: 1; 1. A modified human CD20 protein comprising: The present invention provides a modified human CD20 protein, wherein at least one mutation defined by (i) and (ii) causes reduced or abrogated intracellular signaling when the modified CD20 protein is bound to or associated with the membrane of a cell.

[0012] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) truncation of amino acid residues 1 to 50 shown in SEQ ID NO: 1; and (ii) at least one mutation to S225, S231, and T239 as set forth in SEQ ID NO: 1 1. A modified human CD20 protein comprising: The present invention provides a modified human CD20 protein, wherein at least one mutation defined by (i) and (ii) causes reduced or abrogated intracellular signaling when the modified CD20 protein is bound to or associated with the membrane of a cell.

[0013] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) truncation of amino acid residues 1 to 50 shown in SEQ ID NO: 1; and (ii) at least one mutation to S225, S231, and T239 of SEQ ID NO: 1, including one or more of S225A, S231A, and T239A; 1. A modified human CD20 protein comprising: The present invention provides a modified human CD20 protein, wherein at least one mutation defined by (i) and (ii) causes reduced or abrogated intracellular signaling when the modified CD20 protein is bound to or associated with the membrane of a cell.

[0014] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) at least one mutation to threonine or serine located within amino acid residues 1 to 56 of SEQ ID NO: 1; and (ii) truncation of amino acid residues 253 to 297 of SEQ ID NO: 1 1. A modified human CD20 protein comprising: The present invention provides a modified human CD20 protein, wherein at least one mutation defined by (i) and (ii) causes reduced or abrogated intracellular signaling when the modified CD20 protein is bound to or associated with the membrane of a cell.

[0015] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) at least one mutation to T2, T3, S7, T11, S35, S36, and T51 of SEQ ID NO: 1; and (ii) truncation of amino acid residues 253 to 297 of SEQ ID NO: 1 1. A modified human CD20 protein comprising: The present invention provides a modified human CD20 protein, wherein at least one mutation defined by (i) and (ii) causes reduced or abrogated intracellular signaling when the modified CD20 protein is bound to or associated with the membrane of a cell.

[0016] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) at least one mutation to T2, T3, S7, T11, S35, S36, and T51 of SEQ ID NO: 1, including one or more of T2A, T3A, S7A, T11A, S35A, S36A, and T51A; and (ii) truncation of amino acid residues 253 to 297 of SEQ ID NO: 1 1. A modified human CD20 protein comprising: The present invention provides a modified human CD20 protein, wherein at least one mutation defined by (i) and (ii) causes reduced or abrogated intracellular signaling when the modified CD20 protein is bound to or associated with the membrane of a cell.

[0017] In yet another aspect, the present invention provides a modified human CD20 protein comprising the sequence shown in SEQ ID NO:2 or a variant sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity to SEQ ID NO:2.

[0018] In yet another aspect, the present invention provides a modified human CD20 protein comprising the sequence shown in SEQ ID NO:3, or a variant sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity to SEQ ID NO:3.

[0019] In yet another aspect, the present invention provides a modified human CD20 protein comprising the sequence shown in SEQ ID NO:4, or a variant sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity to SEQ ID NO:4.

[0020] In yet another aspect, the present invention provides a modified human CD20 protein comprising the sequence shown in SEQ ID NO:5, or a variant sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity to SEQ ID NO:5.

[0021] In yet another aspect, the present invention provides a modified human CD20 protein comprising the sequence shown in SEQ ID NO:6, or a variant sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity to SEQ ID NO:6.

[0022] In yet another aspect, the present invention provides a modified human CD20 protein comprising the sequence shown in SEQ ID NO:7, or a variant sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity to SEQ ID NO:7.

[0023] In yet another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) at least one mutation that results in truncation to any one of amino acid residues 1 to 56 of SEQ ID NO: 1; (ii) at least one mutation that results in truncation to any one of amino acid residues 210 to 297 of SEQ ID NO: 1; (iii) at least one mutation to threonine or serine located within amino acid residues 1 to 56 of SEQ ID NO: 1; (iv) at least one mutation to threonine or serine located within amino acid residues 210 to 297 of SEQ ID NO: 1; or (v) a combination comprising any one of (i) to (iv); and (vi) at least one mutation to any one of amino acid residues 142 to 188 of SEQ ID NO: 1 that eliminates binding by the monoclonal antibody obinutuzumab 1. A modified human CD20 protein comprising: The present invention provides a modified human CD20 protein, which, when the modified CD20 protein binds to or associates with the membrane of a cell, causes any one of (i) to (v) to reduce or inhibit intracellular signaling.

[0024] In yet another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) at least one mutation that results in truncation to any one of amino acid residues 1 to 56 of SEQ ID NO: 1; (ii) at least one mutation that results in truncation to any one of amino acid residues 210 to 297 of SEQ ID NO: 1; (iii) at least one mutation to threonine or serine located within amino acid residues 1 to 56 of SEQ ID NO: 1; (iv) at least one mutation to threonine or serine located within amino acid residues 210 to 297 of SEQ ID NO: 1; or (v) a combination comprising any one of (i) to (iv); and (vi) at least one mutation to any one of amino acid residues 142 to 188 of SEQ ID NO: 1 that eliminates binding by the monoclonal antibody rituximab 1. A modified human CD20 protein comprising: The present invention provides a modified human CD20 protein, which, when the modified CD20 protein binds to or associates with the membrane of a cell, causes any one of (i) to (v) to reduce or inhibit intracellular signaling.

[0025] In a further aspect, the invention provides a cell that expresses any of the modified human CD20 proteins described herein.

[0026] In a further aspect, the invention provides a T cell that expresses any modified human CD20 protein described herein.

[0027] In a further aspect, the present invention provides natural killer (NK) cells that express any modified human CD20 protein described herein.

[0028] In a further aspect, the invention provides a B cell that expresses any modified human CD20 protein described herein.

[0029] In a further aspect, the invention provides a myeloid cell that expresses any modified human CD20 protein described herein.

[0030] In a further aspect, the invention provides pluripotent cells that express any modified human CD20 protein described herein.

[0031] In a further aspect, the invention provides hematopoietic stem cells that express any modified human CD20 protein described herein.

[0032] In a further aspect, the invention provides a hematopoietic stem cell line that expresses any modified human CD20 protein described herein.

[0033] In a further aspect, the present invention provides a non-hematopoietic stem cell line that expresses any modified human CD20 protein described herein.

[0034] In a further aspect, the invention provides a human cell line expressing any modified human CD20 protein described herein.

[0035] In still a further aspect, the present invention provides nucleic acid molecules, such as deoxyribonucleic acid molecules (DNA), messenger ribonucleic acid molecules (mRNA) and complementary deoxyribonucleic acid molecules (cDNA), encoding any of the modified human CD20 proteins described herein.

[0036] In yet another aspect, the invention provides a vector comprising a nucleic acid described herein.

[0037] In yet another aspect, the invention provides an expression vector comprising a nucleic acid described herein. [Brief explanation of the drawings]

[0038] [Figure 1] Flow cytometry plots of HEK293 cells transfected with CD20-GFP and various versions of CD20-GFP (diagonal lines indicate cells producing both GFP and CD20). The Raji B cell line was used as a positive control for membrane detection of CD20. [Figure 2]

[0039] Figure 1 shows predicted phosphorylation sites of CD20. Residues in bold / underlined are amino acids that are identical to the mouse sequence and have a NetPhos score greater than 0.7; residues in (parentheses) are amino acids identified as identical to the mouse sequence; residues in [square brackets] are amino acids with a NetPhos score greater than 0.7; residues in gray are amino acids identified by the crystal structure; and residues in gray and underlined are amino acids identified using all three methods (see Example 2). [Figure 3]Detection of surface CD20WT or modified CD20 on HEK293 cell lines (top panel) and primary T cells (bottom panel) is shown, as assessed by labeling the cells with rituximab conjugated to AF405. [Figure 4A] Figure 1 shows total rituximab-mediated cell death of CD20KO HG3 cells that were either untransduced or transduced with CD20WT, C1C3, or C1C252. [Figure 4B] Figure 1 shows the transduction efficiency of CD20WT, C1C3 or C1C252 in CD20KO HG3 cells compared to untransduced cells. [Figure 5] Figure 1 shows the GFP+ cells (% of CD45.1 cells) remaining in mouse blood after treatment with 2H7 antibody or isotype control. Bars represent the average % of 5 mice, and error bars represent SEM. [Figure 6] 1 shows detection of cell surface CD20 by obinutuzumab, rituximab, or L27 antibody in CD20KO HG3 cells transduced with CD20WT, C1C3, or C1C252, and in untransduced control cells. [Figure 7] Figure 1 shows Ca2+ influx induced by hypercrosslinking of CD20WT, C1C3, and C1C252 in CD20KO HG3 cells compared to untransduced controls, as assessed by flow cytometry. Bars represent MFI of triplicate samples, and error bars represent SEM. [Figure 8A] Figure 1 shows the global phosphorylation status of CREB, WINK, GSK, ERK, STAT5 and 6, Lyn, p53 and RSK upon CD20 activation by rituximab binding as measured by phospho-Western Blot. [Figure 8B] Differences in phosphorylation of kinases relevant to both B and T cell activation are shown. Bars represent mean differences, dots represent individual measurements repeated twice per condition. [Figure 9A]Based on published information [8], we present the identification of key binding residues in the CD20 epitope required for rituximab and obinutuzumab. Individual asparagine (N) residues were replaced with alanine (A) to specifically abrogate binding of either antibody. [Figure 9B] 1 shows detection of surface CD20 by rituximab or obinutuzumab in HEK293 cells transduced with CD20WT, Δobinutuzumab mutant (N176A substitution to specifically abrogate obinutuzumab binding), and Δrituximab mutant (N171A substitution to specifically abrogate rituximab binding). [Figure 10] The amino acid sequences of exemplary modified human CD20 proteins of the present invention are shown: X = any naturally occurring or non-naturally occurring amino acid residue. DETAILED DESCRIPTION OF THE INVENTION

[0039] General definition Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs (e.g., in immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0040] Unless otherwise indicated, the recombinant protein and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, vols. 1 and 2, IRL Press (1991); D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, vols. 1-4, IRL Press (1995 and 1996); and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed. They are described and explained throughout the literature in sources such as Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JE Coligan et al., (editors), Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0041] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as expressly endorsing both meanings or either meaning.

[0042] The terms "a" or "an" refer to one or more than one of a specified entity; for example, "a receptor" or "a nucleic acid molecule" can refer to one or more receptors or nucleic acid molecules, or at least one receptor or nucleic acid molecule. Thus, the terms "a" or "an," "one or more," and "at least one" can be used interchangeably herein.

[0043] Throughout this specification, unless specifically stated otherwise or otherwise required by context, references to a single step, composition, group of steps or group of compositions shall be interpreted as encompassing one and more than one (i.e., one or more) such step, composition, group of steps or group of compositions.

[0044] Reference to a numerical range disclosed herein (e.g., 1-10) is also intended to incorporate reference to all associated numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and all rational ranges within that range (e.g., 2-8, 1.5-5.5, and 3.1-4.7), and thus all subranges of all ranges explicitly disclosed herein are specifically disclosed. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited should be considered to be equally explicitly stated in this application.

[0045] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0046] Those skilled in the art will appreciate that the invention described herein is capable of variations and modifications other than those specifically described. The invention is to be understood to include all such variations and modifications. The invention also includes all steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations of any two or more of such steps or features.

[0047] The present invention is not to be limited in scope by the specific embodiments described herein, which are for purposes of illustration only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0048] Any example or embodiment described herein shall be construed as applying mutatis mutandis to any other example or embodiment, unless specifically stated otherwise.

[0049] Selected Definitions The term "amino acid residue" or "amino acid" includes reference to an amino acid incorporated into a protein, polypeptide, or peptide. The term "polypeptide" includes any polymer of amino acids or amino acid residues. The term "polypeptide sequence" refers to the series of amino acids or amino acid residues that physically make up a polypeptide. A "protein" is a macromolecule comprising one or more polypeptides or polypeptide "chains." A "peptide" is a small polypeptide, typically less than 15-20 total amino acid residues in size. The term "amino acid sequence" refers to the series of amino acids or amino acid residues that physically make up a peptide or polypeptide, depending on its length. Unless otherwise indicated, polypeptide and protein sequences disclosed herein are written from left to right and represent that order from the amino terminus to the carboxy terminus.

[0050] The terms "amino acid," "amino acid residue," "amino acid sequence," or polypeptide sequence include naturally occurring amino acids (including L- and D-stereoisomers) and, unless otherwise limited, known analogs of naturally occurring amino acids that can function in a manner similar to the naturally occurring amino acids, such as selenocysteine, pyrrolysine, N-formylmethionine, γ-carboxyglutamate, hydroxyproline, hypusine, pyroglutamic acid, and selenomethionine. The amino acids referred to herein are set forth in Table A by their abbreviated names as follows: [Table 1]

[0051] The term "antibody" refers to an immunoglobulin molecule capable of selectively binding to a target, such as human CD20, via an antigen-binding site contained within at least one variable region. This term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant or engineered antibodies (e.g., chimeric antibodies, humanized antibodies, primatized antibodies, de-immunized antibodies, half antibodies, bispecific antibodies), and single-domain antibodies, such as domain antibodies and heavy-chain-only antibodies (e.g., camelid antibodies or cartilaginous fish immunoglobulin new antigen receptors (IgNARs)). Antibodies generally contain a constant domain, which may be located in the constant region or constant fragment or crystallizable fragment (Fc). Preferred forms of antibodies contain a four-chain structure as their basic unit. Full-length antibodies contain two covalently linked heavy chains (approximately 50-70 kDa) and two light chains (approximately 23 kDa each). Light chains generally contain a variable region and a constant domain, and in mammals are either kappa or lambda light chains. Heavy chains generally contain a variable region and one or two constant domains linked to an additional constant domain by a hinge region. Mammalian heavy chains are of one of the following types: α, δ, ε, γ, or μ. Each light chain is also covalently linked to one of the heavy chains. For example, two heavy chains and heavy and light chains are linked by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds can vary among different types of antibodies. Each chain has an N-terminal variable region (VH or VL, each approximately 110 amino acids long) and one or more C-terminal constant domains. The light chain constant domain (approximately 110 amino acids long, CL) is disulfide-bonded to the first heavy chain constant domain (approximately 330-440 amino acids long, CH). The light chain variable region is aligned with the heavy chain variable region. Antibody heavy chains may contain two or more additional CH domains (e.g., CH2, CH3, etc.) and may include a hinge region that may be identified between the CH1 and Cm constant domains. Antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.In one example, the antibody is a murine (mouse or rat) or primate (preferably human) antibody. The term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, but also variants, fusion proteins comprising an antibody portion having an antigen-binding site, humanized antibodies, human antibodies, chimeric antibodies, primatized antibodies, deimmunized antibodies, or veneered antibodies.

[0052] The term "conservative substitution" with respect to a polypeptide refers to a change in the amino acid composition of the polypeptide that does not substantially alter the function and structure of the polypeptide as a whole (see Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, New York (2nd ed., 1992)).

[0053] As used herein, the terms "expressed," "expressing," or "express" refer to the transcription and / or translation of a polynucleotide or nucleic acid into a polypeptide or protein. The produced polypeptide or protein may remain intracellular, may become a component of the cell surface membrane, or may be secreted into the extracellular space.

[0054] As used herein, cells that express a significant amount of CD20 on at least one cell surface are referred to as "CD20 positive cells" or "CD20 + cells" that are physically bound to significant amounts of the extracellular target biomolecule CD20.

[0055] The term "encode," as used herein, refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, as a template in biological processes for the synthesis of other polymers and macromolecules having a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties derived therefrom. Thus, a gene, cDNA, or RNA encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to the gene. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, which serves as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of the gene or cDNA.

[0056] As used herein, the term "epitope" refers to a portion of an antigen (e.g., human CD20) that specifically interacts with an antibody molecule. These portions are referred to herein as epitopic determinants and typically include or are portions of elements such as amino acid side chains or sugar side chains. Epitopes can be defined, for example, by methods known in the art or disclosed herein, such as by crystallography or hydrogen-deuterium exchange. At least one or a portion of the portion of an antibody molecule that specifically interacts with an epitopic determinant is typically located in a CDR. Typically, an epitope has specific three-dimensional structural characteristics. Typically, an epitope has a specific charge profile. Some epitopes are linear epitopes, while others are conformational epitopes. An exemplary epitope of the invention is that defined by amino acid residues 167-183 of SEQ ID NO: 1, i.e., CEPANPSEKNSPSTQYC (SEQ ID NO: 13), which incorporates amino acid residues essential for binding of the clinically approved monoclonal antibodies rituximab and obinutuzumab.

[0057] The term "encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to the gene. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of the gene or cDNA.

[0058] As used herein, the term "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0059] As used herein, the term "exogenous" refers to any substance introduced into or produced outside of an organism, cell, tissue, or system.

[0060] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0061] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0062] The term "identity" refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as between two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit site in both molecules is occupied by the same monomer subunit; for example, if a position in each of two polypeptide or protein molecules is occupied by serine, they are homologous or identical at that position. Homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the sites in two sequences are the same (e.g., 5 positions in a polymer 10 subunits long), the two sequences are 50% homologous; if 90% of the sites (e.g., 9 out of 10) are matching or homologous, the two sequences are 90% identical.

[0063] The term "intracellular signaling domain," as used herein, refers to the intracellular portion of a molecule, e.g., CD20. The intracellular signaling domain(s) generate a signal that enhances the immune effector function of a CAR-containing cell, such as a CAR T cell (for example). Examples of immune effector functions of CAR T cells and the like include cytolysis and helper activity, including cytokine secretion. In certain instances, the intracellular signaling domain is a portion of a protein that transmits an effector function signal and directs a cell to perform a specialized function. While the entire intracellular signaling domain can be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the entire chain, provided that it transmits the effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0064] The term "isolated," when applied to protein or polypeptide sequences disclosed herein, is used to refer to sequences that have been removed from their native cellular or other naturally occurring biological environment. Isolated molecules can be obtained by any method or combination of methods, including biochemical, recombinant, and synthetic techniques. Polypeptide sequences can be prepared by at least one purification step.

[0065] The term "lentivirus" refers to a member of the retrovirus family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are examples of lentiviruses.

[0066] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, and specifically includes self-inactivating lentiviral vectors such as those provided in

[44] . Other examples of lentiviral vectors that can be used in the clinic include, but are not limited to, those from Oxford Biomedica Gene Delivery Technology or the Lentigen Vector System. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.

[0067] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and polymers thereof, in either single- or double-stranded form. The term "nucleic acid" includes a gene, cDNA, or mRNA. In one example, a nucleic acid molecule is synthetic (e.g., chemically synthesized) or recombinant. Unless otherwise limited, the term includes nucleic acids containing analogs or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions can be achieved by generating sequences in which three positions of one or more selected (or all) codons are substituted with mixed and / or deoxyinosine groups.

[0068] The terms "nucleic acid encoding an amino acid sequence" or "nucleic acid encoding a protein" include all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence or protein. In certain instances, a nucleic acid that encodes a polypeptide or protein may contain intron(s).

[0069] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, with no limit on the maximum number of amino acids that may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (commonly referred to in the art as peptides, oligopeptides, and oligomers, for example) and longer chains (commonly referred to in the art as proteins, of which there are various types). "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. A polypeptide can be a natural peptide, a recombinant peptide, or a combination thereof.

[0070] The term "polynucleotide," as used herein, is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, the terms "nucleic acid" and "polynucleotide" may be used interchangeably herein. Those skilled in the art will understand from their technical knowledge that a nucleic acid is a polynucleotide, which can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques, PCR, etc., as well as by synthetic means.

[0071] The term "promoter" refers to a DNA sequence that is recognized or introduced into the synthetic machinery of a cell and is necessary to initiate the specific transcription of a polynucleotide sequence.

[0072] The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, the sequence may be a core promoter sequence, and in other cases, the sequence may also include an enhancer sequence and other control elements required for expression of the gene product. The promoter / regulatory sequence may be, for example, a sequence that directs expression of the gene product in a tissue-specific manner.

[0073] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a binding partner (e.g., a stimulatory tumor antigen) present in a sample, but does not substantially recognize or bind to other molecules in the sample.

[0074] As used herein, reference to "at least one mutation to a serine or threonine" may include reference to (for example) any one or more of T2, T3, S7, T11, S25, S35, S36, T41, S43, S49, T51, S221, S225, S231, T239, T250, T252, S253, S254, T275, T277, S288, S289, S295 and S296 of SEQ ID NO: 1, including the threonine at position 2, the threonine at position 3, the serine at position 7, the threonine at position 11, the serine at position 12, the threonine at position 13, the serine at position 14, the threonine at position 15, the threonine at position 16, the threonine at position 17, the threonine at position 18, the threonine at position 19, the threonine at position 20, the threonine at position 21, the threonine at position 22, the threonine at position 23, the serine at position 25, the threonine at position 26, the threonine at position 27, the threonine at position 28, the threonine at position 29, the threonine at position 31, the threonine at position 29, the threonine at position 29, the threonine at position 21, the threonine at position 22, the threonine at position 23, the threonine at position 24, the threonine at position 25, the threonine at position 26, the threonine at position 27 These include substitution, deletion, or insertion mutations involving amino acid residues equivalent to threonine at position 25, serine at position 25, serine at position 35, serine at position 36, threonine at position 41, serine at position 43, serine at position 49, threonine at position 51, serine at position 221, serine at position 225, serine at position 231, threonine at position 239, threonine at position 250, threonine at position 252, serine at position 253, serine at position 254, threonine at position 275, threonine at position 277, serine at position 288, serine at position 289, serine at position 295, and serine at position 296.

[0075] The term "variant," as used herein, refers to a protein or polypeptide sequence that differs from a specifically identified sequence, for example, by the deletion, substitution, or addition of one or more amino acid residues. Variants may be naturally occurring allelic variants or non-naturally occurring variants. Variants may be from the same species or from other species and may include homologs, paralogs, and orthologs. In certain embodiments, variants of polypeptides useful in the present invention have biological activity, including signal peptide activity or antigen-binding properties, that is the same or similar to that of the parent polypeptide. The term "variant" in reference to a polypeptide encompasses all forms of the polypeptide as defined herein.

[0076] Variant polypeptide sequences exhibit at least about 50%, at least about 60%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a sequence of the invention. For polypeptides, identity is observed over a comparison window of at least 297 amino acid positions.

[0077] Polypeptide variants also include those that exhibit similarity to one or more of the specifically identified sequences, including those that could not reasonably be expected to have arisen by chance, and which may preserve the functional equivalence of those sequences.

[0078] Polypeptide sequence identity and similarity can be determined in the following manner: A subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTp (BLAST suite of programs, from version 2.2.18 [April 2008]) in bl2seq, publicly available from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ). The default parameters of bl2seq are used, except that filtering of low-complexity regions should be turned off.

[0079] Polypeptide sequence similarity can be examined using the following UNIX command line parameters: bl2seq -i peptideseq1 -j peptideseq2 -F Fp blastp. The parameter -FF turns off filtering of low complexity segments. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between sequences and, in each such region, reports an "E-value," which is the predicted number of times one would expect to find such a match by chance in a database of a fixed reference size containing random sequences. For small E-values, much less than 1, this is approximately the probability of such a random match. Variant polypeptide sequences generally have a similarity of less than 1 x 10 when compared to any one of the specifically identified sequences. -5 Less than 1×10 -6 Less than 1×10 -9 Less than 1×10 -12 Less than 1×10 -15 Less than 1×10 -18 Less than or 1 x 10 -21The E value is less than 0. Polypeptide sequence identity can also be calculated over the entire length of the overlap between the candidate and subject polypeptide sequences using a global sequence alignment program. As mentioned above, EMBOSS-needle (available at http: / / www.ebi.ac.uk / emboss / align / ) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235) are also suitable global sequence alignment programs for calculating polypeptide sequence identity. The use of BLASTp is preferred for use in determining polypeptide variants of the present invention.

[0080] The term "vector," as used herein, refers to a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes self-replicating plasmids or viruses. The term "vector" should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and the like.

[0081] The acronym "WT" or "WT," as used herein, is intended to mean "wild-type," and when used in the context of (for example) human CD20, is intended to mean the composition of the CD20 protein typically found in situ.

[0082] Mutant (human) CD20-cytoplasmic domain modification The physiological role, regulation, and ligands of CD20 are unknown [9], but functional studies suggest that CD20 is required for efficient B cell receptor signaling

[10] and is directly involved in calcium influx, the proper function of which depends on its association with lipid rafts

[11] .

[0083] CD20 is the target of clinically approved monoclonal antibodies, including rituximab and obinutuzumab, which are routinely used to deplete lymphoid cells in the treatment of B-cell cancers and autoimmune diseases and have an established safety profile.

[0084] Rapid elimination of transfected or transgenic cells may be necessary in the event of significant on-target or off-tissue toxicity. However, binding of rituximab or obinutuzumab can lead to transient activation of CD20-expressing cells via calcium influx and the initiation of intracellular signaling cascades, such as kinase phosphorylation

[12] ,

[60] ,

[61] . This risks paradoxical exacerbation of toxicity by the genetically modified cells, particularly among recipients with defects in complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) due to recent cytotoxic or immunosuppressive drug use, or in patients with underlying immunosuppressive disorders.

[0085] Although the primary mechanisms of anti-CD20 antibody-mediated cell killing are CDC and ADCC

[13] , one proposed mechanism for therapeutic B cell apoptosis is direct cytotoxicity mediated via src family kinases as a result of lipid raft clustering

[14] . Assuming that both the N- and C-termini are located intracellularly (i.e., as cytoplasmic domains), applicants designed a series of CD20 truncation and chimeric proteins in an attempt to abrogate CD20 signaling but retain antibody binding and preserve antibody-dependent apoptosis.

[0086] The first strategy employed was to truncate most of the cytoplasmic domain and combine the cytoplasmic and transmembrane domains of CD20. Significant modifications to the transmembrane 4a (MS4a) protein can affect protein structure and its ability to translocate and correctly associate with the cell membrane.

[0087] Applicants also combined the minimal antibody-binding epitope of CD20 with various signal peptides, transmembrane domains, and cytoplasmic domains known to promote good surface protein trafficking and expression on the cell membrane.

[15] Because truncating the intracellular domain of the epidermal growth factor receptor (EGFR) results in a membrane-bound protein, Applicants first tested the human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) signal peptide and the EGFR transmembrane domain.

[15]

[0088] Next, we combined various lengths of the antibody-binding region of CD20 with the CD28 signal peptide and the transmembrane and cytoplasmic domains from CD28 and contactin-associated protein-like 2 (CASPR2) to generate chimeric proteins, since constructs incorporating CD28 and CASPR2 transmembrane / cytoplasmic domains yield membrane-bound proteins [16, 17].

[0089] The various sequence constructs developed for these initial experiments are shown in Table 1 of Example 2.

[0090] The initial results shown in Figure 1 were unexpected: none of the constructs shown in Table 1 were detectable on the membrane of the HEK293 cell line. However, it is important to note that expression of the transgene encoding CD20 was detected in these cells, as reflected by the production of a green fluorescent protein tag.

[0091] By way of example only, neither the CD20t v4 nor the CD20t v5.3 constructs, in which amino acids from the extracellular domain of CD20 were fused to different signal peptides (e.g., GM-CSFR and CD28) and transmembrane / cytoplasmic domains (e.g., EGFR and CASPR2), resulted in membrane expression in the HEK293 cell line. This is particularly surprising given previous reports in the literature documenting successful transmembrane transport / expression of proteins incorporating these domains [4, 16, 17].

[0092] In response to these unexpected observations, Applicants modified their approach to developing membrane-bound, non-signaling CD20 molecules. See the alternative strategies outlined in Examples 3-6, read in conjunction with Figures 2-5.

[0093] In summary, because CD20 is highly phosphorylated on serine and threonine residues in normal and malignant B cells, and this process is linked to B cell proliferation, our alternative approach focused on targeting phosphorylated residues within the cytoplasmic domain of CD20

[18] . Rituximab binding to CD20 initiates a cascade of signals that may play a role in antibody-mediated cytotoxicity. CD20 associates with lyn, fyn, lck, and p75 / 85 kinases

[19] , and this engagement leads to activation of PLCy via Src family kinases

[20] . The cytoplasmic sequence of CD20 contains 15 serine residues, 11 threonine residues, and no tyrosine residues (see Figure 2). Thus, CD20 has 26 potential phosphorylation sites, although direct evidence for only two has been reported in the literature

[21] .

[0094] Thus, in one aspect of the present invention, (i) at least one mutation that results in truncation to any one or more of amino acid residues 1 to 56 set forth in SEQ ID NO: 1; (ii) at least one mutation that results in truncation to any one or more of amino acid residues 210 to 297 set forth in SEQ ID NO: 1; (iii) at least one mutation to threonine or serine located within amino acid residues 1 to 56 set forth in SEQ ID NO: 1; (iv) at least one mutation to threonine or serine located within amino acid residues 210 to 297 of SEQ ID NO: 1; or (v) A combination including any one of (i) to (iv). 1. A modified human CD20 protein comprising: A modified human CD20 protein is provided, in which any one of (i) to (v) causes a reduction or inhibition of intracellular signaling when the modified CD20 protein binds to or associates with the membrane of a cell.

[0095] In one example of this and all other aspects of the invention, the at least one mutation associated with feature (i) is at the following amino acid residues shown in SEQ ID NO: 1: the first amino acid residue defined by 5'-M-3' (SEQ ID NO: 26), the first and second amino acid residues defined by 5'-MT-3' (SEQ ID NO: 27), the first to third amino acid residues defined by 5'-MTT-3' (SEQ ID NO: 28), the first to fourth amino acid residues defined by 5'-MTTP-3' (SEQ ID NO: 29), the first to fourth amino acid residues defined by 5'-MTTPR-3' (SEQ ID NO: 30). the 1st to 5th amino acid residues defined by 5'-MTTPRN-3' (SEQ ID NO: 31), the 1st to 6th amino acid residues defined by 5'-MTTPRNS-3' (SEQ ID NO: 32), the 1st to 8th amino acid residues defined by 5'-MTTPRNSV-3' (SEQ ID NO: 33), the 1st to 9th amino acid residues defined by 5'-MTTPRNSVN-3' (SEQ ID NO: 34), the 1st to 10th amino acid residues defined by 5'-MTTPRNSVNG-3' (SEQ ID NO: 35) amino acid residues, the 1st to 11th amino acid residues defined by 5'-MTTPRNSVNGT-3' (SEQ ID NO: 36), the 1st to 12th amino acid residues defined by 5'-MTTPRNSVNGTF-3' (SEQ ID NO: 37), the 1st to 13th amino acid residues defined by 5'-MTTPRNSVNGTFP-3' (SEQ ID NO: 38), the 1st to 14th amino acid residues defined by 5'-MTTPRNSVNGTFPA-3' (SEQ ID NO: 39), the 1st to 14th amino acid residues defined by 5'-MTTPRNSVNGTFPAE-3' (SEQ ID NO: 40) the 1st to 15th amino acid residues defined by 5'-MTTPRNSVNGTFPAEP-3' (SEQ ID NO: 41), the 1st to 16th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPM-3' (SEQ ID NO: 42), the 1st to 18th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMK-3' (SEQ ID NO: 43), and the 1st to 19th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKG-3' (SEQ ID NO: 44);The 1st to 20th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGP-3' (SEQ ID NO: 45), the 1st to 21st amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPI-3' (SEQ ID NO: 46), the 1st to 22nd amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIA-3' (SEQ ID NO: 47), the 1st to 23rd amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAM-3' (SEQ ID NO: 48), 5'-MTT The 1st to 24th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQ-3' (SEQ ID NO: 49), the 1st to 25th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQS-3' (SEQ ID NO: 50), the 1st to 26th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSG-3' (SEQ ID NO: 51), the 1st to 27th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGP-3' (SEQ ID NO: 52), amino acid residues, the 1st to 28th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPK-3' (SEQ ID NO: 53), the 1st to 29th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKP-3' (SEQ ID NO: 54), the 1st to 30th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPL-3' (SEQ ID NO: 55), the 1st to 31st amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLF-3' ( the 1st to 31st amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFR-3' (SEQ ID NO: 56), the 1st to 32nd amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFR-3' (SEQ ID NO: 57), the 1st to 33rd amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRR-3' (SEQ ID NO: 58), the 1st to 34th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRM-3' (SEQ ID NO: 59),The 1st to 35th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMS-3' (SEQ ID NO: 60), the 1st to 36th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSS-3' (SEQ ID NO: 61), the 1st to 37th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSL-3' (SEQ ID NO: 62), The 1st to 38th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLV-3' (SEQ ID NO: 63), the 1st to 39th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVG-3' (SEQ ID NO: 64), the 1st to 40th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGP-3' (SEQ ID NO: 65), The 1st to 41st amino acid residues defined by PMKGPIAMQSGPKPLFRRMSSLVGPT-3' (SEQ ID NO: 66), the 1st to 42nd amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQ-3' (SEQ ID NO: 67), the 1st to 43rd amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQS-3' (SEQ ID NO: 68), 1 to 44 amino acid residues defined by TFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSF-3' (SEQ ID NO: 69), 1 to 45 amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFF-3' (SEQ ID NO: 70), 1 to 46 amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFM-3' (SEQ ID NO: 71),The 1st to 47th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMR-3' (SEQ ID NO: 72), the 1st to 48th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRE-3' (SEQ ID NO: 73), the 1st to 49th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRES-3' (SEQ ID NO: 74), the 1st to 50th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESK-3' (SEQ ID NO: 75), the 1st to 51st amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKT-3' (SEQ ID NO: 76), This causes truncation of any one of the following: the 1st to 52nd amino acid residues defined by MQSGPKPLFRRMSSLVGPTQSFFMRESKTL-3' (SEQ ID NO: 77), the 1st to 53rd amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLG-3' (SEQ ID NO: 78), the 1st to 54th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGA-3' (SEQ ID NO: 79), the 1st to 55th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAV-3' (SEQ ID NO: 80), and the 1st to 56th amino acid residues defined by 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQ-3' (SEQ ID NO: 81).

[0096] In another example of this and other aspects of the invention, at least one mutation associated with feature (i) causes truncation of amino acid residues 1 to 50 shown in SEQ ID NO: 1, i.e., 5'-MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESK-3' (SEQ ID NO: 75).

[0097] In one example of this and other aspects of the invention, the at least one mutation associated with feature (i) is at least one of the following amino acid residues shown in SEQ ID NO: 1: amino acid residue 297 defined by 5'-P-3' (SEQ ID NO: 82), amino acid residues 296-297 defined by 5'-SP-3' (SEQ ID NO: 83), amino acid residues 295-297 defined by 5'-SSP-3' (SEQ ID NO: 84), amino acid residues 294-297 defined by 5'-DSSP-3' (SEQ ID NO: 85), amino acid residues 295-297 defined by 5'-NDSSP-3' (SEQ ID NO: 86), amino acid residues 296-297 defined by 5'-NDSSP-3' (SEQ ID NO: 87), amino acid residues 297-297 defined by 5'-NDSSP-3' (SEQ ID NO: 88), amino acid residues 298-297 defined by 5'-NDSSP-3' (SEQ ID NO: 89), amino acid residues 299-300 defined by 5'-NDSSP-3' (SEQ ID NO: 90), amino acid residues 299-301 defined by 5'-NDSSP-3' (SEQ ID NO: 91), amino acid residues 299-302 defined by 5'-NDSSP-3' (SEQ ID NO: 92), amino acid residues 299-303 defined by 5'-NDSSP-3' (SEQ ID NO: 93), amino acid residues 299-304 defined by 5'-NDSSP-3' (SEQ ID NO: 94), amino acid residues 299-305 defined by 5'-NDSSP-3' (SEQ ID NO: 95), amino acid the 293rd to 297th amino acid residues defined by 5'-ENDSSP-3' (SEQ ID NO: 86), the 292nd to 297th amino acid residues defined by 5'-ENDSSP-3' (SEQ ID NO: 87), the 291st to 297th amino acid residues defined by 5'-IENDSSP-3' (SEQ ID NO: 88), the 290th to 297th amino acid residues defined by 5'-PIENDSSP-3' (SEQ ID NO: 89), the 289th to 297th amino acid residues defined by 5'-SPIENDSSP-3' (SEQ ID NO: 90), amino acid residues at positions 288 to 297 defined by 5'-ESSPIENDSSP-3' (SEQ ID NO: 91), amino acid residues at positions 287 to 297 defined by 5'-ESSPIENDSSP-3' (SEQ ID NO: 92), amino acid residues at positions 286 to 297 defined by 5'-QESSPIENDSSP-3' (SEQ ID NO: 93), amino acid residues at positions 285 to 297 defined by 5'-DQESSPIENDSSP-3' (SEQ ID NO: 94), amino acid residues at positions 284 to 297 defined by 5'-QDQESSPIENDSSP-3' (SEQ ID NO: 95) the 7th amino acid residue, the 283rd to 297th amino acid residues defined by 5'-PQDQESSPIENDSSP-3' (SEQ ID NO: 96), the 282nd to 297th amino acid residues defined by 5'-PPQDQESSPIENDSSP-3' (SEQ ID NO: 97), the 281st to 297th amino acid residues defined by 5'-EPPQDQESSPIENDSSP-3' (SEQ ID NO: 98), the 280th to 297th amino acid residues defined by 5'-PEPPQDQESSPIENDSSP-3' (SEQ ID NO: 99),The 279th to 297th amino acid residues defined by 5'-FPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 100), the 278th to 297th amino acid residues defined by 5'-NFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 101), the 277th to 297th amino acid residues defined by 5'-TNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 102), the 276th to 297th amino acid residues defined by 5'-ETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 103), amino acid residues at positions 275 to 297 defined by 5'-TETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 104); amino acid residues at positions 274 to 297 defined by 5'-ETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 105); amino acid residues at positions 273 to 297 defined by 5'-EETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 106); the 272nd to 297th amino acid residues defined by 5'-EEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 107); the 271st to 297th amino acid residues defined by 5'-EEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 108); the 270th to 297th amino acid residues defined by 5'-EEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 109); the 271st to 297th amino acid residues defined by 5'-EEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 110); the 269th to 297th amino acid residues, the 268th to 297th amino acid residues defined by 5'-QEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 111), the 267th to 297th amino acid residues defined by 5'-IQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 112), the 266th to 297th amino acid residues defined by 5'-PIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 113), The 265th to 297th amino acid residues defined by 5'-IPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 114), the 264th to 297th amino acid residues defined by 5'-IIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 115), the 263rd to 297th amino acid residues defined by 5'-EIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 116), EEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 117), the 262nd to 297th amino acid residues defined by 5'-DIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 118), the 261st to 297th amino acid residues defined by 5'-EDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 119), the 260th to 297th amino acid residues defined by 5'-EEDIEIIPIQEEEEEEE The 259th to 297th amino acid residues defined by 5'-ETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 120), the 258th to 297th amino acid residues defined by 5'-NEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 121), the 257th to 297th amino acid residues defined by 5'-KNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 122), and the 5'-PKNEEDIEII the 256th to 297th amino acid residues defined by 5'-QPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 123), the 255th to 297th amino acid residues defined by 5'-QPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 124), the 254th to 297th amino acid residues defined by 5'-SQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 125),Amino acid residues 253 to 297 defined by 5'-SSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 126); The 252nd to 297th amino acid residues defined by 5'-TSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 127), the 251st to 297th amino acid residues defined by 5'-ETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 128), and the 252nd to 297th amino acid residues defined by 5'-TETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 129) ' (SEQ ID NO: 129), the 250 to 297 amino acid residues defined by 5'-LTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 130), the 249 to 297 amino acid residues defined by 5'-GLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 131), the 248 to 297 amino acid residues defined by 5'-VGLTETSSQ The 247th to 297th amino acid residues defined by PKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 132), 5'-VVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 133), and the 246th to 297th amino acid residues defined by 5'-EVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP the 245th to 297th amino acid residues defined by 5'-EEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 134), the 244th to 297th amino acid residues defined by 5'-KEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 135), the 243rd to 297th amino acid residues defined by 5'-KEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 136),The 242nd to 297th amino acid residues defined by 5'-IKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 137), the 241st to 297th amino acid residues defined by 5'-EIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 138), and the 242nd to 297th amino acid residues defined by 5'-IKEEVVGLTETSSQPKNEEDIEIIPIQ The amino acid residues 240 to 297 defined by 5'-TIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 139), the amino acid residues 239 to 297 defined by 5'-QTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIEND the 238th to 297th amino acid residues defined by 5'-EQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 141); the 237th to 297th amino acid residues defined by 5'-EQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 142); the 236th to 297th amino acid residues defined by 5'-KKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 144); the 235th to 297th amino acid residues defined by 5'-EKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 145);The 233rd to 297th amino acid residues defined by 5'-EEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 146), the 232nd to 297th amino acid residues defined by 5'-AEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 147), and the 233rd to 297th amino acid residues defined by 5'-SAEEKKEQTIEIKEEVVGLTETSSQPKNEED The amino acid residues 231 to 297 defined by 5'-LSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 148), the amino acid residues 230 to 297 defined by 5'-LLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSP the 229th to 297th amino acid residues defined by 5'-VLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 150); the 228th to 297th amino acid residues defined by 5'-IVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 152); the amino acid residues at positions 227 to 297 defined by 5'-NIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 153); the amino acid residues at positions 225 to 297 defined by 5'-SNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 154);The 224th to 297th amino acid residues defined by 5'-KSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 155), the 223rd to 297th amino acid residues defined by 5'-PKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 156), and 5'-RPK Amino acid residues 222 to 297 defined by SNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 157), 5'-SRPK, amino acid residues 221 to 297 defined by SNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 158), 5'-CSRPK the amino acid residues 220 to 297 defined by SNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 159), 5'-TCSRPK, the amino acid residues 219 to 297 defined by SNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 160), 5'-RTC the 218th to 297th amino acid residues defined by 5'-SRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 161); the 217th to 297th amino acid residues defined by 5'-KRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 162);amino acid residues 216 to 297 defined by 5'-WKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 163), and amino acid residues 216 to 297 defined by 5'-EWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 164) and amino acid residues 215 to 297 defined by 5'-NEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 165). The amino acid residues 213 to 297 defined by ENDSSP-3' (SEQ ID NO: 166) are 5'-VENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPI, and the amino acid residues 212 to 297 defined by ENDSSP-3' (SEQ ID NO: 167) are 5'-IVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIE This causes truncation of any one of the amino acid residues 211 to 297 defined by 5'-GIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 168) and the amino acid residues 210 to 297 defined by 5'-GIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 169).

[0098] In another example of this and other aspects of the invention, at least one mutation associated with feature (ii) causes a truncation of amino acid residues 253 to 297 of SEQ ID NO: 1, i.e., 5'-SSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 126).

[0099] In yet another example of this and other aspects of the present invention, the at least one mutation to threonine or serine located within amino acid residues 1 to 56 of SEQ ID NO: 1 includes mutations T2, T3, S7, T11, S25, S35, S36, T41, S43, S49, and T51.

[0100] In yet another example, at least one mutation to threonine or serine located within amino acid residues 210 to 297 of SEQ ID NO: 1 includes mutations T83, S221, S225, S231, T239, T250, T252, S253, S254, T275, T277, S288, S289, S295 and S296.

[0101] To reduce the number of potential mutagenesis targets and therefore the changes in protein structure, Applicants employed three different predictors to select potential phosphorylation sites for modification: (i) NetPhos3.1, which uses an entire neural network to predict S, T, or Y phosphorylation sites in eukaryotic proteins

[22] ; (ii) the published crystal structure of the CD20 and rituximab complex

[23] ; and (iii) sequence identity between human and mouse CD20 (BLASTp), based on the hypothesis that important functional residues would be conserved.

[0102] This approach identified seven (7) serine and threonine residues in the N-terminal cytoplasmic domain ("C1") of CD20. These residues are set forth below in SEQ ID NO:11: [ka] In the sequences shown in Figure 1, the sequences are shown in bold and underlined.

[0103] This approach also identified three (3) serine and threonine residues in the C-terminal cytoplasmic domain ("C3") of CD20. These residues are set forth below in SEQ ID NO: 12: [ka] In the sequences shown in Figure 1, the sequences are shown in bold and underlined.

[0104] Thus, in another example of this and other aspects of the invention, the at least one mutation associated with feature (iii) comprises a mutation in at least one amino acid residue selected from T2, T3, S7, T11, S35, S36, and T51 as set forth in SEQ ID NO:1.

[0105] In yet another example of this aspect of the invention, the at least one mutation associated with feature (iv) comprises a mutation of at least one amino acid residue selected from S225, S231, and T239 as set forth in SEQ ID NO:1.

[0106] In yet another example of this and other aspects of the invention, the at least one mutation associated with feature (v) is ·Feature (i) and feature (iv); ·Feature (ii) and feature (iii); · feature (i) and mutation to S225 of SEQ ID NO: 1; · feature (i) and mutation to S231 of SEQ ID NO: 1; · feature (i) and mutation of SEQ ID NO: 1 to T239; · feature (i) and mutations to S225 and S231 of SEQ ID NO: 1; · feature (i) and mutations to S225 and T239 of SEQ ID NO: 1; · feature (i) and mutations to S231 and T239 of SEQ ID NO: 1; · feature (i) and mutations to S225, S231 and T239 of SEQ ID NO: 1; · feature (ii) and mutation of SEQ ID NO: 1 to T2; · feature (ii) and mutation of SEQ ID NO: 1 to T3; · feature (ii) and mutation of SEQ ID NO: 1 to S7; · feature (ii) and mutation of SEQ ID NO: 1 to T11; · feature (ii) and a mutation to S35 of SEQ ID NO: 1; · feature (ii) and mutation to S36 of SEQ ID NO: 1; · feature (ii) and mutation of SEQ ID NO: 1 to T51; · feature (ii) and mutation of SEQ ID NO: 1 to T2 and T3; · feature (ii) and mutation of SEQ ID NO: 1 to T2 and S7; · feature (ii) and mutation of SEQ ID NO: 1 to T2 and T11; · feature (ii) and mutations to T2 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to T2 and S36 of SEQ ID NO: 1; · feature (ii) and mutation of SEQ ID NO: 1 to T2 and T51; · feature (ii) and mutation of SEQ ID NO: 1 to T3 and S7; · feature (ii) and mutation of SEQ ID NO: 1 to T3 and T11; · feature (ii) and mutations to T3 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to T3 and S36 of SEQ ID NO: 1; · feature (ii) and mutation of SEQ ID NO: 1 to T3 and T51; · feature (ii) and mutations to S7 and T11 of SEQ ID NO: 1; · feature (ii) and mutations to S7 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to S7 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to S7 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T11 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to T11 and S36 of SEQ ID NO: 1; · feature (ii) and mutations of SEQ ID NO: 1 to T11 and T51; · feature (ii) and mutations to S35 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to S35 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to S36 and T51 of SEQ ID NO: 1; · feature (ii) and mutations of SEQ ID NO: 1 to T2, T3 and S7; · feature (ii) and mutation of SEQ ID NO: 1 to T2, T3 and T11; · feature (ii) and mutations to T2, T3 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to T2, T3 and S36 of SEQ ID NO: 1; · feature (ii) and mutations of SEQ ID NO: 1 to T2, T3 and T51; · feature (ii) and mutations of SEQ ID NO: 1 to T2, S7 and T11; · feature (ii) and mutations to T2, S7 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to T2, S7 and S36 of SEQ ID NO: 1; · feature (ii) and mutations of SEQ ID NO: 1 to T2, S7 and T51; · feature (ii) and mutations to T2, T11 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to T2, T11 and S36 of SEQ ID NO: 1; · feature (ii) and mutations of SEQ ID NO: 1 to T2, T11 and T51; · feature (ii) and mutations to T2, S35 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to T2, S35 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T2, S36 and T51 of SEQ ID NO: 1; · feature (ii) and mutations of SEQ ID NO: 1 to T3, S7 and T11; · feature (ii) and mutations to T3, S7 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to T3, S7 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to T3, S7 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T3, T11 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to T3, T11 and S36 of SEQ ID NO: 1; · feature (ii) and mutations of SEQ ID NO: 1 to T3, T11 and T51; · feature (ii) and mutations to T3, S35 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to T3, S35 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T3, S36 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to S7, T11 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to S7, T11 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to S7, T11 and T51 of SEQ ID NO: 1; feature (ii) and mutations to S7, S35 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to S7, S35 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to S7, S36 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T11, S35 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to T11, S35 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T11, S36 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to S35, S36 and T51 of SEQ ID NO: 1; · feature (ii) and mutations of SEQ ID NO: 1 to T2, T3, S7 and T11; · feature (ii) and mutations to T2, T3, S7 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to T2, T3, S7 and S36 of SEQ ID NO: 1; · feature (ii) and mutations of SEQ ID NO: 1 to T2, T3, S7 and T51; · feature (ii) and mutations to T2, T3, T11 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to T2, T3, T11 and S36 of SEQ ID NO: 1; · feature (ii) and mutations of SEQ ID NO: 1 to T2, T3, T11 and T51; · feature (ii) and mutations to T2, T3, S35 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to T2, T3, S35 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T2, T3, S36 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T2, S7, T11 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to T2, S7, T11 and S36 of SEQ ID NO: 1; · feature (ii) and mutations of SEQ ID NO: 1 to T2, S7, T11 and T51; · feature (ii) and mutations to T2, S7, S35 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to T2, S7, S35 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T2, S7, S36 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T2, T11, S35 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to T2, T11, S35 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T2, T11, S36 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T2, S35, S36 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T3, S7, T11 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to T3, S7, T11 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to T3, S7, T11 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T3, S7, S35 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to T3, S7, S35 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T3, S7, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T3, T11, S35 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to T3, T11, S35 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T3, T11, S36 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T3, S35, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to S7, T11, S35 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to S7, T11, S35 and T51 of SEQ ID NO: 1; feature (ii) and mutations to S7, T11, S36 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to S7, S35, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T11, S35, S36 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T2, T3, S7, T11 and S35 of SEQ ID NO: 1; · feature (ii) and mutations to T2, T3, S7, T11 and S36 of SEQ ID NO: 1; feature (ii) and mutations of SEQ ID NO: 1 to T2, T3, S7, T11 and T51; · feature (ii) and mutations to T2, T3, S7, S35 and S36 of SEQ ID NO: 1; feature (ii) and mutations to T2, T3, S7, S35 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T2, T3, S7, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T2, T3, T11, S35 and S36 of SEQ ID NO: 1; feature (ii) and mutations to T2, T3, T11, S35 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T2, T3, T11, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T2, T3, S35, S36 and T51 of SEQ ID NO: 1; · feature (ii) and mutations to T2, S7, T11, S35 and S36 of SEQ ID NO: 1; feature (ii) and mutations to T2, S7, T11, S35 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T2, S7, T11, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T2, S7, S35, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T2, T11, S35, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T3, S7, T11, S35 and S36 of SEQ ID NO: 1; · feature (ii) and mutations to T3, S7, T11, S35 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T3, S7, T11, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T3, S7, S35, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T3, T11, S35, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to S7, T11, S35, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T2, T3, S7, T11, S35 and S36 of SEQ ID NO: 1; feature (ii) and mutations to T2, T3, S7, T11, S35 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T2, T3, S7, T11, S35 and S36 of SEQ ID NO: 1; feature (ii) and mutations to T2, T3, S7, T11, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T2, T3, S7, S35, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T2, T3, T11, S35, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T2, S7, T11, S35, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1; feature (ii) and mutations to T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1; and Features (iii) and (iv) and any of the subcombinations presented immediately above.

[0107] In yet another example of this and other aspects of the present invention, the at least one mutation to a serine or threonine amino acid residue located within amino acid residues 1 to 56 and 210 to 297, respectively, as set forth in SEQ ID NO: 1, comprises a substitution mutation, a deletion mutation, or an insertion mutation.

[0108] In related examples of this and other aspects of the invention, at least one mutation to serine or threonine located within amino acid residues 1-56 and 210-297, respectively, set forth in SEQ ID NO: 1 comprises a conservative or non-conservative substitution, including a naturally occurring or non-naturally occurring amino acid residue.

[0109] Examples of naturally occurring amino acids include, but are not limited to, alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamate (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

[0110] Examples of unnatural amino acids are reviewed in (47).

[0111] Specific examples of modified CD20 proteins that, when expressed by cells, reduce or abrogate intracellular signaling are outlined in Table 2 of Example 3. Specific examples of sequences include, but are not limited to, SEQ ID NOs: 2-7, including combinations of truncations and / or serine / threonine substitution mutations described herein.

[0112] Thus, in yet another aspect, the present invention provides a modified CD20 protein comprising or consisting of a sequence as set forth in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, or a variant sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, respectively.

[0113] From the data shown in Example 3 and Figure 3, C1C3 (SEQ ID NO: 7) and C1C 252 (SEQ ID NO: 5) mutant proteins bound or associated with the membranes of both HEK293 cells and T cell lines at levels similar to those observed for the wild-type CD20 protein (SEQ ID NO: 1). 252 (SEQ ID NO: 5) variant was carried forward for further analysis in cell signaling assays.

[0114] These data are presented in Examples 7 and 8 and demonstrate that phosphorylation of CD20 + This further reflects the potential for important roles in membrane trafficking and intracellular signaling pathways in cells. Specifically, the differential phosphorylation of all measured proteins and the Ca 2+ In the HG3 CD20KO cell line, C1C3 (SEQ ID NO: 7) or C1C 252 Rituximab stimulation of (SEQ ID NO: 5) induced impaired signal transduction (FIGS. 7 and 8).

[0115] Thus, in a further aspect, the present invention provides a method for treating a vascular endothelial cell comprising: (i) at least one mutation to threonine or serine located within amino acid residues 1 to 56 set forth in SEQ ID NO: 1; and (ii) truncation of amino acid residues 253 to 297 shown in SEQ ID NO: 1 1. A modified human CD20 protein comprising: The present invention provides a modified human CD20 protein, wherein at least one mutation defined by (i) and (ii) causes reduced or abrogated intracellular signaling when the modified CD20 protein is bound to or associated with the membrane of a cell.

[0116] In a still further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) at least one mutation to threonine or serine located within amino acid residues 1 to 56 set forth in SEQ ID NO: 1; and (ii) at least one mutation to threonine or serine located within amino acid residues 210 to 297 of SEQ ID NO: 1; 1. A modified human CD20 protein comprising: The present invention provides a modified human CD20 protein, wherein at least one mutation defined by (i) and (ii) causes reduced or abrogated intracellular signaling when the modified CD20 protein is bound to or associated with the membrane of a cell.

[0117] Mutant (human) CD20 - Cytoplasmic and extracellular loop domain modifications Preliminary data presented in Examples 4 and 5, read in conjunction with Figures 4 and 5, indicate that mutations to predicted phosphorylation sites enable rituximab-induced lethality in the HG3 cell line and primary T cells expressing the modified human CD20 proteins described herein. Thus, transgenes incorporating nucleic acids encoding the modified human CD20 proteins of the invention incorporated for expression in (for example) CAR T cell constructs may be utilized as genetic biomarkers and / or safety / suicide switches to avoid problems with cytotoxicity in patients receiving CAR T cell therapy.

[0118] Thus, the modified human CD20 proteins of the present invention may be further modified in the extracellular loops to induce changes in the epitope recognition sequences targeted by clinically approved monoclonal antibodies, including but not limited to, rituximab, obinutuzumab, and ocrelizumab, to engineer selective binding (or exclusion of binding) by these therapeutic molecules. See Example 10.

[0119] Thus, in yet another aspect of the present invention, (i) at least one mutation that results in truncation to any one or more of amino acid residues 1 to 56 set forth in SEQ ID NO: 1; (ii) at least one mutation that results in truncation to any one or more of amino acid residues 210 to 297 set forth in SEQ ID NO: 1; (iii) at least one mutation to threonine or serine located within amino acid residues 1 to 56 set forth in SEQ ID NO: 1; (iv) at least one mutation to threonine or serine located within amino acid residues 210 to 297 set forth in SEQ ID NO: 1; (v) a combination comprising any one of (i) to (iv); and (vi) at least one mutation to an amino acid within amino acid residues 142 to 188 of SEQ ID NO: 1 1. A modified human CD20 protein comprising: A modified human CD20 protein is provided, in which any one of (i) to (v) causes a reduction or inhibition of intracellular signaling when the modified CD20 protein binds to or associates with the membrane of a cell.

[0120] In one example of this aspect of the present invention, the at least one mutation to an amino acid within amino acid residues 142 to 188 of SEQ ID NO: 1 includes, but is not limited to, N173, S174, and N176.

[0121] The data presented in Examples 3-5 and referred to immediately above when read in conjunction with the data presented in Figure 7 identify specific constructs that meet this objective, namely, C1-C3 and C1-C4, in addition to mutations to the extracellular loop domains. 252 This facilitates the identification of constructs that incorporate mutations in the cytoplasmic domain defined by

[0122] Thus, in a still further aspect, the present invention provides a method for treating a vascular endothelial cell comprising: (i) at least one mutation to T2, T3, S7, T11, S35, S36, and T51 as set forth in SEQ ID NO: 1; (ii) at least one mutation to N173, S174, and N176 as set forth in SEQ ID NO: 1; and (iii) truncation of amino acid residues 253 to 297 shown in SEQ ID NO: 1 The present invention provides a modified human CD20 protein comprising:

[0123] In yet another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) at least one mutation to T2, T3, S7, T11, S35, S36, and T51 as set forth in SEQ ID NO: 1; (ii) at least one mutation to N173, S174, and N176 as set forth in SEQ ID NO: 1; and (iii) at least one mutation to S225, S231, and T239 as set forth in SEQ ID NO: 1 The present invention provides a modified human CD20 protein comprising:

[0124] Expression of mutant (human) CD20 The modified human CD20 proteins of the present invention may be expressed by one or more cells, alone or within a cell population, for utility (for example) as a transgene selectable marker comprising the modified human CD20 protein, or as a suicide / safety switch to manage adverse effects in therapies involving gene transfer or transfection of cells, or as a marker to facilitate detection of modified cells within a recipient of the therapy.

[0125] Thus, in yet another aspect, the invention provides a cell expressing a modified human CD20 protein described herein, wherein the modified human CD20 protein is translocated to, bound to, or associated with the membrane of the cell.

[0126] In one example of this and other aspects of the invention, the cells are selected from T cells, natural killer (NK) cells, B cells, myeloid cells, pluripotent stem cells, non-hematopoietic cell lines, and hematopoietic stem cells.

[0127] The present invention further contemplates specific cell lines, such as hematopoietic stem cell lines or leukemia-derived cell lines, that have been modified to express the human CD20 protein described herein. For example, one method of generating an "off-the-shelf" CAR therapy involves taking a non-hematopoietic cell line (e.g., K562) and genetically modifying it to reduce alloreactivity and express a CAR as a therapeutic agent.

[0128] Nucleic acid encoding mutant human CD20 Nucleic acids, (for example) deoxyribonucleic acid (DNA), messenger ribonucleic acid (mRNA) or complementary deoxyribonucleic acid (cDNA), encoding the modified human CD20 proteins described herein are also contemplated by the present invention.

[0129] Accordingly, in yet another aspect, the invention provides an isolated nucleic acid molecule encoding any modified human CD20 protein described herein.

[0130] Methods for introducing nucleic acids into cells include physical, biological, and chemical methods. Physical methods for introducing polynucleotides such as RNA into host cells include calcium phosphate precipitation, lipofection, biolistics, microinjection, and electroporation. Commercially available methods, including electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM830(BTX) (Harvard Instruments, Boston, Massachusetts) or Gene Pulser II (BioRad, Denver, Colorado), and Multiporator (Eppendort, Hamburg, Germany) can be used to introduce RNA into target cells. RNA can also be introduced into cells using cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or biolistics particle delivery systems such as "gene guns" (e.g.,

[24] ).

[0131] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, and adeno-associated viruses, etc. (e.g., [48, 49]).

[0132] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0133] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristoyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the occurrence of enclosed lipid bilayers or aggregates. Liposomes can be characterized as vesicular structures with a phospholipid bilayer and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of a closed structure, encapsulating water and dissolved solutes between the lipid bilayers.

[50] However, compositions with structures in solution that differ from the usual vesicular structure are also encompassed. For example, lipids may assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0134] Regardless of the method used to introduce exogenous nucleic acid into a host cell, various assays can be performed, including "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR, and "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the present invention.

[0135] In one example, the nucleic acid introduced into a cell (e.g., a T cell) is RNA. In another example, the RNA is mRNA, including in vitro transcribed RNA or synthetic RNA. RNA is generated by in vitro transcription using a template generated by polymerase chain reaction (PCR). DNA of interest from any source can be directly converted into a template for in vitro mRNA synthesis by PCR using appropriate primers and RNA polymerase. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable source of DNA. A desired template for in vitro transcription is a modified membrane protein or chimeric membrane protein, such as the modified human CD20 protein described herein.

[0136] PCR can be used to generate templates for in vitro transcription of mRNA, which is then introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have a region substantially complementary to a region of DNA used as a template for PCR. "Substantially complementary," as used herein, refers to a sequence of nucleotides in which most or all of the bases in the primer sequence are complementary, or in which one or more bases are non-complementary or mismatched. A substantially complementary sequence is capable of annealing or hybridizing with the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify a portion of a gene that is normally transcribed in cells (open reading frame), including the 5' and 3' UTRs. Primers can also be designed to amplify a portion of a gene encoding a specific domain of interest. In one example, primers are designed to amplify the coding region of a human cDNA, including all or part of the 5' and 3' UTRs. Primers useful for PCR are generated by synthetic methods well known in the art. A "forward primer" is a primer that contains a region of nucleotides upstream of the DNA sequence to be amplified that are substantially complementary to nucleotides on a DNA template. "Upstream" is used herein to refer to the 5' side (location 5) of the DNA sequence to be amplified, relative to the coding strand. A "reverse primer" is a primer that contains a region of nucleotides downstream of the DNA sequence to be amplified that are substantially complementary to a double-stranded DNA template. "Downstream" is used herein to refer to the 3' side of the DNA sequence to be amplified, relative to the coding strand.

[0137] Chemical structures capable of enhancing RNA stability and / or translation efficiency may also be used. The RNA preferably has 5' and 3' UTRs. In one example, the 5' UTR is between 0 and 3,000 nucleotides in length. The length of the 5' and 3' UTR sequences added to the coding region can be varied by various methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTR. Using this approach, one skilled in the art can modify the lengths of the 5' and 3' UTRs necessary to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0138] The 5' and 3' UTRs may be the native, endogenous 5' and 3' UTRs of the gene of interest. Alternatively, a non-endogenous UTR sequence may be added to the gene of interest by incorporating the UTR sequence into the forward and reverse primers or by any other modification of the template. The use of a non-endogenous UTR sequence to the gene of interest may be useful for modifying RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in the 3' UTR sequence can reduce mRNA stability. Therefore, the 3' UTR may be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs known in the art.

[0139] In one example, the 5'UTR may contain the Kozak sequence of the endogenous gene. Alternatively, if a 5'UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence may be redesigned by adding a 5'UTR sequence. While the Kozak sequence may increase the efficiency of translation of some RNA transcripts, it does not appear to be necessary for all RNAs to enable efficient translation. The requirement for a Kozak sequence for many mRNAs is known in the art. In another example, the 5'UTR may be derived from an RNA virus whose RNA genome is stable in cells. In another example, various nucleotide analogs may be used in the 3' or 5'UTR to prevent exonuclease degradation of the mRNA.

[0140] To enable RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter should be attached to the DNA template upstream of the sequence to be transcribed. If a sequence that functions as an RNA polymerase promoter is added to the 5' end of the forward primer, the RNA polymerase promoter will be incorporated into the PCR product upstream of the open reading frame to be transcribed. In one example, as described elsewhere herein, the promoter is a T7 polymerase promoter. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3, and SP6 promoters are known in the art.

[0141] In one example, mRNA has both a 5'-end cap and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability in cells. On circular DNA templates, such as plasmid DNA, RNA polymerase generates long concatemeric products that are not suitable for expression in eukaryotic cells. Even when post-transcriptionally polyadenylated, transcription of plasmid DNA linearized at the end of the 3' UTR generates mRNA of a normal size that is not effective in eukaryotic transfection.

[0142] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (e.g., [25, 26]).

[0143] The traditional method for incorporating a polyA / T stretch into a DNA template is molecular cloning. However, polyA / T sequences incorporated into plasmid DNA can cause plasmid instability, and therefore, plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other abnormalities. This makes the cloning procedure not only tedious and time-consuming, but also often unreliable. This is why a method that allows the construction of DNA templates with a polyA / T 3' stretch without cloning is highly desirable.

[0144] Poly(A) / T segments of transcription DNA templates can be generated during PCR by using a reverse primer containing a poly(T) tail, such as a 100T tail (which can range in size from 50 to 5000T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also confer stability to RNA and reduce RNA degradation. Generally, the length of the poly(A) tail positively correlates with the stability of the transcribed RNA. In one example, the poly(A) tail is between 100 and 5000 adenosines.

[0145] The poly(A) tail of RNA can be further extended after in vitro transcription using a poly(A) polymerase, such as Escherichia coli (E. coli) poly(A) polymerase (E-PAP). In one example, increasing the length of the poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides increases the RNA translation efficiency by approximately two-fold. Furthermore, attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachments can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase RNA stability.

[0146] The 5' cap also provides stability to the RNA molecule. In a preferred example, the RNA produced by the methods disclosed herein includes a 5' cap. The 5' cap is provided using techniques known in the art and described herein (e.g.,

[59] ).

[0147] The RNA produced by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and facilitates translation initiation. Any solute suitable for cell electroporation may be included, which may contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and detergents.

[0148] In some examples, RNA encoding the transgene is electroporated into the cell. In one example, the RNA encoding the transgene is in vitro transcribed RNA.

[0149] The method also provides the ability to control the level of expression over a wide range, for example, by varying the promoter or the amount of input RNA, allowing expression levels to be individually controlled. Furthermore, PCR-based techniques for mRNA production greatly facilitate the design of mRNAs with different structures and combinations of their domains.

[0150] One advantage of RNA transfection methods is that they are essentially transient and vector-free. RNA transgenes can be delivered to and expressed in lymphocytes after a short period of in vitro cell activation as a minimal expression cassette without the need for any additional viral sequences. Under these conditions, the likelihood of transgene integration into the host cell genome is low. Due to the efficiency of RNA transfection and its ability to uniformly modify the entire lymphocyte population, cell cloning is not required.

[0151] Genetic modification of cells with in vitro transcribed RNA (IVT-RNA) utilizes two different strategies, both of which have been successively tested in various animal models: Cells are transfected with the in vitro transcribed RNA by lipofection or electroporation. To achieve long-term expression of the transferred IVT-RNA, it is desirable to stabilize the IVT-RNA using various modifications.

[0152] Several IVT vectors are known in the literature and are used in a standardized manner as templates for in vitro transcription, genetically modified to produce stabilized RNA transcripts. Currently, protocols used in the art are based on a plasmid vector with the following structure: a 5' RNA polymerase promoter that enables RNA transcription, followed by a gene of interest flanked by untranslated regions (UTRs) on either the 3' and / or 5' ends, and a 3' polyadenylation cassette containing 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylation cassette by a type II restriction enzyme (the recognition sequence corresponds to the cleavage site). Thus, the polyadenylation cassette corresponds to the subsequent poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization, extending or masking the poly(A) sequence at the 3' end.

[0153] Gene expression from RNA sources does not require transcription, and protein products are rapidly produced after transfection. Furthermore, RNA only needs to access the cytoplasm, not the nucleus, thus allowing for extremely high transfection rates with typical transfection methods. Furthermore, plasmid-based approaches require that the promoter driving the expression of the gene of interest be active in the cells under study.

[0154] In another embodiment, RNA constructs are delivered to cells by electroporation. See, for example, the formulations and methodologies for electroporating nucleic acid constructs into mammalian cells taught in

[53] . The various parameters, including the electric field strength, required for electroporation of any known cell type are generally known in the relevant research literature and numerous patents and applications in the art

[54] . Devices for therapeutic applications of electroporation are commercially available, e.g., the MedPulser™ DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, CA), and are described in patents such as

[55] ; electroporation can also be used for in vitro cell transfection, e.g., as described in

[56] . Electroporation can also be used to deliver nucleic acids to cells in vitro. Thus, electroporation-mediated administration of nucleic acids, including expression constructs, to cells using any of the many available devices and electroporation systems known to those skilled in the art provides an exciting new means for delivering RNA of interest to target cells.

[0155] The invention is further described with reference to the following examples, it will be recognized that the invention as claimed is in no way intended to be limited by these examples. [Example]

[0156] Example 1: Methodology Plasmid constructs and bacterial strains All gene fragments were designed in silico using Benchling (Benchling, 2022) and synthesized de novo by IDT (Integrated DNA Technologies, Inc.). EF1 αFragments for testing in human cell lines or primary cells were cloned into the LeGO plasmid backbone

[27] using PmeI / BamHI restriction enzymes (New England Biolabs) under the promoter. Fragments for testing in mouse cell lines or primary cells were cloned into the pmX_GFP plasmid backbone using XhoI / PacI enzymes. Plasmids were propagated in chemically competent Stable3 E. coli (F - mcrB mrrhsdS20(r B - , m B - )recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20(Str R )xyl-5 λ - leumtl-1) (ThermoFisher Scientific).

[0157] Cell lines and primary cells HEK293 HEK293 cell lines for CD20 truncation / mutation screening and lentiviral packaging were obtained from Thermofisher Scientific.

[0158] HG3 The HG3 B cell line CD20 knockout (CD20KO), along with the CD20+HG3 control cell line, were generated in the laboratory of Michal Smida at the Central European University of Technology and kindly provided.

[0159] Platinum-E Platinum-E cells, a retroviral packaging cell line, were obtained from Cell Biolabs.

[0160] Human primary T cells Primary T cells were isolated from buffy coats obtained from the New Zealand Blood Services (University of Otago Ethics Committee (Health) H20-173). Peripheral blood mononuclear cells (PBMCs) were isolated using SepMate columns and Lymphoprep (Stemcell Technologies), and T cells were selected and activated for 24 hours using anti-CD3 / CD28 Dynabeads (Thermo Fisher Scientific, 11141D) prior to transduction.

[0161] HEK295FT transfection For all experimental screening for CD20 detection on the cell membrane, HEK293 cells were transfected with the LeGO_CD20 construct using Lipofectamine 3000 (ThermoFisher Scientific, L3000015).

[0162] Lentivirus generation Lentiviral particles were packaged in HEK293 cells transfected with LeGO_CD20 and the third-generation lentiviral packaging / structural plasmids pMD2-G, pMDLg, and pRSV (Adgene). 48 hours after transfection, lentiviral particles were collected from the growth medium by centrifugation and frozen at -70°C until use.

[0163] Human B cell transduction The HG3 cell line was transduced with lentiviral particles containing CD20 phospho-mutations / truncations by spinfection using polybrene (Sigma-Aldrich, TR-1003-G).

[0164] Flow cytometry All flow cytometry experiments were performed on a 5-laser (UV-VB-YG-R) Cytek Aurora flow cytometer.

[0165] Detection of surface CD20 Surface CD20 was detected using 2H7 conjugated to APC / Fire750 (Biolegend, 302357), rituximab conjugated to Alexa Fluor 405 (Novus Biologicals), obinutuzumab conjugated to APC (Leinco, LT907), or BD Quantibrite™ PE mouse anti-human CD20 (L27-PE) (BD Biosciences, 347201).

[0166] In vitro CD20-mediated lethality CD20KO HG3 cells and CD20WT, C1C3, or C1C 252 Construct-transduced CD20KO HG3 cells were incubated in a U-bottom 96-well plate with 10 μg rituximab (Abcam, ab275973) per mL of serum-free RPMI for 30 minutes at 37°C. Cells were washed to remove unbound rituximab and incubated with 25% baby rabbit complement (BioRad, C12CA) diluted in RPMI for 1.5 hours. Cells were then stained with Zombie NIR (Biolegend, BIO0423105), and cell viability was assessed by flow cytometry.

[0167] Mouse T cell transduction pmX_hCD20WT-GFP, pmX_C1C3-GFP or pmX_C1C 252 Retroviruses containing -GFP were packaged in Platinum-E cells and transfected into C57 mice (B6.SJL-Ptprc a , CD45.2+). Five million transduced cells were used to transduce T cells isolated from each mouse (B6.SJL-Ptprc b , CD45.1+) were intravenously transferred into a control group, which received sham-transduced cells. Each group contained five mice.

[0168] In vivo CD20+ T cell depletion One day after adoptive transfer, hCD20WT-GFP, C1C3-GFP, C1C 252The hCD20WT-GFP and sham groups were intraperitoneally injected with 500 μg of 2H7 mouse anti-human CD20 antibody (BioXCell, BE0276), followed by a second injection of 250 μg of 2H7 the following day. As a control, one group of mice receiving hCD20WT-GFP T cells received the same dose of isotype control antibody (BioXCell, BE0086). One day after the second dose, up to 200 μL of blood was collected from each mouse (Animal Ethics Committee 30148). Lymphocytes were purified and stained with anti-CD45.1-APC Fire750 (Biolegend, 110752), anti-CD19-EF450 (eBioscience, 48-0193-82), anti-TCRβ-PE (BD, 553172), and Zombie NIR (Biolegend, BIO0423105). GFP production was used as a surrogate for hCD20 expression. Cells were analyzed by flow cytometry to assess the percentage of adoptively transferred GFP+ T cells.

[0169] CD20+ cell sorting WT, C1C3 and C1C 252 CD20-transduced CD20KO HG3 cells were selected using the EasySep™ PE Positive Selection Kit according to the manufacturer's instructions. Briefly, nonspecific antibody binding sites were blocked, and cells were incubated with BD™ PE mouse anti-human CD20. After addition of the selection cocktail, samples were incubated with RapidSpheres™ and subjected to magnetic separation in an EasyEights™ magnet. Selected cells were expanded in RPMI medium supplemented with 10% FBS and 1% penicillin / streptomycin (complete RPMI) at 37°C and 5% CO2 and frozen in liquid nitrogen until use.

[0170] Assessment of CD20-mediated signaling Ca 2+ Flux assay Intracellular Ca2+ induced by rituximab cross-linking 2+Mobilization was measured by flow cytometry using a calcium flux assay kit (Abcam, 233472). 252 CD20KO HG3 cells transduced with and without Zombie NIR and 520AM Ca 2+ The cells were stained with the dye for 10 minutes at 37°C. The cells were washed, nonspecific antibody binding sites were blocked, and then incubated with 30 μg / mL rituximab (RIXIMYO®) for 30 minutes. Unbound rituximab was washed off, and the cells and buffer were left at 37°C for 45 minutes. Basal Ca 2+ The associated fluorescence intensity was measured, and 200 μg / mL of goat anti-human IgG hyper-crosslinking antibody (Invitrogen, 62-8400) was added to measure the peak Ca 2+ The associated fluorescence intensity was measured.

[0171] CD20-mediated phosphorylation One million HG3 CD20KO cells transduced with CD20 mutants were stimulated with 10 μg / mL rituximab (RIXIMYO, 2583917) for 24 hours. CD20-mediated phosphorylation was assessed using the Proteome Profiler Human Phosphokinase Array Kit (R&D systems, ARY003C), and pixel intensity of each array dot was analyzed using Image Lab software according to the manufacturer's instructions.

[0172] statistical analysis Data were analyzed using GraphPad Prism 10. Values ​​are expressed as mean ± standard error of the mean (SEM). Statistical significance was assessed using a multiple comparison two-way ANOVA and was considered at p < 0.05.

[0173] Example 2: Truncated / chimeric CD20 not detectable on the cell membrane The initial strategies for designing modified CD20 molecules with abrogated intracellular signaling were to (i) truncate most of the cytoplasmic domain and (ii) combine the cytoplasmic and transmembrane domains of CD20. The various sequence constructs developed for these initial experiments are shown in Table 1 and include constructs designated CD20t, CD20raft, CD20loop, CD20raft v2, CD20loop v2, CD20t v3.1, and CD20t v3.2.

[0174] Because significant modifications to the transmembrane 4a (MS4a) protein can affect protein structure and its ability to translocate and correctly bind or associate with the cell membrane, Applicants sought to examine the effects of truncating various CD20 domains. [Table 2] TIFF2025540894000006.tif149161

[0175] Applicants also combined the minimal antibody-binding epitope of CD20 with various signal peptides, transmembrane domains, and cytoplasmic domains known to promote good surface protein trafficking and expression.

[15] Because truncating the intracellular domain of EGFR results in a membrane-bound protein, Applicants first tested the human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) signal peptide and epidermal growth factor receptor (EGFR) transmembrane domain.[4] See constructs designated CD20vt v4 and CD20t v5.1.

[0176] Applicants then combined various lengths of the antibody-binding region of CD20 with the CD28 signal peptide and the transmembrane and cytoplasmic domains from CD28 and contactin-associated protein-like 2 (CASPR2) to generate chimeric proteins, since constructs incorporating CD28 and CASPR2 transmembrane / cytoplasmic domains yield membrane-bound proteins [16, 17]. See constructs designated CD20t v5.2, CD20t v5.3, and CD20t v5.4.

[0177] Referring to the data shown in Figure 1, no truncated or chimeric CD20 molecules were detected on the HEK293 cell membrane, but the CD20 gene was expressed as reflected by the production of green fluorescent protein.

[0178] Example 3: Impaired phosphorylation of CD20 is detected on the cell membrane Since no truncated or chimeric forms of CD20 were detected on the HEK293 cell membrane, Applicants modified their approach to produce a membrane-bound, non-signaling CD20 molecule.

[0179] In normal and malignant B cells, CD20 is highly phosphorylated on serine and threonine residues, a process linked to B cell proliferation

[18] . Rituximab binding to CD20 initiates a cascade of signals that may play a role in antibody-mediated cell killing. CD20 associates with lyn, fyn, lck, and p75 / 85 kinases

[19] , and this engagement leads to activation of PLCy via src family kinases

[20] .

[0180] Although reports regarding the function of CD20 are conflicting, it seems clear that phosphorylation and / or association with kinases are important mechanisms for its activation; therefore, CD20 molecules with mutated phosphorylation sites will be unable to signal but will retain the antibody-binding domain.

[0181] Most phosphorylation events in eukaryotic cells occur at serine (S), threonine (T), and tyrosine (Y) residues

[28] . The cytoplasmic sequence of CD20 contains 15 serine, 11 threonine, and no tyrosine residues (Figure 2). Therefore, CD20 has 26 potential phosphorylation sites, but direct evidence exists for only two

[21] . To reduce the number of modifications introduced and therefore the changes in protein structure, we employed three different predictors to select potential phosphorylation sites for modification: (i) NetPhos3.1, which uses a neural network ensemble to predict S, T, or Y phosphorylation sites in eukaryotic proteins

[22] ; (ii) the published crystal structure of the CD20 and rituximab complex

[21] ; and (iii) sequence identity between human CD20 and mouse CD20 (BLASTp), based on the assumption that important functional residues would be conserved.

[0182] The combination of these methods led to the identification of putative phosphorylation sites in cytoplasmic domains 1 and 3 (Fig. 2).

[0183] Ten amino acids that were phosphorylated in the crystal structure and / or identified by both the NetPhos predicted and mouse sequences were substituted with alanine (A), as this removes the amino acid side chain but does not alter the main-chain conformation or cause major electrostatic or steric changes

[29] .

[0184] CD20 variants containing these phosphomutations and conservative truncations, designed by Julie Deans and colleagues

[19] , were generated to study molecular interactions with CD20. See Table 2 below. [Table 3] TIFF2025540894000008.tif106169

[0185] HEK293 cells were transfected with mutated / truncated CD20 contained in the LeGO plasmid backbone. All combinations of phosphomutated and conservatively truncated CD20 versions were detected on the HEK293 cell membrane at various levels of expression. Applicants then transduced primary T cells using lentiviruses containing the phosphomutated / truncated CD20 transgenes (Figure 3).

[0186] All truncated / mutated CD20 molecules were detected on the HEK293 cell membrane (Fig. 3), but C 252 (SEQ ID NO: 9) and N 51 (SEQ ID NO: 8) protein was not detected on the surface of primary T cells, and truncation of both cytoplasmic chains resulted in N 51 C 252 (SEQ ID NO: 10). Surprisingly, when the single truncations were combined with the C1 and C3 mutations, respectively, membrane detection was restored (FIG. 3).

[0187] C1C3 (SEQ ID NO: 7) and C1 to C 252 Further studies focused on evaluating these two CD20 variants, as the cells producing the protein have membrane levels similar to CD20WT.

[0188] Example 4: Mutations to predicted phosphorylation sites enable rituximab-mediated CDC in CD20 mutant-transduced HG3 cells in vitro LeGO_CD20WT, LeGO_C1C3 or LeGO_C1C 252CD20KO HG3 cells transduced with CD20KO and sham-transduced controls were incubated with rituximab and CDC was induced using baby rabbit complement. Rituximab mediated significant CD20-specific cytotoxicity that was unaffected by mutations in predicted phosphorylation sites or truncation of cytoplasmic domain 3 (Figure 4A). Differences in transduction efficiency for each mutant and CD20WT (Figure 4B) accounted for the differences in cytotoxicity, with less than 100% cytotoxicity observed.

[0189] Example 5: Rituximab surrogates hCD20WT+, C1C3 and C1C 252 -Specifically deplete T cells in vivo hCD20WT-GFP, C1C3-GFP, C1C 252 hCD20WT-GFP mouse T cells were adoptively transferred into mice. Administration of the anti-CD20 antibody 2H7, a suitable rituximab surrogate for use in mice, mediated a significant reduction (approximately 65%) of the hCD20+ cell population in all mice compared to mice treated with the control antibody. Importantly, the depleted fraction consisted of the highest transgene-expressing cells, with C1C3-GFP and C1C3-GFP expressing more than hCD20WT-GFP cells. 252 There was no difference in the loss of -GFP cells, indicating that intracellular mutations and / or truncations in CD20 did not affect 2H7-mediated killing.

[0190] Example 6: C1C3 and C1C4 with various anti-CD20 antibodies 252 is detected on the cell surface The FDA has recommended that long-term follow-up studies for integrating vectors should last 15 years.

[30] The applicants propose that their novel CD20 protein can be used not only as a cell therapy safety switch but also as a selection marker for long-term detection of genetically modified cells. The applicants have developed CD20WT, C1C3, and C1C 252We tested the binding of three anti-CD20 antibodies to CD20+ transduced HG3 cells sorted by ELISA (Figure 6). Obinutuzumab-APC, rituximab-AF405, and L27-PE all detect CD20 wild-type and mutant. More importantly, the clinically relevant L27-PE used to detect CD20+ cells in hospital clinical laboratories can also be used to detect cell therapies containing mutant CD20.

[0191] Example 7: Mutation of phosphorylation sites enhances CD20 engagement-induced Ca 2+ Impairing inflow CD20 hypercrosslinking or crosslinking of CD20-binding rituximab 2+ It stimulates the recruitment of CD20 to lipid rafts, along with increased Ca influx

[31] . 2+ Influx promotes cell activation, proliferation, and / or differentiation

[32] , which may be undesirable when attempting to deplete genetically modified cell populations. 252 Ca induced by hypercrosslinking of CD20 or the absence of CD20 2+ Ca influx was assessed in the CD20KO HG3 B cell line (Figure 7). CD20 mutants uptake Ca at significantly lower levels (P ≤ 0.01) than CD20WT. 2+ Mutation of the intracellular CD20 phosphorylation site significantly increased Ca influx upon CD20 cross-linking, as transduced CD20KO cells induced Ca influx at levels comparable to those of non-transduced CD20KO cells. 2+ This suggests that the inflow and its downstream effects may be prevented.

[0192] Example 8: B cell phosphorylation status is affected by C1C3 activation Phosphokinase activity is a major functional readout for signaling proteins. Akt, p38 MAPK, and ERK are among the common kinases that play key roles in signaling pathways involved in T cell and B cell activation [33, 34]. CD20 is a key signaling protein that regulates B cell activation

[35] and is differentially phosphorylated in resting and activated cells. We evaluated the impact of mutations in predicted phosphorylation sites of CD20 on the overall phosphorylation status of downstream kinases in B cells after CD20 engagement. Rituximab-induced activation of C1C3 led to a phosphorylation status equivalent to that of CD20-deficient B cells (Figure 8), indicating that CD20 regulation of B cell activation can be diminished by impairing CD20 phosphorylation. Despite mutations in the phosphorylation site in cytoplasmic domain 1, C1C3 252 Phosphorylation of cytoplasmic domain 3 appears to be highly relevant, as C1C shows a phosphorylation pattern comparable to that of CD20WT. In particular, phosphorylation of ERK1 / 2 leads to activation of transcription factors involved in cell proliferation and survival in B and T cells. 252 Although C1C3 induces phosphorylation of these kinases comparable to CD20WT, activation of C1C3 leads to approximately threefold less phosphorylation of ERK1 / 2, which may result in lower activation of T cells as well as B cells.

[0193] Example 9: Therapeutic utility of modified CD20 proteins The transgenes encoding the modified CD20 proteins of the present invention can potentially be incorporated into (a) genetically modified T cells, including CAR T cells

[36] , T cells expressing a transgenic T cell receptor (TCR)

[37] , or T cells expressing another transgenic or synthetic protein

[38] ; (b) natural killer (NK) cells

[39] ; (c) B cells

[40] ; (d) myeloid cells, including myeloid cells modified to express a transgenic receptor such as a CAR, TCR, B cell receptor (BCR), or NK receptor, or expressing another transgenic or synthetic protein

[41] ; (e) cell lines, including induced pluripotent stem cell (iPSC)-derived cell lines

[42] , and cell lines of hematopoietic origin, which have been modified to express a transgenic or synthetic protein; and (f) hematopoietic stem cells modified to express a transgenic or synthetic protein or edited to correct an inherited defect, where the transgenic or synthetic protein provides a therapeutic benefit

[43] .

[0194] Additionally, nucleic acid molecules encoding the modified CD20 proteins described herein can be included in RNA or DNA products intended to induce transient transfection or long-term transduction of stem cells, non-hematopoietic stem cells, and immune cells, or in viral vectors intended to induce transient or long-term expression of a transgene, all within a human recipient.

[0195] In each case, the modified CD20 may serve as (i) a tag to identify, select, or purify the transfected cells during and after the cell product manufacturing process; (ii) as a tag that allows for identification and / or characterization of the transfected or gene-transfected cells within patient-derived samples or biopsies, including by flow cytometry or immunohistochemistry; and / or (iii) as a safety switch that allows for rapid depletion of the transfected cells using CD20-targeted therapies, such as rituximab, obinutuzumab, or ocrelizumab.

[0196] Modified CD20 proteins that incorporate only changes to intracellular phosphorylation sites retain the native transmembrane and extracellular CD20 sequence, which reduces the risk of antibody-mediated immunogenicity to transfected or gene-transfected cells compared to modified CD20 proteins that incorporate multiple extracellular mutations.

[0197] Example 10: Extracellular Mutations for Exclusive Binding of Rituximab or Obinutuzumab Circulating levels of rituximab or obinutuzumab could limit the use of CD20 as a safety switch in patients with B-cell malignancies treated with these antibodies prior to gene therapy. Here, Applicants targeted mutations to the second extracellular loop of CD20 to allow exclusive binding of rituximab or obinutuzumab.

[0198] In 2013, Klein et al. outlined the epitope interactions of monoclonal antibodies targeting CD20 [8] (Figure 9).

[0199] Asparagine 171 is essential for rituximab binding to CD20, but obinutuzumab binding is maintained when this amino acid is replaced with almost any other amino acid. Mutation of N176 abolishes obinutuzumab binding but maintains rituximab binding. Conservative substitution of these asparagine residues with aspartic acid (i.e., N171D or N176D, underlined in Figure 9A) can lead to CD20 forms that exclusively bind obinutuzumab or rituximab, respectively. Applicants have synthesized Δobinutuzumab mutants (CD20 N176D and Δrituximab mutants (CD20 A Δ-obinutuzumab mutant (Δrituximab-C1C3) was designed. The Δ-obinutuzumab mutant retained the binding of both rituximab and obinutuzumab, but is more relevant due to the widespread use of rituximab; the Δ-rituximab mutant does not allow rituximab binding but retains obinutuzumab binding. A CD20 molecule combining this extracellular point mutation with an intracellular mutation (C1C3) could be used in patients pretreated with rituximab, a prescribed CAR-T cell therapy containing a safety switch. Detection by anti-CD20 antibodies and CD20-mediated killing by this protein (Δrituximab-C1C3) will be tested in further studies.

[0200] Example 11: Conclusion Although the tertiary structure of CD20 is not maintained by major disruption of its secondary structure, minimal changes can lead to successful production of a protein that is detected on the cell surface. Applicants have engineered putative CD20 phosphomutants that can be detected on the plasma membrane of HEK293 (human embryonic kidney) cell lines, HG3 (B cells), and, most importantly, primary T cells. These phosphomutants are capable of detecting Ca 2+It impairs the CD20 signaling cascade in B cells, exemplified by impaired influx and global kinase phosphorylation, but allows detection by CD20 antibodies and antibody-dependent cellular cytotoxicity in B cells in vitro and T cells in vivo. By mutating key residues within the predicted phosphorylation sites of CD20, Applicants were able to abrogate CD20 signaling function while retaining cell surface expression, detection, and cell death induced by clinically relevant antibodies. This minimizes the potential risk of cell activation caused by engagement of the safety switch. 2+ Although influx was abrogated by both CD20 mutants, only C1C3 affected the phosphorylation status of B cells, highlighting the relevance of phosphorylation sites in cytoplasmic domain 3.

[0201] Therefore, the CD20 phosphovariants described herein are compelling candidates for the development of safety switches and detection tools or as selection markers for genetically modified cell therapy.

[0202] Although the present invention has been described by way of example, it should be recognized that variations and modifications can be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to herein.

[0203] References [Table 4] TIFF2025540894000010.tif217169

Claims

1. (i) at least one mutation that results in truncation to any one or more of amino acid residues 1 to 56 set forth in SEQ ID NO:1; (ii) at least one mutation that results in truncation to any one or more of amino acid residues 210 to 297 set forth in SEQ ID NO:1; (iii) at least one mutation to threonine or serine located within amino acid residues 1 to 56 set forth in SEQ ID NO:1; (iv) at least one mutation to threonine or serine located within amino acid residues 210 to 297 of SEQ ID NO: 1; or (v) A combination comprising any one of (i) to (iv).

1. A human CD20 protein comprising: A human CD20 protein, wherein any one of (i) to (v) causes a reduction or abrogation of intracellular signaling when the modified CD20 protein binds to or associates with the membrane of a cell.

2. (i) at least one mutation that results in truncation to any one or more of amino acid residues 1 to 56 set forth in SEQ ID NO: 1; and (ii) at least one mutation to threonine or serine located within amino acid residues 210 to 297 of SEQ ID NO: 1; The human CD20 protein of claim 1, comprising:

3. (i) at least one mutation to threonine or serine located within amino acid residues 1 to 56 of SEQ ID NO: 1; and (ii) at least one mutation that causes truncation to any one or more of amino acid residues 210 to 297 set forth in SEQ ID NO: 1; The human CD20 protein of claim 1, comprising:

4. (i) at least one mutation to threonine or serine located within amino acid residues 1 to 56 of SEQ ID NO: 1; and (ii) at least one mutation to threonine or serine located within amino acid residues 210 to 297 of SEQ ID NO: 1; The human CD20 protein of claim 1, comprising:

5. The human CD20 protein according to claim 1 or 2, wherein the 1st to 50th amino acid residues shown in SEQ ID NO: 1 are truncated.

6. The human CD20 protein according to claim 1 or 3, wherein the amino acid residues 253 to 297 shown in SEQ ID NO: 1 are truncated.

7. A human CD20 protein described in any one of claims 1, 3 or 4, wherein at least one mutation to threonine or serine located within amino acid residues 1 to 56 of SEQ ID NO: 1 includes a mutation to any one or more of T2, T3, S7, T11, S35, S36 and T51.

8. A human CD20 protein according to any one of claims 1, 2 or 4, wherein at least one mutation to threonine or serine located within amino acid residues 210 to 297 of SEQ ID NO: 1 includes a mutation to any one or more of S225, S231 and T239.

9. The human CD20 protein according to any one of claims 1 to 8, wherein at least one mutation is selected from a substitution mutation, a deletion mutation, and an insertion mutation.

10. The human CD20 protein of claim 9 , wherein the substitution mutation comprises a substitution with at least one natural or non-natural amino acid residue.

11. 8. The human CD20 protein of claim 7, wherein the mutation is selected from any one or more of T2A, T3A, S7A, T11A, S35A, S36A and T51A.

12. The human CD20 protein of claim 8, wherein the mutation is selected from any one or more of S225A, S231A, and T239A.

13. 2. The human CD20 protein of claim 1, comprising or consisting of a sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, or a sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, respectively.

14. 14. The human CD20 protein of any one of claims 1 to 13, further comprising at least one mutation to the extracellular domain defined by amino acid residues 142 to 188, preferably amino acid residues 167 to 183 as set forth in SEQ ID NO: 1, wherein the mutation eliminates binding by rituximab or obinutuzumab or ocrelizumab.

15. 15. The human CD20 protein of claim 14, wherein at least one mutation comprises a mutation to amino acid residues N171, S173 and / or N176 shown in SEQ ID NO:

1.

16. A cell expressing the human CD20 protein according to any one of claims 1 to 15.

17. 17. The cell of claim 16, comprising a T cell, a natural killer cell, a B cell, a myeloid cell, a pluripotent stem cell, a hematopoietic stem cell, a non-hematopoietic stem cell, and a human cell.

18. A nucleic acid comprising a sequence encoding the human CD20 protein according to any one of claims 1 to 15.

19. A vector comprising the nucleic acid of claim 18.

20. The vector of claim 19, which is a viral vector.

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