Vector-free process for manufacturing artificial immune cells

Abstract

Disclosed herein, in certain embodiments, are vector-free methods for producing artificial immune cells. Also disclosed herein, in certain embodiments, are compositions comprising artificial immune cells obtained from the methods and processes described herein. In additional embodiments, disclosed herein are methods and kits for treating disease using artificial immune cells obtained from the methods and processes described herein.

Description

Technical Field

[0001] ·Cross - reference to related applications This application is a PCT application claiming the priority benefit of U.S. Provisional Application No. 63 / 071,236, filed Aug. 27, 2020, the entire content of which is hereby expressly incorporated by reference herein.

Background Art

[0002] Novel therapies using T cells engineered to express chimeric antigen receptors (CARs) or exogenous T cell receptors (TCRs) have provided promising immunotherapies for several types of cancer, mainly hematologic malignancies.

[0003] Chimeric antigen receptor (CAR) or T cell receptor (TCR) T cells are effector immune cells genetically modified to recognize specific tumor - associated antigens and then kill tumor cells. The production of these cells generally involves stimulation of T cells and then transduction of the cells using a viral vector such as a lentiviral vector to express the nucleic acid encoding the CAR or TCR. One limitation of current vector transduction, such as lentiviral vector transduction, is that it cannot efficiently transduce quiescent cells such as primary T cells. Previous studies have shown that stimulation and expansion of T cells result in a more differentiated T cell phenotype. Undifferentiated or unstimulated T cell populations are highly persistent and have high efficacy, but have low transfection efficiency. Furthermore, the production of T cells for adoptive therapies using lentiviral vectors requires up to 10 - 12 days from cell collection to preparation for administration.

[0004] Therefore, there is a need for an improved method of T cell transduction using nucleic acids encoding exogenous immune receptors while maintaining an undifferentiated phenotype and reducing manufacturing time. The present invention provides a method to address these needs.

Summary of the Invention

[0005] In certain embodiments, disclosed herein is a vector-free method and process for manufacturing engineered immune cells. In some embodiments, also disclosed herein are compositions comprising engineered immune cells obtained from the methods and processes described herein. In additional embodiments, disclosed herein are methods and kits for treating diseases using engineered immune cells obtained from the methods and processes described herein.

[0006] In some embodiments, disclosed herein is a vector-free method for preparing a modified unstimulated immune cell population, comprising: (a) delivering a homology-directed repair (HDR) template comprising (i) a gene editing nuclease, (ii) a guide RNA, and (iii) a polynucleotide encoding an antigen-binding polypeptide to an unstimulated immune cell population obtained from a biological sample by a transfection method; and (b) culturing the population for about 72 hours or less under non-expansion conditions, wherein the gene editing nuclease and the guide RNA form a complex and generate a double-stranded break at a target site within at least one unstimulated immune cell and The HDR template promotes HDR at the target site and generates at least one modified non-stimulated immune cell within the population. It is a method.

[0007] In some embodiments, the following are disclosed herein: A vector-free method for preparing a population of modified, unstimulated immune cells, (a) A process to obtain an enriched population of non-stimulated immune cells from a biological sample, (b) A step of delivering a homology-directed repair (HDR) template comprising (i) a gene editing nuclease, (ii) a guide RNA, and (iii) a polynucleotide encoding an antigen-binding polypeptide to the enriched population by transfection, (c) A step of culturing the population under non-expansion conditions, The gene editing nuclease and the guide RNA form a complex that generates double-strand breaks at target sites in multiple unstimulated immune cells within the population. The HDR template promotes HDR at the target site, generates a plurality of modified non-stimulated immune cells within the population, and Approximately 10% or more of the unstimulated immune cells in the aforementioned population are modified. It is a method.

[0008] In some embodiments, the following are disclosed herein: A vector-free method for generating a population of modified, unstimulated immune cells, (a) A step of inducing homology-directed repair (HDR) in approximately 10% or more of an unstimulated immune cell population by the following: (i) A step of contacting the unstimulated immune cell population with a homology-directed repair (HDR) template containing a gene-editing nuclease and a polynucleotide encoding an antigen-binding polypeptide, and (ii) A step of delivering the gene editing nuclease and the HDR template into the unstimulated immune cells by transfection, (b) A step of culturing the unstimulated immune cells under non-expansion conditions for about 72 hours or less to generate the modified unstimulated immune cell population, Methods that include... In some embodiments, this method is (a) A step of bringing the unstimulated immune cell population into contact with guide RNA and delivering the guide RNA to the unstimulated immune cells by transfection, and optionally, (b) A step of causing a double-strand break at a target site in the genome of one or more unstimulated immune cells using the gene editing nuclease and optionally the guide RNA. This may further include: In yet another embodiment, in these methods, the HDR template promotes HDR at the target site in about 10% or more of the unstimulated immune cell population. Furthermore, the unstimulated immune cell population is obtained from a biological sample and / or CD4 + T cells, CD8 + This may include enriched populations of T cells or combinations thereof. In some embodiments, the immune cells include T cells, natural killer cells, natural killer T cells, macrophages, monocytes, B cells, hematopoietic stem cells, or a combination thereof. In some embodiments, the immune cells consist of T cells, natural killer cells, natural killer T cells, macrophages, monocytes, B cells, hematopoietic stem cells, or a combination thereof.

[0009] In some embodiments relating to all of the methods described herein, The aforementioned unstimulated immune cell population may include, for example, approximately 5 million, 10 million, 20 million, 50 million, 100 million, 500 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion cells, or more.

[0010] In some embodiments relating to all of the methods described herein, The transfection method described above is: (a) capable of providing efficiencies of approximately 10% to approximately 99%, approximately 12% to approximately 99%, approximately 13% to approximately 99%, approximately 15% to approximately 99%, approximately 20% to approximately 99%, approximately 30% to approximately 99%, approximately 40% to approximately 99%, approximately 50% to approximately 99%, approximately 60% to approximately 99%, approximately 70% to approximately 99%, approximately 10% to approximately 80%, approximately 12% to approximately 80%, approximately 13% to approximately 80%, approximately 15% to approximately 80%, approximately 20% to approximately 80%, approximately 30% to approximately 80%, approximately 40% to approximately 80%, approximately 50% to approximately 80%, approximately 20% to approximately 70%, or approximately 30% to approximately 60%, and / or (b) May provide efficiencies of approximately 10%, 12%, 13%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.

[0011] In some embodiments relating to all of the methods described herein, The aforementioned method, (a) Approximately 10% to approximately 99%, approximately 12% to approximately 99%, approximately 13% to approximately 99%, approximately 15% to approximately 99%, approximately 20% to approximately 99%, approximately 30% to approximately 99%, approximately 40% to approximately 99%, approximately 50% to approximately 99%, approximately 60% to approximately 99%, approximately 70% to approximately 99%, approximately 10% to approximately 80%, approximately 12% to approximately 80%, approximately 13% to approximately 80%, approximately 15% to approximately 80%, approximately 20% to approximately 80%, approximately 30% to approximately 80%, approximately 40% to approximately 80%, approximately 50% to approximately 80%, approximately 20% to approximately 70%, or approximately 30% to approximately 60%, and / or (b) May provide cell viability of approximately 10%, 12%, 13%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.

[0012] In some embodiments relating to all of the methods described herein, (a) The HDR template, in a volume of approximately 1 pM to approximately 10 mM, is delivered into the unstimulated immune cells, and / or (b) The gene-editing nuclease is delivered into the unstimulated immune cells in a concentration of approximately 1 pM to 10 mM, and / or (c) The guide RNA in a concentration of approximately 1 pM to 10 mM is delivered into unstimulated immune cells.

[0013] In some embodiments relating to all of the methods described herein, (a) The ratio of the gene editing nuclease to the guide RNA is approximately 10:1, approximately 5:1, approximately 2:1, approximately 1:1, approximately 1:2, approximately 1:5, or approximately 1:10, and / or (b) The ratio of the HDR template to the complex formed between the gene editing nuclease and the guide RNA is approximately 5:1, approximately 2:1, approximately 1:1, approximately 1:2 or approximately 1:5, and / or (c) The ratio of the HDR template to the gene editing nuclease is approximately 5:1, approximately 2:1, approximately 1:1, approximately 1:2, or approximately 1:5.

[0014] In some embodiments relating to all of the methods described herein, the complex is a ribonucleoprotein (RNP) complex.

[0015] In some embodiments relating to all of the methods described herein, The aforementioned unstimulated immune cell population is (a) including unstimulated T cells, unstimulated natural killer (NK) cells, unstimulated natural killer T (NKT) cells, or a combination thereof, and / or (b) CD4 + T cells, CD8 + T cells, CD4 + / CD8 + Includes T cells or combinations thereof, includes unstimulated T cells, and / or (c)CD4 + T cells, CD8 + This includes enriched populations of T cells or combinations thereof.

[0016] In some embodiments relating to all of the methods described herein, the enriched population comprises about 90%, about 95%, about 99% or about 100% of CD4 + T cells, CD8 + T cells or a combination thereof.

[0017] In some embodiments relating to all of the methods described herein, the method further comprises incubating the biological sample with a plurality of CD4 and / or CD8 labeled microbeads, optionally magnetic microbeads, prior to generating the population of unstimulated immune cells. In another aspect, the step further comprises (a) incubating the biological sample with a solution comprising albumin, optionally human serum albumin (HSA), and / or (b) a cell selection process for enriching the unstimulated cells in the population, and / or (c) incubating the enriched unstimulated cells in a cell culture medium comprising minimum media, HSA, cytokines, supplements or a combination thereof. comprises.

[0018] In some embodiments relating to all of the methods described herein, the modified unstimulated immune cells are (a) cultured under non-expansion conditions for about 48 hours or less, about 36 hours or less, about 24 hours or less or about 18 hours or less, and / or (b) cultured under non-expansion conditions for about 18 hours, about 24 hours, about 36 hours, about 48 hours or about 72 hours, and / or (c) further resuspended in a cryopreservation solution and cryopreserved.

[0019] In some embodiments relating to all of the methods described herein, The aforementioned transfection method is Electroporation, or Cell squeezing method, Includes.

[0020] In some embodiments relating to all of the methods described herein, The aforementioned antigen-binding polypeptide is (a) Containing an antigen-binding domain, (b) containing a chimeric antigen receptor (CAR), (c) containing a cell surface receptor ligand, or (d) containing a T cell receptor (TCR) and / or (e) Binds to tumor antigens. In another embodiment, (a) The antigen-binding domain includes a full-length antibody or its antigen-binding fragment, Fab, F(ab)2, monospecific Fab2, bispecific Fab2, trispecific Fab2, single-chain variable fragment (scFv), diabody, triabody, minibody, V-NAR or VhH, and / or (b) The CAR comprises the antigen-binding domain, the transmembrane domain, and the intracellular domain, and optionally the CAR includes a hinge region. In a further manner, (a) The transmembrane domain is selected from an artificial hydrophobic sequence, a transmembrane domain of a type I transmembrane protein, the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), ICOS (CD278), CD154, and a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR), and / or (b) The intracellular domain includes a costimulatory signaling domain and / or an intracellular signaling domain, (c) The intracellular domain comprises one or more costimulatory domains of proteins selected from the group consisting of proteins in the TNFR superfamily, CD27, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS (CD278), NKG2C, B7-H3 (CD276), intracellular domains obtained from killer immunoglobulin-like receptors (KIRs), or variants thereof. In yet another embodiment (a) The co-stimulatory domain includes the 4-1BB(CD137) co-stimulatory domain and / or (b) The intracellular signaling domain is the cytoplasmic signaling domain of the human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of the Fc receptor, a cytoplasmic receptor having an immune receptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d, or their variants, and / or (c) The intracellular signaling domain includes a 4-1BB costimulatory domain and a human CD3 zeta chain (CD3ζ) cytoplasmic signaling domain. In yet another embodiment, the cytoplasmic signaling domain includes a human CD3 zeta chain (CD3ζ).

[0021] In a method for binding antigen-binding polypeptides to tumor antigens, The aforementioned tumor antigen (a) May be associated with hematological malignancies, and / or (b) CD19, CD20, CD22 and CD33 / IL3Ra may be selected, and / or (c) potentially related to solid tumors and / or (d) Can be selected from ROR1, mesoserine, c-Met, PSMA, PSCA, folate receptor alpha, folate receptor beta, EGFRvIII, GPC2, TnMUC1, GDNF family receptor alpha-4 (GFRa4), fibroblast activating protein (FAP), and IL13Ra2.

[0022] In a method in which the antigen-binding polypeptide comprises a T cell receptor (TCR), The aforementioned TCR is, May include TCR alpha chains and TCR beta chains, and / or The selection is made from wild-type TCRs, high-affinity TCRs, and chimeric TCRs.

[0023] For all of the methods described herein, in some embodiments, The aforementioned HDR template, (a) The polynucleotide may further include a 5' homology arm upstream of the polynucleotide, and / or (b) The polynucleotide may further include a 3' homology arm downstream of the polynucleotide, and / or (c) may be a double-stranded DNA template, and / or (d) A double-stranded DNA template, the HDR template having a length of approximately 2 kilobase pairs (kb) to approximately 5 kb, approximately 2.3 kb to approximately 5 kb, approximately 3 kb to approximately 5 kb, approximately 3 kb to approximately 4 kb, approximately 2 kb to approximately 4 kb, approximately 2.3 kb to approximately 4 kb, approximately 2 kb to approximately 3 kb, approximately 2.3 kb to approximately 3 kb, or approximately 4 kb to approximately 5 kb, and / or (e) It may be delivered by electroporation. In another embodiment, The aforementioned 5' homology arm is, (a) can be adjacent to the polynucleotide and / or, (b) The target site may be homologous to the genomic region 5', and / or (c) lengths of approximately 50 to 500 nucleotides, approximately 50 to 400 nucleotides, approximately 50 to 300 nucleotides, approximately 50 to 200 nucleotides, approximately 50 to 150 nucleotides, approximately 100 to 500 nucleotides, approximately 100 to 400 nucleotides, approximately 100 to 300 nucleotides, approximately 100 to 200 nucleotides, approximately 200 to 500 nucleotides, approximately 200 to 400 nucleotides, approximately 200 to 300 nucleotides, approximately 300 to 500 nucleotides, or approximately 300 to 400 nucleotides, and / or (d) Nucleotides may be of a length of approximately 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500. In yet another embodiment, The 3' homology arm is, (a) can be adjacent to the polynucleotide and / or, (b) The target site may be homologous to the genomic region 3', and / or (c) A combination of approximately 50 to approximately 500 nucleotides, approximately 50 to approximately 400 nucleotides, approximately 50 to approximately 300 nucleotides, approximately 50 to approximately 200 nucleotides, approximately 50 to approximately 150 nucleotides, approximately 100 to approximately 500 nucleotides, approximately 100 to approximately 400 nucleotides, approximately 100 to approximately 300 nucleotides, approximately 100 to approximately 200 nucleotides, approximately 200 to approximately 500 nucleotides, approximately 200 to approximately 400 nucleotides, approximately 200 to approximately 300 nucleotides, approximately 300 to approximately 500 nucleotides, or a combination of approximately 300 to approximately 400 nucleotides, and / or (d) Nucleotides may be of a length of approximately 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500.

[0024] For all of the methods described herein, in some embodiments, the target site is the TRAC locus and optionally exon 1 of the TRAC locus.

[0025] For all of the methods described herein, in some embodiments, The aforementioned gene editing nuclease is (a) Cas nuclease, and / or (b) Zinc finger nuclease, and / or (c) Contains transcription activator-like effector nuclease (TALEN). In another embodiment, The aforementioned Cas nuclease is, (a) Cas9, optionally SpCas9 or SaCas9, and / or (b) and the guide RNA assemble into a complex before being delivered to unstimulated immune cells, and / or (c) The guide RNA is delivered to unstimulated immune cells and then assembles into a complex.

[0026] For all of the methods described herein, in some embodiments, The method further comprises the step of delivering one or more additional guide RNAs to the unstimulated immune cells.

[0027] For all of the methods described herein, in some embodiments, The aforementioned biological sample is (a) is a blood sample and / or (b) A blood sample, which is a whole blood sample, a peripheral blood mononuclear cell (PBMC) sample, or an apheresis sample, and / or (c) A blood sample, which is an apheresis sample that is cryopreserved, and / or (d) A blood sample, which is a fresh apheresis sample.

[0028] For all of the methods described herein, in some embodiments, the method further includes the steps of stimulating the modified non-stimulated immune cells to generate a population of modified stimulated immune cells, and optionally expanding the population of modified stimulated immune cells. In another embodiment, The modified stimulated immune cell population can be cultured under expanded conditions for approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days or longer.

[0029] In some embodiments, disclosed herein is a population of modified, unstimulated immune cells generated by the method described herein.

[0030] In some embodiments, disclosed herein is a population of modified stimulated immune cells generated by the method described herein.

[0031] In some embodiments, disclosed herein are compositions comprising a population of modified unstimulated immune cells generated by the method herein or a population of modified stimulated immune cells generated by the method herein, and optionally comprising pharmaceutically acceptable excipients.

[0032] In some embodiments, disclosed herein are methods for treating a disease in a subject requiring such treatment, comprising administering to a population of modified unstimulated immune cells produced by the method herein, or to a population of modified stimulated immune cells produced by the method herein, or to administer a composition comprising a population of modified unstimulated immune cells or a population of modified stimulated immune cells.

[0033] In some embodiments, the subject has cancer such as a solid tumor. In another embodiment of the method for treating the disease described herein, the cancer is a hematological malignancy. In yet another embodiment of the method for treating the disease described herein, the antigen-binding domain is specific to the antigen expressed by the cancer. In another embodiment, the biological sample is autologous to the subject. Alternatively, the biological sample may be allogeneic with respect to the subject. Finally, in all methods of treating the disease, the subject is human.

[0034] In some embodiments, disclosed herein are kits comprising a modified unstimulated immune cell population obtained from the method described herein or a modified stimulated immune cell population obtained from the method described herein.

[0035] In some embodiments, the following are disclosed herein: A vector-free method for preparing modified, unstimulated cell populations selected from liver, skin, or pancreatic cells. (a) A process of delivering a homology-directed repair (HDR) template, comprising (i) a gene editing nuclease, (ii) a guide RNA, and (iii) a polynucleotide encoding an antigen-binding polypeptide, to an unstimulated cell population obtained from a biological sample by transfection. (b) The step includes culturing the population under non-expansion conditions for about 72 hours or less, The gene-editing nuclease and the guide RNA form a complex that generates a double-strand break at a target site in at least one unstimulated immune cell. The HDR template promotes HDR at the target site and generates at least one modified non-stimulated immune cell within the population.

[0036] In some embodiments, disclosed herein are vector-free methods for preparing modified, unstimulated cell populations selected from liver, skin, or pancreatic cells. (a) A process to obtain an enriched population of unstimulated cells from a biological sample, (b) A step of delivering a homology-directed repair (HDR) template comprising (i) a gene editing nuclease, (ii) a guide RNA, and (iii) a polynucleotide encoding an antigen-binding polypeptide to the enriched population by transfection, (c) A step of culturing the population under non-expansion conditions, The gene editing nuclease and guide RNA form a complex that generates double-strand breaks at target sites in multiple unstimulated cells within the population. The HDR template promotes HDR at the target site, generating multiple modified non-stimulated cells, with approximately 10% or more of the non-stimulated cells in the population being modified.

[0037] In some embodiments, the hepatocytes may be selected from one or more of hepatocytes, hepatic astrocytes, sinusoidal endothelial cells, and Kupffer cells. In some embodiments, the skin cells may be selected from one or more of keratinocytes, melanosomes, melanocytes, Langerhans cells, and Merkel cells. In some embodiments, the pancreatic cells may be selected from one or more of the following: pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic gamma cells, and pancreatic epsilon cells.

[0038] Both the above summary and the following drawings and detailed descriptions are illustrative and descriptive. They are intended to provide further details of the disclosure, but are not intended to be limiting. Other purposes, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the disclosure. [Brief explanation of the drawing]

[0039] Figures 1A to 1C are schematic diagrams illustrating the vector-free manufacturing processes described herein. Figure 1A illustrates a schematic of the vector-free manufacturing process for artificial immune cells. Figure 1B illustrates a schematic of the manufacturing process for clinical-grade vector-free artificial T cells. Figure 1C shows a schematic diagram of the manufacturing process for allogeneic heterozygous T cells.

[0040] Figures 2A and 2B are illustrative schematic diagrams of the vector-free manufacturing process for artificial T cells. Figure 2A details the manufacturing process on day 0, and Figure 2B details the manufacturing process from day 0 to day 3 / harvest.

[0041] Figures 3A and 3B show the disruption of endogenous TCR expression in unstimulated T cells by CRISPR / Cas9 using TRAC-targeted gRNA. CRISPR / Cas9 gene editing was performed to target the TCR alpha constant (TRAC) gene locus in unstimulated T cells. Two CRISPR / Cas9 ribonucleoprotein (RNP) systems (Truecut.v2 Cas9 (ThermoFisher Scientific) and SpyFi Cas9 (Aldevron)) were tested. Figure 3A depicts the percentage of CD3+TCR+ / live cells for positive control, Truecut, and SpyFi. Figure 3B also shows the percentage of CD3+TCR+ / live cells for positive control, Truecut, and SpyFi.

[0042] Figure 4 is an illustrative schematic diagram of the process of introducing donor DNA containing the EcoRI site into exon 1 of the TRAC gene locus by homology-directed repair of double-strand DNA breaks.

[0043] Figures 5A and 5B show gel electrophoresis images of PCR amplicons digested with EcoRI to demonstrate HDR-mediated insertion into chemically modified ssDNA donors or "ultramers".

[0044] Figure 6 is an illustrative schematic diagram showing a knock-in strategy in which donor DNA encoding the NY-ESO-1 TCR is inserted into the TRAC locus.

[0045] Figure 7 shows targeted insertion of a GFP HDR cassette into the T cell genome. [Modes for carrying out the invention]

[0046] • Detailed explanation 1. Overview CAR-T cells and TCR cells offer exciting prospects for cancer patients. However, several challenges currently exist in the production of CAR-T and TCR cells, and these challenges will affect the likelihood of success for these cancer therapies. First, current CAR-T and TCR manufacturing processes generally utilize viral vector systems for propagation. However, this type of propagation method requires a period of approximately nine days before cell harvesting. This disclosure details a remarkable vector-free method that dramatically reduces the time to harvest CAR-T and / or TCR cells, for example, to about 72 hours or less.

[0047] A second challenge present in current technology and addressed by this disclosure is the need to scale up production. This disclosure details a vector-free process that can be remarkably scaled up with acceptable insertion efficiency, even in large-scale production. In particular, short process times significantly impact the process's ability to scale up. Therefore, it was remarkable that it was possible to design short CAR-T and TCR manufacturing processes that could be carried out at a large-scale production level and had a high insertion efficiency.

[0048] Another surprising aspect of this disclosure is the effect of cultured stimulated versus unstimulated cells. Generally, insertion efficiency is higher with stimulated cells, but the manufacturing process is simpler with unstimulated cells. One problem is that moving cells between culture media results in high cell loss. Furthermore, the use of undifferentiated cells allows for the use of multiple different non-stimulated immune cell populations, such as CD4. + or CD8 + Because it can produce T cells (for example, cells with different, non-uniform characteristics), it is possible to manufacture better products. The methods described herein can be used with stimulated cells (which produce higher insertion efficiency) or unstimulated cells (which can produce a less differentiated, more potent end product).

[0049] These advantages and discoveries are described in more detail below.

[0050] II. Methods for Manufacturing Artificial Immune Cells In one embodiment, disclosed herein is a method for preparing modified immune cells or their precursor cells (e.g., modified T cells, modified natural killer (NK) cells, modified natural killer T (NKT) cells, modified macrophages, modified monocytes, modified B cells, or modified hematopoietic stem cells). In some embodiments, the modified immune cells or their progenitor cells are modified unstimulated immune cells or their progenitor cells that express an exogenous antigen-binding polypeptide (e.g., modified unstimulated T cells, modified unstimulated NK cells, modified unstimulated NKT cells, modified unstimulated macrophages, modified unstimulated monocytes, modified unstimulated B cells, or modified unstimulated hematopoietic stem cells). In some embodiments, the antigen-binding polypeptide comprises a chimeric antigen receptor (CAR) and / or an exogenous T cell receptor (TCR). In some embodiments, the antigen-binding polypeptide comprises an antigen-binding domain, a cell surface receptor ligand, or a polypeptide that binds to a tumor antigen. In some embodiments, the method is a Good Manufacturing Practice (GMP) method, for example, compliant with the regulations of the U.S. Food and Drug Administration (FDA) or compliant with the U.S. FDA equivalent in a foreign jurisdiction.

[0051] In some embodiments, the method for producing artificial cells is not limited to immune cells, but further includes mammalian cells from the liver, skin, and pancreas. Any method used herein as applicable to immune cells is also applicable to cells from the liver, skin, and pancreas. In some embodiments, cells from the liver, skin, and pancreas are not subjected to stimulation. In some embodiments, the hepatocytes may be selected from one or more of hepatocytes, hepatic astrocytes, sinusoidal endothelial cells, and Kupffer cells. In some embodiments, skin cells may be selected from one or more of keratinocytes, melanosomes, melanocytes, Langerhans cells, and Merkel cells. In some embodiments, the pancreatic cells may be selected from one or more of the following: pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic gamma cells, and pancreatic epsilon cells.

[0052] In some embodiments, methods for preparing artificial or modified immune cells or their precursor cells are shown in Figure 1A. As shown in Figure 1, the biological sample 101 was processed through a cell separation system to enrich unstimulated immune cells 102 (e.g., enriched unstimulated CD4). + and CD8 + A population of T cells is generated. Next, the enriched unstimulated immune cells 102 (for example, enriched unstimulated CD4) + and CD8 + T cells are transfected with a gene editing system and template (e.g., HDR template), and then cultured for up to approximately 72 hours to modify unstimulated immune cells 103 (e.g., modified unstimulated CD4). + and CD8 + A population of T cells is generated. Next, the modified unstimulated immune cells 103 are cryopreserved to generate cryopreserved modified unstimulated immune cells 104. Optionally, the modified unstimulated immune cells 104 may be stimulated and expanded for up to about 10 to 12 days to generate modified stimulated immune cells 105. The modified stimulated immune cells 105 can be further cryopreserved to generate cryopreserved modified stimulated immune cells 106. In some embodiments, the manufacturing method is a vector-free method for preparing modified immune cells or their progenitor cells. See also Figures 1B and 1C, which outline the vector-free manufacturing processes for clinical-grade artificial T cells and allogeneic atypical T cells, respectively. Depending on the circumstances, the manufacturing method may improve the efficiency of transfection, the yield of modified non-stimulated or stimulated immune cells, cell viability, or any combination thereof.

[0053] A. Selection and enrichment of non-stimulating immune cells In some embodiments, the unstimulated immune cells described herein are unstimulated T cells. The T cells may be cytotoxic T cells, regulatory T cells, or NKT cells. In an exemplary embodiment, the T cells are CD8 + T cells or CD4 + These are T cells. In some embodiments, populations of unstimulated immune cells are collected from biological samples from a subject, such as tissue, fluid, or other samples from the subject. In some embodiments, the biological sample is a tissue or organ sample, such as liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, neck, testis, ovary, tonsil, spleen, lymph node, or tumor tissue or cells derived therefrom. In some embodiments, the biological sample is a fluid sample, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, or sweat. In some embodiments, the biological sample is a blood sample, which is optionally selected from a whole blood sample, a peripheral blood mononuclear cell (PBMC) sample, or an apheresis sample. In some embodiments, the samples are of heterogeneous origin, such as mice, rats, non-human primates, or pigs.

[0054] In some embodiments, the biological sample is an apheresis sample from a patient. In some embodiments, the apheresis sample is a leukocyte sample. In some embodiments, the apheresis sample (e.g., a leukocyte sample) is cryopreserved before the collection of immune cells or a population of immune cells. In some embodiments, the apheresis sample (e.g., a leukocyte sample) is a fresh apheresis sample from a patient that has not been cryopreserved. In some embodiments, the immune cells or immune cell population are obtained from an apheresis sample (e.g., a leukocyte sample) during a process or protocol that includes a concentration step.

[0055] In some embodiments, unstimulated immune cells, particularly unstimulated T cell populations, are isolated and concentrated in one or more selection steps, for example, one or more depletion steps (e.g., removal of non-immune cells or non-T cells). In some examples, the isolation step further comprises one or more separation steps, each comprising separation based on one or more characteristics such as size, density, sensitivity or resistance to a particular reagent, and / or affinity to an antibody or other binding partner, e.g., immunoaffinity. In some embodiments, the isolation is performed sequentially and / or simultaneously using the same apparatus or equipment in a single process stream. In some embodiments, the isolation, culture, and / or engineering of the different populations are carried out from the same starting composition or material, for example, from the same sample.

[0056] In some embodiments, unstimulated immune cell populations are isolated in a closed system or apparatus and / or in the same vessel or set of vessels, e.g., the same (or the same set of) units, chambers, columns, e.g., magnetic separation columns, tubes, tube sets, culture or cultivation chambers, culture vessels, processing units, cell separation vessels, centrifuge chambers. For example, in some cases, the isolation of the unstimulated immune cell population is carried out in a system or apparatus employing a single or identical isolation or separation container or container set, such as a single column or set of columns and / or the same tube or set of tubes, without requiring, for example, the cell population, composition, or suspension to be transferred from one container, such as a set of tubes, to another container.

[0057] In some embodiments, simultaneous or sequential selection is employed to select multiple different populations of unstimulated immune cells, such as CD4. + or CD8 + T cells are selected, enriched, and / or isolated. In some embodiments, the separation step includes the separation of different cell types based on the expression or presence of one or more specific molecules within the cell, such as surface markers, surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation is affinity-based or immunoaffinity-based. For example, in some embodiments, the separation involves, for example, incubation with an antibody or binding partner that specifically binds to such markers, separating cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, followed generally by a washing step and separation of cells bound to the antibody or binding partner from cells that did not bind to the antibody or binding partner.

[0058] Such a separation step may be based on positive selection to retain the unstimulated immune cells bound to the reagent for further use, and / or negative selection to retain the cells that are not bound to the antibody or binding partner. In some cases, both fractions are retained for further use. In some embodiments, negative selection is particularly useful when antibodies that specifically identify cell types in heterogeneous populations are unavailable, and it appears optimal to perform separation based on markers expressed by cells from populations other than the desired population.

[0059] Isolation does not necessarily result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as those expressing a marker, means increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, such as those expressing a marker, means decreasing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells. For example, in some cases, CD4 + and CD8 + Population selection enriches the population but may also result in the retention or small percentage of other unselected cells. In such cases, the retention percentage of other unselected cells may be less than or equal to about 10%, less than or equal to about 9%, less than or equal to about 8%, less than or equal to about 7%, less than or equal to about 6%, less than or equal to about 5%, less than or equal to about 4%, less than or equal to about 3%, less than or equal to about 2%, less than or equal to about 1%, less than or equal to about 0.5%, less than or equal to about 0.1%, or less than or equal to those percentages.

[0060] In some examples, multiple rounds of separation steps can be performed, where fractions selected as positive or negative from one step are then subjected to another separation step that also selects them as positive or negative. In some cases, cells expressing multiple markers simultaneously can be depleted in a single isolation step by incubating cells with multiple antibodies or binding partners specific to each of the markers targeted for negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressing various cell types.

[0061] In some embodiments, selection and enrichment of unstimulated T cell populations by positive or negative selection can be achieved, for example, by a combination of antibodies directed at surface markers specific to positively or negatively selected cells. In some embodiments, CD4 + A cocktail of monoclonal antibodies directed at cell surface markers present on T cells includes antibodies against CD45RA, CCR7, CD62L, CD127 (IL-7Rα), and / or CD132. In some embodiments, CD8 + A cocktail of monoclonal antibodies directed at cell surface markers present on T cells includes antibodies against CD62L, CCR7, and / or CD127 (IL-7Rα). In an additional embodiment, CD4 + and / or CD8 + A cocktail of monoclonal antibodies directed at cell surface markers present on T cells includes antibodies against CD2, CD3, CD27, and / or TCR. In some embodiments, negative selection leads to CD4 - and CD8 -A cocktail of monoclonal antibodies directed at cell surface markers present on cells includes antibodies against CD14, CD20, CD11b, CD16, and / or HLA-DR.

[0062] In some embodiments, the method is CD4 + and CD8 + This includes the separate selection of T cells. In some embodiments, the method is CD4 + This includes a first positive selection of T cells, in which unselected cells (CD4) from the first selection are removed. - Cells) CD8 + It is used as a cell source for a second positive selection to enrich T cells. In another embodiment, the method is CD8 + This includes a first positive selection of T cells, in which unselected cells (CD8) from the first selection are removed. - Cells) CD4 + It is used as a second cell source for site selection to enrich T cells.

[0063] In some embodiments, the method is CD4 + T cells and CD8 + This includes simultaneous selection of T cells. In some embodiments, the method includes positive selection, in which case CD4 + and CD8 + T cells are selected, and then CD4- and CD8- cells are removed.

[0064] In some embodiments, methods for isolating, selecting, and / or enriching unstimulated immune cells by any of the methods described above, such as positive or negative selection based on cell surface markers or marker expression, may include selection based on immunoaffinity. In some embodiments, immunoaffinity-based selection is CD4 + and CD8 +This involves contacting a sample containing cells, such as primary human T cells, with a cell surface marker or an antibody or binding partner that specifically binds to the marker. In some embodiments, the antibody or binding partner is bound to a solid support or matrix such as spheres or beads, e.g., microbeads, nanobeads containing agarose, magnetic beads, or paramagnetic beads, to enable the separation of cells for positive and / or negative selection. In some embodiments, the spheres or beads can be packed into a column to perform immunoaffinity chromatography, in which case CD4 + and CD8 + Samples containing cells, such as primary human T cells, come into contact with the column matrix and are subsequently eluted or released from it.

[0065] In some embodiments, a sample or composition of unstimulated immune cells to be isolated is incubated with a small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, e.g., paramagnetic beads. The magnetically responsive material, for example, particles, generally consists of cells, molecules present on a cell population or cell group that are to be separated, such as binding partners that specifically bind to surface markers, such as antibodies that are directly or indirectly attached, for example, for negative or positive selection. Such beads are known and, in some embodiments, are commercially available from various sources, including DYNABEADS® (Life Technologies, Carlsbad, Calif.), MACS® beads (Miltenyi Biotec, San Diego, Calif.), or STREPTAMER® bead reagent (IBA, Germany).

[0066] Incubation is generally carried out under conditions such that molecules such as antibodies or binding partners attached to the magnetic particles or beads, or secondary antibodies or other reagents that specifically bind to such antibodies or binding partners, specifically bind to cell surface molecules if they are present on cells in the sample.

[0067] In some embodiments, the sample is placed in a magnetic field, and cells having magnetically responsive or magnetizable particles attached to it will be attracted to the magnet and separated from unlabeled cells. In positive selection, cells attracted to the magnet are retained, while in negative selection, cells not attracted (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selection is performed during the same selection step, and the positive and negative fractions are retained and further processed or subjected to further separation steps.

[0068] In some embodiments, the affinity-based selection is performed via magnetically activated cell sorting (MACS) (Miltenyi Biotech, Auburn, Calif.). The aforementioned magnetic activation cell sorting (MACS) system makes it possible to select cells to which magnetized particles are attached with high purity. In one embodiment, MACS operates in a mode in which non-target and target species are sequentially eluted after the application of an external magnetic field. That is, cells attached to magnetized particles are retained in place while non-attached species are eluted. After this initial elution step is complete, species that were trapped by the magnetic field and whose elution was prevented are released in some way so that they can be eluted and recovered. In one embodiment, non-target cells are labeled and depleted from a heterogeneous population of cells.

[0069] In some embodiments, the antibody that specifically binds to a bead or other surface-associated or coated cell surface marker is either a full-length antibody or its antigen-binding fragment, which may be a (Fab) fragment, an F(ab')2 fragment, a Fab' fragment, an Fv fragment, a single-chain antibody fragment containing a variable heavy chain (VH) region capable of specifically binding to an antigen, a single-chain variable fragment (scFv), or a single-domain antibody (e.g., sdAb, sdFv, nanobody) fragment. In some embodiments, the antibody is a Fab fragment. In some embodiments, the antibody may be monovalent, bivalent, or polyvalent. In some embodiments, antibodies such as Fab are polymers. In some embodiments, antibodies such as Fab multimers form polyvalent complexes with cell surface markers.

[0070] For the isolation of a desired population of unstimulated immune cells by positive or negative selection, the concentrations of cells and surface particles (e.g., beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (for example, by increasing the cell concentration) in order to ensure maximum contact between the cells and the beads. For example, in one embodiment, concentrations of 10 billion cells / ml, 9 billion cells / ml, 8 billion cells / ml, 7 billion cells / ml, 6 billion cells / ml, or 5 billion cells / ml are used. In one embodiment, a concentration of 1 billion cells / ml is used. In another embodiment, concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml are used. In a further embodiment, concentrations of 125 million cells / ml or 150 million cells / ml may be used.

[0071] In some embodiments, the isolation and concentration of unstimulated immune cells are carried out using an automated system, such as a CliniMACS system. In some embodiments, the method uses antibody-conjugated magnetizable particles supplied in a sterile, non-pyrogenic solution. In some embodiments, after labeling cells with magnetic particles, the cells are washed to remove excess particles. Next, a cell preparation bag is connected to a tube set, which in turn is connected to a buffer bag and a cell collection bag. The tube set consists of pre-assembled, sterile tubes, including a pre-column and a separation column, and is for single use only. When the separation program is started, the system automatically applies the cell sample to the separation column. Labeled cells are retained in the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell population for use in the method herein is unlabeled and is not retained in the column. In some embodiments, the cell population for use with the method described herein is labeled and retained within the column. In some embodiments, the cell population for use with the method described herein is eluted from the column after the magnetic field is removed and collected in the cell collection bag.

[0072] In some embodiments, the isolation and concentration of unstimulated immune cells are performed using an automated system such as the CliniMACS Plus (Miltenyi) system combined with a filtration system such as the LOVO Cell Processing System (Fresenius Kabi). In some embodiments, unstimulated immune cells are separated from the supernatant in a rotating membrane filtration step. In particular, cells within a certain cutoff range, e.g., <4 μm, are removed by passing through the membrane pores with the supernatant, while cells larger than 4 μm are retained in the filter chamber and subsequently collected. The collected cells are further processed through the CliniMACS Plus system.

[0073] In some embodiments, the separation and concentration steps are carried out using a system equipped with a cell processing unit that enables automated washing and fractionation of cells by centrifugation. In some embodiments, the separation and concentration steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The system, which includes a cell processing unit, may also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by identifying macroscopic layers of the cell product source. For example, peripheral blood is automatically separated into erythrocyte, leukocyte, and plasma layers. Cell processing systems such as the CliniMACS Prodigy system may also include an integrated cell culture chamber that achieves cell culture protocols such as cell differentiation and expansion, antigen loading, and long-term cell culture. An input port allows for sterile removal and replenishment of the culture medium, and cells may be monitored using an integrated microscope. For example, see Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[0074] In some embodiments, the cell populations described herein are collected and concentrated (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are transported in a fluid flow. In some embodiments, the cell populations described herein are collected and enriched (or depleted) via preparative scale (FACS) sorting. In some embodiments, the cell populations described herein are collected and enriched (or depleted) by the use of a microelectromechanical system (MEMS) chip combined with a FACS-based detection system (see, for example, WO2010 / 033140, Cho et al., Lab Chip, 10:1567-1573 (2010) and Godin et al., J. Biophoton., 1(5):355-376 (2008)). In either case, cells can be labeled with multiple markers, enabling the isolation of highly purified and well-defined T cell subsets.

[0075] In some embodiments, the isolation and concentration of unstimulated immune cells are carried out using an elutriation system that separates and purifies cells based on both size and density. In one embodiment, the fluid passes through a layer of unstimulated immune cells located within a centrifugal field in a separation chamber. By altering the fluid flow in the opposite direction to the centrifugal field, the system aligns and collects particles according to their size and density. In some embodiments, the elution system is either ELUTRA from Terumo BCT or ROTEA from ThermoFisher.

[0076] In some embodiments, unstimulated immune cells are incubated in cell culture medium during one or more isolation and concentration steps. In some embodiments, the cell medium is a complete cell medium. In some embodiments, the cell medium is a fetal bovine serum (FBS)-based medium, for example, containing about 1% to about 10% FBS. In some embodiments, the cell culture medium is a chemically defined culture medium. In some embodiments, the cell medium is a minimal medium. Examples of media for incubating unstimulated immune cells during the separation and concentration steps include, but are not limited to, CliniMACS buffer from Miltenyi Biotech.

[0077] In some embodiments, the cell medium further comprises a protein that coats the inner surface of the culture vessel without interacting with the cultured, unstimulated immune cells. In some embodiments, the culture medium contains approximately 0.1% to approximately 5% w / v or approximately 0.5% to approximately 2% w / v of protein. In some embodiments, the culture medium contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1% w / v of protein. In some embodiments, the protein is an isolated recombinant protein or a fragment thereof. In some embodiments, the protein is an engineered de novo polypeptide. In some embodiments, the protein is a naturally occurring protein or a fragment thereof. In some embodiments, the protein is albumin (e.g., human serum albumin or HSA).

[0078] In some embodiments, the culture medium contains about 0.1% to about 5% w / v or about 0.5% to about 2% w / v albumin (e.g., HSA). In some embodiments, the culture medium contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1% w / v albumin (e.g., HSA). In some embodiments, albumin (e.g., HSA) is full-length albumin. In other embodiments, albumin (e.g., HSA) is a fragment thereof, for example, that does not contain a signaling peptide.

[0079] In some embodiments, the unstimulated immune cells collected from one or more of the above-described separation and concentration steps are resuspended in a cell culture medium. In some embodiments, the cell medium is a complete cell medium, and optionally a completely serum-free and xeno-free medium. In some embodiments, the culture medium is a fetal bovine serum (FBS)-based medium, for example, containing about 1% to about 10% FBS. In some embodiments, the cell culture medium is a chemically defined culture medium. In some embodiments, the cell culture medium is a minimal culture medium. In some embodiments, the cell culture medium further comprises a T cell supplement, human serum, one or more cytokines such as IL-7 and / or IL-15, or a combination thereof. In some embodiments, the T cell supplement is included in the culture medium at a final concentration of approximately 2.4% v / v. In some embodiments, the human serum is included in the culture medium at a final concentration of approximately 5%. In some embodiments, the culture medium optionally contains L-glutamine or an L-glutamine substitute (e.g., L-alanine-L-glutamine) at a final concentration of about 2 mM. In some embodiments, the culture medium contains IL-7 at a final concentration of approximately 5 ng / mL. In some embodiments, the culture medium contains IL-15 at a final concentration of approximately 5 ng / mL. In some embodiments, the cell culture medium includes CTS® OPTMIZER® from ThermoFisher, GlutaMAX® Supplement from ThermoFisher, or a combination thereof.

[0080] In some embodiments, the isolation and enrichment steps of unstimulated immune cells are carried out at a temperature of approximately 2°C to approximately 40°C. In some embodiments, the separation and Enrichment steps are performed at approximately 2°C to 37°C, 2°C to 35°C, 2°C to 33°C, 2°C to 30°C, 2°C to 28°C, 2°C to 26°C, 2°C to 25°C, 2°C to 20°C, 2°C to 18°C, 2°C to 15°C, 2°C to 10°C, 2°C to 8°C, 4°C to 37°C, 4°C to 35°C, 4°C to 33°C, 4°C to 30°C, 4°C to 28°C, and 4°C to 26°C. The experiment is conducted within a temperature range of approximately 4°C to 25°C, approximately 4°C to 20°C, approximately 4°C to 18°C, approximately 4°C to 15°C, approximately 4°C to 10°C, approximately 4°C to 8°C, approximately 20°C to 37°C, approximately 20°C to 35°C, approximately 20°C to 33°C, approximately 20°C to 30°C, approximately 20°C to 28°C, approximately 20°C to 26°C, approximately 20°C to 25°C, approximately 22°C to 25°C, approximately 22°C to 28°C, approximately 24°C to 30°C, approximately 24°C to 28°C, or approximately 25°C to 30°C. In some embodiments, the separation and concentration steps are carried out at a temperature of about 2°C to about 8°C. In some embodiments, the separation and concentration are carried out at a temperature of about 4°C.

[0081] In some embodiments, the unstimulated immune cells are autoimmune cells. In other embodiments, the unstimulated immune cells are allogeneic heterogeneous immune cells.

[0082] In some embodiments, the unstimulated immune cell population comprises multiple T cells, and optionally multiple CD4 cells. + T cells, multiple CD8 + This includes T cells or combinations thereof. In some embodiments, CD4 in the immune cell population + CD8 against T cells + The ratio of T cells is approximately 1:1, 1:2, 1:3, 1:4, 2:1, 3:1, or 4:1. In some embodiments, CD4 in a population of immune cells +CD8 against T cells + The ratio of T cells is approximately 1:1. In some embodiments, CD4 in the immune cell population + CD8 against T cells + The ratio of T cells is approximately 1:2. In some embodiments, CD4 in the immune cell population + CD8 against T cells + The ratio of T cells is approximately 2:1.

[0083] In some embodiments, the methods described herein produce approximately 50 million to approximately 50 billion enriched unstimulated immune cells, optionally approximately 50 million to approximately 1 billion, approximately 100 million to approximately 5 billion, approximately 1 billion to approximately 10 billion, or approximately 1 billion to approximately 50 billion cells. In some embodiments, concentrated, unstimulated immune cells are obtained from a starting sample of approximately 50 to 500 mL of biological sample.

[0084] In some embodiments, the enriched population of unstimulated immune cells is approximately 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% CD4 + T cells, CD8 + This includes T cells, or combinations thereof.

[0085] B. Gene-edited, non-stimulated immune cells In one embodiment, unstimulated immune cells (e.g., unstimulated T cells) are modified by a gene editing system. In some embodiments, the unstimulated immune cells are gene-edited to disrupt the expression of one or more endogenously expressed genes. In some embodiments, the unstimulated immune cells have reduced, deleted, eliminated, knocked out, or destroyed expression of endogenous receptors (e.g., endogenous T cell receptors). In some embodiments, the unstimulated immune cells have exogenous immune receptors (e.g., CARs or TCRs) inserted into endogenous gene loci. In some embodiments, the endogenous gene locus is TRAC.

[0086] In one embodiment, unstimulated immune cells are genetically edited to disrupt the expression of endogenous TCR gene products (e.g., TRAC and TRBC gene products). Without being bound by any theory, disrupting the expression of TRAC and / or TRBCs 1) reduces mispairing between endogenous and exogenous TCRs, thus reducing the risk of autoreactivity, and 2) enhances exogenous TCR expression on the cell surface by reducing mispairing with endogenous TCRs, thus increasing the efficacy of modified cells.

[0087] In some embodiments, unstimulated immune cells undergo genetic modifications that disrupt the endogenous TRAC locus and insert an exogenous antigen-binding immune receptor. In some embodiments, the gene modification for disrupting the endogenous TRAC locus is located in the exons of TRAC. In some embodiments, the gene modification is located in an intron of TRAC. In some embodiments, the gene modification for disrupting the endogenous TRAC locus is located within exon 1 of the TRAC locus. In some embodiments, the exogenous antigen-binding immune receptor is a CAR. In some embodiments, the exogenous antigen-binding immune receptor is a TCR.

[0088] In some embodiments, the gene editing system includes an RNA-inducing nuclease such as a clustered regularly interspered short palindromic nucleic acid (CRISPR)-Cas system. The CRISPR system (also referred herein as the CRISPR-Cas system, Cas system, or CRISPR / Cas system) includes a target gene-specific Cas endonuclease and a guide nucleic acid sequence, which, after introduction into a cell, form a complex that allows the Cas endonuclease to introduce cleavage (e.g., double-strand breaks) into the target gene.

[0089] In some embodiments, the Cas endonuclease includes Cas9 endonuclease. In some embodiments, the Cas9 endonuclease is derived from or based on the Cas9 molecule of, for example, S. pyogenes (e.g., SpCas9), S. thermophiles, Staphylococcus aureus (e.g., SaCas9), or Neisseria meningitides. In some embodiments, the Cas9 endonuclease is, for example, Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhiz obium sp., Brevibacillus latemsporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lad, Candidatus Puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter sliibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus spitorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus、Kingella kingae、Lactobacillus crispatus、Listeria ivanovii、Listeria monocytogenes、Listeriaceae bacterium、Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica. Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tislrella mobilis, Treponema sp., or Verminephrobacter eiseniae.

[0090] In some embodiments, the Cas9 endonuclease is derived from S. pyogenes (e.g., strains SF370, MGAS10270, MGAS10750, MGAS2096, MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131, and SSI-1), S. thermophilus (e.g., strain LMD-9), S. pseudoporcinus (e.g., strain SPIN20026), S. mutans (e.g., strain UA159, strain NN2025), S. macacae (e.g., strain NCTC11558), and S. gallolylicus (e.g., strain UCN34, strain ATCC). It is derived from Ca9 molecules of the following species: BAA-2069 strain, S.equines (e.g., ATCC9812 strain, MGCS124 strain), S.dysdalactiae (e.g., GGS124 strain), S.bovis (e.g., ATCC700338 strain), S.cmginosus (e.g., F0211 strain), S.agalactia (e.g., NEM316, A909 strain), Listeria monocytogenes (e.g., F6854 strain), Listeria innocua (L.innocua, e.g., Clip11262 strain), Enterococcus italicus (e.g., DSM15952 strain), or Enterococcus faecium (e.g., 1,23,408 strain).

[0091] In some embodiments, the endonuclease includes Cas3, Cas8a, Cas8b, Cse1, Csy1, Csn2, Cas4, Cas10 (e.g., Cas10d), Cas12a (or Cpf1), Cas12b (or C2c1), Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas13, Cas14, Cs2, or Cmr5.

[0092] In some embodiments, the guide nucleic acid is a guide RNA (gRNA) molecule that directs the Cas-RNA complex to a target sequence. In some embodiments, orientation is achieved by hybridization of a portion of the gRNA to DNA (e.g., via the gRNA targeting domain) and by binding of a portion of the gRNA molecule to an RNA guide nuclease or other effector molecule (e.g., at least via gRNA tracr). In some embodiments, the gRNA molecule consists of a single, continuous polynucleotide molecule, referred to herein as “single guide RNA” or “sgRNA”. In other embodiments, the gRNA molecule consists of multiple, usually two, polynucleotide molecules that are themselves capable of association, usually through hybridization, and is referred to herein as “dual guide RNA” or “dgRNA”.

[0093] In some embodiments, the gRNA molecule comprises crRNA and tracr, which may optionally be located on a single polynucleotide or on separate polynucleotides. In some embodiments, the crRNA includes a targeting domain and a region that interacts with tracr to form a flagpole region. The tracr includes a portion of a gRNA molecule that binds to a nuclease or other effector molecule. In some embodiments, the tracr consists of a nucleic acid sequence that specifically binds to a Cas endonuclease (e.g., Cas9). In some embodiments, the tracr includes a nucleic acid sequence that forms part of the flagpole. In some embodiments, the targeting domain is a part of a gRNA molecule that recognizes a protospacer sequence in the target DNA, for example, a complementary gRNA molecule.

[0094] A protospacer adjacency motif (PAM) is a 2- to 6 base pair DNA sequence located adjacent to the 3' end of a protospacer and recognized by Cas endonucleases. In some embodiments, each Cas endonuclease recognizes a specific PAM sequence. Exemplary PAM sequences include an NGG sequence recognized by S. pyogenes Cas9 endonuclease or an NGGNG or NNAGAAW sequence (where N is any nucleotide) recognized by S. thermophilus Cas9 endonuclease. Those skilled in the art will understand how to design gRNA molecules based on a specific Cas endonuclease to be used with a PAM sequence that the Cas endonuclease will recognize.

[0095] In some embodiments, one or more, two or more, three or more, or four or more guide nucleic acids (e.g., guide RNA molecules) are transfected into immune cells having a Cas endonuclease. In some embodiments, about one, two, or three guide nucleic acids (e.g., guide RNA molecules) are transfected into immune cells possessing Cas endonucleases. In some embodiments, approximately three guide nucleic acids (e.g., guide RNA molecules) are transfected into immune cells possessing Cas endonucleases. In some embodiments, approximately two guide nucleic acids (e.g., guide RNA molecules) are transfected into immune cells possessing Cas endonucleases. In some embodiments, approximately one guide nucleic acid (e.g., a guide RNA molecule) is transfected into an immune cell possessing a Cas endonuclease.

[0096] In some embodiments, the gene editing system is a TALEN gene editing system. TALEN is artificially produced by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. Transcriptional activator-like effects (TALEs) can be manipulated to bind to target DNA. By combining the manipulated TALE with a DNA cleavage domain, it is possible to produce restriction enzymes specific to any target DNA sequence.

[0097] TALE is a protein secreted by the bacterium Xanthomonas. Its DNA-binding domain contains a repeating, highly conserved sequence of 33-34 amino acids, with the exception of the 12th and 13th amino acids. These two positions show a strong correlation with specific nucleotide recognition and are highly variable, allowing them to be manipulated to bind to target DNA sequences.

[0098] To produce TALENs, the TALE protein is fused with a nuclease (N), such as a wild-type or mutant Fokl endonuclease. The Fokl domain functions as a dimer, requiring two constructions with specific DNA-binding domains positioned appropriately and spaced at the target genomic site. Specificity and off-target effects can be regulated by altering the number of amino acid residues between the TALE DNA-binding domain and the Fokl cleavage domain, and the number of bases between the two individual TALEN binding sites.

[0099] In some embodiments, the gene editing system is a zinc finger nuclease (ZFN) gene editing system. A zinc finger nuclease is an artificial nuclease that can be used to modify one or more nucleic acid sites of a target nucleic acid sequence. Similar to TALEN editing systems, a ZFN comprises a Fokl nuclease domain (or a derivative thereof) fused to a DNA-binding domain. In the case of a ZFN, the DNA-binding domain comprises one or more zinc fingers. A zinc finger is a small protein structural motif stabilized by one or more zinc ions. A zinc finger may consist of, for example, Cys2His2 and be capable of recognizing sequences of approximately 3-bp. By combining various zinc fingers with known specificities, multi-finger polypeptides that recognize sequences of approximately 6, 9, 12, 15, or 18-bp can be generated.

[0100] ZFNs recognize non-palindromic DNA sites. To cleave the target site, a pair of ZFNs dimerize and assemble on the strand opposite the target site. Various selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) that recognize specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.

[0101] In some embodiments, the gene editing system is a meganuclease gene editing system. Meganuclease is an artificial nuclease that recognizes cleavage sites of 15 to 40 base pairs. In some embodiments, meganucleases are grouped into families based on their structural motifs that affect nuclease activity and / or DNA recognition. Members of the LAGLIDADG family are characterized by having one or two copies of the conserved LAGLIDADG motif. In some examples, the LAGLIDADG meganuclease having one copy of the LAGLIDADG motif forms homodimers, while members having two copies of the LAGLIDADG motif are found as monomers. Members of the GIY-YIG family have a GP-YIG module 70–100 residues long and contain four or five conserved sequence motifs with four invariant residues, two of which are required for activity. His-Cys box meganucleases are characterized by a highly conserved series of histidine and cysteine ​​in a region spanning several hundred amino acid residues. The NHN family members are defined by motifs containing two pairs of conserved histidines surrounded by asparagine residues. Strategies for manipulating meganucleases with altered DNA binding specificity, such as strategies for binding to specific nucleic acid sequences, are known in the art, e.g., Chevalier et al. (2002), Mol. Cell, 10:895-905; Epinat et al. (2003) Nucleic Acids Res 31: 2952-62; Silva et al. (2006) J Mol Biol 361: 744-54; Seligman et al. (2002) Nucleic Acids Res 30: 3870-9; Sussman et al. (2004) J Mol Biol 342: 31-41; Rosen et al. (2006) Nucleic Acids Res; Doyon et al. (2006) J. Am Chem Soc 128: 2477-84; Chen et al. (2009) Protein Eng Des It is found in Sel 22: 249-56; Arnould S (2006) J Mol Biol. 355: 443-58; Smith (2006) Nucleic Acids Res. 363(2): 283-94.

[0102] In some embodiments, the meganuclease is a hybrid nuclease called megaTAL, which includes a TALE domain fused to the N-terminus of the meganuclease. In some embodiments, the meganuclease is a member of the LAGLIDADG family.

[0103] C. Homologous Directed Repair (HDR) Template In one embodiment, the unstimulated immune cells described herein are modified by homology-directed repair mechanisms. In some embodiments, a homology-directed repair (HDR) template containing a polynucleotide encoding an antigen-binding polypeptide is transfected into target immune cells under conditions that the polynucleotide is inserted into a target site. In some embodiments, the HDR template includes a 5' homology arm homologous to the genomic sequence upstream, i.e., the 5' portion of the target site, and a 3' homology arm homologous to the genomic sequence downstream, i.e., the 3' portion of the target site. In some embodiments, the 5' and / or 3' homology arms are adjacent to the polynucleotide. In some embodiments, the 5' homology arm is approximately 50 to approximately 1000 nucleotides, approximately 50 to approximately 500 nucleotides, approximately 50 to approximately 400 nucleotides, approximately 50 to approximately 300 nucleotides, approximately 50 to approximately 200 nucleotides, approximately 50 to approximately 150 nucleotides, approximately 100 to approximately 500 nucleotides, approximately 100 to approximately 400 nucleotides, approximately 100 to approximately 300 nucleotides, approximately 100 to approximately 200 nucleotides, approximately 200 to approximately 500 nucleotides, approximately 200 to approximately 400 nucleotides, approximately 200 to approximately 300 nucleotides, approximately 300 to approximately 500 nucleotides, or approximately 300 to approximately 400 nucleotides. In some embodiments, the 5' homology arm is approximately 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides in length. In some embodiments, the 5' homology arm is approximately 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, or 300 nucleotides in length. In some embodiments, the 5' homology arm is approximately 50, 100, 150, 200, 300, 350, 400, 450, or 500 nucleotides long. In some embodiments, the 3' homology arm is approximately 50 to approximately 1000 nucleotides, approximately 50 to approximately 500 nucleotides, approximately 50 to approximately 400 nucleotides, approximately 50 to approximately 300 nucleotides, approximately 50 to approximately 200 nucleotides, approximately 50 to approximately 150 nucleotides, approximately 100 to approximately 500 nucleotides, approximately 100 to approximately 400 nucleotides, approximately 100 to approximately 300 nucleotides, approximately 100 to approximately 200 nucleotides, approximately 200 to approximately 500 nucleotides, approximately 200 to approximately 400 nucleotides, approximately 200 to approximately 300 nucleotides, approximately 300 to approximately 500 nucleotides, or approximately 300 to approximately 400 nucleotides in length. In some examples, the 3' homology arm is approximately 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides long. In some examples, the 3' homology arm is approximately 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, or 300 nucleotides long. In some embodiments, the 3' homology arm is approximately 50, 100, 150, 200, 300, 350, 400, 450, or 500 nucleotides in length. In some embodiments, the length of the HDR template is approximately 2 kilobase pairs (kb) to approximately 5kb, approximately 2.3kb to approximately 5kb, approximately 3kb to approximately 5kb, approximately 3kb to approximately 4kb, approximately 2kb to approximately 4kb, approximately 2.3kb to approximately 4kb, approximately 2kb to approximately 3kb, approximately 2.3kb to approximately 3kb, or approximately 4kb to approximately 5kb. In some examples, the length of the HDR template is approximately 2kb, 2.3kb, 2.5kb, 3kb, 4kb, or 5kb. In some embodiments, the HDR template is also referred to herein as an ultramar. In some embodiments, the antigen-binding polypeptide is an antigen receptor, optionally a CAR, or a TCR.

[0104] In some embodiments, the HDR template is a double-stranded DNA (dsDNA) template. In some embodiments, the dsDNA template includes native nucleotides, modified nucleotides, or combinations thereof. In some embodiments, the dsDNA template includes a 5' homology arm homologous to the genomic sequence upstream, i.e., the 5' portion, of the target site, and a 3' homology arm homologous to the genomic sequence downstream, i.e., the 3' portion, of the target site. In some embodiments, the 5' and / or 3' homology arms are adjacent to the polynucleotide. In some embodiments, the 5' homology arm is approximately 50 to 1000 nucleotides, approximately 50 to 500 nucleotides, approximately 50 to 400 nucleotides, approximately 50 to 300 nucleotides, approximately 50 to 200 nucleotides, approximately 50 to 150 nucleotides, approximately 100 to 500 nucleotides, approximately 100 to 400 nucleotides, approximately 100 to 300 nucleotides, approximately 100 to 200 nucleotides, approximately 200 to 500 nucleotides, approximately 200 to 400 nucleotides, approximately 200 to 300 nucleotides, approximately 300 to 500 nucleotides, or approximately 300 to 400 nucleotides. In some embodiments, the 5' homology arm is approximately 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides in length. In some examples, the 5' homology arm is approximately 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, or 300 nucleotides long. In some examples, the 5' homology arm is approximately 50, 100, 150, 200, 300, 350, 400, 450, or 500 nucleotides long. In some embodiments, the 3' homology arm is approximately 50 to 1000 nucleotides, approximately 50 to 500 nucleotides, approximately 50 to 400 nucleotides, approximately 50 to 300 nucleotides, approximately 50 to 200 nucleotides, approximately 50 to 150 nucleotides, approximately 100 to 500 nucleotides, approximately 100 to 400 nucleotides, approximately 100 to 300 nucleotides, approximately 100 to 200 nucleotides, approximately 200 to 500 nucleotides, approximately 200 to 400 nucleotides, approximately 200 to 300 nucleotides, approximately 300 to 500 nucleotides, or approximately 300 to 400 nucleotides. In some embodiments, the 3' homology arm is approximately 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides in length. In some embodiments, the 3' homology arm is approximately 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, or 300 nucleotides in length. In some examples, the 3' homology arm is approximately 50, 100, 150, 200, 300, 350, 400, 450, or 500 nucleotides long. In some embodiments, the length of the dsDNA template is approximately 2kb to 5kb, approximately 2.3kb to 5kb, approximately 3kb to 5kb, approximately 3kb to 4kb, approximately 2kb to 4kb, approximately 2.3kb to 4kb, approximately 2kb to 3kb, approximately 2.3kb to 3kb, or approximately 4kb to 5kb. In some examples, the length of the dsDNA template is approximately 2kb, 2.3kb, 2.5kb, 3kb, 4kb, or 5kb.

[0105] In some embodiments, the HDR template is a single-stranded DNA (ssDNA) template. In some embodiments, the ssDNA template includes native nucleotides, modified nucleotides, or combinations thereof. In some embodiments, the ssDNA template includes a 5' homology arm homologous to the genomic sequence upstream, i.e., the 5' genomic sequence, relative to the target site, and a 3' homology arm homologous to the genomic sequence downstream, i.e., the 3' genomic sequence, relative to the target site. In some embodiments, the 5' and / or 3' homology arms are adjacent to the polynucleotide. In some embodiments, the 5' homology arm is approximately 50 to 1000 nucleotides, approximately 50 to 500 nucleotides, approximately 50 to 400 nucleotides, approximately 50 to 300 nucleotides, approximately 50 to 200 nucleotides, approximately 50 to 150 nucleotides, approximately 100 to 500 nucleotides, approximately 100 to 400 nucleotides, approximately 100 to 300 nucleotides, approximately 100 to 200 nucleotides, approximately 200 to 500 nucleotides, approximately 200 to 400 nucleotides, approximately 200 to 300 nucleotides, approximately 300 to 500 nucleotides, or approximately 300 to 400 nucleotides in length. In some embodiments, the 5' homology arm is approximately 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides in length. In some examples, the 5' homology arm is approximately 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, or 300 nucleotides long. In some examples, the 5' homology arm is approximately 50, 100, 150, 200, 300, 350, 400, 450, or 500 nucleotides long. In some embodiments, the 3' homology arm is approximately 50 to 1000 nucleotides, approximately 50 to 500 nucleotides, approximately 50 to 400 nucleotides, approximately 50 to 300 nucleotides, approximately 50 to 200 nucleotides, approximately 50 to 150 nucleotides, approximately 100 to 500 nucleotides, approximately 100 to 400 nucleotides, approximately 100 to 300 nucleotides, approximately 100 to 200 nucleotides, approximately 200 to 500 nucleotides, approximately 200 to 400 nucleotides, approximately 200 to 300 nucleotides, approximately 300 to 500 nucleotides, or approximately 300 to 400 nucleotides. In some embodiments, the 3' homology arm is approximately 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides in length. In some examples, the 3' homology arm is approximately 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, or 300 nucleotides long. In some examples, the 3' homology arm is approximately 50, 100, 150, 200, 300, 350, 400, 450, or 500 nucleotides long. In some embodiments, the length of the ssDNA template is approximately 2kb to 5kb, approximately 2.3kb to 5kb, approximately 3kb to 5kb, approximately 3kb to 4kb, approximately 2kb to 4kb, approximately 2.3kb to 4kb, approximately 2kb to 3kb, approximately 2.3kb to 3kb, or approximately 4kb to 5kb. In some examples, the length of the ssDNA template is approximately 2kb, 2.3kb, 2.5kb, 3kb, 4kb, or 5kb.

[0106] In some embodiments, the HDR template is inserted into a plasmid before being introduced into immune cells. Exemplary plasmids include, but are not limited to, pBAD / His, pCal-n, pET-3a-c, pET32a-c, pGEX-2T, pTriEx-1, pUC19, pAd5F35, pAdDeltaF6, or TLCV2.

[0107] D. Transfection In some embodiments, the gene editing system and the HDR template are introduced into unstimulated immune cells via a non-viral delivery method. In a non-viral delivery method, the nucleic acid may be naked DNA or contained in a non-viral plasmid or vector. Exemplary non-viral delivery methods, but not limited to these, include electroporation, cell squeeze, calcium phosphate, heat shock, liposome delivery, cationic polymers, lipid polymers, cell-penetrating peptides, cationic nanocarriers, hydrodynamic delivery, ultrasound, cationic lipids, nanoparticles, ballistic DNA injection, magneto-fection, and photoporation.

[0108] In some embodiments, the gene editing system and HDR template are introduced into unstimulated immune cells by electroporation. In some cases, the gene editing system (e.g., the endonuclease and / or the guide nucleic acid) and the HDR template are simultaneously introduced into unstimulated immune cells by electroporation. In other examples, the gene editing system (e.g., the endonuclease and / or the guide nucleic acid) is introduced into unstimulated immune cells simultaneously by first electroporation followed by a separate electroporation of the HDR template, or alternatively, the HDR template is introduced into unstimulated immune cells simultaneously by first electroporation followed by a separate electroporation of the gene editing system (e.g., the endonuclease and / or the guide nucleic acid). In a further example, the endonuclease, the guide nucleic acid, and the HDR template are sequentially introduced into the unstimulated immune cells by electroporation.

[0109] In some embodiments, the endonuclease is introduced into unstimulated immune cells at a concentration of about 1 pM to about 10 mM. In some embodiments, the concentration of endonuclease is approximately 1 pM to 5 mM, approximately 1 pM to 3 mM, approximately 1 pM to 1 mM, approximately 1 pM to 0.8 mM, approximately 1 pM to 0.5 mM, approximately 1 pM to 0.2 mM, approximately 1 pM to 0.1 mM, approximately 1 pM to 0.09 mM, approximately 1 pM to 0.05 mM, approximately 1 pM to 0.01 mM, approximately 1 pM to 5 μM, approximately 1 pM to 1 μM, approximately 1 pM to 500 nM, approximately 1 pM to 100 nM, approximately 1 nM to 10 mM, approximately 1 nM to 5 mM, approximately 1 nM to 3 mM, approximately 1 nM to 1 mM, approximately 1 nM to 0.8 mM, and approximately 1 nM. These ranges from approximately 0.5 mM, approximately 1 nM to approximately 0.2 mM, approximately 1 nM to approximately 0.1 mM, approximately 1 nM to approximately 0.09 mM, approximately 1 nM to approximately 0.05 mM, approximately 1 nM to approximately 0.01 mM, approximately 1 nM to approximately 5 μM, approximately 1 nM to approximately 1 μM, approximately 1 nM to approximately 500 nM, approximately 1 μM to approximately 10 mM, approximately 1 μM to approximately 5 mM, approximately 1 μM to approximately 3 mM, approximately 1 μM to approximately 1 mM, approximately 1 μM to approximately 0.8 mM, approximately 1 μM to approximately 0.5 mM, approximately 1 μM to approximately 0.2 mM, approximately 1 μM to approximately 0.1 mM, approximately 1 μM to approximately 0.09 mM, approximately 1 μM to approximately 0.05 mM, approximately 1 μM to approximately 0.01 mM, or approximately 1 μM to approximately 5 μM.

[0110] In some embodiments, the guide nucleic acid (e.g., the guide RNA molecule) is introduced into unstimulated immune cells at a concentration of about 1 pM to about 10 mM. In some examples, the concentration of the guide nucleic acid (e.g., the guide RNA molecule) is approximately 1 pM to 5 mM, approximately 1 pM to 3 mM, approximately 1 pM to 1 mM, approximately 1 pM to 0.8 mM, approximately 1 pM to 0.5 mM, approximately 1 pM to 0.2 mM, approximately 1 pM to 0.1 mM, approximately 1 pM to 0.09 mM, approximately 1 pM to 0.05 mM, approximately 1 pM to 0.01 mM, approximately 1 pM to 5 μM, approximately 1 pM to 1 μM, approximately 1 pM to 500 nM, approximately 1 pM to 100 nM, approximately 1 nM to 10 mM, approximately 1 nM to 5 mM, approximately 1 nM to 3 mM, approximately 1 nM to 1 mM, approximately 1 nM to 0.8 mM. Approximately 1 nM to approximately 0.5 mM, approximately 1 nM to approximately 0.2 mM, approximately 1 nM to approximately 0.1 mM, approximately 1 nM to approximately 0.09 mM, approximately 1 nM to approximately 0.05 mM, approximately 1 nM to approximately 0.01 mM, approximately 1 nM to approximately 5 μM, approximately 1 nM to approximately 1 μM, approximately 1 nM to approximately 500 nM, approximately 1 μM to approximately 10 mM, approximately 1 μM to approximately The concentrations are 5 mM, approximately 1 μM to 3 mM, approximately 1 μM to 1 mM, approximately 1 μM to 0.8 mM, approximately 1 μM to 0.5 mM, approximately 1 μM to 0.2 mM, approximately 1 μM to 0.1 mM, approximately 1 μM to 0.09 mM, approximately 1 μM to 0.05 mM, approximately 1 μM to 0.01 mM, or approximately 1 μM to 5 μM.

[0111] In some embodiments, the HDR template is introduced into unstimulated immune cells at a concentration of about 1 pM to about 10 mM. In some embodiments, the density of the HDR template is approximately 1 pM to 5 mM, approximately 1 pM to 3 mM, approximately 1 pM to 1 mM, approximately 1 pM to 0.8 mM, approximately 1 pM to 0.5 mM, approximately 1 pM to 0.2 mM, approximately 1 pM to 0.1 mM, approximately 1 pM to 0.09 mM, approximately 1 pM to 0.05 mM, approximately 1 pM to 0.01 mM, approximately 1 pM to 5 μM, approximately 1 pM to 1 μM, approximately 1 pM to 500 nM, approximately 1 pM to 100 nM, approximately 1 nM to 10 mM, approximately 1 nM to 5 mM, approximately 1 nM to 3 mM, approximately 1 nM to 1 mM, approximately 1 nM to 0.8 mM, and approximately 1 nM. These ranges from approximately 0.5 mM, approximately 1 nM to approximately 0.2 mM, approximately 1 nM to approximately 0.1 mM, approximately 1 nM to approximately 0.09 mM, approximately 1 nM to approximately 0.05 mM, approximately 1 nM to approximately 0.01 mM, approximately 1 nM to approximately 5 μM, approximately 1 nM to approximately 1 μM, approximately 1 nM to approximately 500 nM, approximately 1 μM to approximately 10 mM, approximately 1 μM to approximately 5 mM, approximately 1 μM to approximately 3 mM, approximately 1 μM to approximately 1 mM, approximately 1 μM to approximately 0.8 mM, approximately 1 μM to approximately 0.5 mM, approximately 1 μM to approximately 0.2 mM, approximately 1 μM to approximately 0.1 mM, approximately 1 μM to approximately 0.09 mM, approximately 1 μM to approximately 0.05 mM, approximately 1 μM to approximately 0.01 mM, or approximately 1 μM to approximately 5 μM.

[0112] In some embodiments, the gene editing system is a CRISPR-Cas system. In some cases, Cas (e.g., Cas9) endonucleases are introduced into unstimulated immune cells at concentrations ranging from approximately 1 pM to 10 mM. In some embodiments, the concentration of Cas (e.g., Cas9) endonuclease is approximately 1 pM to 5 mM, approximately 1 pM to 3 mM, approximately 1 pM to 1 mM, approximately 1 pM to 0.8 mM, approximately 1 pM to 0.5 mM, approximately 1 pM to 0.2 mM, approximately 1 pM to 0.1 mM, approximately 1 pM to 0.09 mM, approximately 1 pM to 0.05 mM, approximately 1 pM to 0.01 mM, approximately 1 pM to 5 μM, approximately 1 pM to 1 μM, approximately 1 pM to 500 nM, approximately 1 pM to 100 nM, approximately 1 nM to 10 mM, approximately 1 nM to 5 mM, approximately 1 nM to 3 mM, approximately 1 nM to 1 mM, and approximately 1 nM to 0.8 mM. M, 1nM to approximately 0.5mM, approximately 1nM to approximately 0.2mM, approximately 1nM to approximately 0.1mM, approximately 1nM to approximately 0.09mM, approximately 1nM to approximately 0.05mM, approximately 1nM to approximately 0.01mM, approximately 1nM to approximately 5μM, approximately 1nM to approximately 1μM, approximately 1nM to approximately 500nM, approximately 1μM to approximately 10mM, approximately 1μM to approximately The concentrations are 5 mM, approximately 1 μM to 3 mM, approximately 1 μM to 1 mM, approximately 1 μM to 0.8 mM, approximately 1 μM to 0.5 mM, approximately 1 μM to 0.2 mM, approximately 1 μM to 0.1 mM, approximately 1 μM to 0.09 mM, approximately 1 μM to 0.05 mM, approximately 1 μM to 0.01 mM, or approximately 1 μM to 5 μM.

[0113] In some embodiments, the ratio of the Cas (e.g., Cas9) endonuclease concentration to the guide nucleic acid concentration introduced into unstimulated immune cells is approximately 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some cases, the ratio of the Cas endonuclease to the guide nucleic acid is approximately 1:1. In some cases, the ratio of the Cas endonuclease to the guide nucleic acid is approximately 1:2. In some cases, the ratio of the Cas endonuclease to the guide nucleic acid is approximately 1:3. In some cases, the ratio of the Cas endonuclease to the guide nucleic acid is approximately 2:1. In some cases, the ratio of the Cas endonuclease to the guide nucleic acid is approximately 3:1.

[0114] In some embodiments, the ratio of the CRISPR-Cas system to the HDR template introduced into unstimulated immune cells is approximately 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:18, 1:20, 1:25, 1:30, 1:35, 1:40, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 15:1, 20:1, 30:1, or 40:1. In some examples, the ratio of the CRISPR-Cas system to the HDR template is approximately 1:1. In some examples, the ratio of the CRISPR-Cas system to the HDR template is approximately 1:2. In some examples, the ratio of the CRISPR-Cas system to the HDR template is approximately 2:1.

[0115] In some embodiments, the ratio of the gene-editing nuclease (e.g., endonuclease) to the HDR template introduced into unstimulated immune cells is approximately 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some cases, the ratio of the gene editing nuclease (e.g., endonuclease) to the HDR template is approximately 1:1. In some cases, the ratio of the gene editing nuclease (e.g., endonuclease) to the HDR template is approximately 1:2. In some cases, the ratio of the gene editing nuclease (e.g., endonuclease) to the HDR template is approximately 2:1.

[0116] In some embodiments, the electrophoresis is performed at a temperature of approximately 2°C to approximately 40°C. In some examples, the electrophoresis is observed at approximately 2°C to 37°C, 2°C to 35°C, 2°C to 33°C, 2°C to 30°C, 2°C to 28°C, 2°C to 26°C, 2°C to 25°C, 2°C to 20°C, 2°C to 18°C, 2°C to 15°C, 2°C to 10°C, 2°C to 8°C, 4°C to 37°C, 4°C to 35°C, 4°C to 33°C, 4°C to 30°C, 4°C to 28°C, 4°C to 26°C, and 4°C to 33°C. The tests are conducted at temperatures ranging from 10°C to approximately 25°C, 4°C to approximately 20°C, 4°C to approximately 18°C, 4°C to approximately 15°C, 4°C to approximately 10°C, 4°C to approximately 8°C, 20°C to approximately 37°C, 20°C to approximately 35°C, 20°C to approximately 33°C, 20°C to approximately 30°C, 20°C to approximately 28°C, 20°C to approximately 26°C, 20°C to approximately 25°C, 22°C to approximately 25°C, 22°C to approximately 28°C, 24°C to approximately 30°C, 24°C to approximately 28°C, or 25°C to approximately 30°C. In some cases, the electrophoresis is performed at a temperature of approximately 20°C to 30°C. In some cases, the electrophoresis is performed at a temperature of approximately 25°C to 30°C. In some cases, the electrophoresis is performed at a temperature of approximately 25°C to 28°C.

[0117] Exemplary electroporation technologies include, but are not limited to, Lonza's NUCLEOFECTOR® platform, Lonza's Amaxa NUCLEOFECTOR® II electroporation machine, MaxCyte's FLOW ELECTROPORATION® technology, and Bio-Rad's Gene Pulser MXCELL® electroporation system. Those skilled in the art will understand that the pulse type, pulse duration, voltage, and frequency of use depend on the type of apparatus and the type of cell, and that the optimization of transfection efficiency can be adjusted based on the pulse type, pulse duration, voltage, frequency of use, and the concentrations of endonuclease, guide nucleic acid, and / or HDR template.

[0118] In some embodiments, the HDR template promotes HDR at target sites in about 10% or more of the unstimulated immune cell population. In some cases, the HDR template promotes HDR at target sites in approximately 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the unstimulated immune cell population.

[0119] In some embodiments, the efficiency of the transfection method is approximately 2% to approximately 99%, approximately 5% to approximately 99%, approximately 8% to approximately 99%, approximately 10% to approximately 99%, approximately 12% to approximately 99%, approximately 13% to approximately 99%, approximately 15% to approximately 99%, approximately 20% to approximately 99%, 30% to approximately 99%, approximately 40% to approximately 99%, approximately 50% to approximately 99%, and approximately 60% to approximately 99%. The percentages are approximately 70% to 99%, 2% to 80%, 5% to 80%, 8% to 80%, 10% to 80%, 12% to 80%, 13% to 80%, 15% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 20% to 70%, or 30% to 60%. In some examples, the efficiencies are approximately 2%, 5%, 8%, 10%, 12%, 13%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.

[0120] E. Modified nonstimulated immune cells In some embodiments, non-stimulated immune cells after non-viral delivery of the gene editing system and the HDR template are cultured under non-swelling conditions for up to approximately 72 hours to generate modified non-stimulated immune cells. In some cases, the unstimulated immune cells are cultured for approximately 18 to 72 hours, 18 to 36 hours, 18 to 24 hours, 24 to 72 hours, 24 to 36 hours, or 36 to 72 hours to generate modified unstimulated immune cells. In some cases, the unstimulated immune cells are cultured for approximately 48 hours or less, approximately 36 hours or less, approximately 24 hours or less, or approximately 18 hours or less to generate modified unstimulated immune cells. In some cases, the unstimulated immune cells are cultured for approximately 18, 24, 36, 48, or 72 hours to generate modified unstimulated immune cells. In some cases, the unstimulated immune cells are cultured at approximately 37°C in a culture vessel (e.g., a culture bag or bioreactor). In some cases, the culture vessel contains a humidity of approximately 90%, 95%, or 99%. In some cases, the culture vessel contains about 0.5% carbon dioxide.

[0121] In some embodiments, the cell medium used with modified non-stimulated immune cells includes a complete medium, a chemically defined medium, or a fetal bovine serum (FBS)-based medium, for example, a medium containing about 1% to about 10% FBS. In some cases, the cell culture medium is a minimal medium. Examples of media for culturing the modified non-stimulated immune cells include, but are not limited to, CliniMACS buffer from Miltenyi Biotech and CTS® OPTMIZER® from ThermoFisher.

[0122] In some examples, the cell medium further includes proteins that coat the inner surface of the culture vessel without interacting with the cultured modified non-stimulated immune cells. In some cases, the culture medium contains approximately 0.1% to approximately 5% w / v or approximately 0.5% to approximately 2% w / v of protein. In some cases, the medium contains approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% of protein w / v. In some cases, the protein is an isolated recombinant protein or a fragment thereof. In some cases, the protein is an engineered de novo polypeptide. In some cases, the protein is a naturally occurring protein or a fragment thereof. In some embodiments, the protein is albumin (e.g., human serum albumin or HSA).

[0123] In some embodiments, the cell culture medium contains about 0.1% to about 5% w / v or about 0.5% to about 2% w / v of albumin (e.g., HSA). In some examples, the culture medium contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1% w / v albumin (e.g., HSA). In some cases, albumin (e.g., HSA) is full-length albumin. In other cases, the albumin (e.g., HSA) is, for example, a fragment thereof that does not contain a signaling peptide.

[0124] In some embodiments, the modified unstimulated immune cell population produced by the method described above includes approximately 5 million, 10 million, 20 million, 50 million, 100 million, 500 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 10 billion, or more cells. In some cases, the modified, unstimulated immune cell population produced by the method described above contains approximately 50 million to 5 billion cells. In some cases, the modified, unstimulated immune cell population produced by the method described above contains approximately 50 million to 4 billion cells. F. Cryopreservation

[0125] In some embodiments, the modified, unstimulated immune cells are cultured for up to approximately 72 hours and then cryopreserved. In some cases, the modified, unstimulated immune cells are removed from the culture vessel and resuspended in cryopreservation medium. In some examples, the cryopreservation medium includes CRYOSTOR® CSB medium (BioLife Solutions) and DMSO. In some cases, the DMSO is a 2%, 5%, or 10% v / v solution. In some cases, the DMSO is CRYOSTOR® CS5 or CRYOSTOR® CS10 (BioLife Solutions).

[0126] In some cases, the final concentration of DMSO is approximately 0% v / v to approximately 7.5% v / v. In some cases, the final concentration of DMSO is approximately 2.5% v / v. In some cases, the final concentration of the DMSO is approximately 5% v / v. In some cases, the temperature during resuspension is approximately 24°C to 28°C.

[0127] After resuspending the cells in the aforementioned cryopreservation medium, the modified non-stimulated immune cells are cryopreserved, and during this process, the cell temperature is lowered from the resuspension temperature to approximately -80°C using a stepwise method. In some examples, a decrease of approximately 1 to 5°C per minute is used in the stepwise method. In some examples, a decrease of approximately 1°C per minute is used in the stepwise method. In some examples, the stepwise method includes lowering the temperature from the resuspension temperature to about -80°C within about 30 minutes to about 3 hours. In some cases, the resuspension temperature is approximately 24°C to 28°C, approximately 26°C to 28°C, or approximately 24°C to 26°C.

[0128] In some embodiments, the methods herein that generate freeze-modified unstimulated immune cells provide cell viability of about 10% to about 99%, about 12% to about 99%, about 13% to about 99%, about 15% to about 99%, about 20% to about 99%, 30% to about 99%, about 40% to about 99%, about 50% to about 99%, about 60% to about 99%, about 70% to about 99%, about 10% to about 80%, about 12% to about 80%, about 13% to about 80%, about 15% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 20% to about 70%, or about 30% to about 60%. In some cases, the method provides cell viability of about 10%, about 12%, about 13%, about 15%, about 18%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%.

[0129] G. Modified stimulated immune cells In some embodiments, the modified unstimulated immune cells are optionally stimulated before cryopreservation. In some cases, the modified, unstimulated immune cells are stimulated and enlarged for approximately 5 to 12 days. In some cases, the modified unstimulated immune cells are stimulated and expanded for about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or longer. In some cases, the unstimulated immune cells are stimulated and expanded for about 10 to 12 days.

[0130] In some embodiments, the modified unstimulated immune cells are incubated in the presence of an agent capable of stimulating the unstimulated immune cells. In some cases where the unstimulated immune cells are T cells, the drug can activate the intracellular signaling domain of one or more components of the TCR complex, such as the CD3 zeta chain, or can activate signaling through such complex or components. In some cases, the drug is an anti-CD3 antibody, an anti-CD28 antibody, an anti-4-1BB antibody, or a cytokine such as IL-2, IL-15, IL-7, or IL-21, or an artificial antigen-presenting cell (aAPC). In some cases, the antibody is further bound to or present on the surface of a solid support such as beads. In some cases, the drug is Dynabeads human T-Activator CD3 / CD28 (ThermoFisher), optionally in a bead-to-cell ratio of 3:1, or MACS® GEMP T cell TRANSACT® (Mitenyi Biotec).

[0131] In some embodiments, the modified stimulated immune cell populations generated by the methods described herein include approximately 5 million cells, approximately 10 million cells, approximately 20 million cells, approximately 50 million cells, approximately 100 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 10 billion cells, approximately 20 billion cells, or more.

[0132] In some embodiments, the methods herein that generate freeze-modified stimulated immune cells provide cell viability of about 10% to about 99%, about 12% to about 99%, about 13% to about 99%, about 15% to about 99%, about 20% to about 99%, 30% to about 99%, about 40% to about 99%, about 50% to about 99%, about 60% to about 99%, about 70% to about 99%, about 10% to about 80%, about 12% to about 80%, about 13% to about 80%, about 15% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 20% to about 70%, or about 30% to about 60%. In some cases, the method provides cell viability of about 10%, about 12%, about 13%, about 15%, about 18%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%.

[0133] III. Antigen-binding polypeptide In one embodiment, disclosed herein are modified non-stimulatory immune cells or modified stimulatory immune cells that express a foreign antigen-binding polypeptide. In some examples, the antigen-binding polypeptide is a chimeric antigen receptor (CAR). In other examples, the antigen-binding polypeptide is a T cell receptor (TCR). In additional examples, the antigen-binding polypeptide is a polypeptide that binds to an antigen-binding domain, a cell surface receptor ligand, or a tumor antigen.

[0134] A. Chimeric Antigen Receptor In some embodiments, modified non-stimulatory immune cells or modified stimulatory immune cells, e.g., modified non-stimulatory or stimulatory T cells, express a chimeric antigen receptor (CAR). The CARs of the present invention include an antigen-binding domain, a transmembrane domain, a hinge domain, and an intracellular signaling domain. Any modified cell comprising a CAR comprising any antigen-binding domain, any hinge, any transmembrane domain, any intracellular co-stimulatory domain, and any intracellular signaling domain described herein is envisioned and can be readily understood and produced by one of ordinary skill in the art in view of the disclosure herein.

[0135] The antigen-binding domain can be operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, described elsewhere herein for intracellular expression. In one embodiment, a first nucleic acid sequence encoding an antigen-binding domain is operably linked to a second nucleic acid encoding a transmembrane domain and further operably linked to a third nucleic acid sequence encoding an intracellular domain.

[0136] The antigen-binding domains described herein can be combined with any of the transmembrane domains described herein, any of the intracellular domains or cytoplasmic domains described herein, or any of the other domains described herein that can be included in the CARs of the present invention. The CAR of the present invention may also contain a spacer domain as described herein. In some embodiments, each of the antigen-binding domain, transmembrane domain and intracellular domain is separated by a linker.

[0137] 1. antigen-binding domain The antigen-binding domain of the CAR is the extracellular region of the CAR for binding to a specific target antigen including proteins, carbohydrates and glycolipids. In some embodiments, the CAR includes an affinity for a target antigen (e.g., a tumor-associated antigen) on a target cell (e.g., a cancer cell). The target antigen may include any type of protein associated with the target cell, or an epitope thereof. For example, the CAR may include an affinity for a target antigen on a target cell that indicates a specific state of the target cell.

[0138] As described herein, the CAR of the present disclosure having an affinity for a specific target antigen on a target cell may include a target-specific binding domain. In some embodiments, the target-specific binding domain is a mouse target-specific binding domain, for example, the target-specific binding domain is of mouse origin. In some embodiments, the target-specific binding domain is a human target-specific binding domain, for example, the target-specific binding domain is of human origin.

[0139] The antigen-binding domain can include any domain that binds to an antigen, and can include, but is not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and any fragments thereof. Therefore, in one embodiment, the antigen-binding domain portion includes a mammalian antibody or a fragment thereof. In some embodiments, the antigen-binding domain consists of a full-length antibody. In some embodiments, the antigen-binding domain includes antigen-binding fragments (Fab), such as Fab, Fab', F(ab')2, monospecific Fab2, bispecific Fab2, trispecific Fab2, single-chain variable fragment (scFv), dAb, tandem scFv, VhH, V-NAR, camelid, diabody, minibody, triabody, or tetrabody.

[0140] In some embodiments, the CARs of this disclosure may have affinity for one or more target antigens on one or more target cells. In some embodiments, the CAR may have affinity for one or more target antigens on a single target cell. In such embodiments, the CAR is a bispecific CAR or a multispecific CAR. In some embodiments, the CAR includes one or more target-specific binding domains that confer affinity to one or more target antigens. In some embodiments, the CAR includes one or more target-specific binding domains that confer affinity to the same target antigen. For example, a CAR containing one or more target-specific binding domains that have affinity for the same target antigen can bind to different epitopes of the target antigen. When a CAR has multiple target-specific binding domains, the binding domains are arranged in tandem and can be separated by a linker peptide. For example, in a CAR containing two target-specific binding domains, the binding domains are covalently linked to each other on a single polypeptide chain via a polypeptide linker, an Fc hinge region, or a membrane hinge region.

[0141] As used herein, the terms “single-chain variable fragment” or “scFv” refer to a fusion protein formed by covalently linking the variable regions of heavy chains (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) to form a VH::VL heterodimer. The heavy chains (VH) and light chains (VL) are linked either directly or by a peptide-encoding linker or spacer, with the N-terminus of VH ligating to the C-terminus of VL, or the C-terminus of VH ligating to the N-terminus of VL. In this specification, the terms "linker" and "spacer" are used interchangeably. In some embodiments, the antigen-binding domain (e.g., the Tn-MUC1 binding domain) includes an scFv having a VH-linker-VL configuration from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain (e.g., Tn-MUC1 binding domain, PSMA binding domain) includes an scFv having a VL-linker-VH configuration from the N-terminus to the C-terminus. Those skilled in the art will be able to select a suitable configuration for use in the present invention.

[0142] The linker is typically rich in glycine for flexibility and contains serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of the linker described above are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO2014 / 087010, the contents of which are incorporated herein by reference in their entirety. Various linker sequences are known in this art, and are not limited to (GS) n (GSGGS) n (GGGS) n , and (GGGGS) n Examples of glycine-serine (GS) linkers include (where n represents an integer of at least 1). Exemplary linker sequences may include, but are not limited to, amino acid sequences containing GGSG, GGSGG, GSGSG, GGGGG, GGGSG, GSSSG, GGGGS, GGGGSGGGGSGGGGS, etc. Those skilled in the art will be able to select a suitable linker sequence for use in the present invention. In one embodiment, the antigen-binding domain of the present invention (e.g., Tn-MUC1 binding domain, PSMA binding domain) comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL being separated by a linker sequence having the amino acid sequence GGGGSGGGGSGGGGS, which can be encoded by a nucleic acid sequence containing the nucleotide sequence gggggcggtggctcgggcggtgggtcgggtggcggcggatct.

[0143] Despite the removal of the constant region and the introduction of a linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be expressed from nucleic acids containing VH- and VL-encoding sequences, as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405, 4,956,778, and U.S. Patent Publications 20050196754 and 20050196754. Antagonistic scFv with inhibitory activity has been described (e.g., Zhao et al., Hybridoma (Larchmt), 27(6):455-51 (2008); Peter et al., J. Cachexia Sarcopenia Muscle (Aug. 12, 2012); Shieh et al., J. Imunol., 183(4):2277-85 (2009); Giomarelli et al., Thromb. Haemost., 97(6):955-63 (2007); Fife et al., J. Clin. Invst., 116(8):2252-61 (2006); Brocks et al., Immunotechnology, 3(3):173-84 (1997); Moosmayer et al., Ther. Immunol., 2(10):31-40) See (1995). Agonistic scFv with stimulating activity has been described (see, for example, Peter et al., J. Biol. Chem., 25278(38):36740-7 (2003); Xie et al., Nat. Biotech., 15(8):768-71 (1997); Ledbetter et al., Crit. Rev. Immunol., I(5-6):427-55 (1997); Ho et al., BioChim. Biophys. Acta., 1638(3):257-66 (2003)).

[0144] As used herein, "Fab" refers to a fragment of an antibody structure that binds to an antigen but is monovalent and lacks an Fc region. For example, an antibody digested with the enzyme papain produces two Fab fragments and an Fc fragment (e.g., a constant heavy (H) chain region and an Fc region that does not bind to the antigen).

[0145] In some cases, the antigen-binding domain may originate from the same species in which the CAR is ultimately used. For example, when used in humans, the antigen-binding domain of the CAR may contain a human antibody or a fragment thereof, as described elsewhere in this specification.

[0146] Therefore, immune cells obtained by the methods described herein, for example, can be engineered to express a CAR targeting one of the following cancer-associated antigens (tumor antigens): CD19; CD20; CD22 (Siglec-2); CD37; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL). -1 or CLECL1); CD33; CD133; Epidermal growth factor receptor (EGFR); Epidermal growth factor receptor variant III (EGFRvIII); Human epidermal growth factor receptor (HER1); Ganglioside G2 (GD2); Ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213) A2); Mesothelin; Interleukin-11 receptor alpha (IL-11rRa); Prostate stem cell antigen (PSCA); Proteaseserine 21 (Testisin or PRSS21); Vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen 4 (SSEA-4); Folate receptor alpha; Receptor tyrosine protein kinase ERBB2 (Her2 / neu); Mucin 1, cell surface associated (MUC 1);GalNAca1-O-Ser / Thr(Tn)MUC 1 (TnMUC1); neuronal adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutation (ELF2M); ephrin B2, fibroblast-activating protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100); breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1. 1) Oncogene fusion protein consisting of (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-associated (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); staining Chromophore X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); globoH hexasaccharide moiety of glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); europlakin 2 (UPK2); tyrosine protein kinase Met (c-Met); hepatitis A virus cell receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); panexin 3 (PANX3); G protein-coupled receptor 20 (GPR20);Lymphocyte antigen 6 complex, K9 locus (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGEl); angiopoietin-binding cell surface receptor receptor)2(Tie2); melanoma cancer testicular antigen-1(MAD-CT-1); MAD-CT-2); Fos-related antigen 1; tumor protein p53(p53); p53 variant; prostein; survival; telomerase; prostate cancer tumor antigen-1(PCTA-1 or Galectin8), melanoma antigen 1(MelanA or MARTI) recognized by T cells; rat sarcoma (Ras) variant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma apoptosis inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V(NA17); paired box protein Pax-3(PAX3); androgen receptor; cyclin(Bl); v-myc Avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog) (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P4501B1 (CYP1B 1);CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen 3 (SART3) recognized by T cells; paired-box protein Pax-5 (PAX5); pro-acrosin-binding protein sp32 (OY-TES 1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma; X breakpoint 2 (SSX2); advanced glycation end product receptor (RAGE-1); renal ubiquitous protein 1 (RU1); renal ubiquitous protein 2 (RU2); regmine; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 variant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-2 (GPC2); glypican-3 (GPC3); NKG2D; KRAS; GDNF family receptor alpha 4 (GFRa4); IL13Ra2; Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1). In some embodiments, the immune cells are engineered to express CARs targeting CD19, CD20, CD22, BCMA, CD37, mesoserine, PSMA, PSCA, Tn-MUC1, EGFR, EGFRvIII, c-Met, HER1, HER2, CD33, CD133, GD2, GPC2, GPC3, NKG2D, KRAS, or WT1.

[0147] 2. Transmembrane domain With respect to the transmembrane domain, the CAR can be manipulated to include a transmembrane domain that connects the antigen-binding domain of the CAR to an intracellular domain. The transmembrane domain of the target CAR is a region that can span the plasma membrane of a cell (e.g., an immune cell or its precursor). The transmembrane domain is intended for insertion into a cell membrane, for example, a eukaryotic cell membrane. In some embodiments, the transmembrane domain is interposed between the antigen-binding domain and the intracellular domain of the CAR.

[0148] In one embodiment, the transmembrane domain is naturally associated with one or more domains of the CAR. In some cases, the transmembrane domain may be selected or modified by amino acid substitutions to avoid binding of such domain to the transmembrane domains of the same or different surface membrane proteins, in order to minimize interaction with other members of the receptor complex.

[0149] The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein, such as a type I transmembrane protein. If the source is synthetic, the transmembrane domain can be any artificial sequence that facilitates insertion of the CAR into the cell membrane, such as an artificial hydrophobic sequence. Examples of transmembrane domains particularly useful in the present invention include, but are not limited to, the alpha, beta, or zeta chains of the T cell receptor, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), transmembrane domains derived from Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 (i.e., including at least the transmembrane domain). In some embodiments, the transmembrane domain may be synthetic, in which case it predominantly contains hydrophobic residues such as leucine and valine. In one exemplary embodiment, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0150] The transmembrane domains described herein can be combined with any of the antigen-binding domains described herein, any of the co-stimulatory signaling domains described herein, any of the intracellular signaling domains described herein, or any of the other domains described herein that may be included in the subject CAR.

[0151] In some embodiments, the transmembrane domain further includes a hinge region. The subject CAR of the present invention can also include a hinge region. The hinge region of the CAR is a hydrophilic region located between the antigen-binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR. The hinge region is an optional component of the CAR. The hinge region may include a domain selected from an antibody Fc fragment, an antibody hinge region, an antibody CH2 region, an antibody CH3 region, an artificial hinge sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, the CD8a hinge, or artificial hinges made of polypeptides as small as the CH1 and CH3 domains of glycine (Gly) or IgG (such as human IgG4).

[0152] In some embodiments, the CAR of this disclosure includes a hinge region connecting an antigen-binding domain and a transmembrane domain, which in turn connects to an intracellular domain. In exemplary embodiments, the hinge region can support the recognition and binding of the target antigen on the target cell by the antigen-binding domain (see, for example, Hudecek et al., Cancer Immunol. Res., 3(2): 125-135 (2015)). In some embodiments, the hinge region is flexible, allowing the antigen-binding domain to have a structure that optimally recognizes the specific structure and density of a target antigen on cells such as tumor cells. Due to the flexibility of the hinge region, it can assume various conformations.

[0153] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a receptor-derived hinge region polypeptide (e.g., a CD8-derived hinge region).

[0154] The hinge region can have a length of about 4 to about 50 amino acids, for example, about 4 to about 10 amino acids, about 10 to about 15 amino acids, about 15 to about 20 amino acids, about 20 to about 25 amino acids, about 25 to about 30 amino acids, about 30 to about 40 amino acids, or about 40 to about 50 amino acids.

[0155] A suitable hinge region can be easily selected and may include any of a number of suitable lengths, for example, from about 1 amino acid (e.g., .Gly) to about 20 amino acids, from about 2 amino acids to about 15 amino acids, from about 3 amino acids to about 12 amino acids, from about 4 amino acids to about 10 amino acids, from about 5 amino acids to about 9 amino acids, from about 6 amino acids to about 8 amino acids, or from about 7 amino acids to about 8 amino acids, and may be about 1, about 2, about 3, about 4, about 5, about 6, or about 7 amino acids.

[0156] For example, the hinge region is made of glycine polymer (G) n , glycine-serine polymer (e.g., (GS) n (GSGGS) n (Sequence No. 47) and (GGGS) n (SEQ ID NO: 48) (where n is at least an integer of 1), and includes glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine or glycine-serine polymers can be used, and both Gly and Ser are relatively unstructured and can therefore function as neutral tethers between components. Glycine polymers can be used, as glycine has access to considerably more phi-psi space than alanine and is far less restrictive than residues with long side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions may consist of amino acid sequences such as GGSG (SEQ ID NO: 29), GGSGG (SEQ ID NO: 30), GSGSG (SEQ ID NO: 31), GSGGG (SEQ ID NO: 32), GGGSG (SEQ ID NO: 33), and GSSSG (SEQ ID NO: 34), but are not limited to these.

[0157] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Amino acid sequences of immunoglobulin hinge regions are known in the art, such as Tan et al., Proc. Natl. Acad. Sci. USA, 87(1):162-166 (1990); and Huck et al., Nucleic Acids Res., 14(4): 1779-1789 (1986). As a non-limiting example, an immunoglobulin hinge region may contain one of the following amino acid sequences: DKTHT (SEQ ID NO. 35); CPPC (SEQ ID NO. 36); CPEPKSCDTPPPCPR (SEQ ID NO. 37) (e.g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); ELKTPLGDTTHT (SEQ ID NO. 38); KSCDKTHTCP (SEQ ID NO: 39); KCCVDCP (SEQ ID NO. 40); KYGPPCP (SEQ ID NO. 41); EPKSCDKTHTCPPCP (SEQ ID NO. 42) (Human IG1 hinge); ERKCCVECPPCP (SEQ ID NO. 43) (Human IG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO. 44) (Human IG3 hinge); SPNMVPHAHHAQ (SEQ ID NO. 45) (Human IG4 hinge).

[0158] The hinge region may include the amino acid sequence of the hinge region of human IgG1, IgG2, IgG3, or IgG4. In one embodiment, the hinge region may contain one or more amino acid substitutions and / or insertions and / or deletions compared to the wild-type (naturally occurring) hinge region. For example, His229 in the human IgG1 hinge can be replaced with Tyr such that the hinge region contains the sequence EPKSCDKTYTCPPCP (sequence number 46); see, for example, Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one embodiment, the hinge region may consist of an amino acid sequence derived from human CD8 or a variant thereof.

[0159] In one embodiment, the transmembrane domain includes a CD8α transmembrane domain. In some embodiments, the target CAR comprises a CD8α transmembrane domain containing the amino acid sequence described in SEQ ID NO: 23, which may be encoded by a nucleic acid sequence containing the nucleotide sequence described in SEQ ID NO: 24.

[0160] In another embodiment, the target CAR includes a CD8α hinge domain and a CD8α transmembrane domain. In one embodiment, the CD8α hinge domain comprises the amino acid sequence described in SEQ ID NO: 25, which can be encoded by a nucleic acid sequence comprising the nucleotide sequence described in SEQ ID NO: 26.

[0161] In one embodiment, the transmembrane domain consists of a CD28 transmembrane domain. In some embodiments, the target CAR comprises a CD28 transmembrane domain containing the amino acid sequence described in SEQ ID NO: 27, which may be encoded by a nucleic acid sequence containing the nucleotide sequence described in SEQ ID NO: 28.

[0162] Acceptable variations of the transmembrane domain and / or hinge domain will be known to those skilled in the art while maintaining their intended function. For example, in some embodiments, the transmembrane domain or hinge domain contains an amino acid sequence having sequence identity of at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% with any of the amino acid sequences described in SEQ ID NOs. For example, in some embodiments, the transmembrane domain or hinge domain is encoded by a nucleic acid sequence containing a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% sequence identity with any of the nucleotide sequences described in SEQ ID NOs.

[0163] The transmembrane domain may be combined with any hinge domain and / or may include one or more transmembrane domains as described herein.

[0164] The transmembrane domains described herein, for example, the transmembrane regions of the α, β, or zeta chains of T cell receptors, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9 can be combined with any of the antigen-binding domains described herein, any of the co-stimulatory signaling domains, intracellular domains, or cytoplasmic domains described herein, or any of the other domains described herein that may be included in CAR.

[0165] In one embodiment, the transmembrane domain may be synthetic, in which case it mainly consists of hydrophobic residues such as leucine and valine. In exemplary embodiments, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0166] In some embodiments, the target CAR may further include a spacer domain between the extracellular domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR. As used herein, the term “spacer domain” generally means any oligo or polypeptide having the function of linking the transmembrane domain to the extracellular or intracellular domain of a polypeptide chain. The spacer domain may contain up to about 300 amino acids, for example, about 10 to about 100 amino acids, or about 25 to about 50 amino acids. In some embodiments, the spacer domain may be a short oligo or polypeptide linker, for example, between about 2 and about 10 amino acids in length. For example, a glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain of the target CAR.

[0167] Therefore, the subject CAR of this disclosure may include any of the transmembrane domains, hinge domains, or spacer domains described herein.

[0168] 3. intracellular domain The subject CAR of the present invention also includes an intracellular domain. The intracellular domain of the CAR plays a role in activating at least one effector function of the cell expressing the CAR (e.g., an immune cell). The intracellular domain transmits effector function signals, instructing the cell (e.g., an immune cell) to perform its specific function, such as harming and / or destroying target cells.

[0169] The intracellular domain of the CAR, or otherwise its cytoplasmic domain, is responsible for activating the cell expressing the CAR. Examples of intracellular domains used in the present invention include, but are not limited to, the cytoplasmic portion of a surface receptor, a costimulatory molecule, and any molecule that acts in coordination to initiate signal transduction in T cells, as well as any derivatives or variants of these elements, and any synthetic sequence having the same functional capacity.

[0170] In one embodiment, the intracellular domain includes a co-stimulus signaling domain. In one embodiment, the intracellular domain includes an intracellular signaling domain. In one embodiment, the intracellular domain includes a co-stimulatory signaling domain and an intracellular signaling domain. In one embodiment, the intracellular domain includes 4-1BB and CD3 zeta. In one embodiment, the co-stimulus signaling domain includes 4-1BB. In one embodiment, the intracellular signaling domain includes CD3 zeta.

[0171] In one embodiment, the intracellular domain of CAR includes a co-stimulatory signaling domain that comprises any portion of one or more co-stimulatory molecules, such as at least one signaling domain of an intracellular domain derived from a protein of the TNFR superfamily, CD27, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS (CD278), NKG2C, B7-H3 (CD276), or a killer immunoglobulin-like receptor (KIR, its derivatives or variants, and its synthetic sequences having the same functional capacity, and combinations thereof).

[0172] Examples of the intracellular signaling domains mentioned above include, but are not limited to, the zeta chain of the T cell receptor complex or its homologs, such as the η chain, FcsRIγ and β chains, MB1(Iga) chain, B29(Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (Δ, δ, ε), syk family tyrosine kinases (Syk, ZAP70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), CD2, CD5, CD28, and other molecules involved in T cell transduction. In one embodiment, the intracellular signaling domain may be a human CD3 zeta chain, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) having a cytoplasmic receptor, or a combination thereof.

[0173] Other examples of the intracellular domains mentioned above include, but are not limited to, one or more molecules or receptors, such as TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma R11a, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, and lymphocytes. Ligands that specifically bind to globular function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1Id, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CDlib, ITGAX, CD11c, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, D NAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NT B-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other costimulatory molecules described herein, their derivatives, variants or fragments, synthetic sequences of costimulatory molecules having the same functional ability, and combinations thereof. Examples of originating fragments or domains include those derived from these sources.

[0174] Examples of additional intracellular domains include, but are not limited to, several types of intracellular signaling domains of various other immune signaling receptors, including CD3, B7 family costimulatory, and first, second, and third generation T cell signaling proteins, including tumor necrosis factor receptor (TNFR) superfamily receptors (see, for example, Park and Brentjens, J. Clin. Oncol., 33(6): 651-653 (2015)). Furthermore, the intracellular signaling domains may also include signaling domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol., 6: 195 (2015)), such as NKp30(B7-H6) (see, e.g., Zhang et al., J. Immunol., 189(5): 2290-2299 (2012)), and DAP12 (see, e.g., Topfer et al., J. Immunol., 194(7): 3201-3212 (2015)), as well as NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0175] The intracellular signaling domains suitable for use in the target CAR of the present invention include any desired signaling domains that provide a clear and detectable signal (e.g., increased production of one or more cytokines by a cell, alteration of transcription of a target gene, alteration of protein activity; alteration of cell behavior, e.g., cell death; cell proliferation; cell differentiation; cell survival; regulation of cell signaling responses, etc., in response to CAR activation (i.e., activation by an antigen and a dimerizer)). In some embodiments, the intracellular signaling domain includes at least one ITAM motif (e.g., 1, 2, 3, 4, 5, 6, etc.) as described below. In some embodiments, the intracellular signaling domain includes a DAP10 / CD28 type signaling chain. In some embodiments, the intracellular signaling domain is not covalently bound to the membrane-bound CAR, but instead diffuses into the cytoplasm.

[0176] The intracellular signaling domain suitable for use in the target CAR of the present invention includes an immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptide. In some embodiments, the ITAM motif is repeated twice in the intracellular signaling domain, and the first and second instances of the ITAM motif are separated from each other by 6 to 8 amino acids. In one embodiment, the intracellular signaling domain of the target CAR contains three ITAM motifs. In some embodiments, the intracellular signaling domain includes a signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine-based activation motif (ITAM) such as Fc-gamma-RI, Fc-gamma-RIIA, Fc-gamma-RIIC, Fc-gamma-RIIIIA, or FcRL5 (see, for example, Gillis et al., Front. Immunol., 5:254 (2014)).

[0177] A suitable intracellular signaling domain can be an ITAM motif-containing portion derived from an ITAM motif-containing polypeptide. For example, a suitable intracellular signaling domain may be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Therefore, a suitable intracellular signaling domain does not need to include the entire sequence of the entire protein from which it originates. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fcε receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0178] In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR-binding protein; TYRO protein tyrosine kinase binding protein; killer-activated receptor binding protein; killer-activated receptor binding protein, etc.). In one embodiment, the intracellular signal transduction domain is derived from FCER1G (FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc epsilon RI gamma; fcR gamma; fceR1 gamma; also known as high affinity immunoglobulin ε receptor subunit γ; immunoglobulin E receptor, high affinity gamma chain, etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain, etc.). In one embodiment, the intracellular signal transduction domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). In one embodiment, the intracellular signal transduction domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-gamma, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signal transduction domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-zeta, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signal transduction domain is derived from CD79A (also known as B cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; Ig-alpha; membrane-bound immunoglobulin-associated protein; surface IGM-associated protein, etc.). In one embodiment, an intracellular signaling domain suitable for use in the target CAR of this disclosure includes a DAP10 / CD28 type signaling chain. In one embodiment, the intracellular signaling domain suitable for use in the CARs of this disclosure includes the ZAP70 polypeptide. In some embodiments, the intracellular signaling domain includes the cytoplasmic signaling domains of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in CAR includes the cytoplasmic signaling domain of human CD3 zeta.

[0179] Typically, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire chain. When using a truncated portion of the intracellular signaling domain, that portion can be used in place of the intact chain, as long as it transmits the effector functional signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain that is sufficient to transmit the effector functional signal.

[0180] The intracellular signaling domains described herein may be combined with any of the costimulatory signaling domains described herein, any of the antigen-binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that may be included in a CAR.

[0181] Furthermore, mutant intracellular signaling domains suitable for use in target CARs are known in the art. The YMFM motif is an SH2-binding motif present in ICOS that recruits both the p85 and p50 alpha subunits of PI3K, thereby enhancing AKT signaling. See, for example, Simpson et al. (2010) Curr. Opin. Immunol., 22:326-332. In one embodiment, a CD28 intracellular domain mutant may be generated to include a YMFM motif.

[0182] In one embodiment, the intracellular domain of the target CAR includes a 4-1BB costimulatory domain containing the amino acid sequence described in SEQ ID NO: 1, which may be encoded by a nucleic acid sequence containing the nucleotide sequence described in SEQ ID NO: 2 or 3. In one embodiment, the intracellular domain of the target CAR includes a CD28 costimulatory domain containing the amino acid sequence described in SEQ ID NO: 4, which may be encoded by a nucleic acid sequence containing the nucleotide sequence described in SEQ ID NO: 5. In one embodiment, the intracellular domain of the target CAR includes a CD28(YMFM) costimulatory domain containing the amino acid sequence described in SEQ ID NO: 6, which may be encoded by a nucleic acid sequence containing the nucleotide sequence described in SEQ ID NO: 7. In one embodiment, the intracellular domain of the target CAR includes an ICOS costimulatory domain comprising the amino acid sequence described in SEQ ID NO: 8, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence described in SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the intracellular domain of the target CAR includes an ICOS(YMNM) costimulatory domain containing the amino acid sequence described in SEQ ID NO: 11, which may be encoded by a nucleic acid sequence containing the nucleotide sequence described in SEQ ID NO: 12. In one embodiment, the intracellular domain of the target CAR includes a CD2 costimulatory domain comprising the amino acid sequence described in SEQ ID NO: 13, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence described in SEQ ID NO: 14. In one embodiment, the intracellular domain of the target CAR includes a CD27 costimulatory domain comprising the amino acid sequence described in SEQ ID NO: 15, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence described in SEQ ID NO: 16. In one embodiment, the intracellular domain of the target CAR includes an OX40 costimulatory domain comprising the amino acid sequence described in SEQ ID NO: 17, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence described in SEQ ID NO: 18.

[0183] In one embodiment, the intracellular domain of the target CAR includes a CD3 zeta intracellular signaling domain comprising the amino acid sequence described in SEQ ID NO: 19 or SEQ ID NO: 21, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence described in SEQ ID NO: 20 or SEQ ID NO: 22, respectively.

[0184] Acceptable variations of the intracellular domain while maintaining specific activity will be known to those skilled in the art. For example, in some embodiments, the intracellular domain includes an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with either of the amino acid sequences described in SEQ ID NO: 19 or 21. For example, in some embodiments, the intracellular domain is encoded by a nucleic acid sequence containing a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with one of the nucleotide sequences described in SEQ ID NO: 20 or 22.

[0185] In one embodiment, the intracellular domain of the target CAR includes an ICOS costimulatory domain and a CD3 zeta intracellular signaling domain. In one embodiment, the intracellular domain of the target CAR includes a CD28 costimulatory domain and a CD3 zeta intracellular signaling domain. In one embodiment, the intracellular domain of the target CAR includes a CD28 YMFM mutant costimulatory domain and a CD3 zeta intracellular signaling domain. In one embodiment, the intracellular domain of the target CAR includes a CD27 costimulatory domain and a CD3 zeta intracellular signaling domain. In one embodiment, the intracellular domain of the target CAR includes an OX40 costimulatory domain and a CD3 zeta intracellular signaling domain. In one exemplary embodiment, the intracellular domain of the target CAR includes a 4-1BB costimulatory domain and a CD3 zeta intracellular signaling domain. In one exemplary embodiment, the intracellular domain of the target CAR includes a CD2 costimulatory domain and a CD3 zeta intracellular signaling domain.

[0186] Table 1 shows exemplary sequences of CAR domains as described herein. [Table 1A] [Table 1B] [Table 1C]

[0187] BT cell receptor In some embodiments, modified non-stimulated immune cells or modified stimulated immune cells (e.g., modified non-stimulated or stimulated T cells) express exogenous T cell receptors (TCRs). Any modified cells containing TCRs having affinity for any antigen (e.g., solid tumor antigens) are conceivable and can be readily understood and manufactured by those skilled in the art in view of the disclosure herein.

[0188] In some embodiments, the target cells are altered to include specific T cell receptor (TCR) genes (e.g., TRAC and TRBC genes). The TCR or its antigen-binding moiety includes those that recognize peptide epitopes or T cell epitopes of target polypeptides, such as tumor, viral, or autoimmune protein antigens. In some embodiments, the TCR has binding specificity to tumor-associated antigens. In some embodiments, exemplary tumor-associated antigens include NY-ESO-1 (LAGE2, LAGE2B, or cancer / testicular antigen 1), melanoma-associated antigen (MAGE), and H3.3K27M.

[0189] TCRs are disulfide-bonded heterodimer proteins consisting of six different membrane-bound chains, and are involved in the activation of T cells in response to antigens. Alpha / beta TCRs and gamma / delta TCRs exist. Alpha / beta TCRs contain TCR alpha chains and TCR beta chains. T cells expressing a TCR containing TCR alpha chains and TCR beta chains are generally called alpha / beta T cells. Gamma / delta TCRs contain TCR gamma chains and TCR delta chains. T cells expressing a TCR consisting of TCR gamma chains and TCR delta chains are generally called gamma / delta T cells. The TCRs of this disclosure are TCRs containing TCR alpha chains and TCR beta chains. The TCR alpha chain and TCR beta chain each consist of two extracellular domains: a variable region and a constant region. The TCR alpha chain variable region and TCR beta chain variable region are regions necessary for the TCR to become compatible with the target antigen. Each variable region contains three hypervariable or complementarity-determining regions (CDRs) that provide binding to the target antigen. The constant regions of the TCR alpha chain and the TCR beta chain are located near the cell membrane. The TCR further includes a transmembrane region and a short cytoplasmic tail. The CD3 molecule combines with the TCR heterodimer. The CD3 molecule contains a characteristic sequence motif for tyrosine phosphorylation known as the immune receptor tyrosine-based activation motif (ITAM). Since nearby signaling events occur via the CD3 molecule, the interaction of the TCR-CD3 complex plays a crucial role in mediating cell recognition events.

[0190] Stimulation of the transcatheter cell (TCR) is triggered by major histocompatibility complex (MHC) molecules on antigen-presenting cells that present antigen peptides to T cells, and these MHC molecules interact with the TCR to induce a series of intracellular signaling cascades. TCR involvement initiates both positive and negative signaling cascades, leading to cell proliferation, cytokine production, and activation-induced cell death.

[0191] The TCR of the present invention may be a wild-type TCR, a high-affinity TCR, and / or a chimeric TCR. A high-affinity TCR may be the result of modification of a wild-type TCR that confers a higher affinity to a target antigen compared to a wild-type TCR. A high-affinity TCR may be an affinity-matured TCR. Methods for modifying the affinity maturation of a TCR and / or a TCR are known to those skilled in the art. Techniques for engineering the expression of TCRs include, but are not limited to, the production of TCR heterodimers containing native disulfide bridges that link each subunit (Garboczi, et al., (1996), Nature 384(6605): 134-41; Garboczi, et al., (1996), J Immunol 157(12): 5403-10; Chang et al., (1994), PNAS USA 91: 1 1408-1 1412; Davodeau et al., (1993), J. Biol. Chem. 268(21): 15455-15460; Golden et al., (1997), J. Imm. Meth. 206: 163-169; US Pat. No. 6,080,840).

[0192] In some embodiments, the exogenous TCR is a complete TCR or its antigen-binding portion or antigen-binding fragment. In some embodiments, the TCR is an intact or full-length TCR, including an ab-type or gd-type TCR. In some embodiments, the TCR is less than the full-length TCR, but is an antigen-binding moiety that binds to a specific peptide bound to an MHC molecule, such as by binding to an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of the TCR may contain only a portion of the structural domain of the full-length or intact TCR, yet it may still be able to bind peptide epitopes, such as MHC-peptide complexes, to which the complete TCR would normally bind. In some cases, the antigen-binding site includes variable domains of the TCR, such as the variable a-chain and variable b-chain of the TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chain of the TCR contains complementarity-determining regions (CDRs) that are involved in the recognition of peptides, MHCs, and / or MHC-peptide complexes.

[0193] In some embodiments, the variable domain of the TCR includes a hypervariable loop, or CDR, which generally contributes primarily to antigen recognition and binding ability and specificity. In some embodiments, the CDRs of a TCR, or combinations thereof, form all or substantially all of the antigen-binding sites of a given TCR molecule. The various CDRs within the variable region of the TCR chain are generally separated by a framework region (FR), which generally exhibits less variation among TCR molecules compared to the CDRs (see, for example, Jores et al, PNAS, 87:9138 (1990); Chothia et al., EMBO J., 7:3745 (1988) or Lefranc et al., Dev. Comp. Immunol., 27:55 (2003)). In some embodiments, CDR3 is the primary CDR responsible for antigen binding or specificity, or is the most important of three CDRs on a given TCR variable region for antigen recognition and / or interaction with the treated peptide portion of the peptide-MHC complex. In some situations, the CDR1 in the alpha chain can interact with the N-terminal portion of certain antigenic peptides. In some situations, the CDR1 in the beta chain can interact with the C-terminal portion of the peptide. In some situations, CDR2 is the primary CDR that most strongly contributes to, or is responsible for, the interaction with or recognition of the MHC portion of the MHC-peptide complex. In some embodiments, the variable region of the b chain may include a further hypervariable region (CDR4 or HVR4) that is generally involved in superantigen binding but not in antigen recognition (Kotb, Clinical Microbiology Reviews, 8:41 1-426 (1995)).

[0194] In some embodiments, the TCR comprises a variable alpha domain (Va) and / or a variable beta domain (Vp) or antigen-binding fragments thereof. In some embodiments, the alpha and / or beta chains of the TCR may also include a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, for example, Janeway et al., Immunobiology: The Immune System in Health and Disease, 3 Ed., Current Biology Publications, p. 4:33 (1997)).

[0195] In some embodiments, the alpha chain constant domain is encoded by the TRAC gene (IMGT nomenclature) or a variant thereof. In some embodiments, the beta-chain constant region is encoded by the TRBC1 or TRBC2 gene (IMGT nomenclature) or a variant thereof. In some embodiments, the constant domain is adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two strands includes two membrane-proximal constant domains and two membrane-distal variable domains, each of which contains a CDR.

[0196] Determining or identifying the various domains or regions of a TCR is within the scope of the skills of the art. In some embodiments, TCR residues can be identified by known or according to the International Immunogenetic Information System (IMGT) numbering system (see, e.g., www.imgt.org; see also, Lefranc et al., Developmental and Comparative Immunology, 2:55-77 (2003), and The T Cell Factsbook 2nd Edition, Lefranc and LeFranc Academic Press 2001). Using this system, the CDR1 sequence in the TCR Va chain and / or nb chain corresponds to amino acids located between residue numbers 27 and 38 (including both ends), the CDR2 sequence in the TCR Va chain and / or nb chain corresponds to amino acids located between residue numbers 56 and 65 (including both ends), and the CDR3 sequence in the TCR Va chain and / or nb chain corresponds to amino acids located between residue numbers 105 and 117 (including both ends).

[0197] In some embodiments, the TCR may be a heterodimer of two chains a and b (or optionally g and d) linked by one disulfide bond or multiple disulfide bonds. In some embodiments, the constant domain of the TCR may include a short ligation sequence in which cysteine ​​residues form disulfide bonds, thereby linking two chains of the TCR. In some embodiments, the TCR may have additional cysteine ​​residues in each of the a-chain and b-chain such that the TCR contains two disulfide bonds in the constant domain. In some embodiments, each of the constant domain and the variable domain includes a disulfide bond formed by a cysteine ​​residue.

[0198] In some embodiments, the TCRs for manipulating cells as described are generated from known TCR sequences, such as na and b-chain sequences, for which substantially full-length encoding sequences are readily available. Methods for obtaining full-length TCR sequences, including the v-chain sequence, from cell sources are well known. In some embodiments, the nucleic acid encoding the TCR can be obtained from various sources, such as polymerase chain reaction (PCR) amplification of the TCR-encoding nucleic acid isolated from or within a given single or multiple cells, or synthesis of a publicly available TCR DNA sequence. In some embodiments, the TCR is obtained from a biological source, such as cells from T cells (e.g., cytotoxic T cells), T cell hybridomas, or other known sources. In some embodiments, the T cells can be obtained from cells isolated in vivo. In some embodiments, the T cells may be cultured T cell hybridomas or clones. In some embodiments, the TCR or its antigen-binding portion can be synthesized from knowledge of the TCR sequence. In some embodiments, high-affinity T cell clones for a target antigen (e.g., a cancer antigen) are identified, isolated from the patient, and introduced into cells. In some embodiments, the TCR clone of the target antigen is generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system, or HLA). For example, tumor antigens can be referenced (see, e.g., Parkhurst et al., Clin Cancer Res., 15: 169-180 (2009) and Cohen et al., J. Immunol., 175:5799-5808 (2005)). In some embodiments, phage display is used to isolate TCRs against target antigens (see, for example, Varela-Rohena et al., Nat. Med., 14: 1390-1395 (2008) and Li, Nat. Biotechnol., 23:349-354 (2005)). In some embodiments, the TCR or its antigen-binding portion is modified or manipulated. In some embodiments, directed evolution is used to generate TCRs with altered properties, such as higher affinity for specific MHC-peptide complexes. In some embodiments, directed evolution is achieved by display methods, including, but not limited to, yeast display (Holler et al., Nat. Immunol., 4:55-62 (2003); Holler et al., PNAS USA, 97: 5387-92 (2000)), phage display (Li et al., Nat. Biotechnol., 23: 349-54 (2005)), or T cell display (Chervin et al., J. Immunol. Methods, 339:175-84 (2008)). In some embodiments, the display approach involves manipulating or modifying a known, parental, or reference TCR. For example, in some cases, a wild-type TCR can be used as a template to produce a mutagenized TCR in which one or more residues of the CDR are mutated, and a variant having the desired alteration characteristics, such as high affinity for a desired target antigen, is selected.

[0199] In some embodiments as described, the TCR may include one or more introduced disulfide bonds. In some embodiments, native disulfide bonds are absent. In some embodiments, one or more native cysteine ​​residues that form native interchain disulfide bonds (e.g., in the constant domains of the a and b chains) are replaced with other residues such as serine or alanine. In some embodiments, the introduced disulfide bond can be formed by mutating non-cysteine ​​residues in the alpha and beta chains, for example, in the constant domains of the a and b chains, to cysteine. Exemplary non-native disulfide bonds of the TCR are described in WO2006 / 000830 and WO2006 / 037960. In some embodiments, cysteine ​​may be introduced at the Thr48 residue of the alpha chain and the Ser57 residue of the beta chain, the Thr45 residue of the alpha chain and the Ser77 residue of the beta chain, the TyrIO residue of the alpha chain and the Serl7 residue of the beta chain, the Thr45 residue of the alpha chain and the Asp59 residue of the beta chain and / or the Serl5 residue of the alpha chain and the Gulul5 residue of the beta chain. In some embodiments, the presence of non-native cysteine ​​residues (e.g., resulting in one or more non-native disulfide bonds) in recombinant TCRs can favor the production of the desired recombinant TCR in the introduced cells compared to the expression of mismatched TCR pairs containing native TCR chains.

[0200] In some embodiments, the TCR chain includes a transmembrane domain. In some embodiments, the transmembrane domains are positively charged. In some cases, the TCR chain may include a cytoplasmic tail. In some embodiments, each chain of the TCR (e.g., alpha or beta) may possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short C-terminal cytoplasmic tail. In some embodiments, the TCR is associated with an invariant protein of the CD3 complex involved in signal transduction, for example, via the cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules such as CD3 or its subunits. For example, a TCR containing a constant domain with a transmembrane region can anchor the protein to the cell membrane and associate with an invariant subunit of the CD3 signaling machinery or complex. The intracellular tail of CD3 signaling subunits (CD3Y, CD36, CD3s, O-upsilon 3z chain, etc.) contains one or more immunoreceptor tyrosine-based activation motifs or ITAMs that are involved in the signaling ability of the TCR complex.

[0201] In some embodiments, the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-stranded TCR (sc-TCR). The TCR may be cell-bound or soluble. In some embodiments, for the purposes of the provided method, the TCR is a cell-bound type expressed on the surface of a cell. In some embodiments, the dTCR comprises a first polypeptide in which a sequence corresponding to the TCR a-chain variable region sequence is fused to the N-terminus of a sequence corresponding to the TCR a-chain constant region extracellular sequence, and a second polypeptide in which a sequence corresponding to the TCR b-chain variable region sequence is fused to the N-terminus of a sequence corresponding to the TCR b-chain constant region extracellular sequence, wherein the first and second polypeptides are linked by a disulfide bond. In some embodiments, the bond may correspond to the native interchain disulfide bond present in the native dimer ab TCR. In some embodiments, the interchain disulfide bond is absent in the native TCR. For example, in some embodiments, one or more cysteines can be incorporated into the extracellular sequence of the constant region of the dTCR polypeptide pair. In some cases, both native and non-native disulfide bonds may be desirable. In some embodiments, the TCR includes a transmembrane array for anchoring to the membrane. In some embodiments, the dTCR comprises a TCR a-chain containing a variable a-domain, a constant a-domain, and a first dimerization motif attached to the C-terminus of the constant a-domain, and a TCR b-chain containing a variable b-domain, a constant b-domain, and a first dimerization motif attached to the C-terminus of the constant b-domain, wherein the first and second dimerization motifs readily interact to form a covalent bond between the amino acids of the first dimerization motif and the amino acids of the second dimerization motif, linking the TCR a-chain and TCR b-chain together.

[0202] In some embodiments, the TCR is an scTCR, which is a single amino acid chain comprising an α chain and a β chain that can bind to an MHC-peptide complex.

[0203] Typically, scTCR can be produced using methods known to those skilled in the art; see, for example, WO1996 / 13593, WO1996 / 18105, WO1999 / 18129, WO2004 / 033685, WO2006 / 037960, WO2011 / 044186, U.S. Patent No. 7,569,664 and Schlueter et al. J. Mol. Biol., 256:859 (1996).

[0204] In some embodiments, the transmembrane domain may be a Ca or CP transmembrane domain. In some embodiments, the transmembrane domain may be a non-TCR-derived transmembrane region, such as CD3z, CD28, or B7.1. In some embodiments, the TCR includes a sequence corresponding to a cytoplasmic sequence. In some embodiments, the TCR includes a CD3z signaling domain. In some embodiments, the TCR can form a TCR complex with CD3. In some embodiments, the TCR or its antigen-binding portion may be a recombinantly produced natural protein or a variant thereof, in which one or more properties such as binding characteristics have been altered. In some embodiments, the TCR may be derived from one of various animal species, such as humans, mice, rats, or other mammals.

[0205] In some embodiments, the TCR comprises an affinity for a target antigen on the target cell. The target antigen may include any type of protein or epitope associated with the target cell. For example, the TCR may comprise an affinity for a target antigen on the target cell that indicates a specific disease state of the target cell. In some embodiments, the target antigen is treated with MHC and presented.

[0206] In some embodiments, the TCR is encoded by a nucleic acid construct that sequentially encodes a first heterologous TCR subunit chain from the N-terminus to the C-terminus, the TCR subunit chain including a variable region and a constant region of the TCR subunit chain, as well as a variable region and a variable region of a second heterologous TCR subunit chain. The construct further encodes a first self-cleaving peptide preceding the variable region of the first heterologous TCR subunit chain, and a second self-cleaving peptide between the first heterologous TCR subunit chain and the second TCR subunit. In some embodiments, the first heterogeneous TCR subunit chain is a TCRα chain, and the second heterogeneous TCR subunit chain is a TCRβ chain. In some embodiments, the first heterogeneous TCR subunit chain is a TCRβ chain, and the second heterogeneous TCR subunit chain is a TCRα chain.

[0207] Examples of self-cleaving peptides, but not limited to these, include self-cleaving viral 2A peptides, such as porcine tescovirus-1 (P2A) peptide, Thosea asigna (T2A) peptide, equine rhinitis A virus (E2A) peptide, or foot-and-mouth disease virus (F2A) peptide. Self-cleaving 2A peptides can express multiple gene products from a single construct (see, for example, Chng et al. "Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells," MAbs, 7(2): 403-412 (2015)). In some embodiments, the first and second self-cleaving peptides are the same. In some embodiments, the first and second self-cleaving peptides are different.

[0208] C. Addition of antigen-binding polypeptides In some embodiments, the modified non-stimulated immune cells or modified stimulated immune cells (e.g., modified non-stimulated or stimulated T cells) express a polypeptide that binds to an antigen-binding domain, a cell surface receptor ligand, or a tumor antigen. In some examples, the antigen-binding domain includes an antibody that recognizes a cell surface protein or a receptor expressed on tumor cells. In some examples, the antigen-binding domain includes an antibody that recognizes a tumor antigen. In some examples, the antigen-binding domain includes a full-length antibody or its antigen-binding fragment, Fab, F(ab)2, monospecific Fab2, bispecific Fab2, trispecific Fab2, single-chain variable fragment (scFv), diabody, triabody, minibody, V-NAR, or VhH.

[0209] In some embodiments, the modified unstimulated immune cells or modified stimulated immune cells (e.g., modified unstimulated or stimulated T cells) express cell surface receptor ligands. In some cases, the ligand binds to cell surface receptors expressed on tumor cells. In some cases, the ligand includes a wild-type protein or a variant thereof that binds to the cell surface receptor. In some examples, the ligand includes a full-length protein or a functional fragment thereof that binds to the cell surface receptor. In some cases, the functional fragment contains approximately 90%, 80%, 70%, 60%, 50%, or 40% of the length of the full-length version of the protein, but its binding to the cell surface receptor is retained. In some cases, the ligand is a de novo engineered protein that binds to the cell surface receptor. Exemplary ligands, but not limited to these, include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or Wnt3A.

[0210] In some embodiments, the modified non-stimulated immune cells or modified stimulated immune cells (e.g., modified non-stimulated or stimulated T cells) express polypeptides that bind to tumor antigens. In some cases, the tumor antigens are associated with hematological malignancies. Exemplary tumor antigens include, but are not limited to, CD19, CD20, CD22, CD33 / IL3Ra, ROR1, mesoserine, c-Met, PSMA, PSCA, folate receptor alpha, folate receptor beta, EGFRvIII, GPC2, Tn-MUC1, GDNF family receptor alpha 4 (GFRa4), fibroblast-activating protein (FAP), and IL13Ra2. In some embodiments, the tumor antigen includes CD19, CD20, CD22, BCMA, CD37, mesoserine, PSMA, PSCA, Tn-MUC1, EGFR, EGFRvIII, c-Met, HER1, HER2, CD33, CD133, GD2, GPC2, GPC3, NKG2D, KRAS, or WT1. In some embodiments, the polypeptide is a ligand for the tumor antigen, for example, a full-length protein that binds to the tumor antigen, a functional fragment thereof, or a de novo-modified ligand that binds to the tumor antigen. In some cases, the polypeptide is an antibody that binds to the tumor antigen.

[0211] IV. Composition The compositions of the present invention may comprise modified non-stimulated immune cells or modified stimulated immune cells as described herein, optionally in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, adjuvants, or excipients. Such compositions may comprise buffers such as neutral buffered saline or phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran; mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for parenteral administration (e.g., intravenous administration).

[0212] V. How to use In some embodiments, disclosed herein are methods for treating a disease in a subject, which include administering to a modified non-stimulated immune cell population or a modified stimulated immune cell population as described herein. In some embodiments, the disease is cancer, optionally a solid tumor, or a hematological malignancy. In some embodiments, modified non-stimulated immune cells or modified stimulated immune cells each express an antigen-binding domain that is specific to the antigen expressed by cancer.

[0213] In some embodiments, the cancer is a solid tumor. Examples of solid tumors, but not limited to these, include bladder cancer, bone cancer, brain tumors (e.g., glioma, glioblastoma, neuroblastoma), breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer. In some cases, the solid tumors are brain tumors (e.g., glioma, glioblastoma, neuroblastoma), breast cancer, lung cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, or prostate cancer. In some cases, the solid tumor is metastatic cancer. In some cases, the solid tumors are recurrent or refractory solid tumors.

[0214] In some embodiments, cancer is a hematological malignancy. In some embodiments, the hematological malignancy is a B-cell malignancy or a T-cell malignancy. In some embodiments, the hematological malignancy is lymphoma, leukemia, or myeloma. In some embodiments, the hematological malignancy is Hodgkin lymphoma or non-Hodgkin lymphoma. Examples of hematological malignancies include, but are not limited to, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, intranodal marginal zone B-cell lymphoma, and Burkittlin's disease. Examples include peritoneum, non-Burkitt high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-lymphocytic lymphoma, B-cell lymphocytic leukemia, lymphocytic plasma lymphoma, splenic marginal zone lymphoma, plasmacytosis myeloma, plasmacytoma, large mediastinal (thymic) B-cell lymphoma, intravascular large B-cell lymphoma, primary pleural effusion lymphoma, or lymphamatoid granulomatosis. In some cases, the hematological malignancies are metastatic hematological malignancies. In some cases, the hematological malignancies are recurrent or refractory hematological malignancies.

[0215] In some embodiments, a method for treating a disease further includes administering an additional therapeutic agent to the subject or providing additional treatment.

[0216] In some cases, the additional therapeutic agents disclosed herein include chemotherapeutic agents, immunotherapeutic agents, targeted therapies, radiotherapy, or combinations thereof. Exemplary additional therapeutic agents include, but are not limited to, altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, or thiotepa; and antimetabolites such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, phloxlysine, fludarabine, gemcitabine, hydroxyurea, methotrexate, or pemetrexed. Examples include anthracyclines such as daunorubicin, doxorubicin, epirubicin, or idarubicin; topoisomerase I inhibitors such as topotecan or irinotecan (CPT-11); topoisomerase II inhibitors such as etoposide (VP-16), teniposide, or mitocoxantrone; mitotic inhibitors such as docetaxel, estramustine, ixabepyrone, paclitaxel, vinblastine, vincristine, or vinorelbine; or corticosteroids such as prednisone, methylprednisolone, or dexamethasone.

[0217] In some cases, additional therapeutic agents include first-line therapy. As used herein, “first-line therapy” includes primary treatment for subjects with cancer. In some cases, the cancer is a primary cancer. In other cases, the cancer is metastatic or recurrent. In some cases, the first-line therapy includes chemotherapy. In other cases, the first-line treatment includes radiotherapy. Those skilled in the art will readily understand that different first-line treatments may be applicable to different types of cancer.

[0218] In some embodiments, the additional therapeutic agent comprises an immune checkpoint inhibitor. In some examples, the immune checkpoint inhibitors include inhibitors such as antibodies or their fragments (e.g., monoclonal antibodies, human, humanized anti, or chimeric antibodies), RNAi molecules, or small molecules against PD-1, PD-L1, CTLA4, PD-L2, LAG3, B7-H3, KIR, CD137, PS, TFM3, CD52, CD30, CD20, CD33, CD27, OX40, GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM.

[0219] Examples of checkpoint inhibitors include pembrolizumab, nivolumab, tremelimumab, or ipilimumab.

[0220] In some embodiments, the additional therapy includes radiotherapy.

[0221] In some embodiments, additional therapy includes surgery.

[0222] VI. Kits and manufactured products In some embodiments, the kits or products described herein include one or more populations of modified unstimulated immune cells (e.g., modified unstimulated T cells) or one or more populations of modified stimulated immune cells (e.g., modified stimulated T cells). In some examples, the kits or products described herein further include a carrier, package, or container partitioned to accept one or more containers such as vials or tubes, each of which contains one of the separate elements used in the method herein. Suitable containers include, for example, bottles, vials, syringes, test tubes, etc. In one embodiment, the container is formed from various materials such as glass or plastic.

[0223] The manufactured articles provided herein include packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging materials suitable for the selected formulation and intended mode of administration and treatment.

[0224] The kit typically includes a label listing the contents and / or instructions for use, and an insert in the packaging with instructions for use. Instructions are also usually included.

[0225] VII.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by an ordinary person skilled in the art to which this disclosure belongs. In the implementation or testing of this disclosure, similar or equivalent methods and materials may be used, but preferred methods and materials are described herein.

[0226] As used herein, the singular forms "a," "an," and "the" include multiple references unless the context explicitly indicates otherwise. For example, the term "a cell" includes multiple cells, including mixtures thereof.

[0227] As used herein, the term “about” is used to indicate that a value includes the standard deviation of error in the instrument or method employed to determine the value. When used before a numerical specification, such as temperature, time, quantity, or concentration (including range), the term “about” indicates an approximation that may vary by (+) or (-) 15%, 10%, 5%, 3%, 2%, or 1%.

[0228] Unless otherwise specified, the implementation of this disclosure will employ prior art in tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the scope of the art of those skilled in the art. For example, Green and Sambrook eds. (2012) Molecular Cloning: A Laboratory Manual, 4th edition; the series Ausubel et al. eds. (2015) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (2015) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; McPherson et al. (2006) PCR: The Basics (Garland Science); Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Greenfield ed. (2014) Antibodies, A Laboratory Manual; Freshney (2010) Culture of Animal Cells: A Manual of Basic Technique, 6th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Herdewijn ed. (2005) Oligonucleotide Synthesis: Methods and Applications; Hames and Higgins eds. (1984) Transcription and Translation; Buzdin and Lukyanov ed.(2007) Nucleic Acids Hybridization: Modern Applications; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi ed. (2007) In Vitro Transcription and Translation Protocols, 2nd edition; Guisan ed. (2006) Immobilization of Enzymes and Cells; Perbal (1988) A Practical Guide to Molecular Cloning, 2nd edition; Miller and Calos eds, (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Lundblad and Macdonald eds. (2010) Handbook of Biochemistry and Molecular Biology, 4th edition; and Herzenberg et See al. eds (1996) Weir's Handbook of Experimental Immunology, 5th edition.

[0229] "Allogeneic" refers to any material derived from different animals of the same species.

[0230] As used herein, “antibody” refers to such an assembly (e.g., intact antibody molecules, immune adhesins, or variants thereof) that possesses significant known specific immune response activity against an antigen of interest (e.g., tumor-associated antigen). Antibodies and immunoglobulins contain light and heavy chains, with or without interchain covalent bonds. The basic structures of vertebrate immunoglobulins are relatively well understood.

[0231] The term "antibody fragment" refers to a portion of an intact antibody, specifically the antigenicity-determining variable region of an intact antibody. Examples of antibody fragments, but not limited to these, include Fab, Fab', F(ab')2, Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0232] For example, the antigen-binding domain of a chimeric antigen receptor may contain antibody variants. As used herein, the term “antibody variant” includes antibodies in forms synthesized and manipulated to not exist in nature, such as antibodies containing at least two heavy chain portions but not two complete heavy chains (e.g., domain-deleted antibodies or minibodies); multispecific forms of antibodies modified to bind to two or more different antigens or different epitopes on a single antigen (e.g., bispecific, trispecific, etc.); and antibodies in which a heavy chain molecule is conjugated to an scFv molecule, etc. Furthermore, the term “antibody variant” also includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three, four or more copies of the same antigen).

[0233] As used herein, the terms “antigen” or “Ag” are defined as molecules that trigger an immune response. This immune response may involve antibody production, activation of specific immune cells, or both. Those skilled in the art will understand that virtually any macromolecule, including proteins or peptides, can function as an antigen. Furthermore, antigens may be derived from recombinant DNA or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that triggers an immune response, therefore, encodes an “antigen” as the term is used herein. Furthermore, those skilled in the art will understand that antigens do not need to be encoded by the full-length nucleotide sequence of a gene alone. The present invention is not limited to these, but it is readily apparent that partial nucleotide sequences of one or more genes, and these nucleotide sequences, can be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that antigens do not need to be encoded by a “gene” at all. It is readily apparent that antigens can be synthesized and produced, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0234] As used herein, the term “autologous” is intended to refer to any material originating from the same individual that may later be reintroduced into that individual.

[0235] As used herein, the terms “chimeric antigen receptor” or “CAR” refer to an artificial T cell receptor that is expressed on immune effector cells or their precursor cells and designed to specifically bind to an antigen. CARs can be used in adoptive cell therapy via adoptive cell transplantation. In some embodiments, adoptive cell transplantation (or therapy) involves removing T cells from a patient and modifying the T cells to express receptors specific to a particular antigen. In some embodiments, the CAR has specificity for a selected target, such as PSMA or MUC1. The CAR may also include an intracellular activation domain, a transmembrane domain, and an extracellular domain including an antigen-binding region.

[0236] An "expression vector" is a vector containing recombinant polynucleotides that include an expression regulatory sequence operatively linked to the nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements necessary for expression. Other elements for expression can be supplied by host cells or an in vitro expression system. Expression vectors include all known in the field, such as cosmids, plasmids (e.g., naked or liposome-containing) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) that incorporate recombinant polynucleotides.

[0237] The term "cleavage" refers to the breaking of covalent bonds, such as the hydrolysis of the backbone or peptide bonds of nucleic acid molecules. Cleavage can be initiated by a variety of methods, including, but not limited to, the enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand cleavage are possible. Double-strand cleavage can occur as a result of two different single-strand cleavage events. DNA cleavage can produce blunt or staggered ends.

[0238] As used herein, “homologous” refers to the subunit sequence identity between two polymer molecules, for example, between two nucleic acid molecules, for example, between two DNA molecules or two RNA molecules, or between two polypeptide molecules. Two molecules are homologous at a given position if the positions of both subunits are occupied by the same monomer subunit, for example, if the positions of each of the two DNA molecules are occupied by adenine. The homology of two sequences is a direct function of the number of positions that match or are identical. For example, if half of the positions of two sequences are identical (e.g., five positions in a polymer of length 10 subunits), then the two sequences are 50% identical, and if 90% of the positions (e.g., nine out of ten) match or are identical, then the two sequences are 90% identical.

[0239] The term "non-homologous end joining" or NHEJ refers to the process by which a cleaved or damaged end of a DNA strand is directly ligated without the need for a homologous template nucleic acid. NHEJ repair can result in the addition, deletion, substitution, or combination thereof of one or more nucleotides at the repair site.

[0240] The term "homology-directed repair" or HDR refers to the process by which a cleaved or damaged end of a DNA strand is repaired by the insertion of a homologous template or donor nucleic acid. In this process, the original DNA sequence is replaced with homologous template DNA. The homologous template nucleic acid can be provided by a homologous sequence from another location in the genome (sister chromosome, homologous chromosome, or repeating region on the same or different chromosome). By introducing an exogenous template nucleic acid, specific changes in the sequence at the target site can be obtained by HDR. This allows for the introduction of specific mutations or transgenes at the cleavage site. The exogenous template may be a single-stranded DNA (ssDNA) template or a double-stranded DNA (dsDNA) template that encodes the transgene or mutation introduced by HDR. In some cases, the ssDNA or dsDNA template includes two homology regions, for example, a 5' end and a 3' end, flanking the region containing the heterologous sequence to be inserted into the target cut or insertion site.

[0241] As used herein, the term “upstream” refers to the nucleic acid sequence found at a specific site or locus within the genome, for example, at the 5' end of a cleavage site catalyzed by a genome editing system. As used herein, the term “downstream” refers to the nucleic acid sequence found at a specific site or locus within the genome, at the 3' end of a locus.

[0242] As used interchangeably herein, “effective amount” or “therapeutic effective amount” refers to the amount of a compound, formulation, material, pharmaceutical agent, or composition described herein that is effective in achieving a desired physiological, therapeutic, or prophylactic outcome in a subject requiring it. Such outcomes may include, but are not limited to, an amount that, when administered to a mammal, elicits a detectable level of immune response compared to the immune response detected in the absence of the composition of the present invention. Such immune response can be readily assessed by many technically recognized methods. Those skilled in the art will understand that the amount of composition administered herein varies and can be readily determined based on many factors, such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, and the specific compound administered. The effective amount may vary between subjects depending on the health and physical condition of the subject being treated, the taxonomic group of the subject being treated, the formulation of the composition, the assessment of the subject's condition, and other relevant factors.

[0243] "Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, and mRNA, to serve as a template for the synthesis of other polymers and macromolecules in a biological process that has either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the resulting biological properties. Thus, a gene codes for a protein when the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand used as a template for the transcription of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA.

[0244] As used herein, “endogenous” means any substance that originates from or is produced within an organism, cell, tissue, or system.

[0245] As used herein, the term “epitope” is defined as a small chemical molecule on an antigen that can induce an immune response, such as a B and / or T cell response. An antigen can have one or more epitopes. Most antigens have many epitopes, i.e., are polyvalent. Generally, epitopes are about the size of 10 amino acids or sugars. In certain exemplary embodiments, the epitopes are approximately 4–18 amino acids, approximately 5–16 amino acids, approximately 6–14 amino acids, approximately 7–12 amino acids, approximately 10–12 amino acids, or approximately 8–10 amino acids. Those skilled in the art will understand that the overall three-dimensional structure, rather than a specific linear sequence of the molecule, is the primary criterion for antigen specificity, and therefore distinguishes one epitope from another. Based on this disclosure, the peptides used in the present invention may be epitopes.

[0246] As used herein, the term “exogenous” refers to any material introduced or produced from outside an organism, cell, tissue, or system.

[0247] As used herein, the term "expand" means an increase in number, such as an increase in the number of T cells. In one embodiment, the number of T cells expanded ex vivo increases relatively compared to the number originally present in the culture. In another embodiment, T cells grown in vitro are more numerous than other cell types in culture. As used herein, “ex vivo” refers to cells removed from a living organism (e.g., a human) and grown in vitro (e.g., in a culture dish, test tube, or bioreactor).

[0248] As used herein, the term “expression” is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0249] As used herein, "identity" refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules. This identity occurs when two amino acid sequences have the same residue at the same position. For example, if each position in two polypeptide molecules is occupied by arginine, then they are identical at that position. In alignment, the identity or degree of identity between two amino acid sequences having the same residue at the same position is often expressed as a percentage. The identity of two amino acid sequences is a direct function of the number of positions that match or are identical. For example, if half of the positions of the two sequences (e.g., five positions in a polymer of length 10 amino acids) are identical, then the two sequences are 50% identical, and if 90% of the positions (e.g., nine out of ten) match or are identical, then the two amino acid sequences are 90% identical.

[0250] "Isolated" means that something has been altered or removed from its natural state. For example, naturally occurring nucleic acids and peptides in living organisms are not "isolated," but the same nucleic acids or peptides that have been partially or completely separated from their naturally occurring coexisting substances are "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form, or they may exist in a non-natural environment, such as a host cell.

[0251] As used herein, the term "knockdown" refers to a reduction in the gene expression of one or more genes.

[0252] As used herein, the term "knockout" means the ablation of the gene expression of one or more genes.

[0253] As used herein, "lentivirus" refers to a genus of the family Retroviridae. Lentiviruses are characterized by their ability to infect non-dividing cells and deliver large amounts of genetic information to the host cell's DNA, making them one of the most efficient methods of gene transfer using vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means of achieving significant levels of gene transfer in vivo.

[0254] As used herein, the term "modified" refers to an altered state or structure of the molecule or cell of the present invention. Molecules may be modified in various ways, including chemically, structurally, and functionally. Cells can be modified by introducing nucleic acids.

[0255] As used herein, the term “modulating” means mediating a detectable increase or decrease in the level of response in a subject compared to the level of response in the subject in the absence of the treatment or compound, and / or compared to the level of response in another identical but untreated subject. This term includes interfering with and / or influencing a native signal or response, thereby mediating a beneficial therapeutic response in the subject (e.g., human).

[0256] In the context of this invention, the following abbreviations for commonly existing nucleic acid bases are used: "A" for adenosine, "C" for cytosine, "G" for guanosine, "T" for thymidine, and "U" for uridine.

[0257] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns to the extent that a nucleotide sequence encoding a protein may contain introns in some versions.

[0258] As used herein, the term “polynucleotide” is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. Those skilled in the art have general knowledge that nucleic acids are polynucleotides that can be hydrolyzed to monomeric “nucleotides.” Such monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, any nucleic acid sequences available in the art obtained by recombinant means, i.e., cloning and synthesis of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and polymerase chain reactions, etc.

[0259] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that a protein or peptide sequence may contain. A polypeptide includes peptides or proteins in which two or more amino acids are linked by peptide bonds. As used herein, this term refers to both short chains, commonly called peptides, oligopeptides, and oligomers in the art, and long chains, commonly called proteins in the art, and many types exist. Examples of “polypeptides” include biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0260] The term "specificity" refers to the ability to specifically bind (e.g., trigger an immune response) to a given target antigen (e.g., a human target antigen). A chimeric antigen receptor may be monospecific, containing one or more binding sites that specifically bind to a target, or it may be polyspecific, containing two or more binding sites that specifically bind to the same or different targets. In one embodiment, the chimeric antigen receptor is specific to two different (e.g., non-overlapping) parts of the same target. In one embodiment, the chimeric antigen receptor is specific to one or more targets.

[0261] As used herein with respect to antibodies, the term "specifically binds" means an antibody or its binding fragment (e.g., scFv) that recognizes a specific antigen in a sample but substantially does not recognize or bind to other molecules. For example, an antibody that specifically binds to a certain antigen may also bind to that antigen from one or more species. However, such interspecies reactivity does not in itself change the classification of antibody specificity. In another example, an antibody that specifically binds to an antigen may also bind to different alleles of that antigen. However, such cross-reactivity does not, in itself, change the classification of the antibody's specificity. In some cases, the terms "specific binding" or "specific binding" can be used in reference to the interaction between an antibody, protein, chimeric antigen receptor, or peptide and a second chemical species, meaning that the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenic determinant or epitope). For example, chimeric antigen receptors generally recognize and bind to specific protein structures, rather than proteins themselves. If an antibody is specific to epitope "A", then in a reaction involving label "A" and the antibody, the presence of a molecule containing epitope "A" (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0262] As used herein, the term “unstimulated” means a state of immune cells (e.g., T cells) that have not been stimulated or induced by the binding of a stimulating molecule (e.g., the TCR / CD3 complex) to its homologous ligand. Unstimulated T cells are also known as “quiescent” or “naive” T cells and have not been stimulated by means known in the art. Unstimulated cells express markers that may differentiate them from other cells in the population (e.g., stimulated T cells), including but not limited to CD45RA and CD62L.

[0263] As used herein, "quiescent" refers to a cell, preferably a T cell, that is in a reversible state of not dividing but retains the ability to re-enter cell proliferation. Quiescent T cells are characterized by their small cell size, low proliferative capacity, and low basal metabolic program. Quiescent cells can be stimulated to divide and proliferate. Quiescent cells are a population of cells that are substantially not proliferating. Quiescence can be measured by many means, including, but not limited to, cell proliferation assays such as the BrdU assay, EdU assay, MTT cell proliferation assay, XTT cell proliferation assay, and WST-1 cell proliferation assay, and the measurement of markers such as Ki67 and proliferating cell nuclear antigen (PCNA).

[0264] The term "stimulation" refers to a primary response triggered by the binding of a stimulating molecule (e.g., the TCR / CD3 complex) to its homologous ligand, thereby mediating a signaling event, such as, but not limited to, the TCR / CD3 complex-mediated signaling. Stimulation mediates changes in the expression of specific molecules, such as the downregulation of TGF-beta and / or cytoskeletal rearrangement, clonal expansion, and differentiation into different subsets.

[0265] As used herein, "stimulatory molecule" refers to a molecule on a T cell that specifically binds to a similar stimulatory ligand present on an antigen-presenting cell.

[0266] As used herein, "stimulatory ligand" refers to a ligand that, when present on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.), can specifically bind to a congenital binding partner on T cells (referred to here as a "stimulatory molecule"), thereby mediating a primary response by T cells, including (but not limited to) activation, initiation of an immune response, and proliferation. Stimulatory ligands are well known in the art and include, in particular, MHC class I molecules carrying peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0267] As used herein, the term “non-stimulated conditions” in relation to the culture of unstimulated immune cells (e.g., modified unstimulated immune cells) means culture conditions that do not stimulate the immune cells. In some embodiments, the culture medium does not contain any stimulating molecules or ligands that induce a stimulating response in unstimulated immune cells.

[0268] As used herein, the terms “subject” and “patient” are interchangeable. As used herein, the subject may be a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) or a mammal (e.g., monkeys and humans, etc.). In some embodiments, the term “subject” as used herein refers to vertebrates such as mammals. Mammals include, but are not limited to, humans, non-human primates, wild animals, feral animals, farm animals, sports animals, pets, etc. Any organism in which an immune response can be elicited may be a subject or a patient. In one exemplary embodiment, the subject is a human being.

[0269] A "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines the portion of the nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0270] As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. The TCR is responsible for recognizing antigens bound to the major histocompatibility complex molecule. The TCR is composed of an alpha (a) chain and a beta (b) chain heterodimer, although in some cells it is composed of gamma and delta (γ / δ) chains. The alpha / beta and gamma / delta forms of the TCR are structurally similar but differ in anatomical location and function. Each chain consists of two extracellular domains: a variable domain and a constant domain. In some embodiments, the TCR may be modified on any cell containing the TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells.

[0271] As used herein, the term “therapeutic” means treatment and / or prevention. A therapeutic effect is achieved by suppression, remission, or eradication of a disease state.

[0272] As used herein, the term “therapy” means any protocol, method and / or agent (e.g., CAR-T) that can be used in the prevention, management, treatment and / or improvement of a disease or its associated symptoms. In some embodiments, the terms “therapies” and “therapy” refer to biological therapies (e.g., adoptive cell therapy), supportive therapies (e.g., lymphectomy), and / or other therapies that are useful for the prevention, management, treatment, and / or improvement of a disease or related symptoms, as known to those skilled in the art, such as healthcare professionals.

[0273] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process by which an exogenous nucleic acid is transferred to or introduced into a host cell. A “transfected,” “transformed,” or “transduced” cell is one that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include the primary target cell and its offspring.

[0274] As used herein, the terms “treat,” “treatment,” and “to treat” refer to a reduction or improvement in the progression, severity, frequency, and / or duration of a disease or associated symptom resulting from the administration of one or more therapies (including, but not limited to, CAR-T therapies directed towards the treatment of solid tumors). As used herein, the term “to treat” may also refer to altering the course of the disease being treated. Therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of direct or indirect pathological consequences of the disease, slowing of disease progression, improvement or alleviation of the disease state, remission, or improvement of prognosis.

[0275] A "vector" is a composition of substances containing isolated nucleic acids that can be used to deliver the isolated nucleic acids into the interior of a cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides conjugated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" should be interpreted to include autonomously replicating plasmids or viruses, as well as non-plasmid and non-viral compounds that facilitate the translocation of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and lentiviral vectors.

[0276] As used herein, the term "based on" with respect to a Cas endonuclease means a Cas molecule having approximately 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to a reference or target Cas endonuclease of interest.

[0277] As used herein, the terms “complete media” and “complete medium” mean a cell culture medium optimized for the proliferation of immune cells (e.g., T cells). In some cases, complete culture media may contain proteins, inorganic salts, trace elements, vitamins, amino acids, lipids, carbohydrates, cytokines, and / or growth factors, with the ratio of each component optimized for cell proliferation. Exemplary proteins include albumin, transferrin, fibronectin, and insulin. Exemplary carbohydrates include glucose. Exemplary inorganic salts include sodium, potassium, and calcium ions. Exemplary trace elements include zinc, copper, selenium, and tricarboxylic acids. Exemplary amino acids include essential amino acids such as L-glutamine (e.g., alanyl-L-glutamine or glycyl-L-glutamine) or non-essential amino acids (NEAAs) such as glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and / or L-serine. In some embodiments, the complete medium is sodium bicarbonate (NaHCO3). 3 It may also contain one or more of the following: ), HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), phenol red, antibiotics, and / or β-mercaptoethanol. In some embodiments, the complete medium is serum-free medium. In some cases, the complete medium is a xenofree medium.

[0278] As used herein, the term "chemically defined media" means a cell culture medium in which the composition and concentration of all components are known. It differs from complete media in that complete media may contain components of unknown composition and / or concentration, such as animal-derived components.

[0279] In some cases, "xeno-free" culture media do not contain animal-derived (non-human) components. In some cases, xenofree media contain one or more human-derived components, such as human serum, growth factors, and insulin.

[0280] In some embodiments, the "serum-free" medium does not contain serum or plasma, but may contain components derived from serum or plasma. In some cases, "serum-free" media contain animal-derived components such as bovine serum albumin (BSA).

[0281] In some embodiments, the “minimum” medium contains the minimum necessary for the proliferation of target cells. In some examples, the minimal culture medium includes inorganic salts, a carbon source, and water. In some examples, supplements, cytokines, and / or proteins such as albumin (e.g., HSA) are added to the minimal medium. As used herein, supplements include trace elements, vitamins, amino acids, lipids, carbohydrates, cytokines, growth factors, or combinations thereof.

[0282] ·range Throughout this disclosure, various aspects of the invention may be presented in range form. It should be understood that the range form is merely for convenience and conciseness and should not be interpreted as arbitrarily limiting the scope of the invention. Therefore, a range description should be considered to specifically disclose not only the individual numbers within that range, but also all possible subranges. For example, a range description such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. [Examples]

[0283] These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

[0284] Example 1 - A general vector-free method for preparing modified unstimulated T cells Modified unstimulated T cells were prepared based on the method shown in Figure 2.

[0285] Simply put, unstimulated T cells were processed from fresh leukocyte samples. T cell selection from leukocytes was based on the manufacturer's protocol, specifically CD4. + and CD8 + CD4 and CD8 microbeads were used for positive selection of T cells in CliniMACS Prodigy. Enriched CD4 + and CD8 + The T cells were then resuspended in electroporation buffer.

[0286] Cas9 endonuclease, gRNA, and HDR templates targeting the TCR alpha constant (TRAC) gene locus were incubated and subsequently transfected into a population of unstimulated T cells using a 4D Nucleofector (Lonza) or an ATx or GTx electroporator (MaxCyte). The ratio of Cas9 endonuclease, gRNA, and HDR template was 1:1:1.

[0287] Next, the transfected T cells were cultured for approximately 72 hours before harvesting and then frozen at -80°C.

[0288] Example 2 - Disruption of endogenous TCR expression in unstimulated T cells using CRISPR / Cas9 with TRAC-targeting gRNA. CRISPR / Cas9 gene editing was performed to target the TCR alpha constant (TRAC) gene locus in unstimulated T cells.

[0289] Two CRISPR / Cas9 ribonucleoprotein (RNP) systems were tested: Truecut.v2 Cas9 (ThermoFisher Scientific) and SpyFi Cas9 (Aldevron). T cells subjected to mock electroporation without RNPs were used as a positive control. 72 hours after electroporation with RNP complexes, cells were analyzed by flow cytometry for CD3 epsilon and TCR alpha / beta expression.

[0290] Figures 3A and 3B illustrate that the percentage of cells expressing endogenous TCRs is significantly lower in cells with either Truecut or SpyFi CRISPR / Cas9 RNPs compared to positive control cells that were not electroporated for RNPs.

[0291] DNA donor templates were introduced to test HDR-mediated insertion into the disrupted TRAC locus. Single-stranded DNA (ssDNA) ultramer oligonucleotides and chemically modified ssDNA donors were designed with an EcoRI restriction site flanked by 5' homology arms and 3' homology arms (Figure 4). The phasic arms were designed to be homologous upstream and downstream of the CRISPR / Cas9 cut site of the TRAC locus. The ssDNA donors were introduced into cells by electroporation. To verify the presence or absence of HDR-mediated insertion, primers were designed for the genomic sequences upstream and downstream of the insertion, including the phasic arms, and the insertion site was amplified by polymerase chain reaction (PCR).

[0292] Next, the PCR products were incubated with EcoRI, and the resulting DNA fragments were separated and visualized by agarose electrophoresis.

[0293] Figure 5 shows the results of digestion with EcoRI. Successful insertion into the DNA donor is indicated by the low molecular weight bands in the lanes where both CRISPR / Cas9 editing and digestion with EcoRI were performed.

[0294] Example 3-NY-ESO-1: Knock-in strategy involving insertion of donor DNA encoding the TCR into the TRAC locus. Figure 6 shows an HDR-mediated strategy involving the insertion of an artificial TCR specific to NY-ESO-1 into the TRAC gene locus.

[0295] We designed a DNA donor containing complete TCRα and TCRβ (VJ region only) strands. After CRISPR-mediated cleavage of one TRAC exon, the phasic arms on both sides of the payload allow for the insertion of exogenous TCRs. The self-cleaving peptides (T2A and P2A) are removed post-translation, and the full-length TCRα and TCRβ chains of the NY-ESO-1 TCR are constitutively expressed.

[0296] Example 4 - Insertion of a DNA donor encoding a target protein into a specific gene locus in the T cell genome. A DNA donor encoding green fluorescent protein (GFP HDR cassette) was designed and inserted into a specific target region within the T cell genome. As described above, the GFP gene was flanked by a 5' phase arm and a 3' phase arm. Figure 7 shows the GFP gene depending on the amount of DNA donor used in each reaction. + This indicates that the detection rate of T cells differs.

[0297] While certain embodiments have been illustrated and described, it should be understood that modifications and alterations can be made thereto in accordance with the ordinary art of those skilled in the art, without departing from the broader aspects of the art as defined in the following claims.

[0298] The embodiments described herein as illustrative can also be suitably implemented without one or more elements or limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted broadly and not limited. Furthermore, the terms and expressions used herein are for illustrative purposes only, not limiting ones, and the use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or any part thereof, although it should be acknowledged that various modifications are possible within the scope of the claimed technology. Additionally, the expression “consisting essentially of” should be understood to include the specifically mentioned elements and any additional elements that do not materially affect the fundamental and novel characteristics of the claimed technology. The expression “consisting of” excludes elements not specified.

[0299] This disclosure should not be limited in respect to any particular embodiment described herein. As will be apparent to those skilled in the art, many modifications and variations can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and compositions within the scope of this disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be within the scope of the appended claims. This disclosure is limited only by the conditions of the appended claims and is limited in accordance with the entire scope of the equivalents to which such claims are entitled. It should be understood that this disclosure is not limited to any particular method, reagent, compound, or composition, and these may, of course, change. It should also be understood that the terms used herein are for illustrative purposes only and are not intended to limit any particular embodiment.

[0300] Furthermore, if any feature or aspect of the present disclosure is described from the perspective of the Markush Group, a person skilled in the art will recognize that the present disclosure is also described from the perspective of any individual member or subgroup of the members of the Markush Group.

[0301] As will be understood by those skilled in the art, for all purposes, particularly in terms of providing written explanations, all scopes disclosed herein also encompass all possible subranges and combinations of subranges, including the endpoint. It will be readily apparent that any scope listed can be adequately explained and made possible to decompose the same scope into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-restrictive example, each scope discussed herein can be readily decomposed into lower thirds, middle thirds, upper thirds, etc. Also, as will be understood by those skilled in the art, all language such as “up to,” “at least,” “greater than,” and “less than” refers to a scope that includes the number mentioned and can then be decomposed into subranges, as discussed above. Finally, as will be understood by those skilled in the art, a scope includes individual numerical members.

[0302] All publications, patent applications, issued patents, and other documents referenced herein are incorporated by reference in the same manner as if each individual publication, patent application, issued patent, or other document were specifically and individually indicated to be incorporated by reference in whole. Definitions contained in the incorporated text are excluded to the extent that they conflict with the definitions in this disclosure.

[0303] Other embodiments are described in the following claims.

Claims

1. A virus vector-free method for preparing a population of modified, unstimulated T cells, (a) A step of delivering a homology-directed repair (HDR) template containing (i) a gene-editing nuclease, (ii) a guide RNA, and (iii) an antigen-binding polypeptide to an unstimulated T cell population obtained from a biological sample by transfection, and (b) A step of culturing the unstimulated T cell population under non-expansion conditions for 36 to 72 hours, wherein the non-expansion conditions include a culture medium that does not contain stimulating molecules or stimulating ligands that induce a stimulating response in the unstimulated T cells. The gene editing nuclease and the guide RNA form a complex that generates a double-strand break at a target site in at least one unstimulated T cell, where the target site is exon 1 of the TRAC gene locus, and The HDR template promotes HDR at the target site and generates at least one modified unstimulated T cell expressing the antigen-binding polypeptide within the population. method.

2. The method according to claim 1, wherein 10% or more of the unstimulated T cells in the population are modified to express the antigen-binding polypeptide.

3. A virus vector-free method for generating a population of modified, unstimulated T cells, (a) A step of inducing homology-directed repair (HDR) in target sites of 10% or more of the unstimulated T cell population by the following: (i) The step of contacting the unstimulated T cell population with a homology-directed repair (HDR) template containing a gene-editing nuclease and a polynucleotide encoding an antigen-binding polypeptide, (ii) A step of delivering the gene editing nuclease and the HDR template into the unstimulated T cell by transfection, wherein the target site is exon 1 of the TRAC gene locus, and (b) A step of culturing the unstimulated T cells under non-expansion conditions for 36 to 72 hours to generate the modified unstimulated T cell population that expresses the antigen-binding polypeptide, Herein, the non-expansion condition is a method comprising a culture medium that does not contain a stimulating molecule or stimulating ligand that induces a stimulating response in the unstimulated T cells.

4. The method according to claim 3, wherein the gene editing nuclease is delivered to the unstimulated T cell together with the guide RNA by the transfection method, and the guide RNA forms a complex with the gene editing nuclease, causing a double-strand break at the target site.

5. The method according to claim 3, wherein the unstimulated T cell population is obtained from a biological sample.

6. (a) The HDR template in a concentration of 1 pM to 10 mM is delivered into the unstimulated T cell, and / or (b) The gene editing nuclease is delivered to the unstimulated T cell in a concentration of 1 pM to 10 mM, and / or (c) The method according to claim 1 or 3, wherein a guide RNA in a concentration of 1 pM to 10 mM is delivered into an unstimulated T cell.

7. (a) The ratio of the gene editing nuclease to the guide RNA is 2:1 to 1:2, and / or (b) The ratio of the HDR template to the complex formed between the gene editing nuclease and the guide RNA is 2:1 to 1:

2. (c) The method according to claim 1 or 4, wherein the ratio of the HDR template to the gene editing nuclease is 2:1 to 1:

2.

8. (a) The ratio of the gene editing nuclease to the guide RNA is 1:1, and / or (b) The ratio of the HDR template to the complex formed between the gene editing nuclease and the guide RNA is 1:

1. (c) The method according to claim 7, wherein the ratio of the HDR template to the gene editing nuclease is 1:

1.

9. The method according to claim 1 or 4, wherein the complex is a ribonucleoprotein (RNP) complex.

10. The aforementioned population of unstimulated T cells is CD4 ＋ T cells, CD8 ＋ T cells, CD4 ＋ / CD8 ＋ The method according to claim 1 or 3, comprising T cells or a combination thereof.

11. The aforementioned population of unstimulated T cells is CD4 ＋ T cells, CD8 ＋ The method according to claim 10, wherein the method is an enriched population of T cells or combinations thereof.

12. The aforementioned concentrated population contains at least 90% CD4 ＋ T cells, CD8 ＋ The method according to claim 11, comprising T cells or a combination thereof.

13. The method according to claim 1 or 5, comprising the step of incubating the biological sample with a plurality of anti-CD4 and / or anti-CD8 labeled microbeads in order to obtain an enriched population of unstimulated T cells.

14. The method according to claim 1 or 3, wherein the unstimulated T cells are cultured under non-swelling conditions for 48 hours or 72 hours after transfection.

15. The modified unstimulated T cells are cultured under non-expansion conditions, then resuspended in a cryopreservation solution and cryopreserved, according to claim 1 or 3.

16. The transfection method described above (a) Electroporation, or (b) Cell squeeze method, The method according to claim 1 or 3, including the method described in claim 1 or 3.

17. The aforementioned antigen-binding polypeptide (a) containing an antigen-binding domain, and (b) is a chimeric antigen receptor (CAR), or (c) is a T cell receptor (TCR), and (d) The method according to claim 1 or 3, wherein the tumor antigen is bound.

18. where the antigen-binding domain is a Fab, F(ab) ２ , single-chain variable fragment (scFv), diabody, triabody, minibody, V-NAR or VhH, the method according to claim 17

19. The antigen-binding polypeptide is a CAR, and the CAR is (a) A transmembrane domain selected from the group consisting of artificial hydrophobic sequences, transmembrane domains of type I transmembrane proteins, alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), ICOS (CD278), CD154, and transmembrane domains derived from killer immunoglobulin-like receptors (KIRs), and (b) an intracellular domain including a co-stimulus signaling domain and an intracellular signaling domain, The method according to claim 17.

20. The intracellular domain comprises one or more costimulatory domains of proteins selected from the group consisting of proteins of the TNFR superfamily, CD27, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS (CD278), NKG2C, B7-H3 (CD276), and intracellular domains derived from killer immunoglobulin-like receptors (KIRs), or functional variants thereof. The method according to claim 19.

21. (a) The CAR comprises a 4-1BB (CD137) costimulatory domain and / or (b) The method according to claim 19, wherein the CAR comprises an intracellular signaling domain selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

22. The method according to claim 21, wherein the CAR comprises a 4-1BB (CD137) costimulatory domain and a human CD3 zeta chain (CD3ζ) cytoplasmic signaling domain.

23. The antigen-binding polypeptide is a TCR containing an antigen-binding domain that binds to a tumor antigen, and the tumor antigen is (a) related to hematological malignancies and / or (b) related to solid tumors and / or (c) The method according to claim 17, selected from the group consisting of CD19, CD20, CD22, CD33, ROR1, mesothelin, c-Met, PSMA, PSCA, folate receptor alpha, folate receptor beta, EGFRvIII, GPC2, TnMUC1, GDNF family receptor alpha-4 (GFRa4), fibroblast activating protein (FAP), and IL13Ra2.

24. The aforementioned TCR is, (a) comprising TCR alpha chain and TCR beta chain, and / or (b) The method according to claim 17, selected from wild-type TCR, high-affinity TCR and chimeric TCR.

25. The aforementioned HDR template, (a) The polynucleotide having a 5' homology arm upstream of the genomic region 5' of the target site, and / or (b) The polynucleotide having a 3' homology arm downstream of the genomic region 3' of the target site, and / or (c) A double-stranded DNA template, and / or (d) The method according to claim 1 or 3, delivered by electroporation.

26. The aforementioned gene editing nuclease is (a) Cas nuclease, or (b) Zinc finger nuclease, or (c) The method according to claim 3, wherein the transcription activator-like effector nuclease (TALEN).

27. The method according to claim 1, wherein the gene editing nuclease is a Cas nuclease.

28. The aforementioned Cas nuclease is, (a) Cas9 and / or, (b) The method according to claim 26 or 27, wherein the guide RNA and the Cas nuclease form an RNP complex before being delivered to the unstimulated T cells.

29. The method according to claim 28, wherein the Cas9 nuclease is SpCas9 or SaCas9.

30. The method according to claim 1 or 4, comprising the step of delivering a second guide RNA to the unstimulated T cells.

31. The aforementioned biological sample (a) a blood sample and / or, (b) A blood sample, which is a whole blood sample, a peripheral blood mononuclear cell (PBMC) sample, or an apheresis sample, and / or (c) A blood sample, which is a cryopreserved apheresis sample, and / or (d) The method according to claim 1 or 5, wherein the blood sample is a fresh apheresis sample.

32. The method according to claim 1 or 3, further comprising the step of culturing the unstimulated T cells for 36 to 72 hours, and then stimulating the modified unstimulated T cells to generate a population of modified stimulated T cells.

33. The method according to claim 32, further comprising the step of expanding the modified stimulated T cell population.

34. The method according to claim 33, wherein the modified stimulated T cell population is cultured under expanded conditions for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days.

35. The method according to claim 1 or 3, wherein the T cells are human T cells.

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