Chimeric antigen receptors targeting GPC-3, and immune cells expressing such chimeric antigen receptors for therapeutic use

Abstract

Genetically engineered T cells that express a chimeric antigen receptor (CAR) targeting GPC3 and optionally have multiple gene edits (including a disrupted TRAC gene, a disrupted β2M gene, a disrupted Cas1 gene, a disrupted TGFbRII gene, or combinations thereof). Also provided herein are methods of making such gene-edited T cells and methods of using the gene-edited T cells in the treatment of cancer.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Patent Application No. 63 / 356,671, filed on June 29, 2022, the content of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in XML format, the content of which is incorporated herein by reference in its entirety. The XML copy (created on June 12, 2023) is named "095136 - 0766 - 067WO1_SEQ.xml" and is 93,246 bytes in size.

Background Art

[0003] Glypican - 3 (GPC3), a proteoglycan with heparan sulfate, is overexpressed in many malignant tumors (e.g., hepatocellular carcinoma, lung squamous cell carcinoma, ovarian cancer, and melanoma), but is absent or has very low expression in normal Roswell Park tissues and normal liver tissues or liver cirrhosis. Capurro et al. found that 72% of HCC patients express GPC3, and GPC3 serum levels are significantly increased in 53% of HCC patients. The function of GPC3 is proposed to promote the development of HCC by activating the Wnt signaling pathway. Morgan et al. discovered that when GPC3 on HCC cells is silenced, the proliferation and invasion ability of HCC cells are impaired, suggesting that the expression of GPC3 on HCC cells is involved in the proliferation and invasion of HCC cells. In addition, the overexpression of GPC3 is also a predictor of poor prognosis. As a result, GPC3 is used as a target for the diagnosis and treatment of HCC due to its strong specificity and high sensitivity.

[0004] Chimeric antigen receptor (CAR) T cell therapy uses genetically modified T cells to more specifically and efficiently target and kill target cells such as target cancer cells. After collecting T cells from the blood, the cells are engineered to contain CAR on the surface of the T cells. This CAR can be introduced into T cells using CRISPR / Cas9 gene editing technology. When these CAR T cells are injected into a patient, the receptor enables the T cells to kill the target cells.

Summary of the Invention

Means for Solving the Problems

[0005] The present disclosure relates to gene-edited anti-GPC CAR-T cells that may carry one or more additional gene edits, such as a disrupted Regnase 1 (Reg1) gene, a disrupted transforming growth factor beta receptor II (TGFbRII) gene, a disrupted Casitas B-lineage lymphoma proto-oncogene-B (cbl-b) gene, a disrupted T cell receptor alpha chain constant region (TRAC) gene, and / or a disrupted beta-2-microglobulin (β2M) gene, and to an effective method for producing such gene-edited T cells by CRISPR / Cas-mediated gene editing using the guide RNAs disclosed herein, and is at least partially based on the development thereof.

[0006] Accordingly, in some aspects, the present disclosure provides a population of genetically engineered T cells, wherein the genetically engineered T cells comprise: (a) a nucleic acid encoding a chimeric antigen receptor (CAR) that binds to glypican 3 (GPC3) (anti-GPC3 CAR); and (b) one or more disrupted genes, wherein the disrupted genes comprise (i) a disrupted T cell receptor alpha constant region (TRAC) gene, (ii) a disrupted beta-2-microglobulin (β2M) gene, (iii) a disrupted transforming growth factor beta receptor II (TGFbRII) gene, (iv) a disrupted Regnase-1 (Reg1) gene, (v) a disrupted Casitas B-lineage lymphoma proto-oncogene-B (CBLB) gene, or (vi) any combination of (i) to (v). Optionally, the T cells are human T cells. In some examples, the T cells include primary human T cells.

[0007] In some embodiments, the anti-GPC3 CAR comprises: (a) an ectodomain that binds to GPC3; (b) a transmembrane domain; and (c) an endodomain that comprises (i) a co-stimulatory signaling domain and (ii) a CD3ζ cytoplasmic signaling domain.

[0008] Optionally, the ectodomain comprises an anti-GPC3 fragment, and the anti-GPC3 fragment is an anti-GPC3 single-domain antibody (e.g., VHH). Such an anti-GPC3 single-domain antibody may comprise complementarity-determining regions (CDRs) identical to those of SEQ ID NO: 9. In one example, the anti-GPC3 VHH comprises the amino acid sequence of SEQ ID NO: 9.

[0009] Optionally, the ectodomain comprises an anti-GPC3 fragment, and the anti-GPC3 fragment is an anti-GPC3 single-chain variable fragment (scFv). In some examples, the ectodomain comprises an anti-GPC3 scFv, and the anti-GPC3 scFv comprises a heavy-chain variable (V H ) region that comprises CDRs identical to those of SEQ ID NO: 22 and a light-chain variable (VL ) includes a region. In a specific example, this anti-GPC3 scFv is the V described in SEQ ID NO: 22 H and the V described in SEQ ID NO: 21 L and includes. Such an anti-GPC3 scFv may include the amino acid sequence of SEQ ID NO: 23. Alternatively, this anti-GPC3 scFv may include the amino acid sequence of SEQ ID NO: 24.

[0010] Any of the anti-GPC3 CARs disclosed herein may include a co-stimulatory domain. In some examples, this co-stimulatory domain is the CD28 co-stimulatory domain. In other examples, this co-stimulatory domain is the 4-1BB co-stimulatory domain. Alternatively, or in addition, this anti-GPC3 CAR may include a transmembrane domain, which may be the CD8 transmembrane domain. In a specific example, this anti-GPC3 CAR includes the amino acid sequence of SEQ ID NO: 11, 14, 26, 29, 32, or 35. In one example, this anti-GPC3 CAR includes the amino acid sequence of SEQ ID NO: 11. In another example, the anti-GPC3 CAR includes the amino acid sequence of SEQ ID NO: 26.

[0011] In some embodiments, the genetically engineered T cell may include a disrupted TRAC gene and a disrupted β2M gene. Such a genetically engineered T cell may further include a disrupted TGFBRII gene, a disrupted Reg-1 gene, a disrupted CBLB gene, or a combination thereof. For example, this genetically engineered T cell may further include (i) a disrupted TGFBRII gene and a disrupted Reg-1 gene. Alternatively, this genetically engineered T cell may further include a disrupted TGFBRII gene and a disrupted CBLB gene.

[0012] In other embodiments, the genetically engineered T cell may include a disrupted TGFBRII gene, a disrupted Reg-1 gene, a disrupted CBLB gene, or a combination thereof. In some examples, such a genetically engineered T cell may include a wild-type TRAC gene, a wild-type β2M gene, or a combination thereof.

[0013] In some cases, the nucleic acid encoding the anti-GPC3 CAR can be inserted into the endogenous locus of the genetically engineered T cell. For example, this endogenous locus is present within the disrupted TRAC gene, disrupted β2M gene, disrupted TGFbRII gene, disrupted Reg-1 gene, or disrupted CBLB gene. In some examples, this endogenous locus is present within the disrupted TRAC gene. In a specific example, this disrupted TRAC gene contains the deletion of SEQ ID NO: 58, and this deletion can be replaced with the nucleotide sequence encoding the anti-GPC3 CAR.

[0014] In other aspects, provided herein is a method of producing a population of genetically engineered T cells, comprising: (a) delivering to a population of T cells: (i) one or more RNA-guided nucleases; (ii) one or more guide RNAs targeting the T cell receptor alpha constant region (TRAC) gene (TRAC gRNA), beta-2-microglobulin (β2M) gene (β2M gRNA), TGFbRII gene (TGFBRII gRNA), Regnase-1 (Reg1) gene (Reg1 gRNA), and / or Casitas B-lineage lymphoma proto-oncogene B (CBLB) gene (CBLB gRNA); and (iii) a vector comprising a nucleic acid encoding the anti-GPC3 CAR (e.g., as described herein); and (b) producing a population of engineered T cells that express the anti-GPC3 CAR and contain one or more of the disrupted TRAC gene, β2M gene, TGFBRII gene, Reg1 gene, and CBLB gene.

[0015] In some examples, this population of T cells includes human T cells, for example, human primary T cells. In some examples, this population of T cells is obtained from one or more healthy human donors. Alternatively, this population of T cells is obtained from a human patient with GPC3+ cancer.

[0016] In some embodiments, step (a) comprises delivering the TRAC gRNA and the β2M gRNA to a population of T cells. Optionally, step (a) further comprises delivering the TGFBRII gRNA, the Reg1 guide, the CBLB guide, or a combination thereof to the population of T cells. For example, step (a) may further comprise delivering (i) the TGFBRII gRNA and the Reg1 guide to the population of T cells. Alternatively, step (a) may comprise delivering the TGFBRII guide and the CBLB guide to the population of T cells. In other cases, step (a) comprises (e.g., consists of) delivering the TGFBRII gRNA, the Reg1 guide, the CBLB guide, or a combination thereof to the population of T cells.

[0017] In some examples, the TRAC guide is specific for a TRAC gene target sequence comprising the nucleotide sequence of SEQ ID NO: 58. Such a TRAC guide may comprise a spacer comprising the nucleotide sequence of SEQ ID NO: 39. Optionally, this TRAC guide may comprise a scaffold sequence (e.g., as disclosed herein). Optionally, this TRAC guide may comprise one or more modifications. Exemplary TRAC guides may comprise the nucleotide sequence of SEQ ID NO: 37 or SEQ ID NO: 38.

[0018] In some examples, the β2M guide is specific for a β2M gene target sequence comprising the nucleotide sequence of SEQ ID NO: 60. Such a β2M guide may comprise a spacer comprising the nucleotide sequence of SEQ ID NO: 43. Optionally, this β2M guide may comprise a scaffold sequence (e.g., as disclosed herein). Optionally, this β2M guide may comprise one or more modifications. Exemplary β2M guides may comprise the nucleotide sequence of SEQ ID NO: 41 or SEQ ID NO: 42.

[0019] In some examples, the TGFBRII guide is specific to a TGFBRII gene target sequence comprising the nucleotide sequence of SEQ ID NO: 62. Such a TGFBRII guide may comprise a spacer comprising the nucleotide sequence of SEQ ID NO: 47. Optionally, this TGFBRII guide may comprise a scaffold sequence (e.g., as disclosed herein). Optionally, this TGFBRII guide may comprise one or more modifications. Exemplary TGFBRII guides may comprise the nucleotide sequence of SEQ ID NO: 45 or SEQ ID NO: 46.

[0020] In some examples, the Reg1 guide is specific to a Reg1 gene target sequence comprising the nucleotide sequence of SEQ ID NO: 64. Such a Reg1 guide may comprise a spacer comprising the nucleotide sequence of SEQ ID NO: 51. Optionally, this Reg1 guide may comprise a scaffold sequence (e.g., as disclosed herein). Optionally, this Reg1 guide may comprise one or more modifications. Exemplary Reg1 guides may comprise the nucleotide sequence of SEQ ID NO: 49 or SEQ ID NO: 50.

[0021] In some examples, the CBLB guide is specific to a CBLB gene target sequence comprising the nucleotide sequence of SEQ ID NO: 66. Such a CBLB guide may comprise a spacer comprising the nucleotide sequence of SEQ ID NO: 55. Optionally, this CBLB guide may comprise a scaffold sequence (e.g., as disclosed herein). Optionally, this CBLB guide may comprise one or more modifications. Exemplary CBLB guides may comprise the nucleotide sequence of SEQ ID NO: 53 or SEQ ID NO: 54.

[0022] In any of the methods disclosed herein, the one or more RNA-guided nucleases may comprise a Cas9 nuclease. In some examples, this Cas9 nuclease is a S. pyogenes Cas9 nuclease.

[0023] Alternatively, or in addition, the vector of (a)(iii) for use in any of the methods disclosed herein may comprise a donor template in which a nucleic acid encoding an anti-GPC3 CAR is flanked by an upstream fragment and a downstream fragment, the upstream and downstream fragments being homologous to an endogenous locus of a T cell, thereby enabling insertion of the nucleic acid encoding the anti-GPC3 CAR into the endogenous locus. Exemplary endogenous loci include, but are not limited to, a disrupted TRAC gene, a disrupted β2M gene, a disrupted TGFbRII gene, a disrupted Reg-1 gene, or a disrupted CBLB gene. In some examples, the endogenous locus is present within a disrupted TRAC gene. In such cases, the upstream fragment may be SEQ ID NO: 71 and / or the downstream fragment may be SEQ ID NO: 74.

[0024] In some examples, the vector of (a)(iii) is a viral vector, such as an adeno-associated virus (AAV) vector or a lentiviral vector.

[0025] In another aspect, provided herein is a method of treating cancer in a subject, the method comprising administering to a subject in need thereof any of the populations of genetically engineered T cells disclosed herein. In some embodiments, the subject is a human patient having a GPC3+ cancer. Exemplary target cancers include, but are not limited to, liver cancer, gastric cancer, colorectal cancer, lung cancer, ovarian cancer, skin cancer, or thyroid cancer. In one example, the target cancer is HCC.

[0026] In some examples, a population of genetically engineered T cells is syngeneic with a human patient. Such a population of genetically engineered T cells can have a disrupted TRAC gene and / or a disrupted β2M gene, and optionally, a disrupted TGFBRII gene, a disrupted Reg-1 gene, and / or a disrupted cbl-b gene. Alternatively, a population of genetically engineered T cells is autologous to a human patient. Such a population of genetically engineered T cells can have a disrupted TGFBRII gene, a disrupted Reg-1 gene, and / or a disrupted cbl-b gene, and optionally, a wild-type TRAC gene and / or β2M gene.

[0027] Furthermore, the present disclosure provides a chimeric antigen receptor (anti-GPC3 CAR) that binds to glypican 3 (GPC3) disclosed herein. Optionally, this anti-GPC3 CAR can further include an N-terminal signal peptide. Exemplary anti-GPC3 CAR polypeptides (with or without this signal peptide) are shown in Table 1, all of which are within the scope of the present disclosure. Also provided herein are nucleic acids encoding this anti-GPC3 CAR and host cells containing such nucleic acids. Optionally, this nucleic acid is located within a suitable vector, for example, within a viral vector such as an AAV vector or a lentiviral vector.

[0028] In addition, provided herein is any one of genetically engineered T cells that express an anti-GPC3 CAR for use in inhibiting GPC3+ disease cells such as cancer cells and for treating diseases associated with such disease cells (e.g., various cancers disclosed herein). Also within the scope of the present disclosure are genetically engineered T cells that express an anti-GPC3 CAR in the manufacture of a medicament for use in an intended therapeutic application.

[0029] Details of one or more embodiments of the present invention are set forth in the following description. Other features or advantages of the present invention will become apparent from the following drawings and detailed description of several embodiments, and will also become apparent from the appended claims.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0031] The present disclosure aims to establish genetically engineered anti-GPC3 CAR-T cells having improved characteristics (e.g., proliferative activity, persistence, reduction of T cell exhaustion, and / or enhanced efficacy). The anti-GPC3 CAR-T cells disclosed herein are for generating cells suitable for either allogeneic or autologous immunotherapy and for achieving characteristics that may improve treatment effects, including multiple gene edits to endogenous genes (e.g., disruption of the TRAC gene, β2M gene, TGFBRII gene, Reg1 gene, and / or cbl-b gene). For example, disruption of β2M may reduce the risk of host-versus-graft reaction or prevent this reaction, and disruption of TRAC may reduce the risk of graft-versus-host reaction or prevent this reaction. Accordingly, the anti-GPC3 CAR-T cells disclosed herein (having a disrupted TRAC gene and / or a disrupted β2M gene) may be suitable for use in allogeneic cell therapy. Alternatively, or in addition, disruption of TGFBRII may reduce the immunosuppressive effect of transforming growth factor β (TGF-β) in the tumor microenvironment, and disruption of Reg1 may improve the functionality of CAT-T cells by long-term persistence with robust effector function. Furthermore, the CAR-T cells having a disrupted cblb gene disclosed herein also showed enhanced anti-tumor activity and long-term survival rate. In some cases, anti-GPC3 CAR-T cells having a disrupted TGFBRII gene, Reg1 gene, and / or cbl-b gene, together with wild-type TRAC and β2M genes, may be suitable for use in autologous cell therapy.

[0032] As provided herein, the anti-GPC3 CAR-T cells disclosed herein exhibit high editing efficiency of target genes such as TRAC, β2M, Reg-1, TGFBRII, and / or cbl-b, and do not show significant interference with the expression of immune checkpoint molecules such as PD1 and LAG-3. Furthermore, this anti-GPC3 CAR-T cell showed high cytotoxicity against GPC3+ cells and GPC3+ tumors both in vitro and in vivo. Further gene editing such as disruption of the Reg-1 gene, TGFBRII gene, and / or cbl-b gene did not show an impact on the characteristics of CAR-T cells such as cell proliferation and CD4:CD8 cell ratio. CAR-T cells with further gene editing have shown good proliferation and specific cytotoxicity against the target, and the phenotype was considered normal. Furthermore, such CAR-T cells showed enhanced anti-tumor activity when observed in animal models. Therefore, the anti-GPC3 CAR-T cells disclosed herein (for example, those having one or more of gene editing (disruption of the Reg-1 gene, TGFBRII gene, and / or cbl-b gene)) are expected to achieve excellent anti-tumor efficacy in either autologous cell therapy or allogeneic cell therapy.

[0033] Such T cells can use primary T cells as starting materials, for example, non-transformed T cells, terminally differentiated T cells, T cells with a stable genome, and / or T cells that are cytokine- and growth factor-dependent for proliferation and expansion. Alternatively, such T cells can use T cells generated from progenitor cells such as hematopoietic stem cells (e.g., iPSCs) in, for example, in vitro cultures. The T cells disclosed herein can confer one or more benefits in both the manufacture and clinical application of CAR-T cells.

[0034] Other advantageous features related to the disruption of the Reg1 gene and / or TGFBRII gene can be found in WO 2022 / 064428 pamphlet, and this related disclosure is incorporated by reference for the subject matter and purposes mentioned herein.

[0035] Accordingly, provided herein are anti-GPC3 CAR-T cells with improved persistence and potentially enhanced anti-tumor activity, methods of manufacturing such T cells, and therapeutic applications of such T cells in the elimination of GPC3+ disease cells such as cancer cells (e.g., GPC3+ hepatocellular carcinoma cells).

[0036] I. Anti-GPC3 CAR-T Cells with Enhanced Characteristics Glypican-3 (GPC3) is known to be an important cell surface marker in many malignant tumors such as hepatocellular carcinoma, lung squamous cell carcinoma, ovarian cancer, and melanoma. Due to its strong specificity and high sensitivity, GPC3 has been used as a target for the diagnosis and treatment of GPC3+ tumors such as liver cancer, gastric cancer, colorectal cancer, lung cancer, ovarian cancer, skin cancer, and thyroid cancer.

[0037] In some embodiments, provided herein are anti-GPC3 CAR-T cells that may have multiple genetic modifications to improve the functionality of the CAR-T cells, thereby improving the therapeutic efficacy of either allogeneic or autologous cell therapy. The genetically engineered T cells provided herein express a chimeric antigen receptor (CAR) that binds to GPC3 and, optionally, has multiple gene edits to endogenous genes (e.g., the TRAC gene, β2M gene, Reg1 gene, TGFBRII gene, or combinations thereof). In some cases, the anti-GPC3 CAR-T cells disclosed herein contain disrupted TRAC and β2M genes, as well as the Reg1 gene, TGFBRII gene, and / or cbl-b gene. In other cases, the anti-GPC3 CAR-T cells disclosed herein may contain disrupted Reg1, TGFBRII, and / or cbl-b genes and have wild-type TRAC and β2M genes.

[0038] The genetically engineered T cells can be derived from parental T cells (e.g., unedited wild-type T cells) obtained from a suitable source (e.g., one or more mammalian donors). In some examples, the parental T cells are primary T cells (e.g., T cells that have not been transformed or terminally differentiated) obtained from one or more human donors. Alternatively, the parental T cells can be differentiated from precursor T cells obtained from one or more suitable donors or stem cells (e.g., hematopoietic stem cells or induced pluripotent stem cells (iPSCs)) that can be cultured in vitro.

[0039] Any of the genetically engineered T cells can be generated via gene editing (including genome editing), a type of genetic manipulation in which nucleotides / nucleic acids are inserted, deleted, and / or substituted in a DNA sequence in the genome of the target cell. Targeted gene editing enables insertion, deletion, and / or substitution at a preselected site in the genome of the target cell (e.g., in a target gene or target DNA sequence). When the sequence of an endogenous gene is edited, e.g., by nucleotide / nucleic acid deletion, insertion, or substitution, the endogenous gene containing the affected sequence can be knocked out due to this sequence change. Thus, targeted editing can be used to disrupt the expression of an endogenous gene. "Targeted integration" refers to a process involving the insertion of one or more exogenous sequences, with or without deletion of the endogenous sequence at the insertion site. Targeted integration can result from targeted gene editing when a donor template containing the exogenous sequence is present.

[0040] A. Genetically Edited Gene In some embodiments, the disclosure provides genetically engineered T cells that can include a disrupted Reg1 gene, a disrupted TGFBRII gene, a disrupted cbl-b gene, a disrupted TRAC gene, and / or a disrupted β2M gene.

[0041] As used herein, "disrupted gene" refers to a gene that contains an insertion, deletion, or substitution such that the expression of a functional protein from this endogenous gene is reduced or inhibited as compared to the endogenous gene. As used herein, "disrupting a gene" refers to a method of inserting, deleting, or substituting at least one nucleotide / nucleic acid in an endogenous gene such that the expression of a functional protein from this endogenous gene is reduced or inhibited. Methods of disrupting genes are known to those of skill in the art and are described herein.

[0042] In some embodiments, a cell containing a disrupted gene does not express (e.g., on the cell surface) a detectable level of the protein encoded by this gene (e.g., a level detectable in an immunoassay using an antibody that binds to the encoded protein, or a level detectable by flow cytometry). A cell that does not express a detectable level of protein can be referred to as a knockout cell.

[0043] Reg1 gene editing In some embodiments, the genetically engineered T cells can contain a disrupted gene involved in mRNA decay. Such a gene can be Reg1. Reg1 contains a zinc finger motif, binds to RNA, and exhibits ribonuclease activity. Reg1 plays a role in both immune and non-immune cells, and its expression can be rapidly induced under various conditions such as microbial infection, treatment with inflammatory cytokines, and chemical or mechanical stimuli. The human Reg1 gene is located on chromosome 1p34.3. Further information can be found in GenBank under Gene ID: 80149.

[0044] In some examples, the genetically engineered T cells may contain a disrupted Reg1 gene such that the expression of Reg1 in the T cells is substantially reduced or completely abolished. The disrupted Reg1 gene may include one or more gene edits that disrupt the expression of the Reg1 gene at one or more suitable target sites (e.g., target sites in the coding region or target sites in non-coding regulatory regions such as the promoter region). Such target sites can be identified based on gene editing approaches used for the generation of the genetically engineered T cells. Exemplary target sites for gene editing may include exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or combinations thereof. In some examples, one or more gene edits may occur in exon 2 or exon 4. Such gene edits can be induced by CRISPR / Cas technology using a suitable guide RNA (e.g., those listed in Table 2). Reference is also made to International Publication No. WO 2022 / 064428, the relevant disclosure of which is incorporated herein by reference for the subject matter and purposes mentioned herein.

[0045] Disruption of the Reg1 gene can enhance long-term persistence and maintain robust effector function, thereby improving the functionality of the T cells.

[0046] TGFBRII gene editing In some embodiments, the genetically engineered T cells may contain a disrupted TGFBRII gene that encodes transforming growth factor receptor type II (TGFBRII). The TGFBRII receptor is a family of serine / threonine kinase receptors involved in the TGFβ signaling pathway. This receptor binds to TGFβ family growth factors and cytokine signaling proteins, such as TGFβ (TGFβ1, TGFβ2, and TGFβ3), bone morphogenetic protein (BMP), growth differentiation factor (GDF), activin and inhibin, myostatin, anti-Müllerian hormone (AMH), and NODAL.

[0047] In some examples, the genetically engineered T cell may contain a disrupted TGFBRII gene such that the expression of TGFBRII in the T cell is substantially reduced or completely lost. The disrupted TGFBRII gene may include one or more gene edits that disrupt the expression of the TGFBRII gene at one or more suitable target sites (e.g., target sites in the coding region or non-coding regulatory regions such as the promoter region). Such target sites can be identified based on the gene editing approach used to generate the genetically engineered T cell. Exemplary target sites for gene editing can include exon 1, exon 2, exon 3, exon 4, exon 5, or combinations thereof. In some examples, one or more gene edits can occur in exon 4 and / or exon 5. Such gene edits can be induced by gene editing techniques (e.g., CRISPR / Cas technology) using a suitable guide RNA (e.g., those listed in Table 2). Reference is also made to International Publication No. WO 2022 / 064428, the relevant disclosure of which is incorporated by reference for the subject matter and purposes mentioned herein.

[0048] Disruption of the TGFBRII gene can result in the loss of surface expression of TGFBRII and can reduce the immunosuppressive effect of transforming growth factor β (TGF-β) in the tumor microenvironment.

[0049] cbl-b gene editing In some embodiments, the genetically engineered T cells can comprise a disrupted Cbl proto-oncogene B (CBL-b) gene. The CBLB protein contains zinc finger motifs, binds to RNA, and exhibits ribonuclease activity. CBLB plays roles in both immune and non-immune cells, and its expression can be rapidly induced under various conditions such as microbial infection, treatment with inflammatory cytokines, and chemical or mechanical stimuli. The human cbl-b gene is located on chromosome GRCh38.p13. Further information can be found in GenBank under Gene ID:868.

[0050] In some examples, the genetically engineered T cells can comprise a disrupted cbl-b gene such that the expression of cbl-b in the T cells is substantially reduced or completely abolished. This disrupted cbl-b gene can include one or more gene edits that disrupt the expression of the cbl-b gene at one or more suitable target sites (e.g., target sites in the coding region or non-coding regulatory regions such as the promoter region). Such target sites can be identified based on the gene editing approach used for the generation of the genetically engineered T cells. Exemplary target sites for gene editing can include exon 2, exon 7, exon 9, exon 11, exon 12, or combinations thereof. In some examples, one or more gene edits can occur at exon 2. In other examples, one or more gene edits can occur at exon 7. In still other examples, one or more gene edits can occur at exon 9. Such gene edits can be induced by CRISPR / Cas technology using a suitable guide RNA (e.g., those listed in Table 2). Reference is also made to U.S. Provisional Patent Application No. 63 / 292,715, the relevant disclosure of which is incorporated by reference for the subject matter and purposes mentioned herein.

[0051] β2M gene editing In some embodiments, the genetically engineered T cells disclosed herein may further comprise a disrupted β2M gene. β2M is a common (invariant) component of the MHC I complex. Disruption of its expression by gene editing prevents the host's allogeneic T cell response to the treatment, resulting in increased persistence of the allogeneic T cells. In some embodiments, the expression of the endogenous β2M gene is abolished, preventing the host-versus-graft reaction.

[0052] In some embodiments, the edited β2M gene may comprise a nucleotide sequence selected from the sequences in Table 2 below. It is known to those skilled in the art that various nucleotide sequences in edited genes such as the edited β2M gene can be generated from a single gRNA (β2M-1) such as those listed in Table 2. Reference is also made to International Publication No. WO 2019 / 097305, the relevant disclosure of which is incorporated herein by reference for the subject matter and purposes referred to herein.

[0053] TRAC gene editing In some embodiments, the genetically engineered T cells disclosed herein may further comprise a disrupted TRAC gene. This disruption results in loss of TCR function, rendering the engineered T cells non-alloreactive and suitable for allogeneic transplantation, minimizing the risk of graft-versus-host disease. In some embodiments, the expression of the endogenous TRAC gene is abolished, preventing the graft-versus-host reaction. Reference is also made to International Publication No. WO 2019 / 097305, the relevant disclosure of which is incorporated herein by reference for the purposes and subject matter referred to herein.

[0054] Gene editing of such TRAC genes can be induced by gene editing techniques (e.g., CRISPR / Cas technology) using suitable guide RNAs (e.g., those listed in Table 2).

[0055] It should be understood that multiple suitable target sites / gRNAs can be used for each target gene disclosed herein (e.g., those known in the art or disclosed herein). Further examples can be found, for example, in WO 2019 / 097305 pamphlet, and the relevant disclosure of this pamphlet is incorporated herein by reference for the purposes and subject matter referred to herein.

[0056] In some cases, the nucleic acid encoding the anti-GPC3 CAR can be inserted into the TRAC gene, thereby inhibiting the expression of this TRAC gene. For example, the nucleic acid encoding the CAR can replace the target site of the gRNA used in gene editing by CRISPR / Cas9 (e.g., replace the fragment containing SEQ ID NO: 58 in the TRAC gene).

[0057] B. Anti-GPC3 Chimeric Antigen Receptor (CAR) A chimeric antigen receptor (CAR) refers to an artificial immune cell receptor engineered to recognize and bind to an antigen expressed by unwanted cells (e.g., diseased cells such as cancer cells). T cells expressing the CAR polypeptide are referred to as CAR T cells. CAR has the ability to re-induce T cell specificity and reactivity against a selected target in a manner not restricted by MHC. Antigen recognition not restricted by MHC confers on CAR-T cells the ability to recognize antigens independent of antigen processing, thereby bypassing a major mechanism of tumor escape. Further, when expressed in T cells, CAR advantageously does not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0058] There are various generations of CARs, each of which contains different components. The first-generation CARs link a single-chain variable fragment (scFv) derived from an antibody to the CD3 zeta (ζ or z) intracellular signaling domain of the T cell receptor via a hinge domain and a transmembrane domain. The second-generation CARs incorporate an additional co-stimulatory domain (e.g., CD28, 4-1BB (41BB), or ICOS) to provide co-stimulatory signals. The third-generation CARs contain two co-stimulatory domains (e.g., a combination of CD27, CD28, 4-1BB, ICOS, or OX40) fused to the CD3ζ chain of the TCR (Maude et al., Blood. 2015;125(26):4017-4023; Kakarla and Gottschalk, Cancer J. 2014;20(2):151-155). All of the various generations of CAR constructs are within the scope of the present disclosure.

[0059] Generally, a CAR is a fusion polypeptide that includes an extracellular domain that recognizes a target antigen (e.g., a single-chain fragment of an antibody (scFv) or other antibody fragment), an intracellular domain that includes a signaling domain of the T cell receptor (TCR) complex (e.g., CD3ζ), and, in most cases, a co-stimulatory domain. (Enblad et al., Human Gene Therapy. 2015;26(8):498-505). The CAR construct may further include a hinge domain and a transmembrane domain between the extracellular domain and the intracellular domain and may include a signal peptide at the N-terminus for surface expression. Exemplary signal peptides are shown in Table 1. Other signal peptides may be used.

[0060] (i) Antigen-binding extracellular domain The antigen-binding extracellular domain is the region of the CAR polypeptide that is exposed to the extracellular fluid when the CAR is expressed on the cell surface. In some cases, the signal peptide is located at the N-terminus and can promote expression on the cell surface. In some cases, the extracellular antigen-binding domain can be an antibody fragment (e.g., a single-chain variable fragment (scFv)) that binds to GPC3 (e.g., human GPC3), or a single-domain antibody fragment (e.g., a heavy-chain-only antibody fragment (VHH)).

[0061] Anti-GPC3 ScFv fragment In some embodiments, the antigen-binding domain can comprise a single-chain variable fragment (scFv, the variable region of the antibody heavy chain (V H ) and the variable region of the antibody light chain (V L ), in either orientation). In some cases, the V H fragment and the V L fragment can be linked via a peptide linker. As this linker, in some embodiments, there are a series of glycines and serines for mobility and a series of hydrophilic residues having glutamates and lysines for imparting solubility. This scFv fragment retains the antigen-binding specificity of the parent antibody from which the scFv fragment is derived. In some embodiments, this scFv can comprise humanized V H domains and / or V L domains. In other embodiments, the V H domains and / or V L domains of this scFv are fully human.

[0062] In some embodiments, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds to the GPC3 antigen disclosed herein. This scFv can optionally comprise the variable region of the antibody heavy chain (V H ) and the variable region of the antibody light chain (V L ) connected via a flexible peptide linker. In some cases, this scFv is (from the N-terminus to the C-terminus) V H to V LIt may have a direction towards. Alternatively, this scFv may have a direction from V (from the N-terminus to the C-terminus) to V L to V H It may have a direction towards.

[0063] In some examples, the antigen-binding extracellular domain may be a single-chain variable fragment (scFv) that binds to human GPC3. In some cases, the anti-GPC3 scFv may include (i) a heavy-chain variable region (V H ) that contains the same heavy-chain complementarity-determining regions (CDRs) as those of SEQ ID NO: 22, and (ii) a light-chain variable region (V L ) that contains the same light-chain CDRs as those of SEQ ID NO: 21. See Table 1 below. In some specific examples, the anti-GPC3 antibodies disclosed herein may include heavy-chain CDR1, heavy-chain CDR2, and heavy-chain CDR3 as described in SEQ ID NOs: 18-20, respectively, when determined by the Kabat method. Alternatively, or additionally, the anti-GPC3 antibodies disclosed herein may include light-chain CDR1, light-chain CDR2, and light-chain CDR3 as described in SEQ ID NOs: 15-17 when determined by the Kabat method. In one specific example, this anti-GPC3 scFv may include V H that contains the amino acid sequence of SEQ ID NO: 22, and V L contains the amino acid sequence of SEQ ID NO: 21. See Table 1 below.

[0064] The same V H CDR and / or V LTwo antibodies having CDRs means that these CDRs are identical when determined by the same approach (e.g., the Kabat approach, Chothia approach, AbM approach, Contact approach, or IMGT approach known in the art). For example, see the following: Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs.bioinf.org.uk / abs / .

[0065] In other embodiments, the anti-GPC3 scFv can be a functional variant derived from the exemplary anti-GPC3 antibodies listed in Table 1. Such functional variants are substantially similar in both structure and function to this exemplary anti-GPC3 antibody. The functional variant has V H CDRs and V L CDRs that are substantially identical to those of Ab-1 or Ab-2. For example, the functional variant can contain only mutations of up to 8 (e.g., 8, 7, 6, 5, 4, 3, 2, or 1) amino acid residues in the entire CDR region relative to the exemplary anti-GPC3 antibody and have a substantially similar affinity (e.g., within the same order of K Dbinds to the same epitope of GPC3 by value. In some cases, this functional variant may have the same heavy chain CDR3 as this exemplary anti-GPC3 antibody and, optionally, the same light chain CDR3 as this exemplary anti-GPC3 antibody. Such anti-GPC3 scFv has mutations in the CDR amino acid residues only in the heavy chain CDR1 and / or CDR2 (e.g., up to 5, e.g., 5, 4, 3, 2, and 1) compared to this exemplary anti-GPC3 antibody and contains a V H fragment. Alternatively, or in addition, this anti-scFv antibody may further contain a V L fragment with mutations in the CDR amino acid residues only in the light chain CDR1 and / or CDR2 (e.g., up to 5, e.g., 5, 4, 3, 2, and 1) compared to this exemplary anti-GPC3 antibody. In some examples, the mutations of the amino chain residues can be conservative amino acid residue substitutions.

[0066] In some examples, any of the mutations in one or more of the CDR regions can be conservative substitutions. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for changing polypeptide sequences known to those of skill in the art, as found in the following references that summarize such methods: for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made among the amino acids within the following groups: (a) M, I, L, V, (b) F, Y, W, (c) K, R, H, (d) A, G, (e) S, T, (f) Q, N, and (g) E, D.

[0067] In some embodiments, the anti-GPC3 ScFv derived from an exemplary anti-GPC3 antibody can be in the form of V H -linker-V L from the N-terminus to the C-terminus. In some examples, this anti-GPC3 scFv comprises the V H fragment of SEQ ID NO: 22 and / or the VL fragment of SEQ ID NO: 21. In a specific example, the anti-GPC3 scFv in any of the anti-GPC3 CARs can comprise the amino acid sequence of SEQ ID NO: 23. Alternatively, the anti-GPC3 ScFv derived from an exemplary anti-GPC3 antibody can be in the form of V L -linker-V H from the N-terminus to the C-terminus. In some examples, the anti-GPC3 scFv in any of the anti-GPC3 CARs can comprise the amino acid sequence of SEQ ID NO: 24. In some cases, the anti-GPC3 scFv can share at least 85% sequence identity (e.g., at least 90%, at least 95%, or higher) with SEQ ID NO: 23 or SEQ ID NO: 24.

[0068] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. The BLAST protein search can be carried out with the XBLAST program, score = 50, word length = 3 to obtain an amino acid sequence homologous to the target protein molecule. When there are gaps between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used.

[0069] Anti-GPC3 single domain antibody fragment In some embodiments, the antigen-binding domain can be a single domain antibody fragment. A single domain antibody, also known as a nanobody, is a small antigen-binding fragment that contains only one of the heavy chain variable region or the light chain variable region (in contrast to conventional antibodies that have both a heavy chain variable region and a light chain variable region). In some cases, the single domain antibodies provided herein are heavy chain only antibodies (VHH antibodies) that contain a single heavy chain variable region.

[0070] Similar to conventional antibodies, single-domain antibodies such as VHH antibodies contain regions of hypervariability known as "complementary determining regions" ("CDRs") interspersed with more conserved regions known as "framework regions" ("FRs"). VHH antibodies typically consist of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The ranges of the framework regions and CDRs can be accurately identified using methodologies known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are known in the art. See, for example, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs.

[0071] In some embodiments, the anti-GPC3 antibody portions disclosed herein may share the same complementarity-determining regions (CDRs) as the exemplary anti-GPC3 VHH antibodies shown in Table 1 herein. Such anti-GPC3 VHH antibodies may have CDR1, CDR2, and CDR3, respectively, that include SEQ ID NOs: 6-8 (according to the Kabat definition). In some embodiments, the anti-GPC3 antibody portions disclosed herein may share a certain level of sequence identity (e.g., at least 80%, e.g., at least 85%, at least 90%, at least 95%, or higher) compared to the exemplary anti-GPC3 VHH antibodies. In some embodiments, the anti-GPC3 antibody portions disclosed herein may have one or more amino acid mutations (e.g., up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) relative to the exemplary anti-GPC3 VHH antibodies. The mutations of amino acid residues disclosed in the present disclosure (e.g., mutations in the framework region and / or CDR) may be conservative amino acid residue substitutions. In one example, the anti-GPC3 antibody portion may include the amino acid sequence of SEQ ID NO: 9.

[0072] (ii) Transmembrane domain The anti-GPC3 CAR polypeptides disclosed herein may include a transmembrane domain that can be a hydrophobic α-helix spanning the membrane. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable within the cell membrane (preferably a eukaryotic cell membrane). The transmembrane domain may provide stability to the anti-GPC3 CAR including itself.

[0073] In some embodiments, the transmembrane domain of the anti-GPC3 CAR provided herein can be the CD8 transmembrane domain. In other embodiments, this transmembrane domain can be the CD28 transmembrane domain. In still other embodiments, this transmembrane domain is a chimera of the CD8 and CD28 transmembrane domains. Other transmembrane domains may be used as provided herein. In some embodiments, this transmembrane domain is the CD8a extracellular + transmembrane domain containing the sequence of SEQ ID NO: 2 shown in Table 1 below. Other transmembrane domains may be used.

[0074] (iii) Hinge domain In some embodiments, the hinge domain can be located between the extracellular domain (including the antigen-binding domain) and the transmembrane domain of the anti-GPC3 CAR, or between the cytoplasmic domain and the transmembrane domain of the anti-GPC3 CAR. The hinge domain can be any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or cytoplasmic domain in the polypeptide chain. The hinge domain can function to confer mobility to the anti-GPC3 CAR or its domain, or prevent steric hindrance of the anti-GPC3 CAR or its domain.

[0075] In some embodiments, the hinge domain can contain up to 300 amino acids (e.g., 10 - 100 amino acids, or 5 - 20 amino acids). In some embodiments, one or more hinge domains can be included in other regions of the anti-GPC3 CAR. In some embodiments, this hinge domain can be the CD8 hinge domain. Other hinge domains can be used.

[0076] (iv) Intracellular signaling domain All of the anti-GPC3 CAR constructs herein include one or more intracellular signaling domains (e.g., CD3ζ, and optionally one or more costimulatory domains) that are the functional termini of the receptor. After antigen recognition, the receptor clusters and signals are transmitted to the cell.

[0077] CD3ζ is the cytoplasmic signaling domain of the T cell receptor complex. CD3ζ contains three immunoreceptor tyrosine-based activation motifs (ITAMs) that transmit activation signals to T cells after the T cells associate with allogeneic antigens. In many cases, CD3ζ provides an activation signal to naive T cells, but not a fully competent activation signal and requires costimulatory signaling.

[0078] In some embodiments, the anti-GPC3 CAR polypeptides disclosed herein may further comprise one or more costimulatory signaling domains. For example, the costimulatory domains of CD28 and / or 4-1BB may be used together with the primary signaling mediated by CD3ζ to transmit sufficient proliferation / survival signals. In some examples, the CARs disclosed herein comprise a CD28 costimulatory molecule. In other examples, the CARs disclosed herein comprise a 4-1BB costimulatory molecule. In some embodiments, the CAR comprises a CD3ζ signaling domain and a CD28 costimulatory domain. In other embodiments, the CAR comprises a CD3ζ signaling domain and a 4-1BB costimulatory domain. In still other embodiments, the CAR comprises a CD3ζ signaling domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain.

[0079] Table 1 provides examples of signaling domains derived from 4-1BB, CD28, and CD3-zeta that may be used herein.

[0080] (v) Exemplary anti-GPC3 CAR polypeptides Exemplary anti-GPC3 CAR polypeptides are shown in Table 1 below, all of which are within the scope of the present disclosure (including both mature anti-GPC3 CAR (i.e., without the N-terminal signal peptide) and precursor anti-GPC3 CAR (i.e., with the N-terminal signal peptide)). Also within the scope of the present disclosure are nucleic acids encoding any of the anti-GPC3 CAR constructs disclosed herein (e.g., those disclosed in Table 1). Such nucleic acids can be located in a suitable vector, for example, in a viral vector such as an AAV vector or a lentiviral vector. Host cells containing such nucleic acids or vectors are also within the scope of the present disclosure.

[0081] C. Method for producing genetically engineered T cells The genetically engineered T cells disclosed herein can be prepared by genetically editing parental T cells or their precursor cells by conventional gene editing methods or the methods described herein.

[0082] (a) T cells In some embodiments, T cells can be derived from one or more suitable mammals, for example, from one or more human donors. T cells can be obtained from a number of sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art, such as centrifugation, for example, FICOLL™ separation.

[0083] In some examples, T cells can be isolated from a mixture of immune cells (e.g., those described herein) to produce an isolated population of T cells. For example, after isolation of peripheral blood mononuclear cells (PBMCs), both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subsets, either before or after activation, expansion, and / or genetic modification.

[0084] A specific subpopulation of T cells expressing one or more of the following cell surface markers: TCRαβ, CD3, CD4, CD8, CD27, CD28, CD38, CD45RA, CD45RO, CD62L, CD127, CD122, CD95, CD197, CCR7, KLRG1, MHC-I protein, and / or MHC-II protein can be further isolated by positive or negative selection techniques. In some embodiments, a specific subpopulation of T cells expressing one or more of the markers selected from the group consisting of TCRab, CD4, and / or CD8 is further isolated by positive or negative selection techniques. In some embodiments, a subpopulation of T cells can be isolated by positive or negative selection before and / or after genetic manipulation.

[0085] The isolated population of T cells can express one or more of the T cell markers including, but not limited to, CD3+, CD4+, CD8+, or combinations thereof. In some embodiments, T cells are isolated from a donor or subject, first activated, and stimulated to proliferate in vitro before undergoing gene editing.

[0086] In some cases, the T cell population includes primary T cells isolated from one or more human donors. Such T cells are terminally differentiated, not transformed, cytokine- and / or growth factor-dependent for proliferation, and / or have a stable genome.

[0087] Alternatively, T cells can be derived from stem cells (e.g., HSC or iPSC) through in vitro differentiation.

[0088] T cells derived from a suitable source can be subjected to one or more stimulations, activations, and / or expansions. T cells can generally be activated and expanded using the methods described, for example, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041. In some embodiments, the T cells can be activated and expanded for about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 3 days, about 2 days to about 4 days, about 3 days to about 4 days, or about 1 day, about 2 days, about 3 days, or about 4 days prior to the introduction of the genome editing composition into the T cells.

[0089] In some embodiments, the T cells are activated and expanded for about 4 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours prior to the introduction of the gene editing composition into the T cells. In some embodiments, the T cells are activated simultaneously with the introduction of the genome editing composition into the T cells. Optionally, the T cell population can be expanded and / or activated after the gene editing disclosed herein. T cell populations or isolated T cells generated by any of the gene editing methods described herein are also within the scope of the present disclosure.

[0090] (b) Gene editing method Any of the genetically engineered T cells can be prepared using a conventional gene editing method for editing one or more of the target genes disclosed herein or those described herein (targeted editing). Targeted editing can be achieved via either a nuclease-independent approach or a nuclease-dependent approach. In a nuclease-independent targeted editing approach, homologous recombination is induced by homologous sequences flanking an exogenous polynucleotide that is introduced into the endogenous sequence via the enzymatic machinery of the host cell. This exogenous polynucleotide can introduce nucleotide deletions, insertions, or substitutions into this endogenous sequence.

[0091] Alternatively, a nuclease-dependent approach can achieve targeted editing at a relatively high frequency via the specific introduction of double-strand breaks (DSBs) by a specific rare-cutting nuclease (e.g., an endonuclease). Such nuclease-dependent targeted editing also utilizes the DNA repair machinery, for example, non-homologous end joining (NHEJ) that occurs in response to DSBs. DNA repair by NHEJ often causes random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast to NHEJ-mediated repair, repair can also occur by homologous recombination repair (HDR). If a donor template containing exogenous gene material flanked by pairs of homology arms is present, this exogenous gene material can be introduced into the genome by HDR, which results in the targeted integration of this exogenous gene material.

[0092] In some embodiments, gene disruption can occur by deletion of a genomic sequence using two guide RNAs. Methods of using CRISPR-Cas gene editing technology to effect genomic deletions in cells (e.g., knocking out a gene in a cell) are known (Bauer DE et al. Vis. Exp. 2015;95:e52118).

[0093] Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR-Cas9 nucleases (CRISPR / Cas9; clustered regularly interspaced short palindromic repeats associated protein 9). In addition, the DICE (dual integrase cassette exchange) system that utilizes phiC31 integrase and Bxb1 integrase can also be used for targeted integration. Some exemplary approaches are disclosed in detail below.

[0094] CRISPR-Cas9 gene editing system The CRISPR-Cas9 system is a prokaryotic defense mechanism that has been repurposed as an RNA-guided DNA targeting platform for gene editing. The CRISPR-Cas9 system relies on the DNA nuclease Cas9 and two non-coding RNAs, the CRISPR RNA (crRNA) and the trans-activating RNA (tracrRNA), to cleave DNA. CRISPR is an abbreviation for clustered regularly interspaced short palindromic repeats, a family of DNA sequences found in the genomes of bacteria and archaea that contain fragments of DNA (spacer DNA) similar to foreign DNA previously exposed to the cell by, for example, a virus infecting or attacking the prokaryote. For example, upon subsequent attack by a similar virus, these DNA fragments are used by the prokaryote to detect and destroy similar foreign DNA. Transcription of the CRISPR locus results in the formation of an RNA molecule containing the spacer sequence, which associates with a Cas (CRISPR-associated) protein capable of recognizing and cleaving foreign exogenous DNA to target it. Numerous types and classes of CRISPR / Cas systems have been described (see, for example, Koonin et al., (2017) Curr Opin Microbiol 37:67-78).

[0095] crRNA typically drives sequence recognition and specificity of the CRISPR-Cas9 complex by forming Watson-Crick base pairs with a sequence of 20 nucleotides (nt) within the target DNA. By altering the sequence of the 5’ 20 nt within the crRNA, targeting of the CRISPR-Cas9 complex to a specific locus becomes possible. The CRISPR-Cas9 complex binds only to DNA sequences that contain a sequence matching the first 20 nt of the crRNA when the target sequence is followed by a specific short DNA motif, termed the protospacer adjacent motif (PAM), which has the sequence NGG.

[0096] tracrRNA hybridizes with the 3’ end of the crRNA to form an RNA duplex structure to which the Cas9 endonuclease binds, forming a catalytically active CRISPR-Cas9 complex that can then cleave the target DNA.

[0097] When the CRISPR-Cas9 complex binds to DNA at the target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, leaving a double-strand break (DSB) in which both strands of the DNA terminate in base pairs (blunt ends).

[0098] After the CRISPR-Cas9 complex binds to DNA at a specific target site and a site-specific DSB is formed, the next important step is the repair of the DSB. Cells use the following two major DNA repair pathways to repair DSBs: non-homologous end joining (NHEJ) and homologous recombination repair (HDR).

[0099] NHEJ appears to be a robust repair mechanism that is highly active in most cell types, including non-dividing cells. NHEJ is error-prone and can often result in deletions or additions between 1 and several hundred nucleotides at the site of the DSB, although such modifications are typically <20 nt. The resulting insertions and deletions (indels) can disrupt the coding or non-coding regions of genes. Alternatively, HDR uses a long stretch of homologous donor DNA provided endogenously or exogenously to repair DSBs with high fidelity. HDR is only active in dividing cells and occurs at a relatively low frequency in most cell types. In many embodiments of the present disclosure, NHEJ is utilized as a repair operant.

[0100] Endonucleases for use with CRISPR In some embodiments, the Cas9 (CRISPR-associated protein 9) endonuclease is used in the CRISPR method for generating the genetically engineered T cells disclosed herein. This Cas9 enzyme can be derived from Streptococcus pyogenes, although other Cas9 homologs can also be used. As shown herein, it should be understood that wild-type Cas9 may be used, or a modified version of Cas9 (e.g., an evolved version of Cas9, or a Cas9 ortholog or variant) may be used. In some embodiments, Cas9 can be replaced with another RNA-guided endonuclease such as Cpf1 (of the class II CRISPR / Cas system).

[0101] In some embodiments, the CRISPR / Cas system comprises components derived from a type I, II, or III system. The most recent classification scheme for CRISPR / Cas loci defines class 1 and class 2 CRISPR / Cas systems with types I-V or VI (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397). Class 2 CRISPR / Cas systems have a single protein effector. Type II, V, and VI Cas proteins are single-protein RNA-guided endonucleases referred to herein as "class 2 Cas nucleases". Examples of class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. The Cpf1 nuclease (Zetsche et al., (2015) Cell 163:1-13) is homologous to Cas9 and contains an RuvC-like nuclease domain.

[0102] In some embodiments, the Cas nuclease is derived from a type II CRISPR / Cas system (e.g., the Cas9 protein derived from the CRISPR / Cas9 system). In some embodiments, the Cas nuclease is derived from a class 2 CRISPR / Cas system (a single-protein Cas nuclease such as the Cas9 protein or the Cpf1 protein). The Cas9 and Cpf1 families of proteins are enzymes having DNA endonuclease activity, which can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, as further described herein.

[0103] In some embodiments, the Cas nuclease can include multiple nuclease domains. For example, the Cas9 nuclease can include at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease introduces a DSB at the target sequence. In some embodiments, the Cas9 nuclease is modified to include only one functional nuclease domain. For example, the Cas9 nuclease is modified such that one of the nuclease domains is mutated or completely or partially deleted, reducing its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease is modified to not include a functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease is modified to not include a functional HNH-like nuclease domain. In some embodiments where only one of the nuclease domains is functional, the Cas9 nuclease is a nickase capable of introducing a single-strand break (“nick”) at the target sequence. In some embodiments, conserved amino acids within the Cas9 nuclease domain are substituted to reduce or modify nuclease activity. In some embodiments, the Cas nuclease nickase includes an amino acid substitution in the RuvC-like nuclease domain. An exemplary amino acid substitution in the RuvC-like nuclease domain is D10A (based on the S. pyogenes Cas9 nuclease). In some embodiments, this nickase includes an amino acid substitution in the HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 nuclease). An example is shown in Table 2 below (SEQ ID NO: 70).

[0104] In some embodiments, the Cas nuclease is derived from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease is a component of the Cascade complex of the type I CRISPR / Cas system. For example, the Cas nuclease is a Cas3 nuclease. In some embodiments, the Cas nuclease is derived from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type IV CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type V CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type VI CRISPR / Cas system.

[0105] Guide RNA (gRNA) CRISPR technology involves the use of a genome-targeting nucleic acid that can direct an endonuclease to a specific target sequence within a target gene for gene editing at that specific target sequence. This genome-targeting nucleic acid can be RNA. The genome-targeting RNA is referred to herein as "guide RNA" or "gRNA". The guide RNA includes at least one spacer sequence that hybridizes to a target nucleic acid sequence within the target gene for editing and to a CRISPR repeat sequence.

[0106] In type II systems, the gRNA also includes a second RNA called the tracrRNA sequence. In type II gRNAs, the CRISPR repeat sequence and the tracrRNA sequence hybridize to each other to form a duplex. In type V gRNAs, the crRNA forms a duplex. In both systems, the duplex binds to a site-specific polypeptide, resulting in the formation of a complex between the guide RNA and the site-specific polypeptide. In some embodiments, the genome-targeting nucleic acid confers target specificity to the complex by its association with the site-specific polypeptide. Thus, the genome-targeting nucleic acid induces the activity of the site-specific polypeptide.

[0107] As will be understood by those skilled in the art, each guide RNA is designed to include a spacer sequence complementary to its genomic target sequence. See Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011).

[0108] In some embodiments, the genomic targeting nucleic acid (e.g., gRNA) is a bimolecular guide RNA. In some embodiments, the genomic targeting nucleic acid (e.g., gRNA) is a single molecule guide RNA.

[0109] The bimolecular guide RNA comprises two strands of RNA molecules. The first strand includes, in the 5’ to 3’ direction, an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence. The second strand includes a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3’ tracrRNA sequence, and an optional tracrRNA extension sequence.

[0110] The single molecule guide RNA (referred to as “sgRNA”) in type II systems includes, in the 5’ to 3’ direction, an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single molecule guide linker, a minimal tracrRNA sequence, a 3’ tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension may include elements that confer additional functionality (e.g., stability) to the guide RNA. The single molecule guide linker connects the minimal CRISPR repeat and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension includes one or more hairpins. The single molecule guide RNA in type V systems includes, in the 5’ to 3’ direction, a minimal CRISPR repeat sequence and a spacer sequence.

[0111] The spacer sequence within the gRNA is a sequence (e.g., a sequence of 20 nucleotides) that defines the target sequence of the target gene of interest (e.g., a DNA target sequence such as a genomic target sequence). In some embodiments, this spacer sequence is within the range of 15 to 30 nucleotides. For example, this spacer sequence may comprise 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the spacer sequence comprises 20 nucleotides.

[0112] The "target sequence" is a sequence that is present in the target gene adjacent to the PAM sequence and is modified by an RNA-guided nuclease (e.g., Cas9). The "target sequence" is present on the so-called PAM strand within a double-stranded molecule, the "target nucleic acid", which comprises a PAM strand and a complementary non-PAM strand. Those skilled in the art will recognize that the gRNA spacer sequence hybridizes to a complementary sequence located in the non-PAM strand of the target nucleic acid of interest. Therefore, the gRNA spacer sequence is the RNA equivalent of the target sequence. For example, when the target sequence is 5'-AGAGCAACAGTGCTGTGGCC**-3' (SEQ ID NO: 58), the gRNA spacer sequence is 5'-AGAGCAACAGUGCUGUGGCC**-3' (SEQ ID NO: 39). The spacer of the gRNA interacts with the target nucleic acid of interest in a sequence-specific manner via hybridization (i.e., base pairing). Therefore, the nucleotide sequence of the spacer varies depending on the target sequence of the target nucleic acid of interest.

[0113] In the CRISPR / Cas system of this specification, the spacer sequence is designed to hybridize to a region of the target nucleic acid located 5' of the PAM recognizable by the Cas9 enzyme used in this system. The spacer can exactly match the target sequence or can have mismatches. Each Cas9 enzyme has a specific PAM sequence recognized within the target DNA. For example, S. pyogenes recognizes a PAM containing the sequence 5'-NRG-3' (where R includes either A or G, N is any nucleotide, and N is present immediately 3' of the target nucleic acid sequence targeted by the spacer sequence).

[0114] In some embodiments, the target nucleic acid sequence is 20 nucleotides in length. In some embodiments, the target nucleic acid is less than 20 nucleotides in length. In some embodiments, the target nucleic acid is more than 20 nucleotides in length. In some embodiments, the target nucleic acid is at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides in length. In some embodiments, the target nucleic acid is at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides in length. In some embodiments, the target nucleic acid sequence has 20 bases immediately 5' of the first nucleotide of the PAM. For example,

Chemical formula

[0115] The guide RNAs disclosed herein can target any sequence of interest by the spacer sequence within the crRNA. In some embodiments, the degree of complementarity between the spacer sequence of the guide RNA and the target sequence within the target gene can be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the spacer sequence of the guide RNA and the target sequence within the target gene are 100% complementary. In other embodiments, the spacer sequence of the guide RNA and the target sequence within the target gene can include up to 10 mismatches, for example, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 mismatch.

[0116] With respect to any of the gRNA sequences provided herein, those that do not explicitly indicate a modification are meant to encompass both the unmodified sequence and sequences having any suitable modification.

[0117] The length of the spacer sequence in any of the gRNAs disclosed herein can depend on the CRISPR / Cas9 system and the components used to edit any of the target genes also disclosed herein. For example, different Cas9 proteins from different bacterial species have various optimal spacer sequence lengths. Thus, the spacer sequence can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the spacer sequence can be 18 - 24 nucleotides in length. In some embodiments, the targeting sequence can be 19 - 21 nucleotides in length. In some embodiments, the spacer sequence can be 20 nucleotides in length.

[0118] In some embodiments, the gRNA can be a sgRNA, and this sgRNA can include a 20-nucleotide spacer sequence at the 5' end of this sgRNA sequence. In some embodiments, the sgRNA can include a spacer sequence of less than 20 nucleotides at the 5' end of this sgRNA sequence. In some embodiments, the sgRNA can include a spacer sequence of more than 20 nucleotides at the 5' end of this sgRNA sequence. In some embodiments, the sgRNA includes a variable-length spacer sequence having 17 to 30 nucleotides at the 5' end of this sgRNA sequence. Examples are shown in Table 2 below. In these exemplary sequences, the fragment of "n" refers to the spacer sequence at the 5' end.

[0119] In some embodiments, the sgRNA does not contain uracil at the 3' end of this sgRNA sequence. In other embodiments, the sgRNA can contain one or more uracils at the 3' end of this sgRNA sequence. For example, the sgRNA can contain 1 to 8 uracil residues at the 3' end of this sgRNA sequence, for example, can contain 1, 2, 3, 4, 5, 6, 7, or 8 uracil residues at the 3' end of this sgRNA sequence.

[0120] Any of the gRNAs disclosed herein, such as any of the sgRNAs, can be unmodified. Alternatively, this gRNA can include one or more modified nucleotides and / or a modified backbone. For example, a modified gRNA such as sgRNA can include one or more 2'-O-methyl phosphorothioate nucleotides that can be located at either the 5' end, the 3' end, or both.

[0121] In certain embodiments, multiple guide RNAs can be used with a CRISPR / Cas nuclease system. Each guide RNA can contain various targeting sequences such that the CRISPR / Cas system cleaves multiple target nucleic acids. In some embodiments, one or more guide RNAs can have the same or different properties, such as activity or stability, within the Cas9 RNP complex. When multiple guide RNAs are used, each guide RNA can be encoded on the same or different vectors. The promoters used to drive the expression of multiple guide RNAs can be the same or different.

[0122] In some embodiments, the gRNAs disclosed herein target the Reg1 gene, for example, sites within exon 1, exon 2, exon 3, exon 4, exon 5, or exon 6 of the Reg1 gene. Such gRNAs can contain a spacer sequence that is (fully or partially) complementary to a target sequence or a fragment thereof in exon 2 or exon 4 of the Reg1 gene. Exemplary target sequences of Reg1 and exemplary gRNA sequences are shown in Table 2 below.

[0123] In some embodiments, the gRNAs disclosed herein target the TGFBRII gene, for example, sites within exon 1, exon 2, exon 3, exon 4, exon 5, or exon 6 of the TGFBRII gene. Such gRNAs can contain a spacer sequence that is (fully or partially) complementary to a target sequence or a fragment thereof in exon 4 or exon 5 of the TGFBRII gene. Exemplary target sequences of TGFBRII and exemplary gRNA sequences are shown in Table 2 below.

[0124] In some embodiments, the gRNAs disclosed herein target the cbl-b gene, for example, targeting sites within exon 2, exon 7, exon 9, exon 11, or exon 12 of the cbl-b gene. Such gRNAs may include a spacer sequence that is (fully or partially) complementary to the target sequence or a fragment thereof in exon 2 of the cbl-b gene. In other examples, the gRNA may include a spacer sequence that is (fully or partially) complementary to the target sequence or a fragment thereof in exon 7 of the cbl-b gene. Alternatively, the gRNA may include a spacer sequence that is (fully or partially) complementary to the target sequence or a fragment thereof in exon 9 of the cbl-b gene. Exemplary target sequences in the cbl-b gene, and exemplary gRNA sequences, are shown in Table 2 below.

[0125] In some embodiments, the gRNAs disclosed herein target the β2M gene, for example, targeting suitable sites within the β2M gene. Reference is also made to International Publication No. WO 2019 / 097305, the relevant disclosure of which is incorporated herein by reference for the purposes and subject matter referred to herein. Other gRNA sequences may be designed using the β2M gene sequence located on chromosome 15 (GRCh38 coordinates: chromosome 15: 44,711,477-44,718,877; Ensembl: ENSG00000166710). In some embodiments, the gRNA targeting the β2M genomic region and the RNA-guided nuclease cause cleavage in the β2M genomic region, resulting in indels in the β2M gene and disrupting mRNA or protein expression. Exemplary spacer sequences, and gRNAs targeting the β2M gene, are shown in Table 2 below.

[0126] In some embodiments, the gRNAs disclosed herein target the TRAC gene. Reference is also made to International Publication No. WO 2019 / 097305, the relevant disclosure of which is incorporated herein by reference for the subject matter and purposes mentioned herein. Other gRNA sequences can be designed using the TRAC gene sequence located on chromosome 14 (GRCh38: chromosome 14: 22,547,506-22,552,154; Ensembl; ENSG00000277734). In some embodiments, the gRNA targeting the TRAC genomic region, and the RNA-guided nuclease, cause cleavage in the TRAC genomic region to result in indels in the TRAC gene, disrupting mRNA or protein expression. Exemplary spacer sequences, and gRNAs targeting the TRAC gene, are shown in Table 2 below.

[0127] As an example, guide RNAs used in the CRISPR / Cas / Cpf1 system, or other smaller RNAs, can be readily synthesized by chemical means as described below and as described in the art. While chemical synthesis methods continue to expand, purification of such RNAs by methods such as high performance liquid chromatography (HPLC, avoiding the use of gels such as PAGE) tends to become more difficult as the length of the polynucleotide increases significantly beyond 100 nucleotides per molecule. One approach used to generate longer RNAs is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding Cas9 endonuclease or Cpf1 endonuclease, are more readily generated enzymatically. As described in the art, various types of RNA modifications (e.g., modifications that enhance stability, reduce the likelihood or extent of the innate immune response, and / or enhance other properties) can be introduced during and / or after chemical synthesis and / or enzymatic generation of the RNA.

[0128] In some examples, the gRNAs of the present disclosure can be produced by in vitro transcription (IVT), synthesis, and / or chemical synthesis methods, or combinations thereof. Enzymatic (IVT), solid-phase, liquid-phase, composite synthesis methods, small region synthesis, and ligation methods are utilized. In one embodiment, the gRNA is made using an IVT enzymatic synthesis method. Methods for producing polynucleotides by IVT are known in the art and are described in WO 2013 / 151666 pamphlet. Accordingly, the present disclosure also includes polynucleotides, such as DNA constructs, where the vectors are used for in vitro transcription of the gRNAs described herein.

[0129] As described in the art, various types of RNA modifications (e.g., modifications that enhance stability, reduce the likelihood or extent of innate immune responses, and / or enhance other properties) can be introduced during or after the chemical synthesis and / or enzymatic production of RNA. In some embodiments, unnatural modified nucleobases can be introduced into any of the gRNAs disclosed herein during or after synthesis. In certain embodiments, the modification is to the internucleoside linkage, purine or pyrimidine base, or sugar. In some embodiments, the modification is introduced at the ends of the gRNA using chemical synthesis or polymerase enzymes. Examples of modified nucleic acids and their synthesis are disclosed in WO 2013 / 052523 pamphlet. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

[0130] In some embodiments, enzymatic or chemical ligation methods can be used to conjugate polynucleotides or regions thereof with various functional moieties such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).

[0131] In some embodiments of the present disclosure, a CRISPR / Cas nuclease system for use in gene editing of any of the target genes disclosed herein may comprise at least one guide RNA. In some examples, this CRISPR / Cas nuclease system may comprise multiple gRNAs, for example, it may comprise 2, 3, or 4 gRNAs. Such multiple gRNAs may target different sites within the same target gene. Alternatively, these multiple gRNAs may target different genes. In some embodiments, the guide RNA and the Cas protein may form a ribonucleoprotein (RNP), for example, they may form a CRISPR / Cas complex. This guide RNA may direct this Cas protein to a target sequence on one or more target genes such as those disclosed herein, and this Cas protein cleaves the target gene at the target site. In some embodiments, this CRISPR / Cas complex is a Cpf1 / guide RNA complex. In some embodiments, this CRISPR complex is a type II CRISPR / Cas9 complex. In some embodiments, this Cas protein is a Cas9 protein. In some embodiments, this CRISPR / Cas9 complex is a Cas9 / guide RNA complex.

[0132] In some embodiments, the indel frequency (editing frequency) of a particular CRISPR / Cas nuclease system comprising one or more specific gRNAs can be determined using TIDE analysis, which can be used to identify gRNA molecules that are very efficient in editing the target gene. In some embodiments, a very efficient gRNA results in a gene editing frequency higher than 80%. For example, a gRNA is considered very efficient if this gRNA results in a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0133] Delivery of Guide RNA and Nuclease to T Cells The CRISPR / Cas nuclease system disclosed herein, comprising one or more gRNAs disclosed below, and at least one RNA-guided nuclease, optionally including a donor template, can be delivered to target cells (e.g., T cells) by conventional methods to gene-edit a target gene. In some embodiments, the components of the CRISPR / Cas nuclease system disclosed herein can be delivered to target cells individually, either simultaneously or sequentially. In other embodiments, the components of the CRISPR / Cas nuclease system can be delivered into the target together, for example, as a complex. Optionally, the gRNA and the RNA-guided nuclease can be pre-complexed together to form a ribonucleoprotein (RNP) that can be delivered into target cells.

[0134] RNP is useful for gene editing because it minimizes the risk of promiscuous interactions in a nucleic acid-rich cellular environment and protects the RNA from degradation. Methods for forming RNP are known in the art. In some embodiments, an RNP comprising an RNA-guided nuclease (e.g., a Cas nuclease such as Cas9 nuclease) and one or more gRNAs targeting one or more genes of interest can be delivered to cells (e.g., T cells). In some embodiments, the RNP can be delivered to T cells by electroporation.

[0135] In some embodiments, the RNA-guided nuclease can be delivered to cells in a DNA vector that expresses this RNA-guided nuclease intracellularly. In other examples, the RNA-guided nuclease can be delivered to cells in an RNA that encodes this RNA-guided nuclease and expresses this nuclease intracellularly. Alternatively, or in addition, the gRNA targeting a gene can be delivered to cells as an RNA vector or a DNA vector that expresses this gRNA in the cells.

[0136] Delivery of the RNA-guided nuclease, gRNA, and / or RNP can be via direct injection or via cell transfection using known methods (e.g., electroporation or chemical transfection). Other cell transfection methods can be used. In some cases, the Cas9 enzyme can form one RNP with all gRNAs targeting the TRAC gene, β2M gene, Reg1 gene, and TGFBRII gene and can be delivered to T cells via a single electroporation event. Alternatively, the Cas9 enzyme can form two or more RNPs, which together contain all gRNAs targeting the TRAC gene, β2M gene, Reg1 gene, and TGFBRII gene. These multiple RNPs can be delivered to T cells via sequential electroporation events (e.g., two sequential electroporations).

[0137] In other embodiments, a viral vector such as one or more lentiviral vectors can be used to deliver a nucleic acid encoding a nuclease and optionally one or more gRNAs to a target cell (e.g., a T cell) to gene edit one or more of the target genes disclosed herein.

[0138] Other gene editing methods In the generation of the genetically engineered T cells disclosed herein, in addition to the CRISPR methods disclosed herein, additional gene editing methods known in the art can also be used. Some examples include gene editing approaches involving zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), restriction endonucleases, meganuclease homing endonucleases, and the like.

[0139] ZFN is a targeted nuclease that contains a nuclease fused to a zinc finger DNA binding domain (ZFBD), which is a polypeptide domain that binds to DNA in a sequence-specific manner via one or more zinc fingers. A zinc finger is a domain of approximately 30 amino acids within a zinc finger binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. Engineered zinc finger domains are domains that do not occur in nature, primarily resulting from the application of rational criteria for design / composition, such as substitution rules and computer-processed algorithms for processing information in databases that store information on existing ZFP designs and binding data. See, for example, U.S. Patent Nos. 6,140,081; 6,453,242; and 6,534,261; and International Publication Nos. WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536; and WO 03 / 016496. Selected zinc finger domains are domains that do not occur in nature, primarily resulting from experimental processes such as phage display, interaction trap, or hybrid selection. ZFNs are described in more detail in U.S. Patent Nos. 7,888,121 and 7,972,854. The best-known example of a ZFN is a fusion of the FokI nuclease and a zinc finger DNA binding domain.

[0140] TALEN is a targeted nuclease that includes a nuclease fused to a TAL effector DNA binding domain. The "transcription activator-like effector DNA binding domain", "TAL effector DNA binding domain", or "TALE DNA binding domain" is a polypeptide domain of a TAL effector protein that is involved in the binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. This protein enters the nucleus of plant cells, binds to an effector-specific DNA sequence via this DNA binding domain, and activates gene transcription at this sequence via this transcription activation domain. The specificity of the TAL effector DNA binding domain depends on a variable number of imperfect 34-amino acid repeats that are polymorphic at select repeat positions called repeat variable diresidues (RVDs). TALEN is described in more detail in U.S. Patent Application Publication No. 2011 / 0145940. The most well-known example of TALEN in the art is a fusion polypeptide of the FokI nuclease to a TAL effector DNA binding domain.

[0141] Further examples of targeted nucleases suitable for use provided herein include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and Wβ / SPBc / TP901-1, whether for individual use or in combination.

[0142] Any of the nucleases disclosed herein, such as CRISPR / Cas nucleases, can be delivered using vector systems including, but not limited to, plasmid vectors, DNA minicircles, retroviral vectors, lentiviral vectors, adenoviral vectors, poxviral vectors; herpesviral vectors and adeno-associated viral vectors, and combinations thereof.

[0143] Using conventional viral and non-viral gene delivery methods, nucleic acids encoding nucleases and donor templates can be introduced into cells (e.g., T cells). Examples of non-viral vector delivery systems include DNA plasmids, DNA minicircles, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes or poloxamers. Examples of viral vector delivery systems include DNA viruses and RNA viruses that have either an episomal or integrated genome after delivery to the cell.

[0144] Non-viral delivery methods of nucleic acids include electroporation, lipofection, microinjection, gene gun, virosome, liposome, immunoliposome, polycation or lipid:nucleic acid conjugate, naked DNA, naked RNA, capped RNA, artificial virion, and uptake of DNA enhanced with agents. For example, sonoporation using the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. Some specific examples are shown below.

[0145] D Delivery of anti-GPC3 CAR constructs to T cells In some embodiments, the nucleic acid encoding the anti-GPC3 CAR can be introduced into any of the genetically engineered T cells disclosed herein by methods known to those of skill in the art. For example, the coding sequence of the anti-GPC3 CAR can be cloned into a vector that can be introduced into T cells genetically engineered for the expression of the anti-GPC3 CAR. A variety of different methods known in the art can be used to introduce any of the nucleic acids or expression vectors disclosed herein into immune effector cells. Non-limiting examples of methods for introducing nucleic acids into cells include the following: lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral transfection, and nucleofection.

[0146] In some examples, the nucleic acid encoding the anti-GPC3 CAR construct can be delivered to cells using adeno-associated virus (AAV). AAV is a small virus that can site-specifically integrate into the host genome and thus deliver transgenes such as anti-GPC3 CAR. Inverted terminal repeats (ITRs) are present adjacent to the AAV genome and / or the transgene of interest and function as origins of replication. Also present in the AAV genome are the rep and cap proteins that form the capsid for encapsulating the AAV genome and delivering it to target cells upon transcription. The AAV serotype is conferred by surface receptors on these capsids, which determines which target organ the capsid first binds to and thus which cells AAV infects most efficiently. There are currently 12 known human AAV serotypes. In some embodiments, the AAV used for delivery of the nucleic acid encoding anti-GPC3 CAR is AAV serotype 6 (AAV6).

[0147] Adeno-associated virus is one of the viruses most frequently used in gene therapy for several reasons. First, AAV does not induce an immune response when administered to mammals, including humans. Second, AAV is efficiently delivered to target cells, especially when considering the selection of an appropriate AAV serotype. Finally, AAV has the ability to infect both dividing and non-dividing cells because it can persist in host cells without genome integration. This property makes AAV an ideal candidate for gene therapy.

[0148] The nucleic acid encoding the CAR can be designed to insert into the target genomic site within the host T cell. In some embodiments, the target genomic site can be present within a safe harbor locus.

[0149] In some embodiments, a nucleic acid encoding an anti-GPC3 CAR (e.g., via a donor template that can be carried by a viral vector such as an adeno-associated virus (AAV) vector) can be designed to be inserted at a position within the TRAC gene to disrupt the TRAC gene in a genetically engineered T cell and express the CAR polypeptide. Disruption of TRAC results in loss of function of the endogenous TCR. For example, disruption of the TRAC gene can be caused by an endonuclease such as those described herein, and one or more gRNAs targeting one or more TRAC genomic regions. Any of the gRNAs specific for the TRAC gene and target regions disclosed herein can be used for this purpose.

[0150] In some examples, homologous recombination repair, i.e., HDR (e.g., using a donor template that can be part of a viral vector such as an adeno-associated virus (AAV) vector), can result in a genomic deletion within the TRAC gene and replacement with a segment encoding an anti-GPC3 CAR. In some embodiments, disruption of the TRAC gene can be caused by an endonuclease such as those disclosed herein and one or more gRNAs targeting one or more TRAC genomic regions, and by inserting a segment encoding an anti-GPC3 CAR into the TRAC gene.

[0151] The donor templates disclosed herein may contain the coding sequence of an anti-GPC3 CAR. In some examples, this anti-GPC3 CAR coding sequence may be flanked by two homology regions so as to enable efficient HDR at a target genomic location (e.g., the TRAC gene) using gene editing methods known in the art. In some examples, a CRISPR-based method may be used. In this case, the CRISPR Cas9 enzyme induced by a gRNA specific to the target locus may cleave both strands of the DNA at the target locus. Subsequently, HDR occurs to repair the double-strand break (DSB) and insert the donor DNA encoding the CAR. To occur correctly, the donor sequence is designed to have adjacent residues (hereinafter, "homology arms") complementary to the sequence surrounding the DSB site of the target gene such as the TRAC gene. This homology arm functions as a template for DSB repair and enables HDR to be an essentially error-free mechanism. The rate of homologous recombination repair (HDR) is a function of the distance between the mutation site and the cleavage site, and therefore it is important to select overlapping or neighboring target sites. The template may contain an extra sequence adjacent to the homology region or may contain a sequence different from the genomic sequence, thereby enabling sequence editing.

[0152] Alternatively, the donor template may not have a region homologous to the targeted position of the DNA and may be integrated by NHEJ-dependent end ligation after cleavage at the target site.

[0153] The donor template can be single-stranded and / or double-stranded DNA or RNA and can be introduced into cells in linear or circular form. When introduced in linear form, the ends of the donor sequence can be protected by methods known to those skilled in the art (e.g., from degradation by exonucleases). For example, one or more dideoxynucleotide residues can be added to the 3' end of the linear molecule and / or self-complementary oligonucleotides can be ligated to one or both ends. See, for example, Chang et al., (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al., (1996) Science 272:886-889. Further methods for protecting exogenous polynucleotides from degradation include the addition of terminal amino groups and the use of modified internucleotide linkages such as phosphorothioate, phosphoramidate, and O-methyl ribose or deoxyribose residues, among others.

[0154] The donor template can be introduced into cells as part of a vector molecule having additional sequences such as, for example, an origin of replication, a promoter, and a gene encoding antibiotic resistance. Further, the donor template can be introduced into cells as naked nucleic acid, as nucleic acid complexed with an agent such as liposomes or poloxamers, or delivered by a virus (e.g., adenovirus, AAV, herpes virus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV)).

[0155] In some embodiments, the donor template can be inserted at a site (e.g., downstream or upstream) near the endogenous promoter such that its expression can be driven by the endogenous promoter. In other embodiments, the donor template can include an exogenous promoter and / or enhancer to control the expression of the CAR gene, and can include, for example, a constitutive promoter, an inducible promoter, or a tissue-specific promoter. In some embodiments, the exogenous promoter is the EF1α promoter (see, e.g., SEQ ID NO: 72 shown in Table 3 below). Other promoters may be used.

[0156] Furthermore, the exogenous sequence can also include transcriptional or translational control sequences, and can include, for example, a promoter, an enhancer, an insulator, an internal ribosome entry site, a sequence encoding a 2A peptide, and / or a polyadenylation signal.

[0157] In some embodiments, the donor template for delivering an anti-GPC3 CAR can be an AAV vector inserted with a nucleic acid fragment comprising the coding sequence of the anti-GPC3 CAR and optionally a control sequence for the expression of the anti-GPC3 CAR (e.g., a promoter such as the EF1a promoter shown in Table 3), and the coding sequence and the control sequence can be adjacent to homology arms for inserting the coding sequence and the control sequence into the target genomic locus. In some examples, the nucleic acid fragment is inserted into the endogenous TRAC locus, thereby disrupting the expression of the TRAC gene. In a specific example, the nucleic acid can replace a fragment within the TRAC gene, for example, a fragment comprising the nucleotide sequence of SEQ ID NO: 58. In some specific examples, the donor template for delivering an anti-GPC3 CAR can include the nucleotide sequence set forth in SEQ ID NO: 12, 75, 27, 30, 33, or 36, and the nucleotide sequence can be adjacent to upstream and downstream homology arms (e.g., SEQ ID NO: 71 and SEQ ID NO: 74). Optionally, the nucleic acid encoding the CAR can be inserted into the disrupted TRAC gene, for example, the fragment of SEQ ID NO: 58 is replaced.

[0158] In other examples, a nucleic acid encoding an anti-GPC3 CAR construct can be delivered to cells using a lentiviral vector. Lentiviral vectors can infect both dividing and non-dividing cells. Therefore, lentiviral vectors can efficiently deliver transgenes to non-proliferating or slowly proliferating cells and are thus attractive for clinical applications. A lentiviral vector carrying a nucleic acid encoding any of the anti-GPC3 CARs disclosed herein can be constructed according to conventional methods. See also Example 5 below.

[0159] E. Exemplary anti-GPC3 CAR-T cells optionally subjected to multiple gene edits In some embodiments, a population of genetically engineered T cells disclosed herein expresses an anti-GPC3 CAR such as those disclosed herein (e.g., those shown in Table 1). Such genetically engineered T cells can also include a disrupted TRAC gene, a disrupted β2M gene, a disrupted Reg1 gene, a disrupted TGFBRII gene, a disrupted cbl-b gene, or combinations thereof. The nucleotide sequence encoding the anti-GPC3 CAR can be inserted into a target gene site in, for example, the disrupted TRAC gene (e.g., by replacing the site targeted by the sgRNA listed in Table 2 below).

[0160] In some examples, the population of genetically engineered T cells disclosed herein expresses an anti-GPC3 CAR, such as those disclosed herein (e.g., those shown in Table 1), and includes a disrupted TRAC gene and a disrupted β2M gene. The nucleotide sequence encoding the anti-GPC3 CAR can be inserted into the gene site of interest (e.g., the disrupted TRAC gene) (e.g., by replacing the site targeted by the sgRNA listed in Table 2 below). Such a population of genetically engineered T cells can further include a disrupted Reg1 gene, a disrupted TGFBRII gene, a disrupted cbl-b gene, or combinations thereof. For example, such a population of genetically engineered T cells can further include a disrupted Reg1 gene and a disrupted TGFBRII gene. Alternatively, such a population of genetically engineered T cells can further include a disrupted TGFBRII gene and a disrupted cbl-b gene. In other examples, such a population of genetically engineered T cells can further include a disrupted cbl-b gene. Such genetically engineered T cells can further include additional gene editing, e.g., can further include a disrupted Reg-1 and / or TGFBRII gene. Alternatively, such genetically engineered T cells can have a wild-type Reg-1 and / or TGFBRII gene.

[0161] In some embodiments, the population of genetically engineered T cells disclosed herein expresses an anti-GPC3 CAR such as those disclosed herein (e.g., those shown in Table 1), and contains a disrupted Reg1 gene, a disrupted TGFBRII gene, a disrupted cbl-b gene, or a combination thereof. In some examples, this population of genetically engineered T cells expresses an anti-GPC3 CAR and contains a disrupted Reg1 gene and a disrupted TGFBRII gene. Alternatively, this population of genetically engineered T cells may contain a disrupted TGFBRII gene and a disrupted cbl-b gene. In other examples, this population of genetically engineered T cells may further contain a disrupted cbl-b gene. Such genetically engineered T cells may further contain additional gene editing, for example, may further contain a disrupted Reg-1 and / or TGFBRII gene. Alternatively, this genetically engineered T cell may have a wild-type Reg-1 and / or TGFBRII gene. In some cases, this genetically engineered T cell may have a wild-type TRAC gene, a wild-type β2M gene, or both.

[0162] In some examples, this population of genetically engineered T cells contains about 50% - 99% (e.g., about 55% - about 80%) of CAR + T cells, and optionally, about 90% - 99.9% (e.g., about 95% - about 99.7%) of TCR - T cells, about 50% - 90% (e.g., about 60% - about 80%) of β2M - T cells, about 50% - 90% (e.g., about 60% - about 70%) of TGFBRII - T cells, about 50% - 90% (e.g., about 60% - about 70%) of Reg1 - T cells, and / or about 50% - 90% (e.g., about 60% - about 70%) of CBLB - T cells. In other examples, this population of genetically engineered T cells contains about 50% - about 90% (e.g., about 60% - about 70%) of TGFBRII - T cells and about 50% - about 90% (e.g., about 60% - about 70%) of Reg1 -may include T cells. In yet other examples, this population of genetically engineered T cells is about 50% to about 90% (e.g., about 60% to about 70%) TGFBRII - T cells and about 50% to about 90% (e.g., about 60% to about 70%) CBLB - may include T cells.

[0163] It should be understood that gene disruption encompasses gene modification via gene editing (e.g., using CRISPR / Cas gene editing to insert or delete one or more nucleotides). The disrupted gene may contain one or more mutations (e.g., insertions, deletions, or nucleotide substitutions, etc.) relative to the wild-type counterpart such that the activity of the encoded gene product is substantially reduced or completely abolished. This one or more mutations may be located in non-coding regions, for example, in the promoter region, control regions that control transcription or translation, or intron regions. Alternatively, this one or more mutations may be located in the coding region (e.g., in an exon). In some cases, the disrupted gene does not express the encoded protein or expresses the encoded protein at substantially reduced levels. In other cases, the disrupted gene expresses the encoded protein in a mutant form, none of which function or have substantially reduced activity. In some embodiments, the disrupted gene is a gene that does not encode a functional protein. In some embodiments, a cell containing a disrupted gene does not express a detectable level (e.g., by an antibody, e.g., by flow cytometry) of the protein encoded by this gene (e.g., on the cell surface). Cells that do not express a detectable level of protein may be referred to as knockout cells. For example, if the β2M protein cannot be detected on the cell surface using an antibody that specifically binds to the β2M protein, cells in which β2M gene editing has been performed may be considered β2M knockout cells. On the other hand, a cell is considered positive (+) if the surface expression of such a receptor can be detected by conventional methods (e.g., flow cytometry or immunostaining) in the expression of a surface receptor (e.g., anti-GPC3 CAR).

[0164] Any of the anti-GPC3 CAR-T cells disclosed herein can be suspended in a cryopreservation solution (e.g., CryoStor® C55) to form a pharmaceutical composition. The cryopreservation solution for use in the present disclosure may also contain adenosine, dextrose, dextran-40, lactobionic acid, sucrose, mannitol, a buffer, e.g., N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), one or more salts (e.g., calcium chloride, magnesium chloride, potassium chloride, potassium bicarbonate, potassium phosphate, etc.), one or more bases (e.g., sodium hydroxide, potassium hydroxide, etc.), or combinations thereof. The components of the cryopreservation solution can be dissolved in sterile water (for injection quality). Any of the cryopreservation solutions can be substantially serum-free (undetectable by conventional methods).

[0165] II. CAR-T Cell Therapy for GPC3+ Cancer The anti-GPC3 CAR-T cells disclosed herein can be used for the elimination of disease cells expressing GPC3, such as GPC3+ cancer cells. For example, an effective amount of the anti-GPC3 CAR-T cells can be administered to a subject in need of treatment by an appropriate route such as intravenous injection.

[0166] The administration step may include the placement (e.g., transplantation) of the anti-GPC3 CAR-T cells into the subject by a method or route that results in at least partial localization of the CAR T cells at a desired site, such as the tumor site, so as to achieve a desired effect. The anti-GPC3 CAR-T cells can be administered by any suitable route that delivers them to the desired location in the subject, in which case at least a portion of the transplanted cells or components of the cells remain viable. The survival period of the cells after administration to the subject can range from a short period of several hours (e.g., 24 hours) to several days, to a long period of several years, and even up to the lifespan of the subject (i.e., long-term engraftment). For example, in some embodiments described herein, an effective amount of the therapeutic T cells can be administered via a systemic administration route such as intraperitoneal or intravenous route.

[0167] In some embodiments, the anti-GPC3 CAR-T cells are administered systemically, which refers to the administration of a population of cells that enter the subject's circulatory system rather than directly to the target site, tissue, or organ, and are thereby subject to metabolism and other similar processes. Suitable modes of administration include injection, infusion, drip, or oral ingestion. Examples of injections include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injections and infusions. In some embodiments, the route is intravenous.

[0168] The subject can be any subject for which diagnosis, treatment, or therapy is desired. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some cases, the anti-GPC3 CAR-T cells can be autologous (i.e., "self") to the subject, i.e., the cells are derived from the same subject. Alternatively, the anti-GPC3 CAR-T cells can be allogeneic (i.e., "non-self", e.g., allogeneic, syngeneic, or xenogeneic) to the subject. "Allogeneic" means that the anti-GPC3 CAR-T cells are derived from another individual (donor) of the same species as the subject, rather than from the subject being treated. The donor is an individual who is not the subject being treated. The donor is an individual who is not a patient. In some embodiments, the donor is an individual who does not have or is not suspected of having the cancer being treated. In some embodiments, multiple donors (e.g., two or more donors) are used. In some embodiments, the population of anti-GPC3 CAR-T cells administered according to the methods described herein comprises allogeneic T cells obtained from one or more donors (e.g., one or more healthy human donors).

[0169] An effective amount refers to the amount of the anti-GPC3 CAR-T cells disclosed herein that is necessary to prevent or alleviate at least one or more signs or symptoms of a medical condition (e.g., cancer), and relates to a composition in an amount sufficient to produce a desired effect (e.g., to treat a subject having the medical condition). An effective amount also includes an amount sufficient to prevent or delay the onset of symptoms of a disease, alter the course of symptoms of a disease (e.g., but not limited to, slow the progression of symptoms of a disease), or reverse the symptoms of a disease. It is understood that in any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0170] The effectiveness of a treatment using the anti-GPC3 CAR-T cells disclosed herein can be determined by a skilled clinician. A treatment is considered "effective" if any one or all of the signs or symptoms of the functional target, in one example but at a level that is changed in a favorable manner (e.g., increased by at least 10%), or if other clinically recognized symptoms or markers of the disease (e.g., cancer) are improved or alleviated. Effectiveness can also be measured by the subject not worsening when evaluated by the need for hospitalization or medical intervention (e.g., the progression of the disease stops or at least slows down). Methods for measuring these indicators are known to those of skill in the art and / or are described herein. A treatment includes any treatment of a disease in a subject and includes (1) inhibiting the disease, e.g., stopping or slowing the progression of symptoms, or (2) alleviating the disease, e.g., causing regression of symptoms, and (3) preventing or reducing the likelihood of onset of symptoms.

[0171] In some embodiments, the anti-GPC3 CAR-T cells disclosed herein are used to eliminate GPC3 + cancer cells in a human patient and / or to treat GPC3 + cancer. Optionally, the human patient can be a liver cancer (e.g., HCC), gastric cancer, colorectal cancer, lung cancer, ovarian cancer, skin cancer, or thyroid cancer.

[0172] Combination therapies are also encompassed by the present disclosure. For example, the therapeutic T cells disclosed herein can be used in combination with other therapeutic agents to treat the same indication or to improve the efficacy of the therapeutic T cells and / or reduce the side effects of the therapeutic T cells.

[0173] IV. Kits The present disclosure also provides genetically engineered T cells, kits for use in the manufacture of therapeutic T cells, and kits for therapeutic use.

[0174] In some embodiments, the kits provided herein include components for performing gene editing of one or more of the TRAC gene, β2M gene, TGFBRII gene, Reg-1 gene, and clb-b gene, and optionally, a population of immune cells (e.g., leukopak) in which the gene editing is to be performed. The leukopak sample can be an enriched leukocyte harvest product recovered from peripheral blood. This typically includes various blood cells including monocytes, lymphocytes, platelets, plasma, and red blood cells. The components for gene editing one or more of the target genes can include a suitable endonuclease such as an RNA-guided endonuclease and one or more nucleic acid guides that induce cleavage of one or more suitable genomic sites by this endonuclease. For example, the kit can include a Cas enzyme such as Cas9 and one or more gRNAs targeting the cbl-b gene. Any of the gRNAs specific for these target genes can be included in the kit. Such a kit can further include components for further gene editing, for example, gRNAs and optionally, additional endonucleases for editing other target genes such as Reg-1, TGFBRII, clb-b, β2M, and / or TRAC.

[0175] In some embodiments, the kits provided herein can include a population of genetically engineered T cells disclosed herein, as well as one or more components for making the therapeutic T cells also disclosed herein. Such components can include an endonuclease suitable for gene editing and a nucleic acid encoding a CAR construct of interest. The nucleic acid encoding the CAR can be part of a donor template disclosed herein that can include homology arms adjacent to the CAR coding sequence. Optionally, this donor template can be delivered by a viral vector such as an AAV vector or a lentiviral vector.

[0176] The kit can further include a gRNA specific for the TRAC gene for inserting the CAR coding sequence into the TRAC gene. In other examples, the kit can further include a gRNA specific for the β2M gene for inserting the CAR coding sequence into the β2M gene. In other examples, the kit can further include a gRNA specific for the TGFBRII gene for inserting the CAR coding sequence into the TGFBRII gene. In other examples, the kit can further include a gRNA specific for the Reg-1 gene for inserting the CAR coding sequence into the Reg-1 gene. In still other examples, the kit can further include a gRNA specific for the cbl-b gene for inserting the CAR coding sequence into the cbl-b gene.

[0177] In still other embodiments, the kits disclosed herein can include a population of disclosed therapeutic T cells for the intended therapeutic purpose.

[0178] Any of the kits disclosed herein can further include instructions regarding the generation of therapeutic T cells or the therapeutic application of therapeutic T cells. In some examples, the instructions included can include an explanation of the use of gene editing components for genetically engineering one or more of the target genes disclosed herein. In other examples, the instructions included can include an explanation of the method for introducing a nucleic acid encoding a CAR construct into T cells to generate therapeutic T cells.

[0179] Alternatively, the kit may further include instructions regarding the administration of the therapeutic T cells disclosed herein to achieve the intended activity (e.g., elimination of disease cells targeted by a CAR expressed on a therapeutic T cell). The kit may further include an explanation for selecting a subject suitable for treatment based on identifying whether the subject is in need of treatment. Instructions regarding the use of the therapeutic T cells described herein generally include information regarding the dosage, dosing schedule, and route of administration for the intended treatment. The container may be a unit volume, a bulk package (e.g., a multi-dose package), or a sub-unit dose. The instructions provided within the kits of the present disclosure are typically written instructions on a label or package insert. This label or package insert indicates that the therapeutic T cells are used to treat, delay the onset of, and / or alleviate a disease or disorder in a subject.

[0180] The kits provided herein are present within suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Similarly contemplated is a package for use in combination with a specific device such as an infusion device for administering the therapeutic T cells. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). This container may also have a sterile access port.

[0181] The kit may optionally provide additional components such as buffer materials and explanatory information. Usually, the kit includes a container and a label or package insert on or associated with the container. In some embodiments, the present disclosure provides a manufactured article comprising the contents of the kit described above.

[0182] Sequence Listing

[0183]

Table 1

[0184]

Table 2

[0185]

Table 3

[0186]

Table 4

[0187]

Table 5

[0188]

Table 6

[0189]

Table 7

[0190]

Table 8

[0191]

Table 9

[0192]

Table 10

[0193]

Table 11

[0194]

Table 12

[0195]

Table 13

[0196]

Table 14

[0197]

Table 15

[0198] General technology In the practice of the present disclosure, unless otherwise indicated, conventional techniques in the fields of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology within the scope of the art are used. Such techniques are described in detail in the following literature, among others: for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R.I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds. 1993-8) J.Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988 - 1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985>>; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984>>; Animal Cell Culture (R.I. Freshney, ed. (1986>>; Immobilized Cells and Enzymes (lRL Press, (1986>>; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

[0199] Those skilled in the art will be able to make the most of the present invention based on the above description without further elaboration. Therefore, the following specific embodiments should be construed as merely illustrative and not limiting the rest of the present disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter mentioned herein.

Example

[0200] Some aspects of the embodiments discussed above are further disclosed in detail in the following examples, which are not intended to limit the scope of the present disclosure in any way.

[0201] Example 1. TRAC - / B2M - / Production and Characterization of Anti-Glypican 3 (GPC3) CAR+ T Cells This example describes the generation and characterization of allogeneic human T cells that lack the expression of the TRAC gene and the β2 gene and express a chimeric antigen receptor (CAR) targeting glypican 3 (GPC3) (anti-GPC3 CAR).

[0202] Generation of Anti-GPC3 CAR-T Cells Six unique exemplary GPC3 conjugates having either a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain were generated as shown in Table 4.

[0203]

Table 16

[0204] Briefly, PBMCs were thawed and activated with TransAct™. After 0 - 3 days, the cells were electroporated with Cas9:sgRNA RNP complex and transduced with an adeno-associated adenovirus vector (AAV) such that the nucleic acid encoding the anti-GPC3 CAR was inserted at the TRAC locus, genetically engineered TRAC - / B2M - / Anti-GPC3 CAR + T cells were generated.

[0205] This cell was transfected with a sgRNA that forms a RNP with the Cas9 enzyme by one or multiple electroporation events. After this electroporation, this cell was transduced with recombinant AAV to introduce a donor template encoding the anti-GPC3 CAR. Recombinant AAV serotype 6 (AAV6) containing one of the nucleotide sequences encoding the anti-GPC3 CAR listed in Table 1 above was delivered to activated human T cells together with Cas9:sgRNA RNP (1 μM Cas9, 5 μM gRNA). The following sgRNAs were used: TRAC (SEQ ID NO: 38) and β2M (SEQ ID NO: 42). Generally, unmodified versions or modified versions of the sgRNA can be used. Exemplary gRNA sequences are shown in Table 2.

[0206] Evaluation of CAR Expression and Editing Efficiency CAR expression was evaluated by flow cytometry using biotinylated GPC3 antigen (1 μg), followed by incubation with APC-conjugated streptavidin (200 ng). As shown in Figure 1A, CAR expression was similar for all anti-GPC3 CAR constructs. Editing efficiency regarding TRAC and β2M knockout was evaluated by flow cytometry and TIDE analysis. As shown in Figure 1B, similar levels of editing efficiency regarding the TRAC gene and β2M gene were observed in CAR-T cells expressing the anti-GPC3 CAR constructs described above.

[0207] Evaluation of CD4:CD8 T Cell Ratio The frequencies of CD4 T cells and CD8 T cells, as well as the differential profiles of CAR T cells, in these cell cohorts were also determined by flow cytometry. The average frequencies are listed as shown in Figures 2A and 2B. There was no significant change in the CD4+ to CD8+ cell ratio in any of the CAR T cell populations. Figure 2A. All cell populations were highly enriched for central memory cells. Figure 2B.

[0208] Evaluation of Immune Checkpoint Molecules In this cell population, the expression levels of two immune checkpoint molecules, PD1 and Lag3, were evaluated. As an immune checkpoint molecule, PD1 downregulates T cell activity during the immune response to prevent damage to autoimmune tissues (Jubel et al., Front. Immunol. 2020). Lymphocyte activation gene-3 (LAG-3) is an important immune checkpoint associated with cancer, infectious diseases, and autoimmunity (Graydon et al., Front. Immunol. 2021). As a co-inhibitory immune checkpoint, LAG3 inhibits the activation of its host cells and generally promotes a more suppressive immune response. As shown in Figure 3 and Table 5, no significant changes were observed in the PD1 and LAG-3 levels in any of the cell populations.

[0209]

Table 17

[0210] Example 2. In Vitro and In Vivo Cytotoxicity of Anti-GPC3 CAR T Cells This example demonstrates that the anti-GPC3 CAR T cells described in Example 1 have the ability to selectively lyse GPC3 + cancer cells in vitro and in vivo.

[0211] In Vitro Cytotoxicity Anti-GPC3 CAR T cells were seeded at various ratios with HepG2 target cells with high GPC3 expression, A498 cells that do not express GPC3, or Huh7 cells that express GPC3 at low levels. The next day, the surviving target cells and T cells were counted. As shown in Figures 4A - 4C, the CAR T cells specifically killed the target cells (HepG2 cells and Huh7 cells) that express the GPC3 antigen, but did not kill the GPC3-negative target cells (A498 cells). When the target cells expressed high GPC3 levels (e.g., HepG2 target cells), higher cytotoxicity was observed compared to target cells that express low levels of GPC3 (e.g., Huh7 target cells).

[0212] In vivo cytotoxicity Using a mouse xenograft model, the in vivo efficacy of anti-GPC3 CAR T cells in reducing tumor growth was examined.

[0213] Female NSG mice were subcutaneously implanted with HepG2 tumor cells (5×10 6 cells) in 50% Matrigel® / 50% medium in the right flank. When the tumors were palpable, the mice were randomly assigned to 5 groups and injected intravenously with CAR T cells (6×10 6 CAR + cells) per mouse. Tumor volumes were evaluated every few days and are shown in Figure 5 and Table 6. CAR-T cells expressing 1524 and 1525 anti-GPC3 showed the highest anti-tumor activity compared to other anti-GPC3 constructs. N / A indicates that the mice for tumor volume measurement did not survive.

[0214] [Table 18]

[0215] Example 3. Production and Characterization of CAR T Cells with Further Potency Gene Edit This example describes the generation and characterization of allogeneic human TRAC - / B2M - / anti-GPC3 CAR + T cells that were further edited with respect to the TGFBRII gene, the Regnase-1 gene, and the CBLB gene as listed in Table 7.

[0216] [Table 19]

[0217] RAC with further disruption of TGFbRII / Reg-1 or TGFbRII / cbl-b - / B2M - / Generation of anti-GPC3 CAR T cells Briefly, these cells were electroporated with Cas9:sgRNA RNP complexes and transduced with adeno-associated adenovirus vectors (AAV) to generate anti-GPC3 CAR T cells with TRAC, B2M, and TGFbRII knockouts, as well as CLB or Regnase-1 (REG-1) knockouts. An sgRNA that forms an RNP with the Cas9 enzyme was introduced into these T cells by one or more electroporation events. After electroporation, these cells were transduced with recombinant AAV to introduce a donor template encoding anti-GPC3 CAR (see Example 1 and Table 1). Recombinant AAV serotype 6 (AAV6) containing one of the nucleotide sequences encoding one of the anti-GPC3 CARs was delivered to activated human T cells together with Cas9:sgRNA RNP (1 μM Cas9, 5 μM gRNA). The following sgRNAs were used: TRAC (SEQ ID NO: 38), β2M (SEQ ID NO: 42), TGFbRII (SEQ ID NO: 46), REG-1 (SEQ ID NO: 50), and CBLB-T3 (SEQ ID NO: 54). Unmodified or modified versions of the sgRNA can also be used. Exemplary gRNA sequences are shown in Table 2.

[0218] Evaluation of T cell expansion The engineered T cells were counted regularly to evaluate T cell expansion. As shown in Figure 6 and Table 8, knockout of the TGFBRII gene, and the Regnase-1 gene, or the CBLB gene had no effect on cell proliferation because the further edited cells expanded at a similar rate to the TRAC - / B2M - / anti-GPC3 CAR + cells.

[0219]

Table 20

[0220] Evaluation of CAR expression and editing efficiency Flow cytometry using biotinylated GPC3 antigen (1 μg), followed by incubation with APC-conjugated streptavidin (200 ng), was used to evaluate CAR expression. CAR expression was similar for all anti-GPC3 CAR constructs. Figure 7.

[0221] Flow cytometry and TIDE analysis were used to evaluate the editing efficiency of TRAC, β2M, TGFBRII, and Legnase-1 knockouts. Similar levels of editing efficiency were observed for TRAC and β2M in the presence or absence of Legnase-1 and TGFBRII. Figures 8A - 8C. Evaluation of CBLB KO by Western blot analysis showed that CBLB was efficiently knocked out in all anti-GPC3 CAR T cells examined. Figure 9.

[0222] Evaluation of CD4:CD8 T cell ratio The frequencies of CD4 T cells and CD8 T cells in these cell cohorts were also determined by flow cytometry as shown in Figure 10. Disruption of the Legnase-1 gene and TGFBRII gene showed no significant effect on the CD4+ to CD8+ cell ratio in the CAR-T cell population.

[0223] In summary, the results shown in this example indicate that genetic disruption of the TGFbRII gene with only disruption of either the Reg1 gene or the cbl-b gene had no significant effect on the characteristics of CAR-T cells in anti-GPC3 CAR - T cells in which the TRAC gene and B2M gene were disrupted.

[0224] Example 4. In vitro cytotoxicity of anti-GPC3 CAR T cells with effective gene editing This example shows the ability of the anti-GPC3 CAR T cells described in Example 3 above to selectively lyse GPC3+ cancer cells.

[0225] Test 1 Anti-GPC3 CAR T cells were seeded at various ratios with HepG2 target cells with high GPC3 expression or A498 cells that do not express GPC3. The next day, the surviving target cells and T cells were counted. The results are shown in Figures 11A - 11B and Tables 9 - 10 below.

[0226] Considering the loss of target cells during washing after co - culture, the cytotoxicity measurements were normalized as follows: Cytotoxicity CAR-Conc(A) = 100*(1 - (luminescence CAR-Conc(A) / luminescence AAV-Conc(A) )) Where cytotoxicity is the percentage of the reciprocal of the luminescence of "CAR - Conc(A)", which refers to a given ratio of T cells to target cells in the CAR T cell population, divided by the luminescence of "AAV - Conc(A)", which refers to a given ratio of T cells to target cells in the control (AAV -) population. When activated by GPC3+ target cells, an increase in cell expansion was also observed. Cytotoxicity against GPC3 - target cells was not observed as shown in Table 10 below.

[0227]

Table 21

[0228]

Table 22

[0229] Cytokine secretion Cytokine secretion by these CAR T cells in the presence of GPC3 - positive target cells was also measured. As shown in Figures 12A - 12B and Tables 11 - 12, the secretion of IFN - γ and granzyme A showed a dose - dependent increase.

[0230]

Table 23

[0231]

Table 24

[0232] Test 2 The in vitro growth curves of cells in which effective editing has or has not occurred were measured at various days after electroporation. The data are shown in Table 13, and this data indicates that all the cells were in good condition.

[0233] [Table 25]

[0234] Cells in which effective editing has occurred showed lower CAR knock-in and B2M knockout compared to 1524 anti-GPC3 CAR T cells, and - TRAC expression was similar. The ratio of CD4 / CD8 cells in the cell population was similar under all conditions.

[0235] The specific cytotoxicity of these cells against GPC3-expressing cells was measured using HepG2 target cells with high surface expression of GPC3. A498 cells that do not express GPC3 were used as a negative control. Cytotoxicity was measured using chemiluminescence against AAV - cells and is shown in Tables 14 - 15. All cells exhibited good anti-GPC3 specific cytotoxicity. The "E:T ratio" indicates the ratio of effector cells to target cells. "0" indicates that no cytotoxicity was observed.

[0236] [Table 26]

[0237] [Table 27]

[0238] Therefore, all the engineered cells showed good proliferation, specific cytotoxicity against the target, and a normal phenotype when compared to equivalent CAR T cells for in vivo testing.

[0239] Example 5. In Vivo Cytotoxicity of Anti-GPC3 CAR T Cells with Effective Gene Editing The engineered CAR T cells represented in the previous examples were tested in a mouse model of liver cancer.

[0240] Briefly, 10×10 6 Hep3B cancer cells were injected into the left flank of mice (5 mice per cohort), and allowed to grow to approximately 125 mm 3 Once the tumor reached the target volume, 8×10 6 CAR T cells were administered intravenously. These mice were also re-challenged with another administration of tumor cells in the right flank 30 days after CAR T cell administration (day 54 after the start of the test). Any mouse showing a high tumor burden was removed and euthanized. The group with a reduced number of mice is indicated by "*". The tumor volume of the primary tumor was measured regularly and is shown in Table 16. The data indicate that 1524 was effective against the primary tumor compared to the untreated control. The addition of TGFBRII, Legnase-1, or CBLB, which are effective edits, significantly improved the primary tumor control of 1524 CAR T cells.

[0241] [Table 28]

[0242] The tumor volume of the second tumor administered as a re-challenge is shown in Table 17. In this case, a new mouse cohort was used as the untreated control because all the untreated mice bearing the primary tumor were euthanized.

[0243] [Table 29]

[0244] Throughout this experiment, the body weight of the mice was monitored. This data is presented in Table 18 as a measure of the change in body weight from the start of the experiment.

[0245]

Table 30

[0246] Example 6. Generation and Characterization of Anti-GPC3 CAR-T Cells for Autologous Cell Therapy This example describes the construction and characterization of anti-GPC3 CAR-T cells for use in autologous cell therapy.

[0247] To deliver the coding sequence of the anti-GPC3 CAR (see Example 1 and Table 1) to immune cells for the expression of this anti-GPC3 CAR, a lentiviral vector is constructed. Briefly, a pCCL-c-MNDU3-X2 plasmid is constructed that carries the mutated WPRE (mWPRE) and the coding sequence of this anti-GPC3 CAR (as the gene of interest, i.e., GOI). The general structure of this construct is shown below. 5’LTR-Gag’-RRE-cPPT-(promoter)-GOI-mWPRE-3-LTR

[0248] This plasmid carries the Moloney murine leukemia virus (MMLV) LTR (MNDU3) promoter, which is known to be suitable for use in T cells and other white blood cells. This mWPRE can be obtained from a known plasmid that carries it (e.g., pENTR-L5-WPRE-L2, Addgene), or it can be obtained by mutagenesis of the wild-type WPRE.

[0249] The desired cassette carrying "anti-GPC3 CAR-mWPRE" or "MNDU3 promoter-anti-GPC3 CAR-mWPRE" is first constructed on an intermediate plasmid. Briefly, after amplification by PCR and DNA ligation of this cassette with the intermediate plasmid, the ligation mixture is used to transform E. coli cells. The resulting clones are screened to select clones that contain the plasmid carrying the desired cassette. This cassette is removed and cloned into pCCL-c-MNDU3-X2 plasmid. After sequencing to verify the insertion of the desired cassette, the resulting plasmid is used together with a plasmid capable of producing proteins essential for lentivirus packaging (e.g., gag / pol, Reb, and VSV-G) to transduce packaging cells to produce lentivirus particles, which are then harvested.

[0250] Peripheral blood lymphocytes (PBLs) were cultured using GPC3 + Cells are obtained from a human subject that requires cell removal. The cells are electroporated with Cas9:sgRNA RNP complexes for gene editing of TGFbRII, Reg-1, and / or CBLB, and transduced with the lentivirus described above carrying the nucleic acid encoding the anti-GPC3 CAR construct. In some cases, the transduction is performed in the presence of RetroNectin to improve transduction efficiency. The sgRNA sequences for gene editing of TGFbRII gene, REG-1 gene, and CBLB gene are shown in Table 2. The resulting anti-GPC3 CAR-T cells with one or more of the disrupted TGFbRII gene, REG-1 gene, and CBLB gene are collected.

[0251] The ability of these engineered anti-GPC3 CAR-T cells to kill GPC3+ target cells was examined using in vitro proliferation, cytotoxicity, and cytokine release assays and in vivo cytotoxicity, persistence, and cytokine analysis using the NGS mouse model according to the guidelines shown in Examples 2-4 above. Cells recovered from mouse blood, spleen, and other tissues were used to determine the persistence, biodistribution, and phenotype of the engineered anti-GPC3 CAR cells using flow cytometry.

[0252] Example 7. Autologous cell therapy using anti-GPC3 CAR-T cells This example describes autologous cell therapy for a patient with hepatocellular carcinoma (HCC) using anti-GPC3 CAR-T cells prepared from peripheral blood lymphocytes (PBL) obtained from the patient.

[0253] PBL were obtained from a patient with HCC and genetically engineered according to the description shown in Example 6 to produce engineered T cells expressing anti-GPC3 CAR and having one or more of the disrupted TGFbRII gene, REG-1 gene, and CBLB gene.

[0254] The engineered anti-GPC3 CAR-T cells thus generated were administered to the patient. The ability of this anti-GPC3 CAR T cell to reduce tumor burden, persist in vivo, and differentiate in vivo was examined according to conventional practice. Cells recovered from the blood were used to determine the persistence, biodistribution, and phenotype of the engineered anti-GPC3 CAR cells using flow cytometry.

[0255] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless expressly indicated otherwise, each feature disclosed is only an example of a general series of equivalent or similar features.

[0256] From the above description, those skilled in the art can easily grasp the essential characteristics of the present invention and can make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from its spirit and scope. Therefore, other embodiments are also included within the scope of the claims of this patent.

[0257] Equivalents Although several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more of the advantages. Also, each of such changes and / or modifications is to be regarded as being within the scope of the embodiments of the present invention described herein. More broadly, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are intended as examples, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular use for which the teachings of the present invention are used. Those skilled in the art can recognize or confirm many equivalents to the specific embodiments of the present invention described herein using only routine experimentation. Accordingly, the above-described embodiments are presented by way of example only, and it is to be understood that within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced in other ways than specifically described and claimed. Embodiments of the present invention disclosed herein are directed to each individual feature, system, article, material, kit, and / or method described herein. Additionally, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the present invention disclosed herein if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0258] All definitions, when defined and used herein, are to be understood as precedence over dictionary definitions, definitions in incorporated references, and / or ordinary meanings of the defined terms.

[0259] All references, patents, and patent applications disclosed herein are hereby incorporated by reference in their entirety for all purposes to which they are relevant, and may in some cases include the entire document.

[0260] As used in this specification and the claims, the indefinite articles "a" and "an" are to be understood to mean "at least one" unless clearly indicated to the contrary.

[0261] As used in this specification and the claims, the phrase "and / or" is to be understood to mean "either or both" of the elements so conjoined (i.e., elements that are presented in some cases conjunctively and in other cases disjunctively). Multiple elements listed with "and / or" are to be construed in the same manner, i.e., as "one or more" of the elements so conjoined. Other elements may optionally be presented whether or not they are related to the specifically identified elements that are specifically identified by the "and / or" clause. Thus, by way of non-limiting example, a reference to "A and / or B" can, when used in conjunction with open-ended terms such as "comprising", refer, in one embodiment, to only A (optionally including elements other than B), in another embodiment, to only B (optionally including elements other than A), and in yet another embodiment, to both A and B (optionally including other elements).

[0262] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is inclusive, i.e., it should be construed to include one or at least one of the elements of the number or list, but also includes plural and optionally additional unenumerated items. Only terms that clearly indicate the contrary, such as "only one of ~" or "exactly one of ~" or "consisting of ~" when used in the claims, refer to the inclusion of exactly one element of a number of elements or a list of elements. Generally, the term "or" when used in this specification should be construed to indicate only an exclusive alternative when placed before an exclusive term such as "either", "one of ~", "only one of ~", or "exactly one of ~" (i.e., "either one or the other, but not both"). "Consisting essentially of" when used in the claims shall have the ordinary meaning as used in the field of patent law.

[0263] The term "about" as used in this specification means within the tolerance range of a specific value determined by those skilled in the art, which depends in part on the method by which the value is measured or determined (i.e., the limitations of the measurement system). For example, "about" may mean within the acceptable standard deviation according to the practice in the art. Alternatively, "about" may mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and even more preferably up to ±1% of a given value. When a specific value is described in this application and the claims, unless otherwise indicated, the term "about" is implied and means that the specific value is within the tolerance range in relation thereto.

[0264] As used herein, the phrase "at least one" with respect to the recitation of one or more elements in this specification and the claims means at least one element selected from any one or more of the elements in the recitation of the elements, but does not necessarily include at least one of every element specifically recited in the recitation of the elements, and does not necessarily exclude any combination of the elements in the recitation of the elements. By this definition, it is also possible for elements other than those specifically recited in the recitation of the elements referred to by the phrase "at least one" to optionally exist, regardless of whether they are related to those specifically identified elements. Thus, by way of non-limiting example, "at least one of A and B" (or equivalently "at least one of A or B", or equivalently "at least one of A and / or B") can, in one embodiment, refer to at least one that optionally includes a plurality of A and does not include B (and optionally includes elements other than B), in another embodiment, can refer to at least one that optionally includes a plurality of B and does not include A (and optionally includes elements other than A), and in yet another embodiment, can refer to at least one that optionally includes a plurality of A and at least one that optionally includes a plurality of B (and optionally includes other elements), and so on.

[0265] Similarly, unless the contrary is explicitly indicated, in any method claimed herein that includes a plurality of steps or acts, it should also be understood that the order of the steps or acts of this method is not necessarily limited to the order in which the steps or acts of this method are recited.

Claims

**Claim 1** A population of genetically engineered T cells, wherein the genetically engineered T cells comprise: (a) a nucleic acid encoding a chimeric antigen receptor (CAR) that binds to glypican 3 (GPC3) (anti-GPC3 CAR); (b) one or more disrupted genes, (i) a disrupted T cell receptor alpha constant region (TRAC) gene, (ii) a disrupted beta-2-microglobulin (β2M) gene, (iii) a disrupted transforming growth factor beta receptor II (TGFbRII) gene, (iv) a disrupted regnase-1 (Reg1) gene, (v) a disrupted Casitas B-lineage lymphoma proto-oncogene-B (CBLB) gene, or (vi) any combination of one of (i)-(v), and one or more disrupted genes, and a population of genetically engineered T cells. **Claim 2** The anti-GPC3 CAR comprises: (a) an ectodomain that binds to GPC3; (b) a transmembrane domain; and (c) an endodomain comprising (i) a co-stimulatory signaling domain, and (ii) a CD3ζ cytoplasmic signaling domain, and an endodomain, and a population of genetically engineered T cells according to claim 1. **Claim 3** The ectodomain comprises an anti-GPC3 fragment, and the anti-GPC3 fragment is an anti-GPC3 single domain antibody (VHH) or an anti-GPC3 single chain variable fragment (scFv), and a population of genetically engineered T cells according to claim 2. **Claim 4** The ectodomain comprises the anti-GPC3 VHH, and the anti-GPC3 VHH comprises complementarity determining regions (CDRs) identical to those of SEQ ID NO: 9, and a population of genetically engineered T cells according to claim 3. **Claim 5** The anti-GPC3 VHH comprises the amino acid sequence of SEQ ID NO: 9, and a population of genetically engineered T cells according to claim 3. **Claim 6** The ectodomain includes the anti-GPC3 ScFv, and the GPC3 ScFv includes a heavy chain variable (V Ｈ ) region containing CDRs identical to those of SEQ ID NO: 22 and a light chain variable (V Ｌ ) region containing CDRs identical to those of SEQ ID NO: 21, the population of genetically engineered T cells according to claim 3. **Claim 7** The anti-GPC3 scFv comprises V as set forth in SEQ ID NO: 22 Ｈ and V as set forth in SEQ ID NO: 21 Ｌ and a population of genetically engineered T cells according to claim 6. **Claim 8** The anti-GPC3 scFv comprises the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24, and a population of genetically engineered T cells according to claim 7. **Claim 9** The co-stimulatory domain is a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain, and a population of genetically engineered T cells according to any one of claims 2-8. **Claim 10** The transmembrane domain is a CD8 transmembrane domain, and a population of genetically engineered T cells according to any one of claims 2-9. **Claim 11** The anti-GPC3 CAR comprises the amino acid sequence of SEQ ID NO: 11, 14, 26, 29, 32, or 35, and the population of genetically engineered T cells according to claim 2.

12. The one or more disrupted genes in (b) comprise the disrupted TRAC gene and the disrupted β2M gene, and the population of genetically engineered T cells according to any one of claims 1 to 11.

13. The one or more disrupted genes in (b) further comprise the disrupted TGFBRII gene, the disrupted Reg-1 gene, the disrupted CBLB gene, or a combination thereof, and the population of genetically engineered T cells according to claim 12.

14. The one or more disrupted genes in (b) further comprise (i) the disrupted TGFBRII gene and the disrupted Reg-1 gene, or (ii) the disrupted TGFBRII gene and the disrupted CBLB gene, and the population of genetically engineered T cells according to claim 12.

15. The one or more disrupted genes in (b) comprise the disrupted TGFBRII gene, the disrupted Reg-1 gene, the disrupted CBLB gene, or a combination thereof, and the population of genetically engineered T cells according to any one of claims 1 to 11.

16. The population of genetically engineered T cells according to claim 15, comprising the wild-type TRAC gene, the wild-type β2M gene, or a combination thereof.

17. The nucleic acid encoding the anti-GPC3 CAR is inserted into the endogenous locus of the T cells, and the population of genetically engineered T cells according to any one of claims 1 to 16.

18. The endogenous locus is present within the disrupted TRAC gene, the disrupted β2M gene, the disrupted TGFbRII gene, the disrupted Reg-1 gene, or the disrupted CBLB gene, and the population of genetically engineered T cells according to claim 17.

19. The endogenous locus is present within the disrupted TRAC gene, and the population of genetically engineered T cells according to claim 18.

20. The disrupted TRAC gene contains the deletion of SEQ ID NO: 58, and the deletion is optionally replaced with the nucleotide sequence encoding the anti-GPC3 CAR, the population of genetically engineered T cells according to claim 19.

21. The T cells are human T cells, and the human T cells are optionally primary human T cells, the population of genetically engineered T cells according to any one of claims 1 to 20.

22. A method for producing a population of genetically engineered T cells, comprising: (a) to a population of T cells, (i) one or more RNA-guided nucleases, (ii) a T cell receptor alpha chain constant region (TRAC) gene (TRAC gRNA), a beta-2-microglobulin (β2M) gene (β2M gRNA), a TGFbRII gene (TGFBRIIGRNA), a legnase-1 (Reg1) gene (Reg1 gRNA), and / or one or more guide RNAs targeting the Casitas B-lineage lymphoma proto-oncogene-B (CBLB) gene (CBLB gRNA); (iii) delivering a vector containing a nucleic acid encoding the anti-GPC3 CAR; and (b) producing a population of engineered T cells that express the anti-GPC3 CAR and contain one or more of the disrupted TRAC gene, β2M gene, TGFBRII gene, Reg1 gene, and CBLB gene A method comprising.

23. Step (a) comprises delivering the TRAC gRNA and the β2M gRNA to the population of T cells, the method according to claim 22.

24. Step (a) further comprises delivering the TGFBRII gRNA, the Reg1 guide, the CBLB guide, or a combination thereof to the population of T cells, the method according to claim 23.

25. Step (a) further comprises delivering (i) the TGFBRII gRNA and the Reg1 guide, or (ii) the TGFBRII guide and the CBLB guide to the population of T cells, the method according to claim 23.

26. Step (a) comprises delivering the TGFBRII gRNA, the Reg1 guide, the CBLB guide, or a combination thereof to the population of T cells, the method according to claim 22.

27. ​ (i) The TRAC guide is specific to a TRAC gene target sequence comprising the nucleotide sequence of SEQ ID NO: 58; (ii) The β2M guide is specific to a β2M gene target sequence comprising the nucleotide sequence of SEQ ID NO: 60; (iii) The TGFBRII guide is specific to a TGFBRII gene target sequence comprising the nucleotide sequence of SEQ ID NO: 62; (iv) The Reg1 guide is specific to a Reg1 gene target sequence comprising the nucleotide sequence of SEQ ID NO: 64; and / or (v) The CBLB guide is specific to a CBLB gene target sequence comprising the nucleotide sequence of SEQ ID NO: 66, The method according to any one of claims 22 to 26.

28. (i) The TRAC guide comprises a spacer comprising the nucleotide sequence of SEQ ID NO: 39; (ii) The β2M guide comprises a spacer comprising the nucleotide sequence of SEQ ID NO: 43; (iii) The TGFBRII guide comprises a spacer comprising the nucleotide sequence of SEQ ID NO: 47; (iv) The Reg1 guide comprises a spacer comprising the nucleotide sequence of SEQ ID NO: 51; and / or (v) The CBLB guide comprises a spacer comprising the nucleotide sequence of SEQ ID NO: 55, The method according to claim 27.

29. The method according to any one of claims 22 to 28, wherein the TRAC guide, the β2M guide, the TGFBRII guide, the Reg1 guide, and / or the CBLB guide comprises a scaffold sequence.

30. The method according to any one of claims 22 to 29, wherein the TRAC guide, the β2M guide, the TGFBRII guide, the Reg1 guide, and / or the CBLB guide comprises one or more modifications.

31. (i) The TRAC guide comprises the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 38; (ii) The β2M guide comprises the nucleotide sequence of SEQ ID NO: 41 or SEQ ID NO: 42; (iii) The TGFBRII guide comprises the nucleotide sequence of SEQ ID NO: 45 or SEQ ID NO: 46; (iv) The Reg1 guide comprises the nucleotide sequence of SEQ ID NO: 49 or SEQ ID NO: 50; and / or (v) The CBLB guide comprises the nucleotide sequence of SEQ ID NO: 53 or SEQ ID NO: 54, the method according to claim 29 or 30.

32. The one or more RNA-guided nucleases include a Cas9 nuclease, and the Cas9 nuclease is optionally an S. pyogenes Cas9 nuclease, according to any one of claims 22 to 31.

33. The vector of (a)(iii) includes a donor template in which the nucleic acid encoding the anti-GPC3 CAR is adjacent to an upstream fragment and a downstream fragment, and the upstream fragment and the downstream fragment are homologous to the endogenous locus of the T cell, thereby enabling insertion of the nucleic acid encoding the anti-GPC3 CAR into the endogenous locus, according to any one of claims 22 to 32.

34. The endogenous locus is present within the disrupted TRAC gene, the disrupted β2M gene, the disrupted TGFbRII gene, the disrupted Reg-1 gene, or the disrupted CBLB gene, according to claim 33.

35. The endogenous locus is present within the disrupted TRAC gene, according to claim 33.

36. The upstream fragment is set forth in SEQ ID NO: 71, and / or the downstream fragment is set forth in SEQ ID NO: 74, according to claim 35.

37. The anti-GPC3 CAR is as described in any one of claims 2 to 11, according to any one of claims 22 to 36.

38. The vector of (a)(iii) is a viral vector, and the viral vector is optionally an adeno-associated virus (AAV) vector or a lentiviral vector, according to any one of claims 22 to 37.

39. The population of T cells includes human T cells, and the human T cells are optionally human primary T cells, according to any one of claims 22 to 38.

40. The population of T cells is obtained from one or more healthy human donors, according to any one of claims 22 to 39.

41. The population of T cells is obtained from a human patient having GPC3+ cancer, according to any one of claims 22 to 39.

42. A method for treating cancer in a subject, the method comprising administering to a subject in need a population of genetically engineered T cells as described in any one of claims 1 to 21.

43. The method according to claim 42, wherein the subject is a human patient having GPC3+ cancer.

44. The method according to claim 43, wherein the population of genetically engineered T cells is allogeneic to the human patient.

45. The method according to claim 44, wherein the population of genetically engineered T cells is as described in any one of claims 12 to 14.

46. The method according to claim 43, wherein the population of genetically engineered T cells is autologous to the human patient.

47. The method according to claim 46, wherein the population of genetically engineered T cells is as described in claim 15 or 16.

48. The method according to any one of claims 42 to 47, wherein the cancer is liver cancer, gastric cancer, colorectal cancer, lung cancer, ovarian cancer, skin cancer, or thyroid cancer.

49. A chimeric antigen receptor (anti-GPC3 CAR) that binds to glypican 3 (GPC3), the anti-GPC3 CAR as described in any one of claims 2 to 11.

50. The anti-GPC3 CAR according to claim 49, further comprising an N-terminal signal peptide.

51. The anti-GPC3 CAR according to claim 50, comprising the amino acid sequence of SEQ ID NO: 10, 13, 25, 28, 31, or 34.

52. A nucleic acid encoding the anti-GPC3 CAR as described in any one of SEQ ID NOs: 49 to 51.

53. The nucleic acid according to claim 52, which is a vector.

54. The nucleic acid according to claim 53, wherein the vector is a viral vector, and the viral vector is optionally an AAV vector or a lentiviral vector.

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