Fusion proteins and their medical uses

Abstract

This disclosure provides fusion proteins containing modified spacer domains that increase antigen-binding affinity, antibodies against CD19, and their use in the treatment of diseases.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority from the aforementioned international patent applications PCT / CN2023 / 099418 and PCT / CN2024 / 091948, the latter being incorporated into this application in their entirety by reference.

[0002] This disclosure generally relates to fusion proteins containing modified hinge domains and methods for using the same. [Background technology]

[0003] Chimeric antigen receptor (CAR) T cells have shown remarkable response rates in patients with certain B-cell leukemia or lymphoma subtypes, and promising results have also been observed in patients with multiple myeloma. CAR constructs typically include an extracellular antigen-binding domain, a spacer, a transmembrane domain, and an intracellular signaling domain. The spacer, located between the extracellular antigen-binding domain and the transmembrane domain, plays a crucial role in mediating antitumor activity. CARs with improved spacer domains are needed. [Overview of the project] [Means for solving the problem]

[0004] This disclosure provides a novel fusion protein comprising a modified spacer domain having advantageous properties for cell therapy. Specifically, this disclosure relates to a CAR having a modified spacer that allows the CAR to retain its ability to stabilize, maintain the conformation of the antigen-binding domain in the CAR, and / or position the antigen-binding domain at a distance from the target epitope, so that the CAR can effectively interact with target cells.

[0005] In a first aspect, the disclosure provides a fusion protein comprising: a) an extracellular antigen-binding domain; b) a spacer or a functional equivalent thereof consisting of the sequence of Sequence ID No. 28; c) a transmembrane domain; and d) an intracellular signaling domain.

[0006] This specification also provides a fusion protein in which the spacer comprises the amino acid sequence of SEQ ID NO: 31 (EX1KSCDTPPPX2PX3CP) (wherein X1 is S or P, X2 is C or S, and X3 is R or P). In one embodiment, the spacer comprises the amino acid sequence of SEQ ID NO: 31 (EX1KSCDTPPPX2PX3CP) (wherein X1 is S, X2 is C or S, and X3 is R or P). In another embodiment, the spacer comprises the amino acid sequence of SEQ ID NO: 28.

[0007] This specification also provides fusion proteins in which the extracellular antigen-binding domain comprises an antibody containing a heavy chain variable region (VH) and a light chain variable region (VL). In one embodiment, the antibody can bind to the same epitope on CD19 as a reference antibody containing the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 25. In one embodiment, VH comprises HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, and VL comprises LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively. In another embodiment, VH comprises HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, and VL comprises LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0008] This specification also provides a fusion protein in which VH comprises the amino acid sequence of SEQ ID NO: 1, and VL comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, with the amino acids of VL at positions 39 and 80 being R and P, respectively. In one embodiment, VL comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 2, with the amino acids of VL at positions 39 and 80 being R and P, respectively. In one embodiment, the amino acids of VL at positions 100 and 103 are S and R, respectively. In one embodiment, the amino acids of VL at positions 39, 80, 100 and 103 are R, P, S and R, respectively. In one embodiment, VH comprises the amino acid sequence of SEQ ID NO: 1, and VL comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0009] This specification also provides a fusion protein in which the antibody is scFv. In one embodiment, VH is condensed at its N-terminus to the C-terminus of VL via a flexible polypeptide linker. In one embodiment, the flexible polypeptide linker comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20. In one embodiment, scFv comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. In one embodiment, scFv comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23. In one embodiment, scFv comprises the amino acid sequence of SEQ ID NO: 22.

[0010] This specification also provides fusion proteins in which the transmembrane domain comprises a transmembrane domain of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, or PD1. In one embodiment, the transmembrane domain comprises a transmembrane domain of CD8α, CD4, or CD28. In one embodiment, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 32.

[0011] This specification also provides fusion proteins in which the intracellular signaling domain comprises a co-stimulatory signaling domain and / or a primary intracellular signaling domain. In one embodiment, the co-stimulatory signaling domain is condensed at its C-terminus to the N-terminus of the primary intracellular signaling domain. In one embodiment, the co-stimulatory signaling domain is selected from the group consisting of signaling domains of ligands and combinations thereof of CD27, CD28, CD137, OX40, CD30, CD40, CD3, HVEM, ICOS, Myd88, LFA-1, ICOS, CD2, CD7, NKG2C, B7-H3, CD83. In one embodiment, the co-stimulatory signaling domain is a signaling domain of CD28 or CD137. In one embodiment, the co-stimulatory signaling domain is a signaling domain of CD137. In one embodiment, the primary intracellular signaling domain comprises a cytoplasmic signaling domain of CD3ζ. In one embodiment, the co-stimulatory signaling domain is a CD28 or CD137 signaling domain, and the primary intracellular signaling domain includes a CD3ζ cytoplasmic signaling domain. In another embodiment, the co-stimulatory signaling domain is a CD137 signaling domain, and the primary intracellular signaling domain includes a CD3ζ cytoplasmic signaling domain.

[0012] This specification also provides a fusion protein, wherein the fusion protein is a CAR, and the CAR comprises, in order 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 embodiment, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23, and the spacer comprises the amino acid sequence of SEQ ID NO: 28. In one embodiment, the CAR comprises the amino acid sequence of SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37. In one embodiment, the CAR comprises the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0013] A second aspect of this disclosure provides an antibody that binds to CD19, comprising VH and VL, wherein VH comprises the sequence of SEQ ID NO: 1, and VL comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 2, with the amino acids at positions 39 and 80 being R and P, respectively. In one aspect, VL comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 2, with the amino acids at positions 39 and 80 being R and P, respectively. In one aspect, the amino acids at positions 100 and 103 of VL are S and R, respectively. In one aspect, the amino acids at positions 39, 80, 100 and 103 of VL are R, P, S and R, respectively. In one aspect, VH comprises the amino acid sequence of SEQ ID NO: 1, and VL comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0014] This specification also provides antibodies that are scFv. In one embodiment, VH is condensed at its N-terminus to the C-terminus of VL via a flexible polypeptide linker. In one embodiment, the flexible polypeptide linker comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20. In one embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. In one embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23. In one embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 22.

[0015] This disclosure further provides a complex comprising an antibody according to this disclosure as described herein and a heterologous molecule or moiety. In one embodiment, the heterologous molecule or moiety is covalently bound to the antibody. In one embodiment, the complex is a fusion protein comprising, from its N-terminus to its C-terminus, an extracellular antigen-binding domain containing the antibody, a transmembrane domain, and an intracellular signaling domain.

[0016] This disclosure further provides isolated nucleic acids encoding fusion proteins or antibodies as described herein.

[0017] This disclosure further provides vectors comprising nucleic acids as described herein. In one embodiment, the vector is a viral vector. In one embodiment, the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus vector.

[0018] This disclosure further provides cells comprising fusion proteins, antibodies, complexes, isolated nucleic acids, or vectors as described herein. In one embodiment, the cells are lymphocytes. In one embodiment, the cells are NK cells or T cells.

[0019] This disclosure further provides pharmaceutical formulations comprising a fusion protein, antibody, complex, or cell as described herein and a pharmaceutically acceptable carrier.

[0020] This disclosure further provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 31 (EX1KSCDTPPPX2PX3CP) (wherein X1 is S or P, X2 is C or S, and X3 is R or P). In one embodiment, the polypeptide is contained in a fusion protein. In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 31 (EX1KSCDTPPPX2PX3CP) (wherein X1 is S, X2 is C or S, and X3 is R or P). In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 28.

[0021] The present disclosure further provides a method for treating cancer or an autoimmune disease in a subject, the method comprising administering to the subject a fusion protein, an antibody, a complex, a cell or a pharmaceutical formulation according to the present disclosure as described herein. The use of a fusion protein, an antibody, a complex, a cell or a pharmaceutical formulation according to the present disclosure as described herein in the manufacture of a medicament for the treatment of cancer or an autoimmune disease is also provided. The present disclosure further provides a fusion protein, an antibody, a complex, a cell or a pharmaceutical formulation according to the present disclosure as described herein for use as a medicament. In one aspect, the cancer is selected from the group consisting of leukemia, lymphoma, lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell cancer, ovarian cancer, neuroblastoma and rhabdomyosarcoma. In one aspect, the cancer is leukemia or lymphoma. In one aspect, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, lupus nephritis, multiple sclerosis, rheumatoid arthritis, Sjögren's syndrome, idiopathic thrombocytopenic purpura, type 1 diabetes, pemphigus vulgaris, neuromyelitis optica, ANCA vasculitis and myasthenia gravis. In one aspect, the autoimmune disease is systemic lupus erythematosus or lupus nephritis.

Brief Description of the Drawings

[0022] [Figure 1A-1C] Shows the binding constants (Ka), association rates (Kon) and dissociation rates (Koff) of FMC63-scFv, scFv-1, scFv-2 and scFv-3, respectively.

[0023] [Figure 2] Shows the binding affinity of CAR-W and CAR-1-12 on T cells to CD19.

[0024] [Figure 3] Relates to the sustained signaling of CAR-W and CAR-1-12.

[0025] [Figure 4A-4C]Figure 4A shows the activity of CARs including spacer S-3. Figure 4B shows the degree of CD19 expression in different target cells. Figure 4C shows the efficiency of CAR cell death on T cells.

[0026] [Figures 5A-5D] This relates to T cell activation and proliferation after target cell stimulation. Figures 5A, 5B, and 5C show IFN-γ, TNF-α, and IL-2 secreted by CAR-transduced T cells. Figure 5D shows T cell proliferation 7 days after the initial simulation.

[0027] [Figure 6] This shows CAR-T cell death of Raji-CD19Lo target cells on day 3 of the co-culture assay (E:T = 1:1, 1:2, 1:8). [Modes for carrying out the invention]

[0028] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains.

[0029] The terms "a" and "an" refer to one or more (i.e., at least one) grammatical objects of an article. For example, "module" can mean one module or multiple modules.

[0030] As used herein, the terms “contains” and “include” are synonymous with “inclusive,” “includes,” or “contains,” and are inclusive or extensible, and do not exclude any members, elements, or method steps not further enumerated. The terms “contains,” “includes,” etc., also include the term “consisting of.”

[0031] In this specification, the term “antibody” is used in its broadest sense and is not limited to any antibody structure that exhibits the desired antigen-binding activity, but includes a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), intact antibodies, and antibody fragments.

[0032] 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 intact antibody of the IgG class contains two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), followed by three constant domains (CH1, CH2, and CH3), also referred to as the heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), followed by a light chain constant (CL) domain, also referred to as the light chain constant region. The heavy chains of antibodies can be assigned to one of five types, the so-called IgA, IgD, IgE, IgG, or IgM, some of which can be further classified into subtypes, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The light chain of an antibody can be assigned to one of two types, so-called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0033] An "antibody fragment" refers to a molecule other than an intact antibody, including a portion of an intact antibody that binds to an antigen. Examples of antibody fragments, but not limited to, include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFab), single-domain antibodies, and multispecific antibodies formed from antibody fragments.

[0034] A "single-chain Fv" or "scFv" is a fusion protein of the variable regions of a light chain (VL) and a heavy chain (VH), linked by a short linker peptide of approximately 10–25 amino acids. The linker is usually flexible and may connect the N-terminus of VH to the C-terminus of VL, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker.

[0035] The term "antigen-binding domain" refers to a portion of an antibody that includes a region that binds to and is complementary to a part or all of an antigen. The antigen-binding domain may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). In a preferred embodiment, the antigen-binding domain includes an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0036] The term “variable region” or “variable domain” refers to a domain of the antibody heavy or light chain involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and complementarity-determining regions (CDRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated from antigen-binding antibodies using the VH or VL domain, and libraries of complementary VL or VH domains can be screened, respectively. Where used herein in relation to variable region sequences, “Kabat numbering” refers to the numbering scheme presented by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0037] The terms “complementarity-determining region” and “CDR” are known to refer to discontinuous sequences of amino acids within the antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3). “Framework region” and “FR” refer to the non-CDR portions of the heavy and light chain variable regions. Table 1 below lists exemplary positional boundaries of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as identified by the Kabat, Chothia, AbM, IMGT, and Contact schemes, respectively. Unless otherwise indicated, the amino acid sequences of the CDRs presented in this disclosure are determined by Kabat. [Table 1]

[0038] The term "humanized" antibody refers to a chimeric antibody that contains amino acid residues from a non-human CDR and amino acid residues from a human framework. In certain embodiments, the humanized antibody contains at least one, typically two, variable domains, where all or substantially all of the CDRs correspond to the CDRs of the non-human antibody, and all or substantially all of the FRs correspond to the FRs of the human antibody.

[0039] The term "human" antibody refers to an antibody that is produced by a human or human cell, or has an amino acid sequence that corresponds to the amino acid sequence of an antibody derived from a non-human source that utilizes sequences encoding the human antibody repertoire or other human antibodies. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0040] The interchangeable terms “specifically bind” or “bind,” as used herein, mean the ability of a protein to bind to a target protein under specific binding conditions such that its affinity or binding strength is at least five times the average affinity or binding strength of the same protein to a collection of random peptides or polypeptides of sufficient statistical size, but optionally at least 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 250 times, or 500 times, or even at least 1000 times. A specific binding protein does not need to bind exclusively to a single target molecule, but can specifically bind to a non-target molecule due to structural similarity between the target and non-target molecules (e.g., paralogs or orthologs). Those skilled in the art will recognize that specific binding to molecules with the same function in different animal species, or to non-target molecules having substantially similar epitopes to the target molecule, is possible without compromising the specificity of binding determined to a unique, statistically valid collection of non-target molecules. Thus, polypeptides can specifically bind to multiple distinct species of target molecules through cross-reactivity.

[0041] The term “epitope” refers to a portion of an antigen that specifically interacts with an antibody. Such portions, referred to herein as epitope determinants, typically include or are part of elements such as amino acid side chains or sugar side chains. Epitope determinants can be defined, for example, by methods known in the art, such as crystallography or hydrogen-deuterium exchange. At least one or more portions on the antibody molecule that specifically interact with an epitope determinant are typically located in the CDR. Typically, epitopes have specific three-dimensional structural properties and / or specific charge properties. Some epitopes are linear epitopes, while others are stereoconstructive epitopes.

[0042] The term "hinge" refers to the portion of the antibody heavy chain polypeptide that binds the CH1 and CH2 domains in the wild-type antibody heavy chain. In this disclosure, the hinge contained in the fusion protein may include naturally occurring sequences or modified sequences.

[0043] With respect to peptide, polypeptide, or antibody sequences, "amino acid sequence identity percentage (%)" and "homology" are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences as necessary to achieve the maximum sequence identity percentage and introducing gaps, without considering conservative substitutions as part of the sequence identity. Alignment for the purpose of determining amino acid sequence identity percentage can be achieved in various ways within the scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve the maximum alignment over the full length of the sequences being compared.

[0044] In the context of polypeptides (e.g., hinges), the term "fragment" refers to an amino acid sequence of a polypeptide that is shorter than the naturally occurring sequence, lacks the N-terminus and / or C-terminus, or lacks any part of the polypeptide. Thus, a fragment does not necessarily have to be a deletion of only the N-terminal and / or C-terminal amino acids. Polypeptides with deletions of internal amino acids compared to the naturally occurring sequence can also be considered fragments. The term "C-terminal fragment" refers to a fragment of a naturally occurring polypeptide that involves a deletion of only the N-terminal amino acid.

[0045] The term "functional equivalent" refers to a polypeptide that retains polypeptide function. Therefore, in some embodiments, a functional equivalent of a hinge retains the ability to stabilize the CAR molecule, maintain the conformation and valence of the antigen-binding domain (e.g., scFv) in the CAR, and / or position the antigen-binding domain at a distance from the target epitope, so that the CAR can effectively interact with target cells.

[0046] The term "fusion protein" refers to a hybrid (e.g., chimeric, recombinant) polypeptide that contains protein domains from at least two different proteins.

[0047] The term “chimeric antigen receptor” or, instead, “CAR” refers to a genetically engineered fusion protein containing two or more naturally occurring amino acid sequences or engineered amino acid sequences linked together in a manner not naturally occurring in a host cell, which may function as a receptor when present on the surface of a cell. The CARs of this disclosure include an extracellular component comprising an antigen-binding domain (e.g., scFv) linked to a spacer sequence, a transmembrane domain, and one or more intracellular signaling domains (optionally containing a costimulatory domain).

[0048] The terms “extracellular domain” or “external domain,” which may be used interchangeably, refer, as used herein, to a region of a membrane protein, such as a transmembrane protein located outside the vesicle membrane. An external domain often includes a binding domain that specifically binds to a ligand or cell surface receptor, for example, via a binding domain that specifically binds to the ligand or cell surface receptor. The term “extracellular antigen-binding domain” refers to an extracellular domain or a portion of an extracellular domain that can specifically bind to an antigen.

[0049] The interchangeable terms “endodomain,” “intracellular domain,” or “cytoplasmic domain,” as used herein, refer to regions found in certain membrane proteins, such as transmembrane proteins, that extend within the internal space defined by the cell membrane. In some cells, endodomains may interact with intracellular components and play a role in signal transduction, and thus may in some cases be intracellular signal transduction domains.

[0050] The term “transmembrane domain,” as used herein, means a domain found in a membrane protein that extends substantially or completely through a lipid bilayer, such as a lipid bilayer, such as a biological membrane, such as a mammalian cell, or an artificial construct, such as a liposome. A transmembrane protein can pass through both layers of the lipid bilayer one or more times.

[0051] As used herein, the term "spacer" refers to a polypeptide sequence that can covalently link together two separate parts: the antigen-binding domain and the transmembrane domain of a fusion protein (e.g., a CAR).

[0052] As used herein, the term "flexible polypeptide linker" refers to a peptide that binds to other peptides to form a functional protein. In relation to scFv, a flexible polypeptide linker refers to a peptide linker that binds to VH and VL to form an scFv that specifically binds to an antigen.

[0053] The term "self" refers to any material originating from the same individual that is later reintroduced into that individual.

[0054] The term "homogeneous" refers to any material originating from different animals of the same species as the individual into which it is introduced. Two or more individuals are said to be homogeneous if their genes at one or more loci are not identical. In some aspects, homogeneous material from individuals of the same species may be genetically distinct enough to interact antigenically.

[0055] The term "expression" refers to the process by which nucleic acid molecules, such as genes, produce polypeptides based on their coding sequences. This process may include transcription, post-transcriptional regulation, post-transcriptional modification, translation, post-translational regulation, post-translational modification, or any combination thereof.

[0056] The term "encodes" refers to the inherent properties of a specific sequence of a nucleotide, such as a gene, cDNA, or mRNA, in a polynucleotide, which serves as a template for the synthesis of other polymers and macromolecules in biological processes that have a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or amino acids, and the biological properties derived therefrom. Thus, a gene, cDNA, or RNA codes for a protein when the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system.

[0057] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably and refer to compounds containing amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a peptide sequence. The term “polypeptide” is also intended to refer to the products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-natural amino acids. Polypeptides in this disclosure may have sizes of approximately 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, but they do not necessarily have such a structure.

[0058] "Isolated" nucleic acid molecules refer to nucleic acid molecules that have been separated from their natural environment. Isolated nucleic acid molecules include nucleic acid molecules contained in cells that normally contain nucleic acid molecules, but the nucleic acid molecules are located outside of chromosomes or at chromosomal locations different from their natural locations on chromosomes. "Isolated polynucleotide (or nucleic acid) encoding a fusion protein" refers to one or more polynucleotide molecules encoding a fusion protein, including such polynucleotide molecules in a single vector or separate vectors, and such polynucleotide molecules present at one or more locations in a host cell.

[0059] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors can direct the expression of the nucleic acid to which they are functionally linked.

[0060] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include “transformers” and “transformed cells,” including primary transformed cells and their offspring, regardless of the number of passages. Offspring may contain mutations, but may not be completely identical to the parent cells in nucleic acid content. Mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included herein. Host cells are any type of cell line that can be used to produce the antibodies of the present invention. Host cells include cultured cells, e.g., mammalian cultured cells, for example, HEK cells, CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues. In one embodiment, the host cell of the present invention is a eukaryotic cell, particularly a mammalian cell. In another embodiment, the host cell is not a cell from the human body.

[0061] The terms “subject” or “individual” are intended to include living organisms capable of eliciting an immune response (e.g., mammals, humans).

[0062] As used herein, “treatment” (and its grammatical variations, e.g., “to treat” or “to treat”) refers to a clinical intervention in an attempt to alter the natural course of a disease in the treated individual, and may be carried out for preventive purposes or in the course of clinical symptoms. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, achieving remission or mitigation and sedation of the condition, or improving the prognosis.

[0063] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain any further components that are unacceptably toxic to the subject to which the preparation is administered. A pharmaceutical composition usually contains one or more pharmaceutically acceptable carriers. A "pharmaceutically acceptable carrier" refers to a component in the pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers, additives, stabilizers, or preservatives.

[0064] The “effective dose” of a drug, such as a pharmaceutical composition, refers to the amount that is effective in achieving the desired therapeutic or prophylactic outcome at the required dosage for the required duration.

[0065] II. Fusion Proteins This disclosure provides novel fusion proteins having advantageous properties, such as productivity, stability, binding affinity, bioactivity, targeting efficiency, reduced toxicity, an extended dose range that can be administered to patients (e.g., contained in cells), and thereby potentially enhanced efficacy. The novel fusion proteins include spacers that improve antigen binding, CAR stability, and the ability to kill target cells expressing low-density ligands.

[0066] A fusion protein comprising a) an extracellular antigen-binding domain, b) a spacer or a functional equivalent thereof consisting of the amino acid sequence of SEQ ID NO: 28, c) a transmembrane domain, and d) an intracellular signaling domain is provided herein. In some embodiments, the fusion protein is a CAR.

[0067] 2.1 Spacer

[0068] In one embodiment, a fusion protein as defined above in this specification is provided, wherein a fusion protein comprising a spacer consisting of a functional equivalent of SEQ ID NO: 28 exhibits a difference of 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less in CD19 binding compared to a fusion protein comprising a spacer consisting of SEQ ID NO: 28.

[0069] In one embodiment, the spacer comprises at least a portion of the immunoglobulin constant region or a variant or modified version thereof, such as a hinge region. In one embodiment, the spacer comprises at least a portion of the IgG hinge. In one embodiment, the spacer may be a chimeric polypeptide containing one or more fragments of the hinge derived from IgG1, IgG2, IgG3 and / or IgG4. In one embodiment, the spacer may be a chimeric polypeptide containing one or more fragments of the hinge derived from IgG3. In one embodiment, the spacer comprises an amino acid sequence having a length of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues. In some embodiments, the spacer comprises an amino acid sequence having a length of 13 to 17 amino acid residues. In a preferred embodiment, the spacer comprises an amino acid sequence having a length of 15 amino acid residues. In one embodiment, the spacer comprises an amino acid sequence that is at least about 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 28.

[0070] In one embodiment, the spacer consists of the amino acid sequence of Sequence ID No. 31 (EX1KSCDTPPPX2PX3CP) (wherein X1 is S or P, X2 is C or S, and X3 is R or P). In one embodiment, X1 is S, X2 is C or S, and X3 is R or P. In one embodiment, X1 is P, X2 is C or S, and X3 is R or P. In one embodiment, X1 is S or P, X2 is C, and X3 is R or P. In one embodiment, X1 is S or P, X2 is S, and X3 is R or P. In one embodiment, X1 is S or P, X2 is C or S, and X3 is R. In one embodiment, X1 is S or P, X2 is C or S, and X3 is P. In one embodiment, X1 is S, X2 is C, and X3 is R or P. In one embodiment, X1 is S, X2 is S, and X3 is R or P. In one embodiment, X1 is S, X2 is C, and X3 is R. In one embodiment, X1 is S, X2 is C, and X3 is P. In one embodiment, X1 is S, X2 is S, and X3 is R. In one embodiment, X1 is S, X2 is S, and X3 is P. In a particular embodiment, the spacer consists of the amino acid sequence of Sequence ID No. 28.

[0071] 2.2 Extracellular antigen-binding domain

[0072] In one embodiment, a fusion protein as defined above herein is provided, wherein the extracellular antigen-binding domain comprises one or more antibodies. In one embodiment, the antibody is a multispecific antibody formed from Fv, Fab, Fab', Fab'-SH, F(ab')2, a diabody, a linear antibody, a single-chain antibody molecule (e.g., scFv and scFab), a single-domain antibody, or an antibody fragment. In a particular embodiment, the extracellular antigen-binding domain comprises scFv.

[0073] In one embodiment, the extracellular antigen-binding domain can bind to a tumor antigen or a pathogenic antigen. In one embodiment, the tumor antigen is a tumor-specific antigen or a tumor-associated antigen (TAA). Various TAAs are known, some of which are shown in Table 2 below. The antigen-binding domain used in this disclosure is a domain that can bind to the TAAs shown therein. [Table 2]

[0074] In one embodiment, the tumor antigen is CD19. In one embodiment, the fusion protein can bind to the same epitope on CD19 as any anti-CD19 antibody in the art (e.g., FMC63, SC25C1). In one embodiment, the extracellular antigen-binding domain of the fusion protein includes an scFv that can bind to the same epitope on CD19 as a reference antibody containing the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 25. In one embodiment, the scFv includes VH and VL, where VH includes HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, and VL includes LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively. In one embodiment, scFv comprises VH and VL, where VH comprises HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, and VL comprises LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0075] In one embodiment, scFv comprises VH and VL, where VH comprises the amino acid sequence of SEQ ID NO: 1 and VL comprises the amino acid sequence of SEQ ID NO: 2. In one embodiment, VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with respect to SEQ ID NO: 1. In one embodiment, VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with respect to SEQ ID NO: 2. In one embodiment, VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with respect to SEQ ID NO: 2, and the amino acids of VL at positions 39 and 80 are R and P, respectively. In one embodiment, VL contains or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, or 96% sequence identity with SEQ ID NO: 2, and the amino acids of VL at positions 39, 80, 100, and 103 are R, P, S, and R, respectively. In a particular embodiment, VH contains or comprises the amino acid sequence of SEQ ID NO: 1, and VL contains the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0076] In one embodiment, the scFv comprises a humanized antibody or the VH and VL of a human antibody. Exemplary scFvs are described in International Publication Brochure 2010095031, International Publication Brochure 2014153270, International Publication Brochure 2017015783, Chinese Patent No. 107383196A, Chinese Patent No. 111848801A, International Publication Brochure 2018200496, or International Publication Brochure 2022105811.

[0077] In one embodiment, VH and VL are linked via a flexible polypeptide linker. In one embodiment, VH is condensed at its N-terminus to the C-terminus of VL via a flexible polypeptide linker (LH). In one embodiment, VL is condensed at its N-terminus to the C-terminus of VH via a flexible polypeptide linker (HL). In one embodiment, the flexible polypeptide linker is (G4S) n(SG4) n Or G4 (SG4) n (wherein "n" is 1, 2, 3, 4, 5, 6, 7, or 8, particularly 3). In one embodiment, the flexible polypeptide linker comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20. In one embodiment, the scFv comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25. In one embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25. Table 3 provides example scFv SEQ ID NOs. [Table 3]

[0078] 2.3 Transmembrane domain

[0079] In one embodiment, a fusion protein as defined herein is provided, wherein the transmembrane domain can be obtained from a naturally occurring protein or a synthetically produced, naturally occurring protein fragment, such as a hydrophobic protein fragment that is thermodynamically stable in a cell membrane. In one embodiment, the protein fragment has at least approximately 20 amino acids, for example, at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids.

[0080] In one embodiment, the transmembrane domains provided herein are derived from type I single-pass transmembrane proteins. In one embodiment, transmembrane domains from multi-pass transmembrane proteins may also be suitable for use in fusion proteins described herein. Multi-pass transmembrane proteins may contain complex (at least 2, 3, 4, 5, 6, 7 or more) alpha-helix or beta-sheet structures. In one embodiment, the N-terminus and C-terminus of the multi-pass transmembrane protein are located on opposite sides of the lipid bilayer, for example, the N-terminus of the protein is on the cytoplasmic side of the lipid bilayer, and the C-terminus of the protein is on the extracellular side.

[0081] In one embodiment, the transmembrane domain provided herein may include a transmembrane region and a cytoplasmic region located at the C-terminal end of the transmembrane domain. The cytoplasmic region of the transmembrane domain may contain three or more amino acids and, in some embodiments, facilitate the orientation of the transmembrane domain in the lipid bilayer. In one embodiment, one or more cysteine ​​residues are present in the transmembrane region of the transmembrane domain. In one embodiment, one or more cysteine ​​residues are present in the cytoplasmic region of the transmembrane domain. In one embodiment, the cytoplasmic region of the transmembrane domain contains positively charged amino acids. In one embodiment, the cytoplasmic region of the transmembrane domain contains the amino acids arginine, serine, and lysine.

[0082] In one embodiment, the transmembrane region of the transmembrane domain contains hydrophobic amino acid residues. In one embodiment, the transmembrane domain of the CAR provided herein contains an artificial hydrophobic sequence. For example, the phenylalanine, tryptophan, and valine triplet is located at the C-terminus of the transmembrane domain. In one embodiment, the transmembrane region contains mostly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In one embodiment, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region contains a polyleucine-alanine sequence.

[0083] In one embodiment, the transmembrane domain includes the alpha, beta, or zeta chain of the 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 (KLRFl), CD160, GPC3, and IL. -2R Beta, IL-2R Gamma, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGA M, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The transmembrane domain includes a transmembrane domain selected from the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In one embodiment, the transmembrane domain is derived from or includes the transmembrane domains of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some specific embodiments, the transmembrane domain includes an amino acid sequence having at least 88%, 92%, or 96% sequence identity with respect to SEQ ID NO: 32. In certain embodiments, the transmembrane domain includes or consists of the amino acid sequence of SEQ ID NO: 32.

[0084] 2.4 Intracellular signal transduction domains

[0085] In one embodiment, a fusion protein as defined herein is provided, in which the intracellular signaling domain is generally involved in the activation of at least one normal effector function. In one embodiment, the intracellular signaling domain comprises one or two distinct classes of cytoplasmic signaling sequences: one that initiates antigen-dependent primary activation via the TCR (primary intracellular signaling domain) and / or one that acts in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic domain, e.g., co-stimulatory domain).

[0086] In one embodiment, the primary signaling domain modulates the primary activation of the TCR complex in a stimulative or repressive manner. The primary intracellular signaling domain acting in a stimulative manner may contain signaling motifs known as immune receptor tyrosine-based activation motifs (ITAMs). Examples of ITAM-containing primary intracellular signaling domains particularly used in the present invention include those of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FceRI, DAP10, DAP12, and CD66d. In one embodiment, the CAR includes an intracellular signaling domain, such as the primary signaling domain of CD3-zeta. In one embodiment, the primary signaling domain includes a modified ITAM domain, such as a mutant ITAM domain having altered (e.g., increased or decreased) activity compared to a native ITAM domain. In one embodiment, the primary signaling domain includes a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or cleaved ITAM-containing primary intracellular signaling domain. In one embodiment, the CD3-zeta signaling domain is mutant CD3-zeta or wild-type human CD3-zeta. In one embodiment, the primary signaling domain includes one, two, three, four or more ITAM motifs. In one embodiment, the primary intracellular signaling domain is a functional variant of the cytoplasmic signaling domain of CD3ζ containing one or more mutations, e.g., Q65K.

[0087] In one embodiment, intracellular signaling sequences within the cytoplasm may be linked to each other in a random or specific order. In one embodiment, the co-stimulatory molecule is a cell surface molecule other than the antigen receptor or its ligand that is required for the efficient response of lymphocytes to the antigen. Examples of such molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, signaling lymphocyte activating molecules (SEAM proteins), activated NK cell receptors, BTFA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-I, FFA-I (CD11a / CD18), and 4-1BB (CD137). B7-H3, CDS, ICAM-L, ICOS (CD278), GITR, BAFFR, FIGHT, HVEM, KIRDS2, SFAMF7, NKp80 (KFRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 Alpha, CD8 Beta, IF2R Beta, IF2R Gamma, IF7R Alpha, ITGA4, VFA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VFA-6 , CD49f, ITGAD, CD11d, ITGAE, 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 Examples include ligands that specifically bind to CRTAM, 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 CD83.

[0088] In one embodiment, the co-stimulatory signaling domain contains 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 containing one or more amino acid variations may be referred to as a mutant. Amino acid residue mutations in the co-stimulatory signaling domain may result in increased signal transduction and enhanced immune response compared to a non-mutated co-stimulatory signaling domain. Amino acid residue mutations in the co-stimulatory signaling domain may result in decreased signal transduction and reduced immune response compared to a non-mutated co-stimulatory signaling domain.

[0089] In one embodiment, one or more co-stimulatory signaling domains are selected from the group consisting of co-stimulatory signaling domains of ligands that specifically bind to CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, NKG2C, B7-H3, and CD83. In one embodiment, the intracellular signaling domain in the CAR of this disclosure includes a co-stimulatory signaling domain derived from CD137(4-1BB). In one embodiment, the intracellular signaling domain includes a cytoplasmic signaling domain of CD3ζ and a co-stimulatory signaling domain of CD28 or CD137. In a particular embodiment, the intracellular signaling domain includes a cytoplasmic signaling domain of CD3ζ and a co-stimulatory signaling domain of CD137.

[0090] 2.5 CAR

[0091] In one embodiment, a fusion protein as defined herein is provided, the fusion protein being a CAR comprising, in order 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 embodiment, the extracellular antigen-binding domain is an scFv comprising, in order from its N-terminus to its C-terminus, a VL, a linker, and a VH. In one embodiment, the extracellular antigen-binding domain is an scFv comprising or consisting of the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In one embodiment, the spacer consists of the amino acid sequence of SEQ ID NO: 28. In one embodiment, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 32. In one embodiment, the intracellular signaling domain comprises, in order from its N-terminus to its C-terminus, a co-stimulatory signaling domain and a primary intracellular signaling domain. In one embodiment, the intracellular signaling domain comprises, in order from its N-terminus to its C-terminus, a CD28 or CD137 signaling domain and a CD3ζ cytoplasmic signaling domain. In certain embodiments, the CAR comprises or consists of the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0092] III. Antibodies This disclosure also provides novel antibodies having advantageous properties, such as production potential, stability, binding affinity, bioactivity, targeting efficiency, reduced toxicity, an extended dose range that can be administered to patients, and therefore potentially enhanced efficacy.

[0093] In one embodiment, the antibody is a multispecific antibody formed from intact antibodies or antibody fragments, such as Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFab), single-domain antibodies, or antibody fragments.

[0094] In one embodiment, the antibody can bind to CD19. In one embodiment, the antibody can bind to the same epitope on CD19 as any anti-CD19 antibody in the art (e.g., FMC63). In one embodiment, the antibody can bind to the same epitope on CD19 as a reference antibody containing the amino acid sequence of SEQ ID NO: 21. In one embodiment, the antibody comprises VH and VL, where VH comprises HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, and VL comprises LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.

[0095] In one embodiment, the antibody comprises VH and VL, where VH comprises the amino acid sequence of SEQ ID NO: 1, and VL comprises the amino acid sequence of SEQ ID NO: 2. In one embodiment, VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with respect to SEQ ID NO: 1. In one embodiment, VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with respect to SEQ ID NO: 2. In one embodiment, VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with respect to SEQ ID NO: 2, and the amino acids of VL at positions 39 and 80 are R and P, respectively. In one embodiment, VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, or 96% sequence identity with respect to SEQ ID NO: 2, and the amino acids of VL at positions 39, 80, 100, and 103 are R, P, S, and R, respectively. In a particular embodiment, VH comprises or consists of the amino acid sequence of SEQ ID NO: 1, and VL comprises or consists of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0096] In one embodiment, VH and VL are linked via a flexible polypeptide linker. In one embodiment, VH is condensed at its N-terminus to the C-terminus of VL via a flexible polypeptide linker (VL-VH). In one embodiment, VL is condensed at its N-terminus to the C-terminus of VH via a flexible polypeptide linker (VH-VL). In one embodiment, the flexible polypeptide linker is (G4S) n (SG4) n Or G4 (SG4) n (wherein "n" is 1, 2, 3, 4, 5, 6, 7, 8, and especially 3). In one embodiment, the flexible polypeptide linker contains the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20. In one embodiment, the antibody contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In one embodiment, the antibody contains the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. The CDRs and variable regions of exemplary antibodies are shown in columns 2 to 9 of Table 3.

[0097] In one embodiment, the disclosure also provides a complex comprising an antibody as defined herein and a heterologous molecule or moiety. In one embodiment, the heterologous molecule or moiety is covalently bound to the antibody. In one embodiment, the complex is a fusion protein, such as a CAR. In one embodiment, the complex comprises, in order from its N-terminus to its C-terminus, an extracellular antigen-binding domain containing the antibody, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain contains the antibody. The extracellular antigen-binding domain, transmembrane domain, and intracellular signaling domain are listed in Section II.

[0098] IV. Manipulated cells and their preparation 4.1 Cells

[0099] In one embodiment, the Disclosure provides engineered cells, such as engineered immune cells, that contain the above-mentioned fusion protein. The source of the engineered immune cells of the Disclosure may be from the patient being treated (i.e., autologous cells) or from a donor that is not the patient being treated (e.g., allogeneic cells).

[0100] In one embodiment, the cells are immune-responsive cells. In another embodiment, the cells are lymphocytes. In one embodiment, the manipulated immune cells are manipulated T cells. In one embodiment, the T cells are derived from mammalian subjects. In another embodiment, the T cells are derived from primate subjects, such as human subjects. Subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptively regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells. Non-specific examples of commercially available T cell lines include the BCL2(AAA)Jurkat line (ATCC® CRL-2902®), BCL2(S70A)Jurkat line (ATCC® CRL-2900®), BCL2(S87A)Jurkat line (ATCC® CRL-2901®), BCL2Jurkat line (ATCC® CRL-2899®), Neo Jurkat line (ATCC® CRL-2898®), and the TALL-104 cytotoxic human T cell line (ATCC#CRL-11386).Further examples include, but are not limited to, mature T cell lines such as Deglis, EBT-8, HPB-MLp-W, HUT78, HUT102, Karpas384, Ki225, 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, Luucy, MAT, MOLT-1, MOLT3, MOLT-4, MOLT13, MOLT-16, MT-1, MT-ALL, P12 / Ichikawa, Peer, PER0117, PER-255, PF-382, PFI-285, RPMI-8402, ST-4, SUP-T1~T14, TALL -1, TALL-101, TALL-103 / 2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, TK-6, TLBR-1, -2, -3 and -4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCC Examples include 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 such as HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), and HuT102 (ATCC TIB-162). Non-limiting and exemplary sources for such commercially available cell lines include the American Type Culture Collection (ATCC) (Manassas, VA) and the German Collection of Microorganisms and Cell Cultures.

[0101] In certain embodiments, the cells are cytotoxic T cells (also known as TCs, cytotoxic T cells, CTLs, T killer cells, lytic T cells, CD8+ T cells, or killer T cells). In certain embodiments, the T cells are CD4+ T cells. In certain embodiments, the T cells may be CD4+ T cells or CD8+ T cells. In certain embodiments, the cells are tumor-specific T cells.

[0102] In one embodiment, the cells include natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, tumor-infiltrating lymphocytes (TILs), and lymphokine-activated killer (LAK) cells. NK cells can be isolated or obtained from commercially available sources. Non-limiting examples of commercially available NK cell lines include the NK-92 line (ATCC® CRL-2407®) and NK-92MI (ATCC® CRL-2408®). Further examples, but not limited to, include the NK lines HANK1, KHYG-1, NKL, NK-YS, NOI-90, and YT. Non-limiting and exemplary sources for such commercially available cell lines include the American Type Culture Collection (ATCC) (Manassas, VA) and the German Collection of Microorganisms and Cell Cultures.

[0103] In one embodiment, the cells are B cells, monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and / or basophils.

[0104] In certain embodiments, the cells are cells of the myeloid cell lineage. Non-limiting examples of cells of the myeloid cell lineage include monocytes, macrophages, basophils, neutrophils, eosinophils, mast cells, erythrocytes, megakaryocytes, platelets, and stem cells from which myeloid cells can differentiate. In certain embodiments, the stem cells are pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells).

[0105] 4.2. Methods of Genetic Manipulation

[0106] In one embodiment, genetically engineered cells are prepared by various methods of transferring fusion proteins, such as antigen receptors, or polynucleotides encoding CARs. Physical methods include calcium phosphate precipitation, lipofection, particle guns, microinjection, and electroporation. Biological methods include the use of DNA and RNA vectors, such as viral vectors or lentiviral vectors. Chemical methods include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary methods are shown in Table 4. In some embodiments, these well-known methods include transduction via viruses, such as retroviruses or lentiviruses, transposons, and electroporation. [Table 4]

[0107] In one embodiment, recombinant polynucleotides are transmitted into cells using recombinant infectious viral particles, such as adenovirus vectors, AAV vectors, lentiviral vectors, or retroviral vectors, such as gamma-retroviral vectors. In one embodiment, the retroviral vector or lentiviral vector has terminal repeat sequences (LTRs). In one embodiment, the vector is self-inactivating (SIN). In one embodiment, the vector is a conditionally replicating (mobile) vector. In one embodiment, the lentiviral vector is derived from a human, feline, or monkey lentivirus. In one embodiment, the retroviral vector is derived from a mouse retrovirus. In one embodiment, the lentivirus or retrovirus includes those derived from any avian or mammalian cell source. In one embodiment, the lentivirus or retrovirus is amphotropic, meaning that they can infect host cells of several species, including humans. In one embodiment, the expressed gene replaces the gag, pol, and / or env sequences of the retrovirus.

[0108] In one embodiment, a vector containing a fusion protein, such as a polynucleotide encoding a CAR, may contain a promoter and / or enhancer or regulatory element that modulates the expression of the encoded recombinant receptor. In one embodiment, the promoter and / or enhancer or regulatory element may be a condition-dependent promoter, enhancer and / or regulatory element. In one embodiment, the polynucleotide encoding the fusion protein may be operablely coupled to a constitutive promoter. In one embodiment, the promoter is selected from the group consisting of the cytomegalovirus (CMV) promoter, the elongation factor-1 alpha (EF1α) promoter, the ubiquitin C (UbiC) promoter, the phosphoroglycerokinase (PGK) promoter, the monkey virus 40 (SV40) early promoter, and the chicken β-actin promoter (CAGG) coupled with the CMV early enhancer.

[0109] In one embodiment, a polynucleotide is operably linked to an inducible promoter. The inducible promoter can be induced by one or more conditions, such as physical conditions, the microenvironment or physiological state of the engineered immunoeffector cell, an inducer (i.e., an inducer), or a combination thereof. In one embodiment, the inducing conditions do not induce the expression of endogenous genes in the engineered mammalian cell and / or the subject receiving the pharmaceutical composition. In one embodiment, the inducing conditions are selected from the group consisting of an inducer, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and activation state of the engineered mammalian cell.

[0110] In one embodiment, the polynucleotide is operably linked to a Woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) located downstream of the polynucleotide.

[0111] In one embodiment, the vector may contain a single promoter that drives the expression of one or more nucleic acid molecules. In one embodiment, such nucleic acid molecules may be polycistronic. For example, in one embodiment, the transcription unit may be manipulated as a two-cistronic unit containing an IRES (intrasequence ribosome entry site), thereby enabling the simultaneous expression of gene products (e.g., encoding first and second CARs) by a message from a single promoter. In one embodiment, the single promoter may direct the expression of RNA containing two or three genes (e.g., encoding first and second CARs) separated from each other by a sequence encoding a self-cleaving peptide or a protease recognition site in a single open reading frame (ORF). In one embodiment, the self-cleaving peptide is selected from the group consisting of foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), and porcine rhinitis virus-1 (P2A).

[0112] In one embodiment, polynucleotides are transmitted to T cells by electroporation. In another embodiment, polynucleotides are transmitted to T cells by gene transposition. In another embodiment, polynucleotides are delivered using transposons including the Sleeping Beauty transposon system (SB) and / or the piggyBac (PB) transposon system.

[0113] The polynucleotide encoding the fusion protein may associate with a further coding region encoding a secretory or signal peptide that directs the secretion of the fusion protein. For example, if secretion of the fusion protein is desired, the DNA encoding the signal sequence may be located upstream of the fusion protein. Those skilled in the art recognize that polypeptides secreted by vertebrate cells generally have a signal peptide condensed at the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce the secretory or “mature” form of the polypeptide. In one embodiment, the signal peptide includes 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 IgG2H, chymotrypsinogen, trypsinogen-2, HSA, insulin, or tPA signal peptide. An exemplary signal peptide includes or consists of the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

[0114] In one embodiment, the polynucleotide encoding the fusion protein contains nucleic acid sequences encoding one or more markers. In one embodiment, the one or more markers are transduction markers, surrogate markers and / or selection markers. In one embodiment, the polynucleotide encoding the fusion protein contains nucleic acid sequences encoding one or more further fusion proteins that enhance and / or suppress cellular responses through adoptive immunotransduction and ligand encounter.

[0115] In one embodiment, a polynucleotide may also encode one or more alternative markers. In one embodiment, the alternative markers may include cleavage forms of cell surface polypeptides, for example, non-functional cleavage forms that do not or cannot transmit signals or signals normally transmitted by the full-length form of the cell surface polypeptide, and / or do not or cannot translocate internally. In one embodiment, cleavage forms of growth factors or other receptors, for example, cleavage cell surface polypeptides including cleavage human epidermal growth factor receptor 2 (tHER2), cleavage epidermal growth factor receptor (EGFRt), prostate-specific membrane antigen (PSMA), or modified forms thereof. Using EGFRt, cells genetically engineered with EGFRt fusion proteins and encoded foreign proteins can be identified or selected, and / or cells expressing the encoded foreign proteins can be removed or isolated.

[0116] In one embodiment, the marker is a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as superfold 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), as well as species variants, monomer variants, and codon-optimized and / or enhanced variants of the fluorescent protein. In one embodiment, the marker is an enzyme, such as luciferase, the lacZ gene from Escherichia coli (E. coli), alkaline phosphatase, secretory embryonic alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT), or comprising these. Examples of luminescence reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or their variants.

[0117] In one embodiment, the marker is a selection marker. In one embodiment, the selection marker is a polypeptide that confers resistance to an exogenous agonist or drug. In one embodiment, the selection marker is an antibiotic resistance gene. In one embodiment, the selection marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In one embodiment, the selection marker is selected from the group consisting of a puromycin resistance gene, a hygromycin resistance gene, a blastosidine resistance gene, a neomycin resistance gene, a Geneticin resistance gene, or a zeosin resistance gene or a modified form thereof.

[0118] Alternatively, the presence of recombinant DNA sequences in the manipulated cells can be confirmed using various assays, such as Southern and Northern blotting, RT-PCR, and PCR. In one embodiment, the fusion protein may be detected by its ability to recognize target cells or by the release of cytokines (e.g., interferon-γ, granulocyte / monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α), or interleukin-2 (IL-2)). Furthermore, the function of the fusion protein may be evaluated by measuring its cytotoxicity.

[0119] 4.3 Preparation of manipulated cells

[0120] In one embodiment, the present disclosure provides a process for producing engineered cells. In one embodiment, any known method for preparation may be used. In a particular embodiment, the method includes transducing a population of 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 the genetically engineered cells described above.

[0121] In one embodiment, the method includes acquisition, isolation, transduction, and proliferation steps. In a particular embodiment, the method includes (i) acquiring an immune cell population (e.g., blood cells), (ii) isolating a specific cell population (e.g., T cells and / or NK cells), (iii) transduction of the isolated cell population with a polynucleotide encoding a fusion protein, and (iv) proliferation of the subpopulation of the isolated cells that have been successfully transductioned by the nucleic acid sequence in step (iii), thereby producing genetically modified cells. These different steps are described in more detail below.

[0122] 4.3.1 Cell acquisition

[0123] In one embodiment, the subject from whom cells are obtained for the introduction of a fusion protein (e.g., CAR) is a person who has a disease or condition, requires cell therapy, or is being administered cell therapy. In one embodiment, the cells may be derived from a healthy donor.

[0124] In one embodiment, cells can be obtained from a sample, such as a biological sample. In one embodiment, the sample is selected from whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor tissue, leukemia tissue, lymphoma tissue, lymph nodes, gastrointestinal immune tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organs and / or cells derived therefrom. In one embodiment, the cells are primary cells. In one embodiment, cells from the circulating blood of the subject are obtained, for example, by apheresis or leukocyte apheresis. The sample thus obtained includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and / or platelets, and in some embodiments, includes cells other than erythrocytes and platelets.

[0125] 4.3.2 Cell Isolation

[0126] For example, various methods using Life Technologies Dynabeads® system, STEMcell Technologies EasySep®, RoboSep®, RosetteSep®, SepMate®, Miltenyi Biotec MACS® cell isolation kit, cell surface marker expression, and other commercially available cell isolation and separation kits (e.g., ISOCELL from Pierce, Rockford, IL) are readily available for isolating immune cells from a sample. Specific subpopulations of immune cells can be isolated using beads or other binders available in such kits, which are specific to unique cell surface markers. For example, CD4+ and CD8+ T cells can be isolated using MACS® CD4+ and CD8+ microbeads.

[0127] In one embodiment, cell isolation includes one or more cell separation steps that are not based on affinity. In one embodiment, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents to remove, for example, undesirable components, concentrate desirable components, or lyse or remove cells that are sensitive to a particular reagent. In one embodiment, cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity to and / or resistance to a particular component.

[0128] In one embodiment, blood cells collected from the subject are washed to remove, for example, a plasma fraction, and then the cells are placed in a suitable buffer or culture medium for subsequent processing steps. In one embodiment, the cells are washed with phosphate-buffered saline (PBS). In one embodiment, the washing solution is calcium-deficient and may be magnesium-deficient, or may be deficient in many but not all divalent cations. The initial activation step in the absence of calcium may result in increased activation. In one embodiment, the washing step is achieved by a semi-automatic "fluid" centrifuge (e.g., Cobe2991 cell processing device, Baxter) according to the manufacturer's instructions. In one embodiment, the washing step is achieved by tangential flow filtration (TFF) according to the manufacturer's instructions. In one embodiment, the cells are, for example, Ca 2+ / Mg 2+ After washing with PBS free of PBS, the cells are resuspended in various biocompatible buffers. In certain embodiments, components of the blood cell sample are removed and the cells are directly resuspended in culture medium. In one embodiment, isolation includes density-based cell separation methods, such as preparation of leukocytes from peripheral blood by lysing erythrocytes and centrifugation by Percoll or Ficoll gradient.

[0129] In one embodiment, the isolation method includes the separation of different cell types based on the expression or presence of one or more specific molecules in cells, e.g., surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In one embodiment, the separation is based on affinity or immunoaffinity. The separation may be based on positive selection (cells bound to the reagent are retained for further use) and / or negative selection (cells not bound to the antibody or binding partner are retained). The separation does not need to result in 100% enrichment or removal of a specific cell population or cells expressing a specific marker.

[0130] In one embodiment, a single separation step can simultaneously deplete cells expressing multiple markers by incubating cells with multiple antibodies or binding partners specific to each marker that targets negative selection. Similarly, positive selection can be performed simultaneously on multiple cell types by incubating cells with multiple antibodies or binding partners expressed on various cell types. In one embodiment, multiple rounds of separation steps are performed, and fractions that have undergone positive or negative selection from one step are subjected to another separation step, for example, subsequent positive or negative selection.

[0131] In some embodiments, specific subpopulations of T cells, such as those positive for or expressing high levels of one or more surface markers, e.g., CD3+, CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 conjugated particles or beads (e.g., DYNABEADS® M-450CD3 / CD28T Cell Expander, MACSiBeads®, etc.). In one embodiment, the positive selection period is about 30 minutes. In further embodiments, the period is at least 1, 2, 3, 4, 5, or 6 hours. In one embodiment, the period is 10 to 24 hours. In one embodiment, the incubation period is 24 hours. For the isolation of a desired population of cells by positive or negative selection, the concentrations of cells and particles may be varied. In certain embodiments, it may be desirable to significantly reduce the volume at which beads and cells are mixed together (i.e., increasing the cell concentration) to ensure maximum contact between cells and beads. In one embodiment, more than 100 million cells / mL are used.

[0132] In some embodiments, T cells are isolated from the sample by negative selection of non-T cells, e.g., B cells, monocytes, or other leukocytes, e.g., markers expressed on CD14. In some embodiments, CD4+ helper and CD8+ cytotoxic T cells are isolated using a CD4 or CD8 selection step. Such CD4+ and CD8+ populations may be further sorted into subpopulations by positive or negative selection of markers expressed on or relatively more highly expressed on one or more naive, memory, and / or effector T cell subpopulations.

[0133] In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, for example, by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (TCM) cells is performed to increase post-administration efficacy, improving, for example, long-term survival, proliferation, and / or engraftment. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[0134] In some embodiments, memory T cells are present in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted with respect to the CD62L-CD8+ and / or CD62L+CD8+ fractions, for example, using anti-CD8 and anti-CD62L antibodies.

[0135] In some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127. In some embodiments, this is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some embodiments, isolation of a CD8+ population enriched with TCM cells is performed by depletion of cells expressing CD4, CD14, and CD45RA, and positive selection or enrichment for cells expressing CD62L. In one embodiment, enrichment of central memory T (TCM) cells is performed starting with a negative fraction of cells selected based on CD4 expression, which is then subjected to negative selection based on the expression of CD14 and CD45RA and positive selection based on CD62L.

[0136] In one embodiment, NK cell enrichment is based on positive or high surface expression of CD56 and CD16, negative expression of CD3, and / or optionally the presence of NKp46 or NKp30 receptors.

[0137] In one embodiment, 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., Dynabeads® or MACS® beads). The magnetically responsive material, such as particles, is generally attached directly or indirectly to molecules present on the cells or population of cells that are desirable to separate, such as molecules for which negative or positive selection is desired, such as binding partners that specifically bind to surface markers, such as antibodies. In one embodiment, the sample is placed in a magnetic field, and the cells having attached magnetically responsive particles or magnetizable particles are attracted to a magnet and separated from unlabeled cells. For positive selection, cells attracted to the magnet are retained, and for negative selection, cells not attracted (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selection occurs during the same selection step, and the positive and negative fractions are retained for further processing or subject to further separation steps.

[0138] In one embodiment, magnetically responsive particles are attached to cells, which are then incubated, cultured, and / or manipulated. In several embodiments, the particles are attached to cells for administration to a patient. In one embodiment, 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 competing unlabeled antibodies, magnetically responsive particles, or antibodies conjugated to cleavable linkers. In one embodiment, the magnetically responsive particles are biodegradable.

[0139] In one embodiment, affinity-based selection is performed by Magnetically Activated Cell Sorting (MACS®) (Miltenyi Biotec, Auburn, CA). The Magnetically Activated Cell Sorting (MACS®) system can perform high-purity selection of cells having magnetic particles attached to it. In certain embodiments, MACS® operates in a mode in which non-target and target species are sequentially eluted after the application of an external magnetic field. That is, cells attached to magnetic particles are retained in place, while species that are not attached are eluted. Then, after this initial elution step is completed, species that are prevented from eluting are removed in some way so that they can be captured by the magnetic field and eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from heterogeneous populations of cells.

[0140] In one embodiment, the cell population described herein is collected and enriched (or depleted) by flow cytometry, and the cells, stained with multiple cell surface markers, are transported in a flow stream. In another embodiment, the cell population described herein is collected and enriched (or depleted) via experimental scale (FACS) sorting. In a specific embodiment, the cell population described herein is collected and enriched (or depleted) by the use of a microelectromechanical system (MEMS) chip combined with a FACS-based detection system. In any case, the cells may be labeled with multiple markers, enabling the isolation of a distinct T cell subset with high purity.

[0141] In one embodiment, the preparation method includes the step of freezing, for example, cryopreserving, cells before or after isolation, incubation, and / or genetic manipulation. In one embodiment, the freezing and subsequent thawing steps remove granulocytes and, to some extent, monocytes from the cell population. In one embodiment, the cells are suspended in a freezing solution, following a washing step to remove, for example, plasma and platelets. Any of various known freezing solutions and parameters may be used in some embodiments. One example involves using PBS or other suitable cell freezing medium containing 20% ​​DMSO and 8% human serum albumin (HSA). This is then diluted 1:1 with the medium so that the final concentrations of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen at -80°C at a rate of 1°C per minute and stored in the gas phase of a liquid nitrogen storage tank. In one embodiment, the cryopreserved cells are thawed, washed, and allowed to stand at room temperature for 1 hour before activation as described herein.

[0142] 4.3.3 Cell proliferation

[0143] In one embodiment, the method provided includes growth, incubation, culture, and / or genetic engineering steps. In one embodiment, cells are incubated and / or cultured before or in connection with genetic engineering. The incubation step may include culture, growth, stimulation, activation, and / or proliferation. In one embodiment, cells are incubated under stimulating conditions or in the presence of stimulating agents. The conditions may include one or more of a specific medium, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulating elements, e.g., cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells.

[0144] In one embodiment, the stimulating condition or agonist comprises one or more agonists, such as ligands capable of stimulating or activating the intracellular signaling domain of the TCR complex. In some embodiments, the agonist activates or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such agonists may include antibodies, such as those specific to TCR components and / or costimulatory receptors, such as anti-CD3, anti-CD28, and / or one or more cytokines bound to a solid support, such as beads (e.g., Dynabeads®). In one embodiment, cell concentrations of 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 7500, 8000, 8500, 9000, 9500, 10000, 12500, or 150 million cells / mL are used.

[0145] In one embodiment, the mixture may be cultured for a few hours (about 3 hours) to about 14 days or any intermediate integer value in one-hour increments. In another embodiment, the mixture may be cultured for 21 days. In one embodiment, the beads and T cells are cultured together for about 8 days. In another embodiment, the beads and cells are cultured together for 2 to 3 days. Several cycles of stimulation may be desirable so that the T cell culture time can be 60 days or more. Suitable conditions for T cell culture include a suitable medium that may contain factors necessary for proliferation and viability, including interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α or any other additives. Other additives for cell growth include, but are not limited to, surfactants, plasmanates and reducing agents, such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The culture medium may contain RPMI1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo15 and X-Vivo20, and an optimizer (containing added amino acids, sodium pyruvate and vitamins, and either serum-free or supplemented with an appropriate amount of serum (or plasma) or a specified amount of hormones and / or cytokines sufficient for T cell growth and proliferation). Antibiotics, such as penicillin and streptomycin, are included only in the experimental culture and not in the culture of the cells injected into the target. The target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and air (e.g., air plus 5% CO2).

[0146] In one embodiment, the NK cell population can be proliferated in vitro using interleukin-2 (IL-2) IL-15, IL-15 / IL-15RA complex, IL-18, and IL-12. In one embodiment, the NK cells are proliferated ex vivo for at least about 5 days, for example, about 10 days or more, about 15 days or more, or about 20 days or more, before administration to the patient.

[0147] V. Pharmaceutical Compositions The pharmaceutical compositions of this disclosure may comprise engineered cells in combination with one or more pharmaceutically or physiologically acceptable carriers, excipients, or additives. Such compositions may comprise buffers, such as neutral buffered saline or phosphate-buffered saline; 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 embodiment, the compositions of this disclosure are formulated for intravenous administration.

[0148] In one embodiment, the pharmaceutical composition is substantially free from, and does not contain, detectable levels of contaminants selected from the group consisting of, for example, endotoxins, mycoplasma, replicable lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, beads coated with residual anti-CD3 / anti-CD28, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture medium components, vector packaging cells or plasmid components, bacteria, and fungi. In one embodiment, the bacteria is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenzae, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.

[0149] When an "immunologically effective amount", "anti-tumor effective amount", "tumor inhibitory effective amount", or "therapeutic amount" is indicated, the exact amount of the composition of the present disclosure to be administered can be determined by a physician taking into account individual differences in age, body weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). The pharmaceutical composition containing T cells described herein is 10 4 ~10 9 cells / kg body weight, and in some cases 10 5 ~10 6 cells / kg body weight (including all integer values within those ranges) can generally be stated to be administered at a dosage. The T cell composition can be administered one or more times at these dosages. The cells can be administered by using infusion techniques generally known in immunotherapy.

[0150] VII. Methods of Treatment The pharmaceutical compositions of this disclosure may be administered in a manner suitable for the disease being treated (or prevented). The diseases include solid tumors, such as sarcomas and carcinomas, including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synoviomas, 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 carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminomas, bladder cancer, melanoma and CNS tumors (e.g., gliomas (e.g., brainstem gliomas and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) stellate Cell tumors, CNS lymphomas, germ cell tumors, medulloblastomas, schwannomas, craniopharyngiomas, ependymomas, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases), non-solid tumors, such as acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia) This may include leukemia, including chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (slowly progressive and high-grade), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and spinal cord dysplasia.

[0151] The dosage and frequency of administration are determined by the patient's condition and the type and severity of the patient's disease, although the appropriate dosage may be determined by clinical trials. Administration of the composition in question may be carried out in any convenient manner, including aerosol injection, oral ingestion, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient by transarterial, subcutaneous, intradermal, intratumoral, intranodular, intramedullary, intramuscular, intravenous (iv) injection, or intraperitoneal injection. In one embodiment, the T cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In one embodiment, the composition is injected directly into the organ of interest (e.g., the organ affected by the neoplasm). Alternatively, the composition is provided indirectly to the organ of interest, for example, by administration to the circulatory system (e.g., the tumor vascular system). Proliferation and differentiation agents may be provided before, during, or after administration of cells or compositions that increase the production of T cells or NK cells in vitro or in vivo.

[0152] In one embodiment, lymphocyte depletion is performed on the subject, for example, before administering one or more cells as described herein. In one embodiment, lymphocyte depletion includes administering one or more of melphalan, cytoxane, cyclophosphamide, and fludarabine.

[0153] In one embodiment, the manipulated cells are administered, for example, in any order, as part of a combination treatment, either simultaneously or sequentially, with another therapeutic intervention, such as an antibody, or the manipulated cells, receptor, or agonist, such as a cytotoxic agent or therapeutic agent. In some embodiments, the cells or antibodies are administered simultaneously or sequentially in any order, with one or more further therapeutic agents, or in connection with another therapeutic intervention. In one embodiment, the cells are administered concurrently with another treatment in sufficiently close time proximity such that the cell population enhances the effect of one or more further therapeutic agents, or vice versa. In one embodiment, the cells or antibodies are administered before one or more further therapeutic agents. In one embodiment, the cells or antibodies are administered after one or more further therapeutic agents, such as anticancer agents. In connection with this disclosure, cell therapy is intended to be used in conjunction with chemotherapeutic, radiotherapeutic, or immunotherapeutic interventions and apoptotic promoters or cell cycle regulators, such as immune checkpoint inhibitors.

[0154] Alternatively, this treatment may precede or follow treatment with other agents at intervals ranging from a few minutes to several weeks. In embodiments in which other agonists and cells or antibodies of this disclosure are applied to an individual separately, it is generally ensured that no significant time elapses between each delivery so that the agonists and treatments can still exert a favorably matched effect on the cells. In such cases, it is intended that the cells and both modalities may come into contact with each other within approximately 12 to 24 hours, more preferably within approximately 6 to 12 hours. In some cases, it is desirable to significantly extend the time for treatment, but the interval between each administration will be between a few days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks). The treatment cycle is expected to be repeated as needed. It is also intended that various standard therapies and surgical interventions may be applied in combination with cell therapy. VII. Array [Table 5] TIFF2026521498000006.tif255167TIFF2026521498000007.tif138170 [Examples]

[0155] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0156] Recombinant DNA technology

[0157] DNA was manipulated using standard methods as described in Sambrook et al, Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions. General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is presented in Kabat, EA et al, (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No 91-3242.

[0158] DNA sequencing

[0159] The DNA sequence was determined by double-strand sequencing.

[0160] gene synthesis

[0161] Desired gene fragments were generated by PCR using appropriate templates as needed, or synthesized from synthetic oligonucleotides and PCR products by automated gene synthesis. Gene fragments adjacent to a single restriction endonuclease cleavage site were cloned into standard cloning / sequencing vectors. Plasmid DNA was purified from transformed bacteria, and its concentration was determined by UV spectroscopy. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene fragments were designed to have appropriate restriction sites to enable subcloning into their respective expression vectors. All constructs were designed to have a 5' terminal DNA sequence encoding a leader peptide targeting a protein for secretion in eukaryotic cells.

[0162] Protein expression

[0163] The protein was expressed in host cells for several days as needed. Standard cell culture techniques were used, as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J. and Yamada, KM (eds.), John Wiley & Sons, Inc.

[0164] Protein purification

[0165] Proteins were purified from filtered cell culture supernatant according to a standard protocol. In short, antibodies were added to a Protein A Sepharose column and washed with PBS. Antibody elution was performed at a low pH, followed by immediate neutralization of the sample.

[0166] Example 1: Production and evaluation of anti-CD19 antibody Antibodies containing FMC63 scFv, scFv-1, scFv-2, or scFv-3 (sequences shown in Table 3) condensed with human IgG1Fc fragments were synthesized, and their affinity for the congener ligand CD19 was tested by surface plasmon resonance (SPR).

[0167] All SPR experiments were performed on a Biacore T200 (PCytiva) with HBS-EP as the electrophoresis buffer. Antibodies were captured using a protein A tip (LOT#10309880, Cytiva). Soluble CD19 protein (amino acids 20-291) was used as the analyte through a flow cell for 120 seconds at concentrations ranging from 300 nM to 4.69 nM, followed by dissociation analysis for 300 seconds. Binding rate (Kon), dissociation rate (Koff), and constant Ka (Kon / Koff) were fitted to the results. As shown in Figure 1, antibodies containing scFv-1 and scFv-2 exhibited improved affinity compared to other binders.

[0168] Example 2: Production and evaluation of CD19CAR CARs containing the signal peptides shown in Table 6 were formed by fusing FMC63 scFv, scFv-1, scFv-2, or scFv-3, followed by four different spacers and signal peptides (signal peptide-1 for CAR-W, signal peptide-2 for CAR-1 to CAR-13) to the transmembrane domains derived from human CD28 and 41BB and CD3ζ, and to the endodomains encoding signaling motifs. Each CAR was co-expressed with cleaved EGFR (EGFRt, sequence shown in SEQ ID NO: 40) (used as a transduction marker). Both CARs and EGFRt were encoded from a single promoter construct isolated by the self-cleaving T2A peptide. [Table 6]

[0169] Primary human T cell populations expressing various CARs were generated. Nucleic acid molecules encoding each fusion protein were individually cloned into lentiviral vectors. T cells isolated from human PBMC samples obtained from healthy donors were stimulated with CD3 / CD28 beads (DYNABEAD®). One day after stimulation, the cells were transduced with lentiviral vectors and subsequently grown for 6 days. The cells were then fused with EGFR-AF488 and CD19. pro -Stained with APC and tested by flow cytometry. CD19 binding ability was used instead of CAR stability and affinity, and tested by analyzing the mean fluorescence index (MFI) from CD19 staining after gating with EGFRt-positive cells.

[0170] CARs containing spacer S-3 (CAR-3, CAR-7, and CAR-11) exhibit increased binding ability to CD19 compared to other spacers with the same binder (Figure 2). CAR-3 showed the highest binding ability.

[0171] Example 3: Sustained signaling of the CD19CAR construct Certain CARs may exhibit antigen-independent activity or signaling, also known as sustained signaling. Excessive sustained signaling can lead to increased differentiation and T cell depletion. To evaluate the sustained signaling of transduced CAR T cells in Example 2, the cells were left undisturbed in a co-culture for 8 days in the absence of exogenous stimuli. IFNγ concentrations were then measured using the Perkin Elmer AlphaLISA detection kit according to the manufacturer's instructions.

[0172] In Figure 3, CARs containing spacer S-3 (CAR-3, CAR-7, and CAR-11) exhibited low levels of sustained signaling, as indicated by low levels of antigen-independent IFNγ secretion.

[0173] Example 4: Efficiency of CD19CAR construct death and CD69 expression Primary human T cells from four healthy donors were transduced using lentiviral vectors containing polynucleotides encoding the fusion proteins CAR-W, CAR-3, CAR-7, or CAR-11 as in Example 2. Transduction efficiency, as assessed by EGFR staining, was normalized to 50% for all samples by adding untransduced cells. These transduced cells were then exposed to Raji target cells genetically engineered to express approximately 6,704 or 74,602 copies of CD19 on their cell surface, and their cytotoxicity was tested (Figure 4A). Effector cells or vector control cells expressing only EGFRt were co-cultured with target cells in a 1:1 E:T ratio. After 20 hours, the cells were isolated and stained with CD3, EGFR, CD20, and CD69.

[0174] The cell death efficiency for each CAR T cell sample was calculated as the percentage of surviving target cells when co-cultured with T cells transduced by EGFRt alone. As shown in Figure 4B, the cell death efficiencies for CAR-3 and CAR-11 were greater than those observed for CAR-W. All transduced CAR T cells showed comparable levels of CD69 expression (Figure 4C).

[0175] Example 5: Cytokine release and T cell proliferation after target cell stimulation Cytokine release was evaluated using effector cells from Example 4. The supernatant from Example 4 was collected and the accumulated IFN-γ, TNF-α, and IL-2 cytokines were analyzed. The results are shown in Figures 5A-5C. Cells transduced by CAR-3, CAR-7, and CAR-11 showed similar IFNγ and TNFα secretion after exposure to tumor cells. However, CAR-3 and CAR-7 produced more IL-2.

[0176] In the expanded assay of Example 4, T cells were grown for 7 days after initial stimulation with target cells, and the number of CAR T cells was evaluated. As shown in Figure 5D, T cells transduced by CAR-3 and CAR-7 showed a significantly higher number of T cells compared to other CAR constructs.

[0177] Example 6: Killing efficiency of CD19CAR at various E:T ratios Primary human T cells from three healthy donors were transduced using lentiviral vectors (effector cells) containing polynucleotides encoding the fusion proteins CAR-W or CAR-13, as in Examples 1-2, or lentiviral vectors containing polynucleotides encoding EGFRt. Transduction efficiency, as evaluated by EGFR staining, was normalized to 50% for all samples by adding untransduced cells. These effector cells were then subjected to Raji-CD19 staining at various E:T ratios. Lo Cytotoxicity was tested after exposure to target cells. Effector cells expressing only EGFRt or vector control cells were tested for Raji-CD19 in E:T ratios of 1:1, 1:2, and 1:8. Lo The cells were co-cultured with target cells. Cell samples were isolated on day 3 of the co-culture assay and stained with CD3, EGFR, and CD20.

[0178] The cell death efficiency for each CAR-T cell sample was calculated as the percentage of surviving target cells when co-cultured with T cells transduced by EGFRt alone. As shown in Figure 6, the cell death efficiency for both CAR-W and CAR-13 decreases with increasing E:T ratio. However, CAR-13 showed better resistance to Raji-CD19 than CAR-W at E:T ratios of 1:2 (p=0.0129, CAR-13 vs. CAR-W) and 1:8 (p=0.0367, CAR-13 vs. CAR-W). Lo This indicates greater removal of target cells. This data demonstrates enhanced antitumor activity of CAR-T cells via the S-3 spacer.

Claims

1. a) Extracellular antigen-binding domain, b) A spacer consisting of the amino acid sequence of Sequence ID No. 28 or a functional equivalent thereof, c) Transmembrane domain and d) Intracellular signaling domains and A fusion protein containing the above.

2. The spacer has the amino acid sequence (EX) of sequence number 31. １ KSCDTPPPX ２ PX ３ CP) (wherein, X １ is S or P, X ２ is C or S, and X ３ (is R or P) Preferably, the spacer consists of the amino acid sequence of SEQ ID NO: 31 (EX １ KSCDTPPPX ２ PX ３ CP) (wherein X １ is S, X ２ is C or S, and X ３ is R or P). More preferably, the fusion protein according to claim 1, wherein the spacer consists of the amino acid sequence of SEQ ID NO:

28.

3. The extracellular antigen-binding domain comprises an antibody including a heavy chain variable region (VH) and a light chain variable region (VL). Preferably, the antibody can bind to the same epitope on CD19 as the reference antibody containing the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO:

25. More preferably, VH includes HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and VL includes LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or The fusion protein according to claim 1 or 2, wherein VH comprises HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, and VL comprises LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO:

18.

4. The VH comprises the amino acid sequence of SEQ ID NO: 1, the VL comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 2, and the amino acids of the VL at positions 39 and 80 are R and P, respectively. Preferably, the VL contains an amino acid sequence having at least 95% identity with SEQ ID NO: 2, and optionally, the amino acids of the VL at positions 100 and 103 are S and R, respectively. The fusion protein according to claim 3, more preferably, VH comprises the amino acid sequence of SEQ ID NO: 1, and VL comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:

4.

5. The antibody is scFv, and VH is condensed at its N-terminus to the C-terminus of VL via a flexible polypeptide linker. Preferably, the flexible polypeptide linker comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

20. More preferably, the fusion protein according to claim 3 or 4, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO:

23.

6. The transmembrane domain includes the transmembrane domains of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, or PD1. Preferably, the transmembrane domain includes a CD8α, CD4, or CD28 transmembrane domain. More preferably, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 32, the fusion protein according to any one of claims 1 to 5.

7. The intracellular signaling domain includes a co-stimulatory signaling domain, Preferably, the co-stimulatory signaling domain is selected from the group consisting of signaling domains of ligands for CD27, CD28, CD137, OX40, CD30, CD40, CD3, HVEM, ICOS, Myd88, LFA-1, ICOS, CD2, CD7, NKG2C, B7-H3, CD83 and combinations thereof. More preferably, the co-stimulatory signaling domain is a CD28 or CD137 signaling domain, the fusion protein according to any one of claims 1 to 6.

8. The intracellular signaling domain includes a primary intracellular signaling domain. Preferably, the primary intracellular signaling domain includes the cytoplasmic signaling domain of CD3ζ. More preferably, the intracellular signaling domain is a co-stimulatory signaling domain comprising a co-stimulatory signaling domain condensed at its C-terminus to the N-terminus of the primary intracellular signaling domain, wherein the co-stimulatory signaling domain is a CD137 signaling domain, and the primary intracellular signaling domain comprises a CD3ζ cytoplasmic signaling domain, according to any one of claims 1 to 7.

9. A CAR comprising, in order from its N-terminus to its C-terminus, the extracellular antigen-binding domain, the spacer, the transmembrane domain, and the intracellular signal transduction domain, Preferably, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23, and the spacer comprises the amino acid sequence of SEQ ID NO:

28. More preferably, the CAR comprises the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36, the fusion protein according to any one of claims 1 to 8.

10. An antibody that binds to CD19, comprising VH and VL, wherein VH comprises the amino acid sequence of SEQ ID NO: 1, and VL comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 2, and the amino acids of VL at positions 39 and 80 are R and P, respectively. Preferably, the VL contains an amino acid sequence having at least 95% identity with SEQ ID NO: 2, and optionally the amino acids of the VL at positions 39 and 80 are R and P, respectively, and optionally the amino acids of the VL at positions 100 and 103 are S and R, respectively. More preferably, the antibody wherein VH comprises the amino acid sequence of SEQ ID NO: 1, and VL comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:

4.

11. scFv, and VH is condensed at its N-terminus to the C-terminus of VL via a flexible polypeptide linker. Preferably, the flexible polypeptide linker comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

20. More preferably, the antibody according to claim 10, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO:

23.

12. A complex comprising the antibody according to claim 10 or 11 and a heterologous molecule or portion, Preferably, the heterologous molecule or portion is covalently bound to the antibody. More preferably, the complex is a fusion protein comprising, in order from its N-terminus to its C-terminus, an extracellular antigen-binding domain containing the antibody, a transmembrane domain, and an intracellular signaling domain.

13. An isolated nucleic acid encoding a fusion protein according to any one of claims 1 to 9 or an antibody according to claim 10 or 11.

14. A vector comprising the nucleic acid described in claim 13, preferably a viral vector, and more preferably the viral vector being a retrovirus, lentivirus, adenovirus, or adeno-associated virus vector.

15. A cell comprising a fusion protein according to any one of claims 1 to 9, an antibody according to claim 10 or 11, a complex according to claim 12, an isolated nucleic acid according to claim 13, or a vector according to claim 14, preferably a lymphocyte, more preferably an NK cell or a T cell.

16. A pharmaceutical formulation comprising a fusion protein according to any one of claims 1 to 9, an antibody according to claim 10 or 11, a complex according to claim 12, or a cell according to claim 15, and a pharmaceutically acceptable carrier.

17. A method for treating cancer or autoimmune disease in a subject, comprising administering to the subject a fusion protein according to any one of claims 1 to 9, an antibody according to claim 10 or 11, a complex according to claim 12, or a cell according to claim 15, Preferably, the cancer is selected from the group consisting of leukemia, lymphoma, lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, and rhabdomyosarcoma, and the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, lupus nephritis, multiple sclerosis, rheumatoid arthritis, Sjögren's syndrome, idiopathic thrombocytopenic purpura, type 1 diabetes mellitus, pemphigus vulgaris, neuromyelitis optica, ANCA vasculitis, and myasthenia gravis. A method wherein the cancer is leukemia or lymphoma, and the autoimmune disease is systemic lupus erythematosus or lupus nephritis.

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