Recombinant receptor that binds to the receptor for B cell activating factor and its use

Abstract

Disclosed is a recombinant receptor having a binding domain that binds to the B cell activation factor receptor (BAFF-R). The recombinant receptor of the present disclosure comprises a chimeric antigen receptor (CAR) having an anti-BAFF-R binding domain, a transmembrane domain, an intracellular signaling domain of CD3ζ / 4-1BB, and a spacer. Further provided are methods and systems for treating cancers that express BAFF-R, such as mantle cell lymphoma (MCL), multiple myeloma (MM), acute lymphoblastic leukemia (ALL), and diffuse large B cell lymphoma (DLBCL). The recombinant receptor disclosed herein can bind to an antigen and induce a cytotoxic effect even in a state of low antigen density.

Description

Technical Field

[0001] Cross-reference to related applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 365,635, filed on June 1, 2022, the content of which is hereby incorporated by reference in its entirety as if fully set forth herein.

[0002] Reference to the Sequence Listing The Sequence Listing accompanying this application is provided in XML format rather than as a hard copy, and the Sequence Listing is hereby incorporated by reference in this specification. The name of the XML file containing this Sequence Listing is S281-0044PCT.xml. The size of this XML file is 82.6 KB, it was created on May 30, 2023, and it was electronically filed via the Patent Center.

[0003] The present disclosure provides a recombinant receptor having a binding domain that binds to the B cell activating factor receptor (BAFF-R). Further, the present disclosure provides methods and systems for using the recombinant receptors described herein for therapeutic purposes.

Background Art

[0004] According to the World Health Organization, cancer is one of the leading causes of death worldwide, and cancer deaths reached nearly 10 million in 2020.

[0005] Over the years, the treatments selected for cancer have been surgery, chemotherapy, and / or radiation therapy. More recently, targeted advanced treatments that specifically target cancer cells have been developed by identifying and utilizing specific molecular and / or immunophenotypic changes mainly observed in cancer cells. For example, many cancer cells selectively express specific markers on their cell surface, and such markers are utilized as targets for antibody-based therapies.

[0006] The gene manipulation technology of immune cells has made great progress, enabling the targeting and killing of unwanted cells such as cancer cells. Many of these immune cells are T cells genetically modified to express chimeric antigen receptors (CARs). A CAR is a protein that contains several characteristic components that enable the genetically engineered T cells to recognize and kill cancer cells. These components include at least an extracellular portion and an intracellular portion, and these extracellular and intracellular portions are expressed as a single protein or assembled and expressed in the form of functional units. The extracellular portion contains a binding domain that binds to a marker (such as an antigen) selectively present on the surface of unwanted cells. When the binding domain binds to such a marker, a signal is transmitted from the intracellular portion to the T cell to destroy the bound cells. A CAR can further include a transmembrane domain that can link the extracellular portion to the intracellular portion.

[0007] Other components that can enhance the function of the CAR can also be used. For example, a spacer can often enhance the binding ability of the binding domain to the marker of the target cell by further imparting flexibility to the three-dimensional structure of the CAR, resulting in enhanced cell lysis. The appropriate length of the spacer in a particular CAR depends on many factors, including the requirement of how close or far the target marker is from the surface of the cell membrane of the unwanted cell. Therefore, although the general structure of the CAR is known, the ability of a particular CAR to induce cell lysis in vivo and the selection of effective CAR targets still remain as areas that should be vigorously studied and investigated. Also, it is still difficult to induce the cell lysis of the CAR even in a state where the antigen density is low.

Summary of the Invention

Means for Solving the Problems

[0008] As one of the targets of cellular immunotherapy, the B-cell activating factor (BAFF) receptor is of interest. The BAFF ligand is a very important B-cell survival factor that binds to three receptors, BAFF-R, TACI, and BCMA. These receptors are expressed by mature B cells and are also expressed in a wide variety of B-cell neoplasms. The present disclosure provides a recombinant receptor that binds to the B-cell activating factor receptor (BAFF-R) for the treatment of cancers that express BAFF-R. In certain embodiments, when the recombinant receptor of the present disclosure is expressed by a cell, (i) an extracellular portion comprising a binding domain and a spacer that binds to BAFF-R; (ii) an intracellular portion; and (iii) a transmembrane domain that links the extracellular portion to the intracellular portion is included.

[0009] In certain embodiments, the binding domain is derived from the H90 monoclonal antibody. In certain embodiments, the binding domain that binds to BAFF-R comprises a scFV comprising the sequence shown in SEQ ID NO: 1 or 6. In certain embodiments, the binding domain that binds to BAFF-R comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 8 and a light chain variable region comprising the sequence shown in SEQ ID NO: 9. In certain embodiments, the binding domain that binds to BAFF-R comprises a humanized heavy chain variable region comprising the sequence shown in SEQ ID NO: 32, 8, or 34 and a humanized light chain variable region comprising the sequence shown in SEQ ID NO: 35, 9, or 37.

[0010] In certain embodiments, the length of the spacer is 10 - 15 residues long, 110 - 130 residues long, or 230 - 240 residues long. In certain embodiments, the length of the spacer is 12 residues long, 119 residues long, or 229 residues long. In certain embodiments, the spacer comprises the hinge domain of IgG4. In certain embodiments, the spacer further comprises the CH3 domain of IgG4. In certain embodiments, the spacer further comprises the CH2 domain of IgG4. In certain embodiments, the spacer does not comprise the hinge domain of CD8a.

[0011] In certain embodiments, the intracellular portion comprises the signaling domain of CD3ζ or a functional portion thereof. In certain embodiments, the intracellular portion comprises the signaling domain of 4-1BB or a functional portion thereof. In certain embodiments, the intracellular portion comprises i) the signaling domain of CD3ζ and ii) the signaling domain of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, or B7-H3, or a functional portion thereof. In certain embodiments, the intracellular portion comprises the signaling domain of CD3ζ and the signaling domain of 4-1BB or functional portions thereof.

[0012] In certain embodiments, the transmembrane domain is the transmembrane domain of CD28. In some embodiments, the transmembrane domain does not comprise the transmembrane domain of CD8a.

[0013] In certain embodiments, the gene construct encoding the recombinant receptor may further comprise a transduction marker, a selection cassette, a self-cleaving polypeptide, a promoter, a suicide switch, or other regulatory mechanisms, and may further encode any of these.

[0014] Cells genetically modified to express the recombinant receptors disclosed herein can be used for the treatment of cancers expressing BAFF-R, such as mantle cell lymphoma (MCL), multiple myeloma (MM), acute lymphoblastic leukemia (ALL), and diffuse large B-cell lymphoma (DLBCL).

Brief Description of the Drawings

[0015] Part of the drawings submitted in this application is considered to be more understandable in color. The applicants consider the color versions of these drawings to be part of the original application and reserve the right to submit color images of the drawings in subsequent procedures.

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Mode for Carrying Out the Invention

[0026] For many years, the treatments selected for cancer have been surgery, chemotherapy, and / or radiotherapy. More recently, advanced targeted therapies that specifically target cancer cells have been developed by identifying and utilizing specific molecular and / or immunophenotypic changes that are primarily observed in cancer cells. For example, many cancer cells selectively express certain antigens on their cell surface, and such antigens have been successfully used as targets for therapeutic agents.

[0027] B-cell activating factor receptor (BAFF-R), also known as tumor necrosis factor receptor superfamily member 13C (TNFRSF13C) or BLyS receptor 3 (BR3), is a membrane protein that recognizes B-cell activating factor (BAFF). BAFF is an essential ligand for B-cell maturation and survival. Since BAFF-R plays a certain role in B-cell proliferation (Fu et al. Blood. 2009,113(19):4627-4636) and T-cell proliferation (Ye et al. European Journal of Immunology. 2004,34(10):2750-2759), it may be associated with malignancies of these cells. BAFF-R is constitutively saturated in autoimmune and lymphoproliferative diseases (Rodig et al. Human Pathology. 2005,36(10):1113-1119; Carter et al. Arthritis and Rheumatism. 2005,52(12):3943-3954; and Pers et al. Annals of the New York Academy of Sciences. 2005,1050:34-39).

[0028] The present disclosure provides a recombinant receptor that binds to BAFF-R for the treatment of cancers that express BAFF-R.

[0029] In certain embodiments, the recombinant receptor of the present disclosure, when expressed by a cell, (i) An extracellular portion comprising a binding domain and a spacer that binds to BAFF-R; (ii) An intracellular portion; and (iii) A transmembrane domain that links the extracellular portion to the intracellular portion and comprises.

[0030] In certain embodiments, the binding domain is derived from the H90 monoclonal antibody. In certain embodiments, the binding domain that binds to BAFF-R comprises a scFV comprising the sequence shown in SEQ ID NO: 1 or 6. In certain embodiments, the binding domain that binds to BAFF-R comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 8 and a light chain variable region comprising the sequence shown in SEQ ID NO: 9. In certain embodiments, the binding domain that binds to BAFF-R comprises a humanized heavy chain variable region comprising the sequence shown in SEQ ID NO: 32, 8 or 34 and a humanized light chain variable region comprising the sequence shown in SEQ ID NO: 35, 9 or 37.

[0031] In certain embodiments, the length of the spacer is 10-15 residues long, 110-130 residues long or 230-240 residues long. In certain embodiments, the length of the spacer is 12 residues long, 119 residues long or 229 residues long. In certain embodiments, the spacer comprises the hinge domain of IgG4. In certain embodiments, the spacer further comprises the CH3 domain of IgG4. In certain embodiments, the spacer further comprises the CH2 domain of IgG4. In certain embodiments, the spacer does not comprise the hinge domain of CD8a.

[0032] In certain embodiments, the intracellular portion comprises the signaling domain of CD3ζ or a functional portion thereof. In certain embodiments, the intracellular portion comprises the signaling domain of 4-1BB or a functional portion thereof. In certain embodiments, the intracellular portion comprises i) the signaling domain of CD3ζ and ii) the signaling domain of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C or B7-H3, or a functional portion thereof. In certain embodiments, the intracellular portion comprises the signaling domain of CD3ζ and the signaling domain of 4-1BB or functional portions thereof.

[0033] In certain embodiments, the transmembrane domain is the transmembrane domain of CD28. In some embodiments, the transmembrane domain does not include the transmembrane domain of CD8a.

[0034] In certain embodiments, the gene construct encoding the recombinant receptor may further comprise a selection cassette, a transduction marker, a self-cleaving polypeptide, a promoter, a suicide switch or other regulatory mechanisms, and may further encode any of these.

[0035] Cells genetically modified to express the recombinant receptors disclosed herein can be used for the treatment of cancers that express BAFF-R, such as mantle cell lymphoma (MCL), multiple myeloma (MM), acute lymphoblastic leukemia (ALL), and diffuse large B-cell lymphoma (DLBCL). In certain embodiments, the recombinant receptors of the present disclosure confer cytolytic activity even in the presence of low antigen density. For example, the data provided herein shows specific lysis of Raji cells with low antigen density per cell (6,949 antigens per cell). This is a comparison with the K562 BAFF-R cell line expressing 1,608,470 antigens per cell, the NALM 6 parental cell line expressing 57,504 antigens per cell, and the TM-LCL parental cell line expressing 97,838 antigens per cell. Since cytolytic activity can be conferred even in the presence of low antigen density, it provides important clinical utility as a treatment and can be effectively administered before the tumor mass increases and the antigen density becomes high.

[0036] As used herein, the term "low antigen density state" means the expression level of a cancer antigen in which less than 50,000 cancer molecules per diseased cell, less than 40,000 cancer molecules per diseased cell, less than 30,000 cancer molecules per diseased cell, less than 20,000 cancer molecules per diseased cell, less than 10,000 cancer molecules per diseased cell, or less than 7,000 cancer molecules per diseased cell are expressed.

[0037] Also, as used herein, the term "low antigen density state" means the expression level of BAFF-R in which less than 50,000 BAFF-R molecules per diseased cell, less than 40,000 BAFF-R molecules per diseased cell, less than 30,000 BAFF-R molecules per diseased cell, less than 20,000 BAFF-R molecules per diseased cell, less than 10,000 BAFF-R molecules per diseased cell, or less than 7,000 BAFF-R molecules per diseased cell are expressed.

[0038] Various aspects of the present disclosure will be described below in more detail with additional supplementation. Various aspects of the present disclosure include the following items, namely, (I) immune cells; (II) collection of cell samples and concentration of cells; (III) genetic modification of cell populations for expressing recombinant receptors; (III-A) genetic engineering techniques; (III-B) recombinant receptors; (III-B-1) binding domains; (III-B-2) intracellular effector domains; (III-B-3) transmembrane domains; (III-B-4) spacers, linkers and multimerization domains; (III-B-5) control mechanisms including tag cassettes, transduction markers, selection cassettes and / or suicide switches; (IV) characterization of genetically modified cells; (V) cell activation culture conditions; (VI) cell preparations produced ex vivo; (VII) target-directed viral vectors and nanoparticles for modifying cells in vivo; (VIII) kits; (IX) exemplary embodiments; (X) experimental examples; and (XI) conclusions, which will be described along. These headings are shown for the sole purpose of systematic description and do not limit the scope or interpretation of the present disclosure.

[0039] (I) Immune cells The present disclosure relates to cells genetically modified to express recombinant receptors (e.g., CARs and eTCRs). The genetically modified cells may be T cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, lymphocytes, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and / or a mixture of HSCs and HPCs (i.e., HSPCs). In certain embodiments, the genetically modified cells are T cells.

[0040] Several subsets of T cells have been found so far, and each T cell subset has characteristic functions. For example, most T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is composed of two separate peptide chains, which are produced from the T cell receptor alpha gene and the T cell receptor beta gene (TCRα and TCRβ), which are independent genes, and are called the αTCR chain and the βTCR chain, respectively.

[0041] γδ T cells are a small subset of T cells that possess a characteristic T cell receptor (TCR) on their surface. In γδ T cells, the TCR is composed of one γ chain and one δ chain. T cells in this group are not as commonly recognized as αβ T cells (only 2% of all T cells exist).

[0042] CD3 is expressed on all mature T cells. Activated T cells express 4-1BB (CD137), CD69, and CD25. CD5 and the transferrin receptor are also expressed on T cells.

[0043] T cells can be further classified into helper T cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), and cytotoxic T cells include cytolytic T cells. Helper T cells play a role in assisting other white blood cells in immunological processes and perform functions such as the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. Helper T cells are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells are activated by the presentation of peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). When activated, helper T cells rapidly divide and secrete small proteins called cytokines that are responsible for regulating or assisting an active immune response.

[0044] Cytotoxic T cells can destroy virus-infected cells and tumor cells and are also involved in transplant rejection reactions. Cytotoxic T cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. Cytotoxic T cells recognize their targets by binding to antigens bound to MHC class I molecules present on the surface of almost all cells in the body.

[0045] "Central memory" T cells (or "TCM") refer to antigen-experienced cytotoxic T cells that, compared to naive cells, express CD62L or CCR-7 and CD45RO on their surface, do not express CD45RA, or have reduced expression of CD45RA. In certain embodiments, central memory cells are positive for the expression of CD62L, CCR7, CD25, CD127, CD45RO, and CD95 and have reduced expression of CD45RA compared to naive cells.

[0046] "Effector memory" T cells (or "TEM") refer to antigen-experienced T cells that, compared to central memory cells, do not express CD62L or have reduced expression of CD62L on their surface and, compared to naive cells, do not express CD45RA or have reduced expression of CD45RA. In certain embodiments, effector memory cells are negative for the expression of CD62L and CCR7 and positive or negative for the expression of CD28 and CD45RA compared to naive or central memory cells. Also, effector T cells are positive for granzyme B and perforin compared to memory or naive T cells.

[0047] "Naive" T cells refer to T cells that have not experienced antigen and express CD62L and CD45RA and do not express CD45RO compared to central memory or effector memory cells. In certain embodiments, naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T cells, and examples of phenotypic markers of naive T cells include CD62L, CCR7, CD28, CD127, and CD45RA.

[0048] Natural killer cells (also known as NK cells, K cells, and killer cells) are activated in response to interferon or macrophage-derived cytokines. NK cells play a role in suppressing viral infections, while in the acquired immune response, antigen-specific cytotoxic T cells capable of eliminating infections are produced. NK cells express CD8, CD16, and CD56, but do not express CD3.

[0049] NK cells include NK-T cells. NK-T cells are a specialized population of T cells that express a semi-invariant T cell receptor (TCRαβ) and surface antigens normally associated with natural killer cells. NK-T cells contribute to antibacterial and antiviral immune responses and promote tumor-related immune surveillance or tumor-related immunosuppression. Similar to natural killer cells, NK-T cells can also induce cytotoxicity related to perforin, Fas, and TNF. Activated NK-T cells can produce IFN-γ and IL-4. In certain embodiments, NK-T cells are CD3+ / CD56+.

[0050] Macrophages (and their precursor cells, monocytes) are present in all tissues in the body (in some cases, present as microglia, Kupffer cells, and osteoclasts) and phagocytose apoptotic cells, pathogens, and other non-self components. Monocytes / macrophages express CD11b, F4 / 80; CD68; CD11c; IL-4Rα; and / or CD163.

[0051] Immature dendritic cells (i.e., dendritic cells in a pre-activated state) phagocytose peripheral antigens and other non-self components, become activated, migrate to the T cell area of lymphoid tissue, and present antigens to T cells. Dendritic cells express CD1a, CD1b, CD1c, CD1d, CD21, CD35, CD39, CD40, CD86, CD101, CD148, CD209, and DEC-205.

[0052] Hematopoietic stem / progenitor cells, i.e., HSPCs, refer to the combination of hematopoietic stem cells and hematopoietic progenitor cells.

[0053] Hematopoietic stem cells refer to undifferentiated hematopoietic cells that can self-renew in vivo or can self-renew and proliferate substantially without limit in vitro and can differentiate into all other types of hematopoietic cells.

[0054] Hematopoietic progenitor cells are cells derived from hematopoietic stem cells or cells derived from fetal tissues that can further differentiate into mature cells. In certain embodiments, hematopoietic progenitor cells are CD24 lo Lin - CD117 + hematopoietic progenitor cells. HPCs can (i) differentiate into myeloid progenitor cells and ultimately generate monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets or dendritic cells, or (ii) differentiate into lymphoid progenitor cells and ultimately generate T cells, B cells and NK cells.

[0055] HSPCs may be positive for specific markers whose expression levels are increased in HSPCs compared to other types of hematopoietic cells. Such markers include, for example, CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR or combinations thereof. Furthermore, HSPCs may be negative for markers expressed in other types of hematopoietic cells. Such markers include, for example, Lin, CD38 or combinations thereof. In certain embodiments, HSPCs are CD34 + cells.

[0056] The description that a cell or cell population is "positive" for a particular marker, or that a cell or cell population expresses a particular marker, means that a particular detectable marker is present on the surface or inside of the cell. When referring to a cell surface marker, "positive" means, for example, that cell surface expression detected by flow cytometry using, for example, staining with an antibody that binds to the cell surface marker and detecting that antibody is present, and that staining is detected at a substantially higher level than staining detected by the same procedure under the same conditions using an isotype-matched control in flow cytometry, and / or detected at a level substantially comparable to staining of cells known to be positive for that marker, and / or detected at a level substantially higher than staining of cells known to be negative for that marker.

[0057] The description that a cell or cell population is "negative" for a particular marker, or that a cell or cell population does not express a marker, means that no particular detectable marker is substantially present on the surface or inside of the cell. When referring to a cell surface marker, "negative" means, for example, that cell surface expression detected by flow cytometry using, for example, staining with an antibody that binds to the cell surface marker and detecting that antibody is not present, and that staining is not detected at a substantially higher level than staining detected by the same procedure under the same conditions using an isotype-matched control in flow cytometry, and / or detected at a level substantially lower than staining of cells known to be positive for that marker, and / or detected at a level substantially comparable to staining of cells known to be negative for that marker.

[0058] Cells in which a gene is modified in accordance with the teachings of the present disclosure may, where appropriate, be patient-derived cells (autologous cells), cells of the same species as the patient, in vivo cells, or ex vivo cells.

[0059] (II) Collection of cell samples and concentration of cells Methods for collecting samples and methods for concentrating samples are known to those skilled in the art. In certain embodiments, the cells are of human origin, for example, from a patient to be treated. The cells may be from a cell line. In some embodiments, the cells are obtained from a heterologous cell source, for example, from a mouse, rat, non-human primate or pig.

[0060] In some embodiments, the T cells are from samples such as whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy samples, tumors, lymph nodes, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, large intestine, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organs, and / or cells derived from these organs, or isolated from these samples. In certain embodiments, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. In certain embodiments, the sample includes lymphocytes such as T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, HSCs, HPCs, HSPCs, erythrocytes and / or platelets, and in some aspects, the sample includes cells other than erythrocytes and platelets and further processing is required. In certain embodiments, the T cells are from PBMCs.

[0061] In some embodiments, the blood cells collected from the subject are washed, for example, to remove the plasma fraction and to suspend the cells in an appropriate buffer or medium for the next processing step. In certain embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution does not contain calcium and / or magnesium and / or does not contain most or any divalent cations. The washing can be performed using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 Cell Processor, Baxter) according to the manufacturer's instructions. Tangential flow filtration (TFF) can also be performed. In certain embodiments, the washed cells can be resuspended in various types of biocompatible buffers such as Ca++ / Mg++-free PBS.

[0062] Isolation may include one or more of various cell preparation steps and cell separation steps, which include separation based on one or more characteristics such as size, density, sensitivity or resistance to a particular reagent, and / or affinity (e.g., immunological affinity) for an antibody or other binding partner. In certain embodiments, isolation is performed continuously and / or simultaneously in a single manner using one device or one facility. In certain embodiments, the isolation, culture and / or recombination of various different populations is initiated from one starting material such as one sample.

[0063] In certain embodiments, the sample can be used to enrich T cells using cell separation methods and related techniques that utilize density. For example, white blood cells can be separated from other types of cells in peripheral blood by centrifugation using a Percoll density gradient or a Ficoll density gradient after lysing red blood cells.

[0064] In certain embodiments, a bulk T cell population that has not been enriched for a particular type of T cell can be used. In certain embodiments, selected types of T cells can be enriched and / or isolated by positive selection and / or negative selection using cell markers. In positive selection, cells having a cell marker bound to a capture agent are obtained and used for subsequent applications. In negative selection, cells that did not bind to a capture agent, such as an antibody against a cell marker, are obtained and used for subsequent applications. In some examples, both the positively selected fraction and the negatively selected fraction can be obtained and used for subsequent applications. In certain embodiments, CD4+ T cells and / or CD8+ T cells are enriched from PBMCs.

[0065] It is not necessary to achieve 100% enrichment or removal of a particular cell population or cells expressing a particular marker by the above cell separation. For example, positive selection or enrichment of a particular type of cell means increasing the number or proportion of such cells, but it is not necessary to completely remove cells that do not express that marker. Similarly, negative selection, removal or depletion of a particular type of cell means decreasing the number or proportion of such cells, but it is not necessary to completely remove such cells.

[0066] In some examples, by performing the separation step multiple times, a positively selected fraction or a negatively selected fraction from one step is processed in another separation step, such as a subsequent positive selection or negative selection.

[0067] In some embodiments, an antibody or binding domain to a cell marker can be conjugated to a solid support or solid matrix, such as magnetic beads or paramagnetic beads, to separate cells by positive selection and / or negative selection. For example, in some embodiments, immunomagnetic separation techniques (or affinity magnetic separation techniques) are used to separate or isolate cells and cell populations (reviewed in "Methods" in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher (c) Humana Press Inc., Totowa, NJ). See also U.S. Patent Publication No. 4,452,773; U.S. Patent Publication No. 4,795,698; U.S. Patent Publication No. 5,200,084; and European Patent Publication No. 452342.

[0068] In some embodiments, affinity-based selection is performed by magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). The MACS system can select cells bound with magnetic particles with high purity. In certain embodiments, MACS is operated in a mode where non-target species and target species are eluted continuously after an external magnetic field is applied. That is, cells not bound to the magnetic particles are eluted while cells bound to the magnetic particles are retained. Next, after this first elution step is completed, the cells trapped by the magnetic field and prevented from eluting are released by some method that allows for elution and recovery. In certain embodiments, non-target cells are labeled and removed from the heterogeneous cell population.

[0069] In some embodiments, the cell populations described herein are recovered and concentrated (or removed) by flow cytometry, which analyzes cells stained with multiple cell surface markers by loading them onto a fluid flow. In some embodiments, the cell populations described herein are recovered and concentrated (or removed) by fluorescence-activated cell sorting (FACS). In some embodiments, the cell populations described herein are recovered and concentrated (or removed) by using a combination of a microelectromechanical systems (MEMS) chip and a detection system utilizing FACS (see, e.g., WO2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376). In any case, cells can be labeled with multiple markers to isolate well-defined subsets of cells with high purity.

[0070] The cell markers of various T cell subsets are as described above. In certain embodiments, certain T cell subsets, e.g., T cells that are positive or highly expressed for one or more cell surface markers, e.g., CCR7, CD45RO, CD8, CD27, CD28, CD62L, CD127, CD4 and / or CD45RA T cells, are isolated by positive selection techniques or negative selection techniques.

[0071] CD3+CD28+ T cells can be positively selected and expanded using anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0072] In certain embodiments, CD4+ helper T cells and CD8+ cytotoxic T cells are separated by performing a selection step for CD8+ or CD4+. Such CD8+ and CD4+ populations can be further sorted into various subpopulations by performing positive or negative selection of markers expressed in one or more naive T cell subpopulations, memory T cell subpopulations, and / or effector T cell subpopulations, or markers that are relatively highly expressed in these T cell subpopulations.

[0073] In some embodiments, enrichment is performed to obtain central memory T (TCM) cells. In certain embodiments, memory T cells are included in both the CD62L-positive subset and the CD62L-negative subset of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted of the CD62L fraction, the CD8 fraction, and / or the CD62L+CD8+ fraction using, for example, anti-CD8 antibody and anti-CD62L antibody.

[0074] In some embodiments, central memory T (TCM) cells are enriched based on positive or high cell surface expression of CCR7, CD45RO, CD27, CD62L, CD28, CD3, and / or CD127. In some aspects, TCM cells are enriched by negative selection of cells that express or highly express CD45RA and / or granzyme B. In some embodiments, isolation of a CD8+ population enriched for TCM cells is performed by removing cells that express CD4, CD14, and CD45RA and positively selecting or enriching cells that express CCR7, CD45RO, and / or CD62L. In one aspect, enrichment of central memory T (TCM) cells begins with a negative cell fraction selected based on expression of CD4, and this fraction is subjected to negative selection based on expression of CD14 and CD45RA and positive selection based on expression of CD62L. Such selections are performed simultaneously in some aspects and sequentially in other aspects, and may be performed in any order. In some embodiments, a CD4+ cell population or CD4+ cell subpopulation can also be obtained by performing the same selection steps used to prepare the CD8+ cell population or CD8+ cell subpopulation based on expression of CD4, and in this case, both the positive and negative fractions obtained by separation based on CD4 are retained, and one or more additional positive selection steps or negative selection steps may be performed.

[0075] Furthermore, other types of cells can be enriched based on known marker profiles and known techniques. For example, CD34+ HSCs, HSPs, and HSPCs can be enriched using a combination of anti-CD34 antibodies directly or indirectly conjugated to magnetic microparticles and a magnetic cell separator (e.g., CliniMACS® cell separation system (Miltenyi Biotec, Germany, Bergisch Gladbach)).

[0076] (III) Genetic modification of cell populations for expressing recombinant receptors The cell population can be genetically modified to express the recombinant receptor described herein.

[0077] (III - A) Genetic engineering techniques The gene construct encoding the recombinant receptor disclosed in this specification can be introduced into cells by methods known in the art, such as transfection, electroporation, microinjection, lipofection, transfection by the calcium phosphate method, infection with a viral vector or bacteriophage vector containing a gene sequence, cell fusion, gene transfer by chromosomes, gene transfer by the micronucleus cell fusion method, spheroplast fusion, delivery by in vivo nanoparticles, etc. In the art, various techniques for introducing a foreign gene construct into cells are known (see, for example, Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen, et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92), and such techniques may be used as long as the necessary developmental and physiological functions of the recipient cells are not excessively disrupted. These techniques can be utilized to stably transfer a foreign gene construct into cells such that the foreign gene construct is expressed in the cells and, in certain instances, preferably hereditarily and also expressed in its progeny cells.

[0078] "Gene" refers to a nucleic acid sequence encoding a recombinant receptor comprising the anti-BAFF-R binding domain described herein (this term is used interchangeably with "polynucleotide" or "nucleotide sequence"). The definition of this term includes various sequence polymorphisms, mutations and / or sequence variants, such changes having no substantial effect on the function of the encoded recombinant receptor. Also, the term "gene" may include not only the coding sequence but also regulatory regions such as promoters, enhancers, termination regions, etc. The gene sequences encoding these molecules may be DNA or RNA that induces the expression of the recombinant receptor. These nucleic acid sequences may be the DNA strand sequences transcribed into RNA, or the RNA sequences translated into proteins. These sequences may also be sequences that may be introduced to confer codon selectivity in a particular type of cell (e.g., mammalian cells), or may further include degenerate codons of natural sequences. Also, portions of the complete gene sequence are referred to throughout this disclosure as being understood by those skilled in the art.

[0079] Gene sequences encoding recombinant receptors are provided herein and can be readily produced by synthetic or recombinant methods from the relevant amino acid sequences based on the other information provided herein. In some embodiments, the gene sequences encoding any of these sequences may have one or more restriction enzyme sites at the 5' end and / or 3' end of the coding sequence so that the gene sequences encoding these sequences can be readily cleaved and easily replaced with another gene sequence encoding a different sequence.

[0080] "Encoding" refers to the property that a specific nucleotide sequence within a gene, such as cDNA or mRNA, functions as a template for synthesizing another macromolecule such as a defined amino acid sequence. Thus, when mRNA corresponding to a specific gene is transcribed and translated to produce a protein in a cell or other biological system, that gene encodes this protein. "Gene sequences encoding proteins" include all degenerate nucleotide sequences encoding the same amino acid sequence or amino acid sequences having substantially similar forms and functions.

[0081] Multiple polynucleotide gene sequences encoding two or more portions of a recombinant receptor can be operably linked to each other and can also be linked to relevant regulatory sequences. For example, a regulatory sequence and an exogenous nucleic acid sequence may be functionally linked, whereby the exogenous nucleic acid sequence is expressed. In another example, when a first nucleic acid sequence and a second nucleic acid sequence are arranged to be functionally related, the first nucleic acid sequence and the second nucleic acid sequence may be operably linked. For example, when a promoter affects the transcription or expression of a coding sequence, this promoter is operably linked to this coding sequence.

[0082] A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. The vector may be, for example, any of a plasmid (DNA plasmid or RNA plasmid), a transposon-based system, a cosmid, a bacterial artificial chromosome, a virus, or a phage. An "expression vector" is a vector capable of inducing the expression of a protein encoded by one or more genes incorporated into the expression vector when placed in an appropriate environment.

[0083] "Lentivirus" refers to a virus of the genus Retrovirus that can infect both dividing and non-dividing cells. Some examples of lentiviruses include HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0084] A lentiviral vector is a vector derived from at least a part of the lentiviral genome, and in particular, a self-inactivating lentiviral vector as described in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include the LENTIVECTOR® gene delivery technology commercially available from Oxford BioMedica, and LENTIMAX commercially available from Lentigen TM vector systems and the like. Furthermore, non-clinical lentiviral vectors are also available and are known to those skilled in the art. In certain embodiments, a viral vector, transposon vector, integrase vector or mRNA vector is used to genetically modify a cell to express a recombinant receptor (e.g., CAR or eTCR). In certain embodiments, the viral vector includes a lentiviral vector, a retroviral vector, a foamy virus vector or a gamma virus vector. In certain embodiments, the viral vector is a lentiviral vector.

[0085] "Retrovirus" is a virus having an RNA genome. "Gamma retrovirus" refers to a virus belonging to the family Retroviridae. Exemplary gamma retroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus. Foamy virus is a retrovirus useful for the delivery of large transgene cassettes.

[0086] Retroviral vectors can also be used (see Miller, et al., 1993, Meth. Enzymol. 217:581-599). In such embodiments, the gene to be expressed is cloned into the retroviral vector and delivered into the cell. In certain embodiments, the retroviral vector contains all of the cis-acting sequences required for packaging and integration of the viral genome, namely, (a) long terminal repeats (LTRs) or portions thereof located at both ends of the vector, (b) primer binding sites for DNA synthesis of the minus and plus strands, and (c) a packaging signal required for integration of genomic RNA into the virion. A more detailed description of retroviral vectors is provided in Boesen, et al., 1994, Biotherapy 6:291-302; Clowes, et al., 1994, J. Clin. Invest. 93:644-651; Kiem, et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114. Adenoviruses, adeno-associated viruses (AAV), and alphaviruses can also be used.See Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503, Rosenfeld, et al., 1991, Science 252:431-434; Rosenfeld, et al., 1992, Cell 68:143-155; Mastrangeli, et al., 1993, J. Clin. Invest. 91:225-234; Walsh, et al., 1993, Proc. Soc. Exp. Bioi. Med. 204:289-300; and Lundstrom, 1999, J. Recept. Signal Transduct. Res. 19: 673-686. Other gene delivery methods include the use of mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8:351-359); liposomes (Tarahovsky and Ivanitsky, 1998, Biochemistry (Mosc) 63:607-618); ribozymes (Branch and Klotman, 1998, Exp. Nephrol. 6:78-83); and triple helix DNA (Chan and Glazer, 1997, J. Mol. Med. 75:267-282).

[0087] There are numerous virus vectors suitable and available for the present disclosure, including those identified for human gene therapy (see Pfeifer and Verma, 2001, Ann. Rev. Genomics Hum. Genet. 2:177). Methods of using retroviral vectors and lentiviral vectors, as well as methods of packaging cells for transducing mammalian host cells with virus particles containing a transgene of a recombinant receptor (e.g., CAR) are described, for example, in U.S. Patent Publication No. 8,119,772; Walchli, et al., 2011, PLoS One 6:327930; Zhao, et al., 2005, J. Immunol. 174:4415; Engels, et al., 2003, Hum. Gene Ther. 14:1155; Frecha, et al., 2010, Mol. Ther. 18:1748; and Verhoeyen, et al., 2009, Methods Mol. Biol. 506:97. Furthermore, vector constructs of retroviruses and lentiviruses and their expression systems are commercially available.

[0088] Targeted genetic engineering methods may be used. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) nuclease system is a recombinant nuclease system used in genetic engineering utilizing bacterial systems. Information regarding the CRISPR-Cas system and its components is described, for example, in U.S. Patent Publication Nos. 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,889,418; 8,895,308; 8,906,616; 8,932,814; 8,945,839; 8,993,233; and 8,999,641 and their related applications; and WO2014 / 018423; WO2014 / 093595; WO2014 / 093622; WO2014 / 093635; WO2014 / 093655; WO2014 / 093661; WO2014 / 093694; WO2014 / 093701; WO2014 / 093709; WO2014 / 093712; WO2014 / 093718; WO2014 / 145599; WO2014 / 204723; WO2014 / 204724; WO2014 / 204725; WO2014 / 204726; WO2014 / 204727; WO2014 / 204728; WO2014 / 204729; WO2015 / 065964; WO2015 / 089351; WO2015 / 089354; WO2015 / 089364; WO2015 / 089419; WO2015 / 089427; WO2015 / 089462; WO2015 / 089465; WO2015 / 089473; WO2015 / 089486; WO2016205711; WO2017 / 106657; and WO2017 / 127807 and their related applications.

[0089] In certain embodiments, zinc finger nucleases (ZFNs) are used as gene editing agents. ZFNs are a type of site-specific nuclease that has been engineered to bind to and cleave DNA at specific locations. ZFNs are used to introduce double-strand breaks (DSBs) at specific sites in a DNA sequence, and by introducing double-strand breaks, unique sequences within the genomes of various types of cells can be targeted. Zinc fingers are 30-amino acid-long domains within the zinc finger binding domain, and their structure is stabilized by coordination with zinc ions. Examples of zinc fingers include C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. Engineered zinc finger domains are domains that do not exist in nature, and their design / composition is mainly constructed based on rational criteria, for example, by processing information in a database storing information on the design and binding data of existing ZFPs, or by applying substitution rules, etc. A well-known example of a ZFN is a fusion of the FokI nuclease and the zinc finger DNA binding domain.For further information regarding ZFNs and ZFNs useful within the teachings of the present disclosure, see, for example, U.S. Patent Publication No. 6,534,261; U.S. Patent Publication No. 6,607,882; U.S. Patent Publication No. 6,746,838; U.S. Patent Publication No. 6,794,136; U.S. Patent Publication No. 6,824,978; U.S. Patent Publication No. 6,866,997; U.S. Patent Publication No. 6,933,113; U.S. Patent Publication No. 6,979,539; U.S. Patent Publication No. 7,013,219; U.S. Patent Publication No. 7,030,215; U.S. Patent Publication No. 7,220,719; U.S. Patent Publication No. 7,241,573; U.S. Patent Publication No. 7,241,574; U.S. Patent Publication No. 7,585,849; U.S. Patent Publication No. 7,595,376; U.S. Patent Publication No. 6,903,185; U.S. Patent Publication No. 6,479,626; U.S. Patent Publication No. 2003 / 0232410 and U.S. Patent Publication No. 2009 / 0203140, as well as Gaj et al., Nat Methods, 2012, 9(8):805-7; Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7): 1327-33; Urnov et al., Nature Reviews Genetics, 2010, 11 :636-646; Miller, et al. Nature biotechnology 25, 778-785 (2007); Bibikova, et al. Science 300, 764 (2003); Bibikova, et al. Genetics 161, 1169-1175 (2002); Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Kim, et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1160 (1996); and Miller, et al. The EMBO journal 4, 1609-1614 (1985).

[0090] In certain embodiments, transcription activator-like effector nucleases (TALENs) can be used as gene editing agents. A TALEN is a fusion protein that includes a transcription activator-like effector (TALE) DNA binding protein and a DNA cleavage domain. TALENs are used to edit genes and genomes by inducing double-strand breaks in DNA and thereby inducing the cell's repair machinery. Generally, two TALENs bind to opposite sides of a target DNA site, sandwiching the target DNA site and dimerizing the DNA cleavage domains to induce a double-strand break. For further information regarding TALENs, see U.S. Patent Publication Nos. 8,440,431; 8,440,432; 8,450,471; 8,586,363; and 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):49-55; Beurdeley et al., Nat Commun, 2013, 4: 1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011); Christian, et al. Genetics 186, 757-761 (2010); Boch, et al. Science 326, 1509-1512 (2009); and Moscou, & Bogdanove, Science 326, 1501 (2009).

[0091] In certain embodiments, MegaTAL can be used as a gene editing agent. MegaTAL has a nuclease structure that cleaves single strands at extremely rare positions, and a TALE is fused to the DNA cleavage domain of the meganuclease. Meganucleases (also called homing endonucleases) are single-stranded peptides that have both DNA recognition ability and nuclease function in the same domain. Unlike TALEN, megaTAL requires the delivery of a single peptide chain for its functional activity.

[0092] In certain embodiments, to integrate a recombinant receptor construct into cells, a system utilizing a transposon can be used as a gene editing agent. Generally, in such methods, (i) a first vector encoding a transposase (or a transposase polypeptide) and (ii) a second vector encoding a desired gene factor flanked by transposon repeat sequences are introduced into the cells. A transposon, i.e., a transposable element, contains a (short) nucleic acid sequence with terminal repeat sequences upstream and downstream of it, and encodes an enzyme that promotes the cleavage of a target DNA sequence and the insertion of nucleic acids into the target DNA sequence.

[0093] There are several types of transposon / transposase systems constructed to be compatible with the insertion of heterologous DNA sequences. Examples of such transposases include sleeping beauty ("SB", for example, those derived from the genomes of salmonid fish); piggyback (for example, those derived from lepidopteran insect cells and / or the fruit bat); mariner (for example, those derived from Drosophila); frog prince (for example, those derived from Xenopus laevis); Tol1; Tol2 (for example, those derived from medaka); TcBuster (for example, those derived from Tribolium castaneum), Helraiser, Himar1, Passport, Minos, Ac / Ds, PIF, Harbinger, Harbinger3-DR, HSmar1 and spinON. Transposases and transposon systems are also described in U.S. Patent No. 6,489,458; U.S. Patent No. 7,148,203; U.S. Patent No. 8,227,432; and U.S. Patent No. 9,228,180.

[0094] DNA can be integrated into host cells using integrase. For example, phiC31 integrase is a sequence-specific recombinase encoded within the genome of bacteriophage phiC31. phiC31 integrase catalyzes recombination between two 34-base pair sequences, an attachment site (att) within the phage and an attachment site (att) within the host bacterium. This serine integrase has been shown to function efficiently in a number of cell types, including mammalian cells. In the presence of phiC31 integrase, a donor plasmid containing attB recombines with a site having a sequence similar to the native attP site (referred to as a pseudo attP site) and is integrated in one direction into the target genome. phiC31 integrase can integrate plasmids of any size as a single copy and does not require cofactors. The integrated transgene is stably expressed and heritable.

[0095] Nanoparticles that can selectively genetically modify target cells in vivo have been reported and can be used within the scope of the teachings of the present disclosure. In certain embodiments, such nanoparticles may be those described in WO2014153114, WO2017181110, and WO201822672.

[0096] (III - B) Recombinant receptors In certain embodiments, the recombinant receptor is a binding domain that binds to a target antigen or includes a binding domain that binds to a target antigen, and this recombinant receptor is expressed from the cell by artificially introducing a nucleic acid encoding the recombinant receptor into the cell. This recombinant receptor may be, for example, a CAR, a recombinant T cell receptor (eTCR), or a hybrid thereof.

[0097] CAR includes, for example, several characteristic components that enable genetically modified cells (e.g., T cells) to recognize and kill target cells such as cancer cells. These components include at least an extracellular portion and an intracellular portion. The extracellular portion includes a binding domain that binds to a marker that is selectively presented on the surface of unwanted cells. When the binding domain binds to such a marker, the intracellular portion activates the genetically modified cell to destroy the bound cell. CAR can further include a transmembrane domain that links the extracellular portion to the intracellular portion and other components that can enhance the function of the recombinant receptor. For example, by incorporating a spacer sequence and / or one or more linker sequences into the CAR, flexibility can be further imparted to the three-dimensional structure of the recombinant receptor, which often enhances the binding ability of the binding domain to the marker on the target cell.

[0098] (III - B - 1) Binding domain In a specific example, the present disclosure provides a binding domain used for a recombinant receptor (e.g., CAR) based on an antibody that binds to BAFF-R.

[0099] BAFF-R, also known as tumor necrosis factor receptor superfamily member 13C (TNFRSF13C), is a membrane protein that improves B cell survival in vitro and is also a regulator of the peripheral B cell population. In certain embodiments, BAFF-R includes the sequence specified by UniProt reference number Q96RJ3, UniProt reference number Q9D8D0, NCBI reference number GI:16445027, or NCBI reference number GI:16306481, or variants or homologs that are substantially identical thereto.

[0100] An example of a binding domain is an antibody, and examples of such an antibody include a full-length antibody or a binding fragment of an antibody that specifically binds to a cell marker (e.g., BAFF-R), such as those in the form of Fv, Fab, Fab’, F(ab’)2, and single-chain (sc), and fragments thereof. Examples of the antibody or antigen-binding fragment include the full-length or a part of a polyclonal antibody, monoclonal antibody, human antibody, humanized antibody, synthetic antibody, non-human antibody, recombinant antibody, chimeric antibody, bispecific antibody, minibody, or linear antibody.

[0101] Antibodies are generated from two genes, a heavy chain gene and a light chain gene. Antibodies typically contain two identical copies of the heavy chain and two identical copies of the light chain. In the heavy chain variable region and the light chain variable region, segments called complementarity-determining regions (CDRs) determine binding to the epitope. Each heavy chain has three CDRs (i.e., CDRH1, CDRH2, and CDRH3), and each light chain has three CDRs (i.e., CDRL1, CDRL2, and CDRL3). The CDR regions are flanked by framework residues (FR).

[0102] The amino acid assignment to each domain is based on Kabat numbering (Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) (the “Kabat” numbering scheme); Chothia (Al-Lazikani et al., (1997) JMB 273, 927-948 (the “Chothia” numbering scheme)); Martin (Abinandan et al., Mol Immunol. 45:3832-3839 (2008), “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains”); Contact numbering by Gelfand (MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (the Contact numbering scheme)); IMGT (Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77(「IMGT」 numbering scheme)); AHo (Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70(AHo numbering scheme)); North (North et al., J Mol Biol. 406(2):228-256 (2011), “A new clustering of antibody CDR loop conformations”); or it can be performed according to other numbering schemes.

[0103] The boundaries of CDRs or FRs may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Also, the numbering of the Kabat and Chothia schemes is based on the length of the most common antibody region sequences, and insertions are indicated by letters indicating the insertion, such as "30a", and deletions are seen in some antibodies. In these two schemes, the numbering is different because insertions and deletions ("indels") are included at different positions. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects. In certain embodiments, the CDR sequences of the antibodies disclosed herein are shown according to the Kabat numbering. In the North numbering, longer sequences are used than in other schemes in the structural analysis of the three-dimensional structure of the CDR loops. CDR residues can be identified using software programs such as ABodyBuilder.

[0104] In some cases, another scFv based on the binding domains described herein for use in a recombinant receptor (e.g., a CAR) can be prepared according to methods known in the art (see, e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). The scFv molecule can be made by linking the VH and VL regions of the antibody using a flexible polypeptide linker. When a short polypeptide linker (e.g., 5-10 amino acids in length) is used, intra-chain folding is blocked. For inter-chain folding, the two variable regions need to bind to form a binding site for the functional epitope. For examples of linker directionality and size, see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, US Patent Publication No. 2005 / 0100543, US Patent Publication No. 2005 / 0175606, US Patent Publication No. 2007 / 0014794, WO2006 / 020258, and WO2007 / 024715. More specifically, the linker sequence used to link VL and VH of the scFv is typically 5-35 amino acids in length. In certain embodiments, the VL-VH linker comprises 5-35 amino acids, 10-30 amino acids, or 15-25 amino acids. Varying the linker length may retain activity, enhance activity, and thereby provide excellent efficacy in activity assays. Generally, scFv is often used as the binding domain of a CAR. In certain embodiments, the recombinant receptor of the disclosure comprises a binding domain that binds to BAFF-R. In certain embodiments, the binding domain that binds to BAFF-R is an scFv. In certain embodiments, the binding domain that binds to BAFF-R is an scFV derived from the H90 monoclonal antibody.In certain embodiments, examples of the binding domain that binds to BAFF-R include the sequence shown by VQLQESGPGLVKPSQTLSLTCTVSGDSITSGYWNWIRQHPGKGLEYIGYISYSGSTYYNPSLKSRVTISRDTSKNQYSLKLSSVTAADTAVYYCASPNYPFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPATLSLSPGERATLSCRASESVDNYGISFMNWFQQKPGQAPRLLIYAASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSKEVPWTFGGGTKVEIKR (SEQ ID NO: 1).

[0105] In certain embodiments, examples of the binding domain that binds to BAFF-R include the sequence shown by DIVLTQSPATLSLSPGERATLSCRASESVDNYGISFMNWFQQKPGQAPRLLIYAASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSKEVPWTFGGGTKVEIKRGGGGSGGGGSGGGGSVQLQESGPGLVKPSQTLSLTCTVSGDSITSGYWNWIRQHPGKGLEYIGYISYSGSTYYNPSLKSRVTISRDTSKNQYSLKLSSVTAADTAVYYCASPNYPFYAMDYWGQGTLVTVSS (SEQ ID NO: 6).

[0106] In certain embodiments, the binding domain that binds to BAFF-R includes a heavy chain variable region having a complementarity determining region (CDRH)1 containing the sequence GDSITSGY (SEQ ID NO: 2), a CDRH2 containing the sequence ISYSGST (SEQ ID NO: 3), and a CDRH3 containing the sequence ASPNYPFYAMDY (SEQ ID NO: 4), and a light chain variable region having a complementarity determining region (CDRL)1 containing the sequence ESVDNYGISF (SEQ ID NO: 5), a CDRL2 containing the sequence AAS, and a CDRL3 containing the sequence QQSKEVPWT (SEQ ID NO: 7).

[0107] In certain embodiments, the binding domain that binds to BAFF-R comprises a heavy chain variable region comprising the sequence shown in VQLQESGPGLVKPSQTLSLTCTVSGDSITSGYWNWIRQHPGKGLEYIGYISYSGSTYYNPSLKSRVTISRDTSKNQYSLKLSSVTAADTAVYYCASPNYPFYAMDYWGQGTLVTVSS (SEQ ID NO: 8), and a light chain variable region comprising the sequence shown in DIVLTQSPATLSLSPGERATLSCRASESVDNYGISFMNWFQQKPGQAPRLLIYAASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSKEVPWTFGGGTKVEIKR (SEQ ID NO: 9).

[0108] In certain embodiments, the binding domain that binds to BAFF-R is VQLQESGPGLVKPSQTLSLTCTVSGDSITSGYWNWIRQHPGKGLEYIGYISYSGSTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCASPNYPFYAMDYWGQGTLVTVSS (SEQ ID NO: 32), VQLQESGPGLVKPSQTLSLTCTVSGDSITSGYWNWIRQHPGKGLEYIGYISYSGSTYYNPSLKSRVTISRDTSKNQYSLKLSSVTAADTAVYYCASPNYPFYAMDYWGQGTLVTVSS (SEQ ID NO: 8), or VQLQESGPGLVKPSETLSLTCSVSGDSITSGYWNWIRQPPGKGLEYIGYISYSGSTYYNPSLKSRVTISRDTSKNQYSLRLSSVTAADTALYYCASPNYPFYAMDYWGQGTRVTVSS (SEQ ID NO: 34) and comprises a humanized VH domain comprising the sequence comprising; EIVLTQSPATLSLSPGERATLSCRASESVDNYGISFLNWFQQKPGQAPRLLIYAASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSKEVPWTFGGGTKVEIKR (SEQ ID NO: 35), DIVLTQSPATLSLSPGERATLSCRASESVDNYGISFMNWFQQKPGQAPRLLIYAASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSKEVPWTFGGGTKVEIKR (SEQ ID NO:9), or DIVMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSKEVPWTFGQGTKVEIKR (SEQ ID NO:37) comprises a humanized VL domain comprising an array comprising the same.

[0109] In the recombinant receptors (e.g., CARs) disclosed herein, other binding fragments such as Fv, Fab, Fab’, F(ab’)2, etc. can also be used. Another example of an antibody-based binding domain format for use in recombinant receptors includes grababodies made from scFv and soluble VH domain antibodies. These antibodies form the binding region using only the heavy chain variable region. See, for example, Jespers et al., Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.

[0110] In certain embodiments, the binding domain comprises a humanized antibody or a recombinant fragment thereof. In certain embodiments, the non-human antibody is humanized and one or more amino acid residues of the antibody have been modified to increase its similarity to an antibody or fragment thereof that is naturally produced in humans. Such non-human amino acid residues are often referred to as "import" residues and are typically obtained from the "import" variable domain. As provided herein, a humanized antibody or antibody fragment comprises one or more CDRs derived from a non-human immunoglobulin molecule and a framework region, and the amino acid residues comprising the framework are derived solely from the human germ line or are mostly derived from the human germ line.Humanized antibodies can be prepared using a variety of techniques known in the art, such as CDR grafting (see, e.g., European Patent No. 239,400; WO 91 / 09967; and U.S. Patent Publication Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (see, e.g., European Patent Publication Nos. 592,106 and 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973), chain shuffling (see, e.g., U.S. Patent Publication No. 5,565,332), and techniques disclosed, for example, in U.S. Patent Publication Nos. 2005 / 0042664, 2005 / 0048617, 6,407,213, 5,766,886, WO9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994).Framework residues within the framework region are often replaced with the corresponding residues from the CDR donor antibody, for example, to improve binding to BAFF-R. Such replacement of framework residues is identified by methods known in the art, for example, by modeling the interaction between CDR residues and framework residues to identify framework residues important for binding to BAFF-R and by sequence comparison to identify framework residues not normally found at specific positions (see, for example, U.S. Patent Publication No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323).

[0111] Functional variants include the addition or substitution of one or more residues that do not substantially affect the biological action of the protein. Functional fragments also include one or more deletions or truncations that do not substantially affect the biological action of the protein. That there is no substantial effect can be confirmed from the fact that experimentally comparable results are observed in activation tests or binding tests. Functional variants and functional fragments of the intracellular domain (e.g., the intracellular signaling portion) transmit an activation signal or an inhibitory signal comparable to that of the wild-type reference intracellular domain when in the activated state described in the present disclosure. Also, functional variants and functional fragments of the binding domain bind to their cognate antigen or cognate ligand at a level comparable to that of the wild-type reference binding domain.

[0112] In certain embodiments, the VL region of the binding domain of the present disclosure is derived from the VL region of the antibody of the present disclosure or is prepared based on the VL region of the antibody of the present disclosure, and compared to the VL region of the antibody of the present disclosure, contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or combinations of these changes. As long as each CDR contains no changes or contains at most 1, 2, or 3 changes, and the binding domain containing the modified VL region can specifically bind to the target with an affinity comparable to that of the wild-type binding domain, the insertions, deletions, or substitutions may be present at any position in the VL region, including the amino terminus, carboxy terminus, or both termini of the VL region.

[0113] In certain embodiments, the VH region of the binding domain of the present disclosure is derived from the VH region of the antibody of the present disclosure or is prepared based on the VH region of the antibody of the present disclosure, and may contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or combinations of these changes, compared to the VH region of the antibody of the present disclosure. As long as each CDR contains no changes or contains at most 1, 2, or 3 changes, and the binding domain containing the modified VH region can specifically bind to the target with an affinity comparable to that of the wild-type binding domain, the insertions, deletions, or substitutions may be present at any position in the VH region, including the amino terminus, carboxy terminus, or both termini of the VH region.

[0114] In certain embodiments, the binding domain comprises, or is, a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the light chain variable region (VL) and / or the heavy chain variable region (VH), and contains no changes, or at most 1, 2, or 3 changes, in each CDR compared to an antibody, fragment, or derivative thereof of the present disclosure that binds to BAFF-R.

[0115] (III - B - 2) Intracellular effector domain The intracellular effector domain of a recombinant receptor (e.g., a CAR) is responsible for activation of the cell in which the recombinant receptor is expressed. Thus, the term "effector domain" means including a part of the intracellular domain sufficient for transduction of an activation signal. The effector domain can directly or indirectly promote a biological or physiological response of the cell when receiving an appropriate signal. In certain embodiments, the effector domain is part of a protein or protein complex that receives a signal when the recombinant receptor binds to a target molecule, or binds directly to the target molecule, such that a signal is induced from the effector domain. The effector domain may directly promote a cell response when it includes one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM). In another embodiment, the effector domain indirectly promotes a cell response by associating with one or more other proteins that directly promote a cell response, such as a costimulatory domain.

[0116] When a modified cell binds to a cell marker expressed by a cancer cell, the effector domain can activate at least one function of the modified cell. Activation of the modified cell may include one or more of differentiation, proliferation, and / or activation or other effector functions. In certain embodiments, the effector domain may include an intracellular signaling moiety that includes a T cell receptor and a costimulatory domain, and the costimulatory domain may include an intracellular sequence derived from a coreceptor or costimulatory molecule.

[0117] The effector domain may include one, two, or more than two intracellular signaling moieties (e.g., the signaling domain of a receptor or an intracellular signaling sequence), a co-stimulatory domain, or a combination thereof. Exemplary effector domains include signaling domains and / or stimulatory domains selected from 4-1BB (CD137), CARD11, CD3γ, CD3δ, CD3ε, CD3ζ, CD27, CD28, CD79A, CD79B, DAP10, FcRα, FcRβ (FcεR1b), FcRγ, Fyn, HVEM (LIGHTR), ICOS, LAG3, LAT, Lck, LRP, NKG2D, NOTCH1, pTα, PTCH2, OX40, ROR2, Ryk, SLAMF1, Slp76, TCRα, TCRβ, TRIM, Wnt, Zap70, and any combination thereof.In certain embodiments, exemplary effector domains include signaling domains and / or costimulatory domains selected from CD86, FcγRIIa, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that bind to CD83, CDS, ICAM-1, GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, GADS, PAG / Cbp, NKp44, NKp30, NKp46, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In certain embodiments, the effector domain includes the signaling domain of CD3ζ.

[0118] The intracellular signaling subsequence that acts stimulatively may include an immunoreceptor tyrosine-based activation motif (iTAM). Examples of iTAMs containing cytoplasmic major signaling sequences include those derived from CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD66d, CD79a, CD79b, common FcRγ (FCER1G), FcγRlla, FcRβ (Fcε Rib), DAP10, or DAP12. In certain embodiments, the variant of CD3ζ retains at least one, two, three, or all immunoreceptor tyrosine-based activation motif (ITAM) regions.

[0119] In certain embodiments, the effector domain includes an intracellular portion that associates with an intracellular signaling protein, which is a lymphocyte receptor or its signaling domain, a protein comprising a plurality of ITAMs, a costimulatory domain, or any combination thereof.

[0120] Additional examples of the intracellular signaling portion include the intracellular sequence of the CD3ζ chain and / or a coreceptor that acts in concert therewith to initiate signaling subsequent to association with the binding domain.

[0121] A co-stimulatory domain is a domain whose activation is required for an efficient lymphocyte response to binding to a cell marker. Some molecules are replaceable as an intracellular signaling moiety or co-stimulatory domain. Examples of co-stimulatory domains include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3, as well as ligands that bind to CD83. For example, co-stimulation of CD27 has been demonstrated to enhance the proliferation, effector function, and survival of human CAR T cells in vitro and to enhance the persistence and anti-cancer activity of human T cells in vivo (Song et al. Blood. 2012; 119(3):696-706). Further examples of such co-stimulatory domain molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, ITGAM, CDl lb, ITGAX, CDllc, ITGBl, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp and CD19a. In certain embodiments, co-stimulatory domains include CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C and / or B7-H3.In certain embodiments, the co-stimulatory domain includes the signaling domain of 4-1BB.

[0122] In certain embodiments, the nucleic acid sequence encoding the intracellular signaling portion includes the sequence encoding CD3ζ (SEQ ID NO: 23) and the sequence encoding a variant of the signaling portion of 4-1BB (SEQ ID NO: 21). In certain embodiments, the amino acid sequence of the intracellular signaling portion includes an intracellular signaling portion that includes a variant of CD3ζ (SEQ ID NO: 22) and a portion of 4-1BB (SEQ ID NO: 20). In certain embodiments, the amino acid sequence including the intracellular signaling domain of CD3ζ and the intracellular signaling domain of 4-1BB is shown in SEQ ID NO: 24.

[0123] In certain embodiments, the intracellular signaling portion includes (i) all or a portion of the signaling domain of CD3ζ, (ii) all or a portion of the signaling domain of 4-1BB, or (iii) all or a portion of the signaling domains of CD3ζ and 4-1BB.

[0124] The intracellular portion may further include one or more of the proteins selected from the Wnt signaling pathway (e.g., LRP, Ryk or ROR2), the NOTCH signaling pathway (e.g., NOTCH1, NOTCH2, NOTCH3 or NOTCH4), the Hedgehog signaling pathway (e.g., PTCH or SMO), receptor tyrosine kinases (RTK) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor (FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, tropomyosin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE) receptor family, receptor tyrosine kinase-like orphan (ROR) receptor family, discoidin domain (DDR) receptor family, rearranged during transfection (RET) receptor family, tyrosine protein kinase-like (PTK7) receptor family, related to receptor tyrosine kinase (RYK) receptor family, or muscle-specific kinase (MuSK) receptor family); G protein-coupled receptors (GPCR) (Frizzled or Smoothened); serine / threonine kinase receptors (BMPR or TGFR); or cytokine receptors (IL1R, IL2R, IL7R or IL15R).

[0125] (III - B - 3) Transmembrane domain As described herein, the transmembrane domain in a recombinant receptor (e.g., a CAR) serves to link the extracellular portion and the intracellular portion across the cell membrane. The transmembrane domain can anchor the expressed molecule to the cell membrane in the modified cell.

[0126] The transmembrane domain may be of natural origin and / or may be synthetic. When of natural origin, the transmembrane domain may be derived from a membrane-bound protein or may be derived from a transmembrane protein. The transmembrane domain may include at least the transmembrane regions of the α-chain, β-chain or ζ-chain of the T cell receptor, CD28, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 or TLR9. In certain embodiments, the transmembrane domain includes at least, for example, the transmembrane regions of KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C. In certain embodiments, various human hinges such as the hinge of human Ig (immunoglobulin) (e.g., the hinge of IgG4 or IgD), GS linker (e.g., the GS linker described herein), the hinge of KIR2DS2, the hinge of CD8a, etc. can likewise be used.In certain embodiments, the recombinant receptor (e.g., CAR) includes the transmembrane domain of CD28. The transmembrane domain of CD28 has been shown to lower the antigen threshold for activation of second-generation 4-1BB CAR T cells.

[0127] In certain embodiments, the transmembrane domain has a three-dimensional structure that is thermodynamically stable within the cell membrane and is typically 15 to 30 amino acids in length. The structure of the transmembrane domain can include an α-helix, β-barrel, β-sheet, β-helix, or any combination thereof.

[0128] The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids within the extracellular region of the recombinant receptor (e.g., up to 15 amino acids of the extracellular region), and / or one or more additional amino acids within the intracellular region of the recombinant receptor (e.g., up to 15 amino acids of the intracellular portion). In one aspect, the transmembrane domain may be derived from the same protein as the protein from which the signaling domain, co-stimulatory domain, or hinge domain is derived. In another aspect, the transmembrane domain is derived from a protein different from the protein from which the other domains of the recombinant receptor are derived. In some cases, a transmembrane domain may be selected, or the transmembrane domain may be modified by amino acid substitutions such that it is possible to avoid the domains of the recombinant receptor binding to transmembrane domains derived from the same or different surface membrane proteins, thereby minimizing interactions with other unintended members of the receptor complex. In certain embodiments, the transmembrane domain is encoded by the nucleic acid sequence (SEQ ID NO: 18) encoding the transmembrane domain of CD28. In certain embodiments, the transmembrane domain includes the amino acid sequence (SEQ ID NO: 17) of the transmembrane domain of CD28. In certain embodiments, the sequence including the amino acid sequences of the hinge and transmembrane domain of CD8a is shown in SEQ ID NO: 19.

[0129] (III - B - 4) Spacer, linker and multimerization domain Spacers are used to be at an appropriate distance from other components of the recombinant receptor and / or to be more flexible compared to other components. As described herein, in certain embodiments, the length of the spacer is customized so as to bind to the target cells (expressing BAFF-R) and be able to induce cell destruction. In certain embodiments, the length of the spacer can be selected according to the position of the epitope of the cell marker, the affinity of the binding domain for the epitope, and / or the cell destruction inducing ability of the BAFF-R binding factor after binding to BAFF-R.

[0130] Typical spacers include spacers 10 to 250 amino acids in length, spacers 10 to 200 amino acids in length, spacers 10 to 150 amino acids in length, spacers 10 to 100 amino acids in length, spacers 10 to 50 amino acids in length, or spacers 10 to 25 amino acids in length.

[0131] In certain embodiments, the spacer is 5 amino acids in length, 8 amino acids in length, 10 amino acids in length, 12 amino acids in length, 14 amino acids in length, 20 amino acids in length, 21 amino acids in length, 26 amino acids in length, 27 amino acids in length, 45 amino acids in length, 50 amino acids in length or 75 amino acids in length. In certain embodiments, the spacer is 10 to 15 amino acids in length (or 10 to 15 residues in length). In certain embodiments, the spacer is 12 amino acids in length. Such a length of spacer corresponds to a short spacer.

[0132] In certain embodiments, the spacer is 76 amino acids in length, 90 amino acids in length, 100 amino acids in length, 110 amino acids in length, 120 amino acids in length, 125 amino acids in length, 128 amino acids in length, 131 amino acids in length, 135 amino acids in length, 140 amino acids in length, 150 amino acids in length, 160 amino acids in length, 170 amino acids in length, or 179 amino acids in length. In certain embodiments, the spacer is 110-130 amino acids in length (or 110-130 residues in length). In certain embodiments, the spacer is 119 amino acids in length. A spacer of such a length corresponds to a medium-length spacer.

[0133] In certain embodiments, the spacer is 180 amino acids in length, 190 amino acids in length, 200 amino acids in length, 210 amino acids in length, 212 amino acids in length, 214 amino acids in length, 216 amino acids in length, 218 amino acids in length, 220 amino acids in length, 228 amino acids in length, 230 amino acids in length, 240 amino acids in length, 250 amino acids in length, 260 amino acids in length, or 270 amino acids in length. In certain embodiments, the spacer is 230-240 amino acids in length (or 230-240 residues in length). In certain embodiments, the spacer is 229 amino acids in length. A spacer of such a length corresponds to a long spacer.

[0134] Exemplary spacers include the full length or a portion of the hinge region of an immunoglobulin. The hinge region of the immunoglobulin may be the hinge region of a wild-type immunoglobulin or the hinge region of a modified wild-type immunoglobulin. In certain embodiments, the hinge region of the immunoglobulin is the hinge region of a human immunoglobulin. As used herein, "wild-type immunoglobulin hinge region" refers to the amino acid sequence of the hinge that is located between and links the CH1 and CH2 domains of the heavy chain (in the case of IgG, IgA, and IgD), or the amino acid sequence of the hinge that is located between and links the CH1 and CH3 domains of the heavy chain (in the case of IgE and IgM) at the upper and central portions of a native antibody.

[0135] The hinge region of the immunoglobulin may be the hinge region of IgG, IgA, IgD, IgE or IgM. The hinge region of IgG may be the hinge region of IgG1, IgG2, IgG3 or IgG4. The sequences derived from IgG1, IgG2, IgG3, IgG4 or IgD can be used alone, or in combination with the whole or a part of the CH2 region, or in combination with the whole or a part of the CH3 region, or in combination with the whole or a part of the CH2 region and the whole or a part of the CH3 region.

[0136] In certain embodiments, the spacer is a short spacer comprising the hinge region of IgG4. In certain embodiments, the short spacer comprises the sequence shown in SEQ ID NO: 15. In certain embodiments, the short spacer is encoded by the sequence shown in SEQ ID NO: 16. In certain embodiments, the spacer is a medium-length spacer comprising the hinge region of IgG4 and the CH3 region of IgG4. In certain embodiments, the medium-length spacer comprises the sequence shown in SEQ ID NO: 13. In certain embodiments, the medium-length spacer is encoded by SEQ ID NO: 14. In certain embodiments, the spacer is a long spacer comprising the hinge region of IgG4, the CH2 region of IgG4, and the CH3 region of IgG4. In certain embodiments, the long spacer comprises the sequence shown in SEQ ID NO: 11. In certain embodiments, the long spacer is encoded by SEQ ID NO: 12.

[0137] Other examples of hinge regions that can be used in the recombinant receptors described herein include hinge regions.

[0138] As used herein, a linker refers to a chemical moiety that connects two components within a molecule. A linker may only serve the purpose of connecting the components, but many linkers also serve other purposes in addition to connecting. For example, in an scFv, a linker can connect the VL and VH of an antibody-derived binding domain and can function as a connecting amino acid between the components of a recombinant receptor.

[0139] Depending on the desired function of the linker, the linker may be a flexible linker, a rigid linker, or a semi-rigid linker. The linker may further contain connecting amino acids. For example, in certain embodiments, the linker provides flexibility and space for the mobility of the three-dimensional structure between various components of the recombinant receptor. A commonly used flexible linker is the Gly-Ser linker. In certain embodiments, the linker sequence contains a repetitive sequence of glycine and serine, for example, a repetitive sequence consisting of 1 to 10 (Gly x Ser y ) n , where x and y are independently integers from 0 to 10, provided that both x and y being 0 are excluded, and n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. As a specific example, (Gly4Ser) n (SEQ ID NO: 38), (Gly3Ser) n (Gly4Ser) n (SEQ ID NO: 39), (Gly3Ser) n (Gly2Ser) n (SEQ ID NO: 40), or (Gly3Ser) n(Gly4Ser)1 (SEQ ID NO: 41) may be mentioned. In certain embodiments, the linker is (Gly4Ser)4 (SEQ ID NO: 42), (Gly4Ser)3 (SEQ ID NO: 43), (Gly4Ser)2 (SEQ ID NO: 44), (Gly4Ser)1 (SEQ ID NO: 45), (Gly3Ser)2 (SEQ ID NO: 46), (Gly3Ser)1 (SEQ ID NO: 47), (Gly2Ser)2 (SEQ ID NO: 48), (Gly2Ser)1, GGSGGGSGGSG (SEQ ID NO: 49), GGSGGGSGSG (SEQ ID NO: 50) or GGSGGGSG (SEQ ID NO: 51).

[0140] In certain embodiments, the linker region is (GGGGS) n (SEQ ID NO: 38), where n is an integer including an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. In certain embodiments, the spacer is (EAAAK) n (SEQ ID NO: 52), where n is an integer including an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0141] In some cases, flexible linkers may not be able to maintain the distance and position of the recombinant receptor required in certain applications. In such cases, rigid linkers or semi-rigid linkers may be useful. Examples of rigid linkers or semi-rigid linkers include proline-rich linkers. In certain embodiments, a proline-rich linker is a peptide sequence having more proline residues than expected to be included in the sequence depending only on chance. In certain embodiments, a proline-rich linker is a linker in which proline residues account for at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50% or at least 51% of the whole in its sequence. Specific examples of proline-rich linkers include fragments of salivary proline-rich protein (PRP).

[0142] The linker may be sensitive to cleavage such as acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, disulfide bond cleavage, etc. (cleavable linker). Alternatively, the linker may be substantially resistant to cleavage (e.g., a stable linker or non-cleavable linker). In some embodiments, the linker is a linker that is prone to charging, a hydrophilic linker, or a dicarboxylic acid-based linker.

[0143] The linking amino acid may be a linker that can be used for linking sequences when there is no need to provide a distance using a spacer and / or when it is not desirable to provide a distance using a spacer. For example, the linking amino acid may be a short amino acid sequence that can be used for linking intracellular costimulatory signaling moieties. In certain embodiments, the linking amino acid is 9 amino acids or shorter in length (e.g., 2 amino acids in length, 3 amino acids in length, 4 amino acids in length, 5 amino acids in length, 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, or 9 amino acids in length). In certain embodiments, a glycine-serine doublet can be used as a suitable linking amino acid linker. In certain embodiments, a single amino acid such as alanine or glycine can be used as a suitable linking amino acid.

[0144] In certain embodiments, the recombinant receptor may contain a multimerization domain. The biological activity of a protein depends on its tertiary and quaternary structures. The quaternary structure requires physical and chemical interactions with another protein subunit or polypeptide. A "multimerization domain" is a domain that causes two or more proteins (monomers) to interact with each other via covalent and / or non-covalent bonds. When a multimerization domain is present in a protein, protein interactions occur and dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc. can be formed depending on the number of units / monomers incorporated into the multimer.

[0145] (III - B - 5) Control mechanism including promoter, tag cassette, transduction marker, selection cassette and / or suicide switch In certain embodiments, the gene construct can encode one or more regulatory mechanisms. In vitro, in vivo, and / or ex vivo, by utilizing the regulatory mechanisms, activation, proliferation promotion, detection, enrichment, isolation, tracking, removal, and / or elimination of genetically modified cells can be performed.

[0146] Examples of promoters include common promoters, tissue-specific promoters, cell-specific promoters, and / or cytoplasm-specific promoters. Promoters can also include strong promoters, weak promoters, constitutive expression promoters, and / or inducible promoters. Inducible promoters induce expression in response to specific conditions, signals, or cellular events. For example, the promoter may be an inducible promoter that requires a specific ligand, small molecule, transcription factor, or hormone protein for induction of transcription from the promoter. Specific examples of promoters include the EF-1α promoter, CMV promoter, Rho promoter, SV40 early promoter, Hsp68 minimal promoter (proHSP68), and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter.

[0147] A "tag cassette" is a unique synthetic peptide sequence that is added to, fused with, or incorporated as part of a recombinant receptor, and by specifically binding a cognate binding molecule (e.g., ligand, antibody, or other binding partner) to this synthetic peptide sequence, its binding properties are utilized to perform activation, proliferation promotion, detection, enrichment, isolation, tracking, removal, and / or elimination of the tagged protein and / or the cells expressing the tagged protein.

[0148] Examples of tag cassettes that bind to cognate binding molecules include His tag (HHHHHH; SEQ ID NO: 53), Flag tag (DYKDDDDK; SEQ ID NO: 54), Xpress tag (DLYDDDDK; SEQ ID NO: 55), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 56), calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 57), polyglutamic acid tag, HA tag (YPYDVPDYA; SEQ ID NO: 58), Myc tag (EQKLISEEDL; SEQ ID NO: 59), Strep tag (referring to the conventional STREP (registered trademark) tag (WRHPQFGG; SEQ ID NO: 60)), STREP (registered trademark) tag II (WSHPQFEK; SEQ ID NO: 61 (IBA Institut fur Bioanalytik, Germany); see, for example, U.S. Patent Publication No. 7,981,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 62), Softag 3 (TQDPSRVG; SEQ ID NO: 63), and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 64).

[0149] Binding molecules that specifically bind to the tag cassette arrays disclosed in this specification to form a complex are commercially available. For example, His-tag antibodies are commercially available from manufacturers such as Life Technologies, Pierce Antibodies, and GenScript. Flag-tag antibodies are commercially available from manufacturers such as Pierce Antibodies, GenScript, and Sigma-Aldrich. Xpress-tag antibodies are commercially available from manufacturers such as Pierce Antibodies, Life Technologies, and GenScript. Avi-tag antibodies are commercially available from manufacturers such as Pierce Antibodies, IsBio, and Genecopoeia. Calmodulin-tag antibodies are commercially available from manufacturers such as Santa Cruz Biotechnology, Abcam, and Pierce Antibodies. HA-tag antibodies are commercially available from manufacturers such as Pierce Antibodies, Cell Signal, and Abcam. Myc-tag antibodies are commercially available from manufacturers such as Santa Cruz Biotechnology, Abcam, and Cell Signal. Strep-tag antibodies are commercially available from manufacturers such as Abcam, Iba, and Qiagen.

[0150] Transduction markers can also be used for the same purpose, but transduction markers are derived from natural molecules and are often expressed using a skipping element that can separate the transduction marker from other components within the recombinant receptor. The transduction marker may be selected from at least one of truncated CD19 (tCD19; see Budde et al., Blood 122: 1660, 2013); truncated human EGFR (tEGFR or EGFRt; see Wang et al., Blood 118: 1255, 2011); the ECD of human CD34; and / or a target epitope derived from the CD34 antigen (see Fehse et al, Mol. Therapy 1(5 Pt 1);448-456, 2000) and a target epitope derived from the CD20 antigen (see Philip et al, Blood 124: 1277-1278), such as RQR8. Exemplary transduction markers and cognate pairs are described in U.S. Patent Publication No. 13 / 463,247. In certain embodiments, the cells are genetically engineered to express EGFRt.

[0151] In certain embodiments, the selection cassette enables positive or negative selection of the desired cell population. Negative selection is a method of removing some types of cells and leaving the cells of the desired type. Positive selection is a method of targeting the desired cell population and retaining only the desired cells.

[0152] The selection cassette can encode (a) a protein conferring resistance to an antibiotic or other toxin, (b) a protein complementing an auxotrophic deficiency, or (c) a protein supplying an essential nutrient unavailable from complex media, such as a gene encoding D-alanine racemase for Bacillus. The number of selection systems used for the recovery of transformed cells is not particularly limited. In certain embodiments, the positive selection cassette contains a resistance gene to neomycin, hygromycin, ampicillin, puromycin, phleomycin, zeomycin, blasticidin or biomycin. In certain embodiments, the selection cassette contains a DHFR (dihydrofolate reductase) gene or a DHFR double mutant (DHFRdm) gene conferring resistance to methotrexate (MTX), O 6 the MGMT P140K gene responsible for resistance to BG / BCNU, the HPRT (hypoxanthine phosphoribosyl transferase) gene responsible for the conversion of specific bases (aminopterin, hypoxanthine, thymidine) contained in HAT selection medium, or other genes responsible for the detoxification of several types of drugs. In certain embodiments, as the selection agent, neomycin, hygromycin, puromycin, phleomycin, zeomycin, blasticidin, biomycin, ampicillin, O 6 BG / BCNU, MTX, tetracycline, aminopterin, hypoxanthine, thymidine kinase, DHFR, Gln synthetase, or ADA can be mentioned.

[0153] In certain embodiments, the selection cassette contains DHFRdm.

[0154] In certain embodiments, the negative selection cassette contains a gene that converts a substrate contained in the culture medium into a toxic substance for cells expressing itself. Such molecules include the detoxification gene of diphtheria toxin (DTA) (Yagi et al., Anal Biochem. 214(1):77-86, 1993; Yanagawa et al., Transgenic Res. 8(3):215-221, 1999), and the thymidine kinase gene of herpes virus (HSV TK) that is sensitive to ganciclovir or FIAU. Also, by adding 6-thioguanine (6TG) to the medium, the HPRT gene may be used for negative selection. Also, variously derived polyA transcription termination sequences may be used for positive and negative selection, and the most standard ones include those derived from SV40 polyA, or polyA derived from eukaryotic genes (such as bovine growth hormone, rabbit β-globin, etc.).

[0155] In certain embodiments, the gene construct can include a polynucleotide encoding a self-cleaving polypeptide, and the polynucleotide encoding this self-cleaving polypeptide is arranged between a polynucleotide encoding a recombinant receptor and a polynucleotide encoding a transduction marker (e.g., EGFRt) or a selection cassette (e.g., DHFRdm). Exemplary self-cleaving polypeptides include the 2A peptide (P2A) derived from porcine teschovirus-1, the 2A peptide (T2A) derived from Thosea asigna virus, the 2A peptide (E2A) derived from equine rhinitis A virus, the 2A peptide (F2A) derived from foot-and-mouth disease virus, or variants thereof. Furthermore, exemplary nucleic acid sequences and amino acid sequences of the 2A peptide are also described, for example, in Kim et al. (PLOS One 6: e18556 (2011)). In certain embodiments, the cells are genetically modified to contain a self-cleaving polypeptide. In certain embodiments, the self-cleaving polypeptide is T2A. In certain embodiments, the self-cleaving polypeptide is P2A.

[0156] The control mechanism may be contained in multiple copies in the gene construct and can also be expressed as a separate molecule using a skipping factor. For example, the gene construct may express one, two, three, four, or five tag cassettes, one, two, three, four, or five transduction markers, and / or one, two, three, four, or five selection cassettes. For example, embodiments of the present disclosure may include gene constructs having two Myc tag cassettes, a cassette containing His tag and HA tag, a cassette containing HA tag and Softag1 tag, or a cassette containing Myc tag and SBP tag.

[0157] One advantage of incorporating at least one control mechanism into a gene construct is that by using a cognate binding molecule for the tag cassette, the gene construct-expressing cells administered to a subject can be increased or removed. In certain embodiments, the present disclosure provides a method of removing modified cells expressing a gene construct by using an antibody specific for the tag cassette, by using a cognate binding molecule specific for the control mechanism, or by using a second gene construct-expressing modified cell having specificity for the control mechanism. The removal of modified cells may be performed using a removal agent specific for the control mechanism. For example, when using EGFRt, an anti-EGFRt binding domain (such as an antibody or scFv) fused or conjugated to a cytotoxic reagent (such as a toxin or radioactive metal) may be used, or an anti-EGFRt / anti-CD3 bispecific scFv or anti-EGFRt CAR T cell may be used.

[0158] In a common suicide switch for drug-induced cell apoptosis, an inducible caspase suicide gene system using a modified human caspase 9 in a FK506 binding protein (FKBP) that dimerizes in the presence of a small molecule drug has been utilized. This suicide switch is called inducible caspase 9 or iCasp9 (Straathof et al., Blood. 2005, 105(11):4247-4254). Also, the efficacy of this suicide switch has been shown in preclinical and clinical trials (Diaconu et al., Mol Ther. 2017, 25(3):580-592; and Stasi et al., N Engl J Med. 2011, 365(18):1673-1683). In certain embodiments, an FDA-approved small molecule drug such as rapamycin can be used to control the iCasp9 suicide switch (Stavrou et al., mBio. 2018, 9(3):e00923-18).

[0159] The suicide switch can be created by linking the coding sequence of a cell division locus (CDL) and a negative selection marker during transcription. By utilizing the suicide switch, exposure of modified cells to an inducer of the negative selection marker can induce killing of growing host cells containing the suicide switch, or kill at least a portion of the growing cells to inhibit host cell growth. Cells modified to contain the suicide switch can be treated with an inducer (e.g., drug) of the negative selection marker to remove growing cells, or kill at least a portion of the growing cells to inhibit cell growth.

[0160] Examples of CDLs include CDK1, TOP2A, CENPA, BIRC5, and EEF2. Examples of negative selection markers include herpes simplex virus type 1 (HSV) thymidine kinase / ganciclovir (TK / GCV), deaminase / 5-fluorocytosine (CD / 5-FC), and carboxylesterase / irinotecan (CE / CPT-11).

[0161] Host cells modified with the HSV-TK / GCV negative selection marker produce thymidine kinase (TK), which converts GCV to GCV monophosphate, and then the intracellular kinases convert GCV monophosphate to GCV triphosphate. GCV triphosphate is incorporated into replicating DNA during the S phase, causing DNA elongation arrest and cell apoptosis (Halloran and Fenton, 1998, Cancer Res. 58(17): 3855-65).

[0162] The CD / 5-FC negative selection marker system is widely used as a suicide gene system. Cytosine deaminase (CD) is a non-mammalian-derived enzyme that can be obtained from bacteria or yeast (e.g., from Escherichia coli or Saccharomyces cerevisiae) (Ramnaraine et al., 2003). CD catalyzes the conversion of cytosine to uracil and is an important member of the pyrimidine salvage pathway in prokaryotes and fungi but is absent in mammalian cells. 5-Fluorocytosine (5-FC) is an antifungal prodrug that causes low-level cytotoxicity in humans (Denny, 2003, J Biomed Biotechnol). CD catalyzes the conversion of 5-FC to 5-FU, and the genotoxic substance 5-FU exhibits high-level toxicity to humans (Ireton et al., 2002, J Molec. Biol. 315(4):687-697).

[0163] The CE / CPT-11 system is based on the carboxylesterase enzyme, which is a serine esterase found in various tissues of mammalian species (Humerickhouse et al., 2000, Cancer Res. 60(5):1189-92). The anticancer agent CPT-11 is a prodrug that is activated by CE to produce an active drug called 7-ethyl-10-hydroxycamptothecin (SN-38), and this SN-38 is a potent topoisomerase I inhibitor for mammals (Wierdl et al., 2001). SN-38 induces the accumulation of double-strand DNA breaks in dividing cells (Kojima et al., 1998, Anal Chem. 70(13):2446-53).

[0164] In certain embodiments, the suicide switch is an inducible caspase suicide gene system or an HSVTK / GCV suicide gene system.

[0165] In certain embodiments, modified cells expressing a gene construct containing a control mechanism may be detected or tracked in vivo by using an antibody that specifically binds to the control mechanism (e.g., an anti-tag antibody) or other cognate binding molecules that specifically bind to the control mechanism, and the binding partner of this control mechanism is conjugated to a fluorescent dye, a radioactive tracer, iron oxide nanoparticles, or other contrast agents known in the art so that it can be detected by X-ray, CT scan, MRI scan, PET scan, ultrasound, flow cytometry, near-infrared imaging system, or other imaging methods (see, for example, Yu, et al., Theranostics 2:3, 2012).

[0166] Thus, modified cells expressing at least one control mechanism can be more easily identified, isolated, sorted, induced to proliferate, tracked, and / or removed than modified cells into which the tag cassette has not been introduced.

[0167] (IV) Characterization of genetically modified cells In certain embodiments, the genetically modified cells can be evaluated for cell surface expression of a recombinant receptor (e.g., a CAR or eTCR). In certain embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of the genetically modified cells express a detectable level of the recombinant receptor.

[0168] Expression of cell surface proteins can be measured by flow cytometry using methods known in the art. In flow cytometry, a fluorescent molecule is added and the cell population is labeled with an agent (e.g., an antibody) that targets the desired cell surface marker, whereby the cell population that is positive for that cell surface marker can be quantified and the expression level of that cell surface marker can be quantified.

[0169] Integration of a recombinant receptor into the genome of a recombinant cell can be measured by digital droplet PCR (ddPCR). Digital PCR can quantify the DNA concentration in a sample. In digital PCR, a PCR reaction mixture (e.g., containing a nucleic acid molecule sample and multiple copies of a PCR probe) is partitioned into reaction compartments, and multiple compartments containing no PCR probe and multiple compartments containing one or more copies of the PCR probe are generated. PCR amplification of each compartment is performed, and each compartment is analyzed by the PCR reaction. A positive endpoint is obtained from compartments containing one or more probes and one or more target DNA molecules, while a negative endpoint is obtained from compartments containing no PCR probe. Next, the compartments with positive reactions are fitted to a Poisson distribution to determine the absolute copy number of target DNA molecules per volume of the sample before partitioning (i.e., the copy number per 1 μl of the sample) (see Hindson, B. et al., (2011) Anal Chem. 83:8604-8610). Digital droplet PCR is a variant of digital PCR and is characterized by partitioning a nucleic acid sample into droplets using a water-in-oil emulsion. PCR amplification is performed in a batch on the droplets, and a flow path system is used for droplet dispensing and analysis of individual droplets. Those skilled in the art can use ddPCR to perform absolute quantification of DNA in a sample, perform copy number polymorphism analysis, and evaluate the efficiency of genome editing.

[0170] The recombinant cells can also be evaluated for cytokine-independent proliferation. The recombinant cells are expected to proliferate only in the presence of stimulatory cytokines (such as IL-2 and IL-7). Proliferation in the absence of cytokines is an indicator of the presence of tumor-forming ability. In certain embodiments, the recombinant cells are grown for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days in the presence or absence of one or more stimulatory cytokines (such as IL-2 and IL-7). In certain embodiments, conventional methods (such as flow cytometry, microscopy, optical density, metabolic activity) are used to evaluate proliferation from cell number and viability. In certain embodiments, evaluation of proliferation is started on day 1, day 2, day 3, day 4, day 5, or day 6. In certain embodiments, proliferation is evaluated daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or every 8 days. In certain embodiments, at the end of the growth period, proliferation in the absence of cytokines is evaluated. In some specific embodiments, when no proliferation of the recombinant cells is observed in the absence of cytokines, it is defined as having no tumor-forming ability. In certain embodiments, the proliferation of the cell population at the end of the growth period is less than 0.1-fold, less than 0.2-fold, less than 0.3-fold, less than 0.4-fold, less than 0.5-fold, less than 0.6-fold, less than 0.7-fold, less than 0.8-fold, less than 0.9-fold, less than 1.0-fold, less than 1.1-fold, less than 1.2-fold, less than 1.3-fold, less than 1.4-fold, or less than 1.5-fold of that at the start of the growth period, and it is defined as no proliferation being observed. In certain embodiments, the recombinant cells do not proliferate in the absence of cytokine stimulation, growth factor stimulation, and antigen stimulation.

[0171] (V) Cell activation culture conditions The cell population can be grown by incubating it in a starting culture composition. The incubation can be carried out in a culture vessel such as a bag, cell culture plate, flask, chamber, chromatography column, cross-linked gel, cross-linked polymer, column, culture dish, hollow fiber, microtiter plate, silica-coated glass plate, tube, tube set, well, vial, or other culture or cell culture container.

[0172] In certain embodiments, the cell population can be incubated in a starting culture composition before or after genetic recombination. In certain embodiments, the incubation of the cell population can be carried out for 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, or 1 day before genetic recombination. In certain embodiments, the incubation of the cell population can be carried out for 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, or 1 day after genetic recombination. In certain embodiments, the incubation of the cell population can be carried out simultaneously with genetic recombination.

[0173] The culture conditions may include one or more of a specific medium, temperature, oxygen content, carbon dioxide content, time, and an agent (e.g., a nutrient, amino acid, antibiotic, ion, and / or a stimulating factor (e.g., a cytokine, chemokine, antigen, binding partner, fusion protein, recombinant soluble receptor, and other agents designed to activate cells)).

[0174] In some embodiments, the incubation is carried out according to techniques described in U.S. Patent Publication No. 6,040,177, Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0175] Exemplary culture media for culturing T cells include, (i) RPMI supplemented with non-essential amino acids, sodium pyruvate, and penicillin / streptomycin; (ii) RPMI supplemented with HEPES, 5-15% human serum, 1-3% L-glutamine, 0.5-1.5% penicillin / streptomycin, and 0.25×10 -4 ~0.75×10 -4 M β-mercaptoethanol; (iii) RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, 100 U / ml penicillin, and 100 m / mL streptomycin; (iv) DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 100 U / ml penicillin, and 100 m / mL streptomycin; and (v) X-Vivo 15 medium (Lonza, Walkersville, MD) supplemented with 5% human AB serum (Gemcell, West Sacramento, CA), 1% HEPES (Gibco, Grand Island, NY), 1% penicillin / streptomycin (Gibco), 1% GlutaMax (Gibco), and 2% N-acetylcysteine (Sigma-Aldrich, St. Louis, MO). Also included are Furthermore, T cell culture media are also available commercially from HyClone (Logan, UT). Further T cell activation components that can be added to the above culture media are described in more detail below.

[0176] In some embodiments, the proliferation of T cells is achieved by adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the culture starting composition (e.g., at a ratio such that the number of PBMC feeder cells per T lymphocyte is at least 5, 10, 20, or 40 or more in the cell population before proliferation), and then incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some embodiments, the non-dividing feeder cells may include γ-irradiated PBMC feeder cells. In some embodiments, to prevent cell division, PBMCs are irradiated with 3000 - 3600 rad of γ-rays. In some embodiments, the feeder cells are added to the culture medium before adding the T cell population.

[0177] The incubation of T cells may further include the step of adding non-dividing lymphoblastoid cells (LCLs) transformed with EBV as feeder cells. The LCLs may be irradiated with 6000 - 10,000 rad of γ-rays. In some embodiments, the LCL feeder cells are provided in an appropriate amount, for example, provided such that the ratio of LCL feeder cells to T lymphocytes at the start of proliferation is at least 10:1.

[0178] In some embodiments, the stimulation conditions include a temperature suitable for the proliferation of human T lymphocytes, which may be, for example, at least 25°C, at least 30°C, or 37°C.

[0179] The activation culture conditions of T cells include conditions under which the T cells in the starting composition for culture proliferate or expand. The T cell activation conditions may include one or more cytokines, for example, interleukin (IL)-2, IL-7, IL-15, and / or IL-21 may be included. The content of IL-2 may be in the range of 10 to 100 ng / ml (for example, 40 ng / ml, 50 ng / ml, or 60 ng / ml). IL-7, IL-15, and / or IL-21 may each be included in the range of 0.1 to 50 ng / ml (for example, 5 ng / ml, 10 ng / ml, or 15 ng / ml). In certain embodiments, 50 ng / ml of IL-2 is used. In certain embodiments, 10 ng / ml of each of IL-7, IL-15, and IL-21 is used.

[0180] In certain embodiments, the T cell activation culture conditions may include an epitope that stimulates T cells. Examples of epitopes that stimulate T cells include CD3, CD27, CD2, CD4, CD5, CD7, CD8, CD28, CD30, CD40, CD56, CD83, CD90, CD95, 4-1BB (CD137), B7-H3, CTLA-4, Frizzled-1 (FZD1), FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, HVEM, ICOS, IL-1R, LAT, LFA-1, LIGHT, MHCI, MHCII, NKG2D, OX40, ROR2, and RTK.

[0181] CD3 is a major signaling factor of the T cell receptor. As described above, CD3 is expressed in all mature T cells. In certain embodiments, the molecule that stimulates CD3 (i.e., the CD3 binding domain) is the OKT3 antibody (U.S. Patent Publication No. 5,929,212; U.S. Patent Publication No. 4,361,549; ATCC® CRL-8001 TM; see also Arakawa et al., J. Biochem. 120, 657-662 (1996), and may also be derived from the 20G6-F3 antibody, 4B4-D7 antibody, 4E7-C9 antibody, or 18F5-H10 antibody.

[0182] In certain embodiments, the molecule that stimulates CD3 may be included in the culture medium at a concentration of at least 0.25 ng / ml or 0.5 ng / ml, or at a concentration of 2.5 - 10 μg / ml. In certain embodiments, a molecule that stimulates CD3 (e.g., OKT3) is used at a concentration of 5 μg / ml.

[0183] In certain embodiments, the activation molecule associated with the avi tag can be biotinylated and bound to streptavidin beads. By utilizing this method, for example, a removable T cell epitope stimulation activation system can be constructed.

[0184] Exemplary domains that bind to CD28 include TGN1412, CD80, CD86, or the 9D7 antibody, or derivatives thereof. Antibodies that bind to CD28 further include the 9.3 antibody, KOLT-2 antibody, 15E8 antibody, 248.23.2 antibody, EX5.3D10 antibody, and the CD28.3 antibody (deposited as a synthetic single-chain Fv construct in GenBank accession number: AF451974.1; see also Vanhove et al., BLOOD, 15 Jul. 2003, Vol. 102, No. 2, pages 564-570).

[0185] The 4-1BB binding domain can be derived from LOB12, IgG2a, LOB12.3 or IgG1, as described in Taraban et al. Eur J Immunol. 2002 December; 32(12):3617-27. In certain embodiments, the 4-1BB binding domain is derived from the monoclonal antibody described in U.S. Patent Publication No. 9,382,328. Another 4-1BB binding domain is described in U.S. Patent Publication No. 6,569,997, U.S. Patent Publication No. 6,303,121, and Mittler et al. Immunol Res. 2004; 29(1-3):197-208.

[0186] Activation of OX40 (CD134) and / or ICOS may be utilized. The OX40 binding domain is described in U.S. Patent Publication No. 20100196359, U.S. Patent Publication No. 20150307617, WO 2015 / 153513, WO2013 / 038191, and Melero et al. Clin Cancer Res. 2013 Mar. 1; 19(5):1044-53. Exemplary binding domains that can bind to and activate ICOS are described, for example, in U.S. Patent Publication No. 20080279851, and Deng et al. Hybrid Hybridomics. 2004 June; 23(3):176-82.

[0187] When the T cell activator is soluble, it can be conjugated to another molecule such as a polyethylene glycol (PEG) molecule. Any suitable PEG molecule can be used. Typically, PEG molecules with a molecular weight of 1000 Da or less can be dissolved in water or culture medium. In some cases, such PEG-based reagents can be prepared using commercially available activated PEG molecules (e.g., PEG-NHS derivatives available from NOF North America Corporation (Irvine, CA, USA) or activated PEG derivatives available from Creative PEGWorks (Chapel Hill, NC, USA)).

[0188] In certain embodiments, the cell stimulant is immobilized on a solid phase in the culture medium. In certain embodiments, the solid phase is the surface of a culture vessel (e.g., a bag, cell culture plate, chamber, chromatography column, cross-linked gel, cross-linked polymer, column, culture dish, hollow fiber, microtiter plate, silica-coated glass plate, tube, tube set, well, vial, or the surface of other structures or containers for cells or cell culture).

[0189] In certain embodiments, the solid phase can be added to the culture medium. Such solid phases include, for example, beads, hollow fibers, resins, membranes, and polymers.

[0190] Exemplary beads include magnetic beads, polymer beads, and resin beads (e.g., Strep-Tactin® Sepharose, Strep-Tactin Superflow, and Strep-Tactin MacroPrep (IBA GmbH (Göttingen))). Anti-CD3 / anti-CD28 beads are commercially available as a T cell proliferation reagent (Invitrogen). Anti-CD3 / anti-CD28 beads are superparamagnetic non-heat-generating sterilized polystyrene beads with a uniform size of 4.5 μm, and are coated with a mixture of a monoclonal antibody affinity-purified against the CD3 cell surface molecule on human T cells and a monoclonal antibody affinity-purified against the CD28 cell surface molecule. Hollow fibers are available from TerumoBCT (Lakewood, Colorado, USA). Examples of resins include resins for immobilized metal affinity chromatography (IMAC) (e.g., TALON® resin (Westburg (Roosendaal))). Examples of membranes include papers and membrane substrates for chromatographic matrices (e.g., nitrocellulose membranes or polyvinylidene fluoride (PVDF) membranes).

[0191] Exemplary polymers include polysaccharides such as polysaccharide matrices. Such matrices include agarose gels (e.g., Superflow TM agarose or Sepharose® materials, e.g., Superflow commercially available in various bead sizes and pore diameters TM Sepharose), or cross-linked dextran gels. Further specific examples include particulate cross-linked agarose matrices to which dextran is covalently bound, which are commercially available (in various bead sizes and various pore diameters) and are available from GE Healthcare under the trade names Sephadex® or Superdex®.

[0192] Synthetic polymers that may be used include polyacrylamide, polymethacrylate, copolymers of polysaccharides and agarose (e.g., polyacrylamide / agarose composites), or copolymers of polysaccharides and N,N'-methylenebisacrylamide. Examples of copolymers of dextran and N,N'-methylenebisacrylamide include materials of the Sephacryl® series (Pharmacia Fine Chemicals, Piscataway, NJ).

[0193] In certain embodiments, particulate silica bound to a synthetic polymer or particulate silica bound to a natural polymer may be used, such as polysaccharide grafted silica, polyvinylpyrrolidone grafted silica, polyethylene oxide grafted silica, poly(2-hydroxyethyl aspartamide) silica, and poly(N-isopropylacrylamide) grafted silica, among others.

[0194] The cell activator can be immobilized on the solid phase via a covalent bond or can be reversibly immobilized on the solid phase via a non-covalent bond.

[0195] (VI) Cell preparations produced ex vivo In certain embodiments, the genetically modified cells can be recovered from the culture medium, washed, concentrated, and mixed with a carrier in a therapeutically effective amount. Exemplary carriers include saline, buffered saline, physiological saline, water, Hank's solution, Ringer's solution, Normosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Morton Grove, IL), and combinations thereof.

[0196] In certain embodiments, human serum albumin (HSA) or other human serum components or fetal bovine serum can be added to the carrier. In certain embodiments, the carrier for infusion contains buffered saline with 5% HSA or dextrose added. As another isotonic agent, polyhydric alcohols containing trihydric alcohols or polyhydric alcohols with a higher valency than that can be mentioned, for example, glycerin, erythritol, arabitol, xylitol, sorbitol, mannitol, and the like.

[0197] The carrier may contain buffers such as citrate buffer, succinate buffer, tartrate buffer, fumarate buffer, gluconate buffer, oxalate buffer, lactate buffer, acetate buffer, phosphate buffer, histidine buffer and / or trimethylamine salts.

[0198] The stabilizer refers to a wide variety of additives that can have various functions such as bulking agents from additives that can prevent the adhesion of cells to the wall of the container. Typical stabilizers include polyhydric alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine; organic saccharides or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol, cyclitol (for example, inositol); PEG; amino acid polymers; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, sodium thiosulfate; low molecular weight polypeptides (that is, polypeptides with less than 10 residues); proteins such as HSA, bovine serum albumin, gelatin, immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose; trisaccharides such as raffinose; and polysaccharides such as dextran.

[0199] If necessary or beneficial, the formulation may contain a local anesthetic such as lidocaine to relieve pain at the injection site.

[0200] Exemplary preservatives include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halide, hexamethonium chloride, alkyl parabens (e.g., methylparaben and propylparaben), catechol, resorcinol, cyclohexanol and 3-pentanol.

[0201] The therapeutically effective amount of cells contained in the formulation is greater than 10 2 cells, greater than 10 3 cells, greater than 10 4 cells, greater than 10 5 cells, greater than 10 6 cells, greater than 10 7 cells, greater than 10 8 cells, greater than 10 9 cells, greater than 10 10 cells, or greater than 10 11 cells may be.

[0202] In the formulations disclosed herein, the cells are typically contained in a volume of 1 L or less, 500 ml or less, 250 ml or less, or 100 ml or less. Thus, the density of the cells administered is typically greater than 10 4 cells / ml, greater than 10 7 cells / ml, or greater than 10 8 cells / ml.

[0203] In certain embodiments, the formulation may contain one or more genetically modified cells (e.g., modified T cells, modified NK cells or modified stem cells). Also, the formulation may contain different types of genetically modified cells (e.g., a combination of T cells, NK cells and / or stem cells).

[0204] Different types of genetically modified cells or cell subsets (e.g., modified T cells, modified NK cells and / or modified stem cells) can be provided in various ratios, for example, in ratios of 1:1:1, 2:1:1, 1:2:1, 1:1:2, 5:1:1, 1:5:1, 1:1:5, 10:1:1, 1:10:1, 1:1:10, 2:2:1, 1:2:2, 2:1:2, 5:5:1, 1:5:5, 5:1:5, 10:10:1, 1:10:10, 10:1:10 and the like. These ratios are also applicable to the number of cells expressing the same recombinant receptor component or different recombinant receptor components. When only two types of cells are combined, or when the recombinant receptor components contained in the formulation and expressed are only combinations of two types, these ratios may also be combinations of two numerical values that can be created from the combinations of the three numerical values described above. In some embodiments, the combined cell population is tested for efficacy and / or cell proliferation in vitro, in vivo and / or ex vivo, and the ratio of cells that gives efficacy and / or cell proliferation is selected. Certain embodiments include CD4 T cells and CD8 T cells in a ratio of 1:1.

[0205] The cell-based formulations disclosed herein can be prepared, for example, for administration by injection, infusion, perfusion or lavage. This formulation can be further formulated for intramedullary injection, intravenous injection, intradermal injection, intraarterial injection, intranodal injection, intralymphatic injection, intraperitoneal injection, intralesional injection, intraprostatic injection, intravaginal injection, intrarectal injection, intrathecal injection, intratumoral injection, intramuscular injection, intravesical injection and / or subcutaneous injection.

[0206] (VII) Targeted viral vectors and nanoparticles for modifying cells in vivo Immune cells can also be genetically modified in vivo or ex vivo using virus vectors and / or nanoparticles with target-directed properties. Virus vectors that can be used for delivering genes encoding recombinant receptors into cells are described elsewhere in this specification, and target-directed virus vectors (e.g., pseudotyped virus vectors) are well known in the art.

[0207] Exemplary nanoparticles that target cells include nanoparticles having a ligand that targets cells (e.g., CD3, CD4, CD8, CD34) bound to their surface. Since these nanoparticles have a ligand that targets cells bound to their surface, they are selectively taken up by selected types of cells. Next, a gene modification component is delivered by the nanoparticles, and a recombinant receptor (e.g., CAR) is expressed.

[0208] Exemplary nanoparticles include liposomes (tiny vesicles in which at least one lipid bilayer surrounding an aqueous core forms concentric spheres), liposome nanoparticles (liposome structures used to encapsulate another, smaller nanoparticle within their core), and lipid nanoparticles (liposome-like structures that do not have a continuous lipid bilayer characteristic of liposomes). Other polymeric nanoparticles or porous nanoparticles composed of materials capable of forming a porous network can also be used. Exemplary materials include metals, transition metals, and metalloids (e.g., lithium, magnesium, zinc, aluminum, and silica).

[0209] Nanoparticles for the purpose of in vivo delivery and uptake into cells may have an uncharged coating or a negatively charged coating, and the size of these nanoparticles may be 130 nm or less. The dimensions of the nanoparticles can be measured using conventional techniques such as, for example, dynamic light scattering and / or electron microscopy. In certain embodiments, the nanoparticles may be those described in WO2014153114, WO2017181110, and WO201822672.

[0210] The therapeutically effective amount of the vector and / or nanoparticles contained in the formulation may range from 0.1 to 5 μg / kg or 0.5 to 1 μg / kg. In another example, doses include 1 μg / kg, 30 μg / kg, 90 μg / kg, 150 μg / kg, 500 μg / kg, 750 μg / kg, 0.1 to 5 mg / kg or 0.5 to 1 mg / kg. In another example, doses include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or higher doses.

[0211] (viii) Methods of use The methods disclosed herein include treating a subject (human, non-human primate, companion animal (such as dog, cat, reptile, bird, etc.), livestock (such as horse, cow, goat, pig, chicken, etc.) or laboratory animal (such as monkey, rat, mouse, fish, etc.)) using the formulations disclosed herein. Treating the subject includes delivering a therapeutically effective amount. A therapeutically effective amount includes an amount capable of providing an effective amount, prophylactic treatment and / or therapeutic treatment.

[0212] An "effective amount" is the amount of the formulation necessary to effect a desired physiological change in a subject. For example, an effective amount can provide an immunogenic anti-cancer effect. An effective amount is often administered for research purposes. The effective amount disclosed herein is an amount capable of causing a statistically significant effect in an animal model or in vitro assay relevant to the assessment of cancer development or progression. An immunogenic formulation can be provided in an effective amount by which an immune response is stimulated.

[0213] "Preventive treatment" includes treatments performed for the purpose of reducing or mitigating the risk of further progression of cancer in subjects who do not show cancer signs or symptoms or who show only initial signs or symptoms of cancer. Therefore, preventive treatment functions as a treatment for preventing cancers that express BAFF-R. In certain embodiments, the preventive treatment suppresses, delays, or prevents the occurrence of metastasis from the primary cancer tumor site.

[0214] "Therapeutic treatment" includes treatments performed for the purpose of reducing or eliminating cancer signs or symptoms in subjects who show cancer symptoms or signs. By means of therapeutic treatment, it is possible to suppress, control, or eliminate the presence or activity of cancer and / or to suppress, control, or eliminate the side effects of cancer.

[0215] The functions as an effective amount, preventive treatment, or therapeutic treatment are not mutually exclusive, and in certain embodiments, two or more treatments may be performed depending on the administered dose.

[0216] In certain embodiments, an anti-cancer effect can be obtained with a therapeutically effective amount. The anti-cancer effects include a decrease in the number of cancer cells, a decrease in the number of metastases, a decrease in tumor volume, an extension of the average lifespan, induction of chemosensitivity or radiosensitivity in cancer cells, suppression of cancer cell proliferation, suppression of tumor growth, prevention or reduction of metastasis, extension of the subject's lifespan, suppression of cancer-related pain, and / or a decrease in the recurrence or relapse of cancer after treatment. In certain embodiments, a therapeutically effective amount can provide an anti-cancer effect in a state with a low antigen density.

[0217] In certain embodiments, an immune response is induced with a therapeutically effective amount. The immune response may be directed against cancer cells that express BAFF-R.

[0218] "BAFF-R positive cells" refers to cells that express BAFF-R on their surface. "BAFF-R positive cancer cells" refers to cancer cells that express BAFF-R on their surface. In some embodiments, the expression of BAFF-R on the cell surface is measured by using an antibody against BAFF-R in methods such as immunohistochemical analysis and FACS. Alternatively, the expression of BAFF-R mRNA is considered to correlate with the expression of BAFF-R on the cell surface, and the expression of BAFF-R mRNA can be measured by, for example, in situ hybridization and / or RT-PCR (including quantitative RT-PCR).

[0219] Examples of BAFF-R related diseases treatable with the gene constructs disclosed herein include mantle cell lymphoma (MCL), multiple myeloma (MM), acute lymphoblastic leukemia (ALL), and diffuse large B-cell lymphoma (DLBCL).

[0220] As described above, the recombinant receptor disclosed herein can be used for the treatment of subjects having cancer in a state of low antigen density. In a specific example, it includes evaluating the BAFF-R antigen expression level of cancer in a subject and selecting the recombinant receptor of the present disclosure to be used for the treatment of the subject based on the state of low antigen density.

[0221] The recombinant receptor disclosed herein is not limited to the treatment of subjects having cancer in a state of low antigen density, and can also be used for subjects having cancer in a state of high antigen density. Examples of a state of high antigen density include a subject in which the number of BAFF-R molecules per diseased cell exceeds 50,000; a subject in which the number of BAFF-R molecules per diseased cell exceeds 60,000; a subject in which the number of BAFF-R molecules per diseased cell exceeds 70,000; a subject in which the number of BAFF-R molecules per diseased cell exceeds 80,000; a subject in which the number of BAFF-R molecules per diseased cell exceeds 90,000; or a subject in which the number of BAFF-R molecules per diseased cell exceeds 100,000.

[0222] Upon administration, the therapeutically effective amount (also referred to herein as "dose") can initially be estimated based on the results of in vitro assays and / or animal model studies. Using such information, a more accurate determination of the dosage useful for the intended subject can be made. The actual dosage administered to a particular subject can be determined by a physician, veterinarian, or researcher taking into account parameters such as physical and physiological factors including the target, body weight, disease severity, cancer type, cancer stage, past or concurrent therapeutic interventions, the subject's idiopathic diseases, and the route of administration.

[0223] The therapeutically effective amount of a cell-based formulation can be 10 4 ~10 9 cells / kg body weight or 10 3 ~10 11 cells / kg body weight. As the therapeutically effective amount to be administered, a number of cells exceeding 10 2 cells, 10 3 cells, 10 4 cells, 10 5 cells, 10 6 cells, 10 7 cells, 10 8 cells, 10 9 cells, 10 10 cells, or a number of cells exceeding 10 11 cells may be mentioned.

[0224] The therapeutically effective amount of the vector and / or nanoparticles contained in the formulation may be in the range of 0.1 to 5 μg / kg or 0.5 to 1 μg / kg. In another example, the dosage may be 1 μg / kg, 30 μg / kg, 90 μg / kg, 150 μg / kg, 500 μg / kg, 750 μg / kg, 0.1 to 5 mg / kg or 0.5 to 1 mg / kg. In another example, the dosage may be 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or higher dosages.

[0225] The therapeutically effective amount can be achieved by one or multiple administrations during the course of a treatment regimen (e.g., daily, every other day, every three days, every four days, every five days, every six days, once a week, every two weeks, every three weeks, monthly, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or once a year). In certain embodiments, the treatment protocol may be determined according to a clinical trial protocol or a treatment protocol approved by the FDA.

[0226] The therapeutically effective amount can be administered, for example, by injection, infusion, perfusion or washing. Routes of administration include intravenous bolus administration, intradermal administration, intraarterial administration, intraperitoneal administration, intranodal administration, intralymphatic administration, intraperitoneal administration, intralesional administration, intraprostatic administration, intravaginal administration, rectal administration, topical administration, intrathecal administration, intratumoral administration, intramuscular administration, intravesical administration and / or subcutaneous administration.

[0227] In certain embodiments, the formulation and / or composition is administered to a patient in combination with (e.g., before, simultaneously with, or after) a related method of treatment, and the number of such related methods of treatment is not particularly limited. In certain embodiments, the cells of the present disclosure are used in combination with chemotherapy; radiation; immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506; antibodies; other immunosorbents such as CAM PATH; anti-CD3 antibodies; other antibody therapies; cytotoxins; fludarabine; cyclosporine; FK506; rapamycin; mycophenolic acid; steroids; FR901228; cytokines; or radiation therapy.

[0228] In certain embodiments, the cell formulation and / or modified formulation (or formulation) may be co-administered with a chemotherapeutic agent, and the number of chemotherapeutic agents co-administered is not particularly limited. Examples of chemotherapeutic agents include alkylating agents; alkyl sulfonates; aziridines; ethyleneimines and methylamelamines; nitrogen mustards; nitrosoureas; antibiotics; antimetabolites; folic acid analogs; purine analogs; pyrimidine analogs; androgens; adrenal suppressants; folic acid supplements; platinum analogs; retinoic acid; and pharmaceutically acceptable salts, acids, or derivatives thereof. The definition of chemotherapeutic agents further includes antihormonal agents that control or suppress the action of hormones on tumors, and such antihormonal agents include antiestrogens and antiandrogens, and pharmaceutically acceptable salts, acids, or derivatives thereof. Further, combinations of chemotherapeutic agents are administered when deemed appropriate, and these include CHOP therapy, i.e., a combination of cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone.

[0229] In some embodiments, the chemotherapeutic agent is administered concomitantly with the administration of the recombinant cells or nucleic acids of the present disclosure or within one week after the administration of the recombinant cells or nucleic acids of the present disclosure. In another embodiment, the chemotherapeutic agent is administered 1 to 4 weeks, 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after the administration of the recombinant cells or nucleic acids of the present disclosure. In another embodiment, the chemotherapeutic agent is administered at least one month before the administration of the cells or nucleic acids of the present disclosure. In some embodiments, the methods of the present disclosure further comprise administering two or more chemotherapeutic agents.

[0230] In a further embodiment, a formulation comprising immune cells comprising a recombinant receptor can be administered together with an anti-inflammatory agent. Anti-inflammatory agents or anti-inflammatory drugs include steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone and triamcinolone); and non-steroidal anti-inflammatory agents (NSAIDs) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide and mycophenolate.

[0231] In certain embodiments, the formulations and / or compositions described herein are administered in combination with a cytokine. As used herein, "cytokine" refers to a protein released by one cell population that acts as an intercellular mediator on another cell. Examples of cytokines include lymphokines, monokines, and conventional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, human N-methionyl growth hormone, bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH); hepatocyte growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; Mullerian inhibitor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial cell growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGF) such as NGF-β; platelet-derived growth factor; transforming growth factors (TGF) such as TGF-α and TGF-β; insulin-like growth factors I and II; erythropoietin (EPO); osteogenic factor; interferons such as interferon α, interferon β, interferon γ; colony stimulating factors (CSF) such as macrophage CSF (M-CSF); granulocyte macrophage CSF (GM-CSF); granulocyte CSF (G-CSF); interleukins (IL) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15; tumor necrosis factors such as TNF-α and TNF-β; and other polypeptide factors such as LIF and kit ligand (KL). As used herein, "cytokine" also includes naturally-derived proteins or proteins derived from recombinant cell culture, and biologically active equivalents of cytokines having a natural sequence.

[0232] In certain embodiments, an antibody or antigen-binding fragment thereof that binds to a transduction marker expressed by a genetically modified cell can be administered. In certain embodiments, the antibody or antigen-binding fragment thereof may be part of an antibody complex (i.e., an antibody linked to a detectable label or a cytotoxic agent). In certain embodiments, detectable labels include fluorescent proteins (e.g., GFP, YFP, RFP, EGFP, mCherry), radiolabels (e.g., 35 S, 125 I, 32 P, 3 H, 14 C and 131 I), radiophonic labels, enzyme labels (e.g., horseradish peroxidase, hydrolase, luciferase, and alkaline phosphatase), chemiluminescent labels, fluorescent labels (e.g., rhodamine, phycoerythrin, and fluorescein), gold beads, magnetic beads (e.g., Dynabeads TM ), and biotin (for use in combination with labeled avidin or streptavidin). In certain embodiments, cytotoxic agents include toxins (e.g., holotoxins or subtoxins) and drugs (e.g., chemotherapeutic agents).

[0233] In certain embodiments, the genetically modified cells described herein can be further genetically modified to express a second recombinant receptor. This second recombinant receptor can confer additional cancer cell specificity by targeting cells that co-express a cancer antigen or by targeting a cell-specific ligand. In certain embodiments, the second recombinant receptor comprises a binding domain that binds to a second cancer antigen, a B cell-specific ligand, or a small molecule. In certain embodiments, examples of the second cancer antigen include EGFR, B7H3, CD171, ROR1, HER2, IL-13Rα2, GD2, CA-125, MUC-1, EphA2, MAGE-A3, MAGE-A4, MAGE-C2, PRAME, SSX2, adipophilin, AIM2, ALDH1A1, BCLX, EpCAM, CS274, CPSF, cyclin D1, DKK1, ENAH, EPHA3, EZH2, FGF5, G250, HLA-DOB, ID01, IGF2B3, KIF20A, M-CSF, MCSP, MDM2, Meloe, MMP-2, MMP-7, MUC1, MUC5AC, p53, PAX5, PBF, PRAME, PSMA, RAGE-1, RGS5, RhoC, RNF43, RUF43, FU2AS, SOX10, STEAP1, TPBG, VEGF, WT1, or NY-ESO-1. In certain embodiments, examples of the B cell-specific ligand include EGFR, B7H3, CD171, ROR1, or IL-13Rα2. In certain embodiments, examples of the B cell-specific ligand include CD19, CD20, CD22, CD1d, CD5, CD21, CD23, CD24, CD25, CD27, CD32, CD34, CD35, CD38, CD40, CD44, CD45, CD45.1, CD45.2, CD54, CD69, CD72, CD79, CD80, CD84, LFA-1, CALLA, BCMA, the B220 isoform of CD45, CD93, CD84, CD86, TNFSF7, TNFRSF5, ENPP-1, HVEM, BLIMP1, CXCR4, CD148, or CD147. In certain embodiments, examples of the B cell-specific ligand include CD19, CD20, or CD22.In certain embodiments, examples of the small molecule include fluorescein, dinitrophenol, biotin, folic acid, or derivatives thereof.

[0234] (VIII) Kits The present disclosure further includes a kit. The kit may contain various components for implementing the methods disclosed herein. For example, depending on the aspects of the methods of the present disclosure to be implemented, the kit may include one or more nucleic acids encoding a recombinant receptor; one or more nucleic acids encoding a CAR; a nucleic acid encoding a second-generation 4-1BB chimeric antigen receptor (CAR); a lentiviral BAFF-R CAR construct having a short spacer, a medium-length spacer or a long spacer; a nucleic acid encoding a scFv; a nucleic acid encoding a VL; a nucleic acid encoding a VH; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding an EGFRt; a nucleic acid encoding a DHFRdm; a nucleic acid encoding a Her2t; cells (e.g., immune cells, T cells, CD4 T cells, CD8 T cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, lymphocytes, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and / or a mixture of HSCs and HPCs (i.e., HSPCs), non-transduced T cells, BAFF-R-BBζ CAR T cells); cell lines (e.g., NALM6 cell line, TM-LCL cell line, Raji cell line, K562 cell line, lentivirally transduced cell line); tissue samples (e.g., peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy samples, tumors, lymph nodes, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, large intestine, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organs, and / or cells derived from these organs); gene expression components (e.g., genes for expression provided by vectors (e.g., lentiviral vectors or retroviral vectors), components of CRISPR, ZFNs, TALENs, MegaTALs, targeted viral vectors and / or targeted nanoparticles); cell formulation components or cell activation components (e.g., saline, buffered saline, phosphate-buffered saline (PBS)); biocompatible buffers (Ca++ / Mg++-free PBS, saline, water, Hank's solution, Ringer's solution); T cell-stimulating epitopes (e.g., anti-CD3 / anti-CD28 conjugated beads, OKT3, TGN1412);Composition for starting cultivation (RPMI, non-essential amino acids, sodium pyruvate, penicillin / streptomycin, non-dividing lymphoblastoid cells (LCL) transformed with EBV, IL-21, human serum albumin (HSA) or other human serum components or fetal bovine serum, dextrose, stabilizer, preservative); components for combination therapy (e.g., local anesthetic, chemotherapeutic agent, immunosuppressant, anti-inflammatory agent); antibody with fluorescent molecular tag; sequence for PCR amplification; cytokine (e.g., IL-2, IL-7, IL-15, IL-21); culture vessel; GAPDH; enzyme-linked immunosorbent assay (ELISA); culture plate, etc. may be included.;

[0235] The following exemplary embodiments and examples are described to illustrate specific embodiments of the present disclosure. Those skilled in the art who refer to the present disclosure will understand that various changes can be made to the specific embodiments disclosed herein, and that even if such changes are made, similar results or analogous results can be obtained without departing from the gist and scope of the present disclosure.;

[0236] (IX) Exemplary embodiments 1. A nucleic acid comprising a coding sequence of a recombinant receptor, when the recombinant receptor is expressed by a cell, an extracellular portion comprising a binding domain; an intracellular portion comprising an effector domain; and a transmembrane domain that links the extracellular portion to the intracellular portion is included, the binding domain is a heavy chain variable region having complementarity-determining region (CDRH) 1 shown in SEQ ID NO: 2, CDRH2 shown in SEQ ID NO: 3, and CDRH3 shown in SEQ ID NO: 4, a light chain variable region having complementarity-determining region (CDRL) 1 shown in SEQ ID NO: 5, CDRL2 containing SEQ AAS, and CDRL3 shown in SEQ ID NO: 7, a spacer containing the hinge domain of IgG4 and a nucleic acid.; 2. The nucleic acid according to embodiment 1, wherein the intracellular portion comprises a signaling domain of CD3ζ and a co-stimulatory domain of 41-BB. 3. The nucleic acid according to embodiment 2, wherein the signaling domain of CD3ζ is encoded by the sequence shown in SEQ ID NO: 23. 4. The nucleic acid according to embodiment 2 or 3, wherein the co-stimulatory domain of 41-BB is encoded by the sequence shown in SEQ ID NO: 21. 5. A nucleic acid encoding a recombinant receptor, wherein when the recombinant receptor is expressed by a cell, an extracellular portion comprising a binding domain that binds to a B cell activating factor receptor (BAFF-R); an intracellular portion comprising an effector domain; and a transmembrane domain that links the extracellular portion to the intracellular portion is included. 6. The nucleic acid according to embodiment 5, wherein the encoded recombinant receptor is a chimeric antigen receptor (CAR), a recombinant T cell receptor, or a hybrid thereof. 7. The nucleic acid according to embodiment 5 or 6, wherein the encoded recombinant receptor is a CAR. 8. The nucleic acid according to any one of embodiments 5 to 7, wherein the binding domain is derived from the H90 monoclonal antibody. 9. The binding domain is a complementarity-determining region (CDRH) 1 having at least 95% sequence identity with the sequence shown in SEQ ID NO: 2, a CDRH2 having at least 95% sequence identity with the sequence shown in SEQ ID NO: 3, and a CDRH3 having at least 95% sequence identity with the sequence shown in SEQ ID NO: 4, or a heavy chain variable region having a sequence with 0 to 5 conservative amino acid substitutions in these CDR sequences; A light chain variable region having Complementarity Determining Region (CDRL) 1 with at least 95% sequence identity to the sequence shown in SEQ ID NO: 5, CDRL2 with at least 95% sequence identity to sequence AAS, and CDRL3 with at least 95% sequence identity to the sequence shown in SEQ ID NO: 7, or a sequence having 0 to 5 conservative amino acid substitutions in these CDR sequences, and The nucleic acid according to any one of Embodiments 5 to 8, comprising 10. The binding domain is A heavy chain variable region having Complementarity Determining Region (CDRH) 1 shown in SEQ ID NO: 2, CDRH2 shown in SEQ ID NO: 3, and CDRH3 shown in SEQ ID NO: 4, and A light chain variable region having Complementarity Determining Region (CDRL) 1 shown in SEQ ID NO: 5, CDRL2 containing sequence AAS, and CDRL3 shown in SEQ ID NO: 7, and The nucleic acid according to any one of Embodiments 5 to 9, comprising 11. The binding domain is a heavy chain variable region having at least 95% sequence identity to the sequence shown in SEQ ID NO: 8, or a sequence having 0 to 10 conservative amino acid substitutions in the sequence of the heavy chain variable region; and a light chain variable region having at least 95% sequence identity to the sequence shown in SEQ ID NO: 9, or a sequence having 0 to 10 conservative amino acid substitutions in the sequence of the light chain variable region. The nucleic acid according to any one of Embodiments 5 to 10. 12. The nucleic acid according to any one of Embodiments 5 to 11, wherein the binding domain comprises a heavy chain variable region shown in SEQ ID NO: 8 and a light chain variable region shown in SEQ ID NO: 9. 13. The nucleic acid according to any one of Embodiments 5 to 12, wherein the binding domain comprises a humanized amino acid sequence. 14. The binding domain comprises a humanized VH domain comprising a sequence having at least 95% sequence identity to the sequence shown in SEQ ID NO: 32, SEQ ID NO: 8 or SEQ ID NO: 34, and a humanized VL domain comprising a sequence having at least 95% sequence identity to the sequence shown in SEQ ID NO: 35, SEQ ID NO: 9 or SEQ ID NO: 37. The nucleic acid according to any one of Embodiments 5 to 13. 15. The nucleic acid according to any one of Embodiments 5 to 14, wherein the binding domain comprises a humanized VH domain comprising the sequence shown in SEQ ID NO: 32, SEQ ID NO: 8, or SEQ ID NO: 34, and a humanized VL domain comprising the sequence shown in SEQ ID NO: 35, SEQ ID NO: 9, or SEQ ID NO: 37. 16. The nucleic acid according to any one of Embodiments 5 to 15, wherein the binding domain is a scFv. 17. The nucleic acid according to Embodiment 16, wherein the scFv has at least 95% sequence identity with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6. 18. The nucleic acid according to Embodiment 16 or 17, wherein the scFv has 1 to 10 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6. 19. The nucleic acid according to any one of Embodiments 16 to 18, wherein the scFv comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6. 20. The nucleic acid according to any one of Embodiments 5 to 19, wherein the extracellular portion further comprises a spacer. 21. The nucleic acid according to Embodiment 20, wherein the spacer is a long spacer, a medium-length spacer, or a short spacer. 22. The nucleic acid according to Embodiment 20 or 21, wherein the length of the spacer is 10 to 15 residues long, 110 to 130 residues long, or 230 to 240 residues long. 23. The nucleic acid according to Embodiment 21 or 22, wherein the short spacer is 12 residues long. 24. The nucleic acid according to Embodiment 21 or 22, wherein the medium-length spacer is 119 residues long. 25. The nucleic acid according to Embodiment 21 or 22, wherein the long spacer is 229 residues long. 26. The nucleic acid according to any one of Embodiments 20 to 25, wherein the spacer comprises the hinge domain of IgG4. 27. The nucleic acid according to any one of Embodiments 20 to 26, wherein the spacer further comprises the CH3 domain of IgG4. 28. The nucleic acid according to any one of embodiments 20 to 27, wherein the spacer further comprises the CH2 domain of IgG4. 29. The nucleic acid according to any one of embodiments 20 to 25, wherein the spacer comprises the hinge domain of CD8a. 30. The nucleic acid according to any one of embodiments 20 to 29, wherein the spacer comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 15, SEQ ID NO: 13, SEQ ID NO: 11 or SEQ ID NO: 70, or a sequence having 0 to 3 conservative amino acid substitutions in this sequence. 31. The nucleic acid according to any one of embodiments 20 to 30, wherein the spacer comprises the sequence shown in SEQ ID NO: 15, SEQ ID NO: 13, SEQ ID NO: 11 or SEQ ID NO: 70. 32. The nucleic acid according to any one of embodiments 5 to 31, wherein the effector domain comprises the whole or a part of the signaling domain of CD3ζ, CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C and / or B7-H3. 33. The nucleic acid according to any one of embodiments 5 to 32, wherein the effector domain comprises the whole or a part of the signaling domains of CD3ζ and 4-1BB. 34. The nucleic acid according to embodiment 33, wherein the signaling domain of CD3ζ comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 22. 35. The nucleic acid according to embodiment 33 or 34, wherein the signaling domain of CD3ζ has a sequence having 1 to 5 conservative amino acid substitutions compared with SEQ ID NO: 22. 36. The nucleic acid according to embodiment 33 or 34, wherein the signaling domain of CD3ζ comprises the sequence shown in SEQ ID NO: 22. 37. The nucleic acid according to any one of embodiments 33 to 36, wherein the signaling domain of 4-1BB comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 20. 38. The nucleic acid according to any one of embodiments 33 to 37, wherein the signal transduction domain of 4-1BB has 1 to 5 conservative amino acid substitutions as compared with SEQ ID NO: 20. 39. The nucleic acid according to any one of embodiments 33 to 37, wherein the signal transduction domain of 4-1BB comprises the sequence shown in SEQ ID NO: 20. 40. The nucleic acid according to any one of embodiments 5 to 39, wherein the transmembrane domain comprises the transmembrane domain of CD28. 41. The nucleic acid according to embodiment 40, wherein the transmembrane domain of CD28 comprises a sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 17. 42. The nucleic acid according to embodiment 40 or 41, wherein the transmembrane domain of CD28 has 1 to 3 conservative amino acid substitutions as compared with SEQ ID NO: 17. 43. The nucleic acid according to embodiment 40 or 41, wherein the transmembrane domain of CD28 comprises the sequence shown in SEQ ID NO: 17. 44. The nucleic acid according to any one of embodiments 5 to 43, wherein the transmembrane domain comprises the transmembrane domain of CD8a. 45. The nucleic acid according to embodiment 44, wherein the transmembrane domain of CD8a comprises a sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 71. 46. The nucleic acid according to embodiment 44 or 45, wherein the transmembrane domain of CD8a has 1 to 3 conservative amino acid substitutions as compared with SEQ ID NO: 71. 47. The nucleic acid according to embodiment 44 or 45, wherein the CD8a transmembrane domain comprises the sequence shown in SEQ ID NO: 71. 48. The nucleic acid according to any one of embodiments 5 to 47, further comprising a control mechanism selected from a tag cassette, a transduction marker, a selection cassette and / or a suicide switch, or further encoding a control mechanism selected from these. 49. The nucleic acid according to embodiment 48, wherein the transduction marker comprises a truncated EGFR (EGFRt) polypeptide or a truncated Her2 (Her2t). 50. The nucleic acid according to embodiment 48 or 49, wherein the transduction marker comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 29. 51. The nucleic acid according to any one of embodiments 48 to 50, wherein the transduction marker has 1 to 5 conservative amino acid substitutions as compared with SEQ ID NO: 29. 52. The nucleic acid according to any one of embodiments 48 to 50, wherein the transduction marker comprises the sequence shown in SEQ ID NO: 29. 53. The nucleic acid according to any one of embodiments 48 to 52, wherein the transduction marker encodes the sequence shown in SEQ ID NO: 30. 54. The nucleic acid according to embodiment 48 or 49, wherein the transduction marker comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 85. 55. The nucleic acid according to any one of embodiments 48 to 54, wherein the transduction marker has 1 to 5 conservative amino acid substitutions as compared with SEQ ID NO: 85. 56. The nucleic acid according to any one of embodiments 48 to 55, wherein the transduction marker comprises the sequence shown in SEQ ID NO: 85. 57. The nucleic acid according to any one of embodiments 48 to 56, wherein the transduction marker encodes the sequence shown in SEQ ID NO: 86. 58. The nucleic acid according to any one of embodiments 48 to 57, wherein the selection cassette comprises a dihydrofolate reductase double mutant (DHFRdm). 59. The nucleic acid according to embodiment 58, wherein the DHFRdm comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 26. 60. The nucleic acid according to embodiment 58 or 59, wherein the DHFRdm has 1 to 5 conservative amino acid substitutions as compared with SEQ ID NO: 26. 61. The nucleic acid according to embodiment 58 or 59, wherein the DHFRdm comprises the sequence shown in SEQ ID NO: 26. 62. The nucleic acid according to any one of embodiments 48 to 61, wherein the suicide switch comprises a herpes simplex virus thymidine kinase / ganciclovir (HSVTK / GCV) suicide gene system or an inducible caspase suicide gene system. 63. The nucleic acid according to any one of embodiments 5 to 62, further comprising a ribosome skipping factor. 64. The nucleic acid according to embodiment 63, wherein the ribosome skipping factor is T2A, P2A, E2A or F2A. 65. The nucleic acid according to embodiment 63 or 64, wherein the ribosome skipping factor is T2A. 66. The nucleic acid according to any one of embodiments 63 to 65, wherein the ribosome skipping factor is P2A. 67. The nucleic acid sequence according to any one of embodiments 5 to 66, further comprising a promoter operably linked to the coding sequence encoding the recombinant receptor. 68. The nucleic acid sequence according to embodiment 67, wherein the promoter is a constitutive promoter. 69. The nucleic acid according to embodiment 68, wherein the constitutive promoter is an EF1α promoter. 70. The nucleic acid according to any one of embodiments 5 to 69, further comprising an inducible promoter operably linked to the coding sequence encoding the recombinant receptor. 71. The nucleic acid according to any one of embodiments 5 to 70, comprising cDNA. 72. A nanoparticle encapsulating the nucleic acid according to any one of embodiments 5 to 71. 73. A vector comprising the nucleic acid according to any one of embodiments 5 to 71. 74. The vector according to embodiment 73, which is a viral vector, a transposon vector, an integrase vector or an mRNA vector. 75. The vector according to embodiment 74, wherein the viral vector is a lentiviral vector, a foamy virus vector, a retroviral vector or a gammaretroviral vector. 76. The vector according to embodiment 74 or 75, wherein the viral vector is a lentiviral vector. 77. Use of the nanoparticle according to embodiment 72 or the vector according to any one of embodiments 73 to 76 for treating a subject in need of treatment and having a low cell density of BAFF-R antigen. 78. A cell in which a gene is modified to contain the nucleic acid according to any one of embodiments 5 to 71. 79. The cell according to embodiment 78, further comprising a gene construct encoding a second recombinant receptor. 80. The cell according to embodiment 79, wherein the second recombinant receptor comprises an extracellular domain comprising a binding domain that binds to a cancer antigen, a B cell-specific ligand, and / or a small molecule. 81. The cell according to embodiment 80, wherein the cancer antigen is EGFR, B7H3, CD171, ROR1, HER2, IL-13Rα2, GD2, CA-125, MUC-1, EphA2, MAGE-A3, MAGE-A4, MAGE-C2, PRAME, SSX2, adipophilin, AIM2, ALDH1A1, BCLX, EpCAM, CS274, CPSF, cyclin D1, DKK1, ENAH, EPHA3, EZH2, FGF5, G250, HLA-DOB, ID01, IGF2B3, KIF20A, M-CSF, MCSP, MDM2, Meloe, MMP-2, MMP-7, MUC1, MUC5AC, p53, PAX5, PBF, PRAME, PSMA, RAGE-1, RGS5, RhoC, RNF43, RUF43, FU2AS, SOX10, STEAP1, TPBG, VEGF, WT1, or NY-ESO-1. 82. The cell according to embodiment 80 or 81, wherein the B cell-specific ligand is EGFR, B7H3, CD171, ROR1, or IL-13Rα2. 83. The cell according to any one of embodiments 80 to 82, wherein the B cell-specific ligand is CD19, CD20, CD22, CD1d, CD5, CD21, CD23, CD24, CD25, CD27, CD32, CD34, CD35, CD38, CD40, CD44, CD45, CD45.1, CD45.2, CD54, CD69, CD72, CD79, CD80, CD84, LFA-1, CALLA, BCMA, the B220 isoform of CD45, CD93, CD84, CD86, TNFSF7, TNFRSF5, ENPP-1, HVEM, BLIMP1, CXCR4, CD148, or CD147. 84. The cell according to any one of embodiments 80 to 83, wherein the B cell-specific ligand is CD19, CD20 or CD22. 85. The cell according to any one of embodiments 80 to 84, wherein the small molecule is fluorescein, dinitrophenol, biotin, folic acid or a derivative thereof. 86. The cell according to any one of embodiments 80 to 85, wherein the extracellular domain comprises a spacer. 87. The cell according to embodiment 86, wherein the spacer is a long spacer, a medium-length spacer or a short spacer. 88. The cell according to embodiment 86 or 87, wherein the length of the spacer is 10 to 15 residues long, 110 to 130 residues long or 230 to 240 residues long. 89. The cell according to embodiment 87 or 88, wherein the length of the short spacer is 12 residues long. 90. The cell according to embodiment 87 or 88, wherein the length of the medium-length spacer is 119 residues long. 91. The cell according to embodiment 87 or 88, wherein the length of the long spacer is 229 residues long. 92. The cell according to any one of embodiments 86 to 91, wherein the spacer comprises the hinge domain of IgG4. 93. The cell according to any one of embodiments 86 to 92, wherein the spacer further comprises the CH3 domain of IgG4. 94. The cell according to any one of embodiments 86 to 93, wherein the spacer further comprises the CH2 domain of IgG4. 95. The cell according to any one of embodiments 86 to 94, wherein the spacer comprises the hinge domain of CD8a. 96. The cell according to any one of embodiments 86 to 95, wherein the spacer comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 15, SEQ ID NO: 13, SEQ ID NO: 11 or SEQ ID NO: 70, or a sequence having 0 to 3 conservative amino acid substitutions in this sequence. 97. The cell according to any one of embodiments 86 to 96, wherein the spacer comprises the sequence shown in SEQ ID NO: 15, SEQ ID NO: 13, SEQ ID NO: 11 or SEQ ID NO: 70. 98. The cell according to any one of embodiments 78 to 97, which is an autologous cell obtained from a subject or a cell of the same species as the subject. 99. The cell according to any one of embodiments 78 to 98, which is a T cell, B cell, natural killer (NK) cell, NK-T cell, monocyte / macrophage, hematopoietic stem cell (HSC) or hematopoietic progenitor cell (HPC). 100. The cell according to any one of embodiments 78 to 99, which is a T cell selected from CD3+ T cells, CD4+ T cells, CD8+ T cells, central memory T cells, effector memory T cells and / or naive T cells. 101. The cell according to any one of embodiments 78 to 100, which is a CD8+ T cell and / or a CD4+ T cell. 102. The cell according to any one of embodiments 78 to 102, which is an in vivo cell, an in vitro cell or an ex vivo cell. 103. The cell according to any one of embodiments 78 to 102, which is a mammalian cell. 104. The cell according to any one of embodiments 78 to 103, which is a human cell. 105. A formulation comprising the cell according to any one of embodiments 78 to 104 and a pharmaceutically acceptable carrier. 106. A composition, i) A nucleic acid according to any one of Embodiments 5 to 71, a nanoparticle according to Embodiment 72, or a vector according to any one of Embodiments 73 to 76; and ii) a pharmaceutically acceptable carrier A composition comprising the same. 107. A method of treating a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of the formulation according to Embodiment 105 and / or the composition according to Embodiment 106 to treat the subject. 108. The method according to Embodiment 107, wherein the subject has cancer. 109. The method according to Embodiment 108, wherein the cancer comprises BAFF-R+ cells. 110. The method according to Embodiment 108 or 109, wherein the cancer is a B cell cancer. 111. The method according to any one of Embodiments 108 to 110, wherein the cancer is mantle cell lymphoma (MCL), multiple myeloma (MM), acute lymphoblastic leukemia (ALL), and diffuse large B cell lymphoma (DLBCL). 112. The method according to any one of Embodiments 107 to 111, wherein the formulation comprises autologous cells or allogeneic cells. 113. The method according to any one of Embodiments 107 to 112, wherein the subject is a mammal. 114. The method according to any one of Embodiments 107 to 113, wherein the subject is a human. 115. The method according to any one of Embodiments 107 to 114, wherein the cell density of the BAFF-R antigen in the subject is low. 116. The method according to Embodiment 115, wherein the low cell density of the BAFF-R antigen is less than 50,000 BAFF-R molecules per diseased cell. 117. The method according to Embodiment 115 or 116, wherein the low cell density of the BAFF-R antigen is less than 30,000 BAFF-R molecules per diseased cell. 118. The method according to any one of embodiments 115 to 117, wherein the low cell density of the BAFF-R antigen is less than 15,000 BAFF-R molecules per diseased cell. 119. The method according to any one of embodiments 115 to 118, wherein the low cell density of the BAFF-R antigen is less than 10,000 BAFF-R molecules per diseased cell. 120. The method according to any one of embodiments 115 to 119, wherein the low cell density of the BAFF-R antigen is less than 7,000 BAFF-R molecules per diseased cell. 121. The method according to any one of embodiments 107 to 120, further comprising the step of obtaining a sample of the diseased cells and the step of measuring the cell density of the BAFF-R antigen. 122. Use of the cells according to any one of embodiments 78 to 104 in the manufacture of a medicament for treating, suppressing or alleviating a disorder in a subject. 123. Use of the cells according to any one of embodiments 78 to 104 for treating a subject in need of treatment and having a low cell density of the BAFF-R antigen. 124. The cells according to embodiment 123 for use as a medicament. 125. A method of modifying the gene of a cell, comprising the step of introducing into the cell the nucleic acid according to any one of embodiments 5 to 71, the nanoparticle according to embodiment 72, the vector according to any one of embodiments 73 to 76, or the composition according to embodiment 106. 126. The method according to embodiment 125, further comprising the step of stimulating the cell by contacting the cell with a cytokine. 127. The method according to embodiment 126, wherein the cytokine is selected from IL-2, IL-7, IL-15 and IL-21. 128. The method according to embodiment 126 or 127, wherein the stimulating step is performed before the introducing step. 129. The method according to any one of embodiments 126 to 128, further comprising a step of activating the cell by contacting the cell with an anti-CD3 antibody or an antigen-binding fragment thereof and / or an anti-CD28 antibody or an antigen-binding fragment thereof. 130. The method according to embodiment 129, wherein the activation step is performed before the introduction step. 131. The method according to any one of embodiments 126 to 130, further comprising a step of culturing the cells to obtain a plurality of cells in an amount sufficient to administer immunotherapy to the subject. 132. The method according to any one of embodiments 126 to 131, wherein the cells are autologous cells obtained from the subject or cells of the same species as the subject. 133. The method according to any one of embodiments 126 to 132, wherein the cells are T cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, hematopoietic stem cells (HSC) or hematopoietic progenitor cells (HPC). 134. The method according to any one of embodiments 126 to 133, wherein the cells are T cells selected from CD3+ T cells, CD4+ T cells, CD8+ T cells, central memory T cells, effector memory T cells and / or naive T cells. 135. The method according to any one of embodiments 126 to 134, wherein the cells are CD8+ T cells and / or CD4+ T cells. 136. The method according to any one of embodiments 126 to 135, wherein the cells are in vivo cells, in vitro cells or ex vivo cells. 137. The method according to any one of embodiments 126 to 136, wherein the cells are mammalian cells. 138. The method according to any one of embodiments 126 to 137, wherein the cells are human cells. 139. A composition comprising a recombinant receptor encoded by the nucleic acid according to any one of embodiments 5 to 71, wherein the recombinant receptor specifically binds to a BAFF-R polypeptide or a fragment thereof. 140. The composition according to embodiment 139, wherein the recombinant receptor is contained in effector cells. 141. The composition according to embodiment 140, wherein the effector cells are T cells, CD4+ T cells, CD8+ T cells, progenitor T cells or hematopoietic stem cells. 142. The composition according to any one of embodiments 139 to 141, wherein the BAFF-R polypeptide or a fragment thereof is contained in target cells. 143. The composition according to embodiment 142, wherein the target cells are B cells. 144. The composition according to any one of embodiments 140 to 143, wherein the effector cells further comprise a transduction marker. 145. The composition according to embodiment 144, wherein the transduction marker comprises a truncated EGFR (EGFRt) polypeptide or a truncated Her2 (Her2t). 146. The composition according to embodiment 144 or 145, further comprising an antibody or an antigen-binding fragment thereof that specifically binds to the transduction marker. 147. The composition according to embodiment 146, wherein the antibody or the antigen-binding fragment thereof further comprises a detectable label or a cytotoxin.

Examples

[0237] (X) Examples

[0238] Example 1. Anti - BAFF - R CAR The construct shown in FIG. 1 was prepared, and the expression level of BAFF-R was measured in the following cell lines. That is, K562, a human immortalized myeloid leukemia cell line; K562 BAFF-R containing the BAFF-R expression construct; NALM6, a leukemia-derived precursor B cell line; TM-LCL, a lymphoblastoid cell line; and Raji, a lymphoblastoid cell line were used. FIG. 3 shows a bar graph indicating the BAFF-R antigen density on each target cell, showing that BAFF-R is endogenously expressed to varying degrees in the NALM6 cell line, TM-LCL cell line, and Raji cell line, and that K562 cells transduced to express BAFF-R showed a very high expression level compared to other cell lines.

[0239] Example 2. Expression of anti - BAFF - R CAR in effector cells The expression of anti-BAFF-R CAR in effector cells, namely CD4+ T cells and CD8+ T cells, was measured. The anti-BAFF-R CAR contained either an IgG4 hinge polypeptide spacer (short), an IgG4 hinge-CH3 polypeptide spacer (medium), or an IgG4 hinge-CH2-CH3 polypeptide spacer (long). FIGS. 4A and 4B show the percentage of CD8+ T cells or CD4+ T cells expressing anti-BAFF-R CAR (FIG. 4A) and fluorescence-activated cell sorting (FACS) analysis of cells expressing anti-BAFF-R CAR (FIG. 4B).

[0240] Example 3. Specific lysis of target cells by effector cells in vitro An in vitro specific lysis assay of target cells by effector cells expressing anti-BAFF-R CAR was performed. In the presence of effector T cells expressing the anti-BAFF-R CAR described herein, the K562 cell line, K562 OKT3 cell line, K562 BAFF-R cell line, NALM6 cell line, TM-LCL cell line, and Raji cell line were cultured as target cells.

[0241] The cytotoxicity of anti-BAFF-R CAR-T cells was measured by co-culturing effector cells with target cells expressing a fluorescent protein. Data were obtained by monitoring the growth of target cells during the co-culture period using an Incucyte device, and the lytic ability of effector cells expressing each CAR against various target tumor cells was compared.

[0242] Figures 5A-5E show the results of a cytotoxicity assay to examine the specific lysis of target cells by effector cells expressing anti-BAFF-R CAR. In this assay, as target cells, the K562 parental cell line (negative control that does not express BAFF-R), the K562 OKT3+ cell line (positive control that is a target for killing by the T cell receptor), and the K562 BAFF-R+ cell line, TM-LCL cell line, Raji cell line, and NALM6 cell line, which are BAFF-R+ targets, were used. The results are summarized in Figure 5F, which shows a bar graph indicating the specific lysis rate of each target cell by effector cells expressing anti-BAFF-R CAR.

[0243] Example 4. Induction of cytokines in target cells by effector cells in vitro An in vitro cytokine induction assay of target cells by effector cells expressing anti-BAFF-R CAR was performed. In the presence of effector T cells expressing the anti-BAFF-R CAR described herein, the K562 cell line, K562 OKT3 cell line, K562 BAFF-R cell line, NALM6 cell line, TM-LCL cell line, and Raji cell line as target cells were cultured.

[0244] A co-culture assay was used to measure the cytokine production-inducing ability of each CAR in the presence of each target cell. Briefly, effector cells and target cells were mixed in each well of a microplate and incubated for 24 hours. After incubation, interferon γ (IFNγ), interleukin 2 (IL-2), and tumor necrosis factor α (TNFα) in the supernatant obtained from each co-culture were analyzed.

[0245] These results are shown in FIGS. 6A - 6C. FIGS. 6A - 6C show bar graphs depicting the production of each cytokine induced in effector cells expressing anti - BAFF - R CAR in the presence of K562 parental cell line (negative control that does not express BAFF - R), K562 OKT3+ cell line (positive control that is a target for killing by the T - cell receptor), or K562 BAFF - R+ cell line, TM - LCL cell line, Raji cell line or NALM6 cell line as target cells, for each of interferon - γ (IFNγ), interleukin - 2 (IL - 2) and tumor necrosis factor - α (TNFα).

[0246] FIG. 7 shows a series of pie charts (upper panel) showing the percentage of gated cells (gated on lymphocytes / single cells / viable cells / CD8 / EGFRt+), and a bar graph (lower right panel) showing the expression of cytokines (IFNγ, IL - 2 or TNFα) induced in CD8+ T cells expressing anti - BAFF - R CAR in the presence of Raji target cells.

[0247] FIG. 8 shows a series of pie charts (upper panel) showing the percentage of gated cells (gated on lymphocytes / single cells / viable cells / CD8 / EGFRt+), and a bar graph (lower right panel) showing the expression of 4 - 1BB, CD107a or Nur77 induced in CD8+ T cells expressing anti - BAFF - R CAR in the presence of Raji target cells.

[0248] Example 5. In vivo administration of effector cells in a xenograft model The activity of anti-BAFF-R CAR T cells was examined in a xenograft model. Raji cells expressing mCherry and luciferase as reporter genes were administered to mice and treated with T cells expressing anti-BAFF-R CAR. During the test period, D-luciferin was intraperitoneally (i.p.) injected into the mice, and imaging was performed by imaging using an IVIS Spectrum imaging device to detect luminescent tumor cells in the mouse body. During the test period, in the mice, each symptom of tumor mass increase, xenogeneic graft-versus-host disease (xGVHD), and toxicity was evaluated. On two occasions, on day 35 and day 60, a fixed dose of Raji cells was readministered to the mice to measure the durability of T cell transplantation and to evaluate the resistance of T cells with each CAR to exhaustion.

[0249] Figure 9A shows a series of graphs showing bioluminescence in mice, with each line representing an individual subject. Figure 9B shows a graph showing the average bioluminescence of the results shown in Figure 9A. Figure 9C shows the survival rate and tumor-related deaths of the treated mice shown in Figure 9A. As shown in Figures 9A and 9B, unexpectedly, mice contacted with anti-BAFF-R CAR T cells having a CAR containing an IgG4 hinge polypeptide spacer had (i) a lower tumor mass due to a lower flux amount and (ii) an extended survival rate compared to mice treated with anti-BAFF-R CAR T cells having a CAR containing a CD8a hinge polypeptide spacer.

[0250] (XI) Conclusion The nucleic acid sequences and amino acid sequences provided herein are shown in abbreviations used for nucleotide bases and amino acid residues as defined in 37 C.F.R. §§ 1.831 to 1.835 of the United States Patent Law Regulations and as shown in WIPO Standard ST.26 (effective July 1, 2022). Only one strand of each nucleic acid sequence is shown, but if its complementary strand is appropriate, its complementary strand is also included in the embodiments.

[0251] The sequences disclosed in this specification and variants of the cited sequences are also included in the present application. Indicators for determining which amino acid residues can be substituted, inserted or deleted without losing biological activity are well-known computer programs in the art, such as DNASTAR TM software (Madison, Wisconsin, USA) can be used to find. The amino acid changes of the protein variants disclosed in this specification are preferably conservative amino acid changes, that is, substitutions between amino acids with similar charges or substitutions between non-charged amino acids are preferred. Conservative amino acid changes include substitutions by members of amino acid families with related side chains.

[0252] Suitable conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art, and such conservative substitutions can usually be made without changing the biological activity of the ultimately obtained molecule. Those skilled in the art will typically be familiar with the fact that substituting one amino acid in an unimportant region of a polypeptide will not substantially change its biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Natural amino acids are usually classified into conservative substitution families, specifically: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; negatively charged residues with polarity and also classified as their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; positively charged residues with polarity and also classified as such): arginine (Arg), lysine (Lys), and histidine (His); Group 6 (large aliphatic nonpolar residues): isoleucine (Ile), leucine (Leu), methionine (Met), valine (Val), and cysteine (Cys); Group 7 (polar uncharged): tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser, and Thr; Group 8 (large aromatic residues): phenylalanine (Phe), tryptophan (Trp), and Tyr; Group 9 (nonpolar): proline (Pro), Ala, Val, Leu, Ile, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (small aliphatic nonpolar residues or small aliphatic residues with slight polarity): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing residues): Met and Cys. Further information is described in Creighton (1984) Proteins, W.H. Freeman and Company.

[0253] When making such changes, the hydrophobicity index of the amino acids may be taken into account. It is widely understood in the art that the hydrophobicity index of amino acids is important when conferring biological functions that interact with each other on a protein (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). The hydrophobicity indices of each amino acid are: Ile(+4.5); Val(+4.2); Leu(+3.8); Phe(+2.8); Cys(+2.5); Met(+1.9); Ala(+1.8); Gly(-0.4); Thr(-0.7); Ser(-0.8); Trp(-0.9); Tyr(-1.3); Pro(-1.6); His(-3.2); Glutamic acid(-3.5); Gln(-3.5); Aspartic acid(-3.5); Asn(-3.5); Lys(-3.9); and Arg(-4.5).

[0254] It is well known in the art that substituting a particular amino acid with another amino acid having a similar hydrophobicity index or degree of hydrophobicity results in a protein having similar biological activity, i.e., a protein having biologically equivalent functionality. When making such changes, substitutions between amino acids with hydrophobicity indices within the range of ±2 are preferred, substitutions between amino acids with hydrophobicity indices within the range of ±1 are particularly preferred, and substitutions between amino acids with hydrophobicity indices within the range of ±0.5 are even more particularly preferred. Furthermore, it is also well known in the art that substitutions between similar amino acids can be effectively made based on hydrophilicity.

[0255] As detailed in U.S. Patent Publication No. 4,554,101, a hydrophilicity value is assigned to each amino acid residue, and the hydrophilicity values of the respective amino acid residues are: Arg(+3.0); Lys(+3.0); aspartic acid(+3.0±1); glutamic acid(+3.0±1); Ser(+0.3); Asn(+0.2); Gln(+0.2); Gly(0); Thr(-0.4); Pro(-0.5±1); Ala(-0.5); His(-0.5); Cys(-1.0); Met(-1.3); Val(-1.5); Leu(-1.8); Ile(-1.8); Tyr(-2.3); Phe(-2.5); Trp(-3.4). Specific amino acids can be substituted with other amino acids having comparable hydrophilicity values, and it is well known that even when such substitutions are made, biologically equivalent proteins can be obtained, particularly immunologically equivalent proteins. When making such changes, substitutions between amino acids with hydrophilicity values within the range of ±2 are preferred, substitutions between amino acids with hydrophilicity values within the range of ±1 are particularly preferred, and substitutions between amino acids with hydrophilicity values within the range of ±0.5 are even more particularly preferred.

[0256] As outlined above, amino acid substitutions may be made based on the relative similarity of the substituents of the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, size, etc. Also, as noted elsewhere in this specification, variants of gene sequences include codon-optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not have a statistically significant effect on the function of the encoded product.

[0257] Variants of the proteins, nucleic acids, and gene sequences disclosed herein include sequences having at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with the proteins, nucleic acids, or gene sequences disclosed herein.

[0258] "Percent sequence identity" refers to the relatedness of two or more sequences measured by comparing the sequences to each other. In the art, "identity" also means the degree of relatedness between protein sequences, nucleic acid sequences, or gene sequences, measured by matching between protein sequence chains, nucleic acid sequence chains, or gene sequence chains. "Identity" (often called "similarity") can be readily calculated by known methods, such as those described in Computational Molecular Biology (Lesk, A.M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D.W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A.M., and Griffin, H.G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods for measuring identity are designed to obtain the best match between the sequences being tested. Methods for measuring identity and similarity are embodied in publicly available computer programs. The sequence alignment and calculation of identity may be performed using the Megalign program (DNASTAR, Madison, Wis.) included in the LASERGENE suite of bioinformatics calculation software.Multiple alignments of sequences can also be performed using the Clustal alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989), using default parameters (gap penalty = 10, gap length penalty = 10)). Related programs further include the GCG program suite (Wisconsin Package version 9.0, Genetics Computer Group (GCG), Madison, Wis.); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990)); DNASTAR (DNASTAR, Madison, Wis.); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.). In the present disclosure, when sequence analysis software is used for analysis, the analysis results are interpreted based on the "default values" that are the standards of the program. As used herein, "default values" means a series of numerical values or parameters that are pre-registered in the software at the time of software initialization.

[0259] Variants include nucleic acid molecules that hybridize to the sequences disclosed herein under stringent hybridization conditions and have the same function as the reference sequence. Exemplary stringent hybridization conditions include incubation overnight at 42 °C in a solution containing 50% formamide, 5× SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and fragmented and denatured salmon sperm DNA at 20 μg / ml, followed by washing the filter at 50 °C with 0.1× SSC. Alterations in hybridization stringency and signal detection are mainly achieved by adjusting the concentration of formamide (lowering the percentage of formamide results in lower stringency), salt conditions, or temperature. For example, moderately high stringent conditions include incubation overnight at 37 °C in a solution containing 6× SSPE (20× SSPE = 3 M NaCl; 0.2 M NaH2PO4; 0.02 M EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 μg / ml salmon sperm DNA for blocking, followed by washing at 50 °C with 1× SSPE and 0.1% SDS. Further, even lower stringency is achieved by performing the wash after stringent hybridization at a high salt concentration (e.g., 5× SSC). The foregoing conditions can be varied in various ways by adding and / or replacing with another blocking reagent used to reduce the background of the hybridization experiment. Common blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. When adding a specific blocking reagent, it may be necessary to partially modify the foregoing hybridization conditions due to compatibility issues.

[0260] "Binds" means does not significantly bind to other molecules or components in the relevant environmental sample, but has an affinity of 10 5 M -1 or greater, i.e., Ka (i.e., the equilibrium binding constant of a specific binding interaction expressed in units of 1 / M), refers to the binding of a binding domain (e.g., the binding domain of a recombinant receptor) to its cognate binding molecule. The binding domain may be classified as "high affinity" or "low affinity". In certain embodiments, a "high affinity" binding domain has at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 or at least 10 13 M -1 of K a . In certain embodiments, a "low affinity" binding domain has a K 7 M -1 or less, 10 6 M -1 or less or 10 5 M -1 or less of K a . Alternatively, affinity may be defined as the equilibrium dissociation constant (K d ) (units: M) of a specific binding interaction (e.g., 10 -5 M to 10 -13 M). In certain embodiments, a binding domain may have "enhanced affinity", which refers to a selected or engineered binding domain exhibiting stronger binding to its cognate binding molecule than the wild-type (or parental) binding domain. For example, enhanced affinity may be due to a K a (equilibrium binding constant) for its cognate binding molecule being higher than that of a reference binding domain, or a K d (dissociation constant) for its cognate binding molecule being lower than that of a reference binding domain, or a dissociation rate (K off) may also be due to being lower than the binding domain serving as a reference. As assays for detecting a binding domain that specifically binds to a specific cognate binding molecule and assays for measuring binding affinity, various ones such as Western blot, ELISA, BIACORE® analysis, etc. are known (furthermore, for example, see Scatchard, et al., 1949, Ann. N.Y. Acad. Sci. 51:660; and U.S. Patent No. 5,283,173, U.S. Patent No. 5,468,614 or similar publications).

[0261] Unless otherwise stated, the present disclosure can be implemented using prior art related to immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. For example, see Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and R. I. Freshney, ed. Animal Cell Culture (1987).

[0262] As would be understood by those skilled in the art, each embodiment disclosed in this specification includes, consists essentially of, or consists of the specific components, steps, materials, or ingredients described. Therefore, the terms "comprising" or "including" should be construed to have the meaning of "including, consisting essentially of, or consisting of". The transitional phrase "comprising" means, but is not limited to, that components, steps, materials, or ingredients not described are included, even if in large amounts. The transitional phrase "consisting of" excludes all components, steps, materials, and ingredients not described. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the described components, steps, materials, or ingredients, and components, steps, materials, or ingredients that do not materially affect the embodiment. "Materially affect" refers to an effect that statistically significantly increases BAFF-R expressing cells in a subject treated according to the experimental protocols described in this specification.

[0263] Unless otherwise indicated, in this specification and the claims, all numerical values representing amounts of materials, properties such as molecular weights, reaction conditions, etc. are to be construed as being modified in all instances by the term "about". Accordingly, unless otherwise indicated, the numerical parameters set forth in this specification and the appended claims are approximations that vary depending on the desired properties sought to be obtained by the present invention. While not intended to limit the scope of the equivalence principle applicable to the claims, each numerical parameter should be construed in light of at least the reported number of significant digits and by applying ordinary rounding procedures. More specifically, the term "about", when used in conjunction with a recited numerical value or range, has the meaning reasonably construed by one of ordinary skill in the art, i.e., within a range of ±20% of the recited numerical value; within a range of ±19% of the recited numerical value; within a range of ±18% of the recited numerical value; within a range of ±17% of the recited numerical value; within a range of ±16% of the recited numerical value; within a range of ±15% of the recited numerical value; within a range of ±14% of the recited numerical value; within a range of ±13% of the recited numerical value; within a range of ±12% of the recited numerical value; within a range of ±11% of the recited numerical value; within a range of ±10% of the recited numerical value; within a range of ±9% of the recited numerical value; within a range of ±8% of the recited numerical value; within a range of ±7% of the recited numerical value; within a range of ±6% of the recited numerical value; within a range of ±5% of the recited numerical value; within a range of ±4% of the recited numerical value; within a range of ±3% of the recited numerical value; within a range of ±2% of the recited numerical value; or within a range of ±1% of the recited numerical value, indicating that the recited numerical value or range is somewhat more or less than the recited value or range.

[0264] Although the numerical ranges and parameters setting forth the broad scope of the present invention are approximations and approximate ranges, the numerical values set forth in the specific examples are reported as accurately as possible. However, all numerical values inherently contain certain errors necessarily resulting from the standard deviation associated with each test measurement.

[0265] In the description of the present invention (especially in the description of the following claims), the terms "a", "an", "the" and similar indicators are to be construed as encompassing both singular and plural referents, unless otherwise specified or the context clearly dictates a different meaning. The numerical ranges set forth herein are intended to be a shorthand way of referring individually to each numerical value within that range. Unless otherwise specified, each numerical value is to be treated as if it were individually recited herein. Unless otherwise specified or the context clearly dictates a different meaning, any method described herein can be performed in any suitable order. The use of any examples provided herein, or of exemplary language (such as "such as"), is for illustrative purposes only and is not intended to limit the scope of the invention as claimed. Terms described herein should not be construed as indicating essential elements of the invention not recited in the claims.

[0266] Grouping of other components of the invention disclosed herein or grouping of various embodiments of the invention should not be construed as limiting the invention. Members of each group may be individually described in this specification or in the claims, or may be described in this specification or in the claims in combination with other members of the group described herein or with other components. For convenience and / or reasons of patentability, it is contemplated that one or more members of a group may be added to another group or one or more members may be deleted from a group. When such additions or deletions are made, this specification includes groups configured to satisfy the description of all Markush groups recited in the appended claims.

[0267] Certain embodiments of the present invention are described herein, including those embodiments that the inventors recognize as the best mode for carrying out the present invention. It will be readily understood by those skilled in the art that, upon reading the foregoing detailed description, various modifications can be made to the embodiments described herein. The inventors anticipate that those skilled in the art can appropriately adopt such modifications, and it is also intended that the present invention be implemented in ways other than those specifically described herein. Accordingly, the present invention includes, to the extent possible within the scope of the applicable law, any modifications from the subject matter of the present invention described in the appended claims and any equivalents of the subject matter of the present invention. Further, unless otherwise stated or the context clearly indicates a different meaning, any combination of the foregoing components in any variation is also included in the present invention.

[0268] Furthermore, throughout this specification, various patents, publications, journal articles, and other documents (references cited herein) are cited. Each document cited herein is incorporated by reference in its entirety into this specification as if each such document were individually incorporated by reference herein for its cited teachings.

[0269] Finally, the embodiments of the present invention disclosed herein are to be construed as illustrative of the principles of the present invention. Other modifications may be adopted within the scope of the present invention. Thus, by way of example, but not limited thereto, another configuration of the present invention may be used in accordance with the teachings herein. Accordingly, the present invention is not strictly limited to the explicit disclosure and description herein.

[0270] The details described in this specification are examples only, and are intended only to illustrate the preferred embodiments of the present invention, to provide what is considered to be the most useful, and to enable easy understanding of the principles and conceptual aspects of the various embodiments of the present invention. In this regard, the details of the structure of the present invention are not described in more detail than the information necessary for a basic understanding of the present invention, and those skilled in the art will be able to easily understand how some forms of the present invention can be actually embodied by carefully reading the description of the present invention with reference to the drawings and / or examples.

[0271] The definitions and explanations used in this disclosure are intended to control future interpretations, unless clear and explicit changes are made in the examples, or the meaning of the terms becomes meaningless or substantially meaningless due to the meaning of the terms. If the definition of a term does not make sense or is substantially meaningless from the interpretation of the term, it is desired to quote the definition of the term from a dictionary known to those skilled in the art, such as Webster's Dictionary (3rd Edition) or Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

1. A nucleic acid containing the coding sequence for a recombinant receptor, When the recombinant receptor is expressed by a cell, Extracellular component containing a binding domain that binds to the B cell activator receptor (BAFF-R); The intracellular portion including the effector domain; and Transmembrane domain connecting the extracellular portion to the intracellular portion Includes, The aforementioned binding domain, The complementarity-determining region (CDR) H1 shown in SEQ ID NO: 2, CDRH2 shown in SEQ ID NO: 3, CDRH3 shown in SEQ ID NO: 4, CDRL1 shown in SEQ ID NO: 5, CDRL2 including sequence AAS, and CDRL3 shown in SEQ ID NO: 7, and a spacer. Nucleic acid.

2. The aforementioned binding domain, A humanized VH domain containing the sequence shown in SEQ ID NO: 32, SEQ ID NO: 8, or SEQ ID NO: 34, or a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 32, SEQ ID NO: 8, or SEQ ID NO: 34, and retaining the ability to bind to BAFF-R, A humanized VL domain comprising the sequence shown in SEQ ID NO: 35, SEQ ID NO: 9, or SEQ ID NO: 37, or a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 35, SEQ ID NO: 9, or SEQ ID NO: 37, and retaining the ability to bind to BAFF-R, The nucleic acid according to claim 1.

3. The aforementioned binding domain is scFv, The nucleic acid according to claim 1, wherein the scFv has the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6, or has a sequence that has at least 95% sequence identity with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and retains the ability to bind to BAFF-R.

4. The nucleic acid according to claim 1, characterized by any one of the following (i) to (iv): (i) The spacer comprises a hinge domain of IgG4, a CH3 domain of IgG4, a CH2 domain of IgG4, or a hinge domain of CD8a; (ii) The effector domain comprises all or part of the signaling domains of CD3ζ, CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C and / or B7-H3; (iii) The effector domain comprises all or part of the signaling domains of CD3ζ and 4-1BB; (iv) The transmembrane domain includes a transmembrane domain of CD28 or a transmembrane domain of CD8a.

5. The nucleic acid according to claim 1, further comprising, or further encoding, a regulatory mechanism selected from, a tag cassette, a cleaved EGFR (EGFRt) polypeptide, a cleaved Her2 (Her2t) polypeptide, a dihydrofolate reductase double mutant (DHFRdm), a herpes simplex virus thymidine kinase / ganciclovir (HSVTK / GCV) suicide gene system, and an inducible caspase suicide gene system.

6. The nucleic acid according to claim 1, further encoding a self-cleaving peptide comprising T2A, P2A, E2A, or F2A.

7. The nucleic acid according to claim 1, further comprising a promoter operably ligated to the coding sequence encoding the recombinant receptor, wherein the promoter is an EF1α promoter or an inducible promoter.

8. The nucleic acid according to claim 1, which is contained in the vector.

9. A cell genetically modified to contain the nucleic acid described in claim 1, further comprising a gene construct encoding a second recombinant receptor, A cell wherein the second recombinant receptor comprises an extracellular domain including a binding domain, and the binding domain contained in the second recombinant receptor binds to a cancer antigen, a B cell-specific ligand, or a small molecule.

10. A pharmaceutical composition for cancer treatment comprising the cells described in Claim 9 as an active ingredient, comprising a pharmaceutically acceptable carrier, and characterized in that it is used to treat a target by administering a therapeutically effective amount of the cells to the target.

11. The aforementioned cancer contains BAFF-R+ cells, or The pharmaceutical composition according to claim 10, wherein the cancer is B-cell carcinoma, mantle cell lymphoma (MCL), multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or diffuse large B-cell lymphoma (DLBCL).

12. The pharmaceutical composition according to claim 10, comprising autologous cells obtained from the subject.

13. The pharmaceutical composition according to claim 10, wherein the subject is a human.

14. The pharmaceutical composition according to claim 10, wherein the subject has fewer than 50,000 BAFF-R molecules per lesion cell.

15. A method for modifying the genes of cells, A step of contacting the cells with an anti-CD3 antibody or its antigen-binding fragment and / or an anti-CD28 antibody or its antigen-binding fragment; A step of introducing the nucleic acid described in claim 1 into the cells; and The process of culturing the aforementioned cells to obtain a sufficient quantity of multiple cells to administer immunotherapy to the target. Includes, A method wherein the cells are in vitro or ex vivo human cells.

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