Production and / or delivery of a multispecific binding agent

Abstract

Some embodiments of the methods and compositions provided herein relate to methods of preparing cells that express a bispecific T cell engager (BTCE), and to the use of such cells in certain therapies. In some embodiments, the cells are B cells or progenitor B cells.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the priority of U.S. Provisional Patent Application No. 63 / 498,202, entitled "Manufacture and / or Delivery of Multispecific Binding Agents", filed on April 25, 2023, and U.S. Provisional Patent Application No. 63 / 343,537, entitled "Manufacture and / or Delivery of Multispecific Binding Agents", filed on May 18, 2022, the entire disclosures of which are incorporated herein by reference.

[0002] Reference to the Sequence Listing This application is filed with an electronic sequence listing. The sequence listing is provided as a file of approximately 97,869 bytes created on May 8, 2023, with the file name SCRI416WOSEQLIST. The information set forth in this electronic sequence listing is incorporated herein by reference in its entirety.

[0003] Some embodiments of the methods and compositions provided herein relate to methods of preparing cells that express a bispecific T cell engager (BTCE) and the use of such cells in certain therapies. In some embodiments, the cells are B cells or progenitor B cells.

Background Art

[0004] Bispecific antibodies target two epitopes, which are often present on two antigens, and are described as "an important element in next-generation antibody therapies". See, e.g., Wang et al., Antibodies 8:43, 2019. Bispecific antibodies in various formats have been developed. The development of commercial bispecific antibodies has been reported to be "hindered" because "manufacture is extremely difficult".

Summary of the Invention

Means for Solving the Problems

[0005] Some embodiments of the methods and compositions provided herein are systems for modifying cells to express a bispecific T cell engager (BTCE), (a) a nuclease or a nucleic acid encoding the nuclease that can insert an expression cassette into a locus of the genome of the cell; and (b) a first polynucleotide encoding a template for homologous recombination repair (HDR) comprising an expression cassette encoding a bispecific T cell engager (BTCE) comprising a system.

[0006] Some embodiments further comprise the cell, which is a B cell or a pro-B cell. In some embodiments, the cell is selected from hematopoietic stem cells, human embryonic stem cells, induced pluripotent stem cells (iPSCs), naive B cells, memory B cells, plasmablasts, and plasma cells.

[0007] In some embodiments, the nuclease is a Cas nuclease, and the nuclease may be a Cas9 nuclease.

[0008] Some embodiments further comprise a second polynucleotide encoding a guide RNA (gRNA). In some embodiments, the second polynucleotide comprises a DNA sequence or an RNA sequence corresponding to the nucleotide sequence set forth in any one of SEQ ID NOs: 1-27.

[0009] In some embodiments, the locus comprises an endogenous gene that is highly expressed in B cells compared to cells other than B cells. In some embodiments, the locus comprises an endogenous gene that is inactive in B cells. In some embodiments, the locus is selected from the CCR5 gene, the JCHAIN gene, the IGHM locus (also known as E-mu; hg38 genome; chr14:105856225-105863200), the CD19 gene, and the IGHG1 gene.

[0010] In some embodiments, the BTCE comprises a first polypeptide capable of specifically binding to a T cell antigen and a second polypeptide capable of specifically binding to a tumor antigen. In some embodiments, the T cell antigen is CD3. In some embodiments, the tumor antigen is selected from CD33, CD19, CD326 (EpCAM), neural-glial antigen 2 (NG2), HER2, epidermal growth factor receptor (EGFR), CD66e, ephrin type-A receptor 2 (EphA2), CD21, FLT3, gp100, PDL1, and CD22. In some embodiments, the BTCE is AMG 330, blinatumomab, solitomab, or tebentafusp.

[0011] In some embodiments, the expression cassette further comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is the MND promoter, IgVH promoter, EF-1α promoter, or IgHG1 promoter.

[0012] In some embodiments, the expression cassette (i) an enhancer; (ii) a polynucleotide encoding a signal sequence; (iii) a polynucleotide comprising a 5’UTR derived from the IGHV gene or IGHG1 gene; (iv) a polynucleotide comprising a 3’UTR derived from the IGHV gene or IGHG1 gene; and / or (v) a ubiquitous chromatin opening element (UCOE) further comprises, the enhancer may be an Eμ enhancer or an SLC3A2 enhancer, the signal sequence may be an IgHV signal sequence or an IgHG1 signal sequence.

[0013] In some embodiments, the first polynucleotide further comprises a nucleic acid homologous to the locus, and the nucleic acid homologous to the locus may comprise consecutive nucleotides about 200 to 1500 nucleotides in length.

[0014] In some embodiments, the first polynucleotide is contained in a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector. In some embodiments, the AAV vector is an AAV6 vector.

[0015] In some embodiments, the cell is a mammalian cell and may be a human cell. In some embodiments, the cell is an ex vivo cell. In some embodiments, the cell is an autologous cell obtained from a subject. In some embodiments, the cell is a cell of the same species as the subject. In some embodiments, the cell lacks expression of an endogenous protein to which the BTCE can specifically bind.

[0016] Some embodiments are methods of modifying a cell to express a bispecific T cell engager (BTCE), (a) obtaining the system according to any one of claims 2 to 28; and (b) obtaining a modified cell by introducing into the cell a nuclease or a nucleic acid encoding the nuclease contained in the system and the first polynucleotide contained in the system comprising a method comprising.

[0017] Some embodiments further comprise, prior to step (b), a step of activating the cell, which may comprise contacting the cell with oligomerized CD40 ligand (CD40L), CpG and / or IL-21.

[0018] In some embodiments, step (b) includes contacting the cell with a ribonucleoprotein (RNP) comprising the nuclease and the gRNA.

[0019] Some embodiments further include (c) inducing differentiation of the cell.

[0020] Some embodiments include a cell modified by any one of the methods described above.

[0021] Some embodiments include a cell in which the genomic locus has been genetically modified to express a bispecific T cell engager (BTCE).

[0022] In some embodiments, the cell is a B cell or a pro-B cell. In some embodiments, the cell is selected from hematopoietic stem cells, human embryonic stem cells, induced pluripotent stem cells (iPSCs), naive B cells, memory B cells, plasmablasts, and plasma cells.

[0023] In some embodiments, the locus includes an endogenous gene that is highly expressed in B cells compared to cells other than B cells. In some embodiments, the locus includes an endogenous gene that is inactive in B cells. In some embodiments, the locus is selected from the CCR5 gene, the JCHAIN gene, the IGHM locus (also known as E-mu; hg38 genome; chr14:105856225-105863200), the CD19 gene, and the IGHG1 gene.

[0024] In some embodiments, the BTCE comprises a first polypeptide capable of specifically binding to a T cell antigen and a second polypeptide capable of specifically binding to a tumor antigen. In some embodiments, the T cell antigen is CD3. In some embodiments, the tumor antigen is selected from CD33, CD19, CD326 (EpCAM), neural-glial antigen 2 (NG2), HER2, epidermal growth factor receptor (EGFR), CD66e, ephrin type-A receptor 2 (EphA2), CD21, FLT3, gp100, PDL1, and CD22. In some embodiments, the BTCE is AMG 330, blinatumomab, solitomab, or tebentafusp.

[0025] In some embodiments, the genomic locus is modified by inserting an expression cassette comprising a first nucleic acid encoding the BTCE.

[0026] In some embodiments, the first nucleic acid is operably linked to an endogenous promoter of the locus. In some embodiments, the expression cassette further comprises a promoter operably linked to the first nucleic acid. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is an MND promoter, an IgVH promoter, an EF-1α promoter, or an IgHG1 promoter.

[0027] In some embodiments, the expression cassette comprises (i) an enhancer; (ii) a polynucleotide encoding a signal sequence; (iii) a polynucleotide comprising a 5’UTR derived from an IGHV gene or an IGHG1 gene; (iv) a polynucleotide comprising a 3’UTR derived from an IGHV gene or an IGHG1 gene; and / or (v) a ubiquitous chromatin opening element (UCOE) further comprising the enhancer may be an Eμ enhancer or an SLC3A2 enhancer, the signal sequence may be an IgHV signal sequence or an IgHG1 signal sequence.

[0028] In some embodiments, the cell is a mammalian cell and may be a human cell. In some embodiments, the cell is an ex vivo cell. In some embodiments, the cell is an autologous cell obtained from a subject. In some embodiments, the cell is a cell of the same species as the subject.

[0029] Some embodiments include a pharmaceutical composition comprising any one of the cells.

[0030] Some embodiments include a method of treating, alleviating or suppressing a disorder in a subject, the method comprising administering to the subject a cell according to any one of claims 33 - 54. In some embodiments, the cell is administered by a single administration and may be administered by a single bolus administration. In some embodiments, the disorder includes cancer. In some embodiments, the BTCE can specifically bind to a tumor antigen expressed by the cancer. In some embodiments, the cancer is selected from solid tumors and leukemias. In some embodiments, the cancer is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), breast cancer, gastric cancer, malignant melanoma, colorectal cancer, head and neck cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, and pancreatic cancer. In some embodiments, the subject is human.

[0031] Some embodiments include the use of any one of the cells as a pharmaceutical, for example, for the treatment, alleviation or suppression of a disorder or disease in a subject.

[0032] Some embodiments include the use of any one of the cells in the preparation of a medicament for treating, alleviating or suppressing a disorder in a subject.

[0033] In some embodiments, the cells are administered by single administration, and may be administered by a single bolus administration. In some embodiments, the disorder includes cancer. In some embodiments, the BTCE can specifically bind to a tumor antigen expressed by the cancer. In some embodiments, the cancer is selected from solid tumors and leukemia. In some embodiments, the cancer is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), breast cancer, gastric cancer, malignant melanoma, colorectal cancer, colorectal cancer, head and neck cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, and pancreatic cancer. In some embodiments, the subject is human.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0091] Some embodiments of the methods and compositions provided herein relate to methods for preparing cells that express a bispecific T cell engager (BTCE) and the use of such cells in certain therapies. In some embodiments, the cells are B cells or precursor B cells. As used herein, "BTCE" and "BiTE" are used interchangeably and mean bispecific T cell engager.

[0092] Immunotherapies that recruit cytotoxic T cells to kill cancer cells (such as BTCE) have played a major role in improving the survival rate of patients with B cell acute lymphoblastic leukemia (B-ALL). 1-3 Blinatumomab, marketed under the name "Blincyto," is a CD3 / CD19 BTCE approved by the FDA in 2014 for the treatment of relapsed / refractory B-ALL. 4,5 Currently, blinatumomab is used in the treatment of B-ALL in various forms, particularly as a first-line therapy as a bridging treatment for transplantation, consolidation therapy, and a low-toxicity alternative to chemotherapy regimens. 6 However, blinatumomab and other BTCEs have a short half-life and thus require high-dose continuous infusions over a long period. The half-life of blinatumomab is 2.11 hours. 7To maintain therapeutic concentrations and avoid complications associated with administration, blinatumomab is administered for the treatment of minimal residual disease-positive B-ALL by performing four cycles of continuous daily infusions for 28 days followed by a 14-day drug holiday. 5 However, such an intensive dosing schedule is burdensome for patients, especially for those who have difficulty attending the hospital, which is a significant burden. 8,9 。

[0093] Several methods have been developed to directly extend the half-life of biologics 10 , and such formulations include those that form a complex with a small molecule, a crystallizable fragment domain, or an albumin-binding motif. Complexes with an extended half-life have also been applied to BTCE 11 , but it remains unclear whether such BTCE fusions are effective or can avoid the need for multiple consecutive high-dose infusions. Another way to mitigate problems related to the half-life of biologics is to use long-lived recombinant cell therapy to deliver proteins. The use of engineered plasma cells (ePCs) has been investigated, and ePCs are one of the cell therapy modalities used in proof-of-concept studies for the stable production of biologics such as 12-16 anti-pathogen antibodies, 17 immune checkpoint inhibitors, 18 cytokines, 19 protein deficiency-related proteins, etc. Plasma cells are one of the cell therapy modalities with an amazing lifespan 20 (estimated to be 11 to 200 years 21 ) and high secretory capacity (secreting up to 10,000 IgG molecules per second 22,23 ), making them uniquely suitable for the long-term delivery of biologics. Furthermore, ex vivo-generated ePCs closely resemble endogenous plasma cells and can stably secrete therapeutically significant concentrations of IgG in hIL6 humanized mice for over a year 18 . As disclosed herein, plasma cells 24,25 and ePCs 18 are thought to be where B-ALL progenitor cells reside.26 Since ePCs selectively localize to the microenvironment of bone marrow and other tissues, it is predicted that ePCs have the advantage of being able to locally deliver BTCE to the tumor site in B-ALL.

[0094] Some embodiments disclosed herein include homologous recombination repair methods for generating ePCs that produce large amounts of BTCE. From the findings disclosed herein, it has been shown that ePCs secreting BTCE can promote the killing of cell lines, primary cells, and patient-derived B-ALL xenografts by T cells. Some embodiments include the use of ePCs for the stable delivery of blinatumomab to humans in B-ALL; and the use of the ePC platform for delivering biological agents when the half-life is short or when local delivery can reduce off-target adverse effects.

[0095] Depending on its type, BTCE may be rapidly eliminated in a subject, so the half-life of BTCE in a subject may be short. BTCE has conventionally been administered to a subject by multiple administrations over a long period and / or continuous intravenous infusion to maintain its therapeutic concentration (see, for example, Zhu M, et al., (2016) Clin Pharmacokinet. 55:1271-88; and Singh K, et al., (2021) Journal for ImmunoTherapy of Cancer 9:e003679. doi: 10.1136 / jitc-2021-003679). Therefore, the treatment related to BTCE needs to be improved.

[0096] Bispecific antibodies target two epitopes, which are often present on two antigens, and are described as "an important element in next-generation antibody therapy" (see, for example, Wang et al., Antibodies 8:43, 2019). Bispecific antibodies in various formats have been developed. The development of commercial bispecific antibodies has been reported to be "difficult" due to "very difficult manufacturing".

[0097] Some embodiments provided herein relate to the insight that certain challenges associated with the production and / or delivery of bispecific antibodies and / or other multispecific binding agents (multispecific binding agents in various formats) can be overcome by the development of transplantable human cell populations (particularly, transplantable human plasma cell populations) that can express bispecific antibodies and / or other multispecific binding agents.

[0098] Bispecific antibody agents in various formats have been developed and are specifically classified into five types of structures: (i) bispecific IgG (BsIgG), (ii) IgG with an additional antigen-binding portion added, (iii) BsAb fragments, (iv) bispecific fusion proteins, and (v) BsAb complexes. See, for example, Spiess et al. (2015) Mol. Immunol. 67:95-106. As an example of a bispecific antibody format, BTCE can be mentioned, which is a scFv targeting CD3 on T cells (e.g., expressed as a single polypeptide) linked to a scFv targeting an antigen of interest (e.g., a surface antigen present on tumor cells). See, for example, Mack et al. (1995) Proc. Natl. Acad. Sci USA 92:7021. Blinatumomab is an example of a BTCE antibody and has achieved remarkable efficacy in the treatment of B-cell malignancies. See, for example, Zhou et al., (2021) Biomarker Res 9:38. There is a need to promptly provide an improved method for BTCE treatment, particularly for solid tumors that always have poor responses to BTCE treatment. Specific challenges related to achieving effective BTCE treatment include antigen loss and immunosuppressive factors, such as upregulation of immune checkpoints.

[0099] Some embodiments provided herein relate to improved methods for delivering multispecific binder therapy by administering to a subject in need thereof a recombinant cell population that expresses a multispecific binder and / or a recombinant cell population that expresses an antigen on a cell and / or tissue that benefits from targeting of T cells, said therapy specifically including BTCE therapy, said recombinant cell population being, for example, a recombinant mammalian cell population, particularly a recombinant human cell population, said multispecific binder being, for example, a BTCE agent of interest, and said subject being, for example, a subject having cancer, such as a tumor that expresses an antigen targeted by said multispecific binder, or a subject having a tumor that has been found to express such an antigen.

[0100] As described herein, a recombinant plasma cell population can effectively deliver a multispecific binder, such as a BTCE agent, to a subject, such as a mammal, for example a human.

[0101] Some embodiments provided herein include aspects for providing a recombinant plasma cell population as described in U.S. Patent Publication No. 2018 / 0282692, which is hereby incorporated by reference in its entirety.

[0102] In some embodiments, a mammalian cell population (e.g., a human cell population) may be recombined using techniques such as those described in U.S. Patent Publication Nos. 2016 / 0289637, 2019 / 0352614, 20210198344, and WO 2022 / 006309, each of which is hereby expressly incorporated by reference in its entirety, to express a multispecific binder (e.g., a BTCE agent).

[0103] In some embodiments, a construct encoding a multispecific binding agent (e.g., a BTCE agent), and / or a recombinant cell population that expresses a multispecific binding agent or is capable of expressing a multispecific binding agent, when administered and / or transplanted into a recipient mammal, may be characterized, for example, with respect to the expression (e.g., long-term expression) of the multispecific binding agent in the mammal when evaluated in the mouse model that has received the transplantation described herein. Further, in some embodiments, such a construct and / or cell population may be characterized with respect to a decrease in tumor size and / or a decrease in one or more markers of health impairment in the mammal when evaluated in the mouse model that has received the transplantation described herein.

[0104] Some embodiments of the methods and compositions provided herein include a transplantable recombinant mammalian plasmacyte population that expresses a BTCE agent. In some embodiments, the cell population is characterized with respect to the expression of the BTCE agent in the mammal when administered to the mammal. In some embodiments, the cell population is characterized with respect to tumor suppression or treatment success and / or improvement in the health status of the mammal when administered to a mammal having a tumor that expresses the antigen target of the BTCE agent, as compared to in the absence of such administration. Some embodiments of the methods and compositions provided herein include a method of suppressing or treating a tumor that expresses an antigen, the method comprising administering to a mammal having a tumor that expresses an antigen any one of the cell populations. Some embodiments of the methods and compositions provided herein include a construct encoding a BTCE agent, the construct being for expressing a BTCE agent from a recombinant mammalian cell into which the construct has been introduced.

[0105] Definitions As used herein, "B cell" or "B lymphocyte" has its ordinary and general meaning as will be apparent upon reference to this specification, and includes, for example, but is not limited to, a type of white blood cell that is a lymphocyte subtype. B cells, unlike lymphocytes such as T cells and natural killer cells, express B cell receptors on their cell membranes. The B cell receptor enables B cells to bind to specific antigens, thereby initiating an antibody production response. B cells develop from hematopoietic stem cells. As described herein, B cells include progenitor B cells, stem cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, T1 B cells, T2 B cells, marginal zone B cells, mature B cells, naive B cells, activated B cells derived from any B cell population as a starting material, plasmablast (short-lived) cells, GC B cells, memory B cells, long-lived plasma cells and / or short-lived plasma cells, and / or any mixture or combination thereof as appropriate.

[0106] As used herein, the term "precursor B cell" includes cells from which B cells are derived. B cells, like T cells, are lymphoid cells that originate from the bone marrow and may be present in the bone marrow until they mature. The B cells described in the embodiments herein include stem cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, T1 B cells, T2 B cells, marginal zone B cells, mature B cells, naive B cells, plasmablast (short-lived) cells, GC B cells, memory B cells, plasmablast cells and / or long-lived plasma cells. In some embodiments of plasma cells that express molecules such as macromolecules, proteins, peptides, etc., these plasma cells are derived from B cells. In some embodiments, the B cell is a memory B cell. In some embodiments, the B cell is a stem cell, an early pro-B cell, a late pro-B cell, a large pre-B cell, a small pre-B cell, an immature B cell, a T1 B cell, a T2 B cell, a marginal zone B cell, a mature B cell, a naive B cell, a plasmablast (short-lived) cell, a GC B cell, a memory B cell, a plasmablast cell or a long-lived plasma cell. In some embodiments, the B cell includes a precursor B cell, and examples of such precursor B cells include hematopoietic stem cells (HSCs), multipotent progenitor cells (MPPs), common lymphoid progenitor cells (CLPs), naive B cells, GC B cells, plasmablasts, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, T1 B cells, T2 B cells, marginal zone B cells, mature B cells and / or memory B cells. In some embodiments, the macromolecule is a prodrug.

[0107] As used herein, "memory B cell" has its ordinary and common meaning in light of this specification. For example, it is a subtype of B cells formed in the germinal center after primary infection, and when re-infected, it plays an important role in inducing a more potent and accelerated antibody-mediated immune response, but is not limited thereto. B lymphocytes form memory cells, which remember pathogens and produce antibodies when re-infected with the same pathogen. In some embodiments of plasma cells expressing molecules such as macromolecules, proteins, peptides, etc., these plasma cells are derived from B cells. In some embodiments, the B cells are memory B cells. In some embodiments, the macromolecule is a prodrug.

[0108] As used herein, "naive B cell" has its ordinary and common meaning in light of this specification. For example, it includes B cells that have not been exposed to antigens, but is not limited thereto. Naive B cells become memory B cells when exposed to antigens. In some embodiments of plasma cells expressing molecules such as macromolecules, proteins, peptides, etc., these plasma cells are derived from B cells. In some embodiments, the B cells are memory B cells. In some embodiments, the macromolecule is a prodrug. As used herein, "peripheral blood mononuclear cells (PBMC)" are peripheral blood cells having round nuclei. Peripheral blood mononuclear cells consist of lymphocytes (T cells, B cells, NK cells) and monocytes. On the other hand, red blood cells and platelets do not have nuclei, and neutrophils, basophils and eosinophils have segmented nuclei. In the embodiments described herein, the B cells are derived from a subject or are peripheral blood mononuclear cells of the same species as the subject. In some embodiments, the B cells are human B cells derived from blood.

[0109] As used herein, "plasma cell" is also referred to as plasmacyte, plasma cell or effector B cell. "Plasma cell" has its ordinary and common meaning in light of this specification. For example, it includes white blood cells transported by the plasma and lymphatic systems and secreting antibodies, but is not limited thereto.

[0110] As used herein, a "precursor plasma cell" may develop as an immature plasma cell. The most immature blood cells of the plasma cell lineage are called plasmablasts, which can differentiate into fully differentiated mature plasma cells. Plasmablasts can secrete more antibodies than B cells, but the amount is less than that of plasma cells. In some embodiments, a method of producing a plasma cell that expresses a molecule is provided. In some embodiments, the plasma cell is a precursor plasma cell. In some embodiments, the precursor plasma cell is a plasmablast.

[0111] "Genome editing", as used herein, has its ordinary and general meaning, and includes, for example, but is not limited to, processes including genetic engineering methods of inserting, deleting or substituting DNA in the genome of an organism. Editing a gene is also known as gene editing. In some embodiments described herein, there is provided a method of producing a plasma cell or a progenitor cell thereof that expresses a molecule such as a macromolecule, the method comprising performing at least one genome editing on a B cell or a progenitor cell thereof. The method of genome editing may include, but is not limited to, insertion, deletion or substitution of a nucleic acid into the genome of a cell. In some embodiments, a nuclease is used to carry out this method. In some embodiments, the nuclease is an artificial nuclease. In some embodiments, the method includes a step of inducing a double-strand break and performing repair by non-homologous end joining (NHEJ) or homologous recombination (HR). In some embodiments, the genome editing step is carried out by introduction of a single-stranded nucleic acid. In some embodiments, the at least one genome editing further includes cycling the cell cycle of the B cell to perform homologous recombination of a single-stranded DNA oligonucleotide or a candidate locus of a recombinant adeno-associated virus. In some embodiments, the genome editing of the B cell for expressing a protein is carried out without viral integration. In some embodiments, a second genome editing is performed to cleave a specific region. In some embodiments, a third genome editing is performed to express a growth promoting factor that can be activated by a drug. In some embodiments described herein, the genome editing is carried out by targeted homologous recombination editing with a non-pathogenic AAV.

[0112] Genome editing can also be performed by transfection of RNA and proteins. For example, the CRISPR / Cas system can be partially modified and used for genome editing. In this technique, it is necessary to deliver Cas nuclease complexed with synthetic guide RNA (gRNA) into cells, by which the cell genome can be cleaved at specific positions to remove existing genes and / or add new genes to existing genes. CRISPR / Cas and related programmable endonuclease systems have rapidly developed as important genome editing tools in biomedical research, and their usability in gene disruption and / or gene targeting has been demonstrated in various cultured cell lines and model organism systems. In some of the embodiments of the CRISPR / Cas system described herein, the Cas nuclease includes Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 or Cas9.

[0113] The basic components of the CRISPR / Cas system include a target gene, a protospacer adjacent motif (PAM), guide RNA and Cas endonuclease. One of the important points when performing genome editing using CRISPR / Cas is that a system capable of efficiently delivering guide RNA into various types of cells is required. Such a system may include, for example, delivering guide RNA prepared in vitro as a nucleic acid (guide RNA prepared by in vitro transcription or chemical synthesis). In some embodiments, the nucleic acid can be rendered nuclease-resistant by incorporating modified bases.

[0114] The CRISPR-Cas system is classified into two types. In class 1 CRISPR-Cas systems, a complex consisting of multiple Cas proteins degrades foreign nucleic acids. In class 2 CRISPR-Cas systems, a single large Cas protein degrades foreign nucleic acids. There are 93 cas genes classified into 35 families. Eleven of the 35 families form the cas core, which includes the CAS1-CAS9 protein families. As described herein, Cas includes Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, or Cas9.

[0115] Gene editing may be performed by a new gene editing platform that does not use nucleases. So far, new AAV families have been isolated from human hematopoietic stem cells. These non-pathogenic AAVs naturally exist in the bodies of healthy individuals and may have unique gene editing and gene transfer properties. This technique is also called AAV-mediated targeted homologous recombination editing (AmENDR TM ). This method is homologous recombination that utilizes the natural biological mechanism for cells to perform highly accurate DNA repair.

[0116] Targeted homologous recombination editing by AAV starts with designing homologous sequences called "arms" that are specific to a particular region of the genome. When AAV is administered to cells, it can permanently rewrite the DNA. In some embodiments described herein, the gene editing is performed by targeted homologous recombination editing with non-pathogenic AAV. The identification of new AAV genomes has been reported by Smith et al. (Mol Ther. 2014 Sep; 22(9): 1625-1634; this document is incorporated herein by reference in its entirety). The new AAV reported by Smith et al. is a new type of gene vector that can be used for genome engineering of hematopoietic stem cells. Furthermore, these vectors may greatly expand the gene delivery ability to target tissues and cells and can avoid existing immunity to widely spread AAV2, so there is a possibility that genes can be delivered to cells with difficult gene transfer. In some embodiments, the gene editing is performed using non-pathogenic AAV that naturally exists in hematopoietic cells, particularly by targeted homologous recombination editing using non-pathogenic AAV reported by Smith et al.

[0117] "Artificial nuclease" has its ordinary and general meaning in the context of this specification. For example, it includes, but is not limited to, an enzyme artificially synthesized as a hybrid enzyme that can specifically recognize a DNA sequence and efficiently edit the genome by introducing a double-strand break. There are four families, including, but not limited to, meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR-Cas system.

[0118] As used herein, "meganuclease" has its ordinary and general meaning and includes, but is not limited to, for example, an endodeoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence of 12 to 40 base pairs). In some embodiments of the method of producing a plasma cell or a progenitor cell thereof that expresses a molecule such as a macromolecule, the method comprises: (a) isolating B cells; (b) developing the B cells; (c) performing a first genome editing of the B cells without virus integration to express a protein; (d) proliferating the B cells; and optionally, after step (c) or (d), (e) differentiating the B cells, whereby a plasma cell expressing a protein is obtained. In some embodiments, the first genome editing is performed by transfection of RNA and protein. In some embodiments, the nuclease is a meganuclease.

[0119] "Zinc finger nuclease (ZFN)" has its general and ordinary meaning in the context of this specification, and examples include, but are not limited to, artificial restriction enzymes obtained by fusing a zinc finger DNA-binding domain to a DNA cleavage domain. The zinc finger domain can be engineered to target a specific DNA sequence of interest, thereby enabling the zinc finger nuclease to target unique sequences within complex genomes. In some embodiments of the method of generating a plasma cell that expresses a molecule such as a macromolecule, the method comprises: (a) isolating a B cell; (b) developing the B cell; (c) performing a first genome editing of the B cell without viral integration to express a protein; (d) proliferating the B cell; and optionally, after step (c) or (d), (e) differentiating the B cell, whereby a plasma cell that expresses a protein is obtained. In some embodiments, the first genome editing is performed by transfection of RNA and protein. In some embodiments, the nuclease is a zinc finger nuclease.

[0120] "Transcription activator-like effector nuclease (TALEN)" has its ordinary and common meaning in the context of this specification, and examples include, but are not limited to, restriction enzymes that can be recombined to cleave a specific DNA sequence or a specific DNA site. TALEN is produced by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease that cleaves DNA strands). Since transcription activator-like effector (TALE) can be recombined to bind to a desired DNA sequence, it can cleave DNA at a specific position by combining with a nuclease. Therefore, this restriction enzyme can be introduced into cells for use in genome editing or in situ genome editing, and this technology is known as a genome editing technology using artificial nucleases. The use of TALEN is known to those skilled in the art. In some embodiments described herein, there is provided a method for producing a plasma cell or a progenitor cell thereof that expresses a molecule such as a macromolecule, the method comprising performing at least one genome editing on a B cell or a progenitor cell thereof. The genome editing method may include, but is not limited to, insertion, deletion, or substitution of nucleic acids into the genome of the cell. In some embodiments, a nuclease is used to carry out this method. In some embodiments, the nuclease is an artificial nuclease. In some embodiments, the method includes a step of inducing double-strand breaks and performing repair by non-homologous end joining (NHEJ) or homologous recombination (HR). In some embodiments, the method includes a first genome editing or genome editing. In some embodiments, the first genome editing includes a step of delivering a nuclease that targets at least one locus of a B cell. In some embodiments, the at least one locus includes JCHAIN, IGKC, IGMC, PON3, PRG2, FKBP11, SDC1, SLPI, DERL3, EDEM1, LY6C2, CRELD2, REXO2, PDIA4, PRDM1, CARD11, CCR5, or SDF2L1.In some embodiments, the nuclease is a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), a homing endonuclease (HE), a fusion protein of TALEN and HE (megaTAL), or a complex of a synthetic guide RNA targeting clustered regularly interspersed short palindromic repeat (CRISPR) DNA and a Cas nuclease. In some embodiments, the Cas nuclease includes Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, or Cas9. In some embodiments, the first genome editing includes the step of transducing B cells using a recombinant adeno-associated virus vector that functions as a donor template for homologous recombination at a candidate locus. In some embodiments, the recombinant adeno-associated virus vector is single-stranded, double-stranded, or self-complementary.

[0121] As used herein, "differentiation" means the change from one type of cell to another type of cell. B cells can be differentiated, but are not limited to, by exposure to T cell-derived cytokines bound to B cell cytokine receptors. For example, CD40L can function as a stimulatory factor necessary for the activation of B cells by binding to CD40, a B cell surface receptor, and can also affect differentiation thereby. In another method described herein, B cells are induced to differentiate in a culture system consisting of three steps including an activation / proliferation step, a step of inducing differentiation into plasmablasts, and a step of inducing differentiation into plasma cells. In some embodiments, the activation / proliferation step is performed in the presence of any combination of MCD40L (CD40 trimer), CpG, IL-2, IL-10, and / or IL-15. In some embodiments, the step of inducing differentiation into plasmablasts is performed in the presence of any combination of IL-2, IL-6, IL-10, and / or IL-15. In some embodiments, the step of inducing differentiation into plasma cells is performed in the presence of any combination of IL-6, IL-15, APRIL, and / or IFNα.

[0122] Specific methods Some embodiments of the methods and compositions provided herein include methods of modifying cells to express a bispecific T cell engager (BTCE). Examples of methods useful in certain embodiments provided herein are disclosed in U.S. Patent Publication No. 2018 / 0282692, which is incorporated herein by reference in its entirety. In some embodiments, the cells are B cells or progenitor B cells. In some embodiments, the B cells or progenitor B cells are hematopoietic stem cells, human embryonic stem cells, induced pluripotent stem cells (iPSCs), naive B cells, memory B cells, plasmablasts or plasma cells. In some of such embodiments, the cells are primary B cells or primary progenitor B cells.

[0123] In some embodiments, cells are genetically modified to express BTCE. For example, the genome of the cells can be modified by inserting an expression cassette encoding BTCE. In some embodiments, the insertion may occur at a random position in the genome. In some embodiments, the insertion is targeted to a selected position in the genome. Some embodiments include the use of homologous recombination repair (HDR). Some such embodiments may include an endonuclease capable of inserting an expression cassette at a selected position in the genome. Examples of such endonucleases include zinc finger nucleases, transcription activator-like effector nucleases (TALENs), homing endonucleases (HEs), and combinations of TALEN-HE proteins (megaTALs) and the CRISPR / Cas system. In some embodiments, the CRISPR / Cas system includes a Cas nuclease and a guide RNA (gRNA) configured to target a specific locus in the genome, and examples of Cas nucleases include Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, or Cas9. In some embodiments, the Cas nuclease is Cas9.

[0124] In some embodiments, the expression cassette includes a nucleic acid encoding BTCE. In some embodiments, the expression cassette further includes a promoter operably linked to the nucleic acid encoding BTCE. Examples of promoters include the MND promoter, the EF-1α promoter, or the IgHG1 promoter. In some embodiments, the expression cassette further includes enhancer elements and / or a polyadenylation sequence.

[0125] Some embodiments of the methods provided herein for modifying cells to express BTCE (a) Obtaining a first polynucleotide encoding a template for homologous recombination repair (HDR) comprising a nuclease capable of inserting an expression cassette into the genomic locus of a B cell or a nucleic acid encoding the nuclease and an expression cassette encoding a bispecific T cell engager (BTCE); and (b) Obtaining a modified cell by introducing the nuclease or the nucleic acid encoding the nuclease and the first polynucleotide into the B cell comprising. In some embodiments, the B cells are activated prior to step (b). In some such embodiments, this activation may include contacting the B cells with oligomerized CD40 ligand (CD40L), CpG and / or IL-21, and examples of oligomerized CD40L include oligomerized CD40L comprising two linked CD40L trimers. Some such embodiments may include maintaining the B cells in a culture medium containing oligomerized CD40L for a first period. In some embodiments, the first period is 1 to 30 days, 1 to 25 days, 5 to 20 days, or 7 to 20 days. In some embodiments, step (b) includes contacting the B cells with a ribonucleoprotein (RNP) comprising the nuclease and a gRNA.

[0126] Some embodiments further include (c) a step of inducing differentiation of said cells. In some embodiments, step (c) includes a step of inducing differentiation into plasmablasts and / or a step of inducing differentiation into plasma cells. In some of such embodiments, said differentiation induction is carried out in the presence of any combination of oligomerized CD40L, CpG oligodeoxynucleotides, IL-2, IL-10 and IL-15. In some of such embodiments, the step of inducing differentiation into plasmablasts is carried out in the presence of any combination of IL-2, IL-6, IL-10 and IL-15. In some of such embodiments, the step of inducing differentiation into plasma cells is carried out in the presence of any combination of IL-6, IL-15, APRIL and IFNα. In some embodiments, step (c) includes (i) a step of contacting said edited cells with IL-2, IL-6, IL-10 and IL-15 over a second period. In some embodiments, the second period is 1 to 30 days, 1 to 20 days, 1 to 15 days, 1 to 10 days, 1 to 5 days, or 1 to 3 days. Some embodiments further include (ii) a step of contacting said edited cells with IL-6, IL-15 and IFNα over a third period. In some embodiments, the third period is 1 to 30 days, 1 to 20 days, 1 to 15 days, 1 to 10 days, 1 to 5 days, or 1 to 3 days. In some embodiments, step (ii) is carried out after step (i). Examples of methods useful in embodiments of the methods and compositions provided herein are disclosed in Jourdan M., et al. (2009) Blood 114: 5173-5181 (this document is incorporated herein by reference in its entirety).

[0127] In some embodiments, said nuclease is a Cas nuclease, for example, Cas9 nuclease. Some embodiments further include a second polynucleotide encoding a guide RNA (gRNA). In some embodiments, the second polynucleotide includes a DNA sequence or an RNA sequence corresponding to the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 27.

[0128] In some embodiments, the locus comprises an endogenous gene that is highly expressed in B cells as compared to cells other than B cells. In some embodiments, the locus comprises an endogenous gene that is inactive in B cells. In some embodiments, the locus is selected from the CCR5 gene, the JCHAIN gene, the IGHM locus (also known as E-mu; hg38 genome; chr14:105856225-105863200), the CD19 gene, and the IGHG1 gene.

[0129] In some embodiments, the BTCE comprises a first polypeptide that can specifically bind to a T cell antigen and a second polypeptide that can specifically bind to a tumor antigen. In some embodiments, the T cell antigen is CD3. In some embodiments, the tumor antigen is selected from CD33, CD19, CD326 (EpCAM), neural-glial cell antigen 2 (NG2), HER2, epidermal growth factor receptor (EGFR), CD66e, ephrin type-A receptor 2 (EphA2), CD21, FLT3, gp100, PDL1, and CD22. In some embodiments, the BTCE is AMG 330, blinatumomab, solitomab, or tebentafusp.

[0130] In some embodiments, the expression cassette further comprises a promoter operably linked to the nucleic acid encoding the BTCE. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is the MND promoter, the IgVH promoter, the EF-1α promoter, or the IgHG1 promoter. In some embodiments, the expression cassette (i) an enhancer; (ii) a polynucleotide encoding a signal sequence; (iii) a polynucleotide comprising a 5’UTR derived from the IGHV gene or the IGHG1 gene; (iv) A polynucleotide comprising a 3’UTR derived from the IGHV gene or the IGHG1 gene; and / or (v) A ubiquitous chromatin opening element (UCOE) further comprising, the enhancer may be an Eμ enhancer or an SLC3A2 enhancer, the signal sequence may be an IgHV signal sequence or an IgHG1 signal sequence. In some embodiments, the first polynucleotide further comprises a nucleic acid homologous to the locus, and the nucleic acid homologous to the locus may comprise continuous nucleotides about 200 to 1500 nucleotides in length.

[0131] In some embodiments, the first polynucleotide is comprised in a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector. In some embodiments, the AAV vector is an AAV6 vector.

[0132] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an ex vivo cell. In some embodiments, the cell is an in vitro cell. In some embodiments, the cell is an autologous cell obtained from a subject. In some embodiments, the cell is a cell of the same species as the subject. In some embodiments, the cell lacks the expression of an endogenous protein to which the BTCE can specifically bind. For example, the cell can be modified to lack a ligand that can bind to the BTCE. Some such embodiments may be useful for suppressing the killing of the cell by suppressing or preventing the binding of the BTCE to the cell.

[0133] Specific compositions and systems Some embodiments of the methods and compositions provided herein relate to systems for modifying cells to express BTCE. Some such embodiments include (a) a nuclease capable of inserting an expression cassette into a genomic locus of a cell or a nucleic acid encoding the nuclease; and (b) a first polynucleotide encoding a template for homologous recombination repair (HDR) comprising an expression cassette encoding a bispecific T cell engager (BTCE). Including.

[0134] Some embodiments further include the cell, which is a B cell or a pro-B cell. In some embodiments, the cell is selected from hematopoietic stem cells, human embryonic stem cells, induced pluripotent stem cells (iPSCs), naive B cells, memory B cells, plasmablasts, and plasma cells.

[0135] In some embodiments, the nuclease is a Cas nuclease, and the nuclease may be a Cas9 nuclease. Some embodiments further include a second polynucleotide encoding a guide RNA (gRNA). In some embodiments, the second polynucleotide comprises a DNA sequence or an RNA sequence corresponding to the nucleotide sequence shown in any one of SEQ ID NOs: 1-27.

[0136] In some embodiments, the locus comprises an endogenous gene that is highly expressed in B cells compared to cells other than B cells. In some embodiments, the locus comprises an endogenous gene that is inactive in B cells. In some embodiments, the locus is selected from the CCR5 gene, the JCHAIN gene, the IGHM locus (E-mu; hg38 genome; also known as chr14: 105856225-105863200), the CD19 gene, and the IGHG1 gene.

[0137] In some embodiments, the BTCE comprises a first polypeptide capable of specifically binding to a T cell antigen and a second polypeptide capable of specifically binding to a tumor antigen. In some embodiments, the T cell antigen is CD3. In some embodiments, the tumor antigen is selected from CD33, CD19, CD326 (EpCAM), neural-glial antigen 2 (NG2), HER2, epidermal growth factor receptor (EGFR), CD66e, ephrin type-A receptor 2 (EphA2), CD21, FLT3, gp100, PDL1, and CD22. In some embodiments, the BTCE is AMG 330, blinatumomab, solitomab, or tebentafusp.

[0138] In some embodiments, the expression cassette further comprises a promoter operably linked to the nucleic acid encoding the BTCE. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is the MND promoter, IgVH promoter, EF-1α promoter, or IgHG1 promoter. In some embodiments, the expression cassette (i) an enhancer; (ii) a polynucleotide encoding a signal sequence; (iii) a polynucleotide comprising a 5’UTR derived from the IGHV gene or IGHG1 gene; (iv) a polynucleotide comprising a 3’UTR derived from the IGHV gene or IGHG1 gene; and / or (v) a ubiquitous chromatin opening element (UCOE) and further comprises the enhancer may be an Eμ enhancer or an SLC3A2 enhancer, the signal sequence may be the signal sequence of IgHV or the signal sequence of IgHG1.

[0139] In some embodiments, the first polynucleotide further comprises a nucleic acid homologous to the locus, and the nucleic acid homologous to the locus may comprise contiguous nucleotides that are about 200 to 1500 nucleotides in length.

[0140] In some embodiments, the first polynucleotide is comprised in a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector. In some embodiments, the AAV vector is an AAV6 vector.

[0141] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an ex vivo cell. In some embodiments, the cell is an autologous cell obtained from a subject. In some embodiments, the cell is a cell of the same species as the subject. In some embodiments, the cell lacks expression of an endogenous protein to which the BTCE can specifically bind.

[0142] Some embodiments of the methods and compositions provided herein comprise a cell prepared by any one of the methods provided herein.

[0143] Some embodiments of the methods and compositions provided herein comprise a cell in which the genomic locus has been genetically modified to express BTCE. In some embodiments, the cell is a B cell or a pro-B cell. In some embodiments, the cell is selected from hematopoietic stem cells, human embryonic stem cells, induced pluripotent stem cells (iPSCs), naive B cells, memory B cells, plasmablasts, and plasma cells.

[0144] In some embodiments, the locus contains an endogenous gene that is highly expressed in B cells as compared to cells other than B cells. In some embodiments, the locus contains an endogenous gene that is inactive in B cells. In some embodiments, the locus is selected from the CCR5 gene, the JCHAIN gene, the IGHM locus (also known as E-mu; hg38 genome; chr14:105856225-105863200), the CD19 gene, and the IGHG1 gene.

[0145] In some embodiments, the BTCE comprises a first polypeptide capable of specifically binding to a T cell antigen and a second polypeptide capable of specifically binding to a tumor antigen. In some embodiments, the T cell antigen is CD3. In some embodiments, the tumor antigen is selected from CD33, CD19, CD326 (EpCAM), neural-glial antigen 2 (NG2), HER2, epidermal growth factor receptor (EGFR), CD66e, ephrin type-A receptor 2 (EphA2), CD21, FLT3, gp100, PDL1, and CD22. In some embodiments, the BTCE is AMG 330, blinatumomab, solitomab, or tebentafusp.

[0146] In some embodiments, the genomic locus is modified by inserting an expression cassette comprising a first nucleic acid encoding the BTCE. In some embodiments, the first nucleic acid is operably linked to an endogenous promoter of the locus. In some embodiments, the expression cassette further comprises a promoter operably linked to the first nucleic acid. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is the MND promoter, the IgVH promoter, the EF-1α promoter, or the IgHG1 promoter. In some embodiments, the expression cassette comprises (i) an enhancer; (ii) A polynucleotide encoding a signal sequence; (iii) A polynucleotide containing a 5’UTR derived from the IGHV gene or the IGHG1 gene; (iv) A polynucleotide containing a 3’UTR derived from the IGHV gene or the IGHG1 gene; and / or (v) A ubiquitous chromatin opening element (UCOE) further comprising, the enhancer may be an Eμ enhancer or an SLC3A2 enhancer, the signal sequence may be an IgHV signal sequence or an IgHG1 signal sequence.

[0147] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an ex vivo cell. In some embodiments, the cell is an autologous cell obtained from a subject. In some embodiments, the cell is a syngeneic cell to the subject.

[0148] Some embodiments include a pharmaceutical composition comprising any one of the cells provided herein and a pharmaceutically acceptable additive. In some embodiments, the pharmaceutical composition is configured for administration as adoptive cell transfer. In some embodiments, the pharmaceutical composition is configured for administration by injection at a cancer site such as a solid tumor.

[0149] Specific treatment methods Some embodiments of the methods and compositions provided herein include methods of treating, alleviating or suppressing a disorder in a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0150] Some such embodiments include the step of administering to a subject cells that express BTCE. In some embodiments, the cells are cells that have been genetically modified to express BTCE. In some embodiments, the cells are B cells or progenitor B cells. In some embodiments, the B cells or progenitor B cells are hematopoietic stem cells, human embryonic stem cells, induced pluripotent stem cells (iPSCs), naive B cells, memory B cells, plasmablasts or plasma cells. In some such embodiments, the cells are primary B cells or primary progenitor B cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are autologous cells obtained from the subject. In some embodiments, the cells are cells of the same species as the subject. In some embodiments, the cells are prepared by any one of the methods provided herein. In some embodiments, the cells lack the expression of an endogenous protein to which the BTCE can specifically bind.

[0151] In some embodiments, the BTCE comprises a first polypeptide that can specifically bind to a T cell antigen and a second polypeptide that can specifically bind to a tumor antigen. In some embodiments, the T cell antigen is CD3. In some embodiments, the tumor antigen is selected from CD33, CD19, CD326 (EpCAM), neural-glia cell antigen 2 (NG2), HER2, epidermal growth factor receptor (EGFR), CD66e, ephrin type-A receptor 2 (EphA2), CD21, FLT3, gp100, PDL1 and CD22. In some embodiments, the BTCE is AMG 330, blinatumomab, solitomab or tebentafusp.

[0152] In some embodiments, the disease is cancer or an inflammatory disease. In some embodiments, the cancer is a solid tumor or leukemia. In some embodiments, the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), eye cancer, breast cancer, gastric cancer, malignant melanoma, colorectal cancer, colorectal cancer, head and neck cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, or pancreatic cancer.

[0153] In some embodiments, the cancer comprises a tumor antigen. In some embodiments, the tumor antigen is CD33, CD19, CD326 (EpCAM), neural-glial cell antigen 2 (NG2), HER2, epidermal growth factor receptor (EGFR), CD66e, ephrin type A receptor 2 (EphA2), CD21, FLT3, gp100, PDL1 or CD22.

[0154] In some embodiments, the tumor antigen is CD33 and the BTCE is AMG 330. In some of such embodiments, the cancer comprises CD33+ tumor cells. In some of such embodiments, the cancer is acute myeloid leukemia (AML), for example, relapsed / refractory AML.

[0155] In some embodiments, the tumor antigen is CD19 and the BTCE is blinatumomab. In some of such embodiments, the cancer comprises CD19+ tumor cells. In some of such embodiments, the cancer is acute lymphoblastic leukemia (ALL), for example, relapsed / refractory ALL.

[0156] In some embodiments, the tumor antigen is CD326 (EpCAM) and the BTCE is solitomab. In some of such embodiments, the cancer comprises CD326+ tumor cells. In some of such embodiments, the cancer is a gastrointestinal cancer or lung cancer.

[0157] In some embodiments, the tumor antigen is the gp100 / HLA-A*02:01 complex, and the BTCE is tebentafusp. In some such embodiments, the cancer comprises tumor cells that are positive for the gp100 / HLA-A*02:01 complex. In some such embodiments, the cancer is an eye cancer, such as uveal melanoma.

[0158] Depending on its type, BTCE may be rapidly eliminated in a subject, so the half-life of BTCE in a subject may be short. Conventionally, BTCE has been administered to a subject by multiple administrations over a long period and / or continuous intravenous infusion to maintain its therapeutic concentration (see, for example, Zhu M, et al., (2016) Clin Pharmacokinet. 55:1271-88; and Singh K, et al., (2021) Journal for ImmunoTherapy of Cancer 9:e003679. doi: 10.1136 / jitc-2021-003679). Different from the prior art methods, the treatment methods provided herein include administering cells expressing BTCE to a subject at a reduced or limited number of administrations. For example, cells expressing BTCE can be administered to a subject 1 time, 2 times or less, 3 times or less, 4 times or less, or 5 times or less. In some embodiments, cells expressing BTCE can be administered to a subject as a single administration. In some embodiments, different from the prior art methods in which continuous intravenous infusion is performed to maintain its therapeutic concentration, cells expressing BTCE are administered for a short period of time. For example, cells expressing BTCE can be administered in less than 1 hour, less than 30 minutes, less than 20 minutes, less than 10 minutes, or less than 5 minutes.

[0159] Examples of nucleotide sequences of sgRNAs for genes such as the Eμ gene, IGHG1 gene, CCR5 gene, JCHAIN gene, CD19 gene, which are useful in certain embodiments of the methods and compositions provided herein, are listed in Table 1 below. [Table 1]

[0160] Examples of nucleotide sequences of homologous arms for specific loci used in combination with specific HDR templates, which are useful in certain embodiments of the methods and compositions provided herein, are given in Table 2 below. [Table 2] JPEG2025520225000004.jpg244162JPEG2025520225000005.jpg241162JPEG2025520225000006.jpg242162JPEG2025520225000007.jpg131162

[0161] Examples of specific nucleotide sequences useful in certain embodiments of the methods and compositions provided herein are given in Table 3 below. [Table 3] JPEG2025520225000009.jpg239163JPEG2025520225000010.jpg237164JPEG2025520225000011.jpg242163 [Examples]

[0162] Example 1 - Recombination of plasmablasts to secrete anti - cancer biological agents in humanized mice Advances in genomics have enabled the generation of plasma cells (PCs) that secrete large amounts of therapeutic proteins. However, in vivo modeling of non-recombinant and recombinant human plasma cells has a major limitation in that even when plasma cells are transplanted into immunodeficient mouse models such as NSG mice, only short-term investigations can be carried out. Testing human plasma cells in immunodeficient mouse models is difficult, which is partly due to the absence of important factors provided by human bone marrow stromal cells and myeloid cells. For example, the lack of cross-reactivity between species is cited as the reason. In this example, the hypothesis that the engraftment ability of genome-edited human plasma cells is improved in immunodeficient mice humanized by transplantation of human CD34+ peripheral blood hematopoietic stem cells (NSG-huCD34) was tested. Furthermore, this method was predicted to be useful for elucidating important interactions between resident cells (myeloid cells and lymphocytes) in the bone marrow of the human host and human plasma cells. Consistent with this concept, gene-edited autologous plasma cells engrafted more efficiently in NSG-huCD34 mice than in NSG control mice. In addition, gene-edited plasma cells secreted very high concentrations of antibodies in NSG-huCD34 mice (>100 μg / mL over 80 days) and also engrafted into another lymphoid compartment. Furthermore, in vivo imaging using luciferase, ex vivo flow cytometry analysis, and histopathological examination revealed that gene-edited plasma cells migrated to and localized in the bone marrow and spleen within 2 days after transplantation and were retained at these sites. Taken together, these data indicate that transplantation of human autologous hematopoietic cells into NSG-huCD34 mice has established a robust model that enables in vivo study of the ecology of long-lived genome-edited human plasma cells.

[0163] As a characterization of NSG-huCD34 mice, hematoxylin and eosin staining of spleen sections excised from NSG mice and NSG-huCD34 mice (Figure 1A), flow cytometry from spleens obtained from NSG-huCD34 mice (Figure 1B), and measurement of plasma cell-derived cytokines in the peripheral blood sera of NSG-huCD34 mice and NSG mice (Figure 1C) were performed.

[0164] According to the experimental timeline in vitro, primary human B cells were gene-edited and differentiated in vitro to generate primary plasma cells expressing luciferase (Figure 2A). The frequency of alleles edited by homologous recombination repair was measured in primary B cells edited with the luciferase vector or primary B cells not edited with the vector (Figure 2B). Eleven days after gene editing, flow cytometry analysis of viable cell plasma cell staining (CD138+CD38+) (Figure 2C) and IgG staining of plasma cells (Figure 2D) was performed.

[0165] According to the timeline of an in vivo study in which NSG mice or NSG-huCD34 mice were administered gene-edited plasma cells (PC) or PBS control, transplantation of gene-edited human plasma cells into NSG-huCD34 mice was performed (Figure 3A). Engraftment of ffluc-PC was shown from the luminescence images of NSG mice and NSG-huCD34 mice transplanted with bulk plasma cells (Figure 3B). Improvement in engraftment and persistence was shown in humanized mice from the average luminance of NSG mice and NSG-huCD34 mice transplanted with ffluc-PC (Figure 3C). Immunohistological examination of spleens excised from NSG-huCD34 mice transplanted with ffLuc-PC was performed (Figure 3D). Serum IgG titers of NSG mice and NSG-huCD34 mice transplanted with bulk plasma cells were measured (Figure 3E). IgG+ cells were shown in mice transplanted with gene-edited PC from flow cytometry plots of intracellular IgM and intracellular IgG of plasma cells (CD138+, CD38+, hCD45+) (Figure 3F). PCs edited to express BFP were detected in the bone marrow of NSG-huCD34 mice (Figure 3G). 6×10 6Individual gene-edited bulk cells or 1×10 6 Individual enriched ffLuc plasmacytes were transplanted into NSG mice and NSG-huCD34 mice. Engraftment of IgG+ PCs was shown in the bone marrow in both the bulk and enriched groups (Figure 3H), and comparable IgG titers were shown (Figure 3I). Plasmacytes secreting bispecific T cell engager (BiTE, i.e., BTCE) were generated (Figure 4A). Flow cytometry analysis of these gene-edited PCs was performed (Figure 4B). T cells, K562 cells, and K652-CD19+GFP cells were cultured with the supernatant obtained from PCs (Figure 4C). Activation of T cells (Figure 4C) and killing of K562-CD19+ cells (Figure 4D) were measured in the presence of the supernatant obtained from BiTE-PCs or BFP-PCs.

[0166] When at least PCs were tracked by luminescence, the above human autologous plasmacytes gene-edited by homologous recombination repair migrated to the bone marrow of NSG-huCD34 recipient mice and maintained the phenotype of PCs confirmed by flow cytometry.

[0167] In addition, since the human autologous plasmacytes gene-edited by homologous recombination repair showed at least stable luminescence for 100 days in humanized mice, they engrafted more efficiently into NSG-huCD34 recipient mice than into NSG recipient mice.

[0168] In NSG-huCD34 mice transplanted with plasmacytes, high titers of IgG were maintained. In three mice, the IgG titer exceeded 50 μg / mL and remained stable for 100 days.

Number

[0169] It was observed that gene-edited plasmacytes produced approximately 62 pg / cell·day of anti-CD19 BiTE, i.e., anti-CD19 BTCE, per plasmacyte.

[0170] Example 2 - Editing of the IGHG1 locus Multiple sgRNAs were designed to target various positions in exon 1, exon 2, and exon 4 of IGHG1. For each sgRNA, an AAV-based homologous recombination repair (HDR) construct was constructed. This HDR construct contained an MND promoter linked to a GFP reporter gene and was placed between appropriate homologous arms. Figure 5A shows a plot of the target sites of each sgRNA on the structure of the IGHG1 genome. The sequences of the homologous arms of each sgRNA and each repair template are shown in Table 4 below. [Table 4]

[0171] B cells were edited using each sgRNA and each HDR construct using a method substantially similar to the method described in US Patent Publication No. 2018 / 0282692, which is hereby incorporated by reference in its entirety.

[0172] Briefly, B cells were isolated from peripheral blood mononuclear cells (PBMCs) by negative selection and activated using a cytokine cocktail containing oligomerized CD40L, CpG, and IL21. After activating the B cells, editing was performed as follows. That is, ribonucleoprotein particles containing each sgRNA and Cas9 protein were transfected into the cells, and then an AAV6 vector containing an HDR construct was introduced into the B cells. After gene editing, the cells were grown in activation medium for 5 days, and the exchange to differentiation medium was performed in two steps. Flow cytometry analysis was performed to measure the knockout efficiency of IGHG1. As shown in Figure 5B (upper panel), it was observed that the proportion of IGHG+ cells in B cells decreased by approximately 90% in cells treated with RNP containing TH26 sgRNA. The proportion of GFP+ cells in B cells recombined to express GFP at the IGHG1 locus was measured (Figure 5B, lower panel). For live cells, the proportion (%) and mean fluorescence intensity (MFI) of GFP were measured, and the HDR rate for each set of sgRNA / HDR constructs was normalized to the HDR rate observed in sgRNA (TH22) where GFP+ cells were generally observed at 10 - 15%. The normalized proportion of GFP-positive cells is shown in Table 5 below. The normalized MFI values of GFP are shown in Table 6 below.

Table 5

Table 6

[0173] In this experiment, when targeting the fourth exon (TH21) and the second exon (TH26), the transduction rate and MFI value increased.

[0174] Example 3 - In vitro expression of BTCE An expression vector encoding anti-CD19 BTCE (blinatumomab) or an expression vector encoding anti-CD33 BTCE (AMG-330) was transfected into 293T cells. The supernatant was collected, and the expression of BTCE was quantified by Western blot analysis and mass spectrometry. As expected, for both BTCEs, a 50 KDa protein band was generated on the Western blot, and peptides mapped to the expected sequences were produced.

[0175] Example 4 - Editing of the Eμ locus To deliver blinatumomab (anti-CD19) or AMG-330 (anti-CD33) to the Eμ locus, an AAV-based HDR repair template was constructed.

Table 7

[0176] To examine the relative editing rate of each BTCE, B cells were recombined using the above method substantially similar to the method described in U.S. Patent Publication No. 2018 / 0282692, which is hereby incorporated by reference in its entirety. The percentage of GFP cells and the MFI value of GFP were quantified as a percentage of the total live B cells (Figure 6B).

[0177] Example 5 - In vitro activity of B cells expressing BTCE To analyze the effects of each BTCE, the supernatants obtained from cells expressing each BTCE were incubated with autologous PBMCs containing effector T cells, CD33+ monocytes, and CD19+ B cells. After 48 hours of incubation, the effects of each BTCE were quantified by measuring T cell activation (CD69+CD137+ cells in CD3+ T cells), CD33+ monocyte killing, and CD19+ B cell killing (Figure 7). In Figure 7, the left bar is for CD19BiTE, the middle bar is for CD33BiTE, and the right bar is for GFP. As expected, all BTCEs increased T cell activation (left panel). Furthermore, a decrease in CD33+ cells was observed in cells incubated with the supernatant obtained from B cells producing AMG330 (the middle bar in the middle panel), and a decrease in CD19+ cells was observed in cells incubated with the supernatant obtained from B cells producing blinatumomab (the left bar in the right panel). These data demonstrated the effectiveness of the two types of BTCEs secreted by B cells.

[0178] Example 6 - Editing of the Eμ locus and the CCR5 locus As shown in Figure 8A, each HDR template was cloned into an AAV vector. Construct 1 and Construct 2 had homology arms for delivery to the CCR5 locus. Construct 3 and Construct 4 had homology arms for delivery to the Eμ locus. Construct 2 and Construct 4 were negative controls that expressed a fluorescent marker (BFP for Construct 2 and GFP for Construct 4) downstream of the MND promoter. Construct 1 and Construct 3 expressed blinatumomab BTCE in cis to GFP downstream of the MND promoter. In all cases, if HDR was successful, it was predicted that the fluorescent reporter would be expressed and quantification would be possible using the digital droplet PCR assay in our laboratory.

[0179] B cells were recombined using Construct 1, Construct 2, Construct 3, or Construct 4. Figure 8B shows each construct used for editing the CCR5 locus. The sgRNA sequences used are shown in Table 8 below.

Table 8

[0180] After recombining the B cells, the B cells were analyzed to examine the degree of gene editing and the expression of the fluorescent reporter. The expression of the fluorescent reporter was quantified using FACS analysis. As shown in Figure 8C, the highlighted box indicates the expression of the fluorescent reporter in the absence of blinatumomab. The mean fluorescence intensity of the fluorescent reporter was similar at both the Mu locus and the CCR5 locus. Also, as shown in Figure 8D, the highlighted box indicates the expression of the fluorescent reporter expressed downstream of blinatumomab. GFP detected as the percentage of the total cells and the mean fluorescence intensity was significantly higher in gene editing delivery to the Eμ locus than in gene editing delivery to the CCR5 locus.

[0181] In another study, B cells were isolated and compared between B cells edited to express blinatumomab (anti-CD19 BTCE), B cells edited to express AMG-330 (anti-CD33 BTCE), and B cells edited to express BFP when each construct was delivered to the CCR5 locus. Similar to the study described above, the percentage of BFP-positive cells and the mean fluorescence intensity of BFP were higher than those of GFP expressed downstream of each BTCE. To investigate whether this incomplete gene editing is due to expression or a defect in HDR, the degree of integration into the genome was quantified by digital droplet PCR. See, for example, Hung, K.L. et al, (2018) Molecular Therapy, 26:456-467 (this reference is hereby expressly incorporated by reference in its entirety).

[0182] As shown in Fig. 8E, the frequency of alleles in which HDR events occurred with the BFP template was comparable to the proportion of BFP+ cells quantified (e.g., for an allele frequency of 14.5%, the proportion of BFP+ cells was 11.2%; on average, there was a difference of about 1.34-fold). In contrast, the frequency of alleles in which HDR events occurred with each of the two types of BTCE templates was significantly lower than expected (e.g., for an allele frequency of 8.6%, the proportion of GFP+ cells was 2.79%; on average, there was a difference of about 3.45-fold). From these data, it was shown that the BTCE gene is not expressed particularly efficiently at the CCR5 locus.

[0183] Example 7 - Quantification of expressed blinatumomab Fig. 9A is a schematic diagram of the method used for quantification of secreted blinatumomab. In this assay, BTCE or solvent-containing medium was incubated with T cells, a reference cell line (K562; BFP+CD19-) that expresses BFP but not CD19, and a target cell line (K562; GFP+CD19+) that expresses GFP and CD19. The resulting mixture was incubated overnight. After incubation, the activity of BTCE was quantified by measuring the activation of T cells (expression of CD69 and CD137) and the killing by T cells (change in the ratio of target cells (GFP+) to reference cells (BFP+)).

[0184] Furthermore, after incubating the three types of cells described above with recombinant blinatumomab at various concentrations, a titration curve using recombinant blinatumomab was generated by quantifying the killing of target cells (comparison of GFP and BFP). FACS analysis was performed (Fig. 9B). As shown in Fig. 9C, it was shown that as the concentration of blinatumomab increased, the proportion of GFP+ target cells decreased, and reproducibility was also observed.

[0185] After incubating blinatumomab at various concentrations with the above three types of cells, T cell activation was quantified. FACS analysis was performed (Figure 9D). As shown in Figure 9E, it was shown that increasing the concentration of blinatumomab increased the proportion of CD3+ T cells co-expressing CD137 and CD69, and high reproducibility was also observed.

[0186] A quantitative test of the supernatant was performed using the T cell activation / target cell killing assay. As shown in Figure 9F, the activation of T cells induced by the supernatant of B cells was plotted for each sample. In a parallel test, data interpretation was performed using a standard curve prepared with recombinant blinatumomab. Incorporation of the HDR template into the E-mu (Mu) locus secreted a much higher concentration of blinatumomab than incorporation into the CCR5 locus (Figure 9G).

[0187] Example 8 - Editing of the CCR5 locus, the IGHG1 locus and the Eμ locus For the purpose of expressing GFP or blinatumomab_2A_GFP at four different loci, each HDR template was cloned into an AAV vector (Figure 10A). For the CCR5 gene, insertion into the exon was targeted (Figure 10B).

[0188] B cells were edited by the above method using construct 2 containing GFP and homologous arms linked to the MND promoter. Next, FACS analysis was performed. As shown in Figure 10C, HDR to the loci shown in the graph by construct 2 expressing GFP was measured. These data showed that the proportion of edited cells was comparable at the CCR5 locus and two antibody loci (IGHG1 and E-mu). Furthermore, it was also shown that the mean fluorescence intensity of GFP increased in the cells edited at each antibody locus (see the histogram in Figure 10C and the quantitative values shown on the right side thereof). Furthermore, it should also be noted that the editing rates were comparable at all loci. The MFI value of GFP slightly increased at the monoclonal antibody locus (similar to the results of the JCHAIN locus in past experiments). Two peaks were observed at the Eμ locus.

[0189] B cells were edited by the above method using construct 1 in which BTCE linked to the MND promoter was placed cis to GFP and contained homologous arms, or a construct containing GFP linked to a promoter. Next, FACS analysis was performed. As shown in Fig. 10D, HDR to the locus shown in the graph by construct 1 expressing blinatumomab and GFP was measured. From these data, it was shown that the proportion of edited cells reached approximately 5-fold in the editing of two antibody loci (IGHG1 and E-mu). Furthermore, it was also shown that the mean fluorescence intensity of GFP increased significantly in the cells that edited each antibody locus (see the gray histogram in Fig. 10D and the quantitative values shown on the right). It should also be noted that the HDR of the construct encoding each binder was less efficient than in the case of GFP alone. The HDR rate was approximately 10-15%. The best expression was obtained at the IGHG1 locus. The secretion of blinatumomab was comparable at the IGHG1 locus and the Em locus. The data at the JCHAIN locus (not shown) were almost the same as the data at the CCR5 locus. When a T cell activation assay was performed to quantify the amount of blinatumomab produced by B cells, it was found that the amount of blinatumomab increased approximately 8-fold in the cells in which each antibody locus was edited.

[0190] Example 9 - Effect of CD19 inactivation on B cells expressing blinatumomab B cells were edited to express GFP and / or BTCE. CD19 and CD38 (plasma cell marker) in the recombinant cell population were quantified by flow cytometry analysis. Even when CD19 was knocked out, the cell surface expression of CD19 did not disappear, and no significant effect on the differentiation of B cells into plasma cells was observed (see CD38 staining in Fig. 11A). The supernatant was isolated and incubated with CD19+K562 cells, CD19-K562 cells, and T cells in the same manner as above. To measure the activity of blinatumomab in the supernatant of B cells, the activation of T cells in this co-culture was quantified (Fig. 11B). Even when CD19 was knocked out, no significant effect on the production of blinatumomab by the edited B cells was observed.

[0191] Inactivating CD19 in cells is thought to be able to prevent fratricide by recombinant B cells co-expressing CD19.

[0192] Example 10 - In vivo activity of B cells expressing blinatumomab B cells were genetically modified to express blinatumomab (for testing) or GFP (for control). These modified B cells were mixed with Raji cells (CD19+ B cell lymphoma) and autologous T cells modified to express luciferase. This cell mixture was transplanted into the flanks of immunodeficient mice (NSG) (Figure 12A). After transplantation, tumor growth in vivo was measured by measuring luminescence (the blue circular part shown in the mouse) with an IVIS device. In mice transplanted with B cells producing blinatumomab, the in vivo efficacy of B cells producing BTCE was shown because CD19+ tumor cells were eliminated (Figure 12B).

[0193] In another study, humanized immunodeficient mice were generated by transplanting 1 million human CD34 cells into NSG mice at about 12 weeks of age. The transplanted human immune cells were found to differentiate from CD34 cells into various human lymphocytes including monocytes, other myeloid cells, B cells and some T cells over an additional 8 weeks in the body of this mouse. Host-derived T cells were expected to interact with BTCE and induce a cytotoxic response against Raji tumor cells.

[0194] B cells expressing blinatumomab (for testing) or GFP (for control) were mixed with Raji cells expressing luciferase. This cell mixture was transplanted into the above humanized immunodeficient mice. In mice transplanted with B cells secreting blinatumomab, tumor growth was significantly reduced compared to mice transplanted with B cells expressing GFP (Figure 12C).

[0195] Example 11 - Recombinant plasmablasts secrete bispecific T - cell engagers and suppress patient - derived leukemia in vivo Bispecific T cell engagers (BTCEs) are important tools for the management and treatment of human hematological malignancies. However, BTCEs are not commonly used because they have a short serum half-life and off-target toxicity to sites other than the tumor site. Advancements in genome engineering have enabled the generation of human plasma cells that secrete therapeutic proteins in large quantities. Such recombinant cells are capable of long-term in vivo engraftment in humanized mouse models. As a next step towards the clinical application of recombinant plasma cells (ePCs) in cancer therapy, specific embodiments for expressing and secreting BTCEs by human plasma cells are described in this example. As disclosed herein, human plasma cells engineered to express anti-CD19 BTCE (blinatumomab) or anti-CD33 BTCE (AMG 330) were able to induce T cell activation and direct killing of cancer cell lines and primary human cells by T cells in vitro. Furthermore, delivery of BTCE-ePCs locally to sites adjacent to tumor cells implanted in the flank induced tumor eradication in vivo. Additionally, in immunodeficient mice transplanted with ePCs secreting anti-CD19-BTCE and T cells, the in vivo growth of xenografts derived from patients with acute lymphoblastic leukemia expressing CD19 + was inhibited. These findings support the use of ePCs as a durable local delivery system for BTCEs in the treatment of leukemia, lymphoma, and other cancers.

[0196] Human primary B cells recombined by homologous recombination repair using CRISPR Cas9 secrete functional BTCE To integrate the BTCE gene expression cassette into B cells, an AAV-based homology-directed repair (HDR) template was improved, and this template was used to deliver the transgene to the safe harbor gene CCR5 of B cells. 19 This HDR template targeting CCR5 was designed for the delivery of anti-CD19 BTCE blinatumomab or BFP placed in cis to GFP. A previously established protocol for B cell editing 19According to this, primary human B cells were isolated from peripheral blood mononuclear cells (PBMCs) and expanded by culturing for 2 days using a cytokine cocktail. This cytokine cocktail contains oligomerized CD40 ligand, CpG, interleukin 2 (IL-2), IL-10, and IL-15, and has the effect of activating primary B cells (in the growth medium). Next, after transfecting the activated B cells with a Cas9 ribonucleoprotein complex (RNP) containing a guide RNA targeting the sequence within the CCR5 locus, gene editing was initiated by transduction with the above AAV vector. Before analysis, the recombinant B cells were cultured in the growth medium for an additional 5 days (Figure 13A, Figure 17A). When evaluated by digital droplet PCR (ddPCR), it was found that the integration rate by HDR was slightly lower in the vector containing BTCE (Figure 13B, Figure 17). However, despite the similar integration rates, the proportion of cells expressing the fluorescent reporter was significantly reduced in the cells edited using the design containing BTCE, and as a result, the ratio of fluorescent reporter labeling to the integration rate was greatly decreased (Figure 13C - 13D).

[0197] We hypothesized that the expression of the BTCE transgene could be increased by targeting the integration of the transgene into loci that are naturally expressed in B cells or plasma cells. Based on this, three additional AAV-based repair template designs were constructed for the delivery of the transgene cassette to the IGHG1 locus, JCHAIN locus, which are highly expressed in B cells, and the 12μ region adjacent to the heavy chain enhancer (the repair arms and sgRNA are described in the previously reported literature 12; see Figure 13E for the overall schematic diagram of all vectors). The same promoter (the viral promoter MND 27) was used, and when the fluorescent reporter GFP was delivered to these antibody-related loci, it was observed that the proportion of GFP and the mean fluorescence intensity of GFP in B cells increased to varying degrees compared to the expression at the CCR5 locus (Figures 17C - 17D). The integration of BTCE was detected at all loci, but for GFP placed in cis, a significant increase in the mean fluorescence intensity was observed at each antibody locus compared to the expression at the CCR5 locus (Figures 13F - 13G).

[0198] To examine the functionality of anti - CD19 BTCE produced by B cells, an in vitro killing assay was constructed. Briefly, lentivirus was used to stably transduce CD19 and GFP into K562 target cells, and BFP was stably transduced into K562 reference cells. A mixture of CD19 - BFP + reference cells, CD19 + GFP + target cells, and PBMC - derived CD8 + T cells was incubated with recombinant anti - CD19 BTCE or recombinant cell - derived supernatant (Figure 13H). After 48 hours, flow cytometry was used to quantify the degree of T - cell activation (the proportion of CD39 + CD137 +) and the specific lysis of CD19 + target cells (the ratio of GFP to BFP K562 cells) (Figure 18A). Recombinant anti - CD19 BTCE induced a dose - dependent increase in T - cell activation and CD19 - specific lysis (Figure 18B). Also, the supernatant obtained from B cells recombinant to express anti - CD19 BTCE induced strong T - cell activation and lysis specific to CD19 + cells, but such T - cell activation and specific lysis of CD19 + cells were not observed in the supernatant obtained from B cells recombinant to express GFP (Figures 13I - 13J). Using a T - cell activation assay, a standard curve was generated from the data of recombinant BTCE, and using this standard curve, it was found that a high concentration of anti - CD19 BTCE was produced in the supernatant from recombinant B cells (Figure 13K). These findings indicated that primary human B cells can be recombined at various loci to strongly express functional anti - CD19 BTCE.

[0199] Plasmablasts recombined to express BTCE show strong efficacy against common leukemia target antigens in vitro We investigated whether human plasma cells differentiated ex vivo could produce BTCE that specifically targets antigen-expressing cells within a heterogeneous primary human cell population. Using a similar design to the anti-CD19 BTCE (αCD19) targeting the Eμ locus, a repair template for the delivery of anti-CD33 BTCE (αCD33) was additionally constructed (Figure 14A). Each BTCE and GFP control were introduced into B cells by HDR, and each cell was differentiated into ePCs according to the previously reported method (Figure 17A). 19 After gene editing and differentiation, detectable transgene expression was observed in all vector-donor combinations (Figures 14B - 14C). Donor-dependent differences were observed in the expression of the plasma cell differentiation markers CD38 and CD138 (Figure 19B), but the introduction of BTCE did not affect differentiation into plasma cells (plasma cells were defined as CD38++CD138+; Figures 19A - 19B). From the above data, it was shown that plasma cells can be recombined to express BTCE.

[0200] The functionality of BTCE secreted by ePCs was evaluated using a PBMC killing assay. In this assay, autologous PBMCs containing a B cell subset expressing CD19 and a myeloid cell subset expressing CD33 were co-cultured with effector CD8+ T cells. To induce cell killing, recombinant BTCE or ePC-derived supernatants were added (Figure 13D). Forty-eight hours after the addition of each BTCE, the number and phenotype of target cells were quantified by flow cytometry (Figure 19D). As expected, it was found that recombinant αCD19 BTCE induced a dose-dependent decrease in IgM+ B cells, and recombinant αCD33 BTCE induced a dose-dependent decrease in CD14+CD33+ monocytes (Figure 19E). On the other hand, it was found that supernatants obtained from αCD19-ePCs and αCD33-ePCs induced higher T cell activation than supernatants obtained from GFP-ePCs (Figure 14F). Furthermore, the supernatant obtained from αCD19-ePCs specifically killed IgM+ target B cells (Figure 14G), and the supernatant obtained from αCD33-ePCs specifically killed CD33+ CD14+ target monocytes (Figure 14H). Similarly, in a leukemia killing assay, T cell activation (the proportion of CD69+CD137+ cells in CD8+ cells) (Figure 14H); the frequency of NAML-6 (CD19+) (Figure 14I); and the frequency of MOLM-14 (CD33+) (Figure 14J) were quantified by flow cytometry. These data showed that ePCs producing BTCE can induce specific T cell targeting against cells expressing leukemia targets in vitro.

[0201] In plasmablasts recombined to remove autonomously expressed antigens, self - targeting is blocked and BTCE expression is enhanced CAR T cells recombinant to recognize T cell antigens may kill other CAR T cells contained in their own cell products, resulting in a decrease in anti-cancer activity 28,29Since αCD19-ePC expresses CD19, we hypothesized that when it encounters T cells, self-targeting might be induced similarly (Figure 20). To evaluate the degree of self-targeting, GFP-ePC or αCD19-ePC was incubated with autologous T cells (Figure 15A). After 24 hours, the proportion of αCD19-ePC was quantified by flow cytometry. When the assay was initiated using a large number of T cells, the proportion of αCD19-ePC decreased, while the proportion of GFP-ePC did not decrease (Figure 15B, Figure 21), suggesting that CD19-specific self-targeting might affect ePCs secreting BTCE.

[0202] We investigated whether the self-targeting induced by BTCE could be blocked by removing CD19. To knockout CD19, an RNP targeting CD19 and a μ-directed αCD19 BTCE editing reagent were co-delivered. By adding the RNP targeting CD19, the proportion of CD19+ plasma cells decreased by more than 85% (Figure 15C - 15D). Even after knocking out CD19, no obvious effect on the differentiation of edited B cells into blasts or plasma cells in vitro was observed (Figure 22). When these CD19 knockout αCD19-ePCs were exposed to T cells and the number of T cells at the start of the assay was increased, no difference in the proportion of GFP was observed (Figure 15E - 15F). These data suggest that knocking out CD19 protected αCD19-ePCs from killing by self-targeting.

[0203] As another problem regarding the self-expression of CD19, it is mentioned that the αCD19 BTCE produced from recombinant cells is considered to be reduced when it binds to CD19, resulting in less αCD19 BTCE being released from the recombinant cells. Based on this problem, it was investigated whether knocking out CD19 would increase the concentration of αCD19 BTCE detected in the supernatant. To evaluate the free BTCE generated by knocking out CD19, in the K562 cell killing assay, the supernatant derived from BTCE ePC was evaluated in the presence or absence of co-recombination with CD19 RNP (Figure 13E). It was found that in the supernatant derived from CD19KO αCD19-ePC, compared with the supernatant derived from CD19WT αCD19-ePC, the activation of T cells was enhanced, specific lysis was enhanced, and the concentration of αCD19 BTCE increased (Figures 15G - 15I). From the above results, it was shown that knocking out CD19 prevents self-targeting by T cells and significantly increases the αCD19 BTCE concentration.

[0204] Plasmablasts recombined to express BTCE exert an anti - tumor effect in vivo To investigate whether ePC secreting BTCE maintains functionality in vivo, a flank model of B cell leukemia was used. NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ-c(NSG) immunodeficient mice were subcutaneously injected with CD19+ firefly luciferase Raji cells, autologous T cells, and CD19KO ePC expressing GFP or αCD19 BTCE (Figure 16A). It was confirmed that the luciferase-expressing Raji cells engrafted similarly in all groups (at day 1, Figures 16C - 16D). At later time points, in the mice administered with αCD19-ePC, the tumor burden decreased compared with the mice administered with GFP-ePC (Figures 16B - 16C). In the BTCE group, in more than 50% of the mice, the luminescence level reflecting the tumor size decreased below the background within 5 days (Figures 16B - 16C). From these findings, it was shown that αCD19-ePC can promote a strong local anti-tumor response in vivo.

[0205] Blinatumomab is prescribed to B-ALL patients with minimal residual disease as a bridging therapy until transplantation after chemotherapy 30-35 To simulate the condition observed in B-ALL patients after lymphodepletion therapy, a model was constructed as follows to deliver BTCE-ePC before delivering tumor and T cells. NSG mice were intravenously injected with CD19KO GFP-ePC or CD19KO αCD19-ePC. The next day, luciferase-expressing cells obtained from an infiltrating xenograft model (NL482B; gain-of-function mutation in IL7R, SH2B3 deletion) derived from Philadelphia-like B-ALL patients were intravenously injected 36,37 Furthermore, on the day after and 3 days after B-ALL transplantation, effector T cells syngeneic to ePC were retroorbitally injected (Figure 16D). Tumor growth was monitored until the luminescence signal reached saturation in GFP control mice (Figures 16E-16F). In mice administered αCD19-ePC, tumors were almost completely suppressed and the tumor burden was significantly reduced compared to the control group (Figures 16F-16G). Also, 15 days after tumor transplantation, a tendency for a higher frequency of T cells was observed in the peripheral blood of the αCD19-ePC treatment group compared to the GFP control group, consistent with the induction of T cell proliferation by BTCE in vivo (Figure 23B). Furthermore, when sacrificed 34 days after tumor engraftment, the CD19+ cells in the spleen and bone marrow were significantly reduced in the αCD19-ePC treatment group compared to the control group (Figure 23C). BTCE-ePC was able to block patient-derived leukemia seeding in a model that functionally mimics the clinical use of blinatumomab as a bridging therapy in B-ALL patients

[0206] Engineered plasma cells (ePCs) have recently gained attention as a modality for the delivery of therapeutic proteins. This specification discloses the use of ePCs to produce bispecific T cell engagers (BTCEs). By utilizing homology-directed repair (HDR), multiple BTCEs were highly expressed at multiple active loci in primary human B cells. Recombinant B cells engineered to express BTCEs were shown to be capable of differentiating into plasma cells that mediate the killing of primary human cells expressing CD19 or CD33. By knocking out the target antigen CD19, the secretion of αCD19 BTCE by ePCs was enhanced and autotargeting was prevented. Furthermore, BTCE ePCs were shown to be able to induce a potent T cell-dependent antitumor response against transplanted lymphoma cell lines and patient-derived disseminated leukemia xenograft models that reflect BTCE treatment in B-ALL patients.

[0207] In the standard treatment of B-ALL patients, lymphodepleting chemotherapy is first performed, and then consolidation therapy and transplantation are carried out in patients at high risk of refractory disease. 38 Lymphodepleting therapy removes circulating B cells, but in combination with genetic risk factors, it may introduce another genetic abnormality that accelerates the transformation of the remaining B cells 39,40 or ePC products. Another safety concern for B-ALL recipients is that EBV-reactive T cells may be removed after lymphodepleting therapy, resulting in an increased risk of developing EBV-induced lymphoproliferative disease from the remaining recipient B cells or ePC products. 41 For these reasons, αCD19-ePC products useful for the treatment of B-ALL can be produced from cells obtained from EBV-negative allogeneic donors. In addition to alleviating this safety concern, the use of allogeneic donors has two important advantages. As the first advantage, by prioritizing allogeneic donors based on manufacturability for use in multiple recipients, it may be possible to overcome manufacturing concerns such as those seen in other cell therapy modalities for B-ALL patients who are subjected to harsh treatments. 42. As a second advantage, the allogeneic BTCE cell product as a bridging therapy to hematopoietic stem cell transplantation may ensure a safety advantage regarding host rejection by utilizing allogeneic T cells derived from the bone marrow graft. 43,44 . By utilizing allogeneic T cells derived from the bone marrow graft, there is a possibility of ensuring a safety advantage regarding host rejection.

[0208] . As a barrier that may occur when using ePCs in the treatment of leukemia, lymphoma, or multiple myeloma, the expression of cell surface markers (e.g., CD19, CD20, CD38, and BCMA) targeted by many clinically available biological agents is retained in ePCs, which may lead to auto-targeting of ePCs. In fact, the data disclosed herein showed that CD19+ αCD19-ePCs were eliminated when cultured in the presence of T cells. CD19 is not important for the function of plasma cells 18 and CD19 is downregulated in long-lived plasma cells. Therefore, the recombinant method disclosed herein of simultaneously knocking out CD19 and expressing αCD19 BTCE is considered not to negatively affect the function and lifespan of the recombinantly obtained ePCs. On the other hand, in a useful ePC product, it is highly likely that recombination is performed by a single editing for expressing αCD19 BTCE at the CD19 locus in order to avoid oncogenic Eμ translocation. ePCs can be recombined to target several tumor targets expressed on B cells with BTCE or monoclonal antibodies. Similar to the knockout or removal of CD19, knocking out or removing MS4A1 (also known as CD20), which is a target of lymphoma and multiple myeloma, 48,49 or CD38 in plasma cells also does not have a significant impact on the long-term durability of the antibody titer secreted by plasma cells, the accompanying long lifespan of the antibody, and the secretion ability of plasma cells. On the other hand, knocking out TNFRSF17 (also known as BCMA) in mice reduces the survival rate of plasma cells and abolishes the antibody production response. 48,50,51 . Therefore, the recombinant method disclosed herein of simultaneously knocking out CD19 and expressing αCD19 BTCE is considered not to negatively affect the function and lifespan of the recombinantly obtained ePCs. On the other hand, in a useful ePC product, it is highly likely that recombination is performed by a single editing for expressing αCD19 BTCE at the CD19 locus in order to avoid oncogenic Eμ translocation. ePCs can be recombined to target several tumor targets expressed on B cells with BTCE or monoclonal antibodies. Similar to the knockout or removal of CD19, knocking out or removing MS4A1 (also known as CD20), which is a target of lymphoma and multiple myeloma, 52-55 . Therefore, the recombinant method disclosed herein of simultaneously knocking out CD19 and expressing αCD19 BTCE is considered not to negatively affect the function and lifespan of the recombinantly obtained ePCs. On the other hand, in a useful ePC product, it is highly likely that recombination is performed by a single editing for expressing αCD19 BTCE at the CD19 locus in order to avoid oncogenic Eμ translocation. ePCs can be recombined to target several tumor targets expressed on B cells with BTCE or monoclonal antibodies. Similar to the knockout or removal of CD19, knocking out or removing MS4A1 (also known as CD20), which is a target of lymphoma and multiple myeloma, 56-59 or CD38 in plasma cells also does not have a significant impact on the long-term durability of the antibody titer secreted by plasma cells, the accompanying long lifespan of the antibody, and the secretion ability of plasma cells. On the other hand, knocking out TNFRSF17 (also known as BCMA) in mice reduces the survival rate of plasma cells and abolishes the antibody production response. 60,61 or CD38 in plasma cells also does not have a significant impact on the long-term durability of the antibody titer secreted by plasma cells, the accompanying long lifespan of the antibody, and the secretion ability of plasma cells. On the other hand, knocking out TNFRSF17 (also known as BCMA) in mice reduces the survival rate of plasma cells and abolishes the antibody production response. 62, knockout of TNFRSF17 is thought to probably interfere with the lifespan and / or function of ePC products. Therefore, it is considered feasible to engineer ePCs that express biological agents targeting receptors expressed on B cells, and the generation of ePCs for use as BTCEs or monoclonal antibodies in chronic lymphocytic leukemia (CD20; 63 Glofitamab 64 ), non-Hodgkin lymphoma (CD20; rituximab 65 ), ofatumumab 66 ), and multiple myeloma (CD38; daratumumab 67 ), Bi38 68 ) is also considered possible.

[0209] The main advantage of using ePCs for the delivery of biological agents is that the off-target drug effects that occur in healthy tissues due to systemic delivery of high doses of biological agents are reduced. ePCs can localize in the bone marrow 18 and can functionally eliminate B-ALL cells that localize in the bone marrow 26 , and it is thought that ePCs can be locally delivered in other types of liquid cancers and even solid tumors. In addition to B-ALL, some forms of B cell lymphoma 69,70 and myeloid leukemia 71 have pathological foci in the bone marrow and other lymphoid tissues (spleen, lymph nodes) where ePCs can engraft in vivo 18,72 . Regarding BTCEs for acute myeloid leukemia, such as CD33-targeting agents and CD123-targeting agents 73-75 for which the off-target effects on normal cells have been well described, the tissue localization of plasma cells is thought to be particularly useful for the local delivery of BTCEs. Furthermore, plasma cells are generally known to also reside in other tissues such as lung epithelium and intestinal epithelium 76 , and even solid tumors 77-80 . In fact, tracking donor-specific and microbiota-specific IgA produced in infancy in solid organ transplant recipients has shown that antibody-producing plasma cells survive in the intestine for decades 81,82, From this, it is suggested that ePCs delivered to solid tumors in mucosal sites can survive with a comparable lifespan. The half-life of tebentafusp, a BTCE-like biological agent approved for the first time for solid tumors, is only 7.5 hours, and since weekly infusions are required for the treatment of choroidal melanoma with active angiogenesis 83 , it can be said that BTCE is an effective treatment strategy for solid tumors. On the other hand, systemic delivery of BTCE targeting important solid tumor targets such as EGFR may cause harmful off-target off-tumor toxicity 84 . In the field where active research will be conducted in the future, the localization and off-target off-tumor effects of ePCs secreting biological agents for solid tumors after direct delivery to the tumor site will be evaluated.

[0210] Biological agents consisting of therapeutic proteins were the second most approved drugs from 2009 to 2017, but 85 , many of them have the problem that their half-life is below optimal. In clinical applications, if the half-life is short, the use and adoption may be postponed. However, since the lifespan of ePCs in vivo is extended 18 , it is considered that the availability of drugs in clinical applications is also extended. Biological agents used in chronic treatment have particularly many problems because they require frequent administrations (in some cases, daily administrations) throughout life. As such biological agents with a short half-life, enzyme replacement therapy (agalsidase beta; 56 - 76 minutes 86 , factor IX; 18 - 40 hours 87 , laronidase; 1.5 - 3.6 hours 88 ), chronic autoimmune diseases (infliximab; 9.5 days 89 , etanercept; 80 hours 90 ), diabetes (liraglutide; 13 hours 91 ), and human immunodeficiency virus (enfuvirtide; 3.4 hours 92 ) are mentioned. The phenotype (CD38++CD138+) of the BTCE recombinant cells described in this specification is similar to the phenotype of long-lived plasma cells derived from human bone marrow 50、It has been previously shown to maintain survival in humanized mice for over one year. 18 The ability of ePCs to survive over a long period and produce exogenous proteins strongly is considered to be the key to eliciting the therapeutic efficacy of many biological agents and therapeutic peptides with poor pharmacokinetics and insufficient efficacy.

[0211] Experimental methods Production of AAV6 HDR CRISPR Cas9 CCR5, JCHAIN, IgG1, Eμ 12 CRISPR RNAs (crRNAs) targeting CCR5, JCHAIN, IgG1, or CD19 were identified using a tool on the web (http: / / portals.broadinstitute.org / gpp / public / analysis-tools / sgrna-design). These crRNAs are shown in Table 9 below.

Table 9

[0212] crRNAs containing phosphorothioate linkages and 2’O-methyl modifications were synthesized (IDT). A single-guide hybrid consisting of the crRNA and trans-activating crRNA (tracrRNA; IDT) was mixed with 3 μM Cas9 nuclease (Berkeley Labs) at a ratio of 1.2:1 and delivered to cells by electroporation using Lonza 3D (CA-137) or Maxcyte GTX (B cell 3). After electroporation, the cells (1.5 million cells / mL) were transferred to activation medium in the presence of an AAV6 vector (20% v / v AAV) containing a homologous DNA repair template. The medium was changed 24 hours after AAV6 administration. The AAV vectors were produced according to previously reported methods. 19 。

[0213] In vitro injury assay (K562, PBMC, autologous killing) To perform the K562 cell killing assay, K562 cells were obtained from ATCC and transduced with lentivirus to express CD19 cis - linked to GFP (target) via self - cleaving P2A, or BCMA cis - linked to BFP (reference). Transduced cells were purified by sorting with flow cytometry. A mixture of 5×10 3 target cells, 5×10 3 reference cells, and 5×10 4 CD8+ T cells was incubated for 48 hours after adding supernatants from genome - recombined cells diluted at various multiples, or media containing various concentrations of recombinant BTCE (Invivogen, bimab - hcd19cd3) (Figure 13F, Figure 13H, Figure 15G - 15I). Also, to perform the PBMC killing assay, a mixture of 2×10 5 PBMC and 4×10 4 autologous CD8+ T cells was incubated for 48 hours after adding supernatants from genome - recombined cells or media containing recombinant BTCE (Invivogen, bimab - hcd19cd3, AMG330) (Figure 14D - 14G). Furthermore, to perform the autologous killing assay, 2×10 5 genome - recombined B cells and autologous T cells were incubated at various effector:target ratios and cultured for 24 hours (Figure 15B, Figure 15E, Figure 15F). Each assay was performed in duplicate at 37 °C, 5% CO2 with 200 μL per well of a 96 - well plate, using RPMI - 1640 supplemented with 10% FBS as the basal medium. At the end of each assay, duplicate wells were pooled, washed with PBS, stained, and analyzed by flow cytometry.

[0214] NSG mouse model NOD.Cg - Prkdc scid Il2rg tm1Wjl / SzJ - c(NSG) mice were purchased from The Jackson Laboratory. To create a subcutaneous flank model (Figure 16A - 16C), 2.5×10 5 ePC, 5×10 4 autologous T cells, and 2.5×10 4Individual Raji luciferase cells were delivered subcutaneously into the right flank. In the generation of the disseminated NL482 PDX model (Figures 16D - 16G), 2.5×10 6 to 15×10 6 GFP - ePC or BTCE - ePC cells were injected intravenously. The next day, 1×10 5 NL482.ffLuc ALL cells were administered intravenously to the mice. The next day and 3 days later, 1×10 5 or 10×10 5 T cells were administered retro - orbitally to the mice. After subcutaneous injection of luciferin (75 - 150 mg / kg), bioluminescence imaging using an IVIS Lumina S5 (PerkinElmer) was used to monitor tumor engraftment. Blood was collected from the submandibular gland vessels to obtain peripheral blood and serum (BD, 2290057). After euthanizing the mice, the bone marrow and spleen were excised, and after lysing red blood cells (ACK lysis buffer), phenotypic analysis was performed by flow cytometry (Figures 23A - 23C). The mice were housed at ambient temperature and humidity.

[0215] Statistical analysis and data availability Statistical analysis was performed using Prism7 (GraphPad, San Diego, CA). All data were assumed to be normally distributed.

[0216] Cell lines K562 cells and Raji cells were obtained from ATCC and cultured in RPMI 1640 supplemented with 10% fetal bovine serum. K562 cells were transduced with a VSV - g pseudotype virus containing CD19 placed cis to GFP, or BCMA (NP_001183) placed cis to BFP, and then purified using fluorescence - activated cell sorting (FACS) to obtain the K562 CD19 + GFP + target cell line and the K562 BCMA + BFP +Reference cell lines were prepared respectively. Also, using lentivirus, firefly luciferase (AB261984.1) and highly sensitive green fluorescent protein (HM640279.1) were transduced into Raji cells and purified by FACS.

[0217] Flow cytometry Cells were stained with appropriate antibodies. Flow cytometry analysis was performed using an LSR II flow cytometer (BD Biosciences), and events were analyzed using FlowJo software (Tree Star).

[0218] Culture of B cells and differentiation into plasmablasts B cells were isolated from PBMC of healthy human donors using an EasySep human B cell isolation kit (Stem Cell Technologies). B cells with a purity exceeding 95% defined by the CD3 negative rate and CD19 positive rate were obtained. The isolated B cells were cultured in Iscove's modified Dulbecco's medium (Gibco) supplemented with 2-mercaptoethanol (55 μM) and 10% FBS. 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[0219] As used herein, the term "comprising" is synonymous with the terms "including", "containing", or "characterized by", and has an open-ended and inclusive meaning and does not exclude additional elements or steps not described herein.

[0220] The foregoing description discloses some methods and materials of the present invention. The methods and materials of the present invention can be modified, and the manufacturing methods and apparatuses can also be modified. Such modifications can be easily understood by those skilled in the art in consideration of the present disclosure or the practice of the present invention disclosed herein. Accordingly, the present invention is not limited to the specific embodiments disclosed herein, but encompasses all possible modifications and other aspects within the true scope and spirit of the present invention.

[0221] All references, including but not limited to published patent applications, unpublished patent applications, patents, and academic literature, cited herein are hereby incorporated by reference in their entirety and form a part of this specification. In case of any conflict between the incorporated literature, patent, or patent application and the disclosure of this specification, the description of this specification shall be adopted and / or given precedence over such conflicting matters.

Claims

1. A system for modifying cells to express bispecific T cell engagers (BTCEs), (a) a nuclease or nucleic acid encoding said nuclease that can insert an expression cassette into a cellular genomic locus; and (b) a first polynucleotide encoding a homologous recombination repair (HDR) template comprising an expression cassette encoding a bispecific T cell engager (BTCE), The system wherein the nuclease may be a Cas nuclease.

2. The aforementioned cells further comprise The cell is a B cell or a precursor B cell, The system according to claim 1, wherein the cells may be selected from hematopoietic stem cells, human embryonic stem cells, induced pluripotent stem cells (iPSCs), naive B cells, memory B cells, plasmablasts, and plasma cells.

3. The system according to claim 1, further comprising a second polynucleotide encoding a guide RNA (gRNA), wherein the second polynucleotide may include a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 27.

4. The aforementioned seated position (i) containing an endogenous gene that is highly expressed in B cells compared to cells other than B cells, or (ii) containing an endogenous gene that is inactive in B cells, The system according to claim 1.

5. The system according to claim 1, wherein the locus is selected from the CCR5 gene, the JCHAIN ​​gene, the IGHM locus (also known as E-mu; hg38 genome; chr14: 105856225-105863200), the CD19 gene, and the IHG1 gene.

6. The BTCE comprises a first polypeptide that can specifically bind to a T cell antigen and a second polypeptide that can specifically bind to a tumor antigen; The T cell antigen may be CD3, and / or The system according to claim 1, wherein the tumor antigen may be selected from CD33, CD19, CD326 (EpCAM), nerve-glial cell antigen 2 (NG2), HER2, epidermal growth factor receptor (EGFR), CD66e, ephrin type A receptor 2 (EphA2), CD21, FLT3, gp100, PDL1, and CD22.

7. The system according to claim 1, wherein the BTCE is AMG 330, blinatumomab, solitomab, or teventafsp.

8. The system according to claim 1, wherein the expression cassette further comprises one or more selected from (i) to (vi): (i) A promoter which may be a constitutive promoter or an inductive promoter, which may be an MND promoter, an IgVH promoter, an EF-1α promoter or an IgHG1 promoter; (ii) an enhancer, which may be an Eμ enhancer or an SLC3A2 enhancer; (iii) A polynucleotide encoding a signal sequence which may be the signal sequence of IgHV or the signal sequence of IgHG1; (iv) Polynucleotides containing a 5'UTR derived from the IGHV gene or the IGHG1 gene; (v) Polynucleotides containing a 3'UTR derived from the IGHV gene or the IGHG1 gene; (vi) Ubiquitous chromatin opening elements (UCOEs).

9. The first polynucleotide further comprises nucleic acids homologous to the locus, The system according to claim 1, wherein the nucleic acid homologous to the aforementioned locus may contain a sequence of nucleotides having a length of about 200 to about 1500 nucleotides.

10. The first polynucleotide is contained in the vector, The vector may be a viral vector, and the viral vector may be an adeno-associated virus (AAV) vector or a lentiviral vector. The system according to claim 1, wherein the AAV vector may be an AAV6 vector.

11. The system according to claim 1, wherein the cell is characterized by one or more selected from (i) to (iv) below: (i) Mammalian cells, which may be human cells; (ii) Ex vivo cells; (iii) Autologous cells derived from the subject, or cells of the same species as the subject; (iv) Cells lacking expression of an endogenous protein to which the BTCE can specifically bind.

12. A method for modifying cells to express a bispecific T cell engager (BTCE), (a) the step of obtaining the system according to any one of claims 2 to 11; and (b) A step of obtaining modified cells by introducing a nuclease or a nucleic acid encoding the nuclease and a first polynucleotide into the cells. Includes, (i) The step prior to step (b) may further include a step of activating the cells, which may include contacting the cells with oligomerized CD40 ligand (CD40L), CpG and / or IL-21, and / or (ii) The step of inducing differentiation of the cells may further be included, The method wherein step (b) may include contacting the cells with a ribonucleoprotein (RNP) containing the nuclease and gRNA.

13. (a) cells modified by the method of claim 12; and / or (b) cells in which a genomic locus has been genetically modified to express a bispecific T cell engager (BTCE), The aforementioned cells, (i) B cells or precursor B cells, which may be cells selected from hematopoietic stem cells, human embryonic stem cells, induced pluripotent stem cells (iPSCs), naive B cells, memory B cells, plasmablasts and plasma cells; (ii) Mammalian cells, which may be human cells; (iii) Ex vivo cells; (iv) Autologous cells derived from the subject; and / or (v) Cells of the same species as the subject And, A cell may be characterized by one or more of the following (c) to (f): (c) The locus contains an endogenous gene that is highly expressed in B cells compared to cells other than B cells, or contains an endogenous gene that is inactive in B cells. The aforementioned locus may be selected from the CCR5 gene, the JCHAIN ​​gene, the IGHM locus (E-mu; hg38 genome; also known as chr14: 105856225-105863200), the CD19 gene, and the IHG1 gene; (d) The BTCE comprises a first polypeptide that can specifically bind to a T cell antigen and a second polypeptide that can specifically bind to a tumor antigen, (vi) The T cell antigen may be CD3; and / or (vii) The tumor antigen may be selected from CD33, CD19, CD326 (EpCAM), neuronal-glial cell antigen 2 (NG2), HER2, epidermal growth factor receptor (EGFR), CD66e, ephrin type A receptor 2 (EphA2), CD21, FLT3, gp100, PDL1, and CD22; (e) The BTCE is AMG 330, blinatumomab, solitomab, or teventafsp; (f) The genomic locus is modified by inserting an expression cassette containing the first nucleic acid encoding the BTCE, (viii) the first nucleic acid may be operably ligated to an endogenous promoter of the locus, or (ix) the expression cassette may further include a promoter operably ligated to the first nucleic acid. The expression cassette, (A) A promoter operably linked to the first nucleic acid, which may be an inductive promoter or a constitutive promoter, and may be a MND promoter, an IgVH promoter, an EF-1α promoter or an IgHG1 promoter; (B) An enhancer, which may be an Eμ enhancer or an SLC3A2 enhancer; (C) A polynucleotide encoding a signal sequence which may be the signal sequence of IgHV or the signal sequence of IgHG1; (D) Polynucleotides containing a 5'UTR derived from the IGHV gene or the IGHG1 gene; (E) Polynucleotides containing a 3'UTR derived from the IGHV gene or the IGHG1 gene; and / or (F) Ubiquitous chromatin opening element (UCOE) It may also include the following:

14. A pharmaceutical composition comprising the cells described in claim 13.

15. Use of the cells described in claim 13 as a pharmaceutical product for the treatment, palliative care, or suppression of a disorder or disease in the subject, or The use of cells according to claim 13 in the preparation of a pharmaceutical product for the treatment, palliative care, or suppression of a disorder or disease in a subject, It may feature one or more of the following (a) to (d): (a) The cells may be administered as a single dose or as a single bolus dose; (b) The said disorder includes cancer, The aforementioned cancer may be selected from solid tumors and leukemias. The cancers mentioned above may be selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), breast cancer, gastric cancer, malignant melanoma, colorectal cancer, colorectal cancer, head and neck cancer, prostate cancer, ovarian cancer, lung cancer, and pancreatic cancer; (c) The bispecific T cell engager (BTCE) is capable of specifically binding to the tumor antigen expressed by the cancer; (d) The subject is a human being.

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