Fusion protein for treating diseases related to CD20

Abstract

The present disclosure provides a fusion protein against CD20, for example, a CAR, and a use thereof in the treatment of diseases.

Description

Fusion proteins for treating CD20-related diseases

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on and claims the priority of international patent application No. PCT / CN2023 / 109110, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure generally relates to fusion proteins, such as CARs that specifically target CD20. Further provided are immune response cells comprising such CARs, and methods of using such CARs and cells to treat tumors and autoimmune diseases. Background Art

[0004] Chimeric antigen receptors (CARs) are artificial molecules that redirect T cells to specific antigens or their epitopes, which are different in different diseases. CAR constructs typically include an extracellular antigen-binding domain, a spacer, a transmembrane domain, and an intracellular signaling domain. Due to the complexity of the CAR structure, it needs to be optimized based on the antigen. Therefore, it is necessary to develop the best CAR for a specific antigen.

[0005] Summary of the Invention

[0006] The present disclosure provides novel fusion proteins with optimal properties for cell therapy. Specifically, the present disclosure relates to CARs with specific structures that allow the CARs to position the antigen binding domains at a distance from the target epitope that enables the CARs to effectively interact with target cells.

[0007] In one aspect, the present disclosure provides a fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, a spacer located between the extracellular antigen-binding domain and the transmembrane domain, and an intracellular domain, wherein: a) the extracellular antigen-binding domain is capable of specifically binding to the same epitope on CD20 as a reference antibody comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; b) the spacer is composed of a hinge domain from CD28 or a hinge domain from an immunoglobulin, wherein the spacer composed of the hinge domain from an immunoglobulin has a length of 12, 13, 14 or 15 amino acids; and c) the transmembrane domain comprises a transmembrane domain from CD28.

[0008] In one aspect, the present disclosure provides a fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, a spacer located between the extracellular antigen-binding domain and the transmembrane domain, and an intracellular signaling domain, wherein: a) the extracellular antigen-binding domain is capable of specifically binding to a protein comprising SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: NO:3 binds to the same epitope on CD20; b) the spacer consists of a hinge domain from CD28 or a hinge domain from an immunoglobulin, wherein the spacer consisting of a hinge domain from an immunoglobulin is 12, 13, 14 or 15 amino acids in length; c) the transmembrane domain comprises a transmembrane domain from CD28; and d) the intracellular signaling domain comprises a primary intracellular signaling domain and a costimulatory signaling domain located between the transmembrane domain and the primary intracellular signaling domain, wherein the costimulatory signaling domain comprises the signaling domain of CD137 and the primary intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ.

[0009] In one aspect, the extracellular antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL). In one aspect, VH comprises HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and VL comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively. In one aspect, VH comprises HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and VL comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively. In one aspect, the VH comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively. In one aspect, the VH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4, and the VL comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5. In one aspect, the VH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12, and the VL comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. In one aspect, the VH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20, and the VL comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 21. In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 4, and the VL comprises the amino acid sequence of SEQ ID NO: 5. In one aspect, VH comprises the amino acid sequence of SEQ ID NO: 12, and VL comprises the amino acid sequence of SEQ ID NO: 13. In one aspect, VH comprises the amino acid sequence of SEQ ID NO: 20, and VL comprises the amino acid sequence of SEQ ID NO: 21.

[0010] In one aspect, the extracellular antigen binding domain is an scFv. In one aspect, the VH is fused to the N-terminus of the VL via a flexible polypeptide linker at its C-terminus. In one aspect, the extracellular antigen binding domain comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0011] In one aspect, the spacer consists of the hinge domain from CD28. In one aspect, the spacer consists of an amino acid sequence having 90% sequence identity to SEQ ID NO: 30. In one aspect, the spacer consists of the amino acid sequence of SEQ ID NO: 30.

[0012] In one aspect, the spacer consists of a hinge domain from IgG4, wherein the spacer consisting of a hinge domain from IgG4 is 12 amino acids in length. In one aspect, the spacer consists of an amino acid sequence having 90% sequence identity to SEQ ID NO: 31. In one aspect, the spacer consists of the amino acid sequence of SEQ ID NO: 31.

[0013] In one aspect, the transmembrane domain comprises an amino acid sequence having 90% sequence identity to SEQ ID NO: 33. In one aspect, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:33.

[0014] In one aspect, the intracellular signaling domain comprises a primary intracellular signaling domain. In one aspect, the primary intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ. In one aspect, the primary intracellular signaling domain comprises an amino acid sequence having 90% sequence identity to SEQ ID NO: 35. In one aspect, the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 35.

[0015] In one aspect, the intracellular signaling domain comprises a costimulatory signaling domain located between the transmembrane domain and the primary intracellular signaling domain. In one aspect, the costimulatory signaling domain comprises a signaling domain of a protein selected from the group consisting of a ligand of CD27, CD28, CD137, OX40, CD30, CD40, CD3, HVEM, ICOS, Myd88, LFA-1, ICOS, CD2, CD7, NKG2C, B7-H3, CD83, and combinations thereof. In one aspect, the costimulatory signaling domain comprises the signaling domain of CD137. In one aspect, the costimulatory signaling domain comprises an amino acid sequence having 90% sequence identity to SEQ ID NO: 34. In one aspect, the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 34.

[0016] In one aspect, a) the extracellular antigen binding domain is capable of specifically binding to the same epitope on CD20 as a reference antibody comprising the amino acid sequence of SEQ ID NO: 1; b) the spacer region is composed of the hinge domain from CD28; c) the transmembrane domain comprises the transmembrane domain from CD28; d) the intracellular signaling domain comprises the signaling domain of CD137 and the cytoplasmic signaling domain of CD3ζ. In one aspect, a) the extracellular antigen binding domain comprises VH and VL, wherein VH comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, and VL comprises LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; b) the spacer consists of the amino acid sequence of SEQ ID NO: 30; c) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 33; d) the intracellular signaling domain comprises the amino acid sequences of SEQ ID NO: 34 and SEQ ID NO: 35. In one aspect, the fusion protein comprises the amino acid sequence of SEQ ID NO: 36.

[0017] In one aspect, a) the extracellular antigen-binding domain is capable of specifically binding to the same epitope on CD20 as a reference antibody comprising the amino acid sequence of SEQ ID NO: 1; b) the spacer region consists of a hinge domain from IgG4, wherein the spacer region consisting of the hinge domain from IgG4 is 12 amino acids in length; c) the transmembrane domain comprises a transmembrane domain from CD28; d) the intracellular signaling domain comprises the signaling domain of CD137 and the cytoplasmic signaling domain of CD3ζ. In one aspect, a) the extracellular antigen binding domain comprises VH and VL, wherein VH comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, and VL comprises LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; b) the spacer consists of the amino acid sequence of SEQ ID NO: 31; c) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 33; d) the intracellular signaling domain comprises the amino acid sequences of SEQ ID NO: 34 and SEQ ID NO: 35. In one aspect, the fusion protein comprises the amino acid sequence of SEQ ID NO: 38.

[0018] In one aspect, a) the extracellular antigen-binding domain is capable of specifically binding to the same epitope on CD20 as a reference antibody comprising the amino acid sequence of SEQ ID NO: 2; b) the spacer region consists of a hinge domain from IgG4, wherein the spacer region consisting of the hinge domain from IgG4 is 12 amino acids in length; c) the transmembrane domain comprises a transmembrane domain from CD28; d) the intracellular signaling domain comprises the signaling domain of CD137 and the cytoplasmic signaling domain of CD3ζ. In one aspect, a) the extracellular antigen binding domain comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; b) the spacer consists of the amino acid sequence of SEQ ID NO: 31; c) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 33; d) the intracellular signaling domain comprises the amino acid sequences of SEQ ID NO: 34 and SEQ ID NO: 35. In one aspect, the fusion protein comprises the amino acid sequence of SEQ ID NO: 37.

[0019] The present disclosure also provides isolated nucleic acids encoding the fusion proteins described herein.

[0020] The present disclosure also provides vectors comprising nucleic acids as described herein. In one aspect, the vector is a viral vector. In one aspect, the viral vector is a retroviral, lentiviral, adenoviral, or adeno-associated viral vector.

[0021] The present disclosure also provides cells comprising the fusion protein, isolated nucleic acid or vector as described herein. In one aspect, the cell is a lymphocyte. In one aspect, the cell is a NK cell or a T cell.

[0022] The present disclosure also provides a pharmaceutical preparation comprising the fusion protein or cell as described herein and a pharmaceutically acceptable carrier.

[0023] The present disclosure also provides a method for treating a disease or condition associated with CD20 in an individual, comprising administering to the individual a fusion protein, isolated nucleic acid, vector, cell, or pharmaceutical preparation as described herein. In one aspect, the individual suffers from a relapsed or refractory disease or condition associated with CD20, or has previously received treatment with at least one regimen comprising a drug directed against CD20. Also provided is the use of a fusion protein, isolated nucleic acid, vector, cell, or pharmaceutical preparation as described herein in the preparation of a medicament for treating a disease or condition associated with CD20. The present disclosure also provides a fusion protein, isolated nucleic acid, vector, cell, or pharmaceutical preparation as described herein for use as a medicament. In one aspect, the disease or condition is cancer or an autoimmune disease. In one aspect, the cancer is selected from leukemia, lymphoma, lung cancer, liver cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, and rhabdomyosarcoma. In one aspect, the cancer is leukemia or lymphoma. In one aspect, the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, Wegener's disease, inflammatory bowel disease, ulcerative colitis, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, autoimmune thrombocytopenia, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, ANCA-associated vasculitis, diabetes, Reynaud's syndrome, Sjogren's syndrome, neuromyelitis optica, and glomerulonephritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A shows the extent of CD20 expression on various target cells. Figure 1B depicts the results of an activation assay in Jurkat cells.

[0025] Figures 2A to 2C show the KO 、CD20 lo or CD20 WT Percentage of CD25-, CD69-, and Ki67-positive populations in primary T cells after 48 h of Raji cell stimulation.

[0026] Figures 3A to 3B show the secretion of IFN-γ and IL-2 by CAR-transduced T cells upon stimulation with different target cell lines.

[0027] FIG4 shows the 24-hour killing index of T cells against different target cell lines. DETAILED DESCRIPTION

[0028] I. Definition

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0030] The singular forms "a" and "an" refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0031] As used herein, the terms "comprising" and "comprises" are synonymous with "including," "includes," or "containing," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms "comprising" and "comprises" also encompass "consisting of."

[0032] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), whole antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0033] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a natural antibody. For example, an IgG class intact antibody comprises two light chains and two heavy chains connected by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable domain (VH) followed by three constant domains (CH1, CH2, and CH3), also referred to as heavy chain constant regions. Similarly, from N-terminus to C-terminus, each light chain has a variable domain (VL) followed by a constant light chain (CL) domain, also referred to as a light chain constant region. The heavy chains of antibodies can be divided into one of five types, referred to as IgA, IgD, IgE, IgG, or IgM, some of which can be further divided into subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Based on the amino acid sequence of the constant domains of the antibody, the light chains of the antibody can be divided into one of two types, referred to as kappa (κ) and lambda (λ).

[0034] "Antibody fragments" refer to molecules that are not complete antibodies but contain a portion of an intact antibody that binds to an antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, linear antibodies, single-chain antibody molecules (e.g., scFv and scFab), single-domain antibodies, and multispecific antibodies formed from antibody fragments.

[0035] "Single-chain Fv" or "scFv" is a fusion protein of the variable regions of the light chain (VL) and the heavy chain (VH), wherein the VL and VH are connected by a short linker peptide of about 10-25 amino acids. The linker is usually flexible and can connect the N-terminus of VH to the C-terminus of VL and vice versa. Despite the removal of the constant region and the introduction of the linker, this protein retains the specificity of the original antibody.

[0036] The term "antigen binding domain" refers to the portion of an antibody that comprises a region that binds to and is complementary to part or all of an antigen. The antigen binding domain can be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). In a preferred aspect, the antigen binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0037] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding an antibody to an antigen. The variable regions of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs). In some cases, a single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, a VH or VL domain from an antibody that binds to an antigen can be used to screen a library of complementary VL or VH domains, respectively, to isolate antibodies that bind to that specific antigen. As used herein, "Kabat numbering" with respect to variable region sequences refers to the numbering system proposed by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0038] The terms "complementarity determining region" and "CDR" are known to refer to the non-continuous sequence of amino acids in the variable region of an antibody that confer antigen binding specificity and / or binding affinity. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. "Framework region" and "FR" refer to the non-CDR portions of the heavy and light chain variable regions. Table 1 below lists the exemplary positional boundaries of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, as determined by Kabat, Chothia, AbM, IMGT, and Contact schemes, respectively. Unless otherwise noted, the amino acid sequences of the CDRs shown in the present disclosure are determined according to Kabat.

[0039] Table 1. CDR regions according to various numbering systems

[0040] As used herein, the terms "specific binding" or "binding" used interchangeably refer to the ability of a protein to bind to a target protein under specific binding conditions with an affinity or avidity that is at least 5 times greater than the average affinity or avidity of the same protein for a random peptide or polypeptide set of sufficient statistical size, but optionally at least 10, 20, 30, 40, 50, 100, 250 or 500 times, or even at least 1000 times greater. A specific binding protein need not bind only to a single target molecule, but may also specifically bind to non-target molecules if there is a conformational structural similarity between the target molecule and the non-target molecule (e.g., a paralog or ortholog). It will be appreciated by those skilled in the art that specific binding to molecules having the same function in different animal species or to non-target molecules having an epitope that is substantially similar to the target molecule is possible without compromising the binding specificity determined relative to a statistically valid unique non-target set. Thus, due to cross-reactivity, a polypeptide may specifically bind to more than one different target molecule.

[0041] The term "epitope" refers to an antigenic moiety that specifically interacts with an antibody. Such a moiety (referred to herein as an epitope determinant) typically comprises or is a part of an element such as an amino acid side chain or a sugar side chain. An epitope determinant can be defined by methods known in the art, for example, by crystallography or by hydrogen-deuterium exchange. At least one or some of the moieties that specifically interact with an epitope determinant on an antibody molecule are typically located in a CDR. Typically, an epitope has specific three-dimensional structural characteristics and / or specific charge characteristics. Some epitopes are linear epitopes, while others are conformational epitopes.

[0042] The term "hinge" herein refers to a flexible polypeptide linking region that provides structural flexibility and spacing to the attached flanking polypeptide regions and can be composed of natural or synthetic polypeptides.

[0043] "Percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. To determine percent amino acid sequence identity, alignment can be performed using various methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the sequences being compared.

[0044] The term "fusion protein" refers to a hybrid (eg, chimeric, recombinant) polypeptide comprising protein domains from at least two different proteins.

[0045] The term "chimeric antigen receptor" or "CAR" refers to a fusion protein that is engineered to include two or more naturally occurring or engineered amino acid sequences that are linked together in a manner that is not naturally present in a host cell, and when present on the cell surface, the fusion protein can act as a receptor. The CAR of the present disclosure includes an extracellular portion comprising an antigen binding domain (e.g., scFv) connected to a spacer sequence, a transmembrane domain, and one or more intracellular signaling domains (optionally containing a costimulatory domain).

[0046] As used herein, the terms "extracellular domain" or "ectodomain" are used interchangeably to refer to the region of a membrane protein (e.g., a transmembrane protein) located outside the vesicle membrane. The extracellular domain often contains a binding domain that specifically binds to a ligand or cell surface receptor. The term "extracellular antigen binding domain" refers to an extracellular domain or a portion of an extracellular domain that is capable of specifically binding to an antigen.

[0047] As used herein, the terms "endodomain," "intracellular domain," or "cytoplasmic domain," which are used interchangeably, refer to a region found in some membrane proteins (e.g., transmembrane proteins) that extends into the interior space defined by the cell surface membrane. In some cells, the intracellular domain interacts with intracellular components and can play a role in signal transduction, and thus, in some cases, can be an intracellular signaling domain.

[0048] The term "transmembrane domain" as used herein refers to a domain found in a membrane protein that substantially or completely spans a lipid bilayer, such as those in biological membranes (e.g., mammalian cells) or in artificial constructs (e.g., liposomes). A transmembrane protein can cross both layers of the lipid bilayer once or multiple times.

[0049] As used herein, the term "spacer" refers to a polypeptide sequence (e.g., a hinge) that is capable of covalently linking together two spaced-apart portions: an antigen binding domain and a transmembrane domain of a fusion protein (e.g., a CAR).

[0050] The term "flexible polypeptide linker" as used herein refers to a peptide that connects other peptides to form a functional protein. In the context of scFv, a flexible polypeptide linker refers to a peptide linker that connects VH and VL to form an scFv that specifically binds to an antigen.

[0051] The term "expression" refers to the process by which a polypeptide is produced based on the coding sequence of a nucleic acid molecule such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[0052] The term "encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) that enables it to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties. Thus, a gene, cDNA, or RNA encodes a protein if transcription and translation of the mRNA corresponding to the gene in a cell or other biological system can produce the protein.

[0053] The terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that can be contained in a peptide sequence. The term "polypeptide" also refers to products of post-expression modifications of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. The size of the polypeptides of the present disclosure can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. A polypeptide may, but need not, have a defined three-dimensional structure.

[0054] An "isolated" nucleic acid molecule refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid molecule includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. An "isolated polynucleotide (or nucleic acid) encoding a fusion protein" refers to one or more polynucleotide molecules encoding a fusion protein, including such polynucleotide molecule(s) in a single vector or different vectors, and such polynucleotide molecule(s) present at one or more locations in a host cell.

[0055] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked.

[0056] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of these cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages.

[0057] The term "subject" or "individual" is intended to include living organisms (eg, mammals, humans) in which an immune response can be elicited.

[0058] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of a disease in the individual being treated, and can be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis.

[0059] The term "pharmaceutical composition" refers to a preparation that is in a form such that the biological activity of the active ingredient contained therein is effective and that does not contain additional components that are unacceptably toxic to the individual to whom the preparation is administered. Pharmaceutical compositions typically contain one or more pharmaceutically acceptable carriers. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than the active ingredient that is non-toxic to the individual. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0060] An "effective amount" of an active agent (eg, a pharmaceutical composition) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0061] II. Fusion Protein

[0062] The present disclosure provides a new fusion protein with advantageous properties, such as manufacturability, stability, binding affinity, biological activity, targeting efficiency, reduced toxicity, an expanded dosage range that can be given to patients, and thereby potentially enhanced efficacy. Provided herein is a fusion protein comprising a) an extracellular antigen binding domain; b) a spacer; c) a transmembrane domain; and d) an intracellular signaling domain. In some embodiments, the fusion protein is a CAR.

[0063] 2.1 Extracellular antigen-binding domain

[0064] In one aspect, a fusion protein as defined herein is provided, wherein the extracellular antigen binding domain comprises one or more antibodies. In one aspect, the antibody is a Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, linear antibody, single-chain antibody molecule (e.g., scFv and scFab), single domain antibody, or a multispecific antibody formed by an antibody fragment. In a specific embodiment, the extracellular antigen binding domain comprises an scFv.

[0065] In one aspect, the extracellular antigen binding domain is capable of binding to CD20. In one aspect, the fusion protein is capable of binding to the same epitope on CD20 as any anti-CD20 antibody in the art (e.g., Ofatumumab, Ofatumumab, Rituximab, Ocrelizumab, Veltuzumab, Ibritumomab, Tositumumab, Odronextamab, Ublituximab, Leu16, IF5, 1.5.3). In one aspect, the extracellular antigen binding domain is capable of binding to the same epitope on CD20 as a reference antibody comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0066] In one aspect, the extracellular antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL). In one aspect, VH comprises the HCDR1, HCDR2 and HCDR3 contained in SEQ ID NO: 4, and VL comprises the LCDR1, LCDR2 and LCDR3 contained in SEQ ID NO: 5. In one aspect, VH comprises the HCDR1, HCDR2 and HCDR3 contained in SEQ ID NO: 12, and VL comprises the LCDR1, LCDR2 and LCDR3 contained in SEQ ID NO: 13. In one aspect, VH comprises the HCDR1, HCDR2 and HCDR3 contained in SEQ ID NO: 20, and VL comprises the LCDR1, LCDR2 and LCDR3 contained in SEQ ID NO: 21.

[0067] In one aspect, the VH comprises HCDR1, HCDR2, and HCDR3 comprising, or consisting of, the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 comprising, or consisting of, the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively. In one aspect, the VH comprises HCDR1, HCDR2, and HCDR3 comprising, or consisting of, the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 comprising, or consisting of, the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively. In one aspect, VH comprises HCDR1, HCDR2 and HCDR3, which respectively comprise or consist of the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24; VL comprises LCDR1, LCDR2 and LCDR3, which respectively comprise or consist of the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.

[0068] In one aspect, the VH comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to SEQ ID NO: 4, and the VL comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to SEQ ID NO: 5. In one aspect, the VH comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to SEQ ID NO: 12, and the VL comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to SEQ ID NO: 13. In one aspect, the VH comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to SEQ ID NO: 20, and the VL comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to SEQ ID NO: 21. In one aspect, the VH comprises or consists of the amino acid sequence of SEQ ID NO: 4, and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 5. In one aspect, the VH comprises or consists of the amino acid sequence of SEQ ID NO: 12, and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 13. In one aspect, the VH comprises or consists of the amino acid sequence of SEQ ID NO: 20, and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 21.

[0069] In one aspect, VH and VL are connected via a flexible polypeptide linker. In one aspect, VH is fused to the C-terminus of VL via a flexible polypeptide linker via its N-terminus (LH). In one aspect, VL is fused to the C-terminus of VH via a flexible polypeptide linker via its N-terminus (HL). In one aspect, the flexible polypeptide linker is (G4S) n (SEQ ID NO:41), (SG4) n (SEQ ID NO:42) or G4 (SG4) n(SEQ ID NO:43), wherein "n" is 1, 2, 3, 4, 5, 6, 7 or 8, particularly 3. In one aspect, the flexible polypeptide linker comprises or consists of the amino acid sequence of SEQ ID NO:28. In one aspect, the scFv comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. In one aspect, the ScFv comprises or consists of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. Table 2 provides the SEQ ID NOs of exemplary scFvs.

[0070] Table 2. SEQ ID NOs of exemplary scFvs

[0071] 2.2 Spacer

[0072] The spacer or hinge refers to the region where the extracellular antigen binding domain is left from the cell surface to allow cell contact, antigen binding and activation. The spacer in CAR is located between the transmembrane domain and the extracellular antigen binding domain. The exemplary hinge region used in CAR includes a hinge region derived from the extracellular region of type 1 membrane proteins such as CD8 α, CD4, CD28 and CD7, which may be a wild-type hinge region from these proteins or may be modified. In one aspect, the spacer is composed of the hinge domain from CD28. In one aspect, the spacer is composed of the hinge domain (or stem region) from wild-type CD28. In one aspect, the spacer is composed of an amino acid sequence having at least 90% sequence identity to SEQ ID NO:30. In one aspect, the spacer is composed of an amino acid sequence of SEQ ID NO:30.

[0073] In one aspect, the spacer in the fusion protein comprises at least a portion of an immunoglobulin constant region or a variant or modified form thereof, such as a hinge region. In one aspect, the spacer comprises at least a portion of an IgG hinge. In one aspect, the spacer can be a chimeric polypeptide comprising one or more hinge fragments derived from IgG1, IgG2, IgG3, and / or IgG4. In one aspect, the spacer can be a chimeric polypeptide comprising one or more hinge fragments derived from IgG4. In one aspect, the spacer consists of an amino acid sequence of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues in length. In some embodiments, the spacer consists of an amino acid sequence of 12-15 amino acid residues in length. In a preferred embodiment, the spacer consists of an amino acid sequence of 12 amino acid residues in length. In one aspect, the spacer consists of an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 31. In a specific embodiment, the spacer consists of the amino acid sequence of SEQ ID NO: 31.

[0074] 2.3 Transmembrane domain

[0075] In one aspect, a fusion protein as defined herein is provided, wherein the transmembrane domain can be obtained from a naturally occurring protein or a synthetic, non-naturally occurring protein segment, such as a hydrophobic protein segment that is thermodynamically stable in a cell membrane. In one aspect, the protein segment is at least about 20 amino acids, such as at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids.

[0076] In one aspect, the transmembrane domains provided herein are derived from type I single-pass transmembrane proteins. In one aspect, the transmembrane domains from multi-pass transmembrane proteins are also suitable for use in fusion proteins as described herein. Multi-pass transmembrane proteins may comprise complex (at least 2, 3, 4, 5, 6, 7 or more) α-helical or β-sheet structures. In one aspect, the N-terminus and C-terminus of multi-pass transmembrane proteins are present on opposite sides of the lipid bilayer, for example, the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer, and the C-terminus of the protein is present on the extracellular side.

[0077] In one aspect, the membrane-spanning domain provided herein can include a membrane-spanning region and a cytoplasmic region located at the C-terminal side of the membrane-spanning domain. The cytoplasmic region of the membrane-spanning domain can include three or more amino acids, and in some embodiments, contributes to the orientation of the membrane-spanning domain in the lipid bilayer. In one aspect, one or more cysteine ​​residues are present in the membrane-spanning region of the membrane-spanning domain. In one aspect, one or more cysteine ​​residues are present in the cytoplasmic region of the membrane-spanning domain. In one aspect, the cytoplasmic region of the membrane-spanning domain includes positively charged amino acids. In one aspect, the cytoplasmic region of the membrane-spanning domain includes amino acid arginine, serine and lysine.

[0078] In one aspect, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In one aspect, the transmembrane domain of the CAR provided herein comprises an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan and valine can be present at the C-terminus of the transmembrane domain. In one aspect, the transmembrane region mainly comprises hydrophobic amino acid residues such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan or valine. In one aspect, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a polyleucine-alanine sequence.

[0079] In one aspect, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of: the alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, GPC3, IL-13, IL-14, IL-16, IL-17, IL-18, IL-19, IL-20, IL-34, IL-40, IL-54, IL-64, IL-72, IL-84, IL-19, IL-20 -2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C. In one aspect, the transmembrane domain is derived from or comprises the transmembrane domain of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 or PD1. In some specific embodiments, the transmembrane domain comprises an amino acid sequence having at least 88%, 92%, or 96% sequence identity to SEQ ID NO: 32. In a specific embodiment, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 32. In some specific embodiments, the transmembrane domain comprises an amino acid sequence having at least 88%, 92%, or 96% sequence identity to SEQ ID NO: 33. In a specific embodiment, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 33.

[0080] 2.4 Signaling domain

[0081] In one aspect, a fusion protein as defined herein is provided, wherein the intracellular signaling domain is generally responsible for activating at least one normal effector function. In one aspect, the intracellular signaling domain comprises two different types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation by TCR (primary intracellular signaling domain) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, such as costimulatory domains).

[0082] In one aspect, the primary signaling domain regulates the primary activation of the TCR complex in a stimulating manner or in an inhibitory manner. The primary intracellular signaling domain that works in a stimulating manner can contain a signaling motif, which is referred to as an activation motif (ITAM) based on immunoreceptor tyrosine. Examples of particularly useful primary intracellular signaling domains containing ITAM in the present invention include those of TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FceRI, DAP10, DAP12, and CD66d. In one aspect, CAR includes an intracellular signaling domain, such as the primary signaling domain of CD3-ζ. In one aspect, the primary signaling domain includes a modified ITAM domain, for example, an ITAM domain of a mutation whose activity has been changed (eg, increased or decreased) compared to a natural ITAM domain. In one aspect, the primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, such as an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In one aspect, the signaling domain of CD3-ζ is a mutant CD3ζ or wild-type human CD3ζ. In one aspect, the primary signaling domain comprises one, two, three, four or more ITAM motifs. In one aspect, the primary intracellular signaling domain is a functional mutant of the cytoplasmic signaling domain of CD3ζ containing one or more mutations (e.g., Q65K).

[0083] In one aspect, the intracellular signaling sequence in the cytoplasmic part can be connected to each other in a random or specified order. In one aspect, costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens except antigen receptors or their ligands. The example of such molecules includes MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, signaling lymphocyte activation molecules (SEAM proteins), activation NK cell receptors, BTFA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, FFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD2 78), GITR, BAFFR, FIGHT, HVEM, KIRDS2, SFAMF7, NKp80(KFRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, C D8β, IF2Rβ, IF2Rγ, IF7Rα, ITGA4, VFA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VFA-6, CD49f, ITGAD, CD11d, IT GAE, CD103, ITGAF, FFA-l, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, FFA-l, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKF, DNAM1(CD226), SFAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTA M, Fy9 (CD229), CD160 (BY55), PSGF1, CD100 (SEMA4D), CD69, SFAMF6 (NTB-A, Fyl08), SEAM (SFAMF1, CD150, IPO-3), BFAME (SFAMF8), SEFPFG (CD162), FTBR, FAT, GADS, SFP-76, PAG / Cbp, CD19a and ligands that specifically bind to CD83, etc.

[0084] In one aspect, the co-stimulatory signaling domain comprises up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) compared to the wild-type counterpart. Such a co-stimulatory signaling domain comprising one or more amino acid variations can be referred to as a variant. Relative to a co-stimulatory signaling domain that does not comprise a mutation, the mutation of the amino acid residues of the co-stimulatory signaling domain can result in an increase in signal transduction and an enhancement of the stimulation of an immune response. Relative to a co-stimulatory signaling domain that does not comprise a mutation, the mutation of the amino acid residues of the co-stimulatory signaling domain can result in a decrease in signal transduction and a decrease in the stimulation of an immune response.

[0085] In one aspect, one or more costimulatory signaling domains are selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, NKG2C, B7-H3, and a ligand specifically binding to CD83. In one aspect, the intracellular signaling domain in the CAR of the present disclosure includes a costimulatory signaling domain derived from CD137 (4-1BB). In one aspect, the intracellular signaling domain includes a cytoplasmic signaling domain of CD3 ζ and a costimulatory signaling domain of CD28 or CD137. In a specific embodiment, the intracellular signaling domain includes a cytoplasmic signaling domain of CD3 ζ and a costimulatory signaling domain of CD137.

[0086] 2.5CAR

[0087] In one aspect, a fusion protein as defined herein is provided, wherein the fusion protein is a CAR comprising, from its N-terminus to its C-terminus, an extracellular antigen binding domain, a spacer, a transmembrane domain, and an intracellular signaling domain. In one aspect, the extracellular antigen binding domain is an scFv comprising, from its N-terminus to its C-terminus, a VH, a linker, and a VL. In one aspect, the intracellular signaling domain comprises, from its N-terminus to its C-terminus, a signaling domain of CD137 and a cytoplasmic signaling domain of CD3ζ.

[0088] In one aspect, the extracellular antigen-binding domain is capable of specifically binding to the same epitope on CD20 as a reference antibody comprising the amino acid sequence of SEQ ID NO: 1, and the spacer consists of the hinge domain from CD28. In one aspect, the extracellular antigen-binding domain comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; and the spacer consists of the amino acid sequence of SEQ ID NO: 30. In a preferred embodiment, the extracellular antigen-binding domain is a scFv comprising the amino acid sequence of SEQ ID NO: 1, and the spacer consists of the amino acid sequence of SEQ ID NO: 30.

[0089] In one aspect, the extracellular antigen-binding domain is capable of specifically binding to the same epitope on CD20 as a reference antibody comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and the spacer consists of a hinge domain from IgG4, wherein the spacer consisting of a hinge domain from IgG4 is 12 amino acids in length. In one aspect, the extracellular antigen-binding domain comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; and the spacer consists of the amino acid sequence of SEQ ID NO: 31. In one aspect, the extracellular antigen-binding domain comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; and the spacer consists of the amino acid sequence of SEQ ID NO: 31. In a preferred embodiment, the extracellular antigen-binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and the spacer consists of the amino acid sequence of SEQ ID NO: 30.

[0090] In a specific embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38.

[0091] III. Engineered Cells and Their Preparation

[0092] 3.1 Cells

[0093] In one aspect, the present disclosure provides engineered cells comprising the above-mentioned fusion protein, such as engineered immune cells. The source of the engineered immune cells of the present disclosure can be the patient to be treated (i.e., autologous cells) or from a donor who is not the patient to be treated (e.g., allogeneic cells).

[0094] In one aspect, cell is immune response cell. In one aspect, cell is lymphoid lineage cell. In one aspect, engineered immune cell is engineered T cell. In one aspect, T cell is from mammal individual. In one aspect, T cell is from primate individual, such as human individual. T cell and / or CD4+ and / or CD8+ T cell subtype and subpopulation cover immature T (TN) cell, effector T cell (TEFF), memory T cell and its subtype such as stem cell memory T cell (TSCM), central memory T cell (TCM), effector memory T cell (TEM) or terminally differentiated effector memory T cell, tumor infiltrating lymphocyte (TIL), immature T cell, mature T cell, helper T cell, cytotoxic T cell, mucosa associated invariant T cell (MAIT), natural regulatory T (Treg) cell and adaptive regulatory T (Treg) cell, helper T cell such as TH1 cell, TH2 cell, TH3 cell, TH17 cell, TH9 cell, TH22 cell, follicular helper T cell, α / β T cell and δ / γ T cell. Non-limiting examples of commercially available T cell lines include the cell lines BCL2(AAA) Jurkat( CRL-2902 TM )、BCL2(S70A)Jurkat( CRL-2900 TM )、BCL2(S87A)Jurkat( CRL-2901 TM )、BCL2 Jurkat( CRL-2899 TM )、Neo Jurkat( CRL-2898 TM), TALL-104 cytotoxic human T cell line (ATCC #CRL-11386). Other examples include, but are not limited to, mature T cell lines, such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki 225, My-La, Se-Ax, SKW-3, SMZ-1, and T34; and immature T cell lines, such as ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas45, KE-37, KOPT-K1, K-T1, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13. MOLT-16, MT-1, MT-ALL, P12 / Ichikawa, Peer, PER0117, PER-255, PF-382, PFI-285, RPMI-8402, ST-4, SUP-T1 to T14, TALL-1, TALL-101, TALL-1 03 / 2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, TK-6, TLBR-1, TLBR-2, TLBR-3 and TLBR-4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCC TIB-153), J45.01(ATCC CRL-1990), J.CaM1.6(ATCC CRL-2063), RS4;11(ATCC CRL-1873), CCRF-CEM (ATCC CRM-CCL-119); and cutaneous T-cell lymphoma lines, e.g., HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), HuT102 (ATCC TIB-162). Non-limiting exemplary sources of such commercially available cell lines include the American Type Culture Collection (ATCC) (Manassas, Virginia) and the German Collection of Microorganisms and Cell Cultures.

[0095] In some aspects, the cell is a cytotoxic T cell (also referred to as TC, cytotoxic T lymphocyte, CTL, T killer cell, lytic T cell, CD8+T cell or killer T cell). In some aspects, the T cell is a CD4+T cell. In some aspects, the T cell can be a CD4+T cell or a CD8+T cell. In some aspects, the cell is a tumor-specific T cell.

[0096] In one aspect, the cells are natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, tumor infiltrating lymphocytes (TIL), lymphokine-activated killer (LAK) cells, and the like. NK cells can be isolated or obtained from commercially available sources. Non-limiting examples of commercially available NK cell lines include NK-92 ( CRL-2407 TM )、NK-92MI( CRL-2408 TM Other examples include, but are not limited to, the NK lines HANK1, KHYG-1, NKL, NK-YS, NOI-90, and YT. Non-limiting exemplary sources of such commercially available cell lines include the American Type Culture Collection (ATCC) (Manassas, VA) and the German Collection of Microorganisms and Cell Cultures.

[0097] In one aspect, the cell is a B cell, a monocyte, or a granulocyte, such as a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil.

[0098] In some aspects, the cell is a myeloid cell. Non-limiting examples of myeloid cells include monocytes, macrophages, basophils, neutrophils, eosinophils, mast cells, erythrocytes, megakaryocytes, platelets, and stem cells that can differentiate into myeloid cells. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., embryonic stem cell or induced pluripotent stem cell).

[0099] 3.2. Genetic engineering methods

[0100] In one aspect, engineered cells are prepared by various methods of transferring polynucleotides encoding fusion proteins (such as antigen receptors, such as CAR). Physical methods include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Biological methods include the use of DNA and RNA vectors, such as viral vectors, such as lentiviral vectors. Chemical methods include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, microbeads and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. Exemplary methods are shown in Table 3. In some aspects, those well-known methods include transduction by virus (such as retrovirus or lentivirus), transposon and electroporation.

[0101] Table 3. Delivery methods for genome editing systems

[0102] In one aspect, the recombinant polynucleotide is transferred into the cell using recombinant infectious virus particles (e.g., adenovirus vectors, AAV vectors, slow virus vectors, retrovirus vectors such as gamma-retrovirus vectors). In one aspect, the retroviral vector or slow virus vector has a long terminal repeat (LTR). In one aspect, the vector is self-inactivating (SIN). In one aspect, the vector is a conditional replication (mobile) vector. In one aspect, the slow virus vector is derived from a human, cat or ape slow virus. In one aspect, the retroviral vector is derived from a mouse retrovirus. In one aspect, slow virus or retrovirus include slow viruses or retroviruses from any avian or mammalian cell source. In one aspect, slow virus or retrovirus are amphotropic, which means that they can infect host cells of several species (including humans). In one aspect, the gene to be expressed replaces retroviral gag, pol and / or env sequences.

[0103] In one aspect, the vector comprising the polynucleotide encoding the fusion protein (eg, CAR) may include a promoter and / or enhancer or regulatory element to regulate the expression of the encoded recombinant receptor. In one aspect, the promoter and / or enhancer or regulatory element may be a conditional dependent promoter, enhancer and / or regulatory element. In one aspect, the polynucleotide encoding the fusion protein may be operably linked to a constitutive promoter. In one aspect, the promoter is selected from cytomegalovirus (CMV) promoter, elongation factor-1α (EF1α) promoter, ubiquitin C (UbiC) promoter, phosphoglycerol kinase (PGK) promoter, simian virus 40 (SV40) early promoter and chicken beta-actin promoter (CAGG) coupled to CMV early enhancer.

[0104] In one aspect, polynucleotide is operably connected with inducible promoter.Inducible promoter can be induced by one or more conditions, such as physical condition, the microenvironment of engineered immune effector cell, or the physiological state, inducer (i.e., inducer) or its combination of engineered immune effector cell.In one aspect, inducing condition does not induce the expression of endogenous gene in engineered mammalian cell and / or the individual of receiving pharmaceutical composition.In one aspect, inducing condition is selected from: inducer, radiation (such as ionizing radiation, light), temperature (such as heat), redox state, tumor environment and the activation state of engineered mammalian cell.

[0105] In one aspect, the polynucleotide is operably linked to a Woodchuck Hepatitis Virus (WHP) post-transcriptional regulatory element (WPRE) located downstream of the polynucleotide.

[0106] In one aspect, the vector can contain a single promoter driving the expression of one or more nucleic acid molecules. In one aspect, such nucleic acid molecules can be polycistronic. For example, in one aspect, the transcription unit can be engineered to include a bicistronic unit of IRES (internal ribosome entry site), which allows the co-expression of gene products (for example, encoding the first CAR and the second CAR) by the messenger RNA from a single promoter. In one aspect, a single promoter can guide the expression of RNA, which contains two or three genes (for example, encoding the first CAR and the second CAR) separated from each other by the sequence encoding self-cleavage peptides or protease recognition sites in a single open reading frame (ORF). In one aspect, self-cleavage peptides are selected from the self-cleavage peptides of foot-and-mouth disease virus (F2A), horse rhinitis virus (E2A), Thosea asigna virus (T2A) and porcine teschovirus-1 (P2A).

[0107] In one aspect, the polynucleotides are transferred into T cells by electroporation. In one aspect, the polynucleotides are transferred into T cells by transposition. In one aspect, the polynucleotides are delivered by transposons (including the Sleeping Beauty transposon system (SB) and / or the piggyBac (PB) transposon system).

[0108] The polynucleotide encoding the fusion protein can be combined with other coding regions encoding secretory peptides or signal peptides, which guide the secretion of the fusion protein. For example, if the fusion protein needs to be secreted, the DNA encoding the signal sequence can be placed upstream of the fusion protein. It is known to those of ordinary skill in the art that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cut from the translated polypeptide to produce a secreted or "mature" form of the polypeptide. In one aspect, the signal peptide comprises the sequence of human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, CD37, CD45, 4-1BB, GM-CSFR, IL-2, CD33, human IgKVIII, human IgG2 H, chymotrypsinogen, trypsinogen-2, HSA, insulin or tPA signal peptide.

[0109] In one aspect, the polynucleotide encoding the fusion protein contains a nucleic acid sequence encoding one or more markers. In one aspect, the one or more markers are transduction markers, surrogate markers, and / or selection markers. In one aspect, the polynucleotide encoding the fusion protein contains a nucleic acid sequence encoding one or more additional fusion proteins that enhance and / or diminish the response of the cell upon adoptive transfer and encountering a ligand.

[0110] In one aspect, polynucleotides can also encode one or more surrogate markers. In one aspect, surrogate markers can include a truncated form of a cell surface polypeptide, such as a non-functional and non-transducing or non-transducing signal or a signal that is usually transduced by a cell surface polypeptide of full-length form and / or a truncated form that is not internalized or that can not be internalized. In one aspect, the cell surface polypeptide of a truncated form includes a growth factor or other receptors of a truncated form, such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (EGFRt), truncated prostate-specific membrane antigen (PSMA), or a modified form thereof. EGFRt can be used to identify or select cells engineered with EGFRt fusion protein and encoded exogenous protein, and / or for removing or separating cells expressing encoded exogenous protein.

[0111] In one aspect, marker is a fluorescent protein, for example green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP) such as superfolding GFP (sfGFP), red fluorescent protein (RFP) such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP) and yellow fluorescent protein (YFP) and variant thereof, including species variant, monomer variant and codon optimized and / or enhanced variant of fluorescent protein. In one aspect, marker is or comprises enzyme, for example luciferase, from the lacZ gene of Escherichia coli, alkaline phosphatase, secretory embryonic alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT). Exemplary luminescent reporter gene includes luciferase (luc), beta-galactosidase, chloramphenicol acetyltransferase (CAT), beta-glucuronidase (GUS) or its variant.

[0112] In one aspect, the marker is a selective marker. In one aspect, the selective marker is a polypeptide that confers resistance to an exogenous active agent or drug. In one aspect, the selective marker is an antibiotic resistance gene. In one aspect, the selective marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In one aspect, the selective marker is selected from a puromycin resistance gene, a hygromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a zeocin resistance gene or a modified form thereof.

[0113] Alternatively, various assays can be used to confirm the presence of the recombinant DNA sequence in the engineered cells, such as Southern and Northern blotting, RT-PCR and PCR. In one aspect, the fusion protein can be detected by the release of the ability to recognize target cells or cytokines (e.g., interferon-γ, granulocyte / monocyte colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-α) or interleukin 2 (IL-2). In addition, the function of the fusion protein can be evaluated by measuring the cytotoxicity of the cells.

[0114] 3.3 Preparation of engineered cells

[0115] In one aspect, the present disclosure provides a method for manufacturing engineered cells. In one aspect, any known preparation method can be used. In a specific aspect, the method includes transducing isolated cells with a polynucleotide encoding a fusion protein, and selecting a subpopulation of the isolated cells that have been successfully transduced with the polynucleotide, thereby producing genetically modified cells as described above.

[0116] In one aspect, the method includes the steps of collection, isolation, transduction, and amplification. In a specific aspect, the method includes the following steps: (i) collecting an immune cell population (e.g., blood cells); (ii) isolating a specific cell population (e.g., T cells and / or NK cells); (iii) transducing the isolated cell population with a polynucleotide encoding a fusion protein; and (iv) amplifying a subpopulation of the isolated cells that have been successfully transduced with the nucleic acid sequence of step (iii), thereby producing genetically modified cells. These different steps are described in more detail below.

[0117] 3.3.1 Cell collection

[0118] In one aspect, the individual from whom cells are obtained for introduction of fusion proteins (e.g., CARs) is a person who has a disease or disorder or needs a cell therapy or to whom a cell therapy will be administered. In one aspect, cells can be from a healthy donor.

[0119] In one aspect, cell can be obtained from sample, such as biological sample.In one aspect, sample is selected from whole blood, peripheral blood mononuclear cell (PBMC), leukocyte, bone marrow, thymus, tissue biopsy, tumor tissue, leukemia tissue, lymphoma tissue, lymph node, intestinal associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix uteri, testis, ovary, tonsil or other organs and / or the cell derived therefrom.In one aspect, cell is primary cell.In one aspect, for example, by apheresis (apheresis) or cytoremoval (leukapheresis) obtain the cell from individual circulating blood.Gained sample comprises lymphocyte, including T cell, monocyte, granulocyte, B cell, other nucleated leukocytes, red blood cell and / or platelet, and comprises the cell except red blood cell and platelet in some aspects.

[0120] 3.3.2 Cell separation

[0121] There are various methods for conveniently isolating immune cells from samples, such as using Life Technologies System; STEMcell Technologies EasySep TM 、RoboSep TM 、RosetteSep TM SepMate TM ;Miltenyi Biotec MACS TM Cell separation kits; cell surface marker expression and other commercially available cell separation kits (e.g., ISOCELL from Pierce, Rockford, IL). Specific immune cell subsets can be isolated by using microbeads or other binding agents for unique cell surface markers available in such kits. For example, MACS TM CD4+ and CD8+ microbeads can be used to isolate CD4+ and CD8+ T cells.

[0122] In one aspect, the separation of cells comprises one or more non-affinity-based cell separation steps. In one aspect, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, e.g., to remove unwanted components, enrich for desired components, lyse, or remove cells sensitive to a particular reagent. In one aspect, cells are separated based on one or more properties, e.g., density, adhesion properties, size, sensitivity, and / or resistance to a particular component.

[0123] In one aspect, the blood cells collected from the individual are washed, for example, to remove the plasma portion and the cells are placed in a suitable buffer or medium for subsequent processing steps. In one aspect, the cells are washed with phosphate buffered saline (PBS). In one aspect, the washing solution lacks calcium and can lack magnesium, or can lack many (if not all) divalent cations. The initial activation step in the absence of calcium can result in amplified activation. In one aspect, the washing step is completed by a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In one aspect, the washing step is completed by tangential flow filtration (TFF) according to the manufacturer's instructions. In one aspect, the cells can be resuspended in various biocompatible buffers (e.g., calcium-free) after washing. 2+ / Mg 2+ In certain embodiments, the components of the blood cell sample are removed and the cells are directly resuspended in culture medium. In one aspect, separation includes density-based cell separation methods, such as by lysing red blood cells and preparing white blood cells from peripheral blood by Percoll or Ficoll gradient centrifugation.

[0124] In one aspect, separation methods include separating different cell types based on the expression or presence of one or more specific molecules in the cell, such as surface markers, such as surface proteins, intracellular markers, or nucleic acids. In one aspect, separation is based on affinity or immunoaffinity separation. Separation can be based on positive selection and / or negative selection, in which cells that have bound the reagent are retained for further use, and in which cells that have not bound the antibody or binding partner are retained. Separation does not require 100% enrichment or removal of a specific cell population or cells expressing a specific marker.

[0125] In one aspect, a single separation step can remove cells that express multiple markers simultaneously, for example by incubating the cells with multiple antibodies or binding partners, each specific for the marker being targeted for negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating the cells with multiple antibodies or binding partners expressed on various cell types. In one aspect, multiple rounds of separation steps are performed, wherein the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.

[0126] In some aspects, specific T cell subsets are isolated by positive or negative selection techniques, such as cells that are positive for or express high levels of one or more surface markers, such as CD3+, CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells. In one aspect, the T cell subsets are isolated by particles or microbeads (e.g., conjugated to anti-CD3 / anti-CD28) conjugated to anti-CD3 / anti-CD28. M-450CD3 / CD28T Cell Expander, MACSiBeads TM In one embodiment, the T cells are isolated by incubating with the microbeads and cells together (e.g., microbeads ...

[0127] In some aspects, T cells are isolated from a sample by negative selection for markers expressed on non-T cells such as B cells, monocytes, or other leukocytes, such as CD14. In some aspects, a CD4 or CD8 selection step is used to separate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed at a relatively high level on one or more naive, memory, and / or effector T cell subsets.

[0128] In some aspects, for example, by positive or negative selection based on surface antigens associated with the corresponding subpopulations, further enrichment or removal of immature, central memory, effector memory and / or central memory stem cells in the CD8+ cells. In some aspects, enrichment of central memory T (TCM) cells is performed to improve efficacy, for example, to improve long-term survival, expansion and / or implantation after administration. In some aspects, CD8+T cells and CD4+T cells enriched in combination with TCM are further enhanced in efficacy.

[0129] In some aspects, memory T cells are present in CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. For example, using anti-CD8 and anti-CD62L antibodies, CD62L-CD8+ and / or CD62L+CD8+ fractions in PBMCs can be enriched or depleted.

[0130] In some aspects, the enrichment of central memory T (TCM) cells is based on the positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3 and / or CD127. In some aspects, the enrichment is based on the negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, the separation of the CD8+ population of enriched TCM cells is carried out by removing cells expressing CD4, CD14, CD45RA and positively selecting or enriching cells expressing CD62L. In one aspect, the enrichment of central memory T (TCM) cells starts from the negative portion of cells selected based on CD4 expression, followed by negative selection based on the expression of CD14 and CD45RA and positive selection based on CD62L.

[0131] In one aspect, enrichment of NK cells is based on positive or high surface expression of CD56 and CD16 and on negative expression of CD3 and / or optionally on the presence of NKp46 or NKp30 receptors.

[0132] In one aspect, a sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive materials, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g. or Microbeads). The magnetically responsive material (e.g., particles) is typically attached directly or indirectly to a binding partner (e.g., an antibody) that specifically binds to a molecule (e.g., a surface marker) present on a cell, a plurality of cells, or a cell population that needs to be separated (e.g., negative selection or positive selection is required). In one aspect, the sample is placed in a magnetic field, and those cells to which the magnetically responsive or magnetizable particles are attached are attracted to the magnetic object and separated from the unlabeled cells. For positive selection, the cells attracted to the magnetic object are retained; for negative selection, the cells that are not attracted (unlabeled cells) are retained. In some aspects, a combined positive and negative selection is performed during the same selection step, wherein the positive and negative portions are retained and further processed or subjected to a further separation step.

[0133] In one aspect, the magnetically responsive particles are attached to cells that are subsequently incubated, cultured, and / or engineered; in some aspects, the particles are attached to cells that are administered to a patient. In one aspect, the magnetically responsive particles are removed from the cells. Methods for removing magnetically responsive particles from cells are known and include, for example, the use of competitive unlabeled antibodies, magnetically responsive particles, or antibodies coupled to cleavable linkers. In one aspect, the magnetically responsive particles are biodegradable.

[0134] In one aspect, affinity-based selection is by magnetic activated cell sorting (Miltenyi Biotec, Auburn, CA). Magnetic-activated cell sorting The system is capable of selecting cells to which magnetized particles are attached with high purity. In certain embodiments, The system operates in a mode whereby non-target and target substances are sequentially eluted after application of an external magnetic field. That is, cells attached to the magnetized particles are retained, while unattached cells are eluted. Then, after this first elution step is complete, substances captured by the magnetic field but not eluted are released in a manner that allows them to be eluted and recovered. In certain embodiments, non-target cells are labeled and removed from the heterogeneous cell population.

[0135] In one aspect, cell colonies as described herein are collected and enriched (or removed) by flow cytometry, wherein cell staining is carried out for multiple cell surface markers in a fluidic system. In one aspect, cell colonies as described herein are collected and enriched (or removed) by preparation-level scale (FACS) sorting. In certain embodiments, cell colonies as described herein are collected and enriched (or removed) by using a microelectromechanical system (MEMS) chip in conjunction with a FACS-based detection system. In both cases, multiple marker labeling cells can be used, thereby allowing the T cell subpopulations clearly defined to be separated with high purity.

[0136] In one aspect, the preparation method includes the step of freezing (for example, cryopreservation) cells before or after separation, incubation and / or engineering approaches. In one aspect, freezing and subsequent thawing steps can remove the granulocytes in the cell mass and remove monocytes to a certain extent. In one aspect, for example, after a washing step to remove plasma and platelets, the cells are suspended in a freezing solution. In some aspects, any of a variety of known freezing solutions and parameters can be used. An example relates to the use of PBS or other suitable cell freezing culture media containing 20% ​​DMSO and 8% human serum albumin (HSA). It is then diluted with culture medium at 1: 1 so that the final concentrations of DMSO and HSA are 10% and 4% respectively. The cells are then frozen to -80 ° C at a rate of 1 ° C / min and stored in the gas phase of a liquid nitrogen storage tank. In one aspect, the cells of cryopreservation are thawed and washed as described herein, and left to stand at room temperature for 1 hour before activation.

[0137] 3.3.3 Cell Expansion

[0138] In one aspect, provided method comprises cultivation, hatching, culturing and / or genetic engineering step.In one aspect, before genetic engineering or when relevant to genetic engineering, hatch and / or culture cell.Incubation step can comprise cultivation, cultivation, stimulation, activation and / or propagation.In one aspect, incubate cell under stimulating condition or stimulant exists.Described condition can comprise specific culture medium, temperature, oxygen content, carbon dioxide content, time, activating agent (for example nutrient, amino acid, antibiotic, ion) and / or stimulating factor (for example cytokine, chemokine, antigen, binding partner, fusion protein, recombinant soluble receptor) and be designed for activating one or more in any other activating agent of cell.

[0139] In one aspect, the stimulating condition or stimulating agent comprises one or more agents, such as a ligand, which is capable of stimulating or activating the intracellular signaling domain of the TCR complex. In some aspects, the agent turns on or initiates the TCR / CD3 intracellular signaling cascade in the T cell. Such agents may include antibodies, such as those specific for TCR components and / or costimulatory receptors (e.g., anti-CD3, anti-CD28, e.g., bound to a solid support such as microbeads (e.g., In one aspect, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 80, 85, 90, 95, 100, 125, or 150 million cells / mL is used.

[0140] In one aspect, the mixture can be cultivated for several hours (about 3 hours) to about 14 days or any hour integer value therebetween. In another embodiment, the mixture can be cultivated for 21 days. In one embodiment, microbeads and T cells are cultivated together for about 8 days. In another embodiment, microbeads and cells are cultivated together for 2-3 days. Several stimulation cycles may also be required, so that the culture time of T cells can be 60 days or longer. Conditions suitable for T cell culture include appropriate culture medium containing factors necessary for proliferation and vigor or any other additives, including interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α. Other additives for cell growth include but are not limited to surfactants, Plasmanates and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, optimizer, with the addition of amino acids, sodium pyruvate and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a set of determined hormones, and / or some cytokines sufficient to grow and expand T cells. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures and not in cell cultures to be infused into individuals. Target cells are maintained under conditions necessary to support growth, e.g., an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO 2 ).

[0141] In one aspect, NK cell populations are expanded in vitro using interleukin-2 (IL-2), IL-15, IL-15 / IL-15RA complex, IL-18, and IL- 12. In one aspect, NK cells are expanded ex vivo for at least about 5 days, e.g., not less than about 10 days, not less than about 15 days, or not less than about 20 days, prior to administration to a patient.

[0142] IV. Pharmaceutical Compositions

[0143] The pharmaceutical compositions of the present disclosure may comprise engineered cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise: buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In one aspect, the compositions of the present disclosure are formulated for intravenous administration.

[0144] In one aspect, the pharmaceutical composition is substantially free of contaminants, e.g., no detectable levels of contaminants, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3 / anti-CD28 coated microbeads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture medium components, vector packaging cells or plasmid components, bacteria, and fungi. In one aspect, the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.

[0145] When referring to an "immunologically effective amount," "anti-tumor effective amount," "tumor suppressive effective amount," or "therapeutic amount," the precise amount of the composition of the present disclosure to be administered can be determined by the physician taking into account individual differences in age, weight, tumor size, degree and condition of infection or metastasis of the patient (individual). In general, the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 to 10 9 cells / kg body weight, in some cases 10 5 to 10 6 The T cell compositions may be administered at a dose of 10 cells / kg body weight (including all integer values ​​within these ranges). The T cell compositions may also be administered once or multiple times at these doses. The cells may be administered using infusion techniques commonly known in immunotherapy.

[0146] V. Treatment Methods

[0147] The engineered cells or pharmaceutical compositions of the present disclosure can be administered in a manner appropriate to the disease to be treated (or prevented). The diseases may include solid tumors, such as sarcomas and carcinomas, including fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, nipple cancer, thyroid carcinoma ... carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors (e.g., gliomas (e.g., brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germ cell non-solid tumors, such as leukemias, including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, myelomonocytic leukemia, and myeloblastic leukemia); The following are some of the diseases that may be treated: polycythemia vera, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0148] In one aspect, the individual suffers from a relapsed or refractory disease or condition associated with CD20. In one aspect, the individual has previously received treatment with at least one regimen comprising a drug for CD20. In one aspect, the individual is relapsed or refractory to treatment comprising an anti-CD20 monoclonal antibody. In some embodiments, the anti-CD20 monoclonal antibody is ofatumumab, obinutuzumab, or rituximab. In one aspect, the individual has progressed within 6, 12, 18, 24, 30, or 36 months after the start of the previous treatment. In one aspect, the individual has not received treatment with an anti-CD20 monoclonal antibody for at least 4 weeks (e.g., 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, 60 weeks, or longer) prior to administering an effective amount of the engineered cells or pharmaceutical compositions of the present invention.

[0149] Diseases may also comprise autoimmune diseases selected from inflammatory reactions, such as inflammatory skin diseases, including psoriasis and dermatitis (such as atopic dermatitis); systemic scleroderma and sclerosis; reactions associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic diseases, such as eczema and asthma, and other diseases involving T cell infiltration and chronic inflammatory reactions; atherosclerosis; leukocyte adhesion defects; rheumatoid arthritis; arthritis; systemic lupus erythematosus (SLE); Wegener's disease; diabetes mellitus (e.g., type 1 or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes mellitus; and immune reactions associated with cytokine- and T-lymphocyte-mediated acute and delayed hypersensitivity reactions, typically seen in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); and leukocytic edema. diapedesis); central nervous system (CNS) inflammatory diseases; multiple organ damage syndrome; hemolytic anemia; myasthenia gravis; antigen-antibody complex-mediated diseases; anti-glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendotheliosis; Reiter's disease; stiff-man syndrome; Behcet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia. In one embodiment, the medicament is used to treat multiple sclerosis. In a preferred embodiment, the medicament is used to treat secondary progressive multiple sclerosis.

[0150] The amount and frequency of administration will be determined by the type and severity of the patient's condition and the patient's disease, although suitable dosage can be determined by clinical trials. The administration of the composition can be carried out in any convenient manner, including by aerosol injection, intake, infusion, implantation or transplantation. The compositions as described herein can be administered to the patient via artery, subcutaneous, intradermal, intratumoral, intralymph node, intramedullary, intramuscular, by intravenous (iv) injection or intraperitoneal administration. In one aspect, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In one aspect, the composition is injected directly into the organ of interest (for example, an organ affected by a tumor). Alternatively, the composition is indirectly provided to the organ of interest, for example, by being administered to the circulatory system (for example, tumor vasculature). Amplification and differentiation agents can be provided before, during or after administering cells or compositions to increase the production of T cells or NK cells in vitro or in vivo.

[0151] In one aspect, the individual is subjected to lymphodepletion, e.g., prior to administration of one or more cells described herein. In one aspect, lymphodepletion comprises administration of one or more of melphalan, cytoxan, cyclophosphamide, and fludarabine.

[0152] In one aspect, engineered cells are used as a part for combination therapy, for example, with another therapeutic intervention agent (such as antibody or engineered cells or receptor or activating agent, such as cytotoxic agent or therapeutic agent) simultaneously or sequentially in any order. In some embodiments, in a manner of being administered simultaneously or in a manner of being administered sequentially in any order, cells are co-administered with one or more other therapeutic agents or co-administered with another therapeutic intervention. In one aspect, cells are co-administered with another therapy at a sufficiently close time, and the time makes cell mass enhance the effect of one or more other therapeutic agents, or vice versa. In one aspect, cells are administered before one or more other therapeutic agents. In one aspect, cells are administered after one or more other therapeutic agents such as anticancer agents. In the context of the present disclosure, it is expected that cell therapy can be similarly used in combination with chemotherapeutic agents, radiotherapeutic agents or immunotherapy interventions and pro-apoptosis or cell cycle regulators (such as immune checkpoint inhibitors).

[0153] Alternatively, this therapy can be carried out at intervals (ranging from several minutes to several weeks) before or after other active agents are treated. In some embodiments, when other active agents and cells of the present disclosure are applied to individuality respectively, it should generally be ensured that there is no significantly too long time period between the time of each delivery, so that active agent and therapy can still play a favorable combined effect on cells. In this case, it is contemplated that the contact of two treatment modalities with cells occurs within the time of about 12-24 hours, more preferably within the time of about 6-12 hours. However, in some cases, it may be necessary to significantly extend the time period for treatment, wherein between each application, an interval of several days (2,3,4,5,6 or 7 days) to several weeks (1,2,3,4,5,6,7 or 8 weeks) is used. If necessary, it is contemplated that repeated treatment cycles may be used. It is also contemplated that various standard therapies and surgical interventions are combined with cell therapy.

[0154] VI. Sequence

[0155] Table 4. Sequences of the present disclosure

[0156] VIII. Examples

[0157] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention.

[0158] Recombinant DNA technology

[0159] DNA was manipulated using standard methods, such as those described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Molecular biology reagents were used according to the manufacturer's instructions. For general information on the nucleotide sequences of human immunoglobulin light and heavy chains, see: Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No 91-3242.

[0160] DNA sequencing

[0161] The DNA sequence was determined by double-strand sequencing.

[0162] gene synthesis

[0163] If necessary, the desired gene fragment can be generated by PCR using an appropriate template, or synthesized by automated gene synthesis from synthetic oligonucleotides and PCR products. Gene fragments flanked by single restriction endonuclease cleavage sites are cloned into standard cloning / sequencing vectors. Plasmid DNA is purified from transformed bacteria and the concentration is determined by UV spectroscopy. The DNA sequence of the subcloned gene fragment is confirmed by DNA sequencing. The gene fragment is designed with appropriate restriction sites to allow subcloning into the corresponding expression vector. All constructs are designed with a 5'-terminal DNA sequence encoding a leader peptide that directs protein secretion in eukaryotic cells.

[0164] Example 1: Production and evaluation of CD20 CAR in Jurkat cells

[0165] The CAR in Table 5 with a signal peptide is formed by fusing the signal peptide to RTX-scFv, Obin-scFv or Ofa-scFv, and then fusing three different spacers and a transmembrane region derived from human CD28 or CD8 and an intracellular domain encoding a signal motif from 41BB and CD3ζ. Each CAR is co-expressed with a truncated EGFR (EGFRt, sequence as shown in SEQ ID NO: 40), and EGFRt is used as a transduction marker. CAR and EGFRt are encoded by a single promoter construct and separated by a self-cleaving T2A peptide. The sequence of the signal peptide fused to the N-terminus of CAR is shown in SEQ ID NO: 39.

[0166] Table 5. Structures of candidate CARs (from N-terminus to C-terminus)

[0167] Raji cells were purchased from ATCC (Cat No.: CCL-86). CD20 was produced by knocking out MS4A1 (the gene encoding CD20) through CRISPR-Cas9-mediated gene editing. KO Raji cells. Different constructs were transduced into CD20 KO CD20 production in Raji cells lo Raji cells (Figure 1A).

[0168] The Jurkat 76 cell line (Biovector, Cat#: 921816) was engineered to express nanoluciferase under the control of the human IL-2 promoter. The nucleic acid molecules encoding each fusion protein were cloned into lentiviral vectors and transduced into the IL-2 luciferase reporter gene Jurkat cell line. The transduced Jurkat cells were then incubated with two different Raji cells (CD20 KO / CD20 WT ) were co-cultured at an E:T ratio of 5: 1. After 24 hours, luciferase activity in the supernatant was measured using a PE Envision multimode plate reader.

[0169] When co-cultured with CD20-expressing Raji cells, Jurkat cells transduced with CAR-4, CAR-7, and CAR-8 showed higher luciferase activity, indicating higher activation levels (Figure 1B). CAR-8 with a CD28 hinge induced the strongest activation among CARs containing OFA-scFv, while CARs with an IgG4 hinge (CAR1 and CAR4) showed excellent activation signals among CARs containing Obin-scFv or RTX-scFv.

[0170] Example 3: Generation and evaluation of CD20 CAR in primary human T cells

[0171] Generate primary human T cell populations expressing various CARs using CD3 / CD28 beads T cells isolated from human PBMC samples from healthy donors were stimulated. After three days of stimulation, the cells were transduced with a lentiviral vector containing polynucleotides encoding the fusion proteins CAR-4, CAR-7, and CAR-8. The transduction efficiency was determined by EGFR staining 7 days after transduction. The transduced T cells were then compared with three different Raji cells (CD20 WT / CD20 lo / CD20 KO ) were co-cultured at an E:T ratio of 2: 1. After 48 hours, cells were isolated and stained with CD3, EGFR, CD25, CD69, and Ki67.

[0172] In Figures 2A-2B, CAR-4 and CAR-8 showed higher expression of T cell activation markers CD25 and CD69 compared to CAR-7 T cells. lo This was particularly evident when challenged with Raji cells. lo When challenged with Raji cells and stained with the proliferation marker Ki67, CAR-4 and CAR-8 T cells showed slight improvement compared with CAR-7 T cells ( Figure 2C ).

[0173] Example 3: Cytokine release after target cell stimulation

[0174] Cytokine release was assessed using the effector cells of Example 2. The supernatants from Example 2 were collected and analyzed for accumulated IFN-γ and IL-2 cytokines. The results are shown in Figures 3A-3B. Cytokine secretion was positively correlated with antigen expression. After co-culture with wild-type or low CD20-expressing tumor cell lines, T cells transduced with CAR-8 showed the highest IFNγ and IL-2 secretion. In contrast, when challenged with low-expressing targets, CAR-4 had lower cytokine secretion and CAR-7 had cytokine secretion close to background.

[0175] Example 4: Killing efficiency of CD20 CAR constructs

[0176] Primary human T cells from four healthy donors were transduced with a lentiviral vector containing a polynucleotide encoding the fusion proteins CAR-4, CAR-7, or CAR-8 described in Example 2. Three different strains of Raji cells, prestained with CellTrace Far Red, were co-cultured with the transduced T cells at a 2:1 E:T ratio. CellEvent Caspase 3 / 7 Green Detection Reagent was added to the cell mixture to monitor target cell apoptosis. Apoptotic cell signaling was monitored for 24 hours using the Incucyte Live Cell Analysis System SX5, measured every four hours.

[0177] The killing score of each CAR-T was calculated as the ratio of the Caspase3 / 7 signal from the target cell group to the signal from the CAR-T group alone. lo When challenged with Raji cells, CAR-8 showed superior killing scores compared to CAR-4 (Figure 4). lo CAR-7 killing score and background CD20 when co-cultured with Raji target KO Compared with CAR-4 and CAR-7, CAR8 T cells have a high expression of CD20 KO The nonspecific killing score of Raji cells was also slightly lower (Figure 4).

Claims

1. A fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, a spacer between the extracellular antigen binding domain and the transmembrane domain, and an intracellular signaling domain, wherein: a) the extracellular antigen binding domain is capable of specifically binding to the same epitope on CD20 as a reference antibody comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; b) the spacer region consists of a hinge domain from CD28 or a hinge domain from an immunoglobulin, wherein the length of the spacer region consisting of the hinge domain from an immunoglobulin is 12, 13, 14 or 15 amino acids; c) the transmembrane domain comprises a transmembrane domain from CD28; and d) the intracellular signaling domain comprises a primary intracellular signaling domain and a costimulatory signaling domain located between the transmembrane domain and the primary intracellular signaling domain, wherein the costimulatory signaling domain comprises the signaling domain of CD137, and the primary intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ.

2. The fusion protein according to claim 1, wherein the extracellular antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein; The VH comprises HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively; or The VH comprises HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively; or The VH comprises HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively; Preferably, the VH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4, and the VL comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5; or The VH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12, and the VL comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13; or The VH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20, and the VL comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 21; or More preferably, the VH comprises the amino acid sequence of SEQ ID NO: 4, and the VL comprises the amino acid sequence of SEQ ID NO: 5; or The VH comprises the amino acid sequence of SEQ ID NO: 12, and the VL comprises the amino acid sequence of SEQ ID NO: 13; or The VH comprises the amino acid sequence of SEQ ID NO:20, and the VL comprises the amino acid sequence of SEQ ID NO:

21.

3. The fusion protein according to claim 1 or 2, wherein the extracellular antigen binding domain is a scFv; Preferably, the VH is fused to the N-terminus of the VL via a flexible polypeptide linker at its C-terminus; More preferably, the extracellular antigen binding domain comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

4. The fusion protein according to any one of claims 1 to 3, wherein the spacer region consists of a hinge domain from CD28; Preferably, the spacer region consists of the amino acid sequence of SEQ ID NO:

30.

5. The fusion protein according to any one of claims 1 to 3, wherein the spacer consists of a hinge domain from IgG4, wherein the length of the spacer consisting of a hinge domain from IgG4 is 12 amino acids; Preferably, the spacer region consists of the amino acid sequence of SEQ ID NO:

31.

6. The fusion protein according to any one of claims 1 to 5, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:

33.

7. The fusion protein of any one of claims 1 to 6, wherein the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

35.

8. The fusion protein of any one of claims 1 to 7, wherein the co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:

34.

9. The fusion protein according to any one of claims 1 to 4 and 6 to 8, wherein: a) the extracellular antigen binding domain is capable of specifically binding to the same epitope on CD20 as a reference antibody comprising the amino acid sequence of SEQ ID NO: 1; b) the spacer region consists of a hinge domain from CD28; c) the transmembrane domain comprises a transmembrane domain from CD28; and d) the intracellular signaling domain comprises the signaling domain of CD137 and the cytoplasmic signaling domain of CD3ζ; Preferably, a) the extracellular antigen binding domain comprises VH and VL, wherein VH comprises HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and VL comprises LCDR1, LCDR2 and LCDR3 respectively comprising the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; b) the spacer region consists of the amino acid sequence of SEQ ID NO: 30; c) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 33; and d) the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 34 and SEQ ID NO: 35; More preferably, the fusion protein comprises the amino acid sequence of SEQ ID NO:

36.

10. The fusion protein according to any one of claims 1 to 3 and 5 to 8, wherein: a) the extracellular antigen binding domain is capable of specifically binding to the same epitope on CD20 as a reference antibody comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; b) the spacer region consists of a hinge domain from IgG4, wherein the length of the spacer region consisting of the hinge domain from IgG4 is 12 amino acids; c) the transmembrane domain comprises a transmembrane domain from CD28; and d) the intracellular signaling domain comprises the signaling domain of CD137 and the cytoplasmic signaling domain of CD3ζ; Preferably, a) the extracellular antigen binding domain comprises VH and VL, the VH comprises HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively; or The VH comprises HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively; b) the spacer region consists of the amino acid sequence of SEQ ID NO: 31; c) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 33; and d) the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 34 and SEQ ID NO: 35; More preferably, the fusion protein comprises the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:

38.

11. An isolated nucleic acid encoding the fusion protein of any one of claims 1 to 10.

12. A vector comprising the nucleic acid according to claim 11, preferably, the vector is a viral vector, more preferably, the viral vector is a retrovirus, lentivirus, adenovirus or adeno-associated virus vector.

13. A cell comprising the fusion protein of any one of claims 1 to 10, the isolated nucleic acid of claim 11 or the vector of claim 12, preferably, the cell is a lymphocyte, more preferably, the cell is a NK cell or a T cell.

14. A pharmaceutical preparation comprising the fusion protein of any one of claims 1 to 10, the isolated nucleic acid of claim 11, the vector of claim 12 or the cell of claim 13 and a pharmaceutically acceptable carrier.

15. A method for treating a disease or condition associated with CD20 in an individual, comprising administering to the individual the fusion protein of any one of claims 1 to 10, the isolated nucleic acid of claim 11, the vector of claim 12, the cell of claim 13, or the pharmaceutical preparation of claim 14; optionally, the individual suffers from a relapsed or refractory disease or condition associated with CD20, or has previously been treated with at least one regimen comprising a drug directed against CD20; Preferably, the disease or disorder is cancer or an autoimmune disease; More preferably, the cancer is selected from leukemia, lymphoma, lung cancer, liver cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma and rhabdomyosarcoma; and the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, Wegener's disease, inflammatory bowel disease, ulcerative colitis, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, autoimmune thrombocytopenia, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, ANCA-associated vasculitis, diabetes, Raynaud's syndrome, Sjögren's syndrome, neuromyelitis optica and glomerulonephritis.

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